US20030124704A1 - Nogo receptor homologs - Google Patents

Nogo receptor homologs Download PDF

Info

Publication number
US20030124704A1
US20030124704A1 US09/972,546 US97254601A US2003124704A1 US 20030124704 A1 US20030124704 A1 US 20030124704A1 US 97254601 A US97254601 A US 97254601A US 2003124704 A1 US2003124704 A1 US 2003124704A1
Authority
US
United States
Prior art keywords
ngr
polypeptide
seq
protein
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US09/972,546
Inventor
Stephen Strittmatter
Richard Cate
Dinah Sah
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yale University
Biogen MA Inc
Original Assignee
Yale University
Biogen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US09/972,546 priority Critical patent/US20030124704A1/en
Application filed by Yale University, Biogen Inc filed Critical Yale University
Assigned to YALE UNIVERSITY reassignment YALE UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: STRITTMATTER, STEPHEN M.
Assigned to BIOGEN, INC. reassignment BIOGEN, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CATE, RICHARD L., SAH, DINAH W.Y.
Publication of US20030124704A1 publication Critical patent/US20030124704A1/en
Priority to US10/735,256 priority patent/US7173118B2/en
Priority to US11/544,013 priority patent/US7456255B2/en
Assigned to BIOGEN IDEC MA INC. reassignment BIOGEN IDEC MA INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: BIOGEN, INC.
Priority to US12/277,187 priority patent/US20090175850A1/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: YALE UNIVERSITY
Priority to US12/952,747 priority patent/US20110129477A1/en
Assigned to NIH-DEITR reassignment NIH-DEITR CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: YALE UNIVERSITY - NEW HAVEN, CT
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates to neurology and molecular biology. More particularly, the invention relates to CNS neurons and axonal growth
  • Receptors on neurons are particularly important in the development of the nervous system during embryogenesis.
  • the neurons form connections with target cells during development through axonal extension of the neurons toward the target cells in a receptor-mediated process.
  • Axons and dendrites have a specialized region of their distal tips known as the growth cone.
  • Growth cones enable the neuron to sense the local environment through a receptor-mediated process and direct the movement of the axon or dendrite of the neuron toward the neuron's target cell. This process is known as elongation.
  • Growth cones can be sensitive to several guidance cues, for example, surface adhesiveness, growth factors, neurotransmitters and electric fields. The guidance of growth at the cone depends on various classes of adhesion molecules, intercellular signals, as well as factors that stimulate and inhibit growth cones.
  • damaged neurons do not elongate in the central nervous system (CNS) following injury due to trauma or disease, whereas axons in the peripheral nervous system (PNS) regenerate readily.
  • CNS central nervous system
  • PNS peripheral nervous system
  • axon growth inhibitors are predominantly associated with myelin and constitute an important barrier to regeneration.
  • Axon growth inhibitors are present in CNS-derived myelin and the plasma membrane of oligodendrocytes that synthesize myelin in the CNS (Schwab et al., (1993) Annu. Rev. Neurosci. 16, 565-595).
  • Myelin-associated inhibitors appear to be a primary contributor to the failure of CNS axon regeneration in vivo after an interruption of axonal continuity, whereas other non-myelin associated axon growth inhibitors in the CNS may play a lesser role.
  • These inhibitors block axonal regeneration following neuronal injury due to trauma, stroke or viral infection.
  • Nogo is a 250 kDa myelin-associated axon growth inhibitor that was originally characterized based on the effects of the purified protein in vitro and monoclonal antibodies that neutralize the protein's activity (Schwab (1990) Exp. Neurol. 109, 2-5).
  • the Nogo cDNA was first identified through random analysis of brain cDNA and had no suggested function (Nagase et al., (1998) DNA Res. 5, 355-364).
  • Nogo has been shown to be the primary component of CNS myelin responsible for inhibiting axonal elongation and regeneration.
  • Nogo's selective expression by oligodendrocytes and not by Schwann cells is consistent with the inhibitory effects of CNS myelin, in contrast to P.S. myelin (GrandPre et al., (2000) Nature 403, 434-439).
  • Nogo inhibits axonal elongation and causes growth cone collapse (Spillmann et al., (1998) J. Biol. Chem. 272, 19283-19293).
  • Antibodies against Nogo have been shown to block most of the inhibitory action of CNS myelin on neurite growth in vitro (Spillmann et al., (1998) J. Biol. Chem. 272:19283-19293). These experiments indicate that Nogo is the main component of CNS myelin responsible for inhibition of axonal elongation in culture. Furthermore, in vivo, the IN-1 antibody has been shown to enhance axonal regeneration after spinal cord injury, resulting in recovery of behaviors such as contact placing and stride length (Schnell and Schwab (1990) Nature 343, 269-272; Bregman et al., (1995) Nature 378, 498-501). Thus, there is substantial evidence that Nogo is a disease-relevant molecular target. Agents that interfere with the binding of Nogo to its receptor would be expected to improve axonal regeneration in clinical states in which axons have been damaged, and improve patient outcome.
  • NgR2 and NgR3 Genes encoding homologs (NgR2 and NgR3) of a Nogo receptor (NgR1) in mice and humans have been discovered.
  • Various domains in the polypeptides encoded by the NgR2 and NgR3 genes have been identified and compared to domains in mouse and human NgR1 polypeptides. This comparison has led to identification of a consensus sequence (NgR consensus sequence) that characterizes a family of proteins (NgR family). Based on these and other discoveries, the invention features molecules and methods for modulating axonal growth in CNS neurons.
  • the invention provides a polypeptide that contains a polypeptide containing a tryptophan rich LRRCT domain consisting of the amino acid sequence: N X 1 W X 2 C X 3 C R A R X 4 L W X 5 W X 6 X 7 X 8 X 9 R X 10 S S S X 11 V [SEQ ID NO. 19] X 12 C X 13 X 14 P X 15 X 16 X 17 X 18 X 19 X 20 D L X 21 X 22 L X 23 X 24 X 25 D X 26 X 27 X 28 C
  • X is any protein amino acid or a gap, and the polypeptide does not include amino acid sequence from residue 260 to 309 of SEQ ID NO: 5 (human NgR1) or SEQ ID NO: 17 (mouse NgR1).
  • X17 and X23 are (independently) arginine or lysine.
  • the amino acid sequence of the LRRCT domain is residues 261-310 of SEQ ID NO:2, or residues 261-310 of SEQ ID NO: 2 with up to 10 conservative amino acid substitutions.
  • the polypeptide contains the following NTLRRCT amino acid sequence: C P X 1 X 2 C X 3 C Y X 4 X 5 P X 6 X 7 T X 8 S C X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 P [SEQ ID NO: 18] X 17 X 18 X 19 P X 20 X 21 X 22 X 23 R X 24 F L X 25 X 26 N X 27 I X 28 X 29 X 30 X 31 X 32 X 33 X 34 F X 35 X 36 X 37 X 38 X 39 X 40 X 41 X 42 L W X 43 X 44 S N X 45 X 46 X 47 X 48 I X 49 X 50 X 51 X 52 F X 53 X 54 X 55 X 56 X 57 L E X 58 L D L X 59 D N X 60 X 61 L X 62 X 63
  • X is any amino acid residue or a gap and wherein the polypeptide is not the polypeptide of SEQ ID NO: 5 (human NgR1) or SEQ ID NO: 17 (mouse NgR1).
  • X 6 , X 37 and X 38 may represent a gap.
  • Specific examples of polypeptides of the invention are SEQ ID NO: 2 (human NgR2), SEQ ID NO: 4 (mouse NgR3), and SEQ ID NO: 14 (human NgR3).
  • the polypeptide contains: (a) a NTLRRCT domain, and (b) less than a complete CTS domain, provided that a partial CTS domain, if present, consists of no more than the first 39 amino acids of the CTS domain. While the polypeptide may contain a functional GPI domain, a functional GPI domain may be absent, e.g., when a soluble polypeptide is desired.
  • a polypeptide of the invention optionally includes an amino acid sequence of a heterologous polypeptide, e.g., an Fc portion of an antibody.
  • the invention also provides a nucleic acid encoding an above-described polypeptide; a vector containing the nucleic acid, which nucleic acid may be operably linked to an expression control sequence; and a transformed host cell containing the vector.
  • a method of producing a polypeptide of the invention is also provided. The method includes introducing a nucleic acid encoding the above-described polypeptide into a host cell, culturing the cell under conditions suitable for expression of the polypeptide, and recovering the polypeptide.
  • the invention also provides an antisense molecule whose nucleotide sequence is complementary to a nucleotide sequence encoding a polypeptide selected from the group consisting of: a polypeptide consisting of residues 311-395 of SEQ ID NO: 2, a polypeptide consisting of residues 256-396 of SEQ ID NO: 14 and a polypeptide consisting of residues 321-438 of SEQ ID NO: 4, wherein the nucleic acid is from 8 to 100 nucleotides in length, e.g., about 20, 30, 40, 50, 60, 70, 80 or 90 nucleotides.
  • the invention also provides a nucleic acid encoding such an antisense molecule.
  • the invention also provides an antibody that binds to an above-described polypeptide.
  • Polypeptides or antibodies of the invention can be formulated into pharmaceutical compositions containing the polypeptide or antibody and a pharmaceutically acceptable carrier.
  • the invention also provides a method for decreasing inhibition of axonal growth of a CNS neuron.
  • the method includes the step of contacting the neuron with an effective amount of a polypeptide or antibody of the invention.
  • the invention also provides a method for treating a central nervous system disease, disorder or injury.
  • the method includes administering to a mammal, e.g., a human, an effective amount of a polypeptide or antibody of the invention.
  • Exemplary diseases, disorders and injuries that may be treated using molecules and methods of the invention include, but are not limited to, cerebral injury, spinal cord injury, stroke, demyelinating diseases, e.g., multiple sclerosis, monophasic demyelination, encephalomyelitis, multifocal leukoencephalopathy, panencephalitis, Marchiafava-Bignami disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodystrophy and Krabbe's disease.
  • demyelinating diseases e.g., multiple sclerosis, monophasic demyelination, encephalomyelitis, multifocal leukoencephalopathy, panencephalitis, Marchiafava-Bignami disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodystrophy and Krabbe's disease.
  • the invention also provides a method for identifying a molecule that binds a polypeptide of the invention.
  • the method includes the steps of: (a) providing a polypeptide of the invention; (b) contacting the polypeptide with the candidate molecule; and (c) detecting binding of the candidate molecule to the polypeptide.
  • FIGS. 1 A- 1 B shows an alignment of NgR2 (SEQ ID NO:2) and NgR3 (SEQ ID NO:4) with the known NgR, NgR1 (SEQ ID NO:5) and the Consensus Sequence (SEQ ID NO:6).
  • FIG. 2. mNgR3 does not bind hNogoA(1055-1120).
  • COS-7 cells were transfected with vectors encoding myc-NgR1 or myc-NgR3, fixed, and stained with anti-myc antibodies or AP-hNogoA(1055-1120).
  • FIG. 3 An alignment of the amino acid sequences of human NgR1, murine NgR1, murine NgR3, human NgR3 and human NgR2. Numbering begins with amino acid #1 of murine NgR3.
  • the consensus sequence is listed below.
  • the LRR NT domain is indicated by a shaded box; domains LLR 1, LLR 3, LLR 5, and LLR 7 are indicated by open boxes; LLR 2, LLR 4, LLR 6 and LLR 8 are indicated by shaded boxes; and the LLR CT domain is indicated by a shaded box.
  • Amino acids in bold in LLR 8 indicate a conserved glycosylation sites.
  • a dot indicates conserved cystine residue in LRR4. Box at C terminus indicates putative GPI signals.
  • the present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) encoding NgR homologs, referred to herein as NgR.
  • NgR polynucleotides
  • the abbreviation in lower case (NgR) refers to a gene, cDNA, RNA or nucleic acid sequence
  • the upper case version (NgR) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence.
  • NgR2 is a human NgR homolog
  • NgR3 is a murine-derived NgR homolog
  • NgR1 is the known NgR identified by Dr. Stephen Strittmatter.
  • Known NgRs are herein referred to as “NgRs.”
  • DNA polynucleotides of the invention include genomic DNA, cDNA and DNA that has been chemically synthesized in whole or in part.
  • Standard reference works setting forth the general principles of recombinant DNA technology known to those of skill in the art include Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York (1998); Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.
  • the term “axon” refers to a long cellular protrusion from a neuron, whereby action potentials are conducted, either to or from the cell body.
  • axonal growth refers to an extension of the long process or axon, originating at the cell body and proceeded by the growth cone.
  • central nervous system disorder refers to any pathological state associated with abnormal function of the central nervous system (CNS).
  • CNS central nervous system
  • the term includes, but is not limited to, altered CNS function resulting from physical trauma to cerebral tissue, viral infection, autoimmune machanisms and genetic mutation.
  • the term “demyelinating disease” refers to a pathological disorder characterized by the degradation of the myelin sheath of the oligodendrocyte cell membrane.
  • growth cone refers to a specialized region at the tip of a growing neurite that is responsible for sensing the local environment and moving the axon toward its appropriate synaptic target cell.
  • growth cone movement refers to the extension or collapse of the growth cone toward a neuron's target cell.
  • the term “neurite” refers to a process growing out of a neuron. As it is sometimes difficult to distinguish a dendrite from in axon in culture, the term “neurite” is used for both.
  • oligodendrocyte refers to a neuroglial cell of the CNS whose function is to myelinate CNS axons.
  • region is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.
  • domain is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule.
  • Domains may be co-extensive with regions or portions thereof, domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region.
  • NgR protein domains include, but are not limited to, the signal peptide, extracellular (i.e., N-terminal) domain, and leucine-rich repeat domains.
  • the term “activity” refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e., having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event.
  • Such activities may be measured by assays such as competitive inhibition of NgR1 binding to Nogo assays wherein, for example, unlabeled, soluble NgR2 is added to an assay system in increasing concentrations to inhibit the binding of Nogo to NgR1 expressed on the surface of CHO cells.
  • assays such as competitive inhibition of NgR1 binding to Nogo assays wherein, for example, unlabeled, soluble NgR2 is added to an assay system in increasing concentrations to inhibit the binding of Nogo to NgR1 expressed on the surface of CHO cells.
  • assays such as competitive inhibition of NgR1 binding to Nogo assays wherein, for example, unlabeled, soluble NgR2 is added to an assay system in increasing concentrations to inhibit the binding of Nogo to NgR1 expressed on the surface of CHO cells.
  • one may assess the ability of neurons to extend across lesions caused by nerve damage (as in Schnell and Schwab (1990) Nature 343, 269-272) following inhibition of
  • antibody is meant to refer to complete, intact antibodies, and Fab, Fab′, F(ab)2, and other fragments thereof
  • Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies, anti-anti-idiotypic antibodies, and humanized antibodies.
  • binding means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc.
  • the physical interaction, the binding can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates.
  • the term “compound” means any identifiable chemical or molecule, including, but not limited to, small molecules, peptides, proteins, sugars, nucleotides or nucleic acids, and such compound can be natural or synthetic.
  • the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule.
  • the term “contacting” means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention.
  • the polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc.
  • Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoding the NgR or fragment thereof.
  • homologous nucleotide sequence refers to sequences characterized by an identity at the nucleotide level, or a homology at the amino acid level, of at least the specified percentage.
  • Homologous nucleotide sequences include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals.
  • Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence does not, however, include the nucleotide sequence encoding NgR1.
  • Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity.
  • a homologous amino acid sequence does not, however, include the amino acid sequence encoding other known NgRs.
  • Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman ( Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein by reference in its entirety).
  • isolated nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that is substantially free of nucleic acids encoding other proteins with which it is associated in nature, i.e., a nucleic acid that has been removed from its native environment.
  • isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated NgR nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an “isolated” nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
  • heterologous refers to a nucleotide or amino acid sequence that is a different, or non-corresponding sequence, or a sequence derived from a different species.
  • a mouse NgR nucleotide or amino acid sequence is heterologous to a human NgR nucleotide or amino acid sequence
  • a human NgR nucleic or amino acid sequence is heterologous to a human immunoglobulin nucleotide or amino acid sequence.
  • a “soluble NgR polypeptide” is a NgR polypeptide that does not anchor itself in a membrane.
  • Such soluble polypeptides include, for example, NgR2 and NgR3 polypeptides that lack a sufficient portion of their GPI anchor signal to anchor the polypeptide or are modified such that the GPI anchor signal is not adequate to result in replacement of the peptide with a GPI anchor.
  • up to 5, 10, 20 or 25 amino acids are removed from the C-terminus of NgR2 or NgR3 to make the respective proteins soluble.
  • soluble NgR polypeptides include full-length or truncated (e.g., with internal deletions) NgR.
  • Soluble NgR polypeptides may include the entire NgR protein up to the putative GPI signal sequence (e.g., amino acid 1 to about amino acid 395 of NgR2, and from amino acid 1 to about amino acid 438 of NgR3).
  • the signal peptide of the proteins may be removed or truncated (e.g., all or part of the signal sequence of NgR2, which spans amino acid 1 to about amino acid 30 of SEQ ID NO:2, may be removed; all or part of the signal sequence of NgR3, which spans amino acid 1 to about amino acid 40 of SEQ ID NO:4, may be removed).
  • the mature NgR2 (SEQ ID NO:8) and the mature NgR3 (SEQ ID NO:9) are used.
  • Soluble NgR polypeptides include at least one of the putative ligand-binding portions of NgR, including the first cysteine-rich region (SEQ ID NO:10, the leucine repeat region (SEQ ID NO:12) and the second cysteine-rich region (SEQ ID NO:11).
  • soluble NgR polypeptides consist of amino acid 1 through about amino acid 395 of SEQ ID NO:2, or amino acid 1 through about amino acid 438 of SEQ ID NO:4.
  • the soluble NgR polypeptides are fusion proteins that contain amino acids 30 through about amino acid 395 of mature NgR2 or amino acid 40 through about amino acid 438 of NgR3, the C-terminal 10 amino acids of a human IgG 1 hinge region containing the two cysteine residues thought to participate in interchain disulfide bonding, and the CH2 and CH3 regions of a human IgG1 heavy chain constant domain.
  • This type of recombinant protein is designed to modulate inhibition of axonal elongation through inhibition of the Nogo ligand binding to NgR1, or by inhibiting the ligand of the NgR from interacting with cell surface NgR.
  • the NgR portion of the fusion binds to the Nogo ligand and the IgG1 portion binds to the Fc ⁇ RI (macrophage) and Fc ⁇ III (NK cells and neutrophils) receptors.
  • the production of the soluble polypeptides useful in this invention may be achieved by a variety of methods known in the art.
  • the polypeptides may be derived from intact transmembrane NgR molecules by proteolysis using specific endopeptidases in combination with exopeptidases, Edman degradation, or both.
  • the intact NgR molecule in turn, may be purified from its natural source using conventional methods.
  • the intact NgR may be produced by known recombinant DNA techniques using cDNAs, expression vectors and well-known techniques for recombinant gene expression.
  • the soluble polypeptides useful in the present invention are produced directly, thus eliminating the need for an entire NgR as a starting material.
  • This may be achieved by conventional chemical synthesis techniques or by well-known recombinant DNA techniques wherein only those DNA sequences which encode the desired peptides are expressed in transformed hosts.
  • a gene which encodes the desired soluble NgR polypeptide may be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired soluble NgR polypeptide.
  • Specific DNA sequences coding for the desired peptide also can be derived from the full-length DNA sequence by isolation of specific restriction endonuclease fragments or by PCR synthesis of the specified region from cDNA.
  • a nucleic acid molecule of the present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs:1, 3 or a complement of either of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • NgR nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., MOLECULAR CLONING: A LABORATORY MANUAL 2 nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993).
  • a nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to NgR nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • the terms “modulates” or “modifies” means an increase or decrease in the amount, quality, or effect of a particular activity or protein.
  • oligonucleotide refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may be used as probes.
  • probe refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies.
  • preventing refers to decreasing the probability that an organism contracts or develops an abnormal condition.
  • treating refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism.
  • a therapeutic effect refers to the inhibition or activation factors causing or contributing to the abnormal condition.
  • a therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition.
  • a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells.
  • Compounds demonstrating efficacy against abnormal conditions can be identified as described herein.
  • abnormal condition refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism.
  • An abnormal condition can relate to cell proliferation, cell differentiation, cell signaling, or cell survival.
  • An abnormal condition may also include obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid uptake and metabolism.
  • Abnormal cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus and inflammation.
  • Abnormal differentiation conditions include, for example, neurodegenerative disorders, slow wound healing rates and slow tissue grafting healing rates.
  • Abnormal cell signaling conditions include, for example, psychiatric disorders involving excess neurotransmitter activity.
  • Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated.
  • apoptosis programmed cell death
  • a number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death.
  • administering relates to a method of incorporating a compound into cells or tissues of an organism.
  • the abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism.
  • Cells existing outside the organism can be maintained or grown in cell culture dishes.
  • many techniques exist in the art to administer compounds including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications.
  • multiple techniques exist in the art to administer the compounds including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques.
  • the abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism.
  • the effect of administering a compound on organism function can then be monitored.
  • the organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most preferably a human.
  • amplification it is meant increased numbers of DNA or RNA in a cell compared with normal cells. “Amplification” as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA. In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, compared to the basal level.
  • amino acid sequences are presented in the amino to carboxy direction, from left to right.
  • the amino and carboxy groups are not presented in the sequence.
  • the nucleotide sequences are presented by single strand only, in the 5′ to 3′ direction, from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by three letters code.
  • Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or “spliced out.” RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a NgR polypeptide, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts.
  • Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild-type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants arising from in vitro manipulation).
  • the invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding NgR (conventionally followed by second-strand synthesis of a complementary strand to provide a double-stranded DNA).
  • a preferred DNA of the invention comprises a double stranded molecule comprising the coding molecule (i.e., the “coding strand”) along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA.
  • coding strand the coding molecule
  • complementary molecule the “non-coding strand” or “complement” having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA.
  • other polynucleotides encoding NgR polypeptides as shown in SEQ ID NO:3, which comprises murine NgR homolog, NgR3.
  • nucleotide sequences that encode at least a portion of a NgR polypeptide that has at least one biological function of a NgR. More preferred are nucleotide sequences that encode a portion of NgR that encodes at least the mature NgR without the hydrophobic C-terminal GPI signal. Also preferred are nucleotide sequences that encode the portion of NgR that encodes at least the ligand-binding region of NgR.
  • the invention further embraces other species, preferably mammalian, homologs of the human NgR DNA.
  • Species homologs sometimes referred to as “orthologs,” in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with human DNA of the invention.
  • percent sequence “homology” with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the NgR sequences set forth in SEQ ID NOs:1, 3 or 13, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
  • polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art.
  • Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related NgR polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR).
  • related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to NgR and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of NgR.
  • Non-human species genes encoding proteins homologous to NgR can also be identified by Southern and/or PCR analysis and are useful in animal models for NgR disorders. Knowledge of the sequence of a human NgR DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding NgR expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express NgR. Polynucleotides of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a NgR locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies.
  • NgR-encoding polynucleotides comprising at least 6, and preferably at least 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding NgR.
  • fragments of polynucleotides of the invention comprise sequences unique to the NgR-encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., “specifically”) to polynucleotides encoding NgR (or fragments thereof).
  • Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences.
  • Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent and enzymatic labeling.
  • Fragments of polynucleotides are particularly useful as probes for detection of full-length or fragment of NgR polynucleotides.
  • One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding NgR, or used to detect variations in a polynucleotide sequence encoding NgR.
  • the invention also embraces DNAs encoding NgR polypeptides that hybridize under moderately stringent or high stringency conditions to the noncoding strand, or complement, of the polynucleotide in any of SEQ ID NOs:1 or 3.
  • Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3. 1?6.3.6.
  • the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homologous to each other typically remain hybridized to each other.
  • a non-limiting example of stringent hybridization conditions hybridization in a high salt buffer comprising 6 ⁇ SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 mg/ml denatured salmon sperm DNA at 65° C. This hybridization is followed by one or more washes in 0.2 ⁇ SSC, 0.01% BSA at 50° C.
  • An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOs:1 or 3 corresponds to a naturally occurring nucleic acid molecule.
  • a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • stringent hybridization conditions means: 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% (wt/vol) dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1 ⁇ SSC and 1% SDS.
  • vectors or recombinant expression vectors, comprising any of the nucleic acid molecules described above.
  • Vectors are used herein either to amplify DNA or RNA encoding NgR and/or to express DNA which encodes NgR.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector, wherein additional DNA segments can be ligated into the viral genome.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), that serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67, 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione-S-transferase
  • Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69, 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in E. coil is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128.
  • Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coil (Wada et al., (1992) Nucleic Acids Res. 20, 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the NgR expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6, 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30, 933-943), pJRY88 (Schultz et al., (1987) Gene 54, 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).
  • NgR can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al., (1983) Mol. Cell. Biol. 3, 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170, 31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed (1987) Nature 329, 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6, 187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.
  • suitable expression systems for both prokaryotic and eukaryotic cells are examples of cells.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1, 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43, 235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
  • promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249, 374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense NgR mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue-specific or cell-type-specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination).
  • Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses and retroviruses.
  • Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech).
  • expression vectors include, but are not limited to, pSPORTTM vectors, pGEMTM vectors (Promega), pPROEXvectorsTM (LTI, Bethesda, Md.), BluescriptTM vectors (Stratagene), pQETM vectors (Qiagen), pSE420TM (Invitrogen) and pYES2TM(Invitrogen).
  • Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding NgR is operably linked or connected to suitable control sequences capable of effecting the expression of the NgR in a suitable host.
  • DNA regions are operably linked or connected when they are functionally related to each other.
  • a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence.
  • Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen.
  • control sequences include, but are not limited to a transcriptional promoter, enhancers, an optional operator sequence to control transcription, polyadenylation signals, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation.
  • a transcriptional promoter such as a promoter, enhancers, an optional operator sequence to control transcription, polyadenylation signals, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation.
  • regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NgR proteins, mutant forms of NgR, fusion proteins, etc.).
  • Preferred vectors preferably contain a promoter that is recognized by the host organism.
  • the promoter sequences of the present invention may be prokaryotic, eukaryotic or viral.
  • suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (THE BACTERIOPHAGE LAMBDA, Hershey, A. D. (Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; LAMBDA II, Hendrix, R. W. (Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  • Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine and human metallothionein.
  • Additional regulatory sequences can also be included in preferred vectors.
  • Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno sequence of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda.
  • the Shine-Dalgarno sequence may be directly followed by DNA encoding NgR and result in the expression of the mature NgR protein.
  • suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells.
  • the transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra.
  • An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
  • a selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat. No. 4,399,216).
  • Nucleotide sequences encoding NgR may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al., (1983) Mol. Cell. Biol. 3:280, Cosman et al. (1986) Mol. Immunol. 23:935, Cosman et al., (1984) Nature 312:768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.
  • host cells including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded NgR polypeptide.
  • the cell produces little or no endogenous NgR polypeptide.
  • Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector.
  • Methods for introducing DNA into the host cell include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells and protoplasts.
  • Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate and mammalian cells systems.
  • Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with NgR.
  • Host cells of the invention are also useful in methods for the large-scale production of NgR polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like.
  • HPLC high pressure liquid chromatography
  • Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label or chelating moiety that is recognized by a specific binding partner or agent.
  • the purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
  • NgR DNA sequences allows for modification of cells to permit, or increase, expression of endogenous NgR.
  • Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring NgR promoter with all or part of a heterologous promoter so that the cells express NgR at higher levels.
  • the heterologous promoter is inserted in such a manner that it is operatively linked to endogenous NgR encoding sequences.
  • amplifiable marker DNA e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase
  • intron DNA may be inserted along with the heterologous promoter DNA. If linked to the NgR coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the NgR coding sequences in the cells.
  • the DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination or “knock-out” strategies; see Capecchi, Science 244:1288-1292 (1989)) of animals that fail to express functional NgR or that express a variant of NgR.
  • animals especially small laboratory animals such as rats, rabbits and mice
  • Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces and Staphylococcus.
  • eukaryotic cells are cells of higher eukaryotes.
  • Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells.
  • Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, Eds. (1973), which is incorporated herein by reference in its entirety).
  • yeast cell may be employed as a host cell.
  • Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia and Kluveromyces.
  • Preferred yeast hosts are S. cerevisiae and P. pastoris.
  • Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination and a selectable marker gene.
  • ARS autonomously replication sequence
  • Shuttle vectors for replication in both yeast and E. coli are also included herein.
  • insect cells may be used as host cells.
  • the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., Bio/Technology, 1988, 6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, O'Rielly et al. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No. 4,879,236, each of which is incorporated herein by reference in its entirety).
  • the MAXBACTM complete baculovirus expression system can, for example, be used for production in insect cells.
  • Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those that confer resistance to drugs, such as G418, hygromycin, dihydrofolate reductase (DHFR) and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NgR or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • polypeptides of the invention including forms of NgR2 and NgR3, soluble forms of NgR, chimeric NgR polypeptides, NgR/Ig fusions and fragments and variations of each of the above are expressed in Chinese Hamster Ovary (CHO) cells.
  • the vectors for CHO expression include, but are not limited to, pA1-11, pXT1, pRc/CMV, pRc/RSV and pcDNAINeo.
  • the promoter is not specifically limited provided it effectively promotes expression in CHO cells. Examples of suitable promoters are: SR ⁇ , SV40, LTR, CMV, and HSV-TK. Of these, CMV and Sr ⁇ promoters are preferred.
  • the expression vectors may contain enhancers, splicing signals, polyadenylation signals, selectable markers and an SV40 replication origin.
  • selectable markers include, but are not limited to the dihydrofolate reductase (DHFR) gene which provides resistance to methotrexate (MTX), the ampicillin resistance gene, and the neomycin resistance gene.
  • DHFR dihydrofolate reductase
  • Examples of the expression vectors each containing the DNA coding for NgR, portions, fragments and soluble constructs thereof include the vector (such as one described above), into which the promoter is operably linked (preferably upstream) to the nucleotide sequence encoding the desired NgR construct; a polyadenylation signal downstream from the nucleotide sequence encoding the NgR construct; and, preferably, the vector includes an operable DHFR gene.
  • the ampicillin resistant gene is also operably contained in the vector.
  • CHO cell lacking the DHFR gene (Urlaub, G. et al., (1980) Proc. Natl. Acad. Sci. USA 77, 4216-4220) and CHO-K1 ( Proc. Natl. Acad. Sci. USA 60, 1275 (1968)) are suitable for use.
  • NgR expression vectors prepared as above are introduced into CHO cells by any known method, including, but not limited to the calcium phosphate method (Graham and van der Eb (1973) Virol 52, 456-467) and electroporation (Nuemann et al., (1982) EMBO J. 1, 841-845).
  • Transformants carrying the expression vectors are selected based on the above-mentioned selectable markers. Repeated clonal selection of the transformants using the selectable markers allows selection of stable cell lines having high expression of the NgR constructs. Increased MTX concentrations in the selection medium allows gene amplification and greater expression of the desired protein.
  • the CHO cell containing the recombinant NgR can be produced by cultivating the CHO cells containing the NR expression vectors constitutively expressing the NgR constructs.
  • Media used in cultivating CHO cells includes DMEM medium supplemented with about 0.5 to 20% fetal calf serum, DMEM medium and RPMI1640 medium.
  • the pH of the medium is preferably about 6 to 8.
  • Cultivation is preferably at about 30 to 40° C. for about 15 to 72 hours with aeration.
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NgR protein.
  • the invention further provides methods for producing NgR protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NgR has been introduced) in a suitable medium such that NgR protein is produced.
  • the method further comprises isolating NgR from the medium or the host cell.
  • intracellular material can be extracted from the host cell using any standard technique known to one of ordinary skill in the art. Such methods would encompass, by way of example and not by way of limitation, lysing the host cells to release the contents of the periplasm/cytoplasm by French press, homogenization and/or sonication followed by centrifugation.
  • the NgR polypeptide has formed inclusion bodies in the cytosol, such inclusion bodies may frequently bind to the inner and/or outer cellular membranes. Upon centrifugation, the inclusion bodies will be found primarily in the pellet material.
  • the pellet material can then be treated at pH extremes or with one or more chaotropic agents such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris-carboxyethyl phosphine at acid pH to release, break apart and solubilize the inclusion bodies.
  • a reducing agent such as dithiothreitol at alkaline pH or tris-carboxyethyl phosphine at acid pH to release, break apart and solubilize the inclusion bodies.
  • NgR polypeptide can be analyzed using gel electrophoresis, immunoprecipitation or the like.
  • isolation may be accomplished using standard methods such as those set forth below and in Marston et al (1990) Meth. Enzymol. 182, 264-275 (incorporated by reference herein in its entirety).
  • NgR polypeptide is not biologically active following the isolation procedure employed, various methods for “refolding” or converting the polypeptide to its tertiary structure and generating disulfide linkages, can be used to restore biological activity.
  • Methods known to one of ordinary skill in the art include adjusting the pH of the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization but usually at a lower concentration and is not necessarily the same chaotrope as used for the solubilization.
  • a reducing agent or the reducing agent plus its oxidized form in a specific ratio, to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s).
  • Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol (DTT)/dithiane DTT, 2-mercaptoethanol (bME)/dithio-b(ME).
  • GSH glutathione
  • DTT dithiothreitol
  • bME 2-mercaptoethanol
  • a cosolvent such as glycerol, polyethylene glycol of various molecular weights and arginine.
  • the host cells of the invention can also be used to produce non-human transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NgR-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous NgR sequences have been introduced into their genome or homologous recombinant animals in which endogenous NgR sequences have been altered.
  • Such animals are useful for studying the function and/or activity of NgR and for identifying and/or evaluating modulators of NgR activity.
  • a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene.
  • Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NgR gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • a transgenic animal of the invention can be created by introducing NgR-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the human NgR DNA sequence of SEQ ID NOs:1 or 3 can be introduced as a transgene into the genome of a non-human animal.
  • a nonhuman homolog of the human NgR gene such as a mouse NgR gene, can be isolated based on hybridization to the human NgR cDNA (described further above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the NgR transgene to direct expression of NgR protein to particular cells.
  • a transgenic founder animal can be identified based upon the presence of the NgR transgene in its genome and/or expression of NgR mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding NgR can further be bred to other transgenic animals carrying other transgenes.
  • a vector which contains at least a portion of a NgR gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NgR gene.
  • the NgR gene can be a human gene (e.g., SEQ ID NOs:1 or 13), but more preferably, is a non-human homolog of a human NgR gene.
  • a mouse homolog of human NgR gene of SEQ ID NOs:1 or 13 can be used to construct a homologous recombination vector suitable for altering an endogenous NgR gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous NgR gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous NgR gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NgR protein).
  • the altered portion of the NgR gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the NgR gene to allow for homologous recombination to occur between the exogenous NgR gene carried by the vector and an endogenous NgR gene in an embryonic stem cell.
  • flanking NgR nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5′ and 3′ ends
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NgR gene has homologously recombined with the endogenous NgR gene are selected (see e.g., Li et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras.
  • an animal e.g., a mouse
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene.
  • transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene.
  • a system is the cre/loxP recombinase system of bacteriophage P1.
  • Cre/loxP recombinase system of bacteriophage P1.
  • a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) Nature 385:810-813.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
  • the reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • antisense polynucleotides that recognize and hybridize to NgR polynucleotides.
  • Full-length and fragment antisense polynucleotides are provided.
  • Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to NgR RNA (as determined by sequence comparison of DNA encoding NgR to DNA encoding other known molecules). Identification of sequences unique to NgR encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or through use of commercially available sequence comparison programs. After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed. Antisense polynucleotides are particularly relevant to regulating expression of NgR by those cells expressing NgR mRNA.
  • Antisense oligonucleotides, or fragments of a nucleotide sequence set forth in SEQ ID NO:1, 3, 13 or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding NgR are useful as diagnostic tools for probing gene expression in various tissues.
  • tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto.
  • antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NgR coding strand, or to only a portion thereof.
  • Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NgR protein of SEQ ID NO:2, 4 or 14 or antisense nucleic acids complementary to a NgR nucleic acid sequence of SEQ ID NOs:1, 3 or 13 are additionally provided.
  • an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding NgR.
  • the term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human NgR corresponds to the coding region SEQ ID NO:1, 3 or 13).
  • the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding NgR.
  • the term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).
  • Antisense oligonucleotides are preferably directed to regulatory regions of a nucleotide sequence of SEQ ID NO:1, 3, 13 or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of NgR mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NgR mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NgR mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NgR protein to thereby inhibit expression of the protein, e.g. by inhibiting transcription and/or translation. Suppression of NgR expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by aberrant NgR expression.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention.
  • the antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine or cholesterol moieties at their 5′ end.
  • antisense nucleic acid molecules of the invention includes direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule of the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al., (1987) Nucleic Acids Res. 15, 6625-6641).
  • the antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBS Lett. 215, 327-330).
  • the NgR sequences taught in the present invention facilitate the design of novel transcription factors for modulating NgR expression in native cells and animals, and cells transformed or transfected with NgR polynucleotides.
  • the Cys 2 -His 2 zinc finger proteins which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences.
  • These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression.
  • NgR target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries (Segal et al., (1999) Proc. Natl. Acad. Sci. USA 96, 2758-2763; Liu et al., (1997) Proc. Natl. Acad. Sci. USA 94, 5525-5530; Greisman et al. (1997) Science 275, 657-661; Choo et al., (1997) J. Mol. Biol. 273, 525-532).
  • Each zinc finger domain usually recognizes three or more base pairs.
  • a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence (Segal et al., (1999), above).
  • the artificial zinc finger repeats designed based on the promoter of NgR sequences, are fused to activation or repression domains to promote or suppress NgR expression (Liu et al., (1997), above).
  • the promoter of NgR may be obtained by standard methods known to one of ordinary skill in the art with the disclosure contained herein and knowledge of the NgR sequence.
  • the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors (Kim et al., (1997) Proc. Natl. Acad. Sci. USA 94, 3616-3620.
  • TBP TATA box-binding factor
  • Such proteins and polynucleotides that encode them have utility for modulating NgR expression in vivo in both native cells, animals and humans; and/or cells transfected with NgR-encoding sequences.
  • the novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein.
  • Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods (McColl et al., (1997) Proc. Natl. Acad. Sci. USA 96, 9521-9526); Wu et al., (1995) Proc. Natl. Acad. Sci. USA 92, 344-348).
  • the present invention contemplates methods of designing such transcription factors based on the gene sequence of the invention, as well as customized zinc finger proteins, that are useful to modulate NgR expression in cells (native or transformed) whose genetic complement includes these sequences.
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes, described in Haselhoff and Gerlach (1988) Nature 334, 585-591
  • a ribozyme having specificity for a NgR-encoding nucleic acid can be designed based upon the nucleotide sequence of a NgR DNA disclosed herein (i.e., SEQ ID NOs:1, 3 or 13).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NgR-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.
  • NgR mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261, 1411-1418.
  • NgR gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NgR (e.g., the NgR promoter and/or enhancers) to form triple helical structures that prevent transcription of the NgR gene in target cells.
  • nucleotide sequences complementary to the regulatory region of the NgR e.g., the NgR promoter and/or enhancers
  • the NgR promoter and/or enhancers e.g., the NgR promoter and/or enhancers
  • the nucleic acids of NgR can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule.
  • the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., (1996) Bioorg. Med. Chem. Lett. 4, 5-23).
  • peptide nucleic acids refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength.
  • the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al., (1996) above; Perry-O'Keefe et al., (1996) Proc. Natl. Acad. Sci. USA 93,14670-14675.
  • PNAs of NgR can be used in therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication.
  • PNAs of NgR can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup (1996), above); or as probes or primers for DNA sequence and hybridization (Hyrup et al., (1996), above; Perry-O'Keefe (1996), above).
  • PNAs of NgR can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of NgR can be generated that may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), above).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), above and Finn et al. (1996) Nucleic Acids Res. 24, 3357-3363.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl) amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al (1989) Nucleic Acids Res. 17, 973-988). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al (1996), above). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg. Med. Chem. Lett. 5:1119-1124.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86, 6553-6556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84, 648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134).
  • peptides e.g., for targeting host cell receptors in vivo
  • agents facilitating transport across the cell membrane see Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86, 6553-6556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84,
  • oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol et al., (1988) Biotechniques 6, 958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5, 539-549).
  • the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • Automated sequencing methods can be used to obtain or verify the nucleotide sequence of NgR.
  • the NgR nucleotide sequences of the present invention are believed to be 100% accurate. However, as is known in the art, nucleotide sequence obtained by automated methods may contain some errors. Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art.
  • An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation.
  • the invention also provides purified and isolated mammalian NgR polypeptides encoded by a polynucleotide of the invention.
  • a human NgR polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:14.
  • Another preferred embodiment is a mouse NgR polypeptide comprising the amino acid sequence of NgR3, as set forth in SEQ ID NO:4.
  • NgR proteins and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof
  • polypeptide fragments suitable for use as immunogens to raise anti-NgR antibodies are provided.
  • fragments of NgR proteins comprise at least one biological activity of NgR.
  • native NgR proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • NgR proteins are produced by recombinant DNA techniques.
  • a NgR protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • the invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50% or at least 45% identity and/or homology to the preferred polypeptide of the invention.
  • the invention embraces polypeptides having the consensus sequence shown in SEQ ID NO:6, shown in Table 5) excluding the previously characterized NgR (“NgR1”), and polypeptides comprising at least about 90% of the consensus sequence.
  • the term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I, in the case of nucleic acids
  • substantially identical denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region.
  • percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment (Dayhoff, in ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference).
  • a determination of homology or identity is typically made by a computer homology program known in the art.
  • An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) using the default settings, which uses the algorithm of Smith and Waterman ( Adv. Appl. Math., 1981, 2, 482-489, which in incorporated herein by reference in its entirety).
  • GAP software provided in the GCG program package, (see Needleman and Wunsch (1970) J. Mol. Biol.
  • nucleic acid sequence comparison may be used: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOs:1, 3 or 13. BestFit was originally written for Version 1.0 by Paul Haeberli from a careful reading of the papers by Needleman and Wunsch (1970), above, and Smith and Waterman (1981), above.
  • the following Bestfit settings for nucleic acid sequence comparison may be used: GAP creation penalty of 8.0 and GAP extension penalty of 2, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, with the CDS (encoding) part of the amino acid sequence shown in SEQ ID NOs:2, 4 or 14.
  • homology may be determined by hybridization analysis wherein a nucleic acid sequence is hybridized to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., (Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below.
  • Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NgR protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of NgR protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of NgR protein having less than about 30% (by dry weight) of non-NgR protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-NgR protein, still more preferably less than about 10% of non-NgR protein, and most preferably less than about 5% non-NgR protein.
  • non-NgR protein also referred to herein as a “contaminating protein”
  • contaminating protein also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NgR protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of NgR protein having less than about 30% (by dry weight) of chemical precursors or non-NgR chemicals, more preferably less than about 20% chemical precursors or non-NgR chemicals, still more preferably less than about 10% chemical precursors or non-NgR chemicals, and most preferably less than about 5% chemical precursors or non-NgR chemicals.
  • Biologically active portions of a NgR protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the NgR protein, e.g., the amino acid sequence shown in SEQ ID NO:2, 4 or 14 that include fewer amino acids than the full length NgR proteins, and exhibit at least one activity of a NgR protein.
  • biologically active portions comprise a domain or motif with at least one activity of the NgR protein.
  • a biologically active portion of a NgR protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a NgR protein of the present invention may contain at least one of the features that is conserved between the NgR proteins (e.g., a conserved cysteine as the N-terminus of the mature protein, four conserved cysteines in the N-terminus before a leucine-rich region, four conserved cysteines C-terminal with respect to a leucine repeat region, eight leucine-rich repeats, and a hydrophobic C-terminus).
  • An alternative biologically active portion of a NgR protein may contain at least two of the above-identified domains.
  • Another biologically active portion of a NgR protein may contain at least three of the above-identified domains.
  • Yet another biologically active portion of a NgR protein of the present invention may contain at least four of the above-identified domains.
  • the NgR protein has an amino acid sequence shown in SEQ ID NO:2, 4 or 14.
  • the NgR protein is substantially homologous to SEQ ID NO:2, 4 or 14 and retains the functional activity of the protein of SEQ ID NO:2, 4 or 14, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below.
  • the NgR protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:14 and retains the functional activity of the NgR proteins of SEQ ID NO:2, 4 or 14.
  • mammalian host cells Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention.
  • post-translational modifications e.g., glycosylation, truncation, lipidation and phosphorylation
  • Glycosylated and non-glycosylated forms of NgR polypeptides are embraced by the invention.
  • the invention also embraces variant (or analog) NgR polypeptides.
  • insertion variants are provided wherein one or more amino acid residues supplement a NgR amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the NgR amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels.
  • Insertion variants include NgR polypeptides wherein one or more amino acid residues are added to a NgR acid sequence or to a biologically active fragment thereof.
  • Variant products of the invention also include mature NgR products, i.e., NgR products wherein leader or signal sequences are removed, with additional amino terminal residues.
  • the additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins.
  • NgR products with an additional methionine residue at position ⁇ 1 are contemplated, as are variants with additional methionine and lysine residues at positions ⁇ 2 and ⁇ 1 (Met ⁇ 2 -Lys ⁇ 1 -NgR).
  • Variants of NgR with additional Met, Met-Lys, Lys residues are particularly useful for enhanced recombinant protein production in bacterial host cells.
  • the invention also embraces NgR variants having additional amino acid residues which result from use of specific expression systems.
  • a NgR “chimeric protein” or “fusion protein” comprises a NgR polypeptide operatively linked to a non-NgR polypeptide.
  • a “NgR polypeptide” refers to a polypeptide having an amino acid sequence corresponding to NgR
  • a “non-NgR polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not homologous to the NgR protein, e.g., a protein that is different from the NgR protein and that is derived from the same or a different organism.
  • the NgR polypeptide can correspond to all or a portion of a NgR protein.
  • a NgR fusion protein comprises at least one biologically active portion of a NgR protein. In another embodiment, a NgR fusion protein comprises at least two biologically active portions of a NgR protein. In yet another embodiment, a NgR fusion protein comprises at least three biologically active portions of a NgR protein.
  • the term “operatively linked” is intended to indicate that the NgR polypeptide and the non-NgR polypeptide are fused in-frame to each other. The non-NgR polypeptide can be fused to the N-terminus or C-terminus of the NgR polypeptide.
  • a NgR fusion protein comprises a NgR domain operably linked to the extracellular domain of a second protein.
  • Such fusion proteins can be further utilized in screening assays for compounds which modulate NgR activity (such assays are described in detail below).
  • GST glutathione-S-transferase
  • the fusion protein is a NgR protein containing a heterologous signal sequence at its N-terminus.
  • the native NgR signal sequence i.e., amino acids 1-30 of SEQ ID NO:2 and amino acids 1-40 of SEQ ID NO:4
  • the native NgR signal sequence can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion NgR can be increased through use of a heterologous signal sequence.
  • the fusion protein is a NgR-immunoglobulin fusion protein in which the NgR sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family.
  • the NgR-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between NgR ligand and a NgR protein on the surface of a cell, to thereby suppress NgR-mediated signal transduction in vivo.
  • NgR-immunoglobulin fusion proteins can be used to affect the bioavailability of a NgR cognate ligand.
  • NgR-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NgR antibodies in a subject, to purify NgR ligands, and in screening assays to identify molecules that inhibit the interaction of NgR with NgR ligand.
  • a NgR chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992).
  • anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • fusion moiety e.g., a GST polypeptide
  • a NgR-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NgR protein.
  • Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of NgR is/are fused to another polypeptide.
  • the invention provides deletion variants wherein one or more amino acid residues in a NgR polypeptide are removed.
  • Deletions can be effected at one or both termini of the NgR polypeptide, or with removal of one or more non-terminal amino acid residues of NgR.
  • Deletion variants therefore, include all fragments of a NgR polypeptide.
  • the invention also embraces polypeptide fragments of the sequence set forth in SEQ ID NO:2, 4 or 14 wherein the fragments maintain biological (e.g., ligand binding and/or intracellular signaling) immunological properties of a NgR polypeptide. Fragments comprising at least 4, 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of SEQ ID NO:2, 4 or 14 are contemplated by the invention. Preferred polypeptide fragments display antigenic properties unique to, or specific for, human NgR and its allelic and species homologs. Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.
  • the invention provides substitution variants of NgR polypeptides.
  • substitution variants include those polypeptides wherein one or more amino acid residues of a NgR polypeptide are removed and replaced with alternative residues.
  • the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables 2, 3, or 4 below. Table 1.
  • Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention.
  • Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure.
  • a conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties.
  • Exemplary conservative substitutions are set out in Table 2 (from WO 97/09433, page 10, published Mar, 13, 1997 (PCT/GB96/02197, filed 9/6/96), immediately below.
  • Table 2 from WO 97/09433, page 10, published Mar, 13, 1997 (PCT/GB96/02197, filed 9/6/96), immediately below.
  • TABLE 2 Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINO ACID Aliphatic GAP Non-polar ILV Polar-uncharged CSTM NQ Polar-charged DE KR Aromatic HFWY Other NQDE
  • amino acid residues that are conserved among family members of the NgR proteins of the present invention are also predicted to be particularly unamenable to alteration.
  • NgR proteins of the present invention can contain at least one domain that is a typically conserved region in NgRs. Examples of these conserved domains include, e.g., leucine-rich repeat domain. Amino acid residues that are not conserved or are only semi-conserved among members of the NgR proteins may be readily amenable to alteration.
  • Full-length NgRs have an LRR region characterized by the amino acid consensus sequence shown in SEQ ID NO: 19. At least some full-length NgRs also include a CT signaling (CTS) domain and a GPI domain.
  • CTS CT signaling
  • NgR domain designations used herein are defined as follows: hNgR1 mNgR1 hNgR2 hNgR3 SEQ ID: SEQ ID SEQ ID: SEQ ID: mNgR3 Domain 5 NO: 17 2 14 SEQ ID:4 Signal 1-26 1-26 1-30 — 1-40 Seq.
  • the above domains are modified. Modification can be in a manner that preserves domain functionality. Modification can include addition, deletion or substitution of certain amino acids. Exemplary modifications include conservative amino acid substitutions. Preferably such substitutions number 20 or fewer per 100 residues. More preferably, such substitutions number 10 or fewer per 100 residues. Further exemplary modifications include addition of flanking sequences of up to five amino acids at the N terminus and/or C terminus of one or more of the domains.
  • the isolated nucleic acid molecule encodes a polypeptide at least about 70%, 80%, 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO:2, 4 or 14.
  • Mutations can be introduced into SEQ ID NOS:1, 3 or 13 by standard techniques, e.g., site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions can be made at one or more amino acid residues predicted to be non-essential. Alternatively, mutations can be introduced randomly along a NgR coding sequence. This can be accomplished, e.g., by saturation mutagenesis. The resulting mutants can be screened for NgR biological activity.
  • Biological activities of NgR may include but are not limited to: (1) protein:protein interactions, e.g., with other NgRs or other cell-surface proteins involved in Nogo-related signaling; (2) complex formation with a NgR ligand; (3) binding to an anti-NgR antibody.
  • polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.
  • the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties.
  • Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs.
  • the invention further embraces NgR polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol.
  • Variants that display ligand binding properties of native NgR and are expressed at higher levels are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant NgR activity.
  • the polymer may be water soluble to prevent precipitation of the protein in an aqueous environment, such as a physiological environment.
  • Suitable water-soluble polymers may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, a polypropylene oxide/ethylene oxide copolymer polyoxyethylated polyols (e.g.
  • glycerol and polyvinyl alcohol.
  • the selected polymer is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled.
  • Polymers may be of any molecular weight, and may be branched or unbranched, and mixtures of such polymers may also be used. When the chemically modified NgR polymer is destined for therapeutic use, pharmaceutically acceptable polymers will be selected for use.
  • the polymer When the polymer is to be modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, if the polymer is to be modified by reductive alkylation, the polymer should have a single reactive aldehyde group.
  • a preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono Cl-ClO alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714, incorporated by reference herein in its entirety).
  • Pegylation of NgR polypeptides may be carried out by any of the pegylation reactions known in the art, as described, for example, in the following references: Focus on Growth Factors 3, 4-10 (1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated by reference herein in its entirety).
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
  • a preferred water-soluble polymer for pegylation of polypeptides such as NgR is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl-ClO) alkoxy- or aryloxy-polyethylene glycol.
  • NgR polypeptides may be performed under any suitable conditions used to react a biologically active substance with an activated polymer molecule.
  • Methods for preparing pegylated NgR polypeptides will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby NgR polypeptide becomes attached to one or more PEG groups, and (b) obtaining the reaction products. It will be apparent to one of ordinary skill in the art to select the optimal reaction conditions or the acylation reactions based on known parameters and the desired result.
  • Pegylated and other polymer:NgR polypeptides may generally be used to treat conditions that may be alleviated or modulated by administration of the NgR polypeptides described herein.
  • the chemically-derivatized polymer:NgR polypeptide molecules disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to the nonderivatized molecules.
  • the NgR polypeptides, fragments thereof, variants and derivatives may be employed alone, together, or in combination with other pharmaceutical compositions.
  • the cytokines, growth factors, antibiotics, antiinflammatories and/or chemotherapeutic agents as is appropriate for the indication being treated.
  • compositions comprising purified polypeptides of the invention.
  • Preferred compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient or medium. Any diluent known in the art may be used.
  • Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil and cocoa butter.
  • Variants that display ligand binding properties of native NgR and are expressed at higher levels are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant NgR activity.
  • nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode NgR from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference in its entirety.
  • DNA that encodes NgR may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the NgR gene sequence information provided herein. Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al. (1989) above.
  • a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group
  • a nucleic acid molecule comprising any of the NgR nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein.
  • PCR polymerase chain reaction
  • the PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample.
  • the method can be used to amplify either single- or double-stranded DNA.
  • the essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase-mediated replication of a desired nucleic acid molecule.
  • nucleic acid molecules of the present invention are useful for screening for restriction fragment length polymorphism (RFLP) associated with certain disorders, as well as for genetic mapping.
  • RFLP restriction fragment length polymorphism
  • antibodies e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention
  • CDR complementary determining region
  • Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety.
  • Antibody fragments, including Fab, Fab′, F(ab′) 2 , and F v are also provided by the invention.
  • variable regions of the antibodies of the invention recognize and bind NgR polypeptides exclusively (i.e., are able to distinguish NgR polypeptides from other known NgR polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between NgR and such polypeptides).
  • the antigenic peptide of NgR comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2, 4 or 14 and encompasses an epitope of NgR such that an antibody raised against the peptide forms a specific immune complex with NgR.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of NgR that are located on the surface of the protein, e.g., hydrophilic regions.
  • antibodies may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule.
  • Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. in ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y. (1988), Chapter 6.
  • Antibodies that recognize and bind fragments of the NgR polypeptides of the invention are also contemplated, provided that the antibodies are specific for NgR polypeptides.
  • Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
  • polyclonal antibodies For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed NgR protein or a chemically synthesized NgR polypeptide. The preparation can further include an adjuvant.
  • Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum or similar immunostimulatory agents.
  • the antibody molecules directed against NgR can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • the term “monoclonal antibody” or “monoclonal antibody composition,” as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of NgR.
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular NgR protein with which it immunoreacts.
  • any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized.
  • Such techniques include, but are not limited to, the hybridoma technique (see Kohler and Milstein (1975) Nature 256, 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor et al., (1983) Immunol. Today 4, 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole et al., (1985) in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote et al., (1983) Proc. Natl. Acad. Sci. USA 80, 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole et al., (1985), above).
  • NgR NgR protein
  • methods can be adapted for the construction of Fab expression libraries (see e.g., Huse et al., (1989) Science 246, 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a NgR protein or derivatives, fragments, analogs or homologs thereof
  • Fab expression libraries see e.g., Huse et al., (1989) Science 246, 1275-1281
  • Non-human antibodies can be “humanized” by techniques well known in the art. See e.g., U.S. Pat. No. 5,225,539.
  • the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
  • Antibody fragments that contain the idiotypes to a NgR protein may be produced by techniques known in the art including, but not limited to: (i) an F(ab′) 2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′) 2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F fragments.
  • recombinant anti-NgR antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No.
  • a portion of a NgR is joined to an Fc portion of an antibody to form a NgR/Fc fusion protein.
  • the Ig fusion protein is soluble.
  • the NgR/Fc fusion protein may be formed by recombinant techniques as described above.
  • a portion of a NgR including the entire amino acid sequence of NgR except the C-terminal hydrophobic region is fused to an Fc portion of an antibody.
  • the NgR is a human NgR and the Fc is also human. More preferably, the human Fc portion is derived from an IgG antibody. In other embodiments, the N-terminal signal sequence is omitted.
  • Such antibodies are useful in binding Nogo to prevent Nogo signaling through the NgR.
  • methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art.
  • ELISA enzyme-linked immunosorbent assay
  • selection of antibodies that are specific to a particular domain of a NgR protein is facilitated by generation of hybridomas that bind to the fragment of a NgR protein possessing such a domain.
  • Antibodies that are specific for one or more domains within a NgR protein e.g., domains spanning the above-identified conserved regions of NgRs, or derivatives, fragments analogs or homologs thereof, are also provided herein.
  • Anti-NgR antibodies may be used in methods known within the art relating to the localization and/or quantitation of a NgR protein (e.g., for use in measuring levels of the NgR protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • antibodies for NgR proteins, or derivatives, fragments analogs or homologs thereof, that contain the antibody derived binding domain are utilized as pharmacologically-active compounds [hereinafter “Therapeutics”].
  • An anti-NgR antibody (e.g., monoclonal antibody) can be used to isolate NgR by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-NgR antibody can facilitate the purification of natural NgR from cells and of recombinantly produced NgR expressed in host cells.
  • an anti-NgR antibody can be used to detect NgR protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NgR protein.
  • Anti-NgR antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
  • Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • bioluminescent materials include luciferase, luciferin and aequorin, and examples of suitable radioactive material include 125 I, 131
  • Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response.
  • the amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art.
  • An anti-idiotypic antibody is an antibody that recognizes determinants of another antibody (a target antibody). Generally, the anti-idiotypic antibody recognizes determinants of the antigen-binding site of the target antibody. Typically, the target antibody is a monoclonal antibody.
  • An anti-idiotypic antibody is generally prepared by immunizing an animal (particularly, mice) of the same species and genetic type as the source of the target monoclonal antibody, with the target monoclonal antibody. The immunized animal mounts an immune response to the idiotypic determinants of the target monoclonal antibody and produces antibodies against the idiotypic determinants of the target monoclonal antibody.
  • Antibody-producing cells, such as splenic cells, of the immunized animal may be used to generate anti-idiotypic monoclonal antibodies.
  • an anti-idiotypic antibody may also be used to immunize animals to produce anti-anti-idiotypic antibodies. These immunized animals may be used to generate anti-anti-idiotypic monoclonal antibodies using standard techniques.
  • the anti-anti-idiotypic antibodies may bind to the same epitope as the original, target monoclonal antibody used to prepare the anti-idiotypic antibody.
  • the anti-anti-idiotypic antibodies represent other monoclonal antibodies with the same antigen specificity as the original target monoclonal antibody.
  • an anti-idiotypic antibody is an “internal image anti-idiotype” and is capable of inducing an antibody response as if it were the original antigen.
  • Anti-idiotypic antibodies for NgR may be prepared, for example, by immunizing an animal, such as a mouse, with a immunogenic amount of a composition comprising NgR2 (SEQ ID NO:2), NgR3 (SEQ ID NOs:4 or 14), or immunogenic portion thereof, containing at least one antigenic epitope of NgR.
  • the composition may also contain a suitable adjuvant, and any carrier necessary to provide immunogenicity.
  • Monoclonal antibodies recognizing NgR may be prepared from the cells of the immunized animal as described above. A monoclonal antibody recognizing an epitope of NgR is then selected and used to prepare a composition comprising an immunogenic amount of the anti-NgR monoclonal antibody. Typically, a 25 to 200 ⁇ g dose of purified anti-NgR monoclonal would be sufficient in a suitable adjuvant.
  • Animals may be immunized 2-6 times at 14 to 30 day intervals between doses. Typically, animals are immunized by any suitable route of administration, such as intraperitoneal, subcutaneous, intravenous or a combination of these. Anti-idiotypic antibody production may be monitored during the immunization period using standard immunoassay methods. Animals with suitable titers of antibodies reactive with the target monoclonal antibodies may be reimmunized with the monoclonal antibody used as the immunogen three days before harvesting the antibody producing cells. Preferably, spleen cells are used, although other antibody producing cells may be selected. Antibody-producing cells are harvested and fused with myeloma cells to produce Hybridomas, as described above, and suitable anti-idiotypic antibody-producing cells are selected.
  • Anti-anti-idiotypic antibodies are produced by another round of immunization and Hybridoma production by using the anti-idiotypic monoclonal antibody as the immunogen.
  • Antibodies of the invention are useful for, e.g., therapeutic purposes (by modulating activity of NgR), diagnostic purposes to detect or quantitate NgR, and purification of NgR. Therefore, kits comprising an antibody of the invention for any of the purposes described herein are also comprehended.
  • kits including pharmaceutical kits.
  • the kits can comprise any of the nucleic acid molecules described above, any of the polypeptides described above, or any antibody which binds to a polypeptide of the invention as described above, as well appropriate controls, such as positive and/or negative controls.
  • the kit preferably comprises additional components, such as, for example, instructions, solid support, reagents helpful for quantification, and the like.
  • the kit can comprise: a labeled compound or agent capable of detecting NgR protein or mRNA in a biological sample; means for determining the amount of NgR in the sample; and means for comparing the amount of NgR in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which a NgR polypeptide or polynucleotide will interact.
  • Methods to identify binding partner compounds include solution assays, in vitro assays wherein NgR polypeptides are immobilized and cell-based assays. Identification of binding partner compounds of NgR polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with NgR normal and aberrant biological activity.
  • the invention also provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules (e.g., molecules of less than 1,000 Daltons) or other drugs) that bind to NgR proteins or have a stimulatory or inhibitory effect on, for example, NgR expression or NgR activity.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules (e.g., molecules of less than 1,000 Daltons) or other drugs) that bind to NgR proteins or have a stimulatory or inhibitory effect on, for example, NgR expression or NgR activity.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a NgR protein or polypeptide or biologically active portion thereof.
  • the test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12, 145).
  • the invention also provides cell-based assays to identify binding partner compounds of a NgR polypeptide.
  • the invention provides a method comprising the steps of contacting a NgR polypeptide expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the NgR polypeptide.
  • an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NgR protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NgR protein or biologically active portion thereof.
  • an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NgR protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NgR protein determined.
  • the cell for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NgR protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NgR protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting a cell which expresses a membrane-bound form of NgR protein or a biologically active portion thereof, on the cell surface with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the test compound to preferentially bind to NgR or a biologically active portion thereof as compared to the known compound.
  • Determining the ability of the test compound to modulate the activity of NgR or a biologically active portion thereof can be accomplished, for example, by determining the ability of the NgR protein to bind to or interact with a NgR target molecule.
  • a “target molecule” is a molecule with which a NgR protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NgR protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • a NgR target molecule can be a non-NgR molecule or a NgR protein or polypeptide of the present invention.
  • a NgR target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a membrane-bound NgR molecule) through the cell membrane and into the cell.
  • the target for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NgR.
  • the detection comprises detecting a calcium flux or other physiological event in the cell caused by the binding of the molecule.
  • binding molecules including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant NgR products, NgR variants, or preferably, cells expressing such products. Binding partners are useful for purifying NgR products and detection or quantification of NgR products in fluid and tissue samples using known immunological procedures. Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of NgR, especially those activities involved in signal transduction.
  • the invention includes several assay systems for identifying NgR binding partners.
  • methods of the invention comprise the steps of (a) contacting a NgR polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the NgR polypeptide. Identification of the compounds that bind the NgR polypeptide can be achieved by isolating the NgR polypeptide/binding partner complex and separating the binding partner compound from the NgR polypeptide. An additional step of characterizing the physical, biological and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention.
  • the NgR polypeptide/binding partner complex is isolated using an antibody immunospecific for either the NgR polypeptide or the candidate binding partner compound.
  • either the NgR polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation
  • methods of the invention to identify binding partner compounds include a step of isolating the NgR polypeptide/binding partner complex through interaction with the label or tag.
  • An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation.
  • Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention.
  • the invention provides a method comprising the steps of (a) contacting an immobilized NgR polypeptide, or a biologically active fragment thereof with a candidate binding partner compound and (b) detecting binding of the candidate compound to the NgR polypeptide.
  • the candidate binding partner compound is immobilized and binding of NgR is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety.
  • Binding of a test compound to NgR, or interaction of NgR with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants.
  • vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix.
  • GST-NgR fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the test compound or the test compound and either the non-adsorbed target protein or NgR protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • conditions conducive to complex formation e.g., at physiological conditions for salt and pH.
  • the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, and the complexes determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of NgR binding or activity determined using standard techniques.
  • NgR or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated NgR or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with NgR or target molecules can be derivatized to the wells of the plate, and unbound target or NgR trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the NgR or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NgR or target molecule.
  • Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, (v) determining the activity of the NgR, as well as other techniques well known and routinely practiced in the art.
  • Determining the activity of the target molecule may be accomplished by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca 2+ , diacylglycerol, IP 3 , etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NgR-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
  • a cellular second messenger of the target i.e. intracellular Ca 2+ , diacylglycerol, IP 3 , etc.
  • detecting catalytic/enzymatic activity of the target an appropriate substrate detecting the induction of a reporter gene (comprising a NgR-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.
  • the assay comprises contacting the NgR protein or biologically active portion thereof with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the test compound to preferentially bind to NgR or biologically active portion thereof as compared to the known compound.
  • the cell-free assay comprises contacting the NgR protein or biologically active portion thereof with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the NgR protein to modulate the activity of a NgR target molecule.
  • the cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form of NgR.
  • solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, TritonTM X-114, Thesit®, Isotridecypoly(ethylene glycol ether) n , 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.
  • non-ionic detergents such as n-octylglucoside, n
  • Agents that modulate may be identified by incubating a putative modulator with a cell containing a NgR polypeptide or polynucleotide and determining the effect of the putative modulator on NgR activity or expression.
  • the selectivity of a compound that modulates the activity of NgR can be evaluated by comparing its effects on NgR to its effect on other NgR compounds.
  • Selective modulators may include, for example, antibodies and other proteins, peptides or organic molecules which specifically bind to a NgR polypeptide or a NgR-encoding nucleic acid.
  • NgR polynucleotides, polypeptides and modulators may be used in the treatment of such diseases and conditions associated with demyelination.
  • NgR polynucleotides and polypeptides, as well as NgR modulators may also be used in diagnostic assays for such diseases or conditions.
  • Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is carried out in the presence and absence of a candidate modulator.
  • a modulator is identified in those instances where binding between the NgR polypeptide and the binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound.
  • a modulator that increases binding between the NgR polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the NgR polypeptide and the binding partner compound is described as an inhibitor.
  • modulators of NgR expression may be identified in a method wherein a cell is contacted with a candidate compound and the expression of NgR mRNA or protein in the cell is determined. The level of expression of NgR mRNA or protein in the presence of the candidate compound is compared to the level of expression of NgR mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NgR expression based on this comparison. For example, when expression of NgR mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NgR mRNA or protein expression.
  • the candidate compound when expression of NgR mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NgR mRNA or protein expression.
  • the level of NgR mRNA or protein expression in the cells can be determined by methods described herein for detecting NgR mRNA or protein.
  • the invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.) of a NgR polypeptide.
  • HTS assays permit screening of large numbers of compounds in an efficient manner.
  • Cell-based HTS systems are contemplated to investigate NgR receptor-ligand interaction.
  • HTS assays are designed to identify “hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the “hit” or “lead compound” is often based on an identifiable structure/activity relationship between the “hit” and the NgR polypeptide.
  • Another aspect of the present invention is directed to methods of identifying compounds that bind to either NgR or nucleic acid molecules encoding NgR, comprising contacting NgR, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds NgR or a nucleic acid molecule encoding the same.
  • Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Ausubel et al. (Eds.),CURRENT PROTOCOLS TN MOLECULAR BIOLOGY, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety.
  • the NgR proteins for example, can be used as “bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., (1993) Cell 72, 223-232; Madura et al., (1993) J. Biol. Chem.
  • NgR-binding proteins proteins that bind to or interact with NgR
  • Such NgR-binding proteins are also likely to be involved in the propagation of signals by the NgR proteins as, for example, upstream or downstream elements of the NgR pathway.
  • assays may be used to identify specific ligands of a NgR receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods.
  • binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., (1989) Nature 340, 245-246, and Fields et al., (1994) Trends Genet. 10, 286-292, both of which are incorporated herein by reference.
  • the two-hybrid system is a genetic assay based on the modular nature of most transcription factors used for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs.
  • the two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.
  • UAS upstream activation sequence
  • the assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.
  • the DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene.
  • the second hybrid protein which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.
  • the first protein is a NgR gene product, or fragment thereof, that is known to interact with another protein or nucleic acid
  • this assay can be used to detect agents that interfere with the binding interaction.
  • Expression of the reporter gene is monitored as different test agents are added to the system.
  • the presence of an inhibitory agent results in lack of a reporter signal.
  • the compounds to be screened include (which may include compounds that are suspected to bind NgR, or a nucleic acid molecule encoding the same), but are not limited to, extracellular, intracellular, biological or chemical origin.
  • the function of the NgR gene product is unclear and no ligands have yet been found which bind the gene product.
  • the yeast two-hybrid assay is useful to identify proteins that bind to the gene product.
  • a fusion polynucleotide encoding both a NgR receptor (or fragment) and a UAS binding domain i.e., a first protein
  • a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay.
  • the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain.
  • This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein.
  • the system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein.
  • the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
  • the methods of the invention also embrace ligands, especially neuropeptides, that are attached to a label, such as a radiolabel (e.g., 125 I, 35 S, 32 P, 33 P, 3 H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label.
  • a radiolabel e.g., 125 I, 35 S, 32 P, 33 P, 3 H
  • fluorescence label e.g., 125 I, 35 S, 32 P, 33 P, 3 H
  • fluorescence label e.g., 125 I, 35 S, 32 P, 33 P, 3 H
  • chemiluminescent label e.g., chemiluminescent label
  • an enzymic label e.g., a chemiluminescent label
  • an immunogenic label e.g., an immunogenic label.
  • Modulators falling within the scope of the invention include, but are not limited to, non-peptide molecules such
  • the NgR polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell.
  • One skilled in the art can, for example, measure the formation of complexes between NgR and the compound being tested.
  • one skilled in the art can examine the diminution in complex formation between NgR and its substrate caused by the compound being tested.
  • Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of NgR comprising contacting NgR with a compound, and determining whether the compound modifies activity of NgR.
  • the activity in the presence of the test compared is measured to the activity in the absence of the test compound. Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound will have increased activity. Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity.
  • the present invention is particularly useful for screening compounds by using NgR in any of a variety of drug screening techniques.
  • the compounds to be screened include (which may include compounds which are suspected to modulate NgR activity), but are not limited to, extracellular, intracellular, biologic or chemical origin.
  • the NgR polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly.
  • One skilled in the art can, for example, measure the formation of complexes between NgR and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between Nogo-R and its substrate caused by the compound being tested.
  • the activity of NgR polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesized peptide ligands. Alternatively, the activity of the NgR can be assayed by examining their ability to bind calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants and photons. Alternatively, the activity of the NgR can be determined by examining the activity of effector molecules including, but not limited to, adenylate cyclase, phospholipases and ion channels.
  • modulators of NgR activity may alter a NgR receptor function, such as a binding property of a receptor or an activity.
  • the assay may take the form of an ion flux assay, a yeast growth assay, a non-hydrolyzable GTP assay such as a [ 35 S]-GTP S assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca 2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [ 3 H]-arachidonic acid) and an assay for extracellular acidification rates, as well as other binding or function-based assays of NgR activity that are generally known in the art.
  • NgR activity can be determined by methodologies that are used to assay for FaRP activity, which is well known to those skilled in the art.
  • Biological activities of NgR receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of NgRs known in the art.
  • Non-limiting examples of NgR activities include transmembrane signaling of various forms, which may involve phosphatidylinositol (PI) association and/or the exertion of an influence over PI; another exemplary activity of NgRs is the binding of accessory proteins or polypeptides that differ from known GPI proteins.
  • PI phosphatidylinositol
  • the modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural NgR receptor ligands, peptide and non-peptide allosteric effectors of NgR receptors, and peptides that may function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of NgR receptors.
  • the invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries.
  • Recombinant receptors are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson (1992) Bio/Technology 10, 973-980; each of which is incorporated herein by reference in its entirety).
  • a variety of heterologous systems is available for functional expression of recombinant receptors that are well known to those skilled in the art. Such systems include bacteria (Strosberg et al. (1992) Trends Pharmacol. Sci. 13, 95-98), yeast (Pausch (1997) Trends Biotechnol. 15, 487-494), several kinds of insect cells (Vanden Broeck (1996) Int. Rev. Cytol. 164, 189-268), amphibian cells (Jayawickreme et al. (1997) Curr. Opin. Biotechnol. 8, 629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.; see Gerhardt et al. (1997) Eur. J. Pharmacol. 334, 1-23). These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).
  • methods of screening for compounds which modulate NgR activity comprise contacting test compounds with NgR and assaying for the presence of a complex between the compound and NgR.
  • the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to NgR.
  • high throughput screening for compounds having suitable binding affinity to NgR is employed. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test compounds are contacted with NgR and washed. Bound NgR is then detected by methods well known in the art. Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support.
  • an expressed NgR can be used for HTS binding assays in conjunction with its defined ligand.
  • the identified peptide is labeled with a suitable radioisotope, including, but not limited to, 125 I, 3 H, 35 S or 32 P, by methods that are well known to those skilled in the art.
  • the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al. (1994) Drug Dev. Res. 33, 373-398; Rogers (1997) Drug Discov. Today 2, 156-160).
  • Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams (1991) Med. Res. Rev. 11, 147-184; Sweetnam et al. (1993) J. Nat. Prod. 56, 441-455).
  • Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama (1998) Curr. Opin. Drug Disc. Dev. 1, 85-91 Bosse et al. (1998) J. Biomol. Screening 3, 285-292).
  • Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers (1997) Drug Discov. Today 2, 156-160; Hill (1998) Curr. Opin. Drug Disc. Dev. 1, 92-97).
  • FRET fluorescence energy transfer
  • Differophotofluorometric analysis of bound ligand or fluorescence polarization
  • Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch (1997) Trends in Biotechnol. 15, 487-494); changes in intracellular Ca concentration as measured by fluorescent dyes (Murphy et al. (1998) Cur. Opin. Drug Disc. Dev. 1, 192-199). Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder et al. (1996) J. Biomol. Screening 1, 75-80).
  • Preferred methods of HTS employing these receptors include permanently transfected CHO cells, in which agonists and antagonists can be identified by the ability to transduce the signal for the binding of Nogo in membranes prepared from these cells through the putative GPI anchor.
  • permanently transfected CHO cells could be used for the preparation of membranes which contain significant amounts of the recombinant receptor proteins; these membrane preparations would then be used in receptor binding assays, employing the radiolabelled ligand specific for the particular receptor.
  • a functional assay such as fluorescent monitoring of ligand-induced changes in internal Ca 2+ concentration or membrane potential in permanently transfected CHO cells containing each of these receptors individually or in combination would be preferred for HTS.
  • Equally preferred would be an alternative type of mammalian cell, such as HEK293 or COS cells, in similar formats. More preferred would be permanently transfected insect cell lines, such as Drosophila S2 cells. Even more preferred would be recombinant yeast cells expressing the Drosophila melanogaster receptors in HTS formats well known to those skilled in the art (e.g., Pausch (1997), above).
  • the invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to NgR receptors.
  • the NgR receptor is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound.
  • interaction between the NgR receptor and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound.
  • an inhibitor is identified as a compound that decreases binding between the NgR receptor and its binding partner.
  • Another contemplated assay involves a variation of the di-hybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published Aug. 3, 1995.
  • Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors. There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry.
  • Natural product libraries are collections of microorganisms, animals, plants, or marine organisms that are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Cane et al., Science ( 1998) 282, 63-68. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-peptide combinatorial libraries.
  • Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
  • combinatorial chemistry and libraries created therefrom see Myers (1997) Curr. Opin. Biotechnol. 8, 701-707. Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to modulate activity.
  • binding partner as used herein broadly encompasses non-peptide modulators, as well as such peptide modulators as neuropeptides other than natural ligands, antibodies, antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified NgR gene.
  • Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide.
  • the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with NgR. Radiolabeled competitive binding studies are described in Lin et al., (1997) Antimicrob. Agents Chemother. 41, 2127-2131, the disclosure of which is incorporated herein by reference in its entirety.
  • the polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins.
  • Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules. For example, molecules are incubated with the labeled polypeptides, washed to remove unbound polypeptides, and the polypeptide complex is quantified. Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex.
  • Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors.
  • affinity purification a polypeptide is covalently coupled to a chromatography column. Cells and their membranes are extracted, and various cellular subcomponents are passed over the column. Molecules bind to the column by virtue of their affinity to the polypeptide. The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library.
  • compounds may be identified which exhibit similar properties to the ligand for the NgR of the invention, but which are smaller and exhibit a longer half time than the endogenous ligand in a human or animal body.
  • a molecule according to the invention is used as a “lead” compound.
  • the design of mimetics to known pharmaceutically active compounds is a well-known approach in the development of pharmaceuticals based on such “lead” compounds. Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property.
  • structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral or parenteral administration.
  • the compositions, or pharmaceutical compositions, comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's PHARMACEUTICAL SCIENCES, 16th ed., (1980) Osol, A (Ed.), which is incorporated herein by reference in its entirety.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include oral and parenteral (e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal and rectal administration).
  • Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a NgR protein or anti-NgR antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a NgR protein or anti-NgR antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such as
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by any of a number of routes, e.g., as described in U.S. Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91, 3054-3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • compositions can be included in a container, pack or dispenser together with instructions for administration.
  • the dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
  • For treating human or animals between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.
  • NgR nucleotide sequences disclosed herein for identifying homologs of the Nogo-R, in other animals, including but not limited to humans and other mammals and invertebrates. Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs using screening procedures well known to those skilled in the art.
  • homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with NgR sequences can be identified.
  • the present compounds and methods including nucleic acid molecules, polypeptides, antibodies, compounds identified by the screening methods described herein, have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth.
  • the present molecules are used in gene therapy.
  • gene therapy procedures see e.g. Anderson Science (1992) 256, 808-813, which is incorporated herein by reference in its entirety.
  • the present invention also encompasses a method of agonizing (stimulating) or antagonizing a NgR natural binding partner associated activity in a mammal comprising administering to said mammal an agonist or antagonist to one of the above disclosed polypeptides in an amount sufficient to effect said agonism or antagonism.
  • One embodiment of the present invention is a method of treating diseases in a mammal with an agonist or antagonist of the protein of the present invention comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize NgR-associated functions.
  • the proper dosage depends on various factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient.
  • Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC 50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
  • Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder. Such measurements can be carried out. For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI. Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model.
  • Toxicity studies can also be carried out by measuring the blood cell composition.
  • toxicity studies can be carried out in a suitable animal model as follows: (1) the compound is administered to mice (an untreated control mouse should also be used); (2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and (3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymorphonuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present.
  • the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day.
  • Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness. Plasma levels should reflect the potency of the drug. Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy.
  • NgR mRNA transcripts have been found in the brain and heart.
  • SEQ ID NOs: 1 and/or, 3 will, as detailed above, enable screening the endogenous neurotransmitters/hormones/ligands which activate, agonize, or antagonize NgR and for compounds with potential utility in treating disorders including CNS disorders (e.g., stroke) and degenerative disorders such as those associated with demyelination.
  • NgR receptor activation may mediate the prevention of neurite outgrowth. Inhibition would be beneficial in both chronic and acute brain injury. See, e.g., Donovan et al., (1997) J. Neurosci. 17, 5316-5326; Turgeon et al., (1998) J. Neurosci. 18, 6882-6891; Smith-Swintosky et al., (1997) J. Neurochem. 69, 1890-1896; Gill et al., (1998) Brain Res. 797, 321-327; Suidan et al., (1996) Semin. Thromb. Hemost. 22, 125-133.
  • Agents, or modulators that have a stimulatory or inhibitory effect on NgR activity can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., a disease condition such as a demyelination disorder) associated with aberrant NgR activity.
  • disorders e.g., a disease condition such as a demyelination disorder
  • the pharmacogenomics i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
  • the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NgR protein, expression of NgR nucleic acid or mutation content of NgR genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum (1996) Clin. Exp. Pharmacol. Physiol. 23, 983-985 and Linder (1997) Clin. Chem. 43, 254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms.
  • glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
  • oxidant drugs anti-malarials, sulfonamides, analgesics, nitrofurans
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
  • drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
  • NAT 2 N-acetyltransferase 2
  • CYP2D6 and CYP2C19 cytochrome P450 enzymes
  • the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • the activity of NgR protein, expression of NgR nucleic acid, or mutation content of NgR genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
  • pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NgR modulator, such as a modulator identified by one of the exemplary screening assays described herein.
  • NgR e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • agents e.g., drugs, compounds
  • NgR e.g., the ability to modulate aberrant cell proliferation and/or differentiation
  • the effectiveness of an agent determined by a screening assay as described herein to increase NgR gene expression, protein levels or upregulate NgR activity can be monitored in clinical trials of subjects exhibiting decreased NgR gene expression, protein levels, or downregulated NgR activity.
  • the effectiveness of an agent determined by a screening assay to decrease NgR gene expression, protein levels, or downregulate NgR activity can be monitored in clinical trials of subjects exhibiting increased NgR gene expression, protein levels, or upregulated NgR activity.
  • the expression or activity of NgR and, preferably, other genes that have been implicated in, for example, a disease or disorder can be used as a “read out” or markers of the immune responsiveness of a particular cell.
  • genes, including NgR, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NgR activity can be identified.
  • an agent e.g., compound, drug or small molecule
  • NgR activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of NgR and other genes implicated in the disorder.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced by one of the methods as described herein or by measuring the levels of activity of NgR or other genes.
  • the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.
  • the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a NgR protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NgR protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NgR protein, mRNA or genomic DNA in the pre-administration sample with the NgR protein, mRNA or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly.
  • an agent e.g.,
  • increased administration of the agent may be desirable to increase the expression or activity of NgR to higher levels than detected, i.e., to increase the effectiveness of the agent.
  • decreased administration of the agent may be desirable to decrease expression or activity of NgR to lower levels than detected, i.e., to decrease the effectiveness of the agent.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NgR expression or activity.
  • Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, (i) a NgR polypeptide, or analogs, derivatives, fragments or homologs thereof, (ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgR peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to a NgR peptide) are utilized to “knockout” endogenous function of a NgR peptide by homologous recombination (see, e.g., Capecchi (1989) Science 244, 1288-1292); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between a NgR peptide and its binding
  • Therapeutics that increase (i.e., are agonists to) activity may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, a NgR peptide, or analogs, derivatives, fragments or homologs thereof, or an agonist that increases bioavailability.
  • Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a NgR peptide).
  • Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
  • hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NgR expression or activity, by administering to the subject an agent that modulates NgR expression or at least one NgR activity.
  • Subjects at risk for a disease that is caused or contributed to by aberrant NgR expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NgR aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a NgR agonist or NgR antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • Another aspect of the invention pertains to methods of modulating NgR expression or activity for therapeutic purposes.
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NgR protein activity associated with the cell.
  • An agent that modulates NgR protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NgR protein, a peptide, a NgR peptidomimetic, or other small molecule.
  • the agent stimulates one or more NgR protein activity. Examples of such stimulatory agents include active NgR protein and a nucleic acid molecule encoding NgR that has been introduced into the cell.
  • the agent inhibits one or more NgR protein activity.
  • inhibitory agents include antisense NgR nucleic acid molecules and anti-NgR antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NgR protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) NgR expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a NgR protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NgR expression or activity.
  • NgR human disease states Mutations in the NgR gene that result in loss of normal function of the NgR gene product underlie NgR human disease states.
  • the invention comprehends gene therapy to restore NgR activity to treat those disease states. Delivery of a functional NgR gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson (1998) Nature, supplement to 392(6679):25-20.
  • viral vectors e.g., adenovirus, adeno-associated virus, or a retrovirus
  • physical DNA transfer methods e.g., liposomes or chemical treatments. See, for example, Anderson (1998) Nature, supplement to 392(6679):25-20.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NgR expression or activity.
  • Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, (i) a NgR polypeptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgR peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to a NgR peptide) are utilized to “knockout” endogenous function of a NgR peptide by homologous recombination (see, e.g., Capecchi (1989), above); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between a NgR peptide and its binding partner.
  • modulators
  • Therapeutics that increase (i.e., are agonists to) activity may be administered in a therapeutic or prophylactic manner.
  • Therapeutics that may be utilized include, but are not limited to, a NgR peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
  • Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a NgR peptide).
  • Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • immunoassays e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.
  • hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.).
  • the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NgR expression or activity, by administering to the subject an agent that modulates NgR expression or at least one NgR activity.
  • Subjects at risk for a disease that is caused or contributed to by aberrant NgR expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NgR aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • a NgR agonist or NgR antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.
  • the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NgR protein activity associated with the cell.
  • An agent that modulates NgR protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NgR protein, a peptide, a NgR peptidomimetic, or other small molecule.
  • the agent stimulates one or more NgR protein activity. Examples of such stimulatory agents include active NgR protein and a nucleic acid molecule encoding NgR that has been introduced into the cell.
  • the agent inhibits one or more NgR protein activity.
  • inhibitory agents include antisense NgR nucleic acid molecules and anti-NgR antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NgR protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) NgR expression or activity.
  • an agent e.g., an agent identified by a screening assay described herein
  • the method involves administering a NgR protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NgR expression or activity.
  • NgR1 from hu- mans: M K R A S A G G S R L L L A W V L W L Q A W Q V A A P C P G A C C Y N E P K V T T S C P Q Q G L Q A V P V G I P A A S Q R I F L H G N R I S H V P A A S F R A C R N L T I L W L H S N V L A R I D A A A F T G L A L L E Q L D L S D N A Q L R S V D P A T F H G L G R L H T L H L D R C G L Q E L G P G L F R G L A A L Q Y L Y L Q D N A L Q A L P D D T F R D L G N L T H L F L H G N R I S S V P E R A F R G L H S L D R L L L M T L Y L F A N N N
  • X is any amino acid.
  • X where indicated may be no amino acid. Additional features of the invention will be apparent from the following Examples. Examples 1-5 are actual, while the remaining Examples are prophetic.
  • NgR2 and NgR3 have a putative signal sequence, eight leucine-rich repeat domains in a conserved leucine-rich region (SEQ ID NO:12), a conserved cysteine-rich region (SEQ ID NO:10) N-terminal to the leucine-rich region, a second cysteine-rich domain (SEQ ID NO:11) C-terminal to the leucine-rich region, and a putative glycophosphatidylinositol-linkage (GPI-linkage) site.
  • SEQ ID NO:12 conserved leucine-rich region
  • SEQ ID NO:10 conserved cysteine-rich region
  • SEQ ID NO:11 second cysteine-rich domain
  • GPI-linkage putative glycophosphatidylinositol-linkage
  • the protein sequence for the human NgR was used to query the high throughput genomic (HTG) database the use of which is familiar to those skilled in the art.
  • the HTG database is a part of GenBank, a comprehensive NIH genetic sequence database, which includes an annotated collection of all publicly available DNA sequences ( Nucleic Acids Res. (2000) 28, 15-8).
  • the HTG database includes sequences obtained from genomic DNA. Within genomic DNA, genes are typically encoded by multiple segments of DNA called exons. Thus when one aligns a cDNA sequence (or a protein sequence encoded by a cDNA sequence) to a genomic sequence, the sequence will be broken up into segments depending on the number of exons in the gene.
  • the BLAST algorithm which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., (1990) J. Mol. Biol. 215, 403-410, which is incorporated herein by reference in its entirety).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • the basic BLAST algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • HSPs high scoring sequence pair
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached.
  • the Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm Kerlin et al., (1993) Proc. Natl. Acad. Sci. USA 90, 5873-5787, which is incorporated herein by reference
  • Gapped BLAST perform a statistical analysis of the similarity between two sequences.
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a NgR gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a NgR nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the sequence for this cDNA sequence is SEQ ID NO: 1 (nucleotide sequence of human NgR2; AC013606).
  • the translation of this cDNA provides the protein sequence of human NgR2 (SEQ ID NO:2).
  • Another EST (GB_EST26:AI929019) contains 545 nucleotides, part of which corresponds to amino acids 1-75 of the human NgR2 (SEQ ID NO:2). This spans the first intron located between amino acids 10 and 11, and provides positive evidence for the splicing of exons 1 and 2 at the mRNA level.
  • the sequence for this cDNA sequence is SEQ ID NO:3 (nucleotide sequence of mouse NgR3; AC021768).
  • the translation of this cDNA provides the protein sequence of mouse NgR3 (SEQ ID NO:4).
  • FIGS. 1 A- 1 B An alignment between NgR1 and the two new receptors is shown in FIGS. 1 A- 1 B.
  • the similarities between these proteins include:
  • LRRs Leucine Rich Repeats
  • LRRNT leucine rich repeat N-terminal domain
  • LRRCTs of the three NgR proteins can be distinguished from those of other LRR containing proteins, by the pattern of typtophans and cysteines which are completely conserved in this domain.
  • NgR2 and NgR3 have a hydrophobic C-terminus, as does NgR1, an indication that they probably also undergo a modification similar to NgR1, where a GPI moiety is covalently linked to a C-terminal amino acid. This allows the protein to remain tethered to the cell.
  • a Nogo binding assay was developed which utilizes a method widely used in examining semaphorin and ephrin axonal guidance function (Flanagan & Vanderhaeghen (1998) Annu. Rev. Neurosci. 21,309-345; Takahashi et al., (1999) Cell 99, 59-69). It involves fusing a secreted placental alkaline phosphatase (AP) moiety to the ligand in question to provide a biologically active receptor binding agent which can be detected with an extremely sensitive colorimetric assay.
  • AP placental alkaline phosphatase
  • an expression vector is created encoding a signal peptide, a His6 tag for purification, AP, and the 66 amino acid active domain of Nogo.
  • the fusion protein can be purified from the conditioned medium of transfected cells in milligram amounts. This protein is biologically active as a growth cone collapsing agent with an EC 50 of 1 nM.
  • a glutathione-S-transferase Nogo (GST-Nogo) fusion protein may be prepared.
  • an expression vector e.g., a pGEX vector
  • GST-Nogo may be purified from the culture medium and used as a GST fusion protein, or GST may be cleaved from the Nogo portion of the fusion protein with an enzyme that recognizes the specific amino acid cleavage sit engineered between the GST portion and the Nogo portion of the fusion protein.
  • Such sites are part of the commercially available GST vectors.
  • the specific cleavage sites and enzymes may be used in accordance with the Manufacturer's specifications.
  • AP-Nogo is actually slightly more potent than GST-Nogo, perhaps because the protein is synthesized in a eukaryotic rather than a prokaryotic cell.
  • Binding of Nogo to immobilized NgR homologs may be performed in an ELISA-type assay in which AP-Nogo is allowed to react with an immobilized receptor homolog. Specificity of binding may be demonstrated in a competitive binding assay using increasing amounts of GST-Nogo in the type of assay to show a decreasing amount of binding of AP-Nogo (as judged in the calorimetric assay).
  • the homologs of the present invention may be used in transfection studies in COS cells to demonstrate binding of Nogo.
  • nucleotide sequences encoding NgR2 and NgR3 may be transfected into COS cells using a suitable vector.
  • Non-transfected COS-7 cells do not bind AP-Nogo.
  • transfection of COS cells with nucleic acid sequences encoding NgRs will make them capable of binding Nogo.
  • AP alone does not bind with any stable affinity to these transfected cells, indicating that any affinity of Nogo for NgR2 or NgR3 would be due to the 66 amino acids derived from Nogo.
  • NgR2 or NgR3 proteins may be tested in displacement of AP-Nogo assays using GST-Nogo.
  • NgR2 and/or NgR3 may also bind homologs of Nogo, which may also be tested using this assay.
  • a Northern blot is purchased from a commercial source, or RNA samples from cells of interest are run on an agarose gel and blotted to a membrane using any of the well known techniques for Northern blotting.
  • the blot is probed with a fragment of NgR2 (SEQ ID NO:1) or NgR3 (SEQ ID NO:3).
  • the probe is prepared from 50 ng of cDNA labeled by a random-primed method (Feinberg and Vogelstein (1983) Anal. Biochem. 132, 6-13).
  • Hybridization is carried out at 68° C. for 1 hour in ExpressHybTM solution (Clontech, Cat. No.
  • NgR2 and/or NgR3 can be assessed by the presence of an appropriately sized band on the blot.
  • a cDNA library is generated using standard methods from a tissue known to contain NgR2. Such a tissue was identified in Example 2. 1 ⁇ 10 6 plaque forming units from the cDNA library may be screened in duplicate on OPTITRANTM filters. The filters are hybridized with 32 P-labeled oligonucleotides that are generated from the ESTs corresponding to portions of NgR2.
  • the hybridization reaction may consist of 400 mls plaque screen buffer (50 mM Tris pH 7.5, 1M NaCl, 0.1% Sodium pyrophosphate, 0.2% Polyvinylpryolidine and 0.2% Ficoll) containing 10% Dextran sulfate and 100 ⁇ g/ml tRNA and 80 pmol each 32 P-labeled oligonucleotide at 65° C. overnight.
  • the filters are washed twice with 2 ⁇ SSC/1%SDS and twice with 1 ⁇ SSC/1%SDS and exposed to film. Duplicate positives are purified. DNA from each of these clones is analyzed by restriction enzyme digest followed by agarose gel electrophoresis and Southern blotting.
  • the filters are hybridized to the 32 P-labeled oligonucleotides used for the original hybridization to confirm that inserts hybridize to the probe.
  • the insert is then sequenced to confirm that it represents the cDNA for NgR2. Similar methods may be used to generate a full-length clone corresponding to NgR3.
  • NgR2 or NgR3 can be obtained by a person of ordinary skill in the art employing conventional PCR techniques.
  • NgR NgR in mammals
  • mammals such as the rat
  • coronal and sagittal rat brain cryosections (20 ⁇ m thick) are prepared using a Reichert-Jung cryostat. Individual sections are thaw-mounted onto silanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.), and stored at ⁇ 80° C.
  • Sections are processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS), acetylated using acetic anhydride in triethanolamine buffer, and dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at room temperature. Subsequently, sections are delipidated in chloroform, followed by rehydration through successive exposure to 100% and 95% alcohol at room temperature. Microscope slides containing processed cryosections are allowed to air dry prior to hybridization. Other tissues may be assayed in a similar fashion.
  • PBS cold phosphate-buffered saline
  • a NgR-specific probe may be generated using PCR. Following PCR amplification, the fragment is digested with restriction enzymes and cloned into pBluescript II cleaved with the same enzymes. For production of a probe specific for the sense strand of NgR, a cloned NgR fragment cloned in pBluescript II may be linearized with a suitable restriction enzyme, which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase.
  • a suitable restriction enzyme which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase.
  • a probe specific for the antisense strand of NgR may also be readily prepared using the NgR clone in pBluescript II by cleaving the recombinant plasmid with a suitable restriction enzyme to generate a linearized substrate for the production of labeled run-off cRNA transcripts using the T3 promoter and cognate polymerase.
  • the riboprobes may be labeled with [ 35 S]-UTP to yield a specific activity of about 0.40 ⁇ 10 6 cpm/pmol for antisense riboprobes and about 0.65 ⁇ 10 6 cpm/pmol for sense-strand riboprobes.
  • Each riboprobe may be subsequently denatured and added (2 pmol/ml) to hybridization buffer which contains 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 1 ⁇ Denhardt's Solution, and 10 mM dithiothreitol.
  • Microscope slides containing sequential brain cryosections may be independently exposed to 45 ⁇ l of hybridization solution per slide and silanized cover slips may be placed over the sections being exposed to hybridization solution. Sections are incubated overnight (15-18 hours) at 52° C. to allow hybridization to occur. Equivalent series of cryosections are then exposed to sense or antisense NgR-specific cRNA riboprobes.
  • coverslips are washed off the slides in IX SSC, followed by RNase A treatment involving the exposure of slides to 20 ⁇ g/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5 M EDTA, and 0.5 M NaCl for 45 minutes at 37° C.
  • the cryosections are then subjected to three high-stringency washes in 0.1 ⁇ SSC at 52° C. for 20 minutes each.
  • cryosections are dehydrated by consecutive exposure to 70%, 95%, and 100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak BioMaxTM MR-1 film.
  • NgR expression of NgR in the brain provides an indication that modulators of NgR activity have utility for treating neurological disorders.
  • Some other diseases for which modulators of NgR may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like.
  • Use of NgR modulators, including NgR ligands and anti-NgR antibodies, to treat individuals having such disease states is intended as an aspect of the invention.
  • Northern blot hybridizations may be performed to examine the expression of NgR mRNA.
  • a clone containing at least a portion of the sequence of SEQ ID NO:1 may be used as a probe.
  • Vector-specific primers are used in PCR to generate a hybridization probe fragment for 32 P-labeling.
  • the PCR is performed as follows: Mix: 1 ⁇ l NgR-containing plasmid 2 ⁇ l fwd primer (10-50 pM) 2 ⁇ l rev primer (10-50 pM) 10 ⁇ l 10 ⁇ PCR buffer (such as that provided with the enzyme, Amersham Pharmacia Biotech) 1 ⁇ l 10 mM dNTP (such as #1 969 064 from Boehringer Mannheim) 0.5 ⁇ l Taq polymerase (such as #27-0799-62, Amersham Pharmacia Biotech) 83.5 ⁇ l water
  • PCR is performed in a Thermocycler using the following program: 94° C. 5 min
  • the PCR product may be purified using QIAquick PCR Purification Kit (#28104) from Qiagen, and radictively labeled with 32 P-dCTP (#AA0005/250, Amersham Pharmacia Biotech)) may be done by random priming using “Ready-to-go DNA Labeling Beads” (#27-9240-01) from Amersham Pharmacia Biotech. Hybridization is carried out on Human Multiple Tissue Northern Blot from Clontech as described in manufacturer's protocol, or on a Northern Blot prepared by running RNA samples from cells of interest on an agarose gel and blotting to a membrane using any of the known Northern blotting protocols. After exposure overnight on Molecular Dynamics Phosphor Imager screen (#MD146-814) bands of an appropriate size are visualized.
  • a NgR-encoding polynucleotide is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques.
  • a NgR-encoding sequence described in Table 4 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6TM (Boehringer-Mannheim) and the transfection protocol provided in the product insert.
  • Other eukaryotic cell lines including human embryonic kidney (HEK 293) and COS cells, are suitable as well.
  • NgR may be purified from the cells using standard chromatographic techniques. To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the NgR amino acid sequence, and the antisera is used to affinity purify Nogo-R.
  • the NgR also may be expressed in-frame with a tag sequence (e.g., polyhistidine, hemaglutinin, FLAG) to facilitate purification.
  • a tag sequence e.g., polyhistidine, hemaglutinin, FLAG
  • NgR For expression of NgR in Chinese hamster ovary (CHO) cells, a plasmid bearing the relevant NgR coding sequence is prepared, using a vector which also bears the selectable marker dihydrofolate reductase (DHFR). The plasmid is transfected into CHO cells. Selection under MTX drug pressure allows for preparation of stable transformants of a NgR (NgR2 or NgR3) in an expression plasmid carrying a selectable marker such as DHFR.
  • DHFR dihydrofolate reductase
  • NgR For expression of NgR in mammalian cells 293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant NgR coding sequence is prepared, using vector pSecTag2A (Invitrogen).
  • Vector pSecTag2A contains the murine IgK chain leader sequence for secretion, the c-myc epitope for detection of the recombinant protein with the anti-myc antibody, a C-terminal polyhistidine for purification with nickel chelate chromatography, and a Zeocin resistant gene for selection of stable transfectants.
  • the forward primer for amplification of this NgR cDNA is determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce the HindIII cloning site and nucleotides matching the NgR sequence.
  • the reverse primer is also determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce an XhoI restriction site for cloning and nucleotides corresponding to the reverse complement of the NgR sequence.
  • the PCR conditions are 55° C. as the annealing temperature.
  • the PCR product is gel purified and cloned into the HindIII-XhoI sites of the vector.
  • the DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAPTM transfection media (Boehringer Mannheim, Indianapolis, Ind.). Transiently transfected cells are tested for expression after 24 hours of transfection, using western blots probed with anti-His and anti-NgR peptide antibodies. Permanently transfected cells are selected with Zeocin and propagated. Production of the recombinant protein is detected from both cells and media by Western blots probed with anti-His, anti-Myc or anti-NgR peptide antibodies.
  • a polynucleotide molecule having a nucleotide sequence of SEQ ID NO:1 can be cloned into vector p3-CI.
  • This vector is a pUC18-derived plasmid that contains the HCMV (human cytomegalovirus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site.
  • the forward primer is determined by routine procedures and preferably contains a 5′ extension which introduces an XbaI restriction site for cloning, followed by nucleotides which correspond to a nucleotide sequence of SEQ ID NO:1.
  • the reverse primer is also determined by routine procedures and preferably contains 5′-extension of nucleotides which introduces a SalI cloning site followed by nucleotides which correspond to the reverse complement of a nucleotide sequence of SEQ ID NO:1.
  • the PCR consists of an initial denaturation step of 5 min at 95° C., 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C.
  • the PCR product is gel purified and ligated into the XbaI and SalI sites of vector p3-CI.
  • This construct is transformed into E. coli cells for amplification and DNA purification.
  • the DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using LipofectamineTM reagent from BRL, following the manufacturer's protocols. Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.
  • NgR expressed from a COS cell culture can be purified by concentrating the cell-growth media to about 10 mg of protein/ml, and purifying the protein by, for example, chromatography. Purified NgR is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at ⁇ 80° C. NgR3 may also be expressed using this method and the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:13.
  • a polynucleotide molecule having a nucleotide sequence of SEQ ID NO:1, 3 or 13 can be amplified by PCR.
  • the forward primer is determined by routine procedures and preferably contains a 5′ extension which adds the NdeI cloning site, followed by nucleotides which correspond to a nucleotide sequence of SEQ ID NO:1 (or SEQ ID NO:3 or SEQ ID NO:13, respectively).
  • the reverse primer is also determined by routine procedures and preferably contains a 5′ extension which introduces the KpnI cloning site, followed by nucleotides which correspond to the reverse complement of a nucleotide sequence of SEQ ID NO:1 (or SEQ ID NO:3 or SEQ ID NO:13, respectively).
  • the PCR product is gel purified, digested with NdeI and KpnI, and cloned into the corresponding sites of vector pACHTL-A (Pharmingen, San Diego, Calif.).
  • the pAcHTL expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), and a 6XHis tag upstream from the multiple cloning site.
  • a protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein precede the multiple cloning site is also present.
  • baculovirus vectors could be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1.
  • suitable vectors for the expression of NgR polypeptides can be used, provided that the vector construct includes appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required.
  • Such vectors are described in Luckow et al., Virology 170:31-39, among others.
  • the virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et al. (1987) A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555.
  • pAcHLT-A containing NgR gene is introduced into baculovirus using the “BaculoGoldTM” transfection kit (Pharmingen, San Diego, Calif.) using methods established by the manufacturer. Individual virus isolates are analyzed for protein production by radiolabeling infected cells with 35 S-methionine at 24 hours post infection. Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE. Viruses exhibiting high expression levels can be isolated and used for scaled up expression.
  • a polynucleotide molecule having the nucleotide sequence of SEQ ID NO:1 can be amplified by PCR using the primers and methods described above for baculovirus expression.
  • the NgR cDNA is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9 insect.
  • the insert is cloned into the NdeI and KpnI sites, after elimination of an internal NdeI site (using the same primers described above for expression in baculovirus).
  • DNA is purified with Qiagen chromatography columns and expressed in Sf9 cells.
  • Preliminary Western blot experiments from non-purified plaques are tested for the presence of the recombinant protein of the expected size which reacted with the NgR-specific antibody. These results are confirmed after further purification and expression optimization in HiG5 cells.
  • NgR2-Ig fusion protein To generate a NgR2-Ig fusion protein, standard methods may be used as described in the literature (e.g. Sanicola et al. (1997) Proc. Natl. Acad. Sci. USA. 94, 6238-6243).
  • a DNA fragment encoding NgR2 without the sequence encoding the hydrophobic C-terminus (GPI anchor signal) may be ligated to a DNA fragment encoding the Fc domain of IgG1 (which may be human IgG1), and the chimeric fragment may be cloned into an expression vector to generate a plasmid.
  • the plasmid may then be transfected into Chinese hamster ovary cells to generate a stable cell line producing the fusion protein.
  • the fusion protein is then purified from conditioned media using standard methods. For example, clarified conditioned media from the cell line may be loaded by gravity directly onto Protein A Sepharose. The column may then be washed with five column volumes each of PBS, PBS containing 0.5 M NaCl, and 25 mM sodium phosphate, 100 mM NaCl (pH 5.0). The bound protein may then be eluted with 25 mM NaH 2 PO 4 , 100 mM NaCl (pH 2.8) and immediately neutralized with ⁇ fraction (1/10) ⁇ fraction volume of 0. 5 M Na 2 HPO 4 (pH 8.6).
  • the interaction trap/two-hybrid library screening method can be used. This assay was first described in Fields et al. (1989) Nature 340, 245, which is incorporated herein by reference in its entirety. A protocol is published in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1999, John Wiley & Sons, NY and Ausubel, F. M. et al. 1992, SHORT PROTOCOLS IN MOLECULAR BIOLOGY, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety. Kits are available from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3).
  • a fusion of the nucleotide sequences encoding all or partial NgR and the yeast transcription factor GAL4 DNA-binding domain is constructed in an appropriate plasmid (i.e., pGBKT7) using standard subcloning techniques.
  • a GAL4 active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential NgR-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al. 1989, MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety.
  • the DNA-BD/NgR fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis. Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity.
  • Yeast cells are transformed (ca. 105 transformants/mg DNA) with both the NgR and library fusion plasmids according to standard procedure (Ausubel, et al., 1992, SHORT PROTOCOLS IN MOLECULAR BIOLOGY, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety).
  • yeast plasmid reporter genes i.e., lacZ, HIS3, ADE2, LEU2
  • Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes. Colonies are dually assayed for ⁇ -galactosidase activity upon growth in Xgal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) supplemented media (filter assay for b-galactosidase activity is described in Breeden et al., (1985) Cold Spring Harb. Symp. Quant. Biol., 50, 643, which is incorporated herein by reference in its entirety).
  • Standard techniques are employed to generate polyclonal or monoclonal antibodies to the NgR receptor, and to generate useful antigen-binding fragments thereof or variants thereof, including “humanized” variants.
  • Such protocols can be found, for example, in Sambrook et al. (1989), above, and Harlow et al. (Eds.), ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).
  • recombinant NgR polypeptides or cells or cell membranes containing such polypeptides
  • one or more peptides having amino acid sequences corresponding to an immunogenic portion of NgR are used as antigen.
  • Peptides corresponding to extracellular portions of Nogo-R, especially hydrophilic extracellular portions, are preferred.
  • the antigen may be mixed with an adjuvant or linked to a hapten to increase antibody production.
  • recombinant NgR or a synthetic fragment thereof is used to immunize a mouse for generation of monoclonal antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies).
  • peptides are conjugated to Keyhole Limpet Hemocyanin (Pierce), according to the manufacturer's recommendations.
  • the antigen is emulsified with Freund's Complete Adjuvant and injected subcutaneously.
  • additional aliquots of NgR antigen are emulsified with Freund's Incomplete Adjuvant and injected subcutaneously.
  • a serum sample is taken from the immunized mice and assayed by western blot to confirm the presence of antibodies that immunoreact with NgR.
  • Serum from the immunized animals may be used as polyclonal antisera or used to isolate polyclonal antibodies that recognize NgR. Alternatively, the mice are sacrificed and their spleen removed for generation of monoclonal antibodies.
  • the spleens are placed in 10 ml serum-free RPMI 1640, and single cell suspensions are formed by grinding the spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 ⁇ g/ml streptomycin (RPMI) (Gibco, Canada).
  • the cell suspensions are filtered and washed by centrifugation and resuspended in serum-free RPMI.
  • Thymocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a Feeder Layer.
  • NS-1 myeloma cells kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed as well.
  • FBS fetal bovine serum
  • spleen cells from the immunized mice are combined with NS-1 cells and centrifuged, and the supernatant is aspirated.
  • the cell pellet is dislodged by tapping the tube, and 2 ml of 37° C.
  • PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet, followed by the addition of serum-free RPMI.
  • the cells are centrifuged, resuspended in RPMI containing 15% FBS, 100 ⁇ M sodium hypoxanthine, 0.4 ⁇ M aminopterin, 16 ⁇ M thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5 ⁇ 10 6 thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning, N.Y.).
  • NgR-neutralizing antibodies comprise one class of therapeutics useful as NgR antagonists. Following are protocols to improve the utility of anti-NgR monoclonal antibodies as therapeutics in humans by “humanizing” the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-NgR antibodies).
  • a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest with the constant domains of human antibody molecules.
  • the variable domains of NgR-neutralizing anti-NgR antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest.
  • the V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells).
  • CDR complementarity determining regions
  • the B-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody.
  • the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody. See Padlan (1991) Mol. Immunol. 28, 489-498.
  • NgR-neutralizing anti-NgR monoclonal antibodies and the hybridomas that produce them to generate humanized NgR-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein NgR expression or ligand-mediated NgR signaling is detrimental.
  • Human NgR-neutralizing antibodies are generated by phage display techniques such as those described in Aujame et al. (1997) Human Antibodies 8, 155-168; Hoogenboom (1997) TIBTECH 15, 62-70; and Rader et al. (1997), Curr. Opin. Biotechnol. 8, 503-508, all of which are incorporated by reference.
  • antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII. Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody.
  • a phage library comprising such constructs is expressed in bacteria, and the library is screened for NgR-specific phage-antibodies using labeled or immobilized NgR as antigen-probe.
  • modulators agonists and antagonists
  • the modulators that can be identified by these assays are natural ligand compounds of the receptor; synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like. All modulators that bind NgR are useful for identifying NgR in tissue samples (e.g., for diagnostic purposes, pathological purposes, and the like).
  • Agonist and antagonist modulators are useful for up-regulating and down-regulating NgR activity, respectively, to treat disease states characterized by abnormal levels of NgR activity.
  • the assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa).
  • cAMP cyclic adenosine monophosphate
  • Protocols for cAMP assays have been described in the literature. (See, e.g., Sutherland et al., (1968) Circulation 37, 279; Frandsen et al., (1976) Life Sciences 18, 529-541; Dooley et al., (1997) J. Pharmacol. Exp. Therap. 283, 735-41; and George et al., (1997) J. Biomol. Screening 2, 235-40).
  • An exemplary protocol for such an assay using an Adenylyl Cyclase Activation FlashPlate Assay from NENTM Life Science Products, is set forth below.
  • the NgR coding sequence (e.g., a cDNA or intronless genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2 (Invitrogen), and transiently transfected into Chinese Hamster Ovary (CHO) cells using known methods, such as the transfection protocol provided by Boehringer-Mannheim when supplying the FuGENE 6 transfection reagent.
  • Transfected CHO cells are seeded into 96-well microplates from the FlashPlate® assay kit, which are coated with solid scintillant to which antisera to cAMP has been bound.
  • some wells are seeded with wild type (untransfected) CHO cells.
  • Other wells in the plate receive various amounts of a cAMP standard solution for use in creating a standard curve.
  • test compounds i.e., candidate modulators
  • water and/or compound-free medium/diluent serving as a control or controls.
  • cAMP is allowed to accumulate in the cells for exactly 15 minutes at room temperature.
  • the assay is terminated by the addition of lysis buffer containing [ 125 I]-labeled cAMP, and the plate is counted using a Packard TopcountTM 96-well microplate scintillation counter. Unlabeled cAMP from the lysed cells (or from standards) and fixed amounts of [ 125 I]-cAMP compete for antibody bound to the plate.
  • a standard curve is constructed, and cAMP values for the unknowns are obtained by interpolation.
  • Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of NgR modulating activity.
  • Modulators that act as agonists of receptors which couple to the Gs subtype of G proteins will stimulate production of cAMP, leading to a measurable 3-10 fold increase in cAMP levels.
  • Agonists of receptors which couple to the G i/o subtype of G proteins will inhibit forskolin-stimulated cAMP production, leading to a measurable decrease in cAMP levels of 50-100%.
  • Modulators that act as inverse agonists will reverse these effects at receptors that are either constitutively active or activated by known agonists.
  • cells e.g., CHO cells
  • a NgR expression construct e.g., CHO cells
  • apoaquorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium.
  • cytoplasmic intracellular free calcium
  • NgR is subcloned into the commercial expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, Oreg.) into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert.
  • the cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 ⁇ g/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 ⁇ M coelenterazine (Molecular Probes, Eugene, Oreg.). Culturing is then continued for two additional hours at 37° C. Subsequently, cells are detached from the plate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM.
  • MEM Gibco/BRL, Gaithersburg, Md.
  • Dilutions of candidate NgR modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 ⁇ l/well. Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, Va.). The instrument is programmed to dispense 50 ⁇ l cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds. Dose-response curves for the candidate modulators are constructed using the area under the curve for each light signal peak. Data are analyzed with SlideWrite, using the equation for a one-site ligand, and EC 50 values are obtained.
  • Modulators that act as agonists at receptors which couple to the G q subtype of G proteins give an increase in luminescence of up to 100 fold.
  • Modulators that act as inverse agonists will reverse this effect at receptors that are either constitutively active or activated by known agonists.
  • the photoprotein luciferase provides another useful tool for assaying for modulators of NgR activity.
  • Cells e.g., CHO cells or COS 7 cells
  • a NgR expression construct e.g., NgR in pzeoSV2
  • a reporter construct which includes a gene for the luciferase protein downstream from a transcription factor binding site, such as the cAMP-response element (CRE), AP-1, or NF-kappa B.
  • CRE cAMP-response element
  • AP-1 NF-kappa B
  • Luciferase activity may be quantitatively measured using, e.g., luciferase assay reagents that are commercially available from Promega (Madison, Wis.).
  • CHO cells are plated in 24-well culture dishes at a density of 100,000 cells/well one day prior to transfection and cultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 ⁇ g/ml streptomycin.
  • Cells are transiently co-transfected with both a NgR expression construct and a reporter construct containing the luciferase gene.
  • the reporter plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase may be purchased from Stratagene (Legally, Calif.).
  • Transfections are performed using the FuGENE 6 transfection reagent (Boehringer-Mannheim) according to the supplier's instructions. Cells transfected with the reporter construct alone are used as a control. Twenty-four hours after transfection, cells are washed once with PBS pre-warmed to 37° C. Serum-free MEM is then added to the cells either alone (control) or with one or more candidate modulators and the cells are incubated at 37° C. for five hours. Thereafter, cells are washed once with ice-cold PBS and lysed by the addition of 100 ⁇ l of lysis buffer per well from the luciferase assay kit supplied by Promega.
  • Changes in intracellular calcium levels are another recognized indicator of receptor activity, and such assays can be employed to screen for modulators of NgR activity.
  • CHO cells stably transfected with a NgR expression vector are plated at a density of 4 ⁇ 10 4 cells/well in Packard black-walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate. The cells are incubated for 60 minutes at 37° C.
  • D-PBS modified Dulbecco's PBS
  • D-PBS modified Dulbecco's PBS
  • fetal bovine serum containing 36 mg/L pyruvate and 1 g/L glucose
  • 1% fetal bovine serum and one of four calcium indicator dyes (Fluo-3TM AM, Fluo-4TM AM, Calcium GreenTM-1 AM, or Oregon GreenTM 488 BAPTA-1 AM)
  • plates are washed once with modified D-PBS without 1% fetal bovine serum and incubated for 10 minutes at 37° C. to remove residual dye from the cellular membrane.
  • a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response.
  • a calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (10 ⁇ M; positive control), or ATP (4 ⁇ M; positive control). Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488 nm). (See, e.g., Kuntzweiler et al. (1998) Drug Dev. Res. 44,14-20). The F-stop for the detector camera is set at 2.5 and the length of exposure is 0.4 milliseconds. Basal fluorescence of cells is measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level is subtracted from the response signal. The calcium signal is measured for approximately 200 seconds, taking readings every two seconds. Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels. In general, activated NgRs increase the calcium signal at least about 10-15% above baseline signal.
  • NgR signals through a G protein-mediated pathway. Because G protein-coupled receptors signal through intracellular G proteins whose activity involves GTP binding and hydrolysis to yield bound GDP, measurement of binding of the non-hydrolyzable GTP analog [ 35 S]-GTP ⁇ S in the presence and absence of candidate modulators provides another assay for modulator activity. (See, e.g., Kowal et al., (1998) Neuropharmacology 37, 179-187.).
  • cells stably transfected with a NgR expression vector are grown in 10 cm tissue culture dishes to subconfluence, rinsed once with 5 ml of ice-cold Ca 2+ /Mg 2+ -free phosphate-buffered saline, and scraped into 5 ml of the same buffer.
  • Cells are pelleted by centrifugation (500 ⁇ g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 mM EDTA, 5 mM EGTA), and frozen in liquid nitrogen. After thawing, the cells are homogenized using a Dounce homogenizer (1 ml TEE per plate of cells), and centrifuged at 1,000 ⁇ g for 5 minutes to remove nuclei and unbroken cells.
  • the homogenate supernatant is centrifuged at 20,000 ⁇ g for 20 minutes to isolate the membrane fraction, and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA).
  • binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA).
  • the resuspended membranes can be frozen in liquid nitrogen and stored at ⁇ 70° C. until use.
  • NgRs may also potentiate arachidonic acid release in cells, providing yet another useful assay for modulators of NgR activity.
  • CHO cells that are stably transfected with a NgR expression vector are plated in 24-well plates at a density of 15,000 cells/well and grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 ⁇ g/ml streptomycin for 48 hours at 37° C. before use.
  • Cells of each well are labeled by incubation with [ 3 H]-arachidonic acid (Amersham Corp., 210 Ci/mmol) at 0.5 ⁇ Ci/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells are then washed twice with 1 ml of the same buffer.
  • Candidate modulator compounds are added in 1 ml of the same buffer, either alone or with 10 ⁇ M ATP and the cells are incubated at 37° C. for 30 minutes. Buffer alone and mock-transfected cells are used as controls. Samples (0.5 ml) from each well are counted by liquid scintillation spectroscopy. Agonists which activate the receptor will lead to potentiation of the ATP-stimulated release of [ 3 H]-arachidonic acid. This potentiation is blocked by antagonists.
  • CHO cells transfected with a NgR expression vector are seeded into 12 mm capsule cups (Molecular Devices Corp.) at 4 ⁇ 10 5 cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 ⁇ g/ml streptomycin. The cells are incubated in this medium at 37° C. in 5% CO 2 for 24 hours.
  • Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Corp.).
  • the capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 ⁇ g/ml streptomycin, 26 mM NaCl) at a flow rate of 100 ⁇ l/minute.
  • running buffer bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 ⁇ g/ml streptomycin, 26 mM NaCl
  • Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path. During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds.
  • the pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle.
  • the rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program. Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate.
  • the selected instrument detects 61 mV/pH unit. Modulators that act as agonists of the receptor result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist. This response is blocked by modulators which act as antagonists of the receptor.
  • mice NgR3 (hereinafter, mNgR3) for its ability to bind hNogo-A(1055-1120)
  • a cDNA expression vector for a myc epitope-tagged mNgR3protein was created.
  • the mouse NgR3 cDNA was amplified by PCR from mouse adult brain cDNA, from the signal sequence to the stop codon, and ligated into the pSecTag2 vector such that the vector encodes a signal sequence followed by a myc tag followed by the mature mNgR3 sequence.
  • This plasmid was transfected into COS07cells, and expression of a myc-tagged protein of the predicted size was verified by immunoblot analysis. Alkaline phosphatase-hNogo-A(1055-1120) binding studies and myc immunohistology were conducted as described (Fournier et al., supra).
  • the tblastn program was used to search for the human homolog of mouse NgR3.
  • the mouse NgR3 protein sequence (SEQ ID NO:4) was used to query a proprietary human expressed sequence tag (EST) database from Incyte yielding one highly significant hit: Incyte Template ID 190989. 1.
  • EST human expressed sequence tag
  • This sequence (937 nucleotides) contains an open reading frame of 312 amino acids in the second reverse frame that exhibits 88% identity with residues 66 to 381 of mouse NgR3 (SEQ ID NO:4), strongly indicating that it is part of the human NgR3 homolog.
  • a query of SEQ ID NO:4 against the public human EST database in Genbank also produced a hit with a 465-bp EST (Accession number: R35699; Version number: R35699.1; GI: 792600). There are a number of single nucleotide deletions and insertions within this sequence which cause frame shift errors. All of the reliable sequence contained in this public EST is present in the Incyte EST (Template ID 190989.1).
  • the I.M.A.G.E. Consortium clone No. 38319 which corresponds to Genbank accession No. R35699, was purchased from Incyte Genomics Inc. and subjected to further DNA sequence analysis.
  • This clone consists of a NotI/HinD III fragment containing the sequence of interest, cloned into the NotI/HinD III sites of the vector Lafmid BA (http://image.llnl.gov/image/html/libs/lafmidBA.shtml).
  • the clone was received as an agar stab, which was streaked out on LB agar plates containing 50 ug/ml ampicillin to isolate individual colonies. Six colonies were grown in LB medium with antibiotic, and plasmid DNA was prepared using the Promega Wizard Plus Miniprep DNA Purification System (Promega #A7500). These DNAs were subsequently digested with NotI and HinD III restriction enzymes to confirm that the clones contained an insert.
  • the insert of one isolate was sequenced using a combination of vector specific and gene specific primers yielding a partial nucleotide sequence of human NgR3 of 1176 nucleotides (SEQ ID NO:13). A translation of this sequence provides a partial sequence for human NgR3 of 392 amino acids (SEQ ID NO:14).
  • the nucleotide sequence of SEQ ID NO:13 differs from the Incyte EST sequence at three positions. Nucleotide positions 12-13 in SEQ ID NO:13 are CG, whereas the corresponding nucleotides in the Incyte Template ID 190989.1 are GT (i.e., positions 12-13 of the complement of Incyte Template ID 190989.1). In addition, position 641 in SEQ ID NO:13 is a C, whereas the corresponding nucleotide in the Incyte Template ID 190989.1 sequence is an A (i.e., position 641 of the complement of Incyte Template ID 190989.1).
  • SEQ ID NO:14 contains a valine at position 5
  • the ORF encoded by Incyte Template ID 190989.1 contains a leucine
  • SEQ ID NO:14 contains an alanine at position 214
  • the ORF encoded by Incyte Template ID 190989.1 contains a glutamic acid.
  • the nucleotide sequence of SEQ ID NO:13 differs from the public EST (Accession number: R35699; Version number: R35699.1; GI: 792600) sequence at two positions (within the first 200 nucleotides of reliable sequence). Nucleotide positions 12-13 in SEQ ID NO:13 are CG, whereas the corresponding nucleotides in the public EST are GT (i.e., positions 12-13 of the public EST; Accession no: R35699; Version no: R35699.
  • SEQ ID NO:14 contains a valine at position 5, while the ORF encoded by the public EST contains a leucine.
  • a Bestfit analysis of the partial human amino acid sequence with the full-length mouse amino acid sequence indicates that the human NgR3 amino acid sequence is complete at the carboxy terminal end and that they share 89.54% identity.
  • An alignment of all the NgR proteins is shown in FIG. 9. Although the human NgR3 amino acid sequence is missing the first 25 amino acids, it can be determined that the human NgR3 protein contains the following features in common with the other NgR sequences: (1) eight Leucine Rich Repeat (LRR) domains; (2) an LRR carboxy-terminal (LRR-CT) domain; (3) a conserved cysteine in the fourth LRR domain; (4) a conserved potential glycosylation site in the eighth LRR domain; and (5) a hydrophobic carboxyl terminus.
  • LRR Leucine Rich Repeat
  • LRR-CT LRR carboxy-terminal
  • SEQ ID NO:4 a mouse NgR3 amino acid sequence
  • SEQ ID NO:5 a human NgR1 amino acid sequence
  • SEQ ID NO:6 a consensus amino acid sequence for NgRs
  • SEQ ID NO:8 a mature human NgR2 amino acid sequence
  • SEQ ID NO:9 a mature mouse NgR3 amino acid sequence
  • SEQ ID NO:10 a consensus NgR LLRNT amino acid sequence
  • SEQ ID NO:11 a consensus NgR LRRCT domain amino acid sequence
  • SEQ ID NO:12 a consensus NgR LRR domain amino acid sequence
  • SEQ ID NO:13 a partial human NgR3 nucleotide sequence
  • SEQ ID NO:14 a partial human NgR3 amino acid sequence
  • SEQ ID NO:15 a genomic sequence encoding a human NgR2 sequence.
  • SEQ ID NO:16 a genomic sequence (complementary strand) encoding a mouse NgR
  • SEQ ID NO:17 a mouse NgR3 amino acid sequence
  • SEQ ID NO:18 a consensus sequence for the NTLRRCT domain of NgR
  • SEQ ID NO:19 an consensus NgR LRRCT domain amino acid sequence
  • modified_base (1001)..(1100) a, t, c, g, other or unknown 16 ttgggggtat aaacccagaa gtgggattac tgcaccatac aataatcctc taacttcaag 60 caattttcc acaatggttg tatcatttta cattcccact ggctacgaga agggttccca 120 cttctacaca tcttcaccac catttctgttttgttttttg agtaacagct gcctaatgac 180 tgtgaagtgg tatcttatct cagtgtgat ttgcatttct ctgatcatta atgtgggaag 240 gcatcgtttcatta atgtgggaag 240 gcatcgtt

Abstract

The invention relates generally to genes that encode proteins that inhibit axonal growth. The invention relates specifically to genes encoding NgR protein homologs in humans and mice. The invention also includes compositions and methods for modulating the expression and activity of Nogo and the NgR proteins. Specifically, the invention includes peptides, proteins and antibodies that block Nogo-mediated inhibition of axonal extension. The compositions and methods of the invention are useful in the treatment of cranial or cerebral trauma, spinal cord injury, stroke or a demyelinating disease.

Description

    FIELD OF THE INVENTION
  • The invention relates to neurology and molecular biology. More particularly, the invention relates to CNS neurons and axonal growth [0001]
    • BACKGROUND
  • Among the mechanisms through which the cells of an organism communicate with each other and obtain information and stimuli from their environment is through cell membrane receptor molecules expressed on the cell surface. Many such receptors have been identified, characterized, and sometimes classified into major receptor superfamilies based on structural motifs and signal transduction features. The receptors are a first essential link for translating an extracellular signal into a cellular physiological response. [0002]
  • Receptors on neurons are particularly important in the development of the nervous system during embryogenesis. The neurons form connections with target cells during development through axonal extension of the neurons toward the target cells in a receptor-mediated process. Axons and dendrites have a specialized region of their distal tips known as the growth cone. Growth cones enable the neuron to sense the local environment through a receptor-mediated process and direct the movement of the axon or dendrite of the neuron toward the neuron's target cell. This process is known as elongation. Growth cones can be sensitive to several guidance cues, for example, surface adhesiveness, growth factors, neurotransmitters and electric fields. The guidance of growth at the cone depends on various classes of adhesion molecules, intercellular signals, as well as factors that stimulate and inhibit growth cones. [0003]
  • Interestingly, damaged neurons do not elongate in the central nervous system (CNS) following injury due to trauma or disease, whereas axons in the peripheral nervous system (PNS) regenerate readily. The fact that damaged CNS neurons fail to elongate is not due to an intrinsic property of CNS axons, but rather due to the CNS environment that is not permissive for axonal elongation. Classical grafting experiments by Aguayo and colleagues (e.g., Richardson et al., (1980) [0004] Nature 284, 264-265) demonstrated that CNS axons can in fact elongate over substantial distances within peripheral nerve grafts, and that CNS myelin inhibits CNS axon elongation. Therefore, given the appropriate environment, CNS axons can regenerate, implying that CNS axonal injury can potentially be addressed by appropriate manipulation of the CNS environment.
  • The absence of axon regeneration following injury can be attributed to the presence of axon growth inhibitors. These inhibitors are predominantly associated with myelin and constitute an important barrier to regeneration. Axon growth inhibitors are present in CNS-derived myelin and the plasma membrane of oligodendrocytes that synthesize myelin in the CNS (Schwab et al., (1993) [0005] Annu. Rev. Neurosci. 16, 565-595). Myelin-associated inhibitors appear to be a primary contributor to the failure of CNS axon regeneration in vivo after an interruption of axonal continuity, whereas other non-myelin associated axon growth inhibitors in the CNS may play a lesser role. These inhibitors block axonal regeneration following neuronal injury due to trauma, stroke or viral infection.
  • Numerous myelin-derived axon growth inhibitors have been characterized (see, for review, David et al., (1999) WO995394547; Bandman et al., (1999) U.S. Pat. No. 5,858,708; Schwab, (1996) [0006] Neurochem. Res. 21, 755-761). Several components of CNS white matter, NI35, NI250 (Nogo) and Myelin-associated glycoprotein (MAG), which have inhibitory activity for axonal extension, have been described as well (Schwab et al., (1990) WO9005191; Schwab et al., (1997) U.S. Pat. No. 5,684,133). In particular, Nogo is a 250 kDa myelin-associated axon growth inhibitor that was originally characterized based on the effects of the purified protein in vitro and monoclonal antibodies that neutralize the protein's activity (Schwab (1990) Exp. Neurol. 109, 2-5). The Nogo cDNA was first identified through random analysis of brain cDNA and had no suggested function (Nagase et al., (1998) DNA Res. 5, 355-364). The identification of this Nogo cDNA as the cDNA encoding the 250 kDa myelin-associated axon growth inhibitor was discovered only recently (GrandPre et al, (2000) Nature 403, 439-444; Chen et al., (2000) Nature 403, 434-439; Prinjha at al., (2000) Nature 403, 383-384).
  • Importantly, Nogo has been shown to be the primary component of CNS myelin responsible for inhibiting axonal elongation and regeneration. Nogo's selective expression by oligodendrocytes and not by Schwann cells (the cells that myelinate P. S. axons) is consistent with the inhibitory effects of CNS myelin, in contrast to P.S. myelin (GrandPre et al., (2000) [0007] Nature 403, 434-439). In culture, Nogo inhibits axonal elongation and causes growth cone collapse (Spillmann et al., (1998) J. Biol. Chem. 272, 19283-19293). Antibodies (e.g., IN-1) against Nogo have been shown to block most of the inhibitory action of CNS myelin on neurite growth in vitro (Spillmann et al., (1998) J. Biol. Chem. 272:19283-19293). These experiments indicate that Nogo is the main component of CNS myelin responsible for inhibition of axonal elongation in culture. Furthermore, in vivo, the IN-1 antibody has been shown to enhance axonal regeneration after spinal cord injury, resulting in recovery of behaviors such as contact placing and stride length (Schnell and Schwab (1990) Nature 343, 269-272; Bregman et al., (1995) Nature 378, 498-501). Thus, there is substantial evidence that Nogo is a disease-relevant molecular target. Agents that interfere with the binding of Nogo to its receptor would be expected to improve axonal regeneration in clinical states in which axons have been damaged, and improve patient outcome.
  • Modulation of Nogo has been described as a means for treatment of regeneration for neurons damaged by trauma, infarction and degenerative disorders of the CNS (Schwab et al., (1994) WO9417831 Tatagiba et al., (1997) [0008] Neurosurgery 40, 541-546) as well as malignant tumors in the CNS such as glioblastoma (Schwab et al., (1993) U.S. Pat. No. 5,250,414); Schwab et al., (2000) U.S. Pat. No. 6,025,333).
  • Antibodies which recognize Nogo have been suggested to be useful in the diagnosis and treatment of nerve damage resulting from trauma, infarction and degenerative disorders of the CNS (Schnell & Schwab, (1990) [0009] Nature 343, 269-272; Schwab et al., (1997) U.S. Pat. No. 5,684,133). For CNS axons, there is a correlation between the presence of myelin and the inhibition of axon regeneration over long distances (Savio and Schwab (1990) Proc. Natl. Acad. Sci. 87, 4130-4133; Keirstead et al., (1992) Proc. Natl. Acad. Sci. 89, 11664-11668). After Nogo is blocked by antibodies, neurons can again extend across lesions caused by nerve damage (Schnell and Schwab (1990) Nature 343, 269-272).
    • SUMMARY OF THE INVENTION
  • Genes encoding homologs (NgR2 and NgR3) of a Nogo receptor (NgR1) in mice and humans have been discovered. Various domains in the polypeptides encoded by the NgR2 and NgR3 genes have been identified and compared to domains in mouse and human NgR1 polypeptides. This comparison has led to identification of a consensus sequence (NgR consensus sequence) that characterizes a family of proteins (NgR family). Based on these and other discoveries, the invention features molecules and methods for modulating axonal growth in CNS neurons. [0010]
  • The invention provides a polypeptide that contains a polypeptide containing a tryptophan rich LRRCT domain consisting of the amino acid sequence: [0011]
    N X1 W X2 C X3 C R A R X4 L W X5 W X6 X7 X8 X9 R X10 S S S X11 V [SEQ ID NO. 19]
    X12 C X13 X14 P X15 X16 X17 X18 X19 X20 D L X21 X22 L X23 X24 X25 D
    X26 X27 X28 C
  • wherein X is any protein amino acid or a gap, and the polypeptide does not include amino acid sequence from residue 260 to 309 of SEQ ID NO: 5 (human NgR1) or SEQ ID NO: 17 (mouse NgR1). [0012]
  • Preferably, X17 and X23 are (independently) arginine or lysine. In some embodiments, the amino acid sequence of the LRRCT domain is residues 261-310 of SEQ ID NO:2, or residues 261-310 of SEQ ID NO: 2 with up to 10 conservative amino acid substitutions. In some embodiments, the polypeptide contains the following NTLRRCT amino acid sequence: [0013]
    C P X1 X2 C X3 C Y X4 X5 P X6 X7 T X8 S C X9 X10 X11 X12 X13 X14 X15 X16 P [SEQ ID NO: 18]
    X17 X18 X19 P X20 X21 X22 X23 R X24 F L X25 X26 N X27 I X28 X29 X30 X31 X32 X33
    X34 F X35 X36 X37 X38 X39 X40 X41 X42 L W X43 X44 S N X45 X46 X47 X48 I X49
    X50 X51 X52 F X53 X54 X55 X56 X57 L E X58 L D L X59 D N X60 X61 L X62 X63 X64
    X65 P X66 T F X67 G L X68 X69 L X70 X71 L X72 L X73 X74 C X75 L X76 X77 L X78
    X79 X80 X81 F X82 G L X83 X84 L Q Y L Y L Q X85 N X86 X87 X88 X89 L X90 D
    X91 X92 F X93 D L X94 N L X95 H L F L H G N X96 X97 X98 X99 X100 X101 X102
    X103 X104 F R G L X105 X106 L D R L L L H X107 N X108 X109 X110 X111 V H X112
    X113 A F X114 X115 L X116 R L X117 X118 L X119 L F X120 N X121 L X122 X123 L
    X124 X125 X126 X127 L X128 X129 L X130 X131 L X132 X133 L R L N X134 N X135 W
    X136 C X137 C R X138 R X139 L W X140 W X141 X142 X143 X144 R X145 S S S X146
    V X147 C X148 X149 P X150 X151 X152 X153 X154 X155 D L X156 X157 L X158 X159 X160
    D X161 X162 X163 C
  • wherein X is any amino acid residue or a gap and wherein the polypeptide is not the polypeptide of SEQ ID NO: 5 (human NgR1) or SEQ ID NO: 17 (mouse NgR1). For example, X[0014] 6, X37 and X38 may represent a gap. Specific examples of polypeptides of the invention are SEQ ID NO: 2 (human NgR2), SEQ ID NO: 4 (mouse NgR3), and SEQ ID NO: 14 (human NgR3). In some embodiments, the polypeptide contains: (a) a NTLRRCT domain, and (b) less than a complete CTS domain, provided that a partial CTS domain, if present, consists of no more than the first 39 amino acids of the CTS domain. While the polypeptide may contain a functional GPI domain, a functional GPI domain may be absent, e.g., when a soluble polypeptide is desired. A polypeptide of the invention optionally includes an amino acid sequence of a heterologous polypeptide, e.g., an Fc portion of an antibody.
  • The invention also provides a nucleic acid encoding an above-described polypeptide; a vector containing the nucleic acid, which nucleic acid may be operably linked to an expression control sequence; and a transformed host cell containing the vector. A method of producing a polypeptide of the invention is also provided. The method includes introducing a nucleic acid encoding the above-described polypeptide into a host cell, culturing the cell under conditions suitable for expression of the polypeptide, and recovering the polypeptide. [0015]
  • The invention also provides an antisense molecule whose nucleotide sequence is complementary to a nucleotide sequence encoding a polypeptide selected from the group consisting of: a polypeptide consisting of residues 311-395 of SEQ ID NO: 2, a polypeptide consisting of residues 256-396 of SEQ ID NO: 14 and a polypeptide consisting of residues 321-438 of SEQ ID NO: 4, wherein the nucleic acid is from 8 to 100 nucleotides in length, e.g., about 20, 30, 40, 50, 60, 70, 80 or 90 nucleotides. The invention also provides a nucleic acid encoding such an antisense molecule. [0016]
  • The invention also provides an antibody that binds to an above-described polypeptide. Polypeptides or antibodies of the invention can be formulated into pharmaceutical compositions containing the polypeptide or antibody and a pharmaceutically acceptable carrier. [0017]
  • The invention also provides a method for decreasing inhibition of axonal growth of a CNS neuron. The method includes the step of contacting the neuron with an effective amount of a polypeptide or antibody of the invention. The invention also provides a method for treating a central nervous system disease, disorder or injury. The method includes administering to a mammal, e.g., a human, an effective amount of a polypeptide or antibody of the invention. Exemplary diseases, disorders and injuries that may be treated using molecules and methods of the invention include, but are not limited to, cerebral injury, spinal cord injury, stroke, demyelinating diseases, e.g., multiple sclerosis, monophasic demyelination, encephalomyelitis, multifocal leukoencephalopathy, panencephalitis, Marchiafava-Bignami disease, Spongy degeneration, Alexander's disease, Canavan's disease, metachromatic leukodystrophy and Krabbe's disease. [0018]
  • The invention also provides a method for identifying a molecule that binds a polypeptide of the invention. The method includes the steps of: (a) providing a polypeptide of the invention; (b) contacting the polypeptide with the candidate molecule; and (c) detecting binding of the candidate molecule to the polypeptide. [0019]
  • Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In case of conflict, the present application, including definitions, will control. All publications, patent and other references mentioned herein are incorporated by reference. [0020]
  • The materials, methods and examples presented below are illustrative only, and not intended to be limiting. Other features and advantages of the invention will be apparent from the detail description and from the claims.[0021]
    • BRIEF DESCRIPTION OF THE FIGURES
  • FIGS. [0022] 1A-1B shows an alignment of NgR2 (SEQ ID NO:2) and NgR3 (SEQ ID NO:4) with the known NgR, NgR1 (SEQ ID NO:5) and the Consensus Sequence (SEQ ID NO:6).
  • FIG. 2. mNgR3 does not bind hNogoA(1055-1120). COS-7 cells were transfected with vectors encoding myc-NgR1 or myc-NgR3, fixed, and stained with anti-myc antibodies or AP-hNogoA(1055-1120). [0023]
  • FIG. 3. An alignment of the amino acid sequences of human NgR1, murine NgR1, murine NgR3, human NgR3 and human NgR2. Numbering begins with [0024] amino acid #1 of murine NgR3. The consensus sequence is listed below. The LRR NT domain is indicated by a shaded box; domains LLR 1, LLR 3, LLR 5, and LLR 7 are indicated by open boxes; LLR 2, LLR 4, LLR 6 and LLR 8 are indicated by shaded boxes; and the LLR CT domain is indicated by a shaded box. Amino acids in bold in LLR 8 indicate a conserved glycosylation sites. A dot indicates conserved cystine residue in LRR4. Box at C terminus indicates putative GPI signals.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides purified and isolated polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, both single- and double-stranded, including splice variants thereof) encoding NgR homologs, referred to herein as NgR. Unless indicated otherwise, as used herein, the abbreviation in lower case (NgR) refers to a gene, cDNA, RNA or nucleic acid sequence, whereas the upper case version (NgR) refers to a protein, polypeptide, peptide, oligopeptide, or amino acid sequence. Specific proteins are designated by number, e.g., “NgR2” is a human NgR homolog, “NgR3” is a murine-derived NgR homolog, and “NgR1” is the known NgR identified by Dr. Stephen Strittmatter. Known NgRs are herein referred to as “NgRs.” DNA polynucleotides of the invention include genomic DNA, cDNA and DNA that has been chemically synthesized in whole or in part. [0025]
  • Standard reference works setting forth the general principles of recombinant DNA technology known to those of skill in the art include Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York (1998); Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y. (1989); Kaufman et al., Eds., HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE, CRC Press, Boca Raton (1995); McPherson, Ed., DIRECTED MUTAGENESIS: A PRACTICAL APPROACH, IRL Press, Oxford (1991). [0026]
  • As used herein, the term “axon” refers to a long cellular protrusion from a neuron, whereby action potentials are conducted, either to or from the cell body. [0027]
  • As used herein, the term “axonal growth” refers to an extension of the long process or axon, originating at the cell body and proceeded by the growth cone. [0028]
  • As used herein, the term “central nervous system disorder” refers to any pathological state associated with abnormal function of the central nervous system (CNS). The term includes, but is not limited to, altered CNS function resulting from physical trauma to cerebral tissue, viral infection, autoimmune machanisms and genetic mutation. [0029]
  • As used herein, the term “demyelinating disease” refers to a pathological disorder characterized by the degradation of the myelin sheath of the oligodendrocyte cell membrane. [0030]
  • As used herein, the term “growth cone” refers to a specialized region at the tip of a growing neurite that is responsible for sensing the local environment and moving the axon toward its appropriate synaptic target cell. [0031]
  • As used herein, the term “growth cone movement” refers to the extension or collapse of the growth cone toward a neuron's target cell. [0032]
  • As used herein, the term “neurite” refers to a process growing out of a neuron. As it is sometimes difficult to distinguish a dendrite from in axon in culture, the term “neurite” is used for both. [0033]
  • As used herein, the term “oligodendrocyte” refers to a neuroglial cell of the CNS whose function is to myelinate CNS axons. [0034]
  • “Synthesized” as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. “Wholly” synthesized DNA sequences are therefore produced entirely by chemical means, and “partially” synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means. By the term “region” is meant a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein. The term “domain” is herein defined as referring to a structural part of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be co-extensive with regions or portions thereof, domains may also incorporate a portion of a biomolecule that is distinct from a particular region, in addition to all or part of that region. Examples of NgR protein domains include, but are not limited to, the signal peptide, extracellular (i.e., N-terminal) domain, and leucine-rich repeat domains. [0035]
  • As used herein, the term “activity” refers to a variety of measurable indicia suggesting or revealing binding, either direct or indirect; affecting a response, i.e., having a measurable affect in response to some exposure or stimulus, including, for example, the affinity of a compound for directly binding a polypeptide or polynucleotide of the invention, or, for example, measurement of amounts of upstream or downstream proteins or other similar functions after some stimulus or event. Such activities may be measured by assays such as competitive inhibition of NgR1 binding to Nogo assays wherein, for example, unlabeled, soluble NgR2 is added to an assay system in increasing concentrations to inhibit the binding of Nogo to NgR1 expressed on the surface of CHO cells. As another example, one may assess the ability of neurons to extend across lesions caused by nerve damage (as in Schnell and Schwab (1990) [0036] Nature 343, 269-272) following inhibition of Nogo by various forms of NgR2 and/or NgR3 as a biological indicator of NgR function.
  • As used herein, the term “antibody” is meant to refer to complete, intact antibodies, and Fab, Fab′, F(ab)2, and other fragments thereof Complete, intact antibodies include monoclonal antibodies such as murine monoclonal antibodies, chimeric antibodies, anti-idiotypic antibodies, anti-anti-idiotypic antibodies, and humanized antibodies. [0037]
  • As used herein, the term “binding” means the physical or chemical interaction between two proteins or compounds or associated proteins or compounds or combinations thereof. Binding includes ionic, non-ionic, hydrogen bonds, Van der Waals, hydrophobic interactions, etc. The physical interaction, the binding, can be either direct or indirect, indirect being through or due to the effects of another protein or compound. Direct binding refers to interactions that do not take place through or due to the effect of another protein or compound but instead are without other substantial chemical intermediates. [0038]
  • As used herein, the term “compound” means any identifiable chemical or molecule, including, but not limited to, small molecules, peptides, proteins, sugars, nucleotides or nucleic acids, and such compound can be natural or synthetic. [0039]
  • As used herein, the term “complementary” refers to Watson-Crick basepairing between nucleotide units of a nucleic acid molecule. [0040]
  • As used herein, the term “contacting” means bringing together, either directly or indirectly, a compound into physical proximity to a polypeptide or polynucleotide of the invention. The polypeptide or polynucleotide can be in any number of buffers, salts, solutions etc. Contacting includes, for example, placing the compound into a beaker, microtiter plate, cell culture flask, or a microarray, such as a gene chip, or the like, which contains the nucleic acid molecule, or polypeptide encoding the NgR or fragment thereof. [0041]
  • As used herein, the phrase “homologous nucleotide sequence,” or “homologous amino acid sequence,” or variations thereof, refers to sequences characterized by an identity at the nucleotide level, or a homology at the amino acid level, of at least the specified percentage. Homologous nucleotide sequences include those sequences coding for isoforms of proteins. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a species other than humans, including, but not limited to, mammals. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the nucleotide sequence encoding NgR1. Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity. A homologous amino acid sequence does not, however, include the amino acid sequence encoding other known NgRs. Percent homology can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, [0042] Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which is incorporated herein by reference in its entirety).
  • As used herein, the term “isolated” nucleic acid molecule refers to a nucleic acid molecule (DNA or RNA) that is substantially free of nucleic acids encoding other proteins with which it is associated in nature, i.e., a nucleic acid that has been removed from its native environment. Examples of isolated nucleic acid molecules include, but are not limited to, recombinant DNA molecules contained in a vector, recombinant DNA molecules maintained in a heterologous host cell, partially or substantially purified nucleic acid molecules, and synthetic DNA or RNA molecules. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated NgR nucleic acid molecule can contain less than about 50 kb, 25 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized. [0043]
  • As used herein, the term “heterologous” refers to a nucleotide or amino acid sequence that is a different, or non-corresponding sequence, or a sequence derived from a different species. For example, a mouse NgR nucleotide or amino acid sequence is heterologous to a human NgR nucleotide or amino acid sequence, and a human NgR nucleic or amino acid sequence is heterologous to a human immunoglobulin nucleotide or amino acid sequence. [0044]
  • As used herein, a “soluble NgR polypeptide” is a NgR polypeptide that does not anchor itself in a membrane. Such soluble polypeptides include, for example, NgR2 and NgR3 polypeptides that lack a sufficient portion of their GPI anchor signal to anchor the polypeptide or are modified such that the GPI anchor signal is not adequate to result in replacement of the peptide with a GPI anchor. In preferred embodiments, up to 5, 10, 20 or 25 amino acids are removed from the C-terminus of NgR2 or NgR3 to make the respective proteins soluble. As used herein soluble NgR polypeptides include full-length or truncated (e.g., with internal deletions) NgR. [0045]
  • Soluble NgR polypeptides may include the entire NgR protein up to the putative GPI signal sequence (e.g., [0046] amino acid 1 to about amino acid 395 of NgR2, and from amino acid 1 to about amino acid 438 of NgR3). In other embodiments, the signal peptide of the proteins may be removed or truncated (e.g., all or part of the signal sequence of NgR2, which spans amino acid 1 to about amino acid 30 of SEQ ID NO:2, may be removed; all or part of the signal sequence of NgR3, which spans amino acid 1 to about amino acid 40 of SEQ ID NO:4, may be removed). In some embodiments, the mature NgR2 (SEQ ID NO:8) and the mature NgR3 (SEQ ID NO:9) are used.
  • Soluble NgR polypeptides include at least one of the putative ligand-binding portions of NgR, including the first cysteine-rich region (SEQ ID NO:10, the leucine repeat region (SEQ ID NO:12) and the second cysteine-rich region (SEQ ID NO:11). In some embodiments, soluble NgR polypeptides consist of [0047] amino acid 1 through about amino acid 395 of SEQ ID NO:2, or amino acid 1 through about amino acid 438 of SEQ ID NO:4.
  • In other embodiments, the soluble NgR polypeptides are fusion proteins that contain amino acids 30 through about amino acid 395 of mature NgR2 or amino acid 40 through about amino acid 438 of NgR3, the C-terminal 10 amino acids of a [0048] human IgG 1 hinge region containing the two cysteine residues thought to participate in interchain disulfide bonding, and the CH2 and CH3 regions of a human IgG1 heavy chain constant domain. This type of recombinant protein is designed to modulate inhibition of axonal elongation through inhibition of the Nogo ligand binding to NgR1, or by inhibiting the ligand of the NgR from interacting with cell surface NgR. The NgR portion of the fusion binds to the Nogo ligand and the IgG1 portion binds to the FcγRI (macrophage) and FcγIII (NK cells and neutrophils) receptors.
  • The production of the soluble polypeptides useful in this invention may be achieved by a variety of methods known in the art. For example, the polypeptides may be derived from intact transmembrane NgR molecules by proteolysis using specific endopeptidases in combination with exopeptidases, Edman degradation, or both. The intact NgR molecule, in turn, may be purified from its natural source using conventional methods. Alternatively, the intact NgR may be produced by known recombinant DNA techniques using cDNAs, expression vectors and well-known techniques for recombinant gene expression. [0049]
  • Preferably, the soluble polypeptides useful in the present invention are produced directly, thus eliminating the need for an entire NgR as a starting material. This may be achieved by conventional chemical synthesis techniques or by well-known recombinant DNA techniques wherein only those DNA sequences which encode the desired peptides are expressed in transformed hosts. For example, a gene which encodes the desired soluble NgR polypeptide may be synthesized by chemical means using an oligonucleotide synthesizer. Such oligonucleotides are designed based on the amino acid sequence of the desired soluble NgR polypeptide. Specific DNA sequences coding for the desired peptide also can be derived from the full-length DNA sequence by isolation of specific restriction endonuclease fragments or by PCR synthesis of the specified region from cDNA. [0050]
  • A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOs:1, 3 or a complement of either of these nucleotide sequences, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequences of SEQ ID NOs:1 or 3 as a hybridization probe, NgR nucleic acid sequences can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook et al., eds., MOLECULAR CLONING: A [0051] LABORATORY MANUAL 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausubel, et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993).
  • A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to NgR nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. [0052]
  • As used herein, the terms “modulates” or “modifies” means an increase or decrease in the amount, quality, or effect of a particular activity or protein. [0053]
  • As used herein, the term “oligonucleotide” refers to a series of linked nucleotide residues which has a sufficient number of bases to be used in a polymerase chain reaction (PCR). This short sequence is based on (or designed from) a genomic or cDNA sequence and is used to amplify, confirm or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 50 nucleotides, preferably about 15 to 30 nucleotides. They are chemically synthesized and may be used as probes. [0054]
  • As used herein, the term “probe” refers to nucleic acid sequences of variable length, preferably between at least about 10 and as many as about 6,000 nucleotides, depending on use. They are used in the detection of identical, similar or complementary nucleic acid sequences. Longer length probes are usually obtained from a natural or recombinant source, are highly specific and much slower to hybridize than oligomers. They may be single- or double-stranded and carefully designed to have specificity in PCR, hybridization membrane-based, or ELISA-like technologies. [0055]
  • The term “preventing” refers to decreasing the probability that an organism contracts or develops an abnormal condition. [0056]
  • The term “treating” refers to having a therapeutic effect and at least partially alleviating or abrogating an abnormal condition in the organism. [0057]
  • The term “therapeutic effect” refers to the inhibition or activation factors causing or contributing to the abnormal condition. A therapeutic effect relieves to some extent one or more of the symptoms of the abnormal condition. In reference to the treatment of abnormal conditions, a therapeutic effect can refer to one or more of the following: (a) an increase in the proliferation, growth, and/or differentiation of cells; (b) inhibition (i.e., slowing or stopping) of cell death; (c) inhibition of degeneration; (d) relieving to some extent one or more of the symptoms associated with the abnormal condition; and (e) enhancing the function of the affected population of cells. Compounds demonstrating efficacy against abnormal conditions can be identified as described herein. [0058]
  • The term “abnormal condition” refers to a function in the cells or tissues of an organism that deviates from their normal functions in that organism. An abnormal condition can relate to cell proliferation, cell differentiation, cell signaling, or cell survival. An abnormal condition may also include obesity, diabetic complications such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid uptake and metabolism. [0059]
  • Abnormal cell proliferative conditions, for example, include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus and inflammation. [0060]
  • Abnormal differentiation conditions include, for example, neurodegenerative disorders, slow wound healing rates and slow tissue grafting healing rates. [0061]
  • Abnormal cell signaling conditions include, for example, psychiatric disorders involving excess neurotransmitter activity. [0062]
  • Abnormal cell survival conditions may also relate to conditions in which programmed cell death (apoptosis) pathways are activated or abrogated. A number of protein kinases are associated with the apoptosis pathways. Aberrations in the function of any one of the protein kinases could lead to cell immortality or premature cell death. [0063]
  • The term “administering” relates to a method of incorporating a compound into cells or tissues of an organism. The abnormal condition can be prevented or treated when the cells or tissues of the organism exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, injection, and aerosol applications. For cells outside of the organism, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques and carrier techniques. [0064]
  • The abnormal condition can also be prevented or treated by administering a compound to a group of cells having an aberration in a signal transduction pathway to an organism. The effect of administering a compound on organism function can then be monitored. The organism is preferably a mouse, rat, rabbit, guinea pig or goat, more preferably a monkey or ape, and most preferably a human. [0065]
  • By “amplification” it is meant increased numbers of DNA or RNA in a cell compared with normal cells. “Amplification” as it refers to RNA can be the detectable presence of RNA in cells, since in some normal cells there is no basal expression of RNA. In other normal cells, a basal level of expression exists, therefore in these cases amplification is the detection of at least 1-2-fold, and preferably more, compared to the basal level. [0066]
  • The amino acid sequences are presented in the amino to carboxy direction, from left to right. The amino and carboxy groups are not presented in the sequence. The nucleotide sequences are presented by single strand only, in the 5′ to 3′ direction, from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission or (for amino acids) by three letters code. [0067]
  • Nucleic Acids [0068]
  • Genomic DNA of the invention comprises the protein-coding region for a polypeptide of the invention and is also intended to include allelic variants thereof It is widely understood that, for many genes, genomic DNA is transcribed into RNA transcripts that undergo one or more splicing events wherein intron (i.e., non-coding regions) of the transcripts are removed, or “spliced out.” RNA transcripts that can be spliced by alternative mechanisms, and therefore be subject to removal of different RNA sequences but still encode a NgR polypeptide, are referred to in the art as splice variants which are embraced by the invention. Splice variants comprehended by the invention therefore are encoded by the same original genomic DNA sequences but arise from distinct mRNA transcripts. Allelic variants are modified forms of a wild-type gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild-type genes, are naturally occurring sequences (as opposed to non-naturally occurring variants arising from in vitro manipulation). [0069]
  • The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding NgR (conventionally followed by second-strand synthesis of a complementary strand to provide a double-stranded DNA). [0070]
  • Preferred DNA sequences encoding a human NgR polypeptide is set out in SEQ ID NOs:1 and 13. A preferred DNA of the invention comprises a double stranded molecule comprising the coding molecule (i.e., the “coding strand”) along with the complementary molecule (the “non-coding strand” or “complement”) having a sequence unambiguously deducible from the coding strand according to Watson-Crick base-pairing rules for DNA. Also preferred are other polynucleotides encoding NgR polypeptides, as shown in SEQ ID NO:3, which comprises murine NgR homolog, NgR3. [0071]
  • Also preferred are nucleotide sequences that encode at least a portion of a NgR polypeptide that has at least one biological function of a NgR. More preferred are nucleotide sequences that encode a portion of NgR that encodes at least the mature NgR without the hydrophobic C-terminal GPI signal. Also preferred are nucleotide sequences that encode the portion of NgR that encodes at least the ligand-binding region of NgR. [0072]
  • The invention further embraces other species, preferably mammalian, homologs of the human NgR DNA. Species homologs, sometimes referred to as “orthologs,” in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with human DNA of the invention. Generally, percent sequence “homology” with respect to polynucleotides of the invention may be calculated as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the NgR sequences set forth in SEQ ID NOs:1, 3 or 13, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. [0073]
  • The polynucleotide sequence information provided by the invention makes possible large-scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides of the invention also permit identification and isolation of polynucleotides encoding related NgR polypeptides, such as human allelic variants and species homologs, by well-known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR). Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to NgR and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of NgR. Non-human species genes encoding proteins homologous to NgR can also be identified by Southern and/or PCR analysis and are useful in animal models for NgR disorders. Knowledge of the sequence of a human NgR DNA also makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding NgR expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express NgR. Polynucleotides of the invention may also provide a basis for diagnostic methods useful for identifying a genetic alteration(s) in a NgR locus that underlies a disease state or states, which information is useful both for diagnosis and for selection of therapeutic strategies. [0074]
  • The disclosure herein of a full-length polynucleotide encoding a NgR polypeptide makes readily available to the worker of ordinary skill in the art every possible fragment of the full-length polynucleotide. The invention, therefore, provides fragments of NgR-encoding polynucleotides comprising at least 6, and preferably at least 14, 16, 18, 20, 25, 50, or 75 consecutive nucleotides of a polynucleotide encoding NgR. Preferably, fragments of polynucleotides of the invention comprise sequences unique to the NgR-encoding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., “specifically”) to polynucleotides encoding NgR (or fragments thereof). Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full-length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases. Such sequences also are recognizable from Southern hybridization analyses to determine the number of fragments of genomic DNA to which a polynucleotide will hybridize. Polynucleotides of the invention can be labeled in a manner that permits their detection, including radioactive, fluorescent and enzymatic labeling. [0075]
  • Fragments of polynucleotides are particularly useful as probes for detection of full-length or fragment of NgR polynucleotides. One or more polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding NgR, or used to detect variations in a polynucleotide sequence encoding NgR. [0076]
  • The invention also embraces DNAs encoding NgR polypeptides that hybridize under moderately stringent or high stringency conditions to the noncoding strand, or complement, of the polynucleotide in any of SEQ ID NOs:1 or 3. [0077]
  • Stringent conditions are known to those skilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3. 1?6.3.6. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98% or 99% homologous to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions hybridization in a high salt buffer comprising 6× SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and 500 mg/ml denatured salmon sperm DNA at 65° C. This hybridization is followed by one or more washes in 0.2× SSC, 0.01% BSA at 50° C. An isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NOs:1 or 3 corresponds to a naturally occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). As used herein, “stringent hybridization conditions” means: 42° C. in a hybridization solution comprising 50% formamide, 1% SDS, 1 M NaCl, 10% (wt/vol) dextran sulfate, and washing twice for 30 minutes at 60° C. in a wash solution comprising 0.1× SSC and 1% SDS. [0078]
  • Vectors [0079]
  • Another aspect of the present invention is directed to vectors, or recombinant expression vectors, comprising any of the nucleic acid molecules described above. Vectors are used herein either to amplify DNA or RNA encoding NgR and/or to express DNA which encodes NgR. As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), that serve equivalent functions. [0080]
  • Expression of proteins in prokaryotes is most often carried out in [0081] E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67, 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • Examples of suitable inducible non-fusion [0082] E. coli expression vectors include pTrc (Amrann et al, (1988) Gene 69, 301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • One strategy to maximize recombinant protein expression in [0083] E. coil is to express the protein in host bacteria with an impaired capacity to proteolytically cleave the recombinant protein. See, Gottesman, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 119-128. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coil (Wada et al., (1992) Nucleic Acids Res. 20, 2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • In another embodiment, the NgR expression vector is a yeast expression vector. Examples of vectors for expression in yeast [0084] S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6, 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30, 933-943), pJRY88 (Schultz et al., (1987) Gene 54, 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).
  • Alternatively, NgR can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., (1983) [0085] Mol. Cell. Biol. 3, 2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170, 31-39).
  • In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329, 840) and pMT2PC (Kaufman et al. (1987) [0086] EMBO J. 6, 187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells. See, e.g., Chapters 16 and 17 of Sambrook et al., (Eds.) MOLECULAR CLONING: A LABORATORY MANUAL. 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) [0087] Genes Dev. 1, 268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43, 235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8, 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33, 729-740; Queen and Baltimore (1983) Cell 33, 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86, 5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230, 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249, 374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537-546).
  • The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner that allows for expression (by transcription of the DNA molecule) of an RNA molecule that is antisense NgR mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen that direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen that direct constitutive, tissue-specific or cell-type-specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub et al., [0088] Antisense RNA as a molecular tool for genetic analysis, REVIEWS—TRENDS IN GENETICS, Vol. 1(1) 1986.
  • Preferred vectors include, but are not limited to, plasmids, phages, cosmids, episomes, viral particles or viruses and integratable DNA fragments (i.e., fragments integratable into the host genome by homologous recombination). Preferred viral particles include, but are not limited to, adenoviruses, baculoviruses, parvoviruses, herpesviruses, poxviruses, adeno-associated viruses, Semliki Forest viruses, vaccinia viruses and retroviruses. Preferred expression vectors include, but are not limited to, pcDNA3 (Invitrogen) and pSVL (Pharmacia Biotech). Other expression vectors include, but are not limited to, pSPORT™ vectors, pGEM™ vectors (Promega), pPROEXvectors™ (LTI, Bethesda, Md.), Bluescript™ vectors (Stratagene), pQE™ vectors (Qiagen), pSE420™ (Invitrogen) and pYES2™(Invitrogen). [0089]
  • Preferred expression vectors are replicable DNA constructs in which a DNA sequence encoding NgR is operably linked or connected to suitable control sequences capable of effecting the expression of the NgR in a suitable host. DNA regions are operably linked or connected when they are functionally related to each other. For example, a promoter is operably linked or connected to a coding sequence if it controls the transcription of the sequence. Amplification vectors do not require expression control domains, but rather need only the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. The need for control sequences in the expression vector will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include, but are not limited to a transcriptional promoter, enhancers, an optional operator sequence to control transcription, polyadenylation signals, a sequence encoding suitable mRNA ribosomal binding and sequences which control the termination of transcription and translation. Such regulatory sequences are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., NgR proteins, mutant forms of NgR, fusion proteins, etc.). [0090]
  • Preferred vectors preferably contain a promoter that is recognized by the host organism. The promoter sequences of the present invention may be prokaryotic, eukaryotic or viral. Examples of suitable prokaryotic sequences include the PR and PL promoters of bacteriophage lambda (THE BACTERIOPHAGE LAMBDA, Hershey, A. D. (Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1973), which is incorporated herein by reference in its entirety; LAMBDA II, Hendrix, R. W. (Ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1980), which is incorporated herein by reference in its entirety); the trp, recA, heat shock, and lacZ promoters of [0091] E. coli and the SV40 early promoter (Benoist et al., (1981) Nature 290, 304-310, which is incorporated herein by reference in its entirety). Additional promoters include, but are not limited to, mouse mammary tumor virus, long terminal repeat of human immunodeficiency virus, maloney virus, cytomegalovirus immediate early promoter, Epstein Barr virus, Rous sarcoma virus, human actin, human myosin, human hemoglobin, human muscle creatine and human metallothionein.
  • Additional regulatory sequences can also be included in preferred vectors. Preferred examples of suitable regulatory sequences are represented by the Shine-Dalgarno sequence of the replicase gene of the phage MS-2 and of the gene cII of bacteriophage lambda. The Shine-Dalgarno sequence may be directly followed by DNA encoding NgR and result in the expression of the mature NgR protein. [0092]
  • Moreover, suitable expression vectors can include an appropriate marker that allows the screening of the transformed host cells. The transformation of the selected host is carried out using any one of the various techniques well known to the expert in the art and described in Sambrook et al., supra. [0093]
  • An origin of replication can also be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient. Alternatively, rather than using vectors which contain viral origins of replication, one skilled in the art can transform mammalian cells by the method of co-transformation with a selectable marker and NgR DNA. An example of a suitable marker is dihydrofolate reductase (DHFR) or thymidine kinase (see, U.S. Pat. No. 4,399,216). [0094]
  • Nucleotide sequences encoding NgR may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and ligation with appropriate ligases. Techniques for such manipulation are disclosed by Sambrook et al., supra and are well known in the art. Methods for construction of mammalian expression vectors are disclosed in, for example, Okayama et al., (1983) [0095] Mol. Cell. Biol. 3:280, Cosman et al. (1986) Mol. Immunol. 23:935, Cosman et al., (1984) Nature 312:768, EP-A-0367566, and WO 91/18982, each of which is incorporated herein by reference in its entirety.
  • Host Cells and Transformed Host Cells [0096]
  • According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention (or vector of the invention) in a manner that permits expression of the encoded NgR polypeptide. Preferably, the cell produces little or no endogenous NgR polypeptide. Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector. Methods for introducing DNA into the host cell that are well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells and protoplasts. Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, vertebrate and mammalian cells systems. [0097]
  • Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with NgR. Host cells of the invention are also useful in methods for the large-scale production of NgR polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells, or from the medium in which the cells are grown, by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC, and the like. Still other methods of purification include those methods wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or can be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process. [0098]
  • Knowledge of NgR DNA sequences allows for modification of cells to permit, or increase, expression of endogenous NgR. Cells can be modified (e.g., by homologous recombination) to provide increased expression by replacing, in whole or in part, the naturally occurring NgR promoter with all or part of a heterologous promoter so that the cells express NgR at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to endogenous NgR encoding sequences. (See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955.) It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamoyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the NgR coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the NgR coding sequences in the cells. [0099]
  • The DNA sequence information provided by the present invention also makes possible the development (e.g., by homologous recombination or “knock-out” strategies; see Capecchi, [0100] Science 244:1288-1292 (1989)) of animals that fail to express functional NgR or that express a variant of NgR. Such animals (especially small laboratory animals such as rats, rabbits and mice) are useful as models for studying the in vivo activities of NgR and modulators of NgR.
  • Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences. Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces and Staphylococcus. [0101]
  • If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence. Preferably, eukaryotic cells are cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Preferred host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, Eds. (1973), which is incorporated herein by reference in its entirety). [0102]
  • In addition, a yeast cell may be employed as a host cell. Preferred yeast cells include, but are not limited to, the genera Saccharomyces, Pichia and Kluveromyces. Preferred yeast hosts are [0103] S. cerevisiae and P. pastoris. Preferred yeast vectors can contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination and a selectable marker gene. Shuttle vectors for replication in both yeast and E. coli are also included herein.
  • Alternatively, insect cells may be used as host cells. In a preferred embodiment, the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., [0104] Bio/Technology, 1988, 6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL, O'Rielly et al. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No. 4,879,236, each of which is incorporated herein by reference in its entirety). In addition, the MAXBAC™ complete baculovirus expression system (Invitrogen) can, for example, be used for production in insect cells.
  • Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. [0105]
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals. [0106]
  • For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selectable markers include those that confer resistance to drugs, such as G418, hygromycin, dihydrofolate reductase (DHFR) and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding NgR or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die). [0107]
  • In a preferred embodiment, the polypeptides of the invention, including forms of NgR2 and NgR3, soluble forms of NgR, chimeric NgR polypeptides, NgR/Ig fusions and fragments and variations of each of the above are expressed in Chinese Hamster Ovary (CHO) cells. [0108]
  • In order to introduce the DNA fragment coding for the NgR protein or polypeptide into the CHO cell to express the recombinant NgR protein or polypeptide, it is necessary to construct the expression vector. [0109]
  • The vectors for CHO expression include, but are not limited to, pA1-11, pXT1, pRc/CMV, pRc/RSV and pcDNAINeo. The promoter is not specifically limited provided it effectively promotes expression in CHO cells. Examples of suitable promoters are: SRα, SV40, LTR, CMV, and HSV-TK. Of these, CMV and Srα promoters are preferred. [0110]
  • In addition to the above-mentioned promoters, the expression vectors may contain enhancers, splicing signals, polyadenylation signals, selectable markers and an SV40 replication origin. Suitable selectable markers include, but are not limited to the dihydrofolate reductase (DHFR) gene which provides resistance to methotrexate (MTX), the ampicillin resistance gene, and the neomycin resistance gene. [0111]
  • Examples of the expression vectors each containing the DNA coding for NgR, portions, fragments and soluble constructs thereof, include the vector (such as one described above), into which the promoter is operably linked (preferably upstream) to the nucleotide sequence encoding the desired NgR construct; a polyadenylation signal downstream from the nucleotide sequence encoding the NgR construct; and, preferably, the vector includes an operable DHFR gene. Preferably, the ampicillin resistant gene is also operably contained in the vector. [0112]
  • CHO cell lacking the DHFR gene (Urlaub, G. et al., (1980) [0113] Proc. Natl. Acad. Sci. USA 77, 4216-4220) and CHO-K1 (Proc. Natl. Acad. Sci. USA 60, 1275 (1968)) are suitable for use.
  • The NgR expression vectors prepared as above are introduced into CHO cells by any known method, including, but not limited to the calcium phosphate method (Graham and van der Eb (1973) [0114] Virol 52, 456-467) and electroporation (Nuemann et al., (1982) EMBO J. 1, 841-845).
  • Transformants carrying the expression vectors are selected based on the above-mentioned selectable markers. Repeated clonal selection of the transformants using the selectable markers allows selection of stable cell lines having high expression of the NgR constructs. Increased MTX concentrations in the selection medium allows gene amplification and greater expression of the desired protein. The CHO cell containing the recombinant NgR can be produced by cultivating the CHO cells containing the NR expression vectors constitutively expressing the NgR constructs. [0115]
  • Media used in cultivating CHO cells includes DMEM medium supplemented with about 0.5 to 20% fetal calf serum, DMEM medium and RPMI1640 medium. The pH of the medium is preferably about 6 to 8. Cultivation is preferably at about 30 to 40° C. for about 15 to 72 hours with aeration. [0116]
  • A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) NgR protein. Accordingly, the invention further provides methods for producing NgR protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding NgR has been introduced) in a suitable medium such that NgR protein is produced. In another embodiment, the method further comprises isolating NgR from the medium or the host cell. [0117]
  • In situations where the NgR polypeptide will be found primarily intracellularly, intracellular material (including inclusion bodies for Gram-negative bacteria) can be extracted from the host cell using any standard technique known to one of ordinary skill in the art. Such methods would encompass, by way of example and not by way of limitation, lysing the host cells to release the contents of the periplasm/cytoplasm by French press, homogenization and/or sonication followed by centrifugation. [0118]
  • If the NgR polypeptide has formed inclusion bodies in the cytosol, such inclusion bodies may frequently bind to the inner and/or outer cellular membranes. Upon centrifugation, the inclusion bodies will be found primarily in the pellet material. The pellet material can then be treated at pH extremes or with one or more chaotropic agents such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris-carboxyethyl phosphine at acid pH to release, break apart and solubilize the inclusion bodies. Once solubilized, NgR polypeptide can be analyzed using gel electrophoresis, immunoprecipitation or the like. Various methods of isolating the NgR polypeptide would be apparent to one of ordinary skill in the art, for example, isolation may be accomplished using standard methods such as those set forth below and in Marston et al (1990) [0119] Meth. Enzymol. 182, 264-275 (incorporated by reference herein in its entirety).
  • If isolated NgR polypeptide is not biologically active following the isolation procedure employed, various methods for “refolding” or converting the polypeptide to its tertiary structure and generating disulfide linkages, can be used to restore biological activity. Methods known to one of ordinary skill in the art include adjusting the pH of the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization but usually at a lower concentration and is not necessarily the same chaotrope as used for the solubilization. It may be required to employ a reducing agent or the reducing agent plus its oxidized form in a specific ratio, to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridge(s). Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol (DTT)/dithiane DTT, 2-mercaptoethanol (bME)/dithio-b(ME). To increase the efficiency of the refolding, it may be necessary to employ a cosolvent, such as glycerol, polyethylene glycol of various molecular weights and arginine. [0120]
  • Transgenic Animals [0121]
  • The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which NgR-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous NgR sequences have been introduced into their genome or homologous recombinant animals in which endogenous NgR sequences have been altered. Such animals are useful for studying the function and/or activity of NgR and for identifying and/or evaluating modulators of NgR activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A transgene is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops and that remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous NgR gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal. [0122]
  • A transgenic animal of the invention can be created by introducing NgR-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The human NgR DNA sequence of SEQ ID NOs:1 or 3 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homolog of the human NgR gene, such as a mouse NgR gene, can be isolated based on hybridization to the human NgR cDNA (described further above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to the NgR transgene to direct expression of NgR protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866; 4,870,009; and 4,873,191; and Hogan 1986, in MANIPULATING THE MOUSE EMBRYO, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of the NgR transgene in its genome and/or expression of NgR mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding NgR can further be bred to other transgenic animals carrying other transgenes. [0123]
  • To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a NgR gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the NgR gene. The NgR gene can be a human gene (e.g., SEQ ID NOs:1 or 13), but more preferably, is a non-human homolog of a human NgR gene. For example, a mouse homolog of human NgR gene of SEQ ID NOs:1 or 13 can be used to construct a homologous recombination vector suitable for altering an endogenous NgR gene in the mouse genome. In one embodiment, the vector is designed such that, upon homologous recombination, the endogenous NgR gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). [0124]
  • Alternatively, the vector can be designed such that, upon homologous recombination, the endogenous NgR gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous NgR protein). In the homologous recombination vector, the altered portion of the NgR gene is flanked at its 5′ and 3′ ends by additional nucleic acid of the NgR gene to allow for homologous recombination to occur between the exogenous NgR gene carried by the vector and an endogenous NgR gene in an embryonic stem cell. The additional flanking NgR nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the vector. See e.g., Thomas et al. (1987) [0125] Cell 51:503 for a description of homologous recombination vectors. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced NgR gene has homologously recombined with the endogenous NgR gene are selected (see e.g., Li et al. (1992) Cell 69:915).
  • The selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras. See e.g., Bradley 1987, In: TERATOCARCINOMAS AND EMBRYONIC STEM CELLS: A Practical Approach, Robertson, ed. IRL, Oxford, pp. 113-152. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described further in Bradley (1991) [0126] Curr. Opin. Biotechnol. 2:823-829; PCT International Publication Nos.: WO 90/11354; WO 91/01140; WO 92/0968; and WO 93/04169.
  • In another embodiment, transgenic non-humans animals can be produced that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) [0127] Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut et al. (1997) [0128] Nature 385:810-813. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter G0 phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.
  • Antisense [0129]
  • Also provided by the invention are antisense polynucleotides that recognize and hybridize to NgR polynucleotides. Full-length and fragment antisense polynucleotides are provided. Fragment antisense molecules of the invention include (i) those that specifically recognize and hybridize to NgR RNA (as determined by sequence comparison of DNA encoding NgR to DNA encoding other known molecules). Identification of sequences unique to NgR encoding polynucleotides can be deduced through use of any publicly available sequence database, and/or through use of commercially available sequence comparison programs. After identification of the desired sequences, isolation through restriction digestion or amplification using any of the various polymerase chain reaction techniques well known in the art can be performed. Antisense polynucleotides are particularly relevant to regulating expression of NgR by those cells expressing NgR mRNA. [0130]
  • Antisense oligonucleotides, or fragments of a nucleotide sequence set forth in SEQ ID NO:1, 3, 13 or sequences complementary or homologous thereto, derived from the nucleotide sequences of the present invention encoding NgR are useful as diagnostic tools for probing gene expression in various tissues. For example, tissue can be probed in situ with oligonucleotide probes carrying detectable groups by conventional autoradiography techniques to investigate native expression of this enzyme or pathological conditions relating thereto. In specific aspects, antisense nucleic acid molecules are provided that comprise a sequence complementary to at least about 10, 25, 50, 100, 250 or 500 nucleotides or an entire NgR coding strand, or to only a portion thereof. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a NgR protein of SEQ ID NO:2, 4 or 14 or antisense nucleic acids complementary to a NgR nucleic acid sequence of SEQ ID NOs:1, 3 or 13 are additionally provided. [0131]
  • In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding NgR. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the protein coding region of human NgR corresponds to the coding region SEQ ID NO:1, 3 or 13). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding NgR. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions). [0132]
  • Antisense oligonucleotides are preferably directed to regulatory regions of a nucleotide sequence of SEQ ID NO:1, 3, 13 or mRNA corresponding thereto, including, but not limited to, the initiation codon, TATA box, enhancer sequences, and the like. Given the coding strand sequences encoding NgR disclosed herein (e.g., SEQ ID NO:1, 3 or 13), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick or Hoogsteen base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of NgR mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of NgR mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of NgR mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis or enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. [0133]
  • Examples of modified nucleotides that can be used to generate the antisense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection). [0134]
  • The antisense nucleic acid molecules of the invention (preferably oligonucleotides of 10 to 20 nucleotides in length) are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a NgR protein to thereby inhibit expression of the protein, e.g. by inhibiting transcription and/or translation. Suppression of NgR expression at either the transcriptional or translational level is useful to generate cellular or animal models for diseases/conditions characterized by aberrant NgR expression. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. [0135]
  • Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. The antisense oligonucleotides may be further modified by adding poly-L-lysine, transferrin polylysine or cholesterol moieties at their 5′ end. [0136]
  • An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred. [0137]
  • In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al., (1987) [0138] Nucleic Acids Res. 15, 6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res. 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBS Lett. 215, 327-330).
  • The NgR sequences taught in the present invention facilitate the design of novel transcription factors for modulating NgR expression in native cells and animals, and cells transformed or transfected with NgR polynucleotides. For example, the Cys[0139] 2-His2 zinc finger proteins, which bind DNA via their zinc finger domains, have been shown to be amenable to structural changes that lead to the recognition of different target sequences. These artificial zinc finger proteins recognize specific target sites with high affinity and low dissociation constants, and are able to act as gene switches to modulate gene expression. Knowledge of the particular NgR target sequence of the present invention facilitates the engineering of zinc finger proteins specific for the target sequence using known methods such as a combination of structure-based modeling and screening of phage display libraries (Segal et al., (1999) Proc. Natl. Acad. Sci. USA 96, 2758-2763; Liu et al., (1997) Proc. Natl. Acad. Sci. USA 94, 5525-5530; Greisman et al. (1997) Science 275, 657-661; Choo et al., (1997) J. Mol. Biol. 273, 525-532). Each zinc finger domain usually recognizes three or more base pairs. Since a recognition sequence of 18 base pairs is generally sufficient in length to render it unique in any known genome, a zinc finger protein consisting of 6 tandem repeats of zinc fingers would be expected to ensure specificity for a particular sequence (Segal et al., (1999), above). The artificial zinc finger repeats, designed based on the promoter of NgR sequences, are fused to activation or repression domains to promote or suppress NgR expression (Liu et al., (1997), above). The promoter of NgR may be obtained by standard methods known to one of ordinary skill in the art with the disclosure contained herein and knowledge of the NgR sequence. Alternatively, the zinc finger domains can be fused to the TATA box-binding factor (TBP) with varying lengths of linker region between the zinc finger peptide and the TBP to create either transcriptional activators or repressors (Kim et al., (1997) Proc. Natl. Acad. Sci. USA 94, 3616-3620. Such proteins and polynucleotides that encode them, have utility for modulating NgR expression in vivo in both native cells, animals and humans; and/or cells transfected with NgR-encoding sequences. The novel transcription factor can be delivered to the target cells by transfecting constructs that express the transcription factor (gene therapy), or by introducing the protein. Engineered zinc finger proteins can also be designed to bind RNA sequences for use in therapeutics as alternatives to antisense or catalytic RNA methods (McColl et al., (1997) Proc. Natl. Acad. Sci. USA 96, 9521-9526); Wu et al., (1995) Proc. Natl. Acad. Sci. USA 92, 344-348). The present invention contemplates methods of designing such transcription factors based on the gene sequence of the invention, as well as customized zinc finger proteins, that are useful to modulate NgR expression in cells (native or transformed) whose genetic complement includes these sequences.
  • Ribozymes and PNA Moieties [0140]
  • In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes, described in Haselhoff and Gerlach (1988) [0141] Nature 334, 585-591) can be used to catalytically cleave NgR mRNA transcripts to thereby inhibit translation of NgR mRNA. A ribozyme having specificity for a NgR-encoding nucleic acid can be designed based upon the nucleotide sequence of a NgR DNA disclosed herein (i.e., SEQ ID NOs:1, 3 or 13). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a NgR-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, NgR mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al., (1993) Science 261, 1411-1418.
  • Alternatively, NgR gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the NgR (e.g., the NgR promoter and/or enhancers) to form triple helical structures that prevent transcription of the NgR gene in target cells. See generally, Helene (1991) [0142] Anticancer Drug Des. 6: 569-584; Helene. et al., (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) BioEssays 14, 807-815.
  • In various embodiments, the nucleic acids of NgR can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see Hyrup et al., (1996) [0143] Bioorg. Med. Chem. Lett. 4, 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al., (1996) above; Perry-O'Keefe et al., (1996) Proc. Natl. Acad. Sci. USA 93,14670-14675.
  • PNAs of NgR can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. PNAs of NgR can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S1 nucleases (Hyrup (1996), above); or as probes or primers for DNA sequence and hybridization (Hyrup et al., (1996), above; Perry-O'Keefe (1996), above). [0144]
  • In another embodiment, PNAs of NgR can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of NgR can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNase H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup (1996), above). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), above and Finn et al. (1996) [0145] Nucleic Acids Res. 24, 3357-3363. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl) amino-5′-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5′ end of DNA (Mag et al (1989) Nucleic Acids Res. 17, 973-988). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn et al (1996), above). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment. See, Petersen et al. (1975) Bioorg. Med. Chem. Lett. 5:1119-1124.
  • In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see Letsinger et al., (1989) [0146] Proc. Natl. Acad. Sci. USA 86, 6553-6556; Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84, 648-652; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization triggered cleavage agents (see, e.g., Krol et al., (1988) Biotechniques 6, 958-976) or intercalating agents (see, e.g., Zon (1988) Pharm. Res. 5, 539-549). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, etc.
  • Automated sequencing methods can be used to obtain or verify the nucleotide sequence of NgR. The NgR nucleotide sequences of the present invention are believed to be 100% accurate. However, as is known in the art, nucleotide sequence obtained by automated methods may contain some errors. Nucleotide sequences determined by automation are typically at least about 90%, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of a given nucleic acid molecule. The actual sequence may be more precisely determined using manual sequencing methods, which are well known in the art. An error in a sequence which results in an insertion or deletion of one or more nucleotides may result in a frame shift in translation such that the predicted amino acid sequence will differ from that which would be predicted from the actual nucleotide sequence of the nucleic acid molecule, starting at the point of the mutation. [0147]
  • Polypeptides [0148]
  • The invention also provides purified and isolated mammalian NgR polypeptides encoded by a polynucleotide of the invention. Presently preferred is a human NgR polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:14. Another preferred embodiment is a mouse NgR polypeptide comprising the amino acid sequence of NgR3, as set forth in SEQ ID NO:4. [0149]
  • One aspect of the invention pertains to isolated NgR proteins, and biologically active portions thereof, or derivatives, fragments, analogs or homologs thereof Also provided are polypeptide fragments suitable for use as immunogens to raise anti-NgR antibodies. Preferably, fragments of NgR proteins comprise at least one biological activity of NgR. In one embodiment, native NgR proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, NgR proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a NgR protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques. [0150]
  • The invention also embraces polypeptides that have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50% or at least 45% identity and/or homology to the preferred polypeptide of the invention. In addition, the invention embraces polypeptides having the consensus sequence shown in SEQ ID NO:6, shown in Table 5) excluding the previously characterized NgR (“NgR1”), and polypeptides comprising at least about 90% of the consensus sequence. [0151]
  • The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over that region of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I, in the case of nucleic acids) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the region of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The term “substantial identity” as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 80 percent sequence identity, preferably at least 85 percent identity and often 90 to 95 percent sequence identity, more usually at least 99 percent sequence identity as compared to a reference sequence over a comparison region. [0152]
  • In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment (Dayhoff, in ATLAS OF PROTEIN SEQUENCE AND STRUCTURE, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference). [0153]
  • A determination of homology or identity is typically made by a computer homology program known in the art. An exemplary program is the Gap program (Wisconsin Sequence Analysis Package, [0154] Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison, Wis.) using the default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489, which in incorporated herein by reference in its entirety). Employing the GAP software provided in the GCG program package, (see Needleman and Wunsch (1970) J. Mol. Biol. 48, 443-453) the following settings for nucleic acid sequence comparison may be used: GAP creation penalty of 5.0 and GAP extension penalty of 0.3, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, with the CDS (encoding) part of the DNA sequence shown in SEQ ID NOs:1, 3 or 13. BestFit was originally written for Version 1.0 by Paul Haeberli from a careful reading of the papers by Needleman and Wunsch (1970), above, and Smith and Waterman (1981), above. The following Bestfit settings for nucleic acid sequence comparison may be used: GAP creation penalty of 8.0 and GAP extension penalty of 2, the coding region of the analogous nucleic acid sequences referred to above exhibits a degree of identity preferably of at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%, with the CDS (encoding) part of the amino acid sequence shown in SEQ ID NOs:2, 4 or 14.
  • Alternatively, homology may be determined by hybridization analysis wherein a nucleic acid sequence is hybridized to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions. See e.g. Ausubel, et al., (Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1993, and below. [0155]
  • Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. [0156]
  • An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the NgR protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of NgR protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of NgR protein having less than about 30% (by dry weight) of non-NgR protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-NgR protein, still more preferably less than about 10% of non-NgR protein, and most preferably less than about 5% non-NgR protein. When the NgR protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. [0157]
  • The language “substantially free of chemical precursors or other chemicals” includes preparations of NgR protein in which the protein is separated from chemical precursors or other chemicals that are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of NgR protein having less than about 30% (by dry weight) of chemical precursors or non-NgR chemicals, more preferably less than about 20% chemical precursors or non-NgR chemicals, still more preferably less than about 10% chemical precursors or non-NgR chemicals, and most preferably less than about 5% chemical precursors or non-NgR chemicals. [0158]
  • Biologically active portions of a NgR protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the NgR protein, e.g., the amino acid sequence shown in SEQ ID NO:2, 4 or 14 that include fewer amino acids than the full length NgR proteins, and exhibit at least one activity of a NgR protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the NgR protein. A biologically active portion of a NgR protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. [0159]
  • A biologically active portion of a NgR protein of the present invention may contain at least one of the features that is conserved between the NgR proteins (e.g., a conserved cysteine as the N-terminus of the mature protein, four conserved cysteines in the N-terminus before a leucine-rich region, four conserved cysteines C-terminal with respect to a leucine repeat region, eight leucine-rich repeats, and a hydrophobic C-terminus). An alternative biologically active portion of a NgR protein may contain at least two of the above-identified domains. Another biologically active portion of a NgR protein may contain at least three of the above-identified domains. Yet another biologically active portion of a NgR protein of the present invention may contain at least four of the above-identified domains. [0160]
  • Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native NgR protein. [0161]
  • In an embodiment, the NgR protein has an amino acid sequence shown in SEQ ID NO:2, 4 or 14. In other embodiments, the NgR protein is substantially homologous to SEQ ID NO:2, 4 or 14 and retains the functional activity of the protein of SEQ ID NO:2, 4 or 14, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail below. [0162]
  • Accordingly, in another embodiment, the NgR protein is a protein that comprises an amino acid sequence at least about 45% homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:14 and retains the functional activity of the NgR proteins of SEQ ID NO:2, 4 or 14. [0163]
  • Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated forms of NgR polypeptides are embraced by the invention. [0164]
  • The invention also embraces variant (or analog) NgR polypeptides. In one example, insertion variants are provided wherein one or more amino acid residues supplement a NgR amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the NgR amino acid sequence. Insertional variants with additional residues at either or both termini can include, for example, fusion proteins and proteins including amino acid tags or labels. [0165]
  • Insertion variants include NgR polypeptides wherein one or more amino acid residues are added to a NgR acid sequence or to a biologically active fragment thereof. [0166]
  • Variant products of the invention also include mature NgR products, i.e., NgR products wherein leader or signal sequences are removed, with additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from specific proteins. NgR products with an additional methionine residue at position −1 (Met[0167] −2-NgR) are contemplated, as are variants with additional methionine and lysine residues at positions −2 and −1 (Met−2-Lys−1-NgR). Variants of NgR with additional Met, Met-Lys, Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.
  • Polypeptide Variants [0168]
  • The invention also embraces NgR variants having additional amino acid residues which result from use of specific expression systems. [0169]
  • As used herein, a NgR “chimeric protein” or “fusion protein” comprises a NgR polypeptide operatively linked to a non-NgR polypeptide. A “NgR polypeptide” refers to a polypeptide having an amino acid sequence corresponding to NgR, whereas a “non-NgR polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein that is not homologous to the NgR protein, e.g., a protein that is different from the NgR protein and that is derived from the same or a different organism. Within a NgR fusion protein the NgR polypeptide can correspond to all or a portion of a NgR protein. In one embodiment, a NgR fusion protein comprises at least one biologically active portion of a NgR protein. In another embodiment, a NgR fusion protein comprises at least two biologically active portions of a NgR protein. In yet another embodiment, a NgR fusion protein comprises at least three biologically active portions of a NgR protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the NgR polypeptide and the non-NgR polypeptide are fused in-frame to each other. The non-NgR polypeptide can be fused to the N-terminus or C-terminus of the NgR polypeptide. [0170]
  • For example, in one embodiment a NgR fusion protein comprises a NgR domain operably linked to the extracellular domain of a second protein. Such fusion proteins can be further utilized in screening assays for compounds which modulate NgR activity (such assays are described in detail below). [0171]
  • For example, use of commercially available vectors that express a desired polypeptide as part of a glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position −1 after cleavage of the GST component from the desired polypeptide. [0172]
  • In another embodiment, the fusion protein is a NgR protein containing a heterologous signal sequence at its N-terminus. For example, the native NgR signal sequence (i.e., amino acids 1-30 of SEQ ID NO:2 and amino acids 1-40 of SEQ ID NO:4) can be removed and replaced with a signal sequence from another protein. In certain host cells (e.g., mammalian host cells), expression and/or secretion NgR can be increased through use of a heterologous signal sequence. [0173]
  • In yet another embodiment, the fusion protein is a NgR-immunoglobulin fusion protein in which the NgR sequences comprising one or more domains are fused to sequences derived from a member of the immunoglobulin protein family. The NgR-immunoglobulin fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject to inhibit an interaction between NgR ligand and a NgR protein on the surface of a cell, to thereby suppress NgR-mediated signal transduction in vivo. NgR-immunoglobulin fusion proteins can be used to affect the bioavailability of a NgR cognate ligand. Inhibition of the NgR ligand/NgR interaction may be useful therapeutically for both the treatment of proliferative and differentiative disorders, as well as modulating (e.g., promoting or inhibiting) cell survival. Moreover, the NgR-immunoglobulin fusion proteins of the invention can be used as immunogens to produce anti-NgR antibodies in a subject, to purify NgR ligands, and in screening assays to identify molecules that inhibit the interaction of NgR with NgR ligand. [0174]
  • A NgR chimeric or fusion protein of the invention can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, e.g., by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Ausubel et al. (Eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A NgR-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the NgR protein. [0175]
  • Variants resulting from expression in other vector systems are also contemplated. [0176]
  • Insertional variants also include fusion proteins wherein the amino terminus and/or the carboxy terminus of NgR is/are fused to another polypeptide. [0177]
  • In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a NgR polypeptide are removed. Deletions can be effected at one or both termini of the NgR polypeptide, or with removal of one or more non-terminal amino acid residues of NgR. Deletion variants, therefore, include all fragments of a NgR polypeptide. [0178]
  • The invention also embraces polypeptide fragments of the sequence set forth in SEQ ID NO:2, 4 or 14 wherein the fragments maintain biological (e.g., ligand binding and/or intracellular signaling) immunological properties of a NgR polypeptide. Fragments comprising at least 4, 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of SEQ ID NO:2, 4 or 14 are contemplated by the invention. Preferred polypeptide fragments display antigenic properties unique to, or specific for, human NgR and its allelic and species homologs. Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art. [0179]
  • In still another aspect, the invention provides substitution variants of NgR polypeptides. Substitution variants include those polypeptides wherein one or more amino acid residues of a NgR polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables 2, 3, or 4 below. Table 1. [0180]
    TABLE 1
    Xaa#
    (based on a NTLRRCT Column I Column II
    domain) (R1, R2, R3) (R2 + R3 only)
    X1 G, R, M
    X2 A, D, C
    X3 V, T
    X4 N, P, S
    X5 E, A, S
    X6 nothing, K nothing
    X7 V, M, P
    X8 T, V V
    X9 Q, P Q
    X10 Q, A Q
    X11 Q, H, N
    X12 G, N N
    X13 L, F F
    X14 Q, A, S
    X15 A, S
    X16 V, I
    X17 V, T, E, L
    X18 S, G
    X19 L, I
    X20 A, E, V, P
    X21 A, S, D
    X22 S, T
    X23 Q, E
    X24 IVL
    X25 Q,H Q
    X26 N,G N
    X27 R,L
    X28 T,G,R,S
    X29 F,L,T,H
    X30 L,V L
    X31 Q,R,P
    X32 Q,P,A P
    X33 G,A G
    X34 H,T,S
    X35 S,G,R
    X36 P,S,A
    X37 C, nothing nothing
    X38 R, nothing nothing
    X39 A,N
    X40 M,L
    X41 V,L,T
    X42 T, I T
    X43 L,I
    X44 Y,F,H
    X45 N,V N
    X46 I,L
    X47 T,S,A
    X48 F,Y,T,R
    X49 A,H,Y,D
    X50 P,A P
    X51 N,S,G,A
    X52 T,A T
    X53 E,R,T
    X54 G,H
    X55 F,L
    X56 V,Q,H
    X57 H,A,L
    X58 E,Q E
    X59 G,S G
    X60 R,A R
    X61 Q,H Q
    X62 R, H H
    X63 R,S
    X64 L, V L
    X65 A,E,D
    X66 E,D,A
    X67 Q,H Q
    X68 V,E,G
    X69 K,R
    X70 H,Q
    X71 A,S,T
    X72 Y,H
    X73 Y,D Y
    X74 K,R
    X75 G,Q
    X76 S,Q S
    X77 A,S,E
    X78 P,G P
    X79 A,G,P
    X80 G,N
    X81 I,V,L
    X82 G,R
    X83 H,V,A
    X84 S,A S
    X85 D,E
    X86 H,S,A
    X87 I,L
    X88 E,L,Q
    X89 Y,H,A
    X90 Q,P Q
    X91 D, N
    X92 I, L, T
    X93 V,A,R
    X94 V,A,G
    X95 S,T S
    X96 K,R
    X97 L,I L
    X98 W,R,S
    X99 S,L
    X100 L,V L
    X101 G,T,P
    X102 Q,P,E
    X103 G,H,R
    X104 I,T,V,A
    X105 V,G,H
    X106 N,S
    X107 E,G,Q
    X108 Q,R
    X109 L,V
    X110 Q,A
    X111 W,G,H
    X112 H,R,P
    X113 K,A,H
    X114 H,R
    X115 D,G
    X116 H,R,S,G
    X117 T,M
    X118 T,I
    X119 F,Y
    X120 N,A
    X121 S,N
    X122 T,A,S
    X123 E,S,A
    X124 Q,P
    X125 G,T
    X126 D,E D
    X127 C,A
    X128 P,D
    X129 V,G,P,R
    X130 A,S
    X131 E,Q Q
    X132 F,Y F
    X133 G,A,D
    X134 A,P
    X135 D,A,V
    X136 G,D
    X137 A,E
    X138 S,P
    X139 E,A
    X140 L,F
    X141 R,Q
    X142 R,K R
    X143 R,K R
    X144 F,A
    X145 G,V
    X146 A,D,E
    X147 T,P
    X148 A,V,S
    X149 T,S,L
    X150 E,G,P,Q
    X151 L,E,R
    X152 R,L R
    X153 G,D
    X154 Q,H,A
    X155 Q,R
    X156 K,R
    X157 L,A,R
    X158 R,A R
    X159 V,A,E
    X160 E,A,N
    X161 F,L F
    X161 R,Q
    X163 N,A,G
  • Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table 2 (from WO 97/09433, page 10, published Mar, 13, 1997 (PCT/GB96/02197, filed 9/6/96), immediately below. [0181]
    TABLE 2
    Conservative Substitutions I
    SIDE CHAIN
    CHARACTERISTIC AMINO ACID
    Aliphatic GAP
    Non-polar ILV
    Polar-uncharged CSTM
    NQ
    Polar-charged DE
    KR
    Aromatic HFWY
    Other NQDE
  • Alternatively, conservative amino acids can be grouped as described in Lehninger, [BIOCHEMISTRY, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77] as set out in Table 3, immediately below. [0182]
    TABLE 3
    Conservative Substitutions II
    SIDE CHAIN
    CHARACTERISTIC AMINO ACID
    Non-polar (hydrophobic)
    A. Aliphatic: ALIVP
    B. Aromatic: FW
    C. Sulfur-containing: M
    D. Boderline: G
    Uncharged-polar
    A. Hydroxyl: STY
    B. Amides: NQ
    C. Sylihydryl: C
    D. Boderline: G
    Positively Charged (Basic): KRH
    Negatively Charged (Acidic): DE
  • As still another alternative, exemplary conservative substitutions are set out in Table 4, below. [0183]
    TABLE 4
    Conservative Substitutions III
    Original
    Residue Exemplary Substitution
    Ala (A) Val, Leu, Ile
    Arg (R) Lys, Gln, Asn
    Asn (N) Gln, His, Lys, Arg
    Asp (D) Glu
    Cys (C) Ser
    Gln (Q) Asn
    Glu (B) Asp
    His (H) Asn, Gln, Lys, Arg
    Ile (I) Leu, Val, Met, Ala, Phe,
    Leu (L) Ile, Val, Met, Ala, Phe
    Lys (K) Mg, Gln, Asn
    Met (M) Leu, Phe, Ile
    Phe (F) Leu, Val, Ile, Ala
    Pro (P) Gly
    Ser (S) Thr
    Thr (T) Ser
    Trp (W) Tyr
    Tyr (Y) Trp, Phe, Thr, Ser
    Val (V) Ile, Leu, Met, Phe, Ala
  • In addition, amino acid residues that are conserved among family members of the NgR proteins of the present invention, as indicated by the alignment presented herein, are also predicted to be particularly unamenable to alteration. For example, NgR proteins of the present invention can contain at least one domain that is a typically conserved region in NgRs. Examples of these conserved domains include, e.g., leucine-rich repeat domain. Amino acid residues that are not conserved or are only semi-conserved among members of the NgR proteins may be readily amenable to alteration. [0184]
  • Full-length NgRs have an LRR region characterized by the amino acid consensus sequence shown in SEQ ID NO: 19. At least some full-length NgRs also include a CT signaling (CTS) domain and a GPI domain. [0185]
  • The NgR domain designations used herein are defined as follows: [0186]
    hNgR1 mNgR1 hNgR2 hNgR3
    SEQ ID: SEQ ID SEQ ID: SEQ ID: mNgR3
    Domain
    5 NO: 17 2 14 SEQ ID:4
    Signal  1-26  1-26  1-30 —  1-40
    Seq.
    LRRNT 27-56 27-56 31-59 — 41-69
    LRR1 57-81 57-81 60-82  5-27 70-92
    LRR2  82-105  82-105  83-106 28-51  93-106
    LRR3 106-130 106-130 107-131 52-76 106-141
    LRR4 131-154 131-154 132-155 77-100 142-165
    LRR5 155-178 155-178 156-179 101-124 166-189
    LRR6 179-202 179-202 180-203 125-148 190-213
    LRR7 203-226 203-226 204-227 149-172 214-237
    LRR8 227-250 227-250 228-251 173-196 238-261
    LRRCT 260-309 260-309 261-310 206-255 271-320
    CTS 310-445 310-445 311-395 256-396 321-438
    (CT
    Signal-
    ing)
    GPI 446-473 456-473 396-420 370-392 439-462
  • In some embodiments of the invention, the above domains are modified. Modification can be in a manner that preserves domain functionality. Modification can include addition, deletion or substitution of certain amino acids. Exemplary modifications include conservative amino acid substitutions. Preferably such substitutions number 20 or fewer per 100 residues. More preferably, such substitutions number 10 or fewer per 100 residues. Further exemplary modifications include addition of flanking sequences of up to five amino acids at the N terminus and/or C terminus of one or more of the domains. [0187]
  • In some embodiments, the isolated nucleic acid molecule encodes a polypeptide at least about 70%, 80%, 90%, 95%, 98%, and most preferably at least about 99% homologous to SEQ ID NO:2, 4 or 14. [0188]
  • Mutations can be introduced into SEQ ID NOS:1, 3 or 13 by standard techniques, e.g., site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions can be made at one or more amino acid residues predicted to be non-essential. Alternatively, mutations can be introduced randomly along a NgR coding sequence. This can be accomplished, e.g., by saturation mutagenesis. The resulting mutants can be screened for NgR biological activity. Biological activities of NgR may include but are not limited to: (1) protein:protein interactions, e.g., with other NgRs or other cell-surface proteins involved in Nogo-related signaling; (2) complex formation with a NgR ligand; (3) binding to an anti-NgR antibody. [0189]
  • It should be understood that the definition of polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs. Similarly, the invention further embraces NgR polypeptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol. Variants that display ligand binding properties of native NgR and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant NgR activity. [0190]
  • Chemically modified NgR polypeptide compositions in which the NgR polypeptide is linked to a polymer are included within the scope of the present invention. The polymer may be water soluble to prevent precipitation of the protein in an aqueous environment, such as a physiological environment. Suitable water-soluble polymers may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, a polypropylene oxide/ethylene oxide copolymer polyoxyethylated polyols (e.g. glycerol) and polyvinyl alcohol. The selected polymer is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled. Polymers may be of any molecular weight, and may be branched or unbranched, and mixtures of such polymers may also be used. When the chemically modified NgR polymer is destined for therapeutic use, pharmaceutically acceptable polymers will be selected for use. [0191]
  • When the polymer is to be modified by an acylation reaction, the polymer should have a single reactive ester group. Alternatively, if the polymer is to be modified by reductive alkylation, the polymer should have a single reactive aldehyde group. A preferred reactive aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono Cl-ClO alkoxy or aryloxy derivatives thereof (see U.S. Pat. No. 5,252,714, incorporated by reference herein in its entirety). [0192]
  • Pegylation of NgR polypeptides may be carried out by any of the pegylation reactions known in the art, as described, for example, in the following references: [0193] Focus on Growth Factors 3, 4-10 (1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated by reference herein in its entirety). Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). A preferred water-soluble polymer for pegylation of polypeptides such as NgR is polyethylene glycol (PEG). As used herein, “polyethylene glycol” is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl-ClO) alkoxy- or aryloxy-polyethylene glycol.
  • Chemical derivatization of NgR polypeptides may be performed under any suitable conditions used to react a biologically active substance with an activated polymer molecule. Methods for preparing pegylated NgR polypeptides will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby NgR polypeptide becomes attached to one or more PEG groups, and (b) obtaining the reaction products. It will be apparent to one of ordinary skill in the art to select the optimal reaction conditions or the acylation reactions based on known parameters and the desired result. [0194]
  • Pegylated and other polymer:NgR polypeptides may generally be used to treat conditions that may be alleviated or modulated by administration of the NgR polypeptides described herein. However, the chemically-derivatized polymer:NgR polypeptide molecules disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to the nonderivatized molecules. The NgR polypeptides, fragments thereof, variants and derivatives, may be employed alone, together, or in combination with other pharmaceutical compositions. The cytokines, growth factors, antibiotics, antiinflammatories and/or chemotherapeutic agents as is appropriate for the indication being treated. [0195]
  • The present invention provides compositions comprising purified polypeptides of the invention. Preferred compositions comprise, in addition to the polypeptide of the invention, a pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient or medium. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil and cocoa butter. [0196]
  • Variants that display ligand binding properties of native NgR and are expressed at higher levels, as well as variants that provide for constitutively active receptors, are particularly useful in assays of the invention; the variants are also useful in assays of the invention and in providing cellular, tissue and animal models of diseases/conditions characterized by aberrant NgR activity. [0197]
  • With the knowledge of the nucleotide sequence information disclosed in the present invention, one skilled in the art can identify and obtain nucleotide sequences which encode NgR from different sources (i.e., different tissues or different organisms) through a variety of means well known to the skilled artisan and as disclosed by, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference in its entirety. [0198]
  • For example, DNA that encodes NgR may be obtained by screening of mRNA, cDNA, or genomic DNA with oligonucleotide probes generated from the NgR gene sequence information provided herein. Probes may be labeled with a detectable group, such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with procedures known to the skilled artisan and used in conventional hybridization assays, as described by, for example, Sambrook et al. (1989) above. [0199]
  • A nucleic acid molecule comprising any of the NgR nucleotide sequences described above can alternatively be synthesized by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the nucleotide sequences provided herein. See U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis. The PCR reaction provides a method for selectively increasing the concentration of a particular nucleic acid sequence even when that sequence has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single- or double-stranded DNA. The essence of the method involves the use of two oligonucleotide probes to serve as primers for the template-dependent, polymerase-mediated replication of a desired nucleic acid molecule. [0200]
  • A wide variety of alternative cloning and in vitro amplification methodologies are well known to those skilled in the art. Examples of these techniques are found in, for example, Berger et al., Guide to Molecular Cloning Techniques, METHODS IN ENZYMOLOGY 152 Academic Press, San Diego, Calif., which is incorporated herein by reference in its entirety. [0201]
  • The nucleic acid molecules of the present invention, and fragments derived therefrom, are useful for screening for restriction fragment length polymorphism (RFLP) associated with certain disorders, as well as for genetic mapping. [0202]
  • Antibodies [0203]
  • Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and complementary determining region (CDR)-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) specific for NgR or fragments thereof Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fab′, F(ab′)[0204] 2, and Fv, are also provided by the invention. The term “specific for,” when used to describe antibodies of the invention, indicates that the variable regions of the antibodies of the invention recognize and bind NgR polypeptides exclusively (i.e., are able to distinguish NgR polypeptides from other known NgR polypeptides by virtue of measurable differences in binding affinity, despite the possible existence of localized sequence identity, homology, or similarity between NgR and such polypeptides).
  • The antigenic peptide of NgR comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2, 4 or 14 and encompasses an epitope of NgR such that an antibody raised against the peptide forms a specific immune complex with NgR. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. Preferred epitopes encompassed by the antigenic peptide are regions of NgR that are located on the surface of the protein, e.g., hydrophilic regions. [0205]
  • It will be understood that specific antibodies may also interact with other proteins (for example, [0206] S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and, in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. in ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the NgR polypeptides of the invention are also contemplated, provided that the antibodies are specific for NgR polypeptides. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
  • For the production of polyclonal antibodies, various suitable host animals (e.g., rabbit, goat, mouse or other mammal) may be immunized by injection with the native protein, or a synthetic variant thereof, or a derivative of the foregoing. An appropriate immunogenic preparation can contain, for example, recombinantly expressed NgR protein or a chemically synthesized NgR polypeptide. The preparation can further include an adjuvant. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and [0207] Corynebacterium parvum or similar immunostimulatory agents. If desired, the antibody molecules directed against NgR can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • The term “monoclonal antibody” or “monoclonal antibody composition,” as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of NgR. A monoclonal antibody composition thus typically displays a single binding affinity for a particular NgR protein with which it immunoreacts. For preparation of monoclonal antibodies directed towards a particular NgR protein, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture may be utilized. Such techniques include, but are not limited to, the hybridoma technique (see Kohler and Milstein (1975) [0208] Nature 256, 495-497); the trioma technique; the human B-cell hybridoma technique (see Kozbor et al., (1983) Immunol. Today 4, 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole et al., (1985) in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote et al., (1983) Proc. Natl. Acad. Sci. USA 80, 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole et al., (1985), above).
  • According to the invention, techniques can be adapted for the production of single-chain antibodies specific to a NgR protein (see e.g., U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (see e.g., Huse et al., (1989) [0209] Science 246, 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a NgR protein or derivatives, fragments, analogs or homologs thereof Non-human antibodies can be “humanized” by techniques well known in the art. See e.g., U.S. Pat. No. 5,225,539. In one method, the non-human CDRs are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity. Antibody fragments that contain the idiotypes to a NgR protein may be produced by techniques known in the art including, but not limited to: (i) an F(ab′)2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) F fragments.
  • Additionally, recombinant anti-NgR antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in PCT International Application No. PCT/US86/02269; European Patent Application No. 184,187; European Patent Application No. 171,496; European Patent Application No. 173,494; PCT International Publication No. WO 86/01533; U.S. Pat. No. 4,816,567; European Patent Application No. 125,023; Better et al., (1988) [0210] Science 240, 1041-1043; Liu et al., (1987) Proc. Natl. Acad. Sci. USA 84, 3439-3443; Liu et al., (1987) J. Immunol. 139, 3521-3526; Sun et al., (1987) Proc. Natl. Acad. Sci. USA 84, 214-218; Nishimura et al., (1987) Cancer Res. 47, 999-1005; Wood et al., (1985) Nature 314, 446-449; Shaw et al,. (1988) J. Natl. Cancer Inst. 80, 1553-1559); Morrison (1985) Science 229, 1202-1207; Oi et al., (1986) BioTechniques 4, 214; U.S. Pat. No. 5,225,539; Jones et al., (1986) Nature 321, 552-525; Verhoeyan et al., (1988) Science 239, 1534; and Beidler et al., (1988) J. Immunol. 141, 4053-4060.
  • In a preferred embodiment of the invention a portion of a NgR is joined to an Fc portion of an antibody to form a NgR/Fc fusion protein. Preferably, the Ig fusion protein is soluble. The NgR/Fc fusion protein may be formed by recombinant techniques as described above. In one embodiment, a portion of a NgR including the entire amino acid sequence of NgR except the C-terminal hydrophobic region is fused to an Fc portion of an antibody. In preferred embodiments, the NgR is a human NgR and the Fc is also human. More preferably, the human Fc portion is derived from an IgG antibody. In other embodiments, the N-terminal signal sequence is omitted. Such antibodies are useful in binding Nogo to prevent Nogo signaling through the NgR. [0211]
  • In one embodiment, methods for the screening of antibodies that possess the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically-mediated techniques known within the art. In a specific embodiment, selection of antibodies that are specific to a particular domain of a NgR protein is facilitated by generation of hybridomas that bind to the fragment of a NgR protein possessing such a domain. Antibodies that are specific for one or more domains within a NgR protein, e.g., domains spanning the above-identified conserved regions of NgRs, or derivatives, fragments analogs or homologs thereof, are also provided herein. [0212]
  • Anti-NgR antibodies may be used in methods known within the art relating to the localization and/or quantitation of a NgR protein (e.g., for use in measuring levels of the NgR protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies for NgR proteins, or derivatives, fragments analogs or homologs thereof, that contain the antibody derived binding domain, are utilized as pharmacologically-active compounds [hereinafter “Therapeutics”]. [0213]
  • An anti-NgR antibody (e.g., monoclonal antibody) can be used to isolate NgR by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-NgR antibody can facilitate the purification of natural NgR from cells and of recombinantly produced NgR expressed in host cells. Moreover, an anti-NgR antibody can be used to detect NgR protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the NgR protein. Anti-NgR antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin, and examples of suitable radioactive material include [0214] 125I, 131I, 35S or 3H.
  • Another aspect of the present invention is directed to methods of inducing an immune response in a mammal against a polypeptide of the invention by administering to the mammal an amount of the polypeptide sufficient to induce an immune response. The amount will be dependent on the animal species, size of the animal, and the like but can be determined by those skilled in the art. [0215]
  • Another aspect of the invention is directed to anti-idiotypic antibodies and anti-anti-idiotypic antibodies. An anti-idiotypic antibody is an antibody that recognizes determinants of another antibody (a target antibody). Generally, the anti-idiotypic antibody recognizes determinants of the antigen-binding site of the target antibody. Typically, the target antibody is a monoclonal antibody. An anti-idiotypic antibody is generally prepared by immunizing an animal (particularly, mice) of the same species and genetic type as the source of the target monoclonal antibody, with the target monoclonal antibody. The immunized animal mounts an immune response to the idiotypic determinants of the target monoclonal antibody and produces antibodies against the idiotypic determinants of the target monoclonal antibody. Antibody-producing cells, such as splenic cells, of the immunized animal may be used to generate anti-idiotypic monoclonal antibodies. Furthermore, an anti-idiotypic antibody may also be used to immunize animals to produce anti-anti-idiotypic antibodies. These immunized animals may be used to generate anti-anti-idiotypic monoclonal antibodies using standard techniques. The anti-anti-idiotypic antibodies may bind to the same epitope as the original, target monoclonal antibody used to prepare the anti-idiotypic antibody. The anti-anti-idiotypic antibodies represent other monoclonal antibodies with the same antigen specificity as the original target monoclonal antibody. [0216]
  • If the binding of the anti-idiotypic antibody with the target antibody is inhibited by the relevant antigen of the target antibody, and if the anti-idiotypic antibody induces an antibody response with the same specificity as the target antibody, it mimics the antigen of the target antibody. Such an anti-idiotypic antibody is an “internal image anti-idiotype” and is capable of inducing an antibody response as if it were the original antigen. (Bona and Kohler (1984) ANTI-IDIOTYPIC ANTIBODIES AND INTERNAL IMAGE, IN MONOCLONAL AND ANTI-IDIOTYPIC ANTIBODIES: PROBES FOR RECEPTOR STRUCTURE AND FUNCTION, Venter J. C. et al. (Eds), Alan R. Liss, New York, N.Y., pp 141-149, 1984). Vaccines incorporating internal image anti-idiotype antibodies have been shown to induce protective responses against viruses, bacteria, and parasites (Kennedy et al., (1986) 232, 220-223; 1047; McNamara et al., (1985) [0217] Science 226, 1325-1326). Internal image anti-idiotypic antibodies have also been shown to induce immunity to tumor related antigens (Raychauhuri et al., (1986) J. Immunol. 137, 1743-1749; Raychauhuri et al., (1987) J. Immunol. 139, 3902-3910; Bhattacharya-Chatterjee et al., (1987) J. Immunol. 139, 1354-1360; Bhattacharya-Chatterjee et al., (1988) J. Immunol. 141, 1398-1403; Herlyn. et al. (1989) Intern. Rev. Immunol. 4, 347-357; Chen et al. (1990) Cell Imm. Immunother. Cancer 351-359; Herlyn et al., (1991) in vivo 5, 615-624; Furuya et al. (1992) AntiCancer Res. 12, 27-32; Mittelman, A. et al. (1992) Proc. Natl. Acad. Sci., USA 89, 466-470; Durrant. et al., (1994) Cancer Res. 54, 4837-4840; Mittelman. et al. (1994) Cancer Res. 54, 415-421; Schmitt. et al. (1994) Hybridoma 13, 389-396; Chakrobarty. et al. (1995) J. Immunother. 18, 95-103; Chakrobarty. et al. (1995) Cancer Res. 55, 1525-1530; Foon, K. A. et al. (1995) Clin. Cancer Res. 1, 1205-1294; Herlyn et al. (1995) Hybridoma 14, 159-166; Sclebusch et al. (1995) Hybridoma 14, 167-174; Herlyn. et al. (1996) Cancer Immunol Immunother. 43, 65-76).
  • Anti-idiotypic antibodies for NgR may be prepared, for example, by immunizing an animal, such as a mouse, with a immunogenic amount of a composition comprising NgR2 (SEQ ID NO:2), NgR3 (SEQ ID NOs:4 or 14), or immunogenic portion thereof, containing at least one antigenic epitope of NgR. The composition may also contain a suitable adjuvant, and any carrier necessary to provide immunogenicity. Monoclonal antibodies recognizing NgR may be prepared from the cells of the immunized animal as described above. A monoclonal antibody recognizing an epitope of NgR is then selected and used to prepare a composition comprising an immunogenic amount of the anti-NgR monoclonal antibody. Typically, a 25 to 200 μg dose of purified anti-NgR monoclonal would be sufficient in a suitable adjuvant. [0218]
  • Animals may be immunized 2-6 times at 14 to 30 day intervals between doses. Typically, animals are immunized by any suitable route of administration, such as intraperitoneal, subcutaneous, intravenous or a combination of these. Anti-idiotypic antibody production may be monitored during the immunization period using standard immunoassay methods. Animals with suitable titers of antibodies reactive with the target monoclonal antibodies may be reimmunized with the monoclonal antibody used as the immunogen three days before harvesting the antibody producing cells. Preferably, spleen cells are used, although other antibody producing cells may be selected. Antibody-producing cells are harvested and fused with myeloma cells to produce Hybridomas, as described above, and suitable anti-idiotypic antibody-producing cells are selected. [0219]
  • Anti-anti-idiotypic antibodies are produced by another round of immunization and Hybridoma production by using the anti-idiotypic monoclonal antibody as the immunogen. [0220]
  • Antibodies of the invention are useful for, e.g., therapeutic purposes (by modulating activity of NgR), diagnostic purposes to detect or quantitate NgR, and purification of NgR. Therefore, kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. [0221]
  • Kits [0222]
  • The present invention is also directed to kits, including pharmaceutical kits. The kits can comprise any of the nucleic acid molecules described above, any of the polypeptides described above, or any antibody which binds to a polypeptide of the invention as described above, as well appropriate controls, such as positive and/or negative controls. The kit preferably comprises additional components, such as, for example, instructions, solid support, reagents helpful for quantification, and the like. For example, the kit can comprise: a labeled compound or agent capable of detecting NgR protein or mRNA in a biological sample; means for determining the amount of NgR in the sample; and means for comparing the amount of NgR in the sample with a standard. The compound or agent can be packaged in a suitable container. [0223]
  • Screening Assays [0224]
  • The DNA and amino acid sequence information provided by the present invention also makes possible identification of binding partner compounds with which a NgR polypeptide or polynucleotide will interact. Methods to identify binding partner compounds include solution assays, in vitro assays wherein NgR polypeptides are immobilized and cell-based assays. Identification of binding partner compounds of NgR polypeptides provides candidates for therapeutic or prophylactic intervention in pathologies associated with NgR normal and aberrant biological activity. [0225]
  • The invention also provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules (e.g., molecules of less than 1,000 Daltons) or other drugs) that bind to NgR proteins or have a stimulatory or inhibitory effect on, for example, NgR expression or NgR activity. [0226]
  • In one embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a NgR protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the “one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) [0227] Anticancer Drug Des. 12, 145).
  • Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., (1993) [0228] Proc. Natl. Acad. Sci. USA 90, 6909; Erb et al., (1994) Proc. Natl. Acad. Sci. USA 91,11422; Zuckermann et al. (1994) J. Med. Chem 37, 2678; Cho et al., (1993) Science 261, 1303; Carrell et al., (1994) Angew Chem. Int. Ed. Engl. 33, 2059; Carell et al., (1994) Angew Chem. Int. Ed. Engl. 33, 2061; and Gallop et al., (1994) J. Med. Chem 37, 1233.
  • Libraries of compounds may be presented in solution (e.g., Houghten (1992) [0229] BioTechniques 13, 412-421), or on beads (Lam (1991) Nature 354, 82-84), on chips (Fodor (1993) Nature 364, 555-556), bacteria (Ladner, U.S. Pat. No. 5,223,409), spores (Ladner, above), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1865-1869) or on phage (Scott and Smith (1990) Science 249, 386-390; Devlin (1990) Science 249, 404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87, 6378-6382; Felici (1991) J. Mol. Biol. 222, 301-310; Ladner, above).
  • 1. Cell-Based Assays [0230]
  • The invention also provides cell-based assays to identify binding partner compounds of a NgR polypeptide. In one embodiment, the invention provides a method comprising the steps of contacting a NgR polypeptide expressed on the surface of a cell with a candidate binding partner compound and detecting binding of the candidate binding partner compound to the NgR polypeptide. In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a membrane-bound form of NgR protein, or a biologically active portion thereof, on the cell surface with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the NgR protein or biologically active portion thereof. [0231]
  • In one embodiment, an assay is a cell-based assay in which a cell which expresses a membrane-bound form of NgR protein, or a biologically active portion thereof, on the cell surface is contacted with a test compound and the ability of the test compound to bind to a NgR protein determined. The cell, for example, can be of mammalian origin or a yeast cell. Determining the ability of the test compound to bind to the NgR protein can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the NgR protein or biologically active portion thereof can be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with [0232] 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product. In one embodiment, the assay comprises contacting a cell which expresses a membrane-bound form of NgR protein or a biologically active portion thereof, on the cell surface with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the test compound to preferentially bind to NgR or a biologically active portion thereof as compared to the known compound.
  • Determining the ability of the test compound to modulate the activity of NgR or a biologically active portion thereof can be accomplished, for example, by determining the ability of the NgR protein to bind to or interact with a NgR target molecule. As used herein, a “target molecule” is a molecule with which a NgR protein binds or interacts in nature, for example, a molecule on the surface of a cell which expresses a NgR protein, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule. A NgR target molecule can be a non-NgR molecule or a NgR protein or polypeptide of the present invention. In one embodiment, a NgR target molecule is a component of a signal transduction pathway that facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a compound to a membrane-bound NgR molecule) through the cell membrane and into the cell. The target, for example, can be a second intercellular protein that has catalytic activity or a protein that facilitates the association of downstream signaling molecules with NgR. In a preferred embodiment, the detection comprises detecting a calcium flux or other physiological event in the cell caused by the binding of the molecule. [0233]
  • Specific binding molecules, including natural ligands and synthetic compounds, can be identified or developed using isolated or recombinant NgR products, NgR variants, or preferably, cells expressing such products. Binding partners are useful for purifying NgR products and detection or quantification of NgR products in fluid and tissue samples using known immunological procedures. Binding molecules are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of NgR, especially those activities involved in signal transduction. [0234]
  • 2. Cell-Free Assays [0235]
  • (a) Direct Binding: [0236]
  • The invention includes several assay systems for identifying NgR binding partners. In solution assays, methods of the invention comprise the steps of (a) contacting a NgR polypeptide with one or more candidate binding partner compounds and (b) identifying the compounds that bind to the NgR polypeptide. Identification of the compounds that bind the NgR polypeptide can be achieved by isolating the NgR polypeptide/binding partner complex and separating the binding partner compound from the NgR polypeptide. An additional step of characterizing the physical, biological and/or biochemical properties of the binding partner compound is also comprehended in another embodiment of the invention. In one aspect, the NgR polypeptide/binding partner complex is isolated using an antibody immunospecific for either the NgR polypeptide or the candidate binding partner compound. [0237]
  • In still other embodiments, either the NgR polypeptide or the candidate binding partner compound comprises a label or tag that facilitates its isolation, and methods of the invention to identify binding partner compounds include a step of isolating the NgR polypeptide/binding partner complex through interaction with the label or tag. An exemplary tag of this type is a poly-histidine sequence, generally around six histidine residues, that permits isolation of a compound so labeled using nickel chelation. Other labels and tags, such as the FLAG® tag (Eastman Kodak, Rochester, N.Y.), well known and routinely used in the art, are embraced by the invention. [0238]
  • (b) Immobilized NgR [0239]
  • In one variation of an in vitro assay, the invention provides a method comprising the steps of (a) contacting an immobilized NgR polypeptide, or a biologically active fragment thereof with a candidate binding partner compound and (b) detecting binding of the candidate compound to the NgR polypeptide. In an alternative embodiment, the candidate binding partner compound is immobilized and binding of NgR is detected. Immobilization is accomplished using any of the methods well known in the art, including covalent bonding to a support, a bead or a chromatographic resin, as well as non-covalent, high affinity interactions such as antibody binding, or use of streptavidin/biotin binding wherein the immobilized compound includes a biotin moiety. Binding of a test compound to NgR, or interaction of NgR with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, and not by way of limitation, GST-NgR fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, that are then combined with the test compound or the test compound and either the non-adsorbed target protein or NgR protein, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, and the complexes determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of NgR binding or activity determined using standard techniques. [0240]
  • Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either NgR or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated NgR or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with NgR or target molecules, but which do not interfere with binding of the NgR protein to its target molecule, can be derivatized to the wells of the plate, and unbound target or NgR trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the NgR or target molecule, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the NgR or target molecule. [0241]
  • Detection of binding can be accomplished (i) using a radioactive label on the compound that is not immobilized, (ii) using of a fluorescent label on the non-immobilized compound, (iii) using an antibody immunospecific for the non-immobilized compound, (iv) using a label on the non-immobilized compound that excites a fluorescent support to which the immobilized compound is attached, (v) determining the activity of the NgR, as well as other techniques well known and routinely practiced in the art. [0242]
  • Determining the activity of the target molecule, for example, may be accomplished by detecting induction of a cellular second messenger of the target (i.e. intracellular Ca[0243] 2+, diacylglycerol, IP3, etc.), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a NgR-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, cell survival, cellular differentiation, or cell proliferation.
  • (c) Competition Experiments [0244]
  • In yet another embodiment, the assay comprises contacting the NgR protein or biologically active portion thereof with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the test compound to preferentially bind to NgR or biologically active portion thereof as compared to the known compound. [0245]
  • In yet another embodiment, the cell-free assay comprises contacting the NgR protein or biologically active portion thereof with a known compound which binds NgR to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a NgR protein, wherein determining the ability of the test compound to interact with a NgR protein comprises determining the ability of the NgR protein to modulate the activity of a NgR target molecule. [0246]
  • The cell-free assays of the present invention are amenable to use of both the soluble form or the membrane-bound form of NgR. In the case of cell-free assays comprising the membrane-bound form of NgR, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of NgR is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton™ X-114, Thesit®, Isotridecypoly(ethylene glycol ether)[0247] n, 3-(3-cholamidopropyl)dimethylamminiol-1-propane sulfonate (CHAPS), 3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.
  • Modulators [0248]
  • Agents that modulate (i.e., increase, decrease, or block) NgR activity or expression may be identified by incubating a putative modulator with a cell containing a NgR polypeptide or polynucleotide and determining the effect of the putative modulator on NgR activity or expression. The selectivity of a compound that modulates the activity of NgR can be evaluated by comparing its effects on NgR to its effect on other NgR compounds. Selective modulators may include, for example, antibodies and other proteins, peptides or organic molecules which specifically bind to a NgR polypeptide or a NgR-encoding nucleic acid. Modulators of NgR activity will be therapeutically useful in treatment of diseases and physiological conditions in which normal or aberrant NgR activity is involved. NgR polynucleotides, polypeptides and modulators may be used in the treatment of such diseases and conditions associated with demyelination. NgR polynucleotides and polypeptides, as well as NgR modulators, may also be used in diagnostic assays for such diseases or conditions. [0249]
  • Methods of the invention to identify modulators include variations on any of the methods described above to identify binding partner compounds, the variations including techniques wherein a binding partner compound has been identified and the binding assay is carried out in the presence and absence of a candidate modulator. A modulator is identified in those instances where binding between the NgR polypeptide and the binding partner compound changes in the presence of the candidate modulator compared to binding in the absence of the candidate modulator compound. A modulator that increases binding between the NgR polypeptide and the binding partner compound is described as an enhancer or activator, and a modulator that decreases binding between the NgR polypeptide and the binding partner compound is described as an inhibitor. [0250]
  • In another embodiment, modulators of NgR expression may be identified in a method wherein a cell is contacted with a candidate compound and the expression of NgR mRNA or protein in the cell is determined. The level of expression of NgR mRNA or protein in the presence of the candidate compound is compared to the level of expression of NgR mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of NgR expression based on this comparison. For example, when expression of NgR mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of NgR mRNA or protein expression. Alternatively, when expression of NgR mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of NgR mRNA or protein expression. The level of NgR mRNA or protein expression in the cells can be determined by methods described herein for detecting NgR mRNA or protein. [0251]
  • High Throughput Screening [0252]
  • The invention also comprehends high-throughput screening (HTS) assays to identify compounds that interact with or inhibit biological activity (i.e., affect enzymatic activity, binding activity, etc.) of a NgR polypeptide. HTS assays permit screening of large numbers of compounds in an efficient manner. Cell-based HTS systems are contemplated to investigate NgR receptor-ligand interaction. HTS assays are designed to identify “hits” or “lead compounds” having the desired property, from which modifications can be designed to improve the desired property. Chemical modification of the “hit” or “lead compound” is often based on an identifiable structure/activity relationship between the “hit” and the NgR polypeptide. [0253]
  • Another aspect of the present invention is directed to methods of identifying compounds that bind to either NgR or nucleic acid molecules encoding NgR, comprising contacting NgR, or a nucleic acid molecule encoding the same, with a compound, and determining whether the compound binds NgR or a nucleic acid molecule encoding the same. Binding can be determined by binding assays which are well known to the skilled artisan, including, but not limited to, gel-shift assays, Western blots, radiolabeled competition assay, phage-based expression cloning, co-fractionation by chromatography, co-precipitation, cross linking, interaction trap/two-hybrid analysis, southwestern analysis, ELISA, and the like, which are described in, for example, Ausubel et al. (Eds.),CURRENT PROTOCOLS TN MOLECULAR BIOLOGY, 1999, John Wiley & Sons, NY, which is incorporated herein by reference in its entirety. The NgR proteins, for example, can be used as “bait proteins” in a two-hybrid assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al., (1993) [0254] Cell 72, 223-232; Madura et al., (1993) J. Biol. Chem. 268, 12046-12054; Bartel et al., (1993) BioTechniques 14, 920-924; Iwabuchi et al., (1993) Oncogene 8, 1693-1696; and Brent WO 94/10300), to identify other proteins that bind to or interact with NgR (“NgR-binding proteins” or “NgR-bp”) and modulate NgR activity. Such NgR-binding proteins are also likely to be involved in the propagation of signals by the NgR proteins as, for example, upstream or downstream elements of the NgR pathway.
  • Other assays may be used to identify specific ligands of a NgR receptor, including assays that identify ligands of the target protein through measuring direct binding of test ligands to the target protein, as well as assays that identify ligands of target proteins through affinity ultrafiltration with ion spray mass spectroscopy/HPLC methods or other physical and analytical methods. Alternatively, such binding interactions are evaluated indirectly using the yeast two-hybrid system described in Fields et al., (1989) [0255] Nature 340, 245-246, and Fields et al., (1994) Trends Genet. 10, 286-292, both of which are incorporated herein by reference. The two-hybrid system is a genetic assay based on the modular nature of most transcription factors used for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs. The two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast. The assay requires the construction of two hybrid genes encoding (1) a DNA-binding domain that is fused to a first protein and (2) an activation domain fused to a second protein. The DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene. The second hybrid protein, which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the noncovalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene. For example, when the first protein is a NgR gene product, or fragment thereof, that is known to interact with another protein or nucleic acid, this assay can be used to detect agents that interfere with the binding interaction. Expression of the reporter gene is monitored as different test agents are added to the system. The presence of an inhibitory agent results in lack of a reporter signal. The compounds to be screened include (which may include compounds that are suspected to bind NgR, or a nucleic acid molecule encoding the same), but are not limited to, extracellular, intracellular, biological or chemical origin.
  • The function of the NgR gene product is unclear and no ligands have yet been found which bind the gene product. The yeast two-hybrid assay is useful to identify proteins that bind to the gene product. In an assay to identify proteins that bind to a NgR receptor, or fragment thereof, a fusion polynucleotide encoding both a NgR receptor (or fragment) and a UAS binding domain (i.e., a first protein) may be used. In addition, a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay. Typically, the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain. This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein. The system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mRNA that can be repeatedly translated to yield the reporter protein. [0256]
  • Other assays may be used to search for agents that bind to the target protein. One such screening method to identify direct binding of test ligands to a target protein is described in U.S. Pat. No. 5,585,277, incorporated herein by reference. This method relies on the principle that proteins generally exist as a mixture of folded and unfolded states, and continually alternate between the two states. When a test ligand binds to the folded form of a target protein (i.e., when the test ligand is a ligand of the target protein), the target protein molecule bound by the ligand remains in its folded state. Thus, the folded target protein is present to a greater extent in the presence of a test ligand which binds the target protein, than in the absence of a ligand. Binding of the ligand to the target protein can be determined by any method which distinguishes between the folded and unfolded states of the target protein. The function of the target protein need not be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a test ligand, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules. [0257]
  • Another method for identifying ligands of a target protein is described in Wieboldt et al. (1997) [0258] Anal. Chem. 69:1683-1691, incorporated herein by reference. This technique screens combinatorial libraries of 20-30 agents at a time in solution phase for binding to the target protein. Agents that bind to the target protein are separated from other library components by simple membrane washing. The specifically selected molecules that are retained on the filter are subsequently liberated from the target protein and analyzed by HPLC and pneumatically assisted electrospray (ion spray) ionization mass spectroscopy. This procedure selects library components with the greatest affinity for the target protein, and is particularly useful for small molecule libraries.
  • The methods of the invention also embrace ligands, especially neuropeptides, that are attached to a label, such as a radiolabel (e.g., [0259] 125I, 35S, 32P, 33P, 3H), a fluorescence label, a chemiluminescent label, an enzymic label and an immunogenic label. Modulators falling within the scope of the invention include, but are not limited to, non-peptide molecules such as non-peptide mimetics, non-peptide allosteric effectors, and peptides. The NgR polypeptide or polynucleotide employed in such a test may either be free in solution, attached to a solid support, borne on a cell surface or located intracellularly or associated with a portion of a cell. One skilled in the art can, for example, measure the formation of complexes between NgR and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between NgR and its substrate caused by the compound being tested.
  • Another aspect of the present invention is directed to methods of identifying compounds which modulate (i.e., increase or decrease) activity of NgR comprising contacting NgR with a compound, and determining whether the compound modifies activity of NgR. The activity in the presence of the test compared is measured to the activity in the absence of the test compound. Where the activity of the sample containing the test compound is higher than the activity in the sample lacking the test compound, the compound will have increased activity. Similarly, where the activity of the sample containing the test compound is lower than the activity in the sample lacking the test compound, the compound will have inhibited activity. [0260]
  • The present invention is particularly useful for screening compounds by using NgR in any of a variety of drug screening techniques. The compounds to be screened include (which may include compounds which are suspected to modulate NgR activity), but are not limited to, extracellular, intracellular, biologic or chemical origin. The NgR polypeptide employed in such a test may be in any form, preferably, free in solution, attached to a solid support, borne on a cell surface or located intracellularly. One skilled in the art can, for example, measure the formation of complexes between NgR and the compound being tested. Alternatively, one skilled in the art can examine the diminution in complex formation between Nogo-R and its substrate caused by the compound being tested. [0261]
  • The activity of NgR polypeptides of the invention can be determined by, for example, examining the ability to bind or be activated by chemically synthesized peptide ligands. Alternatively, the activity of the NgR can be assayed by examining their ability to bind calcium ions, hormones, chemokines, neuropeptides, neurotransmitters, nucleotides, lipids, odorants and photons. Alternatively, the activity of the NgR can be determined by examining the activity of effector molecules including, but not limited to, adenylate cyclase, phospholipases and ion channels. Thus, modulators of NgR activity may alter a NgR receptor function, such as a binding property of a receptor or an activity. In various embodiments of the method, the assay may take the form of an ion flux assay, a yeast growth assay, a non-hydrolyzable GTP assay such as a [[0262] 35S]-GTP S assay, a cAMP assay, an inositol triphosphate assay, a diacylglycerol assay, an Aequorin assay, a Luciferase assay, a FLIPR assay for intracellular Ca2+ concentration, a mitogenesis assay, a MAP Kinase activity assay, an arachidonic acid release assay (e.g., using [3H]-arachidonic acid) and an assay for extracellular acidification rates, as well as other binding or function-based assays of NgR activity that are generally known in the art. NgR activity can be determined by methodologies that are used to assay for FaRP activity, which is well known to those skilled in the art. Biological activities of NgR receptors according to the invention include, but are not limited to, the binding of a natural or an unnatural ligand, as well as any one of the functional activities of NgRs known in the art. Non-limiting examples of NgR activities include transmembrane signaling of various forms, which may involve phosphatidylinositol (PI) association and/or the exertion of an influence over PI; another exemplary activity of NgRs is the binding of accessory proteins or polypeptides that differ from known GPI proteins.
  • The modulators of the invention exhibit a variety of chemical structures, which can be generally grouped into non-peptide mimetics of natural NgR receptor ligands, peptide and non-peptide allosteric effectors of NgR receptors, and peptides that may function as activators or inhibitors (competitive, uncompetitive and non-competitive) (e.g., antibody products) of NgR receptors. The invention does not restrict the sources for suitable modulators, which may be obtained from natural sources such as plant, animal or mineral extracts, or non-natural sources such as small molecule libraries, including the products of combinatorial chemical approaches to library construction, and peptide libraries. [0263]
  • Other assays can be used to examine enzymatic activity including, but not limited to, photometric, radiometric, HPLC, electrochemical, and the like, which are described in, for example, ENZYME ASSAYS: A PRACTICAL APPROACH, Eisenthal and Danson (Eds.), 1992, Oxford University Press, which is incorporated herein by reference in its entirety. [0264]
  • The use of cDNAs in drug discovery programs is well-known; assays capable of testing thousands of unknown compounds per day in high-throughput screens (HTSs) are thoroughly documented. The literature is replete with examples of the use of radiolabelled ligands in HTS binding assays for drug discovery (see Williams (1991) [0265] Med. Res. Rev., 11, 147-184; Sweetnam et al., (1993) J. Nat. Prod. 56, 441-455 for review). Recombinant receptors are preferred for binding assay HTS because they allow for better specificity (higher relative purity), provide the ability to generate large amounts of receptor material, and can be used in a broad variety of formats (see Hodgson (1992) Bio/Technology 10, 973-980; each of which is incorporated herein by reference in its entirety).
  • A variety of heterologous systems is available for functional expression of recombinant receptors that are well known to those skilled in the art. Such systems include bacteria (Strosberg et al. (1992) [0266] Trends Pharmacol. Sci. 13, 95-98), yeast (Pausch (1997) Trends Biotechnol. 15, 487-494), several kinds of insect cells (Vanden Broeck (1996) Int. Rev. Cytol. 164, 189-268), amphibian cells (Jayawickreme et al. (1997) Curr. Opin. Biotechnol. 8, 629-634) and several mammalian cell lines (CHO, HEK293, COS, etc.; see Gerhardt et al. (1997) Eur. J. Pharmacol. 334, 1-23). These examples do not preclude the use of other possible cell expression systems, including cell lines obtained from nematodes (PCT application WO 98/37177).
  • In preferred embodiments of the invention, methods of screening for compounds which modulate NgR activity comprise contacting test compounds with NgR and assaying for the presence of a complex between the compound and NgR. In such assays, the ligand is typically labeled. After suitable incubation, free ligand is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular compound to bind to NgR. [0267]
  • In another embodiment of the invention, high throughput screening for compounds having suitable binding affinity to NgR is employed. Briefly, large numbers of different small peptide test compounds are synthesized on a solid substrate. The peptide test compounds are contacted with NgR and washed. Bound NgR is then detected by methods well known in the art. Purified polypeptides of the invention can also be coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the protein and immobilize it on the solid support. [0268]
  • Generally, an expressed NgR can be used for HTS binding assays in conjunction with its defined ligand. The identified peptide is labeled with a suitable radioisotope, including, but not limited to, [0269] 125I, 3H, 35S or 32P, by methods that are well known to those skilled in the art. Alternatively, the peptides may be labeled by well-known methods with a suitable fluorescent derivative (Baindur et al. (1994) Drug Dev. Res. 33, 373-398; Rogers (1997) Drug Discov. Today 2, 156-160). Radioactive ligand specifically bound to the receptor in membrane preparations made from the cell line expressing the recombinant protein can be detected in HTS assays in one of several standard ways, including filtration of the receptor-ligand complex to separate bound ligand from unbound ligand (Williams (1991) Med. Res. Rev. 11, 147-184; Sweetnam et al. (1993) J. Nat. Prod. 56, 441-455). Alternative methods include a scintillation proximity assay (SPA) or a FlashPlate format in which such separation is unnecessary (Nakayama (1998) Curr. Opin. Drug Disc. Dev. 1, 85-91 Bosse et al. (1998) J. Biomol. Screening 3, 285-292). Binding of fluorescent ligands can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound ligand, or fluorescence polarization (Rogers (1997) Drug Discov. Today 2, 156-160; Hill (1998) Curr. Opin. Drug Disc. Dev. 1, 92-97).
  • Examples of such biological responses include, but are not limited to, the following: the ability to survive in the absence of a limiting nutrient in specifically engineered yeast cells (Pausch (1997) Trends in [0270] Biotechnol. 15, 487-494); changes in intracellular Ca concentration as measured by fluorescent dyes (Murphy et al. (1998) Cur. Opin. Drug Disc. Dev. 1, 192-199). Fluorescence changes can also be used to monitor ligand-induced changes in membrane potential or intracellular pH; an automated system suitable for HTS has been described for these purposes (Schroeder et al. (1996) J. Biomol. Screening 1, 75-80). Melanophores prepared from Xenopus laevis show a ligand-dependent change in pigment organization in response to heterologous NgR activation; this response is adaptable to HTS formats (Jayawickreme et al. (1997) Curr. Opin. Biotechnol. 8, 629-634). Assays are also available for the measurement of common second messengers, including cAMP, phosphoinositides and arachidonic acid, but these are not generally preferred for HTS.
  • Preferred methods of HTS employing these receptors include permanently transfected CHO cells, in which agonists and antagonists can be identified by the ability to transduce the signal for the binding of Nogo in membranes prepared from these cells through the putative GPI anchor. In another embodiment of the invention, permanently transfected CHO cells could be used for the preparation of membranes which contain significant amounts of the recombinant receptor proteins; these membrane preparations would then be used in receptor binding assays, employing the radiolabelled ligand specific for the particular receptor. Alternatively, a functional assay, such as fluorescent monitoring of ligand-induced changes in internal Ca[0271] 2+ concentration or membrane potential in permanently transfected CHO cells containing each of these receptors individually or in combination would be preferred for HTS. Equally preferred would be an alternative type of mammalian cell, such as HEK293 or COS cells, in similar formats. More preferred would be permanently transfected insect cell lines, such as Drosophila S2 cells. Even more preferred would be recombinant yeast cells expressing the Drosophila melanogaster receptors in HTS formats well known to those skilled in the art (e.g., Pausch (1997), above).
  • The invention contemplates a multitude of assays to screen and identify inhibitors of ligand binding to NgR receptors. In one example, the NgR receptor is immobilized and interaction with a binding partner is assessed in the presence and absence of a candidate modulator such as an inhibitor compound. In another example, interaction between the NgR receptor and its binding partner is assessed in a solution assay, both in the presence and absence of a candidate inhibitor compound. In either assay, an inhibitor is identified as a compound that decreases binding between the NgR receptor and its binding partner. Another contemplated assay involves a variation of the di-hybrid assay wherein an inhibitor of protein/protein interactions is identified by detection of a positive signal in a transformed or transfected host cell, as described in PCT publication number WO 95/20652, published Aug. 3, 1995. [0272]
  • Candidate modulators contemplated by the invention include compounds selected from libraries of either potential activators or potential inhibitors. There are a number of different libraries used for the identification of small molecule modulators, including: (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules. Chemical libraries consist of random chemical structures, some of which are analogs of known compounds or analogs of compounds that have been identified as “hits” or “leads” in other drug discovery screens, some of which are derived from natural products, and some of which arise from non-directed synthetic organic chemistry. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms that are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see Cane et al., [0273] Science (1998) 282, 63-68. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthetic methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers (1997) Curr. Opin. Biotechnol. 8, 701-707. Identification of modulators through use of the various libraries described herein permits modification of the candidate “hit” (or “lead”) to optimize the capacity of the “hit” to modulate activity.
  • Still other candidate inhibitors contemplated by the invention can be designed and include soluble forms of binding partners, as well as such binding partners as chimeric, or fusion, proteins. A “binding partner” as used herein broadly encompasses non-peptide modulators, as well as such peptide modulators as neuropeptides other than natural ligands, antibodies, antibody fragments, and modified compounds comprising antibody domains that are immunospecific for the expression product of the identified NgR gene. [0274]
  • Other embodiments of the invention comprise using competitive screening assays in which neutralizing antibodies capable of binding a polypeptide of the invention specifically compete with a test compound for binding to the polypeptide. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with NgR. Radiolabeled competitive binding studies are described in Lin et al., (1997) [0275] Antimicrob. Agents Chemother. 41, 2127-2131, the disclosure of which is incorporated herein by reference in its entirety.
  • In other embodiments of the invention, the polypeptides of the invention are employed as a research tool for identification, characterization and purification of interacting, regulatory proteins. Appropriate labels are incorporated into the polypeptides of the invention by various methods known in the art and the polypeptides are used to capture interacting molecules. For example, molecules are incubated with the labeled polypeptides, washed to remove unbound polypeptides, and the polypeptide complex is quantified. Data obtained using different concentrations of polypeptide are used to calculate values for the number, affinity, and association of polypeptide with the protein complex. [0276]
  • Labeled polypeptides are also useful as reagents for the purification of molecules with which the polypeptide interacts including, but not limited to, inhibitors. In one embodiment of affinity purification, a polypeptide is covalently coupled to a chromatography column. Cells and their membranes are extracted, and various cellular subcomponents are passed over the column. Molecules bind to the column by virtue of their affinity to the polypeptide. The polypeptide-complex is recovered from the column, dissociated and the recovered molecule is subjected to protein sequencing. This amino acid sequence is then used to identify the captured molecule or to design degenerate oligonucleotides for cloning the corresponding gene from an appropriate cDNA library. [0277]
  • Alternatively, compounds may be identified which exhibit similar properties to the ligand for the NgR of the invention, but which are smaller and exhibit a longer half time than the endogenous ligand in a human or animal body. When an organic compound is designed, a molecule according to the invention is used as a “lead” compound. The design of mimetics to known pharmaceutically active compounds is a well-known approach in the development of pharmaceuticals based on such “lead” compounds. Mimetic design, synthesis and testing are generally used to avoid randomly screening a large number of molecules for a target property. Furthermore, structural data deriving from the analysis of the deduced amino acid sequences encoded by the DNAs of the present invention are useful to design new drugs, more specific and therefore with a higher pharmacological potency. [0278]
  • Comparison of the protein sequence of the present invention with the sequences present in all the available databases showed a significant homology with the transmembrane portion of G protein coupled receptors. Accordingly, computer modeling can be used to develop a putative tertiary structure of the proteins of the invention based on the available information of the transmembrane domain of other proteins. Thus, novel ligands based on the predicted structure of NgR can be designed. [0279]
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein. [0280]
  • Compositions and Pharmaceutical Compositions [0281]
  • In a particular embodiment, the novel molecules identified by the screening methods according to the invention are low molecular weight organic molecules, in which case a composition or pharmaceutical composition can be prepared thereof for oral or parenteral administration. The compositions, or pharmaceutical compositions, comprising the nucleic acid molecules, vectors, polypeptides, antibodies and compounds identified by the screening methods described herein, typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The nature of the carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington's PHARMACEUTICAL SCIENCES, 16th ed., (1980) Osol, A (Ed.), which is incorporated herein by reference in its entirety. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. [0282]
  • A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include oral and parenteral (e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal and rectal administration). Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. [0283]
  • Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. [0284]
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a NgR protein or anti-NgR antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [0285]
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate or orange flavoring. [0286]
  • For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide or a nebulizer. [0287]
  • Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels or creams as generally known in the art. [0288]
  • The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery. [0289]
  • In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved. [0290]
  • The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by any of a number of routes, e.g., as described in U.S. Pat. No. 5,703,055. Delivery can thus also include, e.g., intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or stereotactic injection (see e.g., Chen et al. (1994) [0291] Proc. Natl. Acad. Sci. USA 91, 3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • The pharmaceutical compositions can be included in a container, pack or dispenser together with instructions for administration. [0292]
  • The dosage of these low molecular weight compounds will depend on the disease state or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. For treating human or animals, between approximately 0.5 mg/kg of body weight to 500 mg/kg of body weight of the compound can be administered. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed. [0293]
  • Another aspect of the present invention is the use of the NgR nucleotide sequences disclosed herein for identifying homologs of the Nogo-R, in other animals, including but not limited to humans and other mammals and invertebrates. Any of the nucleotide sequences disclosed herein, or any portion thereof, can be used, for example, as probes to screen databases or nucleic acid libraries, such as, for example, genomic or cDNA libraries, to identify homologs using screening procedures well known to those skilled in the art. Accordingly, homologs having at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably at least 100% homology with NgR sequences can be identified. [0294]
  • The present compounds and methods, including nucleic acid molecules, polypeptides, antibodies, compounds identified by the screening methods described herein, have a variety of pharmaceutical applications and may be used, for example, to treat or prevent unregulated cellular growth, such as cancer cell and tumor growth. In a particular embodiment, the present molecules are used in gene therapy. For a review of gene therapy procedures, see e.g. Anderson [0295] Science (1992) 256, 808-813, which is incorporated herein by reference in its entirety.
  • The present invention also encompasses a method of agonizing (stimulating) or antagonizing a NgR natural binding partner associated activity in a mammal comprising administering to said mammal an agonist or antagonist to one of the above disclosed polypeptides in an amount sufficient to effect said agonism or antagonism. One embodiment of the present invention, then, is a method of treating diseases in a mammal with an agonist or antagonist of the protein of the present invention comprising administering the agonist or antagonist to a mammal in an amount sufficient to agonize or antagonize NgR-associated functions. [0296]
  • Methods of determining the dosages of compounds to be administered to a patient and modes of administering compounds to an organism are disclosed in U.S. application Ser. No. 08/702,282, filed Aug. 23, 1996, and International patent publication number WO 96/22976, published Aug. 1, 1996, both of which are incorporated herein by reference in their entirety, including any drawings, figures or tables. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it. [0297]
  • The proper dosage depends on various factors such as the type of disease being treated, the particular composition being used and the size and physiological condition of the patient. Therapeutically effective doses for the compounds described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC[0298] 50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
  • Plasma half-life and biodistribution of the drug and metabolites in the plasma, tumors and major organs can also be determined to facilitate the selection of drugs most appropriate to inhibit a disorder. Such measurements can be carried out. For example, HPLC analysis can be performed on the plasma of animals treated with the drug and the location of radiolabeled compounds can be determined using detection methods such as X-ray, CAT scan and MRI. Compounds that show potent inhibitory activity in the screening assays, but have poor pharmacokinetic characteristics, can be optimized by altering the chemical structure and retesting. In this regard, compounds displaying good pharmacokinetic characteristics can be used as a model. [0299]
  • Toxicity studies can also be carried out by measuring the blood cell composition. For example, toxicity studies can be carried out in a suitable animal model as follows: (1) the compound is administered to mice (an untreated control mouse should also be used); (2) blood samples are periodically obtained via the tail vein from one mouse in each treatment group; and (3) the samples are analyzed for red and white blood cell counts, blood cell composition and the percent of lymphocytes versus polymorphonuclear cells. A comparison of results for each dosing regime with the controls indicates if toxicity is present. [0300]
  • At the termination of each toxicity study, further studies can be carried out by sacrificing the animals (preferably, in accordance with the American Veterinary Medical Association guidelines Report of the American Veterinary Medical Assoc. Panel on Euthanasia, (1993) [0301] J. Am. Vet. Med. Assoc. 202:229-249). Representative animals from each treatment group can then be examined by gross necropsy for immediate evidence of metastasis, unusual illness or toxicity. Gross abnormalities in tissue are noted and tissues are examined histologically. Compounds causing a reduction in body weight or blood components are less preferred, as are compounds having an adverse effect on major organs. In general, the greater the adverse effect the less preferred the compound.
  • For the treatment of cancers the expected daily dose of a hydrophobic pharmaceutical agent is between 1 to 500 mg/day, preferably 1 to 250 mg/day, and most preferably 1 to 50 mg/day. Drugs can be delivered less frequently provided plasma levels of the active moiety are sufficient to maintain therapeutic effectiveness. Plasma levels should reflect the potency of the drug. Generally, the more potent the compound the lower the plasma levels necessary to achieve efficacy. [0302]
  • NgR mRNA transcripts have been found in the brain and heart. SEQ ID NOs: 1 and/or, 3 will, as detailed above, enable screening the endogenous neurotransmitters/hormones/ligands which activate, agonize, or antagonize NgR and for compounds with potential utility in treating disorders including CNS disorders (e.g., stroke) and degenerative disorders such as those associated with demyelination. [0303]
  • For example, NgR receptor activation may mediate the prevention of neurite outgrowth. Inhibition would be beneficial in both chronic and acute brain injury. See, e.g., Donovan et al., (1997) [0304] J. Neurosci. 17, 5316-5326; Turgeon et al., (1998) J. Neurosci. 18, 6882-6891; Smith-Swintosky et al., (1997) J. Neurochem. 69, 1890-1896; Gill et al., (1998) Brain Res. 797, 321-327; Suidan et al., (1996) Semin. Thromb. Hemost. 22, 125-133.
  • Pharmacogenomics [0305]
  • Agents, or modulators that have a stimulatory or inhibitory effect on NgR activity (e.g., NgR gene expression), as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) disorders (e.g., a disease condition such as a demyelination disorder) associated with aberrant NgR activity. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) of the individual may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of NgR protein, expression of NgR nucleic acid or mutation content of NgR genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. [0306]
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum (1996) [0307] Clin. Exp. Pharmacol. Physiol. 23, 983-985 and Linder (1997) Clin. Chem. 43, 254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is haemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
  • As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification. [0308]
  • Thus, the activity of NgR protein, expression of NgR nucleic acid, or mutation content of NgR genes in an individual can be determined to thereby select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a NgR modulator, such as a modulator identified by one of the exemplary screening assays described herein. [0309]
  • Monitoring Clinical Efficacy [0310]
  • Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of NgR (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase NgR gene expression, protein levels or upregulate NgR activity, can be monitored in clinical trials of subjects exhibiting decreased NgR gene expression, protein levels, or downregulated NgR activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease NgR gene expression, protein levels, or downregulate NgR activity, can be monitored in clinical trials of subjects exhibiting increased NgR gene expression, protein levels, or upregulated NgR activity. In such clinical trials, the expression or activity of NgR and, preferably, other genes that have been implicated in, for example, a disease or disorder, can be used as a “read out” or markers of the immune responsiveness of a particular cell. [0311]
  • For example, genes, including NgR, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) that modulates NgR activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on demyelination disorders, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of NgR and other genes implicated in the disorder. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced by one of the methods as described herein or by measuring the levels of activity of NgR or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent. [0312]
  • In one embodiment, the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a NgR protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the NgR protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the NgR protein, mRNA or genomic DNA in the pre-administration sample with the NgR protein, mRNA or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of NgR to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of NgR to lower levels than detected, i.e., to decrease the effectiveness of the agent. [0313]
  • Methods of Treatment [0314]
  • The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NgR expression or activity. [0315]
  • Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, (i) a NgR polypeptide, or analogs, derivatives, fragments or homologs thereof, (ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgR peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to a NgR peptide) are utilized to “knockout” endogenous function of a NgR peptide by homologous recombination (see, e.g., Capecchi (1989) [0316] Science 244, 1288-1292); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between a NgR peptide and its binding partner.
  • Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a NgR peptide, or analogs, derivatives, fragments or homologs thereof, or an agonist that increases bioavailability. [0317]
  • Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a NgR peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.). [0318]
  • In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NgR expression or activity, by administering to the subject an agent that modulates NgR expression or at least one NgR activity. Subjects at risk for a disease that is caused or contributed to by aberrant NgR expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NgR aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of NgR aberrancy, for example, a NgR agonist or NgR antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. [0319]
  • Another aspect of the invention pertains to methods of modulating NgR expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NgR protein activity associated with the cell. An agent that modulates NgR protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NgR protein, a peptide, a NgR peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NgR protein activity. Examples of such stimulatory agents include active NgR protein and a nucleic acid molecule encoding NgR that has been introduced into the cell. In another embodiment, the agent inhibits one or more NgR protein activity. Examples of such inhibitory agents include antisense NgR nucleic acid molecules and anti-NgR antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NgR protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) NgR expression or activity. In another embodiment, the method involves administering a NgR protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NgR expression or activity. [0320]
  • Gene Therapy [0321]
  • Mutations in the NgR gene that result in loss of normal function of the NgR gene product underlie NgR human disease states. The invention comprehends gene therapy to restore NgR activity to treat those disease states. Delivery of a functional NgR gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson (1998) [0322] Nature, supplement to 392(6679):25-20. For additional reviews of gene therapy technology see Friedmann (1989) Science 244, 1275-1281; Verma (1990) Sci. Am. 68-84; and Miller (1992) Nature 357, 455-460. Alternatively, it is contemplated that in other human disease states, preventing the expression of, or inhibiting the activity of, NgR will be useful in treating disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of NgR.
  • The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant NgR expression or activity. [0323]
  • Diseases and disorders that are characterized by increased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that antagonize (i.e., reduce or inhibit) activity. Therapeutics that antagonize activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, (i) a NgR polypeptide, or analogs, derivatives, fragments or homologs thereof; (ii) antibodies to a NgR peptide; (iii) nucleic acids encoding a NgR peptide; (iv) administration of antisense nucleic acid and nucleic acids that are “dysfunctional” (i.e., due to a heterologous insertion within the coding sequences of coding sequences to a NgR peptide) are utilized to “knockout” endogenous function of a NgR peptide by homologous recombination (see, e.g., Capecchi (1989), above); or (v) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or antibodies specific to a peptide of the invention) that alter the interaction between a NgR peptide and its binding partner. [0324]
  • Diseases and disorders that are characterized by decreased (relative to a subject not suffering from the disease or disorder) levels or biological activity may be treated with Therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered in a therapeutic or prophylactic manner. Therapeutics that may be utilized include, but are not limited to, a NgR peptide, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability. [0325]
  • Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying it in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or mRNAs of a NgR peptide). Methods that are well-known within the art include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, etc.). [0326]
  • In one aspect, the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant NgR expression or activity, by administering to the subject an agent that modulates NgR expression or at least one NgR activity. Subjects at risk for a disease that is caused or contributed to by aberrant NgR expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the NgR aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of NgR aberrancy, for example, a NgR agonist or NgR antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein. [0327]
  • Another aspect of the invention pertains to methods of modulating NgR expression or activity for therapeutic purposes. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of NgR protein activity associated with the cell. An agent that modulates NgR protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of a NgR protein, a peptide, a NgR peptidomimetic, or other small molecule. In one embodiment, the agent stimulates one or more NgR protein activity. Examples of such stimulatory agents include active NgR protein and a nucleic acid molecule encoding NgR that has been introduced into the cell. In another embodiment, the agent inhibits one or more NgR protein activity. Examples of such inhibitory agents include antisense NgR nucleic acid molecules and anti-NgR antibodies. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a NgR protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) NgR expression or activity. In another embodiment, the method involves administering a NgR protein or nucleic acid molecule as therapy to compensate for reduced or aberrant NgR expression or activity. [0328]
  • The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figure. Such modifications are intended to fall within the scope of the appended claims. [0329]
  • The following Table 5 contains the sequences of exemplary polynucleotides and polypeptides of the invention. [0330]
    TABLE 5
    The following DNA sequence NgR2 <SEQ ID NO. 1> was identified
    in humans:
    ATGCTGCCCGGGCTCAGGCGCCTGCTGCAAGCTCCCGCCTCGGCCTGCCTCCTGCTGATG
    CTCCTGGCCCTGCCCCTGGCGGCCCCCAGCTGCCCCATGCTCTGCACCTGCTACTCATCC
    CCGCCCACCGTGAGCTGCCAGGCCAACAACTTCTCCTCTGTGCCGCTGTCCCTGCCACCC
    AGCACTCAGCGACTCTTCCTGCAGAACAACCTCATCCGCACGCTGCGGCCAGGCACCTTT
    GGGTCCAACCTGCTCACCCTGTGGCTCTTCTCCAACAACCTCTCCACCATCTACCCGGGC
    ACTTTCCGCCACTTGCAAGCCCTGGAGGAGCTGGACCTCGGTGACAACCGGCACCTGCGC
    TCGCTGGAGCCCGACACCTTCCAGGGCCTGGAGCGGCTGCAGTCGCTGCATTTGTACCGC
    TGCCAGCTCAGCAGCCTGCCCGGCAACATCTTCCGAGGCCTGGTCAGCCTGCAGTACCTC
    TACCTCCAGGAGAACAGCCTGCTCCACCTACAGGATGACTTGTTCGCGGACCTGGCCAAC
    CTGAGCCACCTCTTCCTCCACGGGAACCGCCTGCGGCTGCTCACAGAGCACGTGTTTCGC
    GGCCTGGGCAGCCTGGACCGGCTGCTGCTGCACGGGAACCGGCTGCAGGGCGTGCACCGC
    GCGGCCTTCCGCGGCCTCAGCCGCCTCACCATCCTCTACCTGTTCAACAACAGCCTGGCC
    TCGCTGCCCGGCGAGGCGCTCGCCGACCTGCCCTCGCTCGAGTTCCTGCGGCTCAACGCT
    AACCCCTGGGCGTGCGACTGCCGCGCGCGGCCGCTCTGGGCCTGGTTCCAGCGCGCGCGC
    GTGTCCAGCTCCGACGTGACCTGCGCCACCCCCCCGGAGCGCCAGGGCCGAGACCTGCGC
    GCGCTCCGCGAGGCCGACTTCCAGGCGTGTCCGCCCGCGGCACCCACGCGGCCGGGCAGC
    CGCGCCCGCGGCAACAGCTCCTCCAACCACCTGTACGGGGTGGCCGAGGCCGGGGCGCCC
    CCAGCCGATCCCTCCACCCTCTACCGAGATCTGCCTGCCGAAGACTCGCGGGGGCGCCAG
    GGCGGGGACGCGCCTACTGAGGACGACTACTGGGGGGGCTACGGGGGTGAGGACCAGCGA
    GGGGAGCAGATGTGCCCCGGCGCTGCCTGCCAGGCGCCCCCGGACTCCCGAGGCCCTGCG
    CTCTCGGCCGGGCTCCCCAGCCCTCTGCTTTGCCTCCTGCTCCTGGTGCCCCACCACCTC
    The following amino acid sequence <SEQ ID NO. 2> is the pre-
    dicted amino acid sequence derived from the DNA sequence of SEQ
    ID No. 1:
    M L P G L R R L L Q A P A S A C L L L M L L A L P L A A P S C
    P M L C T C Y S S P P T V S C Q A N N F S S V P L S L P P S T
    Q R L F L Q N N L I R T L R P G T F G S N L L T L W L F S N N
    L S T I Y P G T F R H L Q A L E E L D L G D N R H L R S L E P
    D T F Q G L E R L Q S L H L Y R C Q L S S L P G N I F R G L V
    S L Q Y L Y L Q E N S L L H L Q D D L F A D L A N L S H L F L
    H G N R L R L L T E H V F R G L G S L D R L L L H G N R L Q G
    V H R A A F R G L S R L T I L Y L F N N S L A S L P G E A L A
    D L P S L E F L R L N A N P W A C D C R A R P L W A W F Q R A
    R V S S S D V T C A T P P E R Q G R D L R A L R E A D F Q A C
    P P A A P T R P G S R A R G N S S S N H L Y G V A E A G A P P
    A D P S T L Y R D L P A E D S R G R Q G G D A P T E D D Y W G
    G Y G G E D Q R G E Q M C P G A A C Q A P P D S R G P A L S A
    G L P S P L L C L L L L V P H H L
    The following DNA sequence NgR3 <SEQ ID NO. 3> was identified
    in mouse:
    ATGTCTTGGCAGTCTGGAACCACAGTGACACAATCTCCCGTGCAGGCTGCTCAGGTCTCA
    GGGTGCTGTGTGGAATTGCTGCTGTTGCTGCTCGCTGGAGAGCTACCTCTGGGTGGTGGT
    TGTCCTCGAGACTGTGTGTGCTACCCTGCGCCCATGACTGTCAGCTGCCAGGCACACAAC
    TTTGCTGCCATCCCGGAGGGCATCCCAGAGGACAGTGAGCGCATCTTCCTGCAGAACAAT
    CGCATCACCTTCCTCCAGCAGGGCCACTTCAGCCCCGCCATGGTCACCCTCTGGATCTAC
    TCCAACAACATCACTTTCATTGCTCCCAACACCTTCGAGGGCTTTGTGCATCTGGAGGAG
    CTAGACCTTGGAGACAACCGACAGCTGCGAACGCTGGCACCCGAGACCTTCCAAGGCCTG
    GTGAAGCTTCACGCCCTCTACCTCTATAAGTGTGGACTGAGCGCCCTGCCCGCAGGCATC
    TTTGGTGGCCTGCACAGCCTGCAGTATCTCTACTTGCAGGACAACCATATCGAGTACCTC
    CAAGATGACATCTTTGTGGACCTGGTCAATCTCAGTCACTTGTTTCTCCATGGTAACAAG
    CTATGGAGCCTGGGCCAAGGCATCTTCCGGGGCCTGGTGAACCTGGACCGGTTGCTGCTG
    CATGAGAACCAGCTACAGTGGGTTCACCACAAGGCTTTCCATGACCTCCACAGGCTAACC
    ACCCTCTTTCTCTTCAACAACAGCCTCACTGAGCTGCAGGGTGACTGTCTGGCCCCCCTG
    GTGGCCTTGGAGTTCCTTCGCCTCAATGGGAATGCTTGGGACTGTGGCTGCCGGGCACGT
    TCCCTGTGGGAATGGCTGCGAAGGTTCCGTGGCTCTAGCTCTGCTGTCCCCTGCGCGACC
    CCCGAGCTGCGGCAAGGCCAGGATCTGAAGCTGCTGAGGGTGGAGGACTTCCGGAACTGC
    ACAGGACCAGTGTCTCCTCACCAGATCAAGTCTCACACGCTTACCACCTCTGACAGGGCT
    GCCCGCAAGGAGCACCATCCGTCCCATGGGGCCTCCAGGGACAAAGGCCACCCACATGGC
    CATCCGCCTGGCTCCAGGTCAGGTTACAAGAAGGCAGGCAAGAACTGCACCAGCCACAGG
    AACCGGAACCAGATCTCTAAGGTGAGCTCTGGGAAAGAGCTTACCGAACTGCAGGACTAT
    GCCCCCGACTATCAGCACAAGTTCAGCTTTGACATCATGCCCACCGCACGACCCAAGAGG
    AAGGGCAAGTGTGCTCGCAGGACCCCCATCCGTGCCCCCAGTGGGGTGCAGCAGGCATCC
    TCAGGCACGGCCCTTGGGGCCCCACTCCTGGCCTGGATACTGGGGCTGGCAGTCACTCTC
    CGC
    The following protein sequence <SEQ ID NO. 4> is deduced pro-
    tein of SEQ ID NO: 3:
    M S W Q S G T T V T Q S P V Q A A Q V S G C C V E L L L L L L
    A G E L P L G G G C P R D C V C Y P A P M T V S C Q A H N F A
    A I P E G I P E D S E R I F L Q N N R I T F L Q Q G H F S P A
    M V T L W I Y S N N I T F I A P N T F E G F V H L E E L D L G
    D N R Q L R T L A P E T F Q G L V K L H A L Y L Y K C G L S A
    L P A G I F G G L H S L Q Y L Y L Q D N H I E Y L Q D D I F V
    D L V N L S H L F L H G N K L W S L G Q G I F R G L V N L D R
    L L L H E N Q L Q W V H H K A F H D L H R L T T L F L F N N S
    L T E L Q G D C L A P L V A L E F L R L N G N A W D C G C R A
    R S L W E W L R R F R G S S S A V P C A T P E L R Q G Q D L K
    L L R V E D F R N C T G P V S P H Q I K S H T L T T S D R A A
    R K E H H P S H G A S R D K G H P H G H P P G S R S G Y K K A
    G K N C T S H R N R N Q I S K V S S G K E L T E L Q D Y A P D
    Y Q H K F S F D I M P T A R P K R K G K C A R R T P I R A P S
    G V Q Q A S S G T A L G A P L L A W I L G L A V T L R
    The following protein sequence <SEQ ID NO. 5> is NgR1 from hu-
    mans:
    M K R A S A G G S R L L A W V L W L Q A W Q V A A P C P G A
    C
    C Y N E P K V T T S C P Q Q G L Q A V P V G I P A A S Q R I
    F L H G N R I S H V P A A S F R A C R N L T I L W L H S N V L
    A R I D A A A F T G L A L L E Q L D L S D N A Q L R S V D P A
    T F H G L G R L H T L H L D R C G L Q E L G P G L F R G L A A
    L Q Y L Y L Q D N A L Q A L P D D T F R D L G N L T H L F L H
    G N R I S S V P E R A F R G L H S L D R L L L H Q N R V A H V
    H P H A F R D L G R L M T L Y L F A N N L S A L P T E A L A P
    L R A L Q Y L R L N D N P W V C D C R A R P L W A W L Q K F R
    G S S S E V P C S L P Q R L A G R D L K R L A A N D L Q G C A
    V A T G P Y H P I W T G R A T D E E P L G L P K C C Q P D A A
    D K A S V L E P G R P A S A G N A L K G R V P P G D S P P G N
    G S G P R H I N D S P F G T L P G S A E P P L T A V R P E G S
    E P P G F P T S G P R R R P G C S R K N R T R S H C R L G Q A
    G S G G G G T G D S E G S G A L P S L T C S L T P L G L A L V
    L W T V L G P C
    The following amino acid sequence <SEQ ID NO: 6> is a Consensus
    Sequence of NgR based on homology with NgR1
    C P X X C X C Y X X P X X T X S C X X X X X X X X P X
    X X P X X X X R X F L X X N X I X X X X X X X F X X X X X X X
    X L W X
    X S N X X X X I X X X X F X X X X X L E X L D L X D N X X L R
    X X X P X T F X G L X X L X L X L X X C X L X X L X X X X F X
    G L X X L Q Y L Y L Q X N X X X X L X D D X F X D L X N L X H
    L F L H G N X X X X X X X X X F R G L X X L D R L L L H X N X
    X X X V H X X A F X X L X R L X X L X L F X N X L X X L X X X
    X L A X L X X L X X L R L N X N X W X C X C R A R X L W X W X
    X X X R X S S S X V X C X X P X X X X G X D L X X L X X X D X
    X X C X X X X X P X X P X X X X X X X X X X X X X X X X X X X
    X X X X X X X X X X X X X X X X X X G X X X X X X X X X X X X
    P P X X X S X X X X X X X X X X X X X X X X X X X X X X X X X
    X X X X X X X X X X X X X X X X X X X X X X X X X X X R X X X
    X X X X X X X X X X X X X X X X X X X X X X X X L X X X X X
    X X X X X L
    The following protein sequence <SEQ ID NO: 7> is the 66 amino
    acid active domain of Nogo:
    R I Y K G V I Q A I Q K S D E G H P F R A Y L E S E V A I S E
    E L V Q K Y S N S A L G H V N C T I K E L R R L F L V D D L V
    D S L K
    The following protein sequence <SEQ ID NO: 8> is the amino acid
    sequence of the mature NgR2:
    C P M L C T C Y S S P P T V S C Q A N N F S S V P L S L P P S
    T Q R L F L Q N N L I R T L R P G T F G S N L L T L W L F S N
    N L S T I Y P G T F R H L Q A L E E L D L G D N R H L R S L E
    P D T F Q G L E R L Q S L H L Y R C Q L S S L P G N I F R G L
    V S L Q Y L Y L Q E N S L L H L Q D D L F A D L A N L S H L F
    L H G N R L R L L T E H V F R G L G S L D R L L L H G N R L Q
    G V H R A A F R G L S R L T I L Y L F N N S L A S L P G E A L
    A D L P S L E F L R L N A N P W A C D C R A R P L W A W F Q R
    A R V S S S D V T C A T P P E R Q G R D L R A L R E A D F Q A
    C P P A A P T R P G S R A R G N S S S N H L Y G V A E A G A P
    P A D P S T L Y R D L P A E D S R G R Q G G D A P T E D D Y W
    G G Y G G E D Q R G E Q M C P G A A C Q A P P D S R G P A L S
    A G L P S P L L C L L L L V P H H L
    The following protein sequence <SEQ ID NO: 9> is the amino acid
    sequence of the mature NgR3:
    C P R D C V C Y P A P M T V S C Q A H N F A A I P E G I P E D
    S E R I F L Q N N R I T F L Q Q G H F S P A M V T L W I Y S N
    N I T F I A P N T F E G F V H L E E L D L G D N R Q L R T L A
    P E T F Q G L V K L H A L Y L Y K C G L S A L P A G I F G G L
    H S L Q Y L Y L Q D N H I E Y L Q D D I F V D L V N L S H L F
    L H G N K L W S L G Q G I F R G L V N L D R L L L H E N Q L Q
    W V H H K A F H D L H R L T T L F L F N N S L T E L Q G D C L
    A P L V A L E F L R L N G N A W D C G C R A R S L W E W L R R
    F R G S S S A V P C A T P E L R Q G Q D L K L L R V E D F R N
    C T G P V S P H Q I K S H T L T T S D R A A R K E H H P S H G
    A S R D K G H P H G H P P G S R S G Y K K A G K N C T S H R N
    R N Q I S K V S S G K E L T E L Q D Y A P D Y Q H K F S F D I
    M P T A R P K R K G K C A R R T P I R A P S G V Q Q A S S G T
    A L G A P L L A W I L G L A V T L R
    The following amino acid sequence <SEQ ID NO: 10> is a con-
    served cysteine motif (Cysteine domain 1) of the NgR and homo-
    logs based on the Consensus Sequence:
    C P X X C X C Y X X P X X T X S C
    The following amino acid sequence <SEQ ID NO: 11> is a con-
    served cysteine motif (Cysteine domain 2) of the NgR and homo-
    logs based on the Consensus Sequence:
    N X W X C X C R A R X L W X W X X X X R X S S S X V X C X X P
    X X X X G X D L X X L X X X D X X X C
    The following amino acid sequence <SEQ ID NO: 12> is a con-
    served Leucine-rich domain of the NgR and homologs based on the
    Consensus Sequence:
    R X F L X X N X I X X X X X X X F X X X X X X X X L W X X S N
    X X X X I X X X X F X X X X X L E X L D L X D N X X L R X X X
    P X T F X G L X X L X L X L X X C X L X X L X X X X F X G L X
    X L Q Y L Y L Q X N X X X X L X D D X F X D L X N L X H L F L
    H G N X X X X X X X X X F R G L X X L D R L L L H X N X X X X
    V H X X A F X X L X R L X X L X L F X N X L X X L X X X X L A
    X L X X L X X L R L
  • Unless otherwise indicated, X is any amino acid. For example, X where indicated may be no amino acid. Additional features of the invention will be apparent from the following Examples. Examples 1-5 are actual, while the remaining Examples are prophetic. [0331]
  • As shown by the following Examples, a gene encoding novel NgRs have been identified by computational analysis of DNA sequence data. The proteins encoded by NgR2 and NgR3 have a putative signal sequence, eight leucine-rich repeat domains in a conserved leucine-rich region (SEQ ID NO:12), a conserved cysteine-rich region (SEQ ID NO:10) N-terminal to the leucine-rich region, a second cysteine-rich domain (SEQ ID NO:11) C-terminal to the leucine-rich region, and a putative glycophosphatidylinositol-linkage (GPI-linkage) site. NgR2 and NgR3 differ from the previously identified NgR sequence. The NgR homologs, when compared to known NgRs, show a consensus sequence (SEQ ID NOs:6). The putative mature NgR2 and NgR3 are shown in Table 5 as SEQ ID NOs: 8 and 9, respectively. [0332]
    • EXAMPLE 1 Tblastn Query of the HTG Database
  • The protein sequence for the human NgR (NgR1) (SEQ ID NO:5) was used to query the high throughput genomic (HTG) database the use of which is familiar to those skilled in the art. The HTG database is a part of GenBank, a comprehensive NIH genetic sequence database, which includes an annotated collection of all publicly available DNA sequences ([0333] Nucleic Acids Res. (2000) 28, 15-8). The HTG database includes sequences obtained from genomic DNA. Within genomic DNA, genes are typically encoded by multiple segments of DNA called exons. Thus when one aligns a cDNA sequence (or a protein sequence encoded by a cDNA sequence) to a genomic sequence, the sequence will be broken up into segments depending on the number of exons in the gene.
  • The BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity (Altschul et al., (1990) [0334] J. Mol. Biol. 215, 403-410, which is incorporated herein by reference in its entirety). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The basic BLAST algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., (1992) Proc. Natl. Acad. Sci. USA 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.
  • The BLAST algorithm (Karlin et al., (1993) [0335] Proc. Natl. Acad. Sci. USA 90, 5873-5787, which is incorporated herein by reference) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a NgR gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a NgR nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • To query the HTG database with the NgR protein sequence, we used a variation of the BLAST algorithm known as the tblastn program, which compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames ([0336] J. Mol. Biol. (1990) 215, 403-410: Nucleic Acids Res. (1997) 25, 3389-3402). The results of the tblastn search indicated the presence of genes in the database with a significant identity to the NgR. In addition to finding hits to genomic clones which contain the human and mouse NgR genes, we found hits to clones where the identity was not as high, but still very significant. Three human clones were found (Accession numbers: AC068514, AC016869, AC013606) with an e-value of 4e-43 and one mouse clone was found (Accession No. AC021768) with an e-value of 1e-78. The three human clones all appeared to encode the same gene, so further analysis was confined to AC013606.
    • EXAMPLE 2 Prediction of the Human NgR2 Protein Sequence (AC013606)
  • The human NgR protein sequence aligned with two regions of translated sequence from nucleotide sequence AC013606, indicating that the new gene was encoded by at least two exons. In order to define the complete gene, we used the computer program GENSCAN ([0337] J. Mol. Biol. (1997) 268, 78-94) which can identify complete exon/intron structures of genes in genomic DNA. The gene prediction by GENESCAN™ contained seven exons. By comparing these predicted exons to the NgR, it was concluded that the new human gene contains two of these exons and a part of another (containing the initiating methionine). The predicted cDNA (mRNA) encoded by these three exons was assembled from AC013606 (HTG11; deposited March 2000; length=143899; GenBank release 118.0; SEQ ID NO: 15) by combining nucleotides from the three exons whose coordinates are: 123292-123322 (exon 1); 130035-130516 (exon 2); and 138589-139335 (exon 3). The sequence for this cDNA sequence is SEQ ID NO: 1 (nucleotide sequence of human NgR2; AC013606). The translation of this cDNA provides the protein sequence of human NgR2 (SEQ ID NO:2).
  • We used the protein sequence of human NgR2 as a query sequence against the human EST database. A number of hits of high significance were found indicating that the NgR2 mRNA is expressed in a number of tissues including fetal brain. Furthermore, two of these ESTs provided support for the exon structure that we deduced. One EST (Accession No: GB_EST19:AI346757) contains 565 nucleotides corresponding to amino acids 84-271 of the human NgR2 (SEQ ID No:4). This spans the second intron located between amino acids 171 and 172, and provides positive evidence for the splicing of [0338] exons 2 and 3 at the mRNA level. Another EST (GB_EST26:AI929019) contains 545 nucleotides, part of which corresponds to amino acids 1-75 of the human NgR2 (SEQ ID NO:2). This spans the first intron located between amino acids 10 and 11, and provides positive evidence for the splicing of exons 1 and 2 at the mRNA level.
    • EXAMPLE 3 Prediction of the Mouse NgR3 Protein Sequence (AC021768)
  • The human NgR protein sequence aligned with only one region of translated sequence from nucleotide sequence AC021768, indicating that most of the new mouse gene was encoded by one large exon. However, upon inspection, the protein encoded by this exon was missing an initiating methionine. In order to define the complete gene, we used the computer program GENSCAN as described above. The gene prediction by GENSCAN contained two exons; the large one found by visual inspection and a short one at the 5′ end which provided an initiating methionine. The predicted cDNA (mRNA) encoded by these two exons was assembled from AC021768 (HTG14; deposited March 2000; length=215980; GenBank release 118.0; SEQ ID NO: 16) by combining nucleotides from the two exons whose coordinates are: the complement of 164265-164325 (exon 1); and the complement of 155671-156992 (exon 2). The sequence for this cDNA sequence is SEQ ID NO:3 (nucleotide sequence of mouse NgR3; AC021768). The translation of this cDNA provides the protein sequence of mouse NgR3 (SEQ ID NO:4). [0339]
  • We used the protein sequence of mouse NgR3 as a query sequence against the mouse EST database. One hit of high significance was found indicating that the NgR2 mRNA is expressed in the heart. This EST (GB_EST20:AI428334) contains 463 nucleotides, part of which correspond to amino acids 45-193 of mouse NgR3 (SEQ ID NO:4). [0340]
    • EXAMPLE 4 Similarity Between the NgRs
  • An alignment between NgR1 and the two new receptors is shown in FIGS. [0341] 1A-1B. The similarities between these proteins include:
  • (1) The SignalP program, which locates the signal sequence cleavage position, predicts a cleavage before the first conserved cysteine in all the proteins. Thus the mature protein in all cases will have a cysteine at the N-terminus. [0342]
  • (2) All proteins contain eight Leucine Rich Repeats (LRR). LRRs are short sequence motifs present in a number of proteins with diverse functions and cellular locations. These repeats are usually involved in protein-protein interactions. Each LRR is composed of a beta-alpha unit. [0343]
  • (3) All three proteins contain a leucine rich repeat N-terminal domain (LRRNT), in which four cysteines are conserved. LRRs are often flanked by cysteine rich domains at both their N and C termini. [0344]
  • (4) All three proteins contain a LRR C-terminal domain (LRRCT). The LRRCTs of the three NgR proteins can be distinguished from those of other LRR containing proteins, by the pattern of typtophans and cysteines which are completely conserved in this domain. [0345]
  • (5) All three proteins contain a conserved cysteine in the fourth LRR domain. [0346]
  • (6) All three proteins contain a conserved potential glycosylation site in the eighth LRR domain. [0347]
  • (7) NgR2 and NgR3 have a hydrophobic C-terminus, as does NgR1, an indication that they probably also undergo a modification similar to NgR1, where a GPI moiety is covalently linked to a C-terminal amino acid. This allows the protein to remain tethered to the cell. [0348]
    • EXAMPLE 5 Preparation of Nogo Proteins
  • A Nogo binding assay was developed which utilizes a method widely used in examining semaphorin and ephrin axonal guidance function (Flanagan & Vanderhaeghen (1998) [0349] Annu. Rev. Neurosci. 21,309-345; Takahashi et al., (1999) Cell 99, 59-69). It involves fusing a secreted placental alkaline phosphatase (AP) moiety to the ligand in question to provide a biologically active receptor binding agent which can be detected with an extremely sensitive colorimetric assay. For Nogo, an expression vector is created encoding a signal peptide, a His6 tag for purification, AP, and the 66 amino acid active domain of Nogo. The fusion protein can be purified from the conditioned medium of transfected cells in milligram amounts. This protein is biologically active as a growth cone collapsing agent with an EC50 of 1 nM.
  • Alternatively, a glutathione-S-transferase Nogo (GST-Nogo) fusion protein may be prepared. For GST-Nogo, an expression vector (e.g., a pGEX vector) is created encoding a signal peptide, GST, and the 66 amino acid active domain of Nogo. GST-Nogo may be purified from the culture medium and used as a GST fusion protein, or GST may be cleaved from the Nogo portion of the fusion protein with an enzyme that recognizes the specific amino acid cleavage sit engineered between the GST portion and the Nogo portion of the fusion protein. Such sites are part of the commercially available GST vectors. The specific cleavage sites and enzymes may be used in accordance with the Manufacturer's specifications. [0350]
  • It has been found that AP-Nogo is actually slightly more potent than GST-Nogo, perhaps because the protein is synthesized in a eukaryotic rather than a prokaryotic cell. [0351]
  • Binding of Nogo to immobilized NgR homologs may be performed in an ELISA-type assay in which AP-Nogo is allowed to react with an immobilized receptor homolog. Specificity of binding may be demonstrated in a competitive binding assay using increasing amounts of GST-Nogo in the type of assay to show a decreasing amount of binding of AP-Nogo (as judged in the calorimetric assay). [0352]
    • EXAMPLE 6 Transfected COS Cell Binding Assays
  • The homologs of the present invention may be used in transfection studies in COS cells to demonstrate binding of Nogo. Specifically, nucleotide sequences encoding NgR2 and NgR3 may be transfected into COS cells using a suitable vector. Non-transfected COS-7 cells do not bind AP-Nogo. However, transfection of COS cells with nucleic acid sequences encoding NgRs will make them capable of binding Nogo. AP alone does not bind with any stable affinity to these transfected cells, indicating that any affinity of Nogo for NgR2 or NgR3 would be due to the 66 amino acids derived from Nogo. Furthemore, specific affinity of Nogo for the NgR2 or NgR3 proteins may be tested in displacement of AP-Nogo assays using GST-Nogo. NgR2 and/or NgR3 may also bind homologs of Nogo, which may also be tested using this assay. [0353]
    • EXAMPLE 7 Expression of NgR in Human Cell Lines Using Northern Blot and a Random-Primed Probe
  • A Northern blot is purchased from a commercial source, or RNA samples from cells of interest are run on an agarose gel and blotted to a membrane using any of the well known techniques for Northern blotting. The blot is probed with a fragment of NgR2 (SEQ ID NO:1) or NgR3 (SEQ ID NO:3). The probe is prepared from 50 ng of cDNA labeled by a random-primed method (Feinberg and Vogelstein (1983) [0354] Anal. Biochem. 132, 6-13). Hybridization is carried out at 68° C. for 1 hour in ExpressHybTM solution (Clontech, Cat. No. 8015-1) followed by washing with 2× SSC/0.05% SDS at room temperature and two washes with 0.1× SSC/0.1% SDS at 50° C. Expression of NgR2 and/or NgR3 can be assessed by the presence of an appropriately sized band on the blot.
    • EXAMPLE 8 Cloning of cDNA Corresponding to NgRs
  • To obtain the full-length clone corresponding to NgR2 from a cDNA library, the following method may be used. A cDNA library is generated using standard methods from a tissue known to contain NgR2. Such a tissue was identified in Example 2. 1×10[0355] 6 plaque forming units from the cDNA library may be screened in duplicate on OPTITRAN™ filters. The filters are hybridized with 32P-labeled oligonucleotides that are generated from the ESTs corresponding to portions of NgR2. The hybridization reaction may consist of 400 mls plaque screen buffer (50 mM Tris pH 7.5, 1M NaCl, 0.1% Sodium pyrophosphate, 0.2% Polyvinylpryolidine and 0.2% Ficoll) containing 10% Dextran sulfate and 100 μg/ml tRNA and 80 pmol each 32P-labeled oligonucleotide at 65° C. overnight. The filters are washed twice with 2× SSC/1%SDS and twice with 1× SSC/1%SDS and exposed to film. Duplicate positives are purified. DNA from each of these clones is analyzed by restriction enzyme digest followed by agarose gel electrophoresis and Southern blotting. The filters are hybridized to the 32P-labeled oligonucleotides used for the original hybridization to confirm that inserts hybridize to the probe. The insert is then sequenced to confirm that it represents the cDNA for NgR2. Similar methods may be used to generate a full-length clone corresponding to NgR3.
  • Alternatively, a full-length clone of NgR2 or NgR3 can be obtained by a person of ordinary skill in the art employing conventional PCR techniques. [0356]
    • EXAMPLE 9 Hybridization Analysis to Demonstrate NgR Expression in the Brain
  • The expression of NgR in mammals, such as the rat, may be investigated by in situ hybridization histochemistry. To investigate expression in the brain, for example, coronal and sagittal rat brain cryosections (20 μm thick) are prepared using a Reichert-Jung cryostat. Individual sections are thaw-mounted onto silanized, nuclease-free slides (CEL Associates, Inc., Houston, Tex.), and stored at −80° C. Sections are processed starting with post-fixation in cold 4% paraformaldehyde, rinsed in cold phosphate-buffered saline (PBS), acetylated using acetic anhydride in triethanolamine buffer, and dehydrated through a series of alcohol washes in 70%, 95%, and 100% alcohol at room temperature. Subsequently, sections are delipidated in chloroform, followed by rehydration through successive exposure to 100% and 95% alcohol at room temperature. Microscope slides containing processed cryosections are allowed to air dry prior to hybridization. Other tissues may be assayed in a similar fashion. [0357]
  • A NgR-specific probe may be generated using PCR. Following PCR amplification, the fragment is digested with restriction enzymes and cloned into pBluescript II cleaved with the same enzymes. For production of a probe specific for the sense strand of NgR, a cloned NgR fragment cloned in pBluescript II may be linearized with a suitable restriction enzyme, which provides a substrate for labeled run-off transcripts (i.e., cRNA riboprobes) using the vector-borne T7 promoter and commercially available T7 RNA polymerase. A probe specific for the antisense strand of NgR may also be readily prepared using the NgR clone in pBluescript II by cleaving the recombinant plasmid with a suitable restriction enzyme to generate a linearized substrate for the production of labeled run-off cRNA transcripts using the T3 promoter and cognate polymerase. The riboprobes may be labeled with [[0358] 35S]-UTP to yield a specific activity of about 0.40×106 cpm/pmol for antisense riboprobes and about 0.65×106 cpm/pmol for sense-strand riboprobes. Each riboprobe may be subsequently denatured and added (2 pmol/ml) to hybridization buffer which contains 50% formamide, 10% dextran, 0.3 M NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 1× Denhardt's Solution, and 10 mM dithiothreitol. Microscope slides containing sequential brain cryosections may be independently exposed to 45 μl of hybridization solution per slide and silanized cover slips may be placed over the sections being exposed to hybridization solution. Sections are incubated overnight (15-18 hours) at 52° C. to allow hybridization to occur. Equivalent series of cryosections are then exposed to sense or antisense NgR-specific cRNA riboprobes.
  • Following the hybridization period, coverslips are washed off the slides in IX SSC, followed by RNase A treatment involving the exposure of slides to 20 μg/ml RNase A in a buffer containing 10 mM Tris-HCl (pH 7.4), 0.5 M EDTA, and 0.5 M NaCl for 45 minutes at 37° C. The cryosections are then subjected to three high-stringency washes in 0.1× SSC at 52° C. for 20 minutes each. Following the series of washes, cryosections are dehydrated by consecutive exposure to 70%, 95%, and 100% ammonium acetate in alcohol, followed by air drying and exposure to Kodak BioMax™ MR-1 film. After 13 days of exposure, the film is developed, and any significant hybridization signal is detected. Based on these results, slides containing tissue that hybridized, as shown by film autoradiograms, are coated with Kodak NTB-2 nuclear track emulsion and the slides are stored in the dark for 32 days. The slides are then developed and counterstained with hematoxylin. Emulsion-coated sections are analyzed microscopically to determine the specificity of labeling. The signal is determined to be specific if autoradiographic grains (generated by antisense probe hybridization) are clearly associated with cresyl violate-stained cell bodies. Autoradiographic grains found between cell bodies indicate non-specific binding of the probe. [0359]
  • In some cases, such as using a probe to detect a NgR homolog in a heterologous species, in order to achieve optimal hybridization, it may be necessary to decrease the stringency conditions. Such conditions are well known to those of ordinary skill in the art and examples are provided above. [0360]
  • Expression of NgR in the brain provides an indication that modulators of NgR activity have utility for treating neurological disorders. Some other diseases for which modulators of NgR may have utility include depression, anxiety, bipolar disease, epilepsy, neuritis, neurasthenia, neuropathy, neuroses, and the like. Use of NgR modulators, including NgR ligands and anti-NgR antibodies, to treat individuals having such disease states is intended as an aspect of the invention. [0361]
    • EXAMPLE 10 Northern Blot Analysis of NgR-RNA With a PCR-Generated Probe
  • Northern blot hybridizations may be performed to examine the expression of NgR mRNA. A clone containing at least a portion of the sequence of SEQ ID NO:1 may be used as a probe. Vector-specific primers are used in PCR to generate a hybridization probe fragment for [0362] 32P-labeling. The PCR is performed as follows:
    Mix: 1 μl NgR-containing plasmid
    2 μl fwd primer (10-50 pM)
    2 μl rev primer (10-50 pM)
    10 μl 10 × PCR buffer (such as that provided with the
    enzyme, Amersham Pharmacia Biotech)
    1 μl 10 mM dNTP (such as #1 969 064 from
    Boehringer Mannheim)
    0.5 μl Taq polymerase (such as #27-0799-62, Amersham
    Pharmacia Biotech)
    83.5 μl water
  • PCR is performed in a Thermocycler using the following program: [0363]
    94° C.  5 min |
    94° C.  1 min |
    55° C.  1 min | 30 cycles
    72° C.  1 min |
    72° C. 10 min |
  • The PCR product may be purified using QIAquick PCR Purification Kit (#28104) from Qiagen, and radictively labeled with [0364] 32P-dCTP (#AA0005/250, Amersham Pharmacia Biotech)) may be done by random priming using “Ready-to-go DNA Labeling Beads” (#27-9240-01) from Amersham Pharmacia Biotech. Hybridization is carried out on Human Multiple Tissue Northern Blot from Clontech as described in manufacturer's protocol, or on a Northern Blot prepared by running RNA samples from cells of interest on an agarose gel and blotting to a membrane using any of the known Northern blotting protocols. After exposure overnight on Molecular Dynamics Phosphor Imager screen (#MD146-814) bands of an appropriate size are visualized.
    • EXAMPLE 11 Recombinant Expression of NgR in Eukaryotic Host Cells
  • A. Expression of NgR in Mammalian Cells [0365]
  • To produce NgR protein, a NgR-encoding polynucleotide is expressed in a suitable host cell using a suitable expression vector and standard genetic engineering techniques. For example, a NgR-encoding sequence described in Table 4 is subcloned into the commercial expression vector pzeoSV2 (Invitrogen, San Diego, Calif.) and transfected into Chinese Hamster Ovary (CHO) cells using the transfection reagent FuGENE6™ (Boehringer-Mannheim) and the transfection protocol provided in the product insert. Other eukaryotic cell lines, including human embryonic kidney (HEK 293) and COS cells, are suitable as well. Cells stably expressing NgR are selected by growth in the presence of 100 μg/ml zeocin (Stratagene, LaJolla, Calif.). As an TM alternative to FuGENE6™, the expression vector may carry the gene for dihydrofolate reductase (dhfr) and selection of clones with methotrexate (MTX) drug pressure allows for stable transformation of CHO cells. Optionally, NgR may be purified from the cells using standard chromatographic techniques. To facilitate purification, antisera is raised against one or more synthetic peptide sequences that correspond to portions of the NgR amino acid sequence, and the antisera is used to affinity purify Nogo-R. The NgR also may be expressed in-frame with a tag sequence (e.g., polyhistidine, hemaglutinin, FLAG) to facilitate purification. Moreover, it will be appreciated that many of the uses for NgR polypeptides, such as assays described below, do not require purification of NgR from the host cell. [0366]
  • B. Expression of NgR in CHO cells [0367]
  • For expression of NgR in Chinese hamster ovary (CHO) cells, a plasmid bearing the relevant NgR coding sequence is prepared, using a vector which also bears the selectable marker dihydrofolate reductase (DHFR). The plasmid is transfected into CHO cells. Selection under MTX drug pressure allows for preparation of stable transformants of a NgR (NgR2 or NgR3) in an expression plasmid carrying a selectable marker such as DHFR. [0368]
  • C. Expression of NgR in 293 Cells [0369]
  • For expression of NgR in mammalian cells 293 (transformed human, primary embryonic kidney cells), a plasmid bearing the relevant NgR coding sequence is prepared, using vector pSecTag2A (Invitrogen). Vector pSecTag2A contains the murine IgK chain leader sequence for secretion, the c-myc epitope for detection of the recombinant protein with the anti-myc antibody, a C-terminal polyhistidine for purification with nickel chelate chromatography, and a Zeocin resistant gene for selection of stable transfectants. The forward primer for amplification of this NgR cDNA is determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce the HindIII cloning site and nucleotides matching the NgR sequence. The reverse primer is also determined by routine procedures and preferably contains a 5′ extension of nucleotides to introduce an XhoI restriction site for cloning and nucleotides corresponding to the reverse complement of the NgR sequence. The PCR conditions are 55° C. as the annealing temperature. The PCR product is gel purified and cloned into the HindIII-XhoI sites of the vector. [0370]
  • The DNA is purified using Qiagen chromatography columns and transfected into 293 cells using DOTAP™ transfection media (Boehringer Mannheim, Indianapolis, Ind.). Transiently transfected cells are tested for expression after 24 hours of transfection, using western blots probed with anti-His and anti-NgR peptide antibodies. Permanently transfected cells are selected with Zeocin and propagated. Production of the recombinant protein is detected from both cells and media by Western blots probed with anti-His, anti-Myc or anti-NgR peptide antibodies. [0371]
  • D. Transient Expression of Nogo-R in COS Cells [0372]
  • For expression of the NgR in COS7 cells, a polynucleotide molecule having a nucleotide sequence of SEQ ID NO:1, for example, can be cloned into vector p3-CI. This vector is a pUC18-derived plasmid that contains the HCMV (human cytomegalovirus) promoter-intron located upstream from the bGH (bovine growth hormone) polyadenylation sequence and a multiple cloning site. [0373]
  • The forward primer is determined by routine procedures and preferably contains a 5′ extension which introduces an XbaI restriction site for cloning, followed by nucleotides which correspond to a nucleotide sequence of SEQ ID NO:1. The reverse primer is also determined by routine procedures and preferably contains 5′-extension of nucleotides which introduces a SalI cloning site followed by nucleotides which correspond to the reverse complement of a nucleotide sequence of SEQ ID NO:1. [0374]
  • The PCR consists of an initial denaturation step of 5 min at 95° C., 30 cycles of 30 sec denaturation at 95° C., 30 sec annealing at 58° C. and 30 sec extension at 72° C., followed by 5 min extension at 72° C. The PCR product is gel purified and ligated into the XbaI and SalI sites of vector p3-CI. This construct is transformed into [0375] E. coli cells for amplification and DNA purification. The DNA is purified with Qiagen chromatography columns and transfected into COS 7 cells using Lipofectamine™ reagent from BRL, following the manufacturer's protocols. Forty-eight and 72 hours after transfection, the media and the cells are tested for recombinant protein expression.
  • NgR expressed from a COS cell culture can be purified by concentrating the cell-growth media to about 10 mg of protein/ml, and purifying the protein by, for example, chromatography. Purified NgR is concentrated to 0.5 mg/ml in an Amicon concentrator fitted with a YM-10 membrane and stored at −80° C. NgR3 may also be expressed using this method and the nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:13. [0376]
  • E. Expression of NgR in Insect Cells [0377]
  • For expression of NgR in a baculovirus system, a polynucleotide molecule having a nucleotide sequence of SEQ ID NO:1, 3 or 13 can be amplified by PCR. The forward primer is determined by routine procedures and preferably contains a 5′ extension which adds the NdeI cloning site, followed by nucleotides which correspond to a nucleotide sequence of SEQ ID NO:1 (or SEQ ID NO:3 or SEQ ID NO:13, respectively). The reverse primer is also determined by routine procedures and preferably contains a 5′ extension which introduces the KpnI cloning site, followed by nucleotides which correspond to the reverse complement of a nucleotide sequence of SEQ ID NO:1 (or SEQ ID NO:3 or SEQ ID NO:13, respectively). [0378]
  • The PCR product is gel purified, digested with NdeI and KpnI, and cloned into the corresponding sites of vector pACHTL-A (Pharmingen, San Diego, Calif.). The pAcHTL expression vector contains the strong polyhedrin promoter of the [0379] Autographa californica nuclear polyhedrosis virus (AcMNPV), and a 6XHis tag upstream from the multiple cloning site. A protein kinase site for phosphorylation and a thrombin site for excision of the recombinant protein precede the multiple cloning site is also present. Of course, many other baculovirus vectors could be used in place of pAcHTL-A, such as pAc373, pVL941 and pAcIM1. Other suitable vectors for the expression of NgR polypeptides can be used, provided that the vector construct includes appropriately located signals for transcription, translation, and trafficking, such as an in-frame AUG and a signal peptide, as required. Such vectors are described in Luckow et al., Virology 170:31-39, among others.
  • The virus is grown and isolated using standard baculovirus expression methods, such as those described in Summers et al. (1987) A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555. [0380]
  • In a preferred embodiment, pAcHLT-A containing NgR gene is introduced into baculovirus using the “BaculoGold™” transfection kit (Pharmingen, San Diego, Calif.) using methods established by the manufacturer. Individual virus isolates are analyzed for protein production by radiolabeling infected cells with [0381] 35S-methionine at 24 hours post infection. Infected cells are harvested at 48 hours post infection, and the labeled proteins are visualized by SDS-PAGE. Viruses exhibiting high expression levels can be isolated and used for scaled up expression.
  • For expression of a NgR polypeptide in a Sf9 cells, a polynucleotide molecule having the nucleotide sequence of SEQ ID NO:1 (or SEQ ID NO:3 or SEQ ID NO:13) can be amplified by PCR using the primers and methods described above for baculovirus expression. The NgR cDNA is cloned into vector pAcHLT-A (Pharmingen) for expression in Sf9 insect. The insert is cloned into the NdeI and KpnI sites, after elimination of an internal NdeI site (using the same primers described above for expression in baculovirus). DNA is purified with Qiagen chromatography columns and expressed in Sf9 cells. Preliminary Western blot experiments from non-purified plaques are tested for the presence of the recombinant protein of the expected size which reacted with the NgR-specific antibody. These results are confirmed after further purification and expression optimization in HiG5 cells. [0382]
  • F. Expression of Soluble Forms of NgR2 and NgR3 as NgR-Ig Fusion Proteins. [0383]
  • To generate a NgR2-Ig fusion protein, standard methods may be used as described in the literature (e.g. Sanicola et al. (1997) [0384] Proc. Natl. Acad. Sci. USA. 94, 6238-6243). For example, a DNA fragment encoding NgR2 without the sequence encoding the hydrophobic C-terminus (GPI anchor signal) may be ligated to a DNA fragment encoding the Fc domain of IgG1 (which may be human IgG1), and the chimeric fragment may be cloned into an expression vector to generate a plasmid. The plasmid may then be transfected into Chinese hamster ovary cells to generate a stable cell line producing the fusion protein. The fusion protein is then purified from conditioned media using standard methods. For example, clarified conditioned media from the cell line may be loaded by gravity directly onto Protein A Sepharose. The column may then be washed with five column volumes each of PBS, PBS containing 0.5 M NaCl, and 25 mM sodium phosphate, 100 mM NaCl (pH 5.0). The bound protein may then be eluted with 25 mM NaH2PO4, 100 mM NaCl (pH 2.8) and immediately neutralized with {fraction (1/10)} fraction volume of 0. 5 M Na2HPO4 (pH 8.6).
  • Similar methods may be used to generate a NgR3-Ig fusion protein. [0385]
    • EXAMPLE 12 Interaction Trap/Two-Hybrid System
  • In order to assay for NgR-interacting proteins, the interaction trap/two-hybrid library screening method can be used. This assay was first described in Fields et al. (1989) [0386] Nature 340, 245, which is incorporated herein by reference in its entirety. A protocol is published in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 1999, John Wiley & Sons, NY and Ausubel, F. M. et al. 1992, SHORT PROTOCOLS IN MOLECULAR BIOLOGY, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety. Kits are available from Clontech, Palo Alto, Calif. (Matchmaker Two-Hybrid System 3).
  • A fusion of the nucleotide sequences encoding all or partial NgR and the yeast transcription factor GAL4 DNA-binding domain (DNA-BD) is constructed in an appropriate plasmid (i.e., pGBKT7) using standard subcloning techniques. Similarly, a GAL4 active domain (AD) fusion library is constructed in a second plasmid (i.e., pGADT7) from cDNA of potential NgR-binding proteins (for protocols on forming cDNA libraries, see Sambrook et al. 1989, MOLECULAR CLONING: A LABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which is incorporated herein by reference in its entirety. The DNA-BD/NgR fusion construct is verified by sequencing, and tested for autonomous reporter gene activation and cell toxicity, both of which would prevent a successful two-hybrid analysis. Similar controls are performed with the AD/library fusion construct to ensure expression in host cells and lack of transcriptional activity. Yeast cells are transformed (ca. 105 transformants/mg DNA) with both the NgR and library fusion plasmids according to standard procedure (Ausubel, et al., 1992, SHORT PROTOCOLS IN MOLECULAR BIOLOGY, fourth edition, Greene and Wiley-interscience, NY, which is incorporated herein by reference in its entirety). In vivo binding of DNA-BD/NgR with AD/library proteins results in transcription of specific yeast plasmid reporter genes (i.e., lacZ, HIS3, ADE2, LEU2). Yeast cells are plated on nutrient-deficient media to screen for expression of reporter genes. Colonies are dually assayed for β-galactosidase activity upon growth in Xgal (5-bromo-4-chloro-3-indolyl-b-D-galactoside) supplemented media (filter assay for b-galactosidase activity is described in Breeden et al., (1985) [0387] Cold Spring Harb. Symp. Quant. Biol., 50, 643, which is incorporated herein by reference in its entirety). Positive AD-library plasmids are rescued from transformants and reintroduced into the original yeast strain as well as other strains containing unrelated DNA-BD fusion proteins to confirm specific NgR/library protein interactions. Insert DNA is sequenced to verify the presence of an open reading frame fused to GAL4 AD and to determine the identity of the NgR-binding protein.
    • EXAMPLE 13 Antibodies to Nogo-R
  • Standard techniques are employed to generate polyclonal or monoclonal antibodies to the NgR receptor, and to generate useful antigen-binding fragments thereof or variants thereof, including “humanized” variants. Such protocols can be found, for example, in Sambrook et al. (1989), above, and Harlow et al. (Eds.), ANTIBODIES A LABORATORY MANUAL; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). In one embodiment, recombinant NgR polypeptides (or cells or cell membranes containing such polypeptides) are used as antigen to generate the antibodies. In another embodiment, one or more peptides having amino acid sequences corresponding to an immunogenic portion of NgR (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acids) are used as antigen. Peptides corresponding to extracellular portions of Nogo-R, especially hydrophilic extracellular portions, are preferred. The antigen may be mixed with an adjuvant or linked to a hapten to increase antibody production. [0388]
  • A. Polyclonal or Monoclonal antibodies [0389]
  • As one exemplary protocol, recombinant NgR or a synthetic fragment thereof is used to immunize a mouse for generation of monoclonal antibodies (or larger mammal, such as a rabbit, for polyclonal antibodies). To increase antigenicity, peptides are conjugated to Keyhole Limpet Hemocyanin (Pierce), according to the manufacturer's recommendations. For an initial injection, the antigen is emulsified with Freund's Complete Adjuvant and injected subcutaneously. At intervals of two to three weeks, additional aliquots of NgR antigen are emulsified with Freund's Incomplete Adjuvant and injected subcutaneously. Prior to the final booster injection, a serum sample is taken from the immunized mice and assayed by western blot to confirm the presence of antibodies that immunoreact with NgR. Serum from the immunized animals may be used as polyclonal antisera or used to isolate polyclonal antibodies that recognize NgR. Alternatively, the mice are sacrificed and their spleen removed for generation of monoclonal antibodies. [0390]
  • To generate monoclonal antibodies, the spleens are placed in 10 ml serum-free RPMI 1640, and single cell suspensions are formed by grinding the spleens in serum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada). The cell suspensions are filtered and washed by centrifugation and resuspended in serum-free RPMI. Thymocytes taken from three naive Balb/c mice are prepared in a similar manner and used as a Feeder Layer. NS-1 myeloma cells, kept in log phase in RPMI with 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah) for three days prior to fusion, are centrifuged and washed as well. [0391]
  • To produce hybridoma fusions, spleen cells from the immunized mice are combined with NS-1 cells and centrifuged, and the supernatant is aspirated. The cell pellet is dislodged by tapping the tube, and 2 ml of 37° C. PEG 1500 (50% in 75 mM HEPES, pH 8.0) (Boehringer-Mannheim) is stirred into the pellet, followed by the addition of serum-free RPMI. Thereafter, the cells are centrifuged, resuspended in RPMI containing 15% FBS, 100 μM sodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco), 25 units/ml IL-6 (Boehringer-Mannheim) and 1.5×10[0392] 6 thymocytes/ml, and plated into 10 Corning flat-bottom 96-well tissue culture plates (Corning, Corning, N.Y.).
  • On [0393] days 2, 4, and 6 after the fusion, 100 μl of medium is removed from the wells of the fusion plates and replaced with fresh medium. On day 8, the fusions are screened by ELISA, testing for the presence of mouse IgG that binds to NgR. Selected fusion wells are further cloned by dilution until monoclonal cultures producing anti-NgR antibodies are obtained.
  • B. Humanization of Anti-NgR Monoclonal Antibodies [0394]
  • The expression pattern of NgR as reported herein and the potential of NgRs as targets for therapeutic intervention suggest therapeutic indications for NgR inhibitors (antagonists). NgR-neutralizing antibodies comprise one class of therapeutics useful as NgR antagonists. Following are protocols to improve the utility of anti-NgR monoclonal antibodies as therapeutics in humans by “humanizing” the monoclonal antibodies to improve their serum half-life and render them less immunogenic in human hosts (i.e., to prevent human antibody response to non-human anti-NgR antibodies). [0395]
  • The principles of humanization have been described in the literature and are facilitated by the modular arrangement of antibody proteins. To minimize the possibility of binding complement, a humanized antibody of the IgG4 isotype is preferred. [0396]
  • For example, a level of humanization is achieved by generating chimeric antibodies comprising the variable domains of non-human antibody proteins of interest with the constant domains of human antibody molecules. (See, e.g., Morrison et al., (1989) [0397] Adv. Immunol., 44, 65-92). The variable domains of NgR-neutralizing anti-NgR antibodies are cloned from the genomic DNA of a B-cell hybridoma or from cDNA generated from mRNA isolated from the hybridoma of interest. The V region gene fragments are linked to exons encoding human antibody constant domains, and the resultant construct is expressed in suitable mammalian host cells (e.g., myeloma or CHO cells).
  • To achieve an even greater level of humanization, only those portions of the variable region gene fragments that encode antigen-binding complementarity determining regions (“CDR”) of the non-human monoclonal antibody genes are cloned into human antibody sequences. (See, e.g., Jones et al., (1986) [0398] Nature 321, 522-525; Riechmann et al., (1988) Nature 332, 323-327; Verhoeyen et al., (1988) Science 239, 1534-1536 ; and Tempest et al., (1991) Bio/Technology 9, 266-271). If necessary, the B-sheet framework of the human antibody surrounding the CDR3 regions also is modified to more closely mirror the three dimensional structure of the antigen-binding domain of the original monoclonal antibody. (See Kettleborough et al., (1991) Protein Engin. 4, 773-783; and Foote et al., (1992) J. Mol. Biol. 224, 487-499).
  • In an alternative approach, the surface of a non-human monoclonal antibody of interest is humanized by altering selected surface residues of the non-human antibody, e.g., by site-directed mutagenesis, while retaining all of the interior and contacting residues of the non-human antibody. See Padlan (1991) [0399] Mol. Immunol. 28, 489-498.
  • The foregoing approaches are employed using NgR-neutralizing anti-NgR monoclonal antibodies and the hybridomas that produce them to generate humanized NgR-neutralizing antibodies useful as therapeutics to treat or palliate conditions wherein NgR expression or ligand-mediated NgR signaling is detrimental. [0400]
  • C. Human NgR-Neutralizing Antibodies from Phage Display [0401]
  • Human NgR-neutralizing antibodies are generated by phage display techniques such as those described in Aujame et al. (1997) [0402] Human Antibodies 8, 155-168; Hoogenboom (1997) TIBTECH 15, 62-70; and Rader et al. (1997), Curr. Opin. Biotechnol. 8, 503-508, all of which are incorporated by reference. For example, antibody variable regions in the form of Fab fragments or linked single chain Fv fragments are fused to the amino terminus of filamentous phage minor coat protein pIII. Expression of the fusion protein and incorporation thereof into the mature phage coat results in phage particles that present an antibody on their surface and contain the genetic material encoding the antibody. A phage library comprising such constructs is expressed in bacteria, and the library is screened for NgR-specific phage-antibodies using labeled or immobilized NgR as antigen-probe.
  • D. Human NgR-Neutralizing Antibodies from Transgenic Mice [0403]
  • Human NgR-neutralizing antibodies are generated in transgenic mice essentially as described in Bruggemann et al. (1996) [0404] Immunol. Today 17, 391-397 and Bruggemann et al. (1997) Curr. Opin. Biotechnol. 8, 455-458. Transgenic mice carrying human V-gene segments in germline configuration and that express these transgenes in their lymphoid tissue are immunized with a NgR composition using conventional immunization protocols. hybridomas are generated using B cells from the immunized mice using conventional protocols and screened to identify hybridomas secreting anti-NgR human antibodies (e.g., as described above).
    • EXAMPLE 14 Assays to Identify Modulators of NgR Activity
  • Set forth below are several nonlimiting assays for identifying modulators (agonists and antagonists) of NgR activity. Among the modulators that can be identified by these assays are natural ligand compounds of the receptor; synthetic analogs and derivatives of natural ligands; antibodies, antibody fragments, and/or antibody-like compounds derived from natural antibodies or from antibody-like combinatorial libraries; and/or synthetic compounds identified by high-throughput screening of libraries; and the like. All modulators that bind NgR are useful for identifying NgR in tissue samples (e.g., for diagnostic purposes, pathological purposes, and the like). Agonist and antagonist modulators are useful for up-regulating and down-regulating NgR activity, respectively, to treat disease states characterized by abnormal levels of NgR activity. The assays may be performed using single putative modulators, and/or may be performed using a known agonist in combination with candidate antagonists (or visa versa). [0405]
  • A. cAMP Assays [0406]
  • In one type of assay, levels of cyclic adenosine monophosphate (cAMP) are measured in NgR-transfected cells that have been exposed to candidate modulator compounds. Protocols for cAMP assays have been described in the literature. (See, e.g., Sutherland et al., (1968) [0407] Circulation 37, 279; Frandsen et al., (1976) Life Sciences 18, 529-541; Dooley et al., (1997) J. Pharmacol. Exp. Therap. 283, 735-41; and George et al., (1997) J. Biomol. Screening 2, 235-40). An exemplary protocol for such an assay, using an Adenylyl Cyclase Activation FlashPlate Assay from NEN™ Life Science Products, is set forth below.
  • Briefly, the NgR coding sequence (e.g., a cDNA or intronless genomic DNA) is subcloned into a commercial expression vector, such as pzeoSV2 (Invitrogen), and transiently transfected into Chinese Hamster Ovary (CHO) cells using known methods, such as the transfection protocol provided by Boehringer-Mannheim when supplying the [0408] FuGENE 6 transfection reagent. Transfected CHO cells are seeded into 96-well microplates from the FlashPlate® assay kit, which are coated with solid scintillant to which antisera to cAMP has been bound. For a control, some wells are seeded with wild type (untransfected) CHO cells. Other wells in the plate receive various amounts of a cAMP standard solution for use in creating a standard curve.
  • One or more test compounds (i.e., candidate modulators) are added to the cells in each well, with water and/or compound-free medium/diluent serving as a control or controls. After treatment, cAMP is allowed to accumulate in the cells for exactly 15 minutes at room temperature. The assay is terminated by the addition of lysis buffer containing [[0409] 125I]-labeled cAMP, and the plate is counted using a Packard Topcount™ 96-well microplate scintillation counter. Unlabeled cAMP from the lysed cells (or from standards) and fixed amounts of [125I]-cAMP compete for antibody bound to the plate. A standard curve is constructed, and cAMP values for the unknowns are obtained by interpolation. Changes in intracellular cAMP levels of cells in response to exposure to a test compound are indicative of NgR modulating activity. Modulators that act as agonists of receptors which couple to the Gs subtype of G proteins will stimulate production of cAMP, leading to a measurable 3-10 fold increase in cAMP levels. Agonists of receptors which couple to the Gi/o subtype of G proteins will inhibit forskolin-stimulated cAMP production, leading to a measurable decrease in cAMP levels of 50-100%. Modulators that act as inverse agonists will reverse these effects at receptors that are either constitutively active or activated by known agonists.
  • B. Aequorin Assays [0410]
  • In another assay, cells (e.g., CHO cells) are transiently co-transfected with both a NgR expression construct and a construct that encodes the photoprotein apoaquorin. In the presence of the cofactor coelenterazine, apoaquorin will emit a measurable luminescence that is proportional to the amount of intracellular (cytoplasmic) free calcium. (See generally, Cobbold, et al. “Aequorin measurements of cytoplasmic free calcium,” In: McCormack J. G. and Cobbold P. H., eds., CELLULAR CALCIUM: A PRACTICAL APPROACH. Oxford:IRL Press (1991); Stables et al., (1997) [0411] Anal. Biochem. 252, 115-26; and Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS. Sixth edition. Molecular Probes, Eugene, Oreg. (1996)).
  • In one exemplary assay, NgR is subcloned into the commercial expression vector pzeoSV2 (Invitrogen) and transiently co-transfected along with a construct that encodes the photoprotein apoaquorin (Molecular Probes, Eugene, Oreg.) into CHO cells using the transfection reagent FuGENE 6 (Boehringer-Mannheim) and the transfection protocol provided in the product insert. [0412]
  • The cells are cultured for 24 hours at 37° C. in MEM (Gibco/BRL, Gaithersburg, Md.) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin, at which time the medium is changed to serum-free MEM containing 5 μM coelenterazine (Molecular Probes, Eugene, Oreg.). Culturing is then continued for two additional hours at 37° C. Subsequently, cells are detached from the plate using VERSEN (Gibco/BRL), washed, and resuspended at 200,000 cells/ml in serum-free MEM. [0413]
  • Dilutions of candidate NgR modulator compounds are prepared in serum-free MEM and dispensed into wells of an opaque 96-well assay plate at 50 μl/well. Plates are then loaded onto an MLX microtiter plate luminometer (Dynex Technologies, Inc., Chantilly, Va.). The instrument is programmed to dispense 50 μl cell suspensions into each well, one well at a time, and immediately read luminescence for 15 seconds. Dose-response curves for the candidate modulators are constructed using the area under the curve for each light signal peak. Data are analyzed with SlideWrite, using the equation for a one-site ligand, and EC[0414] 50 values are obtained. Changes in luminescence caused by the compounds are considered indicative of modulatory activity. Modulators that act as agonists at receptors which couple to the Gq subtype of G proteins give an increase in luminescence of up to 100 fold. Modulators that act as inverse agonists will reverse this effect at receptors that are either constitutively active or activated by known agonists.
  • C. Luciferase Reporter Gene Assay [0415]
  • The photoprotein luciferase provides another useful tool for assaying for modulators of NgR activity. Cells (e.g., CHO cells or [0416] COS 7 cells) are transiently co-transfected with both a NgR expression construct (e.g., NgR in pzeoSV2) and a reporter construct which includes a gene for the luciferase protein downstream from a transcription factor binding site, such as the cAMP-response element (CRE), AP-1, or NF-kappa B. Expression levels of luciferase reflect the activation status of the signaling events. (See generally, George et al. (1997) J. Biomol. Screening 2, 235-240; and Stratowa et al. (1995) Curr. Opin. Biotechnol. 6, 574-581). Luciferase activity may be quantitatively measured using, e.g., luciferase assay reagents that are commercially available from Promega (Madison, Wis.).
  • In one exemplary assay, CHO cells are plated in 24-well culture dishes at a density of 100,000 cells/well one day prior to transfection and cultured at 37° C. in MEM (Gibco/BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin. Cells are transiently co-transfected with both a NgR expression construct and a reporter construct containing the luciferase gene. The reporter plasmids CRE-luciferase, AP-1-luciferase and NF-kappaB-luciferase may be purchased from Stratagene (Legally, Calif.). Transfections are performed using the [0417] FuGENE 6 transfection reagent (Boehringer-Mannheim) according to the supplier's instructions. Cells transfected with the reporter construct alone are used as a control. Twenty-four hours after transfection, cells are washed once with PBS pre-warmed to 37° C. Serum-free MEM is then added to the cells either alone (control) or with one or more candidate modulators and the cells are incubated at 37° C. for five hours. Thereafter, cells are washed once with ice-cold PBS and lysed by the addition of 100 μl of lysis buffer per well from the luciferase assay kit supplied by Promega. After incubation for 15 minutes at room temperature, 15 μl of the lysate is mixed with 50 μl of substrate solution (Promega) in an opaque-white, 96-well plate, and the luminescence is read immediately on a Wallace model 1450 MicroBeta scintillation and luminescence counter (Wallace Instruments, Gaithersburg, Md.).
  • Differences in luminescence in the presence versus the absence of a candidate modulator compound are indicative of modulatory activity. Receptors that are either constitutively active or activated by agonists typically give a 3-20-fold stimulation of luminescence compared to cells transfected with the reporter gene alone. Modulators that act as inverse agonists will reverse this effect. [0418]
  • D. Intracellular Calcium Measurement Using FLIPR [0419]
  • Changes in intracellular calcium levels are another recognized indicator of receptor activity, and such assays can be employed to screen for modulators of NgR activity. For example, CHO cells stably transfected with a NgR expression vector are plated at a density of 4×10[0420] 4 cells/well in Packard black-walled, 96-well plates specially designed to discriminate fluorescence signals emanating from the various wells on the plate. The cells are incubated for 60 minutes at 37° C. in modified Dulbecco's PBS (D-PBS) containing 36 mg/L pyruvate and 1 g/L glucose with the addition of 1% fetal bovine serum and one of four calcium indicator dyes (Fluo-3™ AM, Fluo-4™ AM, Calcium Green™-1 AM, or Oregon Green™ 488 BAPTA-1 AM), each at a concentration of 4 μM. Plates are washed once with modified D-PBS without 1% fetal bovine serum and incubated for 10 minutes at 37° C. to remove residual dye from the cellular membrane. In addition, a series of washes with modified D-PBS without 1% fetal bovine serum is performed immediately prior to activation of the calcium response.
  • A calcium response is initiated by the addition of one or more candidate receptor agonist compounds, calcium ionophore A23187 (10 μM; positive control), or ATP (4 μM; positive control). Fluorescence is measured by Molecular Device's FLIPR with an argon laser (excitation at 488 nm). (See, e.g., Kuntzweiler et al. (1998) [0421] Drug Dev. Res. 44,14-20). The F-stop for the detector camera is set at 2.5 and the length of exposure is 0.4 milliseconds. Basal fluorescence of cells is measured for 20 seconds prior to addition of candidate agonist, ATP, or A23187, and the basal fluorescence level is subtracted from the response signal. The calcium signal is measured for approximately 200 seconds, taking readings every two seconds. Calcium ionophore A23187 and ATP increase the calcium signal 200% above baseline levels. In general, activated NgRs increase the calcium signal at least about 10-15% above baseline signal.
  • E. [[0422] 35S]GTPγS Binding Assay
  • It is also possible to evaluate whether NgR signals through a G protein-mediated pathway. Because G protein-coupled receptors signal through intracellular G proteins whose activity involves GTP binding and hydrolysis to yield bound GDP, measurement of binding of the non-hydrolyzable GTP analog [[0423] 35S]-GTPγS in the presence and absence of candidate modulators provides another assay for modulator activity. (See, e.g., Kowal et al., (1998) Neuropharmacology 37, 179-187.).
  • In one exemplary assay, cells stably transfected with a NgR expression vector are grown in 10 cm tissue culture dishes to subconfluence, rinsed once with 5 ml of ice-cold Ca[0424] 2+/Mg2+-free phosphate-buffered saline, and scraped into 5 ml of the same buffer. Cells are pelleted by centrifugation (500× g, 5 minutes), resuspended in TEE buffer (25 mM Tris, pH 7.5, 5 mM EDTA, 5 mM EGTA), and frozen in liquid nitrogen. After thawing, the cells are homogenized using a Dounce homogenizer (1 ml TEE per plate of cells), and centrifuged at 1,000× g for 5 minutes to remove nuclei and unbroken cells.
  • The homogenate supernatant is centrifuged at 20,000× g for 20 minutes to isolate the membrane fraction, and the membrane pellet is washed once with TEE and resuspended in binding buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM MgCl[0425] 2, 1 mM EDTA). The resuspended membranes can be frozen in liquid nitrogen and stored at −70° C. until use.
  • Aliquots of cell membranes prepared as described above and stored at −70° C. are thawed, homogenized, and diluted into buffer containing 20 mM HEPES, 10 mM MgCl[0426] 2, 1 mM EDTA, 120 mM NaCl, 10 μM GDP, and 0.2 mM ascorbate, at a concentration of 10-50 μg/ml. In a final volume of 90 μl, homogenates are incubated with varying concentrations of candidate modulator compounds or 100 μM GTP for 30 minutes at 30° C. and then placed on ice. To each sample, 10 μl guanosine 5′-O-(3[35S]thio) triphosphate (NEN, 1200 Ci/mmol; [35S]-GTPγS), was added to a final concentration of 100-200 pM. Samples are incubated at 30° C. for an additional 30 minutes, 1 ml of 10 mM HEPES, pH 7.4, 10 mM MgCl2, at 4° C. is added and the reaction is stopped by filtration.
  • Samples are filtered over Whatman GF/B filters and the filters are washed with 20 ml ice-cold 10 mM HEPES, pH 7.4, 10 mM MgCl[0427] 2. Filters are counted by liquid scintillation spectroscopy. Nonspecific binding of [35S]-GTPγS is measured in the presence of 100 μM GTP and subtracted from the total. Compounds are selected that modulate the amount of [35S]-GTPγS binding in the cells, compared to untransfected control cells. Activation of receptors by agonists gives up to a five-fold increase in [35S]-GTPγS binding. This response is blocked by antagonists.
  • F. [[0428] 3H]Arachidonic Acid Release
  • The activation of NgRs may also potentiate arachidonic acid release in cells, providing yet another useful assay for modulators of NgR activity. (See, e.g., Kanterman et al., (1991) [0429] Mol. Pharmacol. 39,364-369.) For example, CHO cells that are stably transfected with a NgR expression vector are plated in 24-well plates at a density of 15,000 cells/well and grown in MEM medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 10 U/ml penicillin and 10 μg/ml streptomycin for 48 hours at 37° C. before use. Cells of each well are labeled by incubation with [3H]-arachidonic acid (Amersham Corp., 210 Ci/mmol) at 0.5 μCi/ml in 1 ml MEM supplemented with 10 mM HEPES, pH 7.5, and 0.5% fatty-acid-free bovine serum albumin for 2 hours at 37° C. The cells are then washed twice with 1 ml of the same buffer.
  • Candidate modulator compounds are added in 1 ml of the same buffer, either alone or with 10 μM ATP and the cells are incubated at 37° C. for 30 minutes. Buffer alone and mock-transfected cells are used as controls. Samples (0.5 ml) from each well are counted by liquid scintillation spectroscopy. Agonists which activate the receptor will lead to potentiation of the ATP-stimulated release of [[0430] 3H]-arachidonic acid. This potentiation is blocked by antagonists.
  • G. Extracellular Acidification Rate [0431]
  • In yet another assay, the effects of candidate modulators of NgR activity are assayed by monitoring extracellular changes in pH induced by the test compounds (see, e.g., Dunlop et al. (1998) [0432] J. Pharmacol. Toxicol. Meth. 40, 47-55). In one embodiment, CHO cells transfected with a NgR expression vector are seeded into 12 mm capsule cups (Molecular Devices Corp.) at 4×105 cells/cup in MEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 U/ml penicillin, and 10 μg/ml streptomycin. The cells are incubated in this medium at 37° C. in 5% CO2 for 24 hours.
  • Extracellular acidification rates are measured using a Cytosensor microphysiometer (Molecular Devices Corp.). The capsule cups are loaded into the sensor chambers of the microphysiometer and the chambers are perfused with running buffer (bicarbonate-free MEM supplemented with 4 mM L-glutamine, 10 units/ml penicillin, 10 μg/ml streptomycin, 26 mM NaCl) at a flow rate of 100 μl/minute. Candidate agonists or other agents are diluted into the running buffer and perfused through a second fluid path. During each 60-second pump cycle, the pump is run for 38 seconds and is off for the remaining 22 seconds. The pH of the running buffer in the sensor chamber is recorded during the cycle from 43-58 seconds, and the pump is re-started at 60 seconds to start the next cycle. The rate of acidification of the running buffer during the recording time is calculated by the Cytosoft program. Changes in the rate of acidification are calculated by subtracting the baseline value (the average of 4 rate measurements immediately before addition of a modulator candidate) from the highest rate measurement obtained after addition of a modulator candidate. The selected instrument detects 61 mV/pH unit. Modulators that act as agonists of the receptor result in an increase in the rate of extracellular acidification compared to the rate in the absence of agonist. This response is blocked by modulators which act as antagonists of the receptor. [0433]
    • EXAMPLE 15 mNgR3 Does Not Bind hNogo-A(1055-1120)
  • To functionally test the mouse NgR3 (hereinafter, mNgR3) for its ability to bind hNogo-A(1055-1120), a cDNA expression vector for a myc epitope-tagged mNgR3protein was created. The mouse NgR3 cDNA was amplified by PCR from mouse adult brain cDNA, from the signal sequence to the stop codon, and ligated into the pSecTag2 vector such that the vector encodes a signal sequence followed by a myc tag followed by the mature mNgR3 sequence. This plasmid was transfected into COS07cells, and expression of a myc-tagged protein of the predicted size was verified by immunoblot analysis. Alkaline phosphatase-hNogo-A(1055-1120) binding studies and myc immunohistology were conducted as described (Fournier et al., supra). [0434]
  • The cells expressing mNgR3 express the myc-tagged protein but binding to AP-hNogo-A(1055-1120) was not observed under the conditions employed (FIG. 8). [0435]
    • EXAMPLE 16 Identification of Partial Human NgR3 cDNA and Protein Sequences
  • The tblastn program was used to search for the human homolog of mouse NgR3. The mouse NgR3 protein sequence (SEQ ID NO:4) was used to query a proprietary human expressed sequence tag (EST) database from Incyte yielding one highly significant hit: Incyte Template ID 190989. 1. This sequence (937 nucleotides) contains an open reading frame of 312 amino acids in the second reverse frame that exhibits 88% identity with residues 66 to 381 of mouse NgR3 (SEQ ID NO:4), strongly indicating that it is part of the human NgR3 homolog. [0436]
  • A query of SEQ ID NO:4 against the public human EST database in Genbank also produced a hit with a 465-bp EST (Accession number: R35699; Version number: R35699.1; GI: 792600). There are a number of single nucleotide deletions and insertions within this sequence which cause frame shift errors. All of the reliable sequence contained in this public EST is present in the Incyte EST (Template ID 190989.1). [0437]
  • To obtain more nucleotide sequence that would extend the amino acid sequence at that carboxy terminal end, the I.M.A.G.E. Consortium clone No. 38319, which corresponds to Genbank accession No. R35699, was purchased from Incyte Genomics Inc. and subjected to further DNA sequence analysis. This clone consists of a NotI/HinD III fragment containing the sequence of interest, cloned into the NotI/HinD III sites of the vector Lafmid BA (http://image.llnl.gov/image/html/libs/lafmidBA.shtml). The clone was received as an agar stab, which was streaked out on LB agar plates containing 50 ug/ml ampicillin to isolate individual colonies. Six colonies were grown in LB medium with antibiotic, and plasmid DNA was prepared using the Promega Wizard Plus Miniprep DNA Purification System (Promega #A7500). These DNAs were subsequently digested with NotI and HinD III restriction enzymes to confirm that the clones contained an insert. The insert of one isolate was sequenced using a combination of vector specific and gene specific primers yielding a partial nucleotide sequence of human NgR3 of 1176 nucleotides (SEQ ID NO:13). A translation of this sequence provides a partial sequence for human NgR3 of 392 amino acids (SEQ ID NO:14). [0438]
  • The nucleotide sequence of SEQ ID NO:13 differs from the Incyte EST sequence at three positions. Nucleotide positions 12-13 in SEQ ID NO:13 are CG, whereas the corresponding nucleotides in the Incyte Template ID 190989.1 are GT (i.e., positions 12-13 of the complement of Incyte Template ID 190989.1). In addition, position 641 in SEQ ID NO:13 is a C, whereas the corresponding nucleotide in the Incyte Template ID 190989.1 sequence is an A (i.e., position 641 of the complement of Incyte Template ID 190989.1). This results in two changes in amino acids when comparing SEQ ID NO:14 to the ORF encoded by Incyte Template 190989.1: SEQ ID NO:14 contains a valine at [0439] position 5, whereas the ORF encoded by Incyte Template ID 190989.1 contains a leucine; SEQ ID NO:14 contains an alanine at position 214, whereas the ORF encoded by Incyte Template ID 190989.1 contains a glutamic acid.
  • The nucleotide sequence of SEQ ID NO:13 differs from the public EST (Accession number: R35699; Version number: R35699.1; GI: 792600) sequence at two positions (within the first 200 nucleotides of reliable sequence). Nucleotide positions 12-13 in SEQ ID NO:13 are CG, whereas the corresponding nucleotides in the public EST are GT (i.e., positions 12-13 of the public EST; Accession no: R35699; Version no: R35699. 1; GI: 792600) This leads to a single amino acid change when comparing SEQ ID NO:14 to the ORF encoded by the public EST: SEQ ID NO:14 contains a valine at [0440] position 5, while the ORF encoded by the public EST contains a leucine.
  • A Bestfit analysis of the partial human amino acid sequence with the full-length mouse amino acid sequence indicates that the human NgR3 amino acid sequence is complete at the carboxy terminal end and that they share 89.54% identity. An alignment of all the NgR proteins is shown in FIG. 9. Although the human NgR3 amino acid sequence is missing the first 25 amino acids, it can be determined that the human NgR3 protein contains the following features in common with the other NgR sequences: (1) eight Leucine Rich Repeat (LRR) domains; (2) an LRR carboxy-terminal (LRR-CT) domain; (3) a conserved cysteine in the fourth LRR domain; (4) a conserved potential glycosylation site in the eighth LRR domain; and (5) a hydrophobic carboxyl terminus. [0441]
  • As those skilled in the art will appreciate, numerous changes and modifications may be made to the preferred embodiments of the invention without departing from the spirit of the invention. It is intended that all such variations fall within the scope of the invention. [0442]
  • The entire disclosure of each publication cited herein is hereby incorporated by reference. This application claims benefit from U.S. provisional application No. 60/238,361, filed Oct. 6, 2000, which is incorporated by reference herein in its entirety. [0443]
  • Key for Sequence Listing: [0444]
  • SEQ ID NO:1 human NgR2 cDNA sequence derived from genomic sequence AC013606 [0445]
  • SEQ ID NO:2 human NgR2 amino acid sequence [0446]
  • SEQ ID NO:3 mouse NgR3 cDNA sequence derived from AC021768 [0447]
  • SEQ ID NO:4 a mouse NgR3 amino acid sequence [0448]
  • SEQ ID NO:5 a human NgR1 amino acid sequence [0449]
  • SEQ ID NO:6 a consensus amino acid sequence for NgRs [0450]
  • SEQ ID NO:7 #1055-1120 amino acid residues of hNogoA (Nogo-66) [0451]
  • SEQ ID NO:8 a mature human NgR2 amino acid sequence [0452]
  • SEQ ID NO:9 a mature mouse NgR3 amino acid sequence [0453]
  • SEQ ID NO:10 a consensus NgR LLRNT amino acid sequence [0454]
  • SEQ ID NO:11 a consensus NgR LRRCT domain amino acid sequence [0455]
  • SEQ ID NO:12 a consensus NgR LRR domain amino acid sequence [0456]
  • SEQ ID NO:13 a partial human NgR3 nucleotide sequence [0457]
  • SEQ ID NO:14 a partial human NgR3 amino acid sequence [0458]
  • SEQ ID NO:15 a genomic sequence encoding a human NgR2 sequence. [0459]
  • SEQ ID NO:16 a genomic sequence (complementary strand) encoding a mouse NgR [0460]
  • SEQ ID NO:17 a mouse NgR3 amino acid sequence [0461]
  • SEQ ID NO:18 a consensus sequence for the NTLRRCT domain of NgR [0462]
  • SEQ ID NO:19 an consensus NgR LRRCT domain amino acid sequence [0463]
  • 1 19 1 1260 DNA Homo sapiens 1 atgctgcccg ggctcaggcg cctgctgcaa gctcccgcct cggcctgcct cctgctgatg 60 ctcctggccc tgcccctggc ggcccccagc tgccccatgc tctgcacctg ctactcatcc 120 ccgcccaccg tgagctgcca ggccaacaac ttctcctctg tgccgctgtc cctgccaccc 180 agcactcagc gactcttcct gcagaacaac ctcatccgca cgctgcggcc aggcaccttt 240 gggtccaacc tgctcaccct gtggctcttc tccaacaacc tctccaccat ctacccgggc 300 actttccgcc acttgcaagc cctggaggag ctggacctcg gtgacaaccg gcacctgcgc 360 tcgctggagc ccgacacctt ccagggcctg gagcggctgc agtcgctgca tttgtaccgc 420 tgccagctca gcagcctgcc cggcaacatc ttccgaggcc tggtcagcct gcagtacctc 480 tacctccagg agaacagcct gctccaccta caggatgact tgttcgcgga cctggccaac 540 ctgagccacc tcttcctcca cgggaaccgc ctgcggctgc tcacagagca cgtgtttcgc 600 ggcctgggca gcctggaccg gctgctgctg cacgggaacc ggctgcaggg cgtgcaccgc 660 gcggccttcc gcggcctcag ccgcctcacc atcctctacc tgttcaacaa cagcctggcc 720 tcgctgcccg gcgaggcgct cgccgacctg ccctcgctcg agttcctgcg gctcaacgct 780 aacccctggg cgtgcgactg ccgcgcgcgg ccgctctggg cctggttcca gcgcgcgcgc 840 gtgtccagct ccgacgtgac ctgcgccacc cccccggagc gccagggccg agacctgcgc 900 gcgctccgcg aggccgactt ccaggcgtgt ccgcccgcgg cacccacgcg gccgggcagc 960 cgcgcccgcg gcaacagctc ctccaaccac ctgtacgggg tggccgaggc cggggcgccc 1020 ccagccgatc cctccaccct ctaccgagat ctgcctgccg aagactcgcg ggggcgccag 1080 ggcggggacg cgcctactga ggacgactac tgggggggct acgggggtga ggaccagcga 1140 ggggagcaga tgtgccccgg cgctgcctgc caggcgcccc cggactcccg aggccctgcg 1200 ctctcggccg ggctccccag ccctctgctt tgcctcctgc tcctggtgcc ccaccacctc 1260 2 420 PRT Homo sapiens 2 Met Leu Pro Gly Leu Arg Arg Leu Leu Gln Ala Pro Ala Ser Ala Cys 1 5 10 15 Leu Leu Leu Met Leu Leu Ala Leu Pro Leu Ala Ala Pro Ser Cys Pro 20 25 30 Met Leu Cys Thr Cys Tyr Ser Ser Pro Pro Thr Val Ser Cys Gln Ala 35 40 45 Asn Asn Phe Ser Ser Val Pro Leu Ser Leu Pro Pro Ser Thr Gln Arg 50 55 60 Leu Phe Leu Gln Asn Asn Leu Ile Arg Thr Leu Arg Pro Gly Thr Phe 65 70 75 80 Gly Ser Asn Leu Leu Thr Leu Trp Leu Phe Ser Asn Asn Leu Ser Thr 85 90 95 Ile Tyr Pro Gly Thr Phe Arg His Leu Gln Ala Leu Glu Glu Leu Asp 100 105 110 Leu Gly Asp Asn Arg His Leu Arg Ser Leu Glu Pro Asp Thr Phe Gln 115 120 125 Gly Leu Glu Arg Leu Gln Ser Leu His Leu Tyr Arg Cys Gln Leu Ser 130 135 140 Ser Leu Pro Gly Asn Ile Phe Arg Gly Leu Val Ser Leu Gln Tyr Leu 145 150 155 160 Tyr Leu Gln Glu Asn Ser Leu Leu His Leu Gln Asp Asp Leu Phe Ala 165 170 175 Asp Leu Ala Asn Leu Ser His Leu Phe Leu His Gly Asn Arg Leu Arg 180 185 190 Leu Leu Thr Glu His Val Phe Arg Gly Leu Gly Ser Leu Asp Arg Leu 195 200 205 Leu Leu His Gly Asn Arg Leu Gln Gly Val His Arg Ala Ala Phe Arg 210 215 220 Gly Leu Ser Arg Leu Thr Ile Leu Tyr Leu Phe Asn Asn Ser Leu Ala 225 230 235 240 Ser Leu Pro Gly Glu Ala Leu Ala Asp Leu Pro Ser Leu Glu Phe Leu 245 250 255 Arg Leu Asn Ala Asn Pro Trp Ala Cys Asp Cys Arg Ala Arg Pro Leu 260 265 270 Trp Ala Trp Phe Gln Arg Ala Arg Val Ser Ser Ser Asp Val Thr Cys 275 280 285 Ala Thr Pro Pro Glu Arg Gln Gly Arg Asp Leu Arg Ala Leu Arg Glu 290 295 300 Ala Asp Phe Gln Ala Cys Pro Pro Ala Ala Pro Thr Arg Pro Gly Ser 305 310 315 320 Arg Ala Arg Gly Asn Ser Ser Ser Asn His Leu Tyr Gly Val Ala Glu 325 330 335 Ala Gly Ala Pro Pro Ala Asp Pro Ser Thr Leu Tyr Arg Asp Leu Pro 340 345 350 Ala Glu Asp Ser Arg Gly Arg Gln Gly Gly Asp Ala Pro Thr Glu Asp 355 360 365 Asp Tyr Trp Gly Gly Tyr Gly Gly Glu Asp Gln Arg Gly Glu Gln Met 370 375 380 Cys Pro Gly Ala Ala Cys Gln Ala Pro Pro Asp Ser Arg Gly Pro Ala 385 390 395 400 Leu Ser Ala Gly Leu Pro Ser Pro Leu Leu Cys Leu Leu Leu Leu Val 405 410 415 Pro His His Leu 420 3 1383 DNA Mus sp. 3 atgtcttggc agtctggaac cacagtgaca caatctcccg tgcaggctgc tcaggtctca 60 gggtgctgtg tggaattgct gctgttgctg ctcgctggag agctacctct gggtggtggt 120 tgtcctcgag actgtgtgtg ctaccctgcg cccatgactg tcagctgcca ggcacacaac 180 tttgctgcca tcccggaggg catcccagag gacagtgagc gcatcttcct gcagaacaat 240 cgcatcacct tcctccagca gggccacttc agccccgcca tggtcaccct ctggatctac 300 tccaacaaca tcactttcat tgctcccaac accttcgagg gctttgtgca tctggaggag 360 ctagaccttg gagacaaccg acagctgcga acgctggcac ccgagacctt ccaaggcctg 420 gtgaagcttc acgccctcta cctctataag tgtggactga gcgccctgcc cgcaggcatc 480 tttggtggcc tgcacagcct gcagtatctc tacttgcagg acaaccatat cgagtacctc 540 caagatgaca tctttgtgga cctggtcaat ctcagtcact tgtttctcca tggtaacaag 600 ctatggagcc tgggccaagg catcttccgg ggcctggtga acctggaccg gttgctgctg 660 catgagaacc agctacagtg ggttcaccac aaggctttcc atgacctcca caggctaacc 720 accctctttc tcttcaacaa cagcctcact gagctgcagg gtgactgtct ggcccccctg 780 gtggccttgg agttccttcg cctcaatggg aatgcttggg actgtggctg ccgggcacgt 840 tccctgtggg aatggctgcg aaggttccgt ggctctagct ctgctgtccc ctgcgcgacc 900 cccgagctgc ggcaaggcca ggatctgaag ctgctgaggg tggaggactt ccggaactgc 960 acaggaccag tgtctcctca ccagatcaag tctcacacgc ttaccacctc tgacagggct 1020 gcccgcaagg agcaccatcc gtcccatggg gcctccaggg acaaaggcca cccacatggc 1080 catccgcctg gctccaggtc aggttacaag aaggcaggca agaactgcac cagccacagg 1140 aaccggaacc agatctctaa ggtgagctct gggaaagagc ttaccgaact gcaggactat 1200 gcccccgact atcagcacaa gttcagcttt gacatcatgc ccaccgcacg acccaagagg 1260 aagggcaagt gtgctcgcag gacccccatc cgtgccccca gtggggtgca gcaggcatcc 1320 tcaggcacgg cccttggggc cccactcctg gcctggatac tggggctggc agtcactctc 1380 cgc 1383 4 461 PRT Mus sp. 4 Met Ser Trp Gln Ser Gly Thr Thr Val Thr Gln Ser Pro Val Gln Ala 1 5 10 15 Ala Gln Val Ser Gly Cys Cys Val Glu Leu Leu Leu Leu Leu Leu Ala 20 25 30 Gly Glu Leu Pro Leu Gly Gly Gly Cys Pro Arg Asp Cys Val Cys Tyr 35 40 45 Pro Ala Pro Met Thr Val Ser Cys Gln Ala His Asn Phe Ala Ala Ile 50 55 60 Pro Glu Gly Ile Pro Glu Asp Ser Glu Arg Ile Phe Leu Gln Asn Asn 65 70 75 80 Arg Ile Thr Phe Leu Gln Gln Gly His Phe Ser Pro Ala Met Val Thr 85 90 95 Leu Trp Ile Tyr Ser Asn Asn Ile Thr Phe Ile Ala Pro Asn Thr Phe 100 105 110 Glu Gly Phe Val His Leu Glu Glu Leu Asp Leu Gly Asp Asn Arg Gln 115 120 125 Leu Arg Thr Leu Ala Pro Glu Thr Phe Gln Gly Leu Val Lys Leu His 130 135 140 Ala Leu Tyr Leu Tyr Lys Cys Gly Leu Ser Ala Leu Pro Ala Gly Ile 145 150 155 160 Phe Gly Gly Leu His Ser Leu Gln Tyr Leu Tyr Leu Gln Asp Asn His 165 170 175 Ile Glu Tyr Leu Gln Asp Asp Ile Phe Val Asp Leu Val Asn Leu Ser 180 185 190 His Leu Phe Leu His Gly Asn Lys Leu Trp Ser Leu Gly Gln Gly Ile 195 200 205 Phe Arg Gly Leu Val Asn Leu Asp Arg Leu Leu Leu His Glu Asn Gln 210 215 220 Leu Gln Trp Val His His Lys Ala Phe His Asp Leu His Arg Leu Thr 225 230 235 240 Thr Leu Phe Leu Phe Asn Asn Ser Leu Thr Glu Leu Gln Gly Asp Cys 245 250 255 Leu Ala Pro Leu Val Ala Leu Glu Phe Leu Arg Leu Asn Gly Asn Ala 260 265 270 Trp Asp Cys Gly Cys Arg Ala Arg Ser Leu Trp Glu Trp Leu Arg Arg 275 280 285 Phe Arg Gly Ser Ser Ser Ala Val Pro Cys Ala Thr Pro Glu Leu Arg 290 295 300 Gln Gly Gln Asp Leu Lys Leu Leu Arg Val Glu Asp Phe Arg Asn Cys 305 310 315 320 Thr Gly Pro Val Ser Pro His Gln Ile Lys Ser His Thr Leu Thr Thr 325 330 335 Ser Asp Arg Ala Ala Arg Lys Glu His His Pro Ser His Gly Ala Ser 340 345 350 Arg Asp Lys Gly His Pro His Gly His Pro Pro Gly Ser Arg Ser Gly 355 360 365 Tyr Lys Lys Ala Gly Lys Asn Cys Thr Ser His Arg Asn Arg Asn Gln 370 375 380 Ile Ser Lys Val Ser Ser Gly Lys Glu Leu Thr Glu Leu Gln Asp Tyr 385 390 395 400 Ala Pro Asp Tyr Gln His Lys Phe Ser Phe Asp Ile Met Pro Thr Ala 405 410 415 Arg Pro Lys Arg Lys Gly Lys Cys Ala Arg Arg Thr Pro Ile Arg Ala 420 425 430 Pro Ser Gly Val Gln Gln Ala Ser Ser Gly Thr Ala Leu Gly Ala Pro 435 440 445 Leu Leu Ala Trp Ile Leu Gly Leu Ala Val Thr Leu Arg 450 455 460 5 473 PRT Homo sapiens 5 Met Lys Arg Ala Ser Ala Gly Gly Ser Arg Leu Leu Ala Trp Val Leu 1 5 10 15 Trp Leu Gln Ala Trp Gln Val Ala Ala Pro Cys Pro Gly Ala Cys Val 20 25 30 Cys Tyr Asn Glu Pro Lys Val Thr Thr Ser Cys Pro Gln Gln Gly Leu 35 40 45 Gln Ala Val Pro Val Gly Ile Pro Ala Ala Ser Gln Arg Ile Phe Leu 50 55 60 His Gly Asn Arg Ile Ser His Val Pro Ala Ala Ser Phe Arg Ala Cys 65 70 75 80 Arg Asn Leu Thr Ile Leu Trp Leu His Ser Asn Val Leu Ala Arg Ile 85 90 95 Asp Ala Ala Ala Phe Thr Gly Leu Ala Leu Leu Glu Gln Leu Asp Leu 100 105 110 Ser Asp Asn Ala Gln Leu Arg Ser Val Asp Pro Ala Thr Phe His Gly 115 120 125 Leu Gly Arg Leu His Thr Leu His Leu Asp Arg Cys Gly Leu Gln Glu 130 135 140 Leu Gly Pro Gly Leu Phe Arg Gly Leu Ala Ala Leu Gln Tyr Leu Tyr 145 150 155 160 Leu Gln Asp Asn Ala Leu Gln Ala Leu Pro Asp Asp Thr Phe Arg Asp 165 170 175 Leu Gly Asn Leu Thr His Leu Phe Leu His Gly Asn Arg Ile Ser Ser 180 185 190 Val Pro Glu Arg Ala Phe Arg Gly Leu His Ser Leu Asp Arg Leu Leu 195 200 205 Leu His Gln Asn Arg Val Ala His Val His Pro His Ala Phe Arg Asp 210 215 220 Leu Gly Arg Leu Met Thr Leu Tyr Leu Phe Ala Asn Asn Leu Ser Ala 225 230 235 240 Leu Pro Thr Glu Ala Leu Ala Pro Leu Arg Ala Leu Gln Tyr Leu Arg 245 250 255 Leu Asn Asp Asn Pro Trp Val Cys Asp Cys Arg Ala Arg Pro Leu Trp 260 265 270 Ala Trp Leu Gln Lys Phe Arg Gly Ser Ser Ser Glu Val Pro Cys Ser 275 280 285 Leu Pro Gln Arg Leu Ala Gly Arg Asp Leu Lys Arg Leu Ala Ala Asn 290 295 300 Asp Leu Gln Gly Cys Ala Val Ala Thr Gly Pro Tyr His Pro Ile Trp 305 310 315 320 Thr Gly Arg Ala Thr Asp Glu Glu Pro Leu Gly Leu Pro Lys Cys Cys 325 330 335 Gln Pro Asp Ala Ala Asp Lys Ala Ser Val Leu Glu Pro Gly Arg Pro 340 345 350 Ala Ser Ala Gly Asn Ala Leu Lys Gly Arg Val Pro Pro Gly Asp Ser 355 360 365 Pro Pro Gly Asn Gly Ser Gly Pro Arg His Ile Asn Asp Ser Pro Phe 370 375 380 Gly Thr Leu Pro Gly Ser Ala Glu Pro Pro Leu Thr Ala Val Arg Pro 385 390 395 400 Glu Gly Ser Glu Pro Pro Gly Phe Pro Thr Ser Gly Pro Arg Arg Arg 405 410 415 Pro Gly Cys Ser Arg Lys Asn Arg Thr Arg Ser His Cys Arg Leu Gly 420 425 430 Gln Ala Gly Ser Gly Gly Gly Gly Thr Gly Asp Ser Glu Gly Ser Gly 435 440 445 Ala Leu Pro Ser Leu Thr Cys Ser Leu Thr Pro Leu Gly Leu Ala Leu 450 455 460 Val Leu Trp Thr Val Leu Gly Pro Cys 465 470 6 440 PRT Artificial Sequence Description of Artificial Sequence Consensus sequence 6 Cys Pro Xaa Xaa Cys Xaa Cys Tyr Xaa Xaa Pro Xaa Xaa Thr Xaa Ser 1 5 10 15 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Pro Xaa Xaa 20 25 30 Xaa Xaa Arg Xaa Phe Leu Xaa Xaa Asn Xaa Ile Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Trp Xaa Xaa Ser 50 55 60 Asn Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Leu Glu Xaa Leu Asp Leu Xaa Asp Asn Xaa Xaa Leu Arg Xaa Xaa Xaa 85 90 95 Pro Xaa Thr Phe Xaa Gly Leu Xaa Xaa Leu Xaa Leu Xaa Leu Xaa Xaa 100 105 110 Cys Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Phe Xaa Gly Leu Xaa Xaa 115 120 125 Leu Gln Tyr Leu Tyr Leu Gln Xaa Asn Xaa Xaa Xaa Xaa Leu Xaa Asp 130 135 140 Asp Xaa Phe Xaa Asp Leu Xaa Asn Leu Xaa His Leu Phe Leu His Gly 145 150 155 160 Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Arg Gly Leu Xaa Xaa 165 170 175 Leu Asp Arg Leu Leu Leu His Xaa Asn Xaa Xaa Xaa Xaa Val His Xaa 180 185 190 Xaa Ala Phe Xaa Xaa Leu Xaa Arg Leu Xaa Xaa Leu Xaa Leu Phe Xaa 195 200 205 Asn Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Leu Ala Xaa Leu Xaa Xaa 210 215 220 Leu Xaa Xaa Leu Arg Leu Asn Xaa Asn Xaa Trp Xaa Cys Xaa Cys Arg 225 230 235 240 Ala Arg Xaa Leu Trp Xaa Trp Xaa Xaa Xaa Xaa Arg Xaa Ser Ser Ser 245 250 255 Xaa Val Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Gly Xaa Asp Leu Xaa 260 265 270 Xaa Leu Xaa Xaa Xaa Asp Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Pro 275 280 285 Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335 Xaa Xaa Xaa Xaa Xaa Pro Pro Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 370 375 380 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg 385 390 395 400 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 405 410 415 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa 420 425 430 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu 435 440 7 66 PRT Homo sapiens 7 Arg Ile Tyr Lys Gly Val Ile Gln Ala Ile Gln Lys Ser Asp Glu Gly 1 5 10 15 His Pro Phe Arg Ala Tyr Leu Glu Ser Glu Val Ala Ile Ser Glu Glu 20 25 30 Leu Val Gln Lys Tyr Ser Asn Ser Ala Leu Gly His Val Asn Cys Thr 35 40 45 Ile Lys Glu Leu Arg Arg Leu Phe Leu Val Asp Asp Leu Val Asp Ser 50 55 60 Leu Lys 65 8 390 PRT Homo sapeins 8 Cys Pro Met Leu Cys Thr Cys Tyr Ser Ser Pro Pro Thr Val Ser Cys 1 5 10 15 Gln Ala Asn Asn Phe Ser Ser Val Pro Leu Ser Leu Pro Pro Ser Thr 20 25 30 Gln Arg Leu Phe Leu Gln Asn Asn Leu Ile Arg Thr Leu Arg Pro Gly 35 40 45 Thr Phe Gly Ser Asn Leu Leu Thr Leu Trp Leu Phe Ser Asn Asn Leu 50 55 60 Ser Thr Ile Tyr Pro Gly Thr Phe Arg His Leu Gln Ala Leu Glu Glu 65 70 75 80 Leu Asp Leu Gly Asp Asn Arg His Leu Arg Ser Leu Glu Pro Asp Thr 85 90 95 Phe Gln Gly Leu Glu Arg Leu Gln Ser Leu His Leu Tyr Arg Cys Gln 100 105 110 Leu Ser Ser Leu Pro Gly Asn Ile Phe Arg Gly Leu Val Ser Leu Gln 115 120 125 Tyr Leu Tyr Leu Gln Glu Asn Ser Leu Leu His Leu Gln Asp Asp Leu 130 135 140 Phe Ala Asp Leu Ala Asn Leu Ser His Leu Phe Leu His Gly Asn Arg 145 150 155 160 Leu Arg Leu Leu Thr Glu His Val Phe Arg Gly Leu Gly Ser Leu Asp 165 170 175 Arg Leu Leu Leu His Gly Asn Arg Leu Gln Gly Val His Arg Ala Ala 180 185 190 Phe Arg Gly Leu Ser Arg Leu Thr Ile Leu Tyr Leu Phe Asn Asn Ser 195 200 205 Leu Ala Ser Leu Pro Gly Glu Ala Leu Ala Asp Leu Pro Ser Leu Glu 210 215 220 Phe Leu Arg Leu Asn Ala Asn Pro Trp Ala Cys Asp Cys Arg Ala Arg 225 230 235 240 Pro Leu Trp Ala Trp Phe Gln Arg Ala Arg Val Ser Ser Ser Asp Val 245 250 255 Thr Cys Ala Thr Pro Pro Glu Arg Gln Gly Arg Asp Leu Arg Ala Leu 260 265 270 Arg Glu Ala Asp Phe Gln Ala Cys Pro Pro Ala Ala Pro Thr Arg Pro 275 280 285 Gly Ser Arg Ala Arg Gly Asn Ser Ser Ser Asn His Leu Tyr Gly Val 290 295 300 Ala Glu Ala Gly Ala Pro Pro Ala Asp Pro Ser Thr Leu Tyr Arg Asp 305 310 315 320 Leu Pro Ala Glu Asp Ser Arg Gly Arg Gln Gly Gly Asp Ala Pro Thr 325 330 335 Glu Asp Asp Tyr Trp Gly Gly Tyr Gly Gly Glu Asp Gln Arg Gly Glu 340 345 350 Gln Met Cys Pro Gly Ala Ala Cys Gln Ala Pro Pro Asp Ser Arg Gly 355 360 365 Pro Ala Leu Ser Ala Gly Leu Pro Ser Pro Leu Leu Cys Leu Leu Leu 370 375 380 Leu Val Pro His His Leu 385 390 9 421 PRT Mus sp. 9 Cys Pro Arg Asp Cys Val Cys Tyr Pro Ala Pro Met Thr Val Ser Cys 1 5 10 15 Gln Ala His Asn Phe Ala Ala Ile Pro Glu Gly Ile Pro Glu Asp Ser 20 25 30 Glu Arg Ile Phe Leu Gln Asn Asn Arg Ile Thr Phe Leu Gln Gln Gly 35 40 45 His Phe Ser Pro Ala Met Val Thr Leu Trp Ile Tyr Ser Asn Asn Ile 50 55 60 Thr Phe Ile Ala Pro Asn Thr Phe Glu Gly Phe Val His Leu Glu Glu 65 70 75 80 Leu Asp Leu Gly Asp Asn Arg Gln Leu Arg Thr Leu Ala Pro Glu Thr 85 90 95 Phe Gln Gly Leu Val Lys Leu His Ala Leu Tyr Leu Tyr Lys Cys Gly 100 105 110 Leu Ser Ala Leu Pro Ala Gly Ile Phe Gly Gly Leu His Ser Leu Gln 115 120 125 Tyr Leu Tyr Leu Gln Asp Asn His Ile Glu Tyr Leu Gln Asp Asp Ile 130 135 140 Phe Val Asp Leu Val Asn Leu Ser His Leu Phe Leu His Gly Asn Lys 145 150 155 160 Leu Trp Ser Leu Gly Gln Gly Ile Phe Arg Gly Leu Val Asn Leu Asp 165 170 175 Arg Leu Leu Leu His Glu Asn Gln Leu Gln Trp Val His His Lys Ala 180 185 190 Phe His Asp Leu His Arg Leu Thr Thr Leu Phe Leu Phe Asn Asn Ser 195 200 205 Leu Thr Glu Leu Gln Gly Asp Cys Leu Ala Pro Leu Val Ala Leu Glu 210 215 220 Phe Leu Arg Leu Asn Gly Asn Ala Trp Asp Cys Gly Cys Arg Ala Arg 225 230 235 240 Ser Leu Trp Glu Trp Leu Arg Arg Phe Arg Gly Ser Ser Ser Ala Val 245 250 255 Pro Cys Ala Thr Pro Glu Leu Arg Gln Gly Gln Asp Leu Lys Leu Leu 260 265 270 Arg Val Glu Asp Phe Arg Asn Cys Thr Gly Pro Val Ser Pro His Gln 275 280 285 Ile Lys Ser His Thr Leu Thr Thr Ser Asp Arg Ala Ala Arg Lys Glu 290 295 300 His His Pro Ser His Gly Ala Ser Arg Asp Lys Gly His Pro His Gly 305 310 315 320 His Pro Pro Gly Ser Arg Ser Gly Tyr Lys Lys Ala Gly Lys Asn Cys 325 330 335 Thr Ser His Arg Asn Arg Asn Gln Ile Ser Lys Val Ser Ser Gly Lys 340 345 350 Glu Leu Thr Glu Leu Gln Asp Tyr Ala Pro Asp Tyr Gln His Lys Phe 355 360 365 Ser Phe Asp Ile Met Pro Thr Ala Arg Pro Lys Arg Lys Gly Lys Cys 370 375 380 Ala Arg Arg Thr Pro Ile Arg Ala Pro Ser Gly Val Gln Gln Ala Ser 385 390 395 400 Ser Gly Thr Ala Leu Gly Ala Pro Leu Leu Ala Trp Ile Leu Gly Leu 405 410 415 Ala Val Thr Leu Arg 420 10 17 PRT Artificial Sequence Description of Artificial Sequence Consensus sequence 10 Cys Pro Xaa Xaa Cys Xaa Cys Tyr Xaa Xaa Pro Xaa Xaa Thr Xaa Ser 1 5 10 15 Cys 11 50 PRT Artificial Sequence Description of Artificial Sequence Consensus sequence 11 Asn Xaa Trp Xaa Cys Xaa Cys Arg Ala Arg Xaa Leu Trp Xaa Trp Xaa 1 5 10 15 Xaa Xaa Xaa Arg Xaa Ser Ser Ser Xaa Val Xaa Cys Xaa Xaa Pro Xaa 20 25 30 Xaa Xaa Xaa Gly Xaa Asp Leu Xaa Xaa Leu Xaa Xaa Xaa Asp Xaa Xaa 35 40 45 Xaa Cys 50 12 196 PRT Artificial Sequence Description of Artificial Sequence Consensus sequence 12 Arg Xaa Phe Leu Xaa Xaa Asn Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Trp Xaa Xaa Ser Asn Xaa 20 25 30 Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Leu Glu 35 40 45 Xaa Leu Asp Leu Xaa Asp Asn Xaa Xaa Leu Arg Xaa Xaa Xaa Pro Xaa 50 55 60 Thr Phe Xaa Gly Leu Xaa Xaa Leu Xaa Leu Xaa Leu Xaa Xaa Cys Xaa 65 70 75 80 Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Phe Xaa Gly Leu Xaa Xaa Leu Gln 85 90 95 Tyr Leu Tyr Leu Gln Xaa Asn Xaa Xaa Xaa Xaa Leu Xaa Asp Asp Xaa 100 105 110 Phe Xaa Asp Leu Xaa Asn Leu Xaa His Leu Phe Leu His Gly Asn Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Arg Gly Leu Xaa Xaa Leu Asp 130 135 140 Arg Leu Leu Leu His Xaa Asn Xaa Xaa Xaa Xaa Val His Xaa Xaa Ala 145 150 155 160 Phe Xaa Xaa Leu Xaa Arg Leu Xaa Xaa Leu Xaa Leu Phe Xaa Asn Xaa 165 170 175 Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Leu Ala Xaa Leu Xaa Xaa Leu Xaa 180 185 190 Xaa Leu Arg Leu 195 13 1176 DNA Homo sapiens 13 gagggcatcc ccgtggacag cgagcgcgtc ttcctgcaga acaaccgcat cggcctcctc 60 cagcccggcc acttcagccc cgccatggtc accctgtgga tctactcgaa caacatcacc 120 tacatccacc ccagcacctt cgagggcttc gtgcacctgg aggagctgga cctcggcgac 180 aaccggcagc tgcggacgct ggcacccgag accttccagg gcctggtgaa gcttcacgcc 240 ctctacctct acaagtgtgg gctcagcgcc ttgccggccg gcgtctttgg cggcctgcac 300 agcctgcagt acctctacct gcaggacaac cacatcgagt acctccagga cgacatcttc 360 gtggacctgg tcaacctcag ccacctgttt ctccacggca acaagctgtg gagtctgggc 420 ccgggcacct tccggggcct ggtgaacctg gaccgtcttt tgctgcacga gaaccagctg 480 cagtgggtcc accacaaggc attccacgac ctccgcaggc tgaccaccct cttcctcttc 540 aacaacagcc tctcggagct gcagggtgag tgcctggccc cgctgggggc cctggagttc 600 ctccgcctca acggcaaccc ctgggactgt ggttgtcgcg cgcgctccct gtgggaatgg 660 ctgcagaggt tccggggctc cagctccgct gtcccctgtg tgtcccctgg gctgcggcac 720 ggccaggacc tgaagctgct gagggccgag gacttccgga actgcacggg accagcgtcc 780 ccgcaccaga tcaagtcaca cacgctcacc accaccgaca gggccgcccg caaggaacac 840 cactcacccc acggccccac caggagcaag ggccacccgc acggcccccg gcccggccac 900 aggaagccgg ggaagaactg caccaacccc aggaaccgca atcagatctc taaggcgggc 960 gccgggaaac aggcccccga gctgccagac tatgccccag actaccagca caagttcagt 1020 tttgacatca tgcctacggc ccggcccaag aggaagggca agtgtgcccg caggaccccc 1080 atccgtgccc ccagcggggt gcagcaggcc tcctcggcca gttccctggg ggcctccctc 1140 ctggcctgga cactggggct ggcggtcact ctccgc 1176 14 392 PRT Homo sapiens 14 Glu Gly Ile Pro Val Asp Ser Glu Arg Val Phe Leu Gln Asn Asn Arg 1 5 10 15 Ile Gly Leu Leu Gln Pro Gly His Phe Ser Pro Ala Met Val Thr Leu 20 25 30 Trp Ile Tyr Ser Asn Asn Ile Thr Tyr Ile His Pro Ser Thr Phe Glu 35 40 45 Gly Phe Val His Leu Glu Glu Leu Asp Leu Gly Asp Asn Arg Gln Leu 50 55 60 Arg Thr Leu Ala Pro Glu Thr Phe Gln Gly Leu Val Lys Leu His Ala 65 70 75 80 Leu Tyr Leu Tyr Lys Cys Gly Leu Ser Ala Leu Pro Ala Gly Val Phe 85 90 95 Gly Gly Leu His Ser Leu Gln Tyr Leu Tyr Leu Gln Asp Asn His Ile 100 105 110 Glu Tyr Leu Gln Asp Asp Ile Phe Val Asp Leu Val Asn Leu Ser His 115 120 125 Leu Phe Leu His Gly Asn Lys Leu Trp Ser Leu Gly Pro Gly Thr Phe 130 135 140 Arg Gly Leu Val Asn Leu Asp Arg Leu Leu Leu His Glu Asn Gln Leu 145 150 155 160 Gln Trp Val His His Lys Ala Phe His Asp Leu Arg Arg Leu Thr Thr 165 170 175 Leu Phe Leu Phe Asn Asn Ser Leu Ser Glu Leu Gln Gly Glu Cys Leu 180 185 190 Ala Pro Leu Gly Ala Leu Glu Phe Leu Arg Leu Asn Gly Asn Pro Trp 195 200 205 Asp Cys Gly Cys Arg Ala Arg Ser Leu Trp Glu Trp Leu Gln Arg Phe 210 215 220 Arg Gly Ser Ser Ser Ala Val Pro Cys Val Ser Pro Gly Leu Arg His 225 230 235 240 Gly Gln Asp Leu Lys Leu Leu Arg Ala Glu Asp Phe Arg Asn Cys Thr 245 250 255 Gly Pro Ala Ser Pro His Gln Ile Lys Ser His Thr Leu Thr Thr Thr 260 265 270 Asp Arg Ala Ala Arg Lys Glu His His Ser Pro His Gly Pro Thr Arg 275 280 285 Ser Lys Gly His Pro His Gly Pro Arg Pro Gly His Arg Lys Pro Gly 290 295 300 Lys Asn Cys Thr Asn Pro Arg Asn Arg Asn Gln Ile Ser Lys Ala Gly 305 310 315 320 Ala Gly Lys Gln Ala Pro Glu Leu Pro Asp Tyr Ala Pro Asp Tyr Gln 325 330 335 His Lys Phe Ser Phe Asp Ile Met Pro Thr Ala Arg Pro Lys Arg Lys 340 345 350 Gly Lys Cys Ala Arg Arg Thr Pro Ile Arg Ala Pro Ser Gly Val Gln 355 360 365 Gln Ala Ser Ser Ala Ser Ser Leu Gly Ala Ser Leu Leu Ala Trp Thr 370 375 380 Leu Gly Leu Ala Val Thr Leu Arg 385 390 15 143899 DNA Homo sapiens modified_base (2044)..(2144) a, t, c, g, other or unknown 15 aagcacatac aggtgacatt acagaactga cagttatgcc aggcactgta cttagcccct 60 ataccatcct caaacagctg tatgatgtag attgggtatt aaccccatta ataacaaaag 120 tacagggaac aaagtgactt tccaaaggtc atgccattca aaggagggtg aatcttaggt 180 tggacgcagg ctgtctgact ctggagtctg aggtgttaat gctgcctcct ccatgggaac 240 agcccaagtg aaaaacagct gatccactct tcatttactt ggcatctgtg ctaagctggt 300 ccctgagcca agctctgagc aacagaaaca gaagctctgc attaggagct tgtgagcatg 360 tcaatgccgg gtaaaggagt gctggaaacc gctgggatgg ccgccgagca ctaggccgtt 420 gaaggtgggc tctgtgtgac tggttcctct acactctggc ctggctgcct gcaggaagaa 480 gatcaagctg agtgggctgg ccctggacca caaggtgaca ggtgacctct tctacaccca 540 tgtgaccacc atgggccaga ggctcagcca gaaggccccc agcctggagg acggttcgga 600 tgccttcatg tcaccccagg atgttcgggg cacctcagaa aaccttcctg agagtgagtg 660 tctggtcaag gtgccggcct tgggggatag tgatggtggg tcctcatatt cagtgagcac 720 tcatggttga gtatttattc gcacccctct tcagtcctta caacacccca tgatgtaggt 780 ggggcatgct cctcatttac agatgggcac atcaaagctc agctaacgct gggaagttca 840 gattcagggt taccctgctg gattcctggg attggggagg gaggagcttc caaaatgggg 900 acaaggtctc tgggcctgtc gggtagctgg tttcctcagg gccccttgca acctctgagc 960 ttattgcatc aggtgcagcc aggcccgtga gcctcctggc aggggtcctc cacacctggc 1020 tgtcttttgc cccctgctgg tcacaggagg agctgcagca cctgcctggg ctgcttctca 1080 ggagggtaca tgaagatccc aggaccgcca gctccatgat aagtggaagg agctccttgg 1140 agtcaggagc gggagttgag gagtttgagt cctgctctcc agttataggc tatgtgactt 1200 gtgtagatca cctaaccttg ctcttgattt ccttacctct taaactagca ctaaaagcac 1260 cccacaaact gtaaagttag ttgtgatgat tgaatgacac catgggtgtg gaagctcttt 1320 gtaaagtgca aaacggtgtg cagtttgagg gtggttaccc ccagtgccga ttctcagagg 1380 gcaacatggc taagggcacg agctggagtt aggctgacct gctgcttcca gccctgtgag 1440 cttgagcaag tcatttaact tcctgagctg cagtttcctc atcagtaaaa tgtgataagg 1500 atagggttgt tgtaagattt tattaaatgg ggtaataaat gtcaagtatg tagcccatag 1560 tgagtgcttc agagtttttt tcttttgttt ctttcccccc cgccccgaga tggagcctta 1620 ctctgttgcc caggctggag tgcagtggca tgatcttggc tcactgcaac ctccgcctcc 1680 cgggttcaag caattctcct gcctcagcct cccaaatagc tgggactaca ggcgtgcacc 1740 accatgctcg gctaattttt gtatctttag tagagacggg gtttcaccat gttggccagg 1800 ctggtctcga actcctgacc tcatgatgct cctgcctcag cccccgaaag ttttgggatt 1860 acaagtgtga gcccccgtgc cctgccaggt tttttttttt tttttttttt tgtaaaacac 1920 ccacagggta ttgctgttgc ctgggctgga gtgcggtagt gcaatcatag ttcactgcag 1980 ccttgacctc ctgggctcaa gtgatcctcc tgcctcagcc tcctgagtag ctgggaatac 2040 aggnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2100 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnttttgta ttttaagtag 2160 agacagggtt ttcccaatgt tggccaaggc tggtctaaaa ctcccaacct caggtgatcc 2220 acccacctca gcctcccaaa gtactgggat tacaggcgtg agacaccgtg cccagccagg 2280 aggcttattt tcttgataaa ttacccagtc tcaggtattt ctctacagcg atgcaagaac 2340 agcctaatac atccaggctc agcatcagtg gacccaggtg ggagagctta agatgtcaag 2400 gtctgaatgc cgcttccaca cacctttggg acctagggac tccctctctt tttctttttt 2460 cagtagaaga tgttatcttc tcctttctct gaccagtagt tggtgatggt ttcagagata 2520 gtttttcagt caagatatat ttcagtggct tcactgagcc caagttccct cgcctctcta 2580 ggactttatt tccttgtttc tagaagaggg ataacacata ttttctaagg tggttgtgag 2640 attaagggag ctggtaccgg gtggtgcata aggacaggat agagcaatgg tgagaccact 2700 caaaaagcga aaagttgacc tgcgagggtg acacttatca aatcagcaca cagtgggagt 2760 ggaaggaatg tccctcatca gttacaatat ttggagagtg caagttatag aaaacccagc 2820 cctggccggg cgcggtgggt catgcctata atcccagcac tttgggaggc tgaggcaggt 2880 ggatcacgag gtcaggagtt caagaccagc ctgaccaacg tggtgaaacc ccacctctac 2940 taaaaataca aaattagctg ggcgtggtgg tgtgtgcctg taatctgagc tactcaggag 3000 gctgaggcac gagaatcact tgaaaccggg aggtggagtt tgcagtgagc cgagatcgca 3060 ccactgcact ccagcctggg caacagagcg agactccatc tcaaacgaaa aaaaaaaaag 3120 aaagaaaacc cagctctaac tggcttaaac agtaagaaga tctattatat tatccatctc 3180 aggcagcagc aagcccagag gtaggggact ccaaggttgg ttgatccagg gcttaacgat 3240 gtcatcaaag acccaggttc tttctgtctc ggcacctctg tctgcagggc cagcttcatc 3300 ctaagccaga ttgttcttgt cttgattaca agttggctgc tgggccagca gacgctgcct 3360 gcctccctgt tcatcttcag aagtagaaag tggcccttcc ccagtcatgg aatgaaagag 3420 tttcctttct gtctgggatt gcttaggtcc acccacctga agccaatgac tgtcaccagg 3480 aaggtaatat acactgattg tcttaagtca gggttcctga gccagtcttg ggcaaggagt 3540 gtgatactgt catgattgtc ttgggctcat cagggcagct ctgcagatga gatcaaactc 3600 caagctacat tattctgaac agtgggaagt aggaaagaga cattttggga gatacaaaac 3660 acaatgtcta tcccatatcc ctaggtccag gtcacagtgt cttggttgga catcaaatgt 3720 agaaaaagaa agactgtcca tccatttatc tacctattca tctggttttt gatttttttt 3780 aaattttatt ttaagacatt ctcactctgt cacccagact ggagtgcagt ggtttgatca 3840 tggctcatgg cagcctcaac ctcccaggct caagtgaccc tcccatgctc aagtgatcct 3900 cctacctcag cctcccaagt agctagaact aaaggtgcat gccaccacgc tcagttaatt 3960 tttgcatttt ttgtagagat ggggtttcgt catgatgccc atgctagtct ggaattcctg 4020 aactcaagca atatgcctgc ctttgcctcc caaaatgctg ggattgtagg catgagccac 4080 tgctcctggc tcatctgttt aataatttat gaaacaacta ctgggtgctg agcacggggc 4140 caggggctgg agatctagca gggaccaggc agatctctgc caagtcgttg gtttcttaaa 4200 ggttttgctc ataattcccc ttttcttttc tctttcgttt tttttctttt ctttctttct 4260 ttctttcttt tttttttttt gagacagagt ctcactctgt tacccaggct ggagtgcagt 4320 ggtgcgatct cagctcactg caacctctgc ctcctgggtt caagcgattc tcctgcctca 4380 gcctcccgag tagctgggac tacaggcgcc tgccaccatg cccggctaat ttttgtgttt 4440 ttagtagaga ctgggtttca ccatattggc caggctggtc ttgaactcct gaccttgtga 4500 tccgcccgct tcggcctccc acagtgctgg gattacaggc gtgagccacg gcgcccagcc 4560 agtttccctt ttcaatgagg cctccctgac ctccatactc tactcctcca cctggcccac 4620 tcagctctac tttttcttcc ccatagcact caagacctcc taacatacta cgtaagttat 4680 ttatttacta ggcttactgt gtattgtctg tcttcctcta ctagaatgta aactccatga 4740 gaatagaaat ttttgccttt ttatttagtg tggtgtctgc agcccctggc ttagtccctg 4800 gcatacaaca gtcactccac ccacagttgc tgaataagtg actaaaggtc cctgccctca 4860 tattgttatg agggagtgtg catgttgtta gagaaaaatc tgaggcacaa taaaatttta 4920 tagagtttaa gttttctttt ttaagcaatc cacgaattgg ggtagtttca gaggtagttt 4980 ttcagtcatg acgtatttca atggcttcac tgagcccaag ttctttcacc tctctaggac 5040 tttatttcct tatttctaga acggggataa cacatagttc ataaggcagt tatgagagta 5100 agggagctgg tatggggtga tgcataagga caggatagag cagtggtgag accgctcaga 5160 tgacaaagcg tcagagacca gtatttacga cggaaatgtg gaagcatgat aaagaaatta 5220 tttgggctgg gcacaatgac tcacaactaa taaaactttg ggaggccaag gtgggaggat 5280 cacttgactt gcagaaggtc aaggctgcag tgagctgtga ttttgccact gcactccagc 5340 ctggtcaaca gagtgagacc ctggctcgaa acgttatttg attggttaca gttatacagt 5400 tgccttattt ggtctattcc atttgaaagt tcctagttct ataattttaa gtttgttggc 5460 tgtttctgat tggttaagct taagttttgt tttcctttaa tacagttaag tgccccataa 5520 tgacattttg gtcaaggaca gaccacatat acagtggtgg tcccataaga ttataatgga 5580 gctgaaacat tcctattgtc tatggcgtag tggtcctgat gttgtagcgc aatgcattag 5640 ttatatgttt gtggcaatgc tggtgtaaac acacctactg cactgccagt gatataaaag 5700 aatagcacat acagttatat atagtacata atatctgata atgataatac ataactatat 5760 tactggttta tatatttact atattattta tctttatttt atttttgaga cagagtctca 5820 ttctgtcacc caggctggag tgcagtggcg cgatcttggc tcaccgcaac ctccgcttcc 5880 tgggttcaag tgattctcct gcctcagtct cctgagtagc tgggattaca ggtgtgcacc 5940 atgacaccct gctaatatgt tttgtatttt tagtagagat ggggtttcac catgttggcc 6000 aggctggtct tgaactactg acctcaagtg atcaccccgc ctcggcttcc caaagtgctg 6060 ggattacagg cgtgagccac cacgcatggc ctatttataa ttattttaga gtgtacgcct 6120 tatacttata aaaaaaagct aactgtcaaa cagcctcggg caggtccttc aacagatatt 6180 ccagaagaca ttgttatcat aggagatgac agctccgtgc atattattgt ccctgaaaac 6240 cttctagtgt ggaagtggaa gacagtgata ttgatgatag gacccagtgt aggcctaggc 6300 taatgtgtgt gtttgtgtct ttgcttttaa caagaaagtt taaaaagtta aaataaaata 6360 caaaaatttt taaatagaaa aaagctgccc aggaacaatg gctcacacct gtaatcccac 6420 cattcgggga ggccaaggtg ggtggattgc ttgagctcag gagttcaaga ccagcctggg 6480 caacatggtg aaaccccatc tctacaaaaa atacaaaaat tagccgggtg tggtggcatg 6540 cggctatagt tccagctaat cgaggggctg aggtgggagg atcactgggg gggaggtggt 6600 tgaggctgna gtgagctgtg attgnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 6720 nnnnatattc ttaaaaaaat ttttttttat ttttgagaca gaatttctct cttgttgccc 6780 aggctggagt gcaatggcgc tatctcagct cagggcaacc tccacctcct gggttcaagc 6840 gattctcctg ccttagcctc ccaggtacag gcgcccgcca ccatgctcgg ctaatttttg 6900 tatttttagt agagatgggg tttcaccatg ttgtccaggc tggtcttgaa atcctgcctc 6960 aggtgatcca cccccctcgg cctcccaaag tgctggaatt tacaggcgtg agccactgtg 7020 cctggcctcc tttacatttt tttaaattta attttaattt tttaattttt aatttctcat 7080 atatatatat ttttaagact agccaagtga agcagtggga gtggaaaagg aactggtttt 7140 gatcaatagg tgtaaacacc actgcactgg gaccagccta ttttacattc ctgttagcag 7200 tgatgagggt tcactttctt tgtagcctca acaatatgtg tcgttgccca tctttttttt 7260 tttttttttt tttttttttg agatggagtc tcactctgtt gcctaggctg gaatgcaatg 7320 gcatgatctc agctcactgc aacctccgcc tcccaggttc aagtgattct tgtgtctcag 7380 cctcctgagt agatgggatt acaggcgtcc accaccacgc ccggctaatt ttttgtattt 7440 tcagtagaga tggggtttca ccatgttggc caggttggtt tcgaactcct gacctcaagt 7500 gatccgccca cctcggcctc ccaaagtgct gggattacag gcatgagcca ccgcgcccgg 7560 cctgcccatc ttttttttgt tatagccatc ctagtggatg taaagttttt ttgtgatttt 7620 gatttgtgtt tccctactga tcaatgatgt tgagcatctt ttcctgtgct tattggcttt 7680 tggtatatct ttggagaaag gtctattcag gtcctttgcc cactttaaaa ttaggttatc 7740 tttctattac tgagatgtaa gagttcttta tgttctagat ataagtctcc tacatatgat 7800 ttgtaaaaat tttccttcca ttattgggtt gtctttcact ttcttttggt gtcctttagt 7860 gcacaacagt ttttaatatt gaagtccaat tttctatttt tctcttttgc cacttgtatc 7920 ttggtgtcat gtttaaggaa ctattgccta atctcaggtc acaaagattt acacctgtgt 7980 ttccttcttt ccttccttcc ttccttcctt ccttctttcc ctccctccct ctctccctcc 8040 ctccctctct ccctccctcc ctccttccct tcctccctcc ctccctcctt ccttccttcc 8100 ttccttcctt ccttccttcc ttccttcctt ccttcctttg tccttctgac ggaatcttgc 8160 tctgtcaccc aggctggagt gtagtggcac gatcttggct cactgcaacc tctgcctcct 8220 gggttcaagc aattctcctg cctcagcctc ctgagtagct gggactacag gcacacacca 8280 ccatgcccag ctaatttttg tatttttagt agagacgggg tttcaccaca ttggccagga 8340 tggtttcgat ctcctgacct cgtgatccac ccgccttggc ctcccaaagt gctgggattg 8400 caggtgtgag ccaccatgcc cggcctgtgt tttcttagag ttttgtagtt ttagctctta 8460 tagttagatc cttgatccat tttgagttga ttttgtatat agtgtgagat atccacctgg 8520 tgttgtaaat tgcccagaag tgggtatgct tctaaatctg gctgttaggg attactagag 8580 gtgaccaaag tgaatttttt ctttgtttct tttttttttt ggagacagag tctccgtcac 8640 ccaggctgga gtgcaatggc ttcatcttgg ctcagtgcaa cctctgcctt ctggtttcaa 8700 gcagttctcc tgcctcagac tcctgagtag ctggtattac aggcgtgtac caccatgctt 8760 ggctaatttt tgtattttta gtaaagatgc agtttcacct gttggccagg cttttctgga 8820 actcccggcc tcaagtgatc catctgcctc tacctcccaa agtgctggga ttacaggtgg 8880 gagccaccgt gcccagtcct tttctcagaa tttatttgtt tttttttgtt ttgtttcatt 8940 tttgagatag ggtctcactc tgtcagctag gcaggagttc agtggtgtga tcattgctgc 9000 agccttgaac ttctggactc acgtgatctt cccacctcag cctcctgagt agctaggatt 9060 acaggcatgt gcttccacac ctggctaatt ttttaatttt ctaggactta tttgtccatt 9120 cttgcaaagc agggtacaac atgcctatct ctacctacct ctcttccctt caagggactc 9180 cagccaaaat ccttgaggct ctcgggctga ctgtgggtgc tgttgcctga tctgcctcag 9240 tcatgctgca tgatcaaaag tgtccgtttt ctgcttcttg gaactttatt cactttgggt 9300 gtcagtcttc ctctgcagtg tcccaagaac acagaattag accaggaatc tgtgttgcca 9360 tagtgtgtgg aaagaggcag acttccaact ccgctatgtg ctgttgggtg attgaagctt 9420 aattttcttt ctatctttct ttcttttctt ttcttttttt ttttttggag atggaatctc 9480 gctctgttgc ccaggctgga gtgcagtggt gcgatctcac ctcactgcaa cctccgcctc 9540 ccaggttcaa gcgattctcc tgcctcagcc tcctgagtag ctgggattac aggtgcatgc 9600 caccatgccc ggctaatttg tgtaatttta gtagaaacag tgtttcacca tattggtcag 9660 gctggtctcg acctcctcac ctcaggtgat ccacccgcct tggcctccca aagtgtcggg 9720 attacaggcg tgagccaccg tgcctggcac ttaattttct taatacctca attaccccat 9780 atggtaaaat gggactagta atccatacct tatagcgctg ttgtgaaaat gaaatgaggg 9840 taagcagata aaatttcaga ctacggatgg gattgttact acattctgaa cctggctttg 9900 ctgttatttg ctatgtgacc ttatcttctc tggatctcca ttctttccaa gtctataaaa 9960 caaagtggac aattgtcaac ctttcttcca aagagcaatg atttaaggat caaatgatgt 10020 catttaacaa aaatatgaag agctcaacaa atgaggaact cattattatt attacaatta 10080 ttattatttt agaaataggg tcttgttctc ttgcctaggc tggagtccag tggtataaac 10140 acagctcaat gcatcttcag cctcctggat acaagtgatc ctcatgtctc atccccctaa 10200 gtagctggga ccacaggcat gtaccaccac gcacggctaa ttttttattt tttattttta 10260 ttttttgaga cagtcttgct ttgtcgccca gactggagtg cagcagcgca atcaccgctc 10320 actgcaacct ccgcctcctg ggttcaagtg attctgctgc ctcaacctcc caagtagctg 10380 ggattacagg cctgtgccac catgcccggc taattttttt gtatttttgg taaagacggg 10440 gtttcaccat gttgcccagg ctgatctaga acccctggcc tcaagtgatc cccctttctt 10500 ggcctcctaa agtgctagga ttacaggcgt gagcctctgc acctggcctc ggctaatttt 10560 ttattttttg tagagacagg ttctcactat gttgccaggg ctggtcttga actcctgggc 10620 tcaagtgatc ttcccacctc agcctcccaa agtgctgaga ttacagatgt gagccactgt 10680 gcctggcctg gaactcatta ttgaagcatt cactagtatc aactttgggg ttacctggcc 10740 acatcctctg acctacctat aagggtatca cagctaacgg agcctctgtt tctcagaatt 10800 taggcagaag cagttcaatt tatcacaaac tactctatat ccagcataag tgcccaaata 10860 aaacaattgc taaagttctt taggcattta ctgtttgtta gttagatatt tagtcctcac 10920 tacaaatctg tgatacaggt attattttta ttaaccccat tttatagaag agaaacctga 10980 agctcagaga tgctaagtaa cttgtgcaag gtcacacagc tagtaaataa agggcagagt 11040 aaagatttag tttcacattg gactccagaa cctttctact gggactcatg ggaatagtgt 11100 ggatgtccct gaccttcagt ggcccagggc tctcctgggg gaatccagcc atagacaaga 11160 caccagcgag agcccaatcc taagattttg tttgtttgtt tttgagacaa ggtctcactc 11220 tgtcaccaga ctggagtgca gtggcatgat caatgctcac tgcaaccttg atctcccagg 11280 ctcaagcaat cctcccacct cagcctcctg agtagcttgg actacaggtg cacaccacca 11340 cacctgacta attttaaaat tttatttaat taattactta ctattatttt ttgagacagg 11400 gtatcacttt gtcacccaag ctggactgca atggtgtggt ctcagctcat tgcgtcctcc 11460 acctcccagg ttcaagtgat cctcccacct cagcctctgg agttgcaggg actgcaggtg 11520 tgcgccacta tgctcagcta atgtttttat tttttgtata gatggggtct cactatgttg 11580 ccagggctag tctcaaactc ttggactcaa gcgatcctcc tgtcttggcc tcccaaagtg 11640 ccgggattac aggcataaac caccacaccc aacccctaag gtgtttttgc tgaatgtgac 11700 catgtcagag gcaggaaagg gaagcatcat ggggttagga aaggaacact gagcagggag 11760 acaaagaaaa tgggatcatt ttgtgagtgt tcgctgtgtg tgtatgtgtg acaattctca 11820 gagccagcct ctcaggtggt tgagaccaca gtccccattt cccagatgag ataatggagc 11880 ctcagagagt ttctgcagca cagctagtgg aattagaatt tgaacccggc tcttccagac 11940 tccaggtgct tcacaaccat cccaaaccta gtcatttgca gtttaccttc atgattttac 12000 catttccctt tgccatagct agtgttattt acttaataat tccttttgaa tcagtctgct 12060 taaaaaaaaa tagcttcatt ctaaagtgta atattcttgg aatatcgggt ttgctgttac 12120 ccacccccac acgttataca tatacatgta tgtttctaat acatatatat gtacgtatat 12180 acgtgtatcg ttttttgtta ttttttttgt tgttgttagt tttttttaga tggagtctct 12240 ctctgtagcc caggctggag tgcagtggtg tgatttcggc tcactggaac ctctgcctcc 12300 tgggttcaag cgattctcct gcctcagcct ctggagtagc tgggattaca ggcacccacc 12360 actacacccg gctaatgttt gtatttttag tagagacagg gtttcaccat gttggccagg 12420 tgggtcttga actcctgatc tcaagtgatc cacctgcttt ggcttcccaa agtgctggga 12480 ttataggtgc gagctactgc ggctggccaa tgtatgtttt taatacacat tcaaataacg 12540 aataactatg aaacctgaaa aactgctcca tgttacttcc tgaacccatc ttgagtgctc 12600 acatgctgtg cataccacat attgggaaac actgctttcc ctggcttcca agcccagctt 12660 aatcactgtc ccatcctatg cttcgcttta tttgtctata aatgttgggg ttgggggttg 12720 atgccaaaga ccttttctgt tgtcattaac atggacacag ctctaagagg tcttggcatc 12780 ttgggctggc tctcctttta gttcagaatt tggattttta tccaactact cagagtgatc 12840 aagccttcct tatgaatgaa ctcgttggtc aaactcataa aaggctgatc gataaaacag 12900 gaatgaatgt atgaattgac actaagtcat tagcatttca cgggaatgga ttctccgtta 12960 gtggaagagc acatgtcctt tctggcactg atgtgtgctt gggaaactta ctgagctaac 13020 tggcccatgt aacacagagg ccctttggtg cagtggaaaa ctgttgactt tggagattat 13080 cttgagtttg aatctgagcc tgcctgtaag aagctggcta actgaattgc tttgcttctt 13140 ggacccttac catttataaa atggggacca ttgtactcac cctttagggt tattgcatgg 13200 attaaatggg attctctata gaaaatattg gcacaaagta ggtgtaaatt tgcacgctag 13260 tgggattgtt tgtgagggaa attgtcattt gattatcaaa gacttaggag caggaacagt 13320 gtctaattca gggactgcaa atggaaatgc cagctgaggc caggcatttg ctaataattg 13380 ggtaaagcag ggcaggtgta gaatagcaat gtctgggaat taaaagagag gtgaggacgt 13440 gtatgacctt gagaaggcaa gccctggcaa aaggggatgg cctccactca gctacagtca 13500 tgcctagatc ttctaacttt ttatttttat ttttattttt tgagacggag tcttgctctg 13560 tcacccaggc tggagtgcag tggcgcgatc tcggctcact gcaagctccg cctcccgggt 13620 tcacgccatt ctcctgcctc agcctcccaa gtagctggga ctacgggcgc ccaccaccat 13680 gcccggctaa tttttttttt gtatttttag tagagatggg gtttcaccgt gttagccagg 13740 atggtctcga tctcctgact ttgtgattta ccctccttgg cctcccaaag tgctgggatt 13800 acaggcttga gccaccgcac ctggccgatc ttctaacttt ttaaagagaa gcaagacatc 13860 tggattttta tgtgataact cctgatttta aactggcacc caattataat ttacaacact 13920 ataagggtca acattgccag cagagcaaaa catgggtggg ggcaactgct ggtcaccggt 13980 gtgcagcctc tggtctaaaa tcatctttgt atttcttctt gctttacgca ttgtcccagc 14040 acagtgctgt tgtatagtaa atatccagta agtgggtgta gaatgaataa accaatgcag 14100 ataaacctgt agagaggccg ggcacagttg ctcatgtctg taatctcagc acnnnnnnnn 14160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 14220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntagtccca gcactttggg aggccaaggt 14280 gggtagatca cctgaggtca ggagttcaag accagcctgg ccaatatagt gaaaccccgt 14340 ctctacaaaa ataaaaaaat tatctgggca tgattgcagg tgcctctaat cccagctact 14400 cgggaggctg aggccggaga attgcttgaa cctgggaggc ggaggttgta gtgagccgag 14460 atcatgccat tgcactccag cctaggtgac ggagcaagat tctgtctcaa aaaaaaaaaa 14520 aaaaaaaaag aaaaaagaaa agaaaaagaa acaatgaatg agtgtgaggc tcatggtagt 14580 attggttcct gagagtagcc aaccttattg gtcatcccag ccacgaagtg aaatggtacc 14640 cctggcttgg gccaatgaat gaggaagaat aatggcaaat gggggtctat gcctccaccc 14700 tccaccacta gggaggtctc aagcttgaaa tccagtgacc aggtttttag gtcctggacc 14760 tggccagtcc tcctacagtc aagtagataa gtggagggtt tggtccgttg ggctacggag 14820 atagtgatca aggccgttac tctgcaatca gactcagaaa tggcctctca gttacttctc 14880 catttgtggg tcttttggaa gagcagagaa gaggaaggaa tttaggtctt ctcaccctct 14940 gggctgcctg tccctgctcc ctgagccatg gagggctggg gtggaatatg gggaataaat 15000 ctgtactttt tttttttttt ttttttgaga cagagtctcg ctccgtcgcc caggctggag 15060 tgccgtggcg tgatctctgc tcacagcagc atctgcctcc cgggttcaag ttattcttcc 15120 acctcagtct cctgagtagc tgggattaca ggtgcccacc accacgcccg gctaattttt 15180 gtatttttag tagagacagg gtttcactgt gttgggcagg ctggtctcaa atacctgacc 15240 tcaggtgatc cacccgcaca tgcctcccaa agtgctggaa ttacaggcat gagccaccgt 15300 gcccggtcct accaatctgc acattttaat tgacaagggt caccctccac tcatgtgcca 15360 ggcatagttc tgagaagcat cccacaagga tgcctctgag ttcaccctga caagtccact 15420 agctcttggc agagacatct ggcaaattca aggcttgaga catgctggcc tctctttaaa 15480 gtgcagcaaa ttttgtctag agcttggtca gttaaaattt tgatgttttg ttttgcatta 15540 atttcaattt ttaagaaatg ttgcattaaa atgttattta tcttgaatag taaatttctt 15600 agtgtcccct taatttctta gtgtgtctga gttgagagcc tcccctgcct gattctagtc 15660 cagaccctgg ggtgacagaa gactggtggg agatgggagg tgaggagggg agtgttggtt 15720 ggagaggatg atctacagag tgctggagag actctgtatg gagcttttca tgctgcctgt 15780 ttgccagccc tgaagctatg ccttgaggtt gggcaaggtg gcatatccta gatcagagat 15840 cctcaactgg ggccattttt ctccccagag gacatttgga aacatgtgga gacatttttg 15900 atcatctgcg ggggtgggga gaggggctac tgacatctgg tgagtagaga ccagagggac 15960 cattaaactt tctacaacgc ccaggacagc ccctccacaa taaagagtta tttgacctca 16020 catattaata gcacaaagtt gaggaacctt gatctagatc cacagcacag aagaaaggat 16080 gtagattttt cacacattaa agatgagaaa gcttgtgcct gtaatccctg tgactcagga 16140 ggctgtggca ggaggattac ttgagcccag gaattcaggg ttacagtgaa ctatcatcgc 16200 agcactgcac tccagcctgg gtgacagagc aagattttgt ctcttaaaaa aaaaaaagat 16260 gaggacaggc acagtggctc atgcctgtaa tcccagcatt ttgggaggcc gaagtgggtg 16320 gatcacgagg tcaggagttc aagaccagcc tggccagcat agtgaaaccc catctctact 16380 aaaaatacaa aaaattagcc agctacttgg gaggctgagg caggagaagc gcttgaaccc 16440 gggaggtgga gcttgcagtg agccaaaatc ttgccattgc actccagcct gggcgacaga 16500 gcaagactcc gtctcaaaaa gaaaaaaaaa aaagatgaga aagaggaagg gagagaaaaa 16560 agagagagag gaaagaaaga gagaaggttt tggagtcaaa aagacttaga aattccagtt 16620 cttccacttc ccatggaacc ttggcaagtt gccttctctc tttctctgaa tctcacattt 16680 tgcctctgtg aagtaggggt ggtacctggt ggagatgatg cggagatgag ggtgaggggt 16740 gtgttgcaca ctatgcccct aggatgggtg agagcttggg agcactgaac ctccctttcc 16800 cctcttgttt cttcccccca ttgtctccca ccagctccct gggatctcca cttcactctc 16860 tgggattcca ccagcaggag gctactcctg gagttaaggc gtgttgttca gactggggca 16920 ttttaggggg cataaataat aattatgcct ggacaatgga cataacatct agggccttct 16980 gaagcaaacc agggtgtggg gtacccaaac aaggcagtag gccccaggag gcaggtccct 17040 gcagtcccag cagagagcag ggcacagggt tgagaagact gagcaaactt cattatcagc 17100 tcctttgtcc cccactctgt cctggagcaa tcattctggc ctcttcccac ttccccaaaa 17160 acccagtata aaggctgctt ctggcccctg aagccagagg cactgagagt ggaggtctca 17220 gactcttgga aggtgagttc ttttctggct gcccaggcag gaccagtgta ggccctggga 17280 agaagcagca cctcataggg caaacacgta ggaggcctgt ccttaggaac atcatagcta 17340 agcagacctg tccccgcagg ggcaggagtc tgggctaagg gtgatactgg agagcagcaa 17400 cggagactgg aagacaaatg aaatttggta cctgagttat ccctcccacc attccttttc 17460 tagactctcc agctcagggt ctgttcatgg caagaggaga aagcaatctt gtttgctctt 17520 taatcaaaca attaaacaaa tattccctct atactatgtg ccaggggcta tactagacac 17580 acaaagacag ccccaagaag gacggtggag tagtgtcctc gctaaaagac agtagatatg 17640 caatgcctct tgctcctgcc ctttctcctg ctgggaacag tttctgctct tcatctgggt 17700 aagtctctcc cttccctcct catgcgtctt tccctttttt cctttttcct acactcccct 17760 ccccccgctt ttatttgcac tcatgaggcc aggaccacag ccttccctct ttagctgata 17820 cagctcatct ccggtaagat atcacttgga ctcagaactg taacctggaa ctttctcttt 17880 tttgtttgat ttttttttgt tgttgttgtt tttgtttttt tttttgtttg ttttttgttt 17940 tgttttgaga cggagtctcg ctctgttgcc caggctggag tgcagtggcg cgatctcggc 18000 tcaccacaaa ctccgcctcc cgggttcaag caattcttct gcctcagcct cctgagtagc 18060 tgggactaca ggcacatgcc accacgcctg gctaatcttt gtatttttag tagagatggg 18120 gtttcaccat atttgccagg ctggtctcaa actcctaacc ttgtgattcg cccgccccgg 18180 cctcccaaag tgctgggatt acaggcgtga gccaccgcac ccggcaaact gtaacctgaa 18240 ctttcagaag gaaaaaccac ccacctgtta agatgaaggg ctggtgactg ccccaggctt 18300 ctcacacgtg ctttctccca ccttcaaaac acacactcgt ggtgtcggcc agaagtcagg 18360 ttcttgtcca tttgtgggtg tgacccgaga gatctctcct tacctaacac caaggaaatc 18420 ctccagtctt gtcttcaggt ggaattccta ggaaagctcg agcgacgttg ctggagctgt 18480 ccacggtgct ggaactagga agctcttgac ctgatggcag gttacctctt cttcccagag 18540 aatgatgccc cccatctgga gagcctagag acacaggcag acctaggcca ggatctggat 18600 agttcaaagg agcaggagag agacttggct ctgacggagg aggtgattca ggcagaggga 18660 gaggaggtca aggcttctgc ctgtcaagac aactttgagg atgaggaagc catggagtcg 18720 gacccagctg ccttagacaa ggacttccag tgccccaggg aagaagacat tgttgaagtg 18780 cagggaagtc caaggtgcaa gatctgccgc tacctattgg tgcggactcc taaaactttt 18840 gcagaagctc aggtaagtag tagggaggct actgcggagg acctggggga aaagagagta 18900 cattcagtct tctgttccct attcatttag gctagtggtt ctcaaagcct cgcatgcatc 18960 agaatcacct ggagttgttg ttaaaacaca gctttctggg cctcacctgc acgacttctg 19020 atttaggagg gctgaggtga agcctgagaa tttgcattta caacaaatcc ccaggtgatg 19080 atgatattgt tggtctgggg agaaccaccg atttaaacaa aaggctttgg tgttagaaac 19140 gcctgtgtta aattctggtt ctgcctttta ttagctgtgt tacctgggca agttgctttg 19200 cctttcaaag ctttagcacc ttcatttgta aaacgaagat atatagcacc aacttcttag 19260 agttgtggtg agcattaaat gagataatac atgaaaagtg tttggaatag tcactgggct 19320 gtaataaact ctcaataagc ggtggttata attattatga gtattatcat ttcctgtagg 19380 attgtcctga cagctaatta agaagcaaaa gataggatta agggaggcaa gtaggtttat 19440 ttttaacctg aaaagggatg ccgggctctt gcctggagac tcagaaactt gaaataaatg 19500 agagggaatt cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 19560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngaattctct 19620 gttagcacat agccagaaca tctagaaggg gtggtaggag tggggattag aggttccagc 19680 tggaggcaat ggcacttgca aaggctttgt tgaagtggcg taagtgtgga ggtggagcat 19740 tcaggaaagg agagcttcag cttcagtgtg gctggagtgc tgggtgtgaa gagaggtgaa 19800 gatgaggctt ggaggctggg cagattttgc tccaaaagag cttggtgaac tgtgataagg 19860 agtttggatt ttctcctact aaggacaaca gcaaactatt gaagagttta aatcgttcag 19920 tgacaatgac acgtttgcgt tttggtggct cactcgagct gccagccagg tagacagtgg 19980 cagaagatgg aagataaagc actaaagggt gatgaggcag gaagccagtg aggagagaaa 20040 ggggacgatg tgagtgacag taaatcattt gttgggttgc tattgtgtgc taagctctgt 20100 gctaaattct tcacgtgtat tatttcagct aatccatcta acaactctgt aaggcaggta 20160 caatcgttcc cagctgaaga agctgaggct ctcaaaagct agtaacttgc ctaagttcat 20220 gcagcatgca agttgtccag ccaggattct aacttagaca ccagaggcca cttttaacca 20280 ctgctctagg actgggggaa atggtcccta gtgagatatg tgtcgagttt catatttcat 20340 tcaacaatat tgttggcctg ctacatgtga agagctgtgg aaagcgccca aagtgagtta 20400 gatccctatg agcaagtggg atgggggtgg agtggacagt aggagggctg gaacacacat 20460 aaaagggtat aagaaataac aattaggccg gccaggggtg gtggctcacg cttttaatcc 20520 cagcactttg ggaggccgag gagggtggat cacttgaggc caggagtttg agaccagccc 20580 ggccaacatg gtaaaacccc atctctacta aaaatgcaaa aattagctgg gctggtggtg 20640 cacgcctgta atcccagcta cttgggaggc tgaggcacga gaatcacttg aacccaggag 20700 gcagaggtta cagtgaactg agattgcacc actctactcc agcctgggag acagagtgtg 20760 accctgtctc aaaaaaagaa aacaaaacaa gtaggtactt tctgccatag ggaggattca 20820 taaactgcta gtcctcaggt gcatttttgc ttatcagttt taaaaatcag agaatgtctc 20880 aaagaattag gatgtcagct tcttttgaaa atttgggcca gaagcggtgg ctcacgcctg 20940 taatcccagc actttgggag gctgaggtgg gtagatcacc cgaggtcagg agttggagac 21000 cagcctgacc aacatggcga aaccccgtat ctactaaaaa tacaaaaatt agctgggctg 21060 gtggtgcatg cctttagttc cagctactca ggatgctgag gcatgagaat cacttgaacc 21120 cgggaggcag gggttacagt gaaatgagat tgcaccactg cactctagcc tgggagacag 21180 agcaagaccc tgcctcgaaa aaaagaaaaa gaaaatttgg aagatctgac aacagttgac 21240 ctgcattcct gctcggcaac agcctgatgg tggatgggca gaggctcagt tgtctgccaa 21300 acctcccatc actgatgtct tccctcgctg tcatcatctg cttgacatgt aggcatttgg 21360 tgtgtgcctt ctgctctggg tgcccagatg aattggatgc tatatgagaa aacattctgt 21420 aaatgtcttg tggtaggcaa cctcaaagat cactggggcc tccaatgatc cctccttcct 21480 ggtattcatg cctgtgtata atcctctccc ttgagtgtgt actacacctg gatacttgct 21540 tctaataaac agaacacagc aagggtaatg ggatgctact tctaaggtta aattacaaga 21600 gtgtaaagtc tgtcttgttt gtttccctct cttgatcttc ctctcattct ctctctctcc 21660 ctctctctca ctttcttact gtcttgtcct tccctttgtt tactctgatg aagcaagcta 21720 gcaagcatcc atgttgtgag ctgacctatg aagaggccca tgtggtggta aggaactgag 21780 ggcagcctct acccagcaag gaactgagtc actcatcata tgggtgagct tggagacaaa 21840 tccttcccca cttgagcttt cagatgacgg cagccctggc tgatgctttg caggcttgtg 21900 agagaccctg agacagaaca ctcagctaag ctatacccta tctcctgaga tagagtataa 21960 tacatgtagt tttaagctac tatgttttgg gataatttgt tactcagcaa tagataacca 22020 atacatatac catgtacata actgtttcag ttgtctgaga ctatatttag tcattttaca 22080 cctacatcaa gaatgtgtca ggcaccattc caggtacttg gaatacatca attaacagaa 22140 taggtaaaga ggccaggcat agggctcaca tctataatcc cagcactttg ggaggcccag 22200 gtgggaggac tgcttgagcc caggagttga gaccagcctg ggtaaaatag tgagacactg 22260 tctcaactaa aaaaaaaaaa aattagttgg gcacagtggc acatgcctgt ggtgccagct 22320 gctcaggagg ctgaggtggg aagatcgctt gagcccagga gtttgaagct ccagtgagcc 22380 acggtcacaa aactgcactc tagcctgagc aacagaaaaa gaccctgtct caattaaaaa 22440 aaaaaaaaaa aaaaggaaag aaagaaaaaa ataggtaaag atccttgatt cttgccctct 22500 tggaacttct attctagagg gggatggttt ttcacagtag aagtctgtgt tgacagcgct 22560 gtttaaagct ccttcagcat ctggggaaaa ggttnnnnnn nnnnnnnnnn nnnnnnnnnn 22620 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 22680 nnnnnnnnnn nnnnattttt tagagatagg gtcttgctat gttgcccacc aggctggtct 22740 tgaactcctg ggctcaagca atcctcctgc ctcagcctcc tgagtagctg ggaatacagg 22800 tgtgcaccac catgcctggc ttatttcata tatatatatt tttatatata tgtatattta 22860 tatatataaa tatatatata atttctgtat ataaataaat aaatatatat atatatatat 22920 ttttagagat agggtcttgc tatgttgacc accaggtctt gaactcctgg gctcaagtga 22980 tcctcctacc tctgcctttc aaagtgttgg gattacaggc gtgagccatg gcacctaact 23040 gagttatttt taccacacga agcataggac atacatccaa aaatgttctg agctgagcaa 23100 gagcctggag gcaagtgaat ctgaactttc ccgtctttga agaaaccagt ctctctccaa 23160 agtcacatag ttagtgtcac tccccccaag aactgcatga gctgggacaa tcagagggca 23220 gtggaaggtc tggggctcag gggcgccccc tgctgtctcc ccagggtctg tccccttacg 23280 caagagcctc tgctccccca ctttcctgtg gagcctcctc accatgggca tgacccagct 23340 gcggatcatc ttctacatgg ctgctgtgaa caagatgctg gagtaccttg tgactggtgg 23400 ccaggagcat ggtgaggcac cgctgaggcc cctgggggtt gggggcacag gcgggtcacc 23460 ctggctgagc tcccctcacc atacgtttcc ctacccacag agacaaatga acagcaacaa 23520 aaggtggcag agacaggtag ggctatgaaa gcagggccct ggctcacgcc caccccactg 23580 caacccgctt ctcagggggc gggactcctc taggcctggg cccacccagg taaccctttt 23640 gtgggatgta agagtctggg ttcagaggaa ggctattttg gtgctctctg gcctccgctg 23700 gaaggggtga tagtgtccac tgagtgccag ttcctgaccc cactgccctt cccatcctgc 23760 ccagttgggt tctactcctc cgtcttcggg gccatgcagc tgttgtgcct tctcacctgc 23820 cccctcattg gctacatcat ggactggcgg atcaaggact gcgtggacgc cccaactcag 23880 ggcactgtcc tcggagatgc caggtgacct gcctgtacag ggatggtgac agcaagtggt 23940 caggcagtgc ttttcatttt ctctgtgcgt ttacatccag cagcttgttg ctttctccca 24000 agaaccctag gagatcaggg gtacctcccc attttacaga tgaggaaact gaggctagga 24060 agggacctgg cttgcttaat aataagaata gctaatgcag agtgctgact gtgcacttgg 24120 caccttgcct tgtttagtcc tacaacacct ctttgaggta gatgcgttaa tatcttcatt 24180 ttgcagttga ggaaaccgag gtacagggtt gcacagttag gtcattcacc caagatcaca 24240 cagctttcag tggcagcctc cagaacctgt gttataaggg tacacgctaa agtcttgtta 24300 gggctagaat aggtagagtt ggtatattag atatttattg ctgtataaca aatcacccca 24360 aggcttggca ttttaaaaca acaaacactt ctcatctcat acagtttctg acagtcagaa 24420 atcagggaga gactcagccg gctgattctg agtcacagtc tctcatgaag acatagtcag 24480 gctgtcagcc agggctgcag tcatctgaag ggctgactgg ggttggagaa tctatgtcag 24540 ttcaattacc cccatggcct ctccataggg ctgctcagga cacagcacct gctttccctt 24600 gagcaagagg gctaagcgac agagaccccg tatcttctct cacataatct cagacgtagc 24660 ataccatcac ttctgttacg ttctattata ggcacagagc aaccctgata tactgtggaa 24720 ggagactgga caaagcaggg gaataccagg aggcaggatc cttgagggct gtcttgttgg 24780 ctggagacca ccattgaggg tttttttttt tttttttatt gagacagtct tgctctgtcg 24840 cccaggctgg agtgcagtgg cacgatctca gctcactgca acctctgcct cccaggttca 24900 agcgattctc ctgcctcagc ctcccgagta gctgggattc accatggagt cttgaaccca 24960 gattctgtga ctgcttttgc tctttttgtg ttcatccaaa cagtccctgt ttatcctaag 25020 aggatgggag aaagagactg ggagagaagg aaatccagtg gcctccctcc ctgctagcag 25080 agcctggccc tggcactgag ccttcctcct ctaccctctg ctcctaatgg tgagggtccc 25140 ctagcagggc ccttctgtcc aggacacatg ggccgcctgt cctcacccca gcctactgac 25200 ctctctcctg ggctggcctc agtgcccttg attgtgccgg agagaggaag cgctggacag 25260 tcaggccaag ctgctgtccc caggagggca tctgcttatg tctagggcag ggacaccttc 25320 ctgaggactt ctgatgagag acggtgtgag agcttcccac ttcccacctt ccttcccatc 25380 cttggttctc aaaccttcaa gtgtgcatga gaatcactta gtgggggata tttgtccaaa 25440 tgcagatttg cagatatccc cgctgagatt ctgagggccg agatgaggcc tgtgaatctg 25500 catgttaaga aagcacccgc tttgatgcgt gtgtcattgg gtaggggagc aacactttga 25560 gaaacatgga gctagagaac gtgggtttct atgggtttcc catagaaaca tggatttctg 25620 tgttttctgc tgccctgaca tcgaaggcac atctgaaggg ggaggggcca ggccaagaac 25680 cagggagtcc tgggaacgta gaggcagcag ccagtgactt cccgtactcc tcagggacgg 25740 ggttgctacc aaatccatca gaccacgcta ctgcaagatc caaaagctca ccaatgccat 25800 cagtgccttc accctgacca acctgctgct tgtgggtttt ggcatcacct gtctcatcaa 25860 caacttacac ctccaggtac ccaccttcat ccttcccctc tccctgcctc ccgaggctcc 25920 tccaaaggga tggtccatcc agcacctgcc ttccaggaag cgcagttctg gtcttctgat 25980 ctggatctat tttccgggtt ctccaggaag tgtttctagt agattgggtt ggcgaggggg 26040 tgggaattga ggcccagttg gcctcttcgc cctacccctc cttcctccag cctccacaca 26100 ctctcctaac ctcttcactc tctctttttg gttttagttt gtgacctttg tcctgcacac 26160 cattgttcga ggtttcttcc actcagcctg tgggagtctc tatgctgcag tgtgagtctg 26220 ttgggctgaa atgccttcct gagctttgca accgtgatca gagaacccca gggaagggtt 26280 gggagggccc caggcatccc ctaatgcacc tctctctgag accctctgat ggcagggagc 26340 tcacttcctt aaaggcagcc tatcctgctg taattgactc cccctgttgg agtcttccct 26400 tagaggaagc tgaaatacct ggcttgatga cactttggtt ctatgtctgc tgtttgaaac 26460 ggcccccaga atggcctccc ctccatgccc accctgaaga aatttcccaa gggcagccat 26520 ttgccttata attttcctct tcatgttgga cagtccccac ttgcatctct ctcctggttt 26580 cccctgctgg gcgctgctga gggactctcc cctgtgtatg tgatggagta acaggacatt 26640 acaataatga tgacaaaatg acaaccatta tcaagtgctc cgttggtgca ggcagcaggc 26700 aggatccttg accatcactc cctgagttca gcctcactgc agcggtctcg gcagagggca 26760 gctctctttc cttcatctgc tcaagccaga accctggagt ttccttgatg tttctctccc 26820 tcacactcca tgttcactcc atcctcagta cagccagcag cagcttctac acaccccaaa 26880 tctgaccctt cttgtcacct ccactgctgc ctctccagtc ctagccacca acatctctag 26940 cctggattat tgtggcagcc tttagtctcc cacatctgcc ctggccccgc tgtctcagtc 27000 tatttttaac acaggggctg cagtcacctg tcaggacata agtctcttca catcactctg 27060 tggtgtcctg tctcatctgt ctcagagtaa aagccaaagg ctttactatg gcctaaaaag 27120 ccctgcaagc tctggcccca gcacttcact cccctctagc tccccctcct ccattgttca 27180 ctctgccaca gccacagtgc ttcctagtgc tccggaagtc tcaagtgtgt tccctgcttg 27240 gcatctttgc atgtactagt ccctgtttct agaacattct tctccagata tctgcaaggt 27300 gcccaatctt accttctctc cttcttcagg tctttccctg actgtcctct tctcagtgag 27360 gcctcccttg gctgtcccat gtacaattgc aacctcccta ctgcccgctt ctctgcttgg 27420 tttttctcag cgtttatcac taacactctg cctatctctt gcttattgtc tgaccgccac 27480 ctgctccatg ggaatgccac ctcctcgatg gcaggaatct gttgacttgc ttgatcgtgg 27540 tatctccagc acctagagca gtgcctggca catagtaggt tctcagctaa atgtttgttg 27600 acagaataca gtggacagtc ctgcgaggtc aatgccatcc ctgttattag tggaggaagt 27660 ggggctcagg gagtttgagc cacttgccaa tatcacacat acaggaggtg tgagaaccca 27720 gctcagtggc cctgaagttg gagcatttgc cctcaaggct ggggaccaaa gagcccatgc 27780 aaagagcccg aacgcttaag caccaccctg cctggccagc ggggnnnnnn nnnnnnnnnn 27840 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 27900 nnnnnnnnnn nnnnnnnnnn nnnncccact gcgcctggcc cattactttt aatggcaaaa 27960 accacaatta cttttgcacc cacataaata gttaccatgg gctgagcatg gtggctcagg 28020 cctgcaatcc cagcactttg ggaggctgag ccaggcggat cacttgaggc caggagttca 28080 agaccagcct ggccaacatg gtgaaacccc gtctccacta aaaaatacaa aaattagctg 28140 ggtgtggtgg cgcgtgcctg taatcccagc tattcaggag gcagaggttg cagttcactg 28200 aaatcatgcc actgcactcc agcctgggcg acagaatgag actctgtctc aaaaataaat 28260 aaataaataa ataaatattt accatgtttt gaccacctgt tatgtgccaa ctgtattact 28320 taaaaacacc catgggaggc tgggcacagt ggctcacgcc tgtaatcgga cactttggaa 28380 gggcaagcgg ggaggatccc ttaaggccag gagttcaaaa ccagcctagg taacacagta 28440 agccctgtct ctacaaaaaa taaaaaaatt aactgggcat ggtggtgtgt gcctgtaacc 28500 ccagctcctc gggaggcaga gggagaggtt cgcttgagcc cagcagtttt aggttgcagt 28560 gagccaggac caagacacta cactccagcc tgagtgacag agcaagacac tgcctctaaa 28620 caaacaaaca aacaaaagcg acctgtgggt aggtaggaac aggctcatag tacagatgag 28680 aaagcagagc ttggagggct caagcgattt gccaagcaga ggtccaagcc gaggtctctc 28740 tgaatccaaa gttaattccg tctatcatat caccacagcc ctctctgccc cagggagagt 28800 ctctgcccac tccagccact cacgtgtaat tgacttcctc aggggcagga aaggcttcga 28860 tgggccagtt gagggtgcag ttcagaaaga taaggcaggc caggccagac caggtgaaca 28920 tgatgaccac gaaggccaca ccggcatcgt agatcagctg tgagaggagg gggcaggccc 28980 gtgggggaga ctgcctggcc ccagacccca ccaaggtaga tcccaggcct cagaggcctt 29040 aaagaagttc tcttctcccc ttgtccttgt gcccaatttg cagatgagga aaccaagacc 29100 agaagtttag agtcagactc agaagaccca tcattccttt ttctttttca cttgaggccc 29160 cctagagagc tatgaaatag tctccacaaa gcctgaagtt gctggccact ggctcaaaat 29220 atctctgaaa tttccattat cttaaaaaaa tacatacatt tttgcctatg actccacaaa 29280 cattcatgtt catgttcgca caaaaatgtc catttcatag tacgtacaaa ggaaacttag 29340 tgctctaggt ttaccgggcc taatcgtgtt tatcctgccc cttcctggca cattccccag 29400 gggaaaaggc aaacccagac tgctcatgct cagccttttc tcacctttcc caggtcctcc 29460 cacgtgcaac aactgggggg gttggggaga gggaggtgca agtgctctgc ccaagggctc 29520 tcaaccccag ggcaggtaag ttctcaattg aatgagattc tgtgcaaatg tgtcagccct 29580 tcttatggaa gaagctgatg caccatctgt cctcttgtcc tccccatacc atctgaccag 29640 gataattaat gtctgctctc ccctcaggct cctgctcaaa cctttttctc tgcagtcttg 29700 gaccttggtg ccttttcctc cctaggggca ggacagagct tcaaagggcc acacccccaa 29760 atgtgtggag gtaagatctg gctcttcaaa cactacttca gttgaaaaga agggagaact 29820 gcccaccctc catgcctgcc caccagaaca actgatggcc cccccaccca tgcgctctct 29880 caaactcctt tggagacact gagcaaaagt accttcttta gtactctttg taaagtgcaa 29940 aacggtatgc agtttggtac tgcccaccgt ggaggttgag gagcatggca tggctcaaag 30000 ggtcctttga tatttgacag aggaaattga ggcccccatc ttgcactgag ctaaaacttt 30060 ggtcccctgg cttcgaggta caccaggttg acctgtccag gatccagcct ggcataaact 30120 cactttgtga ccttggacca aaccacccat cctctctgga aggtgtggaa aaatgtggcc 30180 ccaaaggctg aataaagcca gagagtcagg gaccttgaac gcatgtgaag gggctggact 30240 tgattctgta ggtgaagcta aaccactgaa ggtttttcag cagtgtgtga gccagttccc 30300 catctgagat ctttctggaa gtcacgtgag tgacagagta cagagaaaaa gaatcagagg 30360 cagggagacc agctgagaaa gcttgctgtg gcccaggaga gagggggaag gcctgcattg 30420 ggatgatgac agagaaagga gagcggagaa gtcagacccg tgggtcagca ctagctgctg 30480 ctcactcggc cccacccggt tcttgtgtca agacaaaaag aaaacccagg tggcctcata 30540 ccttgattcc tgggaacgta atggcagaag aggcgtaaga gccaatcatg agggccatta 30600 acgtggagcg caggttccca aacatgttgg gcagctgagg agggaaagca gcacccatga 30660 ggtggggaca ccgtgaccct tgcccagcat tcccagccct gctccataca atagctccag 30720 gagacgcagc agaaaagccc caaggtaaaa caaacagaaa aatcaatgtg ggaaactgta 30780 ctctgccccc tgcctacaca gtcacagtgc cctttagctt caaaaaggct cccagacacc 30840 cctcagagag acattttgtt aattttgttt aattccaggt ttcccaagtt tgttacgtaa 30900 cacctctgaa aaacacatgg aataggtgct taagaaacac tgatcttggc tgggcgcagt 30960 ggctcatgcc tgtaatccca gcactttggg aagccgaagc tggtgggaag cttgaggtca 31020 ggagttcaag accagcctgg acaacatggt gaaaccccat ctccaccaaa aatacaaaaa 31080 ttagctaggc atggtggcat gcgcctgtaa tcccacctac tccagaggct gaggcaggag 31140 aatcgcttga acctgggagg tggaggttgc agtgagccga gatcgcacca ctgcacttta 31200 gcctgggtga cagagcgaga ctatgtcccc accccccaaa aaaaaagaaa agaaaagaaa 31260 gaaacagtga tcttgtccaa cccatttgag atgagacaat tgagacccag ggaggaaaag 31320 tgtactcaag ttcacagagc acattaatgg ctttctcccc attgtcgttg tcccagccct 31380 aacccaaggc tgtgaccatg gctgtgtccc ggtaataggc agtgcctctt aaccctctcg 31440 gttgacgtcc cagcccagtt tctgcctaat caggacaaat cacatcctgg gaggtgaggg 31500 tggaaataag ggagggaact gagccagggc agacagtctc cagaggaggt ggctctgacg 31560 cagagcaggg tcagaaccca caccaggaga gaatttaatt gatcatgtgt tccactcacc 31620 tgcctcagcc aagccctcag ggcaggggaa ggcaaagtca ggatgccctt cgcacacacc 31680 ctcctctggc cccaccatcc tccccaagtc actagatccc acagctgaga aggaccttag 31740 gatccgtaca aagcctaaac acactccaca gagggggaaa ctgagactct gaagggaggc 31800 ctcaacagct ctggtaaaaa aggcgtttag gccgggcgca gtggctcaca cctgtaatcc 31860 cagcactttg ggaggccgag gcgggtggat tgcctgagct caggagttcg cgaccagcct 31920 gagcaacacg gtgaaacccc gtctccacta aaatacgaaa aaattagccg ggcgtggagg 31980 cgtgcacctg tagtcccagc tactcgggag gctgaggcag gagaattgct tgaacctagg 32040 aggcagaggt tgcagtgagc cgagatcgcg ccactgcact ccagcctggg cgacactgcg 32100 agactccgtc tcaacaacaa aaaaaaaaaa atggtgttta aacacatata actaaattat 32160 ccttccccct tcccctgaag tggctggctc aggaaaaacc tctacccact caggcagagg 32220 ttttcctgca ccctgcatcc gtgaggcacc actgccaagg acgccaggga aggctgccag 32280 gcctggagag gggcagggcc ccctcccctc caaggggcca caaacgctgt ctgcgcccag 32340 taccgtgggt aaggcgaggc cggccggcta accccgggct ggcggccttg cagcgtgcgt 32400 ggcaacagca gctgggcccg caagactcag cacgggacgt cctcgtccaa gtctgggcca 32460 agagcagcgg cccagggggc ggggccggcc agagggagcg gggagaggct gaggggcggt 32520 gccagcgccg gaccctgcca ttggctggag attacaggag gcggggacat agcagggagg 32580 agccgctgga caagccccac ccggccgcca gggagggtct gaggtcaaga gccggagaga 32640 agggatttag ggccctgggc caagttgcac agcagggaga aggggctgcg cagaggggcg 32700 gggagaaagg gatccgcttc cttcctttag agctgtgaaa tgtccccggt tggaattaaa 32760 ggcggctgct ggggagaggt gaaattcagc caaaaccacc cagtcaggca gcccttctca 32820 gagataaaca gtccgagcca gcccggccag gaaccttccc ctccaacctc cctaagcctt 32880 taacactcct aagcctttaa cgcgtttaca cactcacata aataaacaca ctttgagcaa 32940 cacacataca ccactcacca catgtaatag gtcaagccat gtgcacgacg aggtgtcgac 33000 aatttcatat ggttcaacct agtacactca caaacacacc taccaactca tggctttcac 33060 agggacgggg tcacacaccc actctcccac gacatggcaa gcgtgcacac gctatctcaa 33120 gctgctccct ccccctcaag atcatgttac ccagttttat tttcttccca gcacctatga 33180 cgactgacat aatttattag tttacttgtt tattgggtta tctgtgcccc tcacccccaa 33240 aatgtaacct ccagcaggga ggatgactcg gtcagtcctg attgtgctgt agtccaggac 33300 ctagaacaga gctccatgga cattcatggg ctctgtacac acaaacacac acattaacat 33360 acaccccgac acacagcctc atccacacac acacagcctc acacctgctc tttgcagcca 33420 cctgcacagt ttctcacaca ctcacttgat ctagtgatct gcgtccacag gcccctcccc 33480 cagcccactc atactgccct caccccactc actctgccct caccccactc gggggaactc 33540 tgctgccagg ccaggcctgt gacactcacc gtgagtgaag tgaacgttag gcagatgcca 33600 ccaaagccat tcagggacag cgccaggaat atcaacggag acagagctgg aaaggggaaa 33660 gcagcagatg agggcatttg gggagctgtg ggaagccaag ggcgggagct ggggtaaaca 33720 tccgccttca tcccacctat tcttttcttg tggggccaca agaggacaga caactcacct 33780 tccacgtccc gggaggccag ggccatgagg gtgcaggacg cagtgaagca ggcactgtgg 33840 agacacaggg aagggcgagg ggttggcctg tgagcacccc ccctcccctc cccctgcagc 33900 acggtccctg tcctcccgtt ccccatagcc cagccacctc acctgccaac cagccgcacg 33960 ggtcgggggc caaagcggtc catgaggatc cccagtggca gggtggtggc gctgagcacg 34020 aaggaaccaa tggtgaagcc caggttgagc atctcgtcct gctggtcaca gcctggccac 34080 ctgcgctgct catcctgggt ggtgttggtg ctgctctcag ctgaggaggg ggaagggagg 34140 gctcagcaca tgacaccagg aacagctggg cacaggagac agcagcccac agtcaggcgg 34200 cctgctttca aatcccatgc caagtgcctt tgggggtacc ctagagtcac atctcctctg 34260 atggggctgc tccagaaatg gcagccatta gtacctgacc ctgggagagt cttgtgcaca 34320 cacagcctga ggcttcaact agctcaaatg aaatactgga cataaaagta tttactaagt 34380 tgtaatatgc actcagtgtc caagcttagg gggttgtgga cccccaacaa gaagtgcccc 34440 catatctaga ggcaaaggca aaggcagtga gtggtactct aatggctata acaagaattc 34500 attaaaatgg cccggcgtgg tagttcatgc ctgtaatccc accactgtgg gaggctgaga 34560 caggcagatc gcttaagcct acaagtttga gaccagcctg ggcaacatgg taaaacccca 34620 tctctaaata aaaaaaagaa atttagaaag aacactaaaa cttagaggaa gctttcccga 34680 taaatgatag tctgataaaa taatagctaa tacttattga gcacttaact atgctccagg 34740 cactgttata agtcagttaa taaagtatcc cgttccctag gtgatgaagc tgaggcacag 34800 aatgagaacc aggcactgcc ctccagtccc ctctagaagt ccacttggag gacttgtcct 34860 taacggtaaa ctgccaactt ggagttgtga caagttaagg agaaaagcta gtgataggag 34920 acaaagggct gcttcgcttt actcaatgct cannnnnnnn nnnnnnnnnn nnnnnnnnnn 34980 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 35040 nnnnnnnnnn nntccctccc tccctccctc cctcccttcc ttccttcctt ccttccttcc 35100 ttccttcctt ccctccatct tctccacacc tggtatcatc atacagaagc agagaggact 35160 gcacttggtg aaagtttcaa ttctcctgtg tggagaggtg agcactgagg aaggggtggg 35220 ggctgtcaaa ggagacttac ccaatctttc cagcccacca atcccttgcc cagtgtttct 35280 ataaaataag ggccttttgc atctgattta agtaggaagc tgattcctga gcccctcaga 35340 tctgctgaat cagatagcta agggggccct ggaatctgca ttttagcaag cggaggtggg 35400 ttatgaagca ctccaaagtt tgagacacgc ctcaaaggtg gagtggttct gtggggggca 35460 gaaaggaaaa tgcaaagggg gaaggggtca cacttgggga aggtttcaga caataccgag 35520 tggaaagggt gatgccaggt gtggggagta acagatagag gaggcaaagt gagtggagac 35580 caagccagac cggggaggag ggggccacag ccaaggtgag acaggtcagc agccagaaac 35640 cgaagcagac acttgcaggg tgcaccccgc cctctcttcg tggcaatctg agaccgagga 35700 cgtggagacc ctggagagcc cccaaccttg tttctggggg gtgggtcaga gaggaagcct 35760 ctcatccccg ggcaccagcg gccttcccgg gaggctcaac acgcagatac ctggataggc 35820 gtccatcact cccccggcca gagcccacca acgctcctcg aggtccgacc ttgtccctcc 35880 ttctacccca cagtccccag tcctagctca tctgcataaa gctccaatta acatgttttt 35940 cctttgctat ttgcgatccc agaactcgtt ccccaccccg agcccgtttc ccgccgcttc 36000 ctcgcccctg ggagggcggc cccattaacc ctcgcgaccc gggccgctcc tggcggtcct 36060 gaccccgcca ccccgtcccg cggcgggggt ctgggggtga ggggcgcgcc ctggggcaga 36120 ggattgcgcg gcagggtctg ccacagggca gaggccaggg ctctccggga aaaaggcagg 36180 cgcatatatg cccccctttc tgggaaaaga cggggagggg ggcttctcct gggagactcc 36240 aggcttcgaa attcctcgtt ccctatcctc cggcccccgc acccctcctc ctccccgcca 36300 cgcaccctct cccctcccca gccatctgtt ccactccgca gcgccgcgac aaacacggct 36360 ccagctcgct tccgcccctg cccagccccc tccccaagcc ccggggagtg ggggagtgag 36420 cagacgccct tctcctagga ggccggaatt tctgcctcca tctcccaccg gggtccggct 36480 ggccagaggc aagcttcgag accccccacc aaccaccacc accgttgcga gggccggtga 36540 ggctgcagat aacgcttgca aggacgggag tcggggaggg tgtagggcga gtttaaagga 36600 cgggcagagc aagccccggg aagaggcagg ggttttccct cccgggtcgc cgccccccgc 36660 accctcggag ccagccgcag ccacgcagcg ccgcctgccg ggcacaccaa ggacctggcg 36720 cgcacgtggc gcttaccccc acccccgggt ccgctcctgg ctcgcgctca gcctccccag 36780 actattcgca aattgaggat cccggacaca gagtgcagag accccggcaa gcctactgaa 36840 agccagccga acccgctggt gggtgctagc caattctgat tttgtacttt acaaaaacaa 36900 aaaaagtcag tgttggaagt cgggagtctg ggctcagagc agcagggatc tgcgatgtga 36960 ctttgccaag tctccagacc cctgaggaca ggttttccta tctgaaaacg gaggggacag 37020 tctctcttat taacttctca agagaaacaa agacaaaggg agggaaaatg gcttagctgg 37080 aatgctgtct tacagagcca acctttggag gtgggggaga tggccaaggc ctctgaggtc 37140 actcttggcc ccaggagcag ctgagaaccg gaaagaagct tgggacctcc tttctgcaga 37200 gctatccttt ccacagactg ccgaggttcc aaattgagct ccaccaccta acactgtgtg 37260 cccttgggtg tgtgccttaa cctctctggg cttgtttcct acagcgacaa gaaagaatga 37320 caacaccaac ctcttaggct atagtttgga taaaatgaga tagctgtgta gaacagacag 37380 atcctaaacc aatgttagtt ttcccttcat ttggggactt gctctaacct ccagggctta 37440 tgtcccagag gcacaagcag gtgcagggct ggataaataa ggtatgtctt tctgcaggat 37500 ctcttgtcct cactgatggt gtcttctctt gatatagata attttaaagc ttcacgttat 37560 ttatttattt actttaaagc ctcactttaa tgttaaaggt aaatgtaaat atagtataac 37620 aaggaagctc aaaatttgca taaagtttta agataaaata ggagactcca aaaaagtgtt 37680 actttcggca ggccctaggg atgctatggt gggaagtttg agtcatacct tagcattctt 37740 tctaaagcat tctgtcctaa tcctctgtat ggagaaaagc cagcttcctg gatgtacccc 37800 aaatcctggg aagtaggggg caggagctgg actccctcca agcactaagg gcagggcatg 37860 gttgggaaca gggaggtgag ccagacagcc agaggcgaac gggctggcat gccaagcgtc 37920 ctagttaatg cccagctgag cctgggtgaa gaaggatggg ggtgtgggga agacaccccc 37980 caccaaccgc caaagacagg cgcacaccag ccagtctctc acttcccttt ttatttcctc 38040 taagacttgc aagcagcagc accagagagg gaacctgccc tcctggccct ggaaggggcc 38100 gacccccaac ccctaaccca ggacacagct ggcacctcag gcccctttcc ttctgaaagg 38160 agggctgtgt ctctctcaca ttcacacata cacagacaca tgcatgtgtg cacactcatg 38220 gcacatggga cctcaggggt agcctgtttg ccgatccccc caagaggtac caggaggcag 38280 accgctagaa ggagataaga ggcaccctgg tctcctccaa cccaaggagg aagaaagctc 38340 aacccctcta ggatagggac tgtcttcagt caatggagcg ttgacttagg gggcgttttt 38400 gaaggttttt tttcctcctt tttgcagtct ttacaaaaat agaacttctc ttggtattta 38460 taaatctacg gccatggctc tatgtgcatg ttacaggtag aaaagccata tggggcactc 38520 cttttggttg ctcaggcctt gattgcctgt catccaggtc ccttggtctg agaagtctat 38580 gcggtcacct cagagccgct aagcaccttc agtgggccca tcccattggc ggcgtactcc 38640 tgctggagcc gggcacggta atagaagagg taggaaggca acaggaatcc caggagtgag 38700 aatagcagga ggcccagatt cacctttagg gcaaggagag agaaacagag tcaagtaggt 38760 agtcatctgc ccttagcctc ccacagggag gagaaggcgg ccatttttct ccaggtcctg 38820 agccagaata aatacagcta gtacttatta tgtgtagtca ttgttccacc agtatctcac 38880 ttaatgttca gcaattctgc aaagtggctg agatgagact tctcaggtat aacaagtggc 38940 agggcctggt gggtgcccac accatatggc actcactagg taggtatgag gaaggcacag 39000 cactgtagga gtctgggctg gtcaggctgc tcccgaaatg gggccttctg ggctcacccc 39060 tctgaccttt ggagatgtta accaatggga tcccgttcag ggtggcgaga ggaggctctc 39120 agacacagtt caaggaactg ggatgcacag cctggtggac agaaggcttg gaaggcccag 39180 gacacgcggg ctctgactcg gttcacatcc cactctgcat tactcactgt gtgactttgg 39240 gcaaataatg gcaattctta ctgagtgcct ccttctcagg gctgttgtgg cgaagatgta 39300 agttaaaaaa aagtatgcat catgcttagc acatagtgag tgcttggtaa atagaagcag 39360 ttatttcatc acaattcttt gggaggaggg tttacgtgtg ggtggcccca cagggcagat 39420 gaaagatcag cgtcagggag gcagatgagt tcaatgtaag gaaaagactt actaacagca 39480 gcagggctgc ctcgtgcagg agtgggtgcc ctaccactga gggtatctaa gctaagaggg 39540 aagggtcccc tttcaggggt gctggagaca ggatcccaca ctaggtagaa ctggattgga 39600 ccaatggtgc ctgaacacag gcccaagagt caggactggc cacttcacaa agcacctgga 39660 gtttactaaa aacagactcc taggaggtca ggcactgtgg ctcacgcctg taaccccagc 39720 actctgggag gccaaggtga gaagatcatt tgaggccagg agtttaagac tagcctgtgc 39780 aacatggcaa gaccctgttt atctgtacaa aatttttttt taaaaaatta gccaggtatg 39840 gtagccatca cctgtggttg cagctactca gaaggctggg gccggaggat cgcttgagcc 39900 caggaatcag aggctgcagt gagctgtgat tttaccaccg cactccagac tgggcaacag 39960 aacaagacac cttctctaca aaaaaaaaaa aacaataggg ccgggcgcgg tggctaaggc 40020 atgtaatccc agcactttgg gaggctgagg agggcagatc acgaggtcgg gagatcgagg 40080 ccatcctggc tagcacggtg aaaccccgtc tctactaaaa atccaaaaaa aaaaaaaaaa 40140 ttagctgggc gtggtggtgg gcgcctgtgg tcccagctac ttgagaggct gaggcaggag 40200 aatggcatga acccgggagg cggagcttgc agtgagccga gatcgcacca ctgcactcca 40260 gcctgggcaa cagaatgaga ctccgtctca aaaaataaaa ataaaaataa ataaataaat 40320 aaaataacaa taaattaaaa acaaaaacag actcctacgg tcaggctgag atatcctgat 40380 tcaggggact ggggaatctg tatttttaac actccgtgag gggttctaaa aggcagacaa 40440 cttggaaacc tgcagattag agacctctga ggtgcctctg gctgagatga gtgagggatg 40500 gcaccacata caaggcccta cccctgcccc caggagagtg gctcctgctc cccccacacc 40560 aaccctcgct ctcacccaga agggctctcc tttcaggggt cccaccatcc ccatgaaaag 40620 tggctgctga agcaaggcga acacagcact ggtgagggac tgcaggcctg tcagcgtccc 40680 aaaaggggtt ggatgggaac ctgtccccaa aacgggagat caaagggtgg tgggggcctt 40740 tcagcccagg caagaacttt ttcttttcct tcccaacatg ggnnnnnnnn nnnnnnnnnn 40800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 40860 nnnnnnnnnn nnnnnnnnnn nncactccag cttgggtgac agagtgaaac cctgtctcaa 40920 aagaaaaaaa aatcttaaag aataaggata taaagaaaga aaatattttt gtgtagctgt 40980 tcaatgtttg tatttcaagc caagtgttat tacaaaacag tcaaaagttt ttaaaaattt 41040 aaaagtttat aaagtaaaaa agctaagtaa gctagggtta atttttttat cgaacaaaga 41100 aaaatatctt tgtataaact tagtgtagtc taagtgtaca ttgtttttat tttatttatt 41160 ttttattttt ttgaaatgga gtttcactct tgttgcccag gctggagtgc aatggcatga 41220 tcttggctca cggcaagctc tgtctcctgg gttcaagcga ttctcctgcc tcagcctccc 41280 aagtagctgg gattataggc acccgccacc atgcatggct agtttctttg catttttttt 41340 ttgaaatgga attttgctct ttgacccagg ctggagtgca atggtgcaat ctgggctaaa 41400 tgcaacctcc acctcccagg ttcaagagat tctcctgcct cagcctcctg agtagctggg 41460 attacaggca tgcaccacca cactcggcta atttttgtat ttttagtaga gacagggttc 41520 tcaactaaag agaaccatgt tggccaggct ggtctagaat tcctgacctc aggtgatcca 41580 cccacctcgg cctcccaaag tgctgggatt gcaggcatga gccaccatgc ccagccagta 41640 tacagtgttt ataaagcctc cagtagtgta cagcaatgtc ctagaccttc acattcactt 41700 actactcact cactcactca cccagagcaa ctgccagtcc tgcaagctgc atgcatgata 41760 agtgccctat ataggtgaac cattttttaa tattttatac tatattttta ctgcaccttt 41820 tctatgatta gctacacaaa tgcttaccat tgtgttacaa ctgcctacag taatcagtac 41880 agtactatgt atgggtttgt agcctaggct ataccatgtt gcctacgtgt gtagtcgtct 41940 atactgtcta gtttgtacac tctatcatgt ttgcataaag ataaaatcac ctaatgacac 42000 atttctctga gtgtattcct gttgttaagc aacacatgta taaacattta caagaaatag 42060 ctcaaatttt tttttctttt gatacagggt cttgctttgt cacccaggct ggagtgcagt 42120 ggcgcaatct cggcgcactg cgacatctac ctccccggtt caatcgattc tccggcctta 42180 gcctcctgag tagttaggac tacaggcacg caccaccacg cctggctaat ttttttgtat 42240 ttttattaag agatggggtt ttgccatgtt ggctaggctg gtctcgaact cctgacctca 42300 ggtgatctgc ccgccttggc ctcccaacat gctgggatta caggcatgag ccaccatgcc 42360 cagccattac gtttttttgg ttgtttaatt tttttttttt taagagacag attctcactc 42420 tgtcatcaag gctggagtgc aatggcacaa ccatagctca ctgcagcctc caactcctgg 42480 gctcaaggga ccctcctgcc tcagccttcc cagtaactga gactacaggt gtgagccacc 42540 atgctcagct aattattttt tatcttttat tttttgtaga gggggggtct ttctatgttg 42600 ctcaggtttg tctcaaactc ctgggctcaa tcaattctcc tgctttggcc tcccaaaggg 42660 ctgggattac aggtgtgagc ctgaaaacct tctagtgtgg aagtggaaga taggcccagg 42720 ccacttatgt tttcaagtta agcaaggttt aggtcactta tgaagcctga ctagttttgt 42780 ttgcttaagg gatctgcagg cctgacctcg gttttcattt gttttaacag tgtctatgtg 42840 tatgtgtgtg tttatgtacg tgcatgatgg ggggaaagct cagaaatcaa gtaagccaaa 42900 cacaaacatg taattataag cagggataaa ttctatgatg aagaagtatg ggccacggga 42960 gagtacttgt gccagtctgg tgatcaggaa caatgtcctt tgggaagtga catttgagcc 43020 atgccctgaa gtacggtagg agttggttag gggtgaggca gtaagaccca gagctggggc 43080 ttcctgcaca agctcagctg ggcactgagg acccagtgga ctctgctaca gggcagtgag 43140 gagcagaaag gctgaggaag gctgggtgtg gtggctcaca cttgtaatcc cagggctttg 43200 agaggctgat gggggaaaat cggtagagct caggagtttg agaccagcct gagcaacata 43260 gcaagactcc atccctgtaa aaagctttta aaaattagct gggtgtggtg gtatgcatct 43320 gcagtctcag ctactcaaga ggctggggta aggattgctt gagcctagga ggtggacgct 43380 gcagtgcgcc acgattgtgc cactgtactc caacctagga gacaaagcga gatcctgtct 43440 caaaactgaa tgaataggct gtgtgcggtg gctcactcct gtaatcccag cacttttgga 43500 ggctgaggtg ggtggatcac ctgtgattgg gagtttgaga ccagcctggc caatatggtg 43560 aaacccgata caaaaattaa ctgggcatgg tggctcacat ctgtaattcc agctactcgg 43620 gaggctgagg catgagaatg tcttgaaccc ggggggcaga gggtgcagtg agctgagatc 43680 gcaccactgc actccagcct gggagacagc gagactccat ctcaaaaaaa aaataataat 43740 aataacaatt aaaaaaaaat taaaaggcca gggagcactg gcagcctgtc caaggtttca 43800 ggtcacttta gtaaagggag aacaatggct cctcccagga cctctgggat ctcagcattg 43860 atacgacagt catggaaatg ctagggccca ggcagaccat ctcagggaaa acaagtggct 43920 ctgccctgcc ttggccactt cctggccctc tgcatgcccc agggtctcag caccaagctg 43980 ttctcagtga gtagctctca tttagtgcca gggctctcgg gcttacatcc tacgatgacg 44040 atggaatgca taaaagatgg ggctgtgata gcccagagct aggggtttga atctcatgag 44100 atgttcatgg agccctggga gggagctcag tgcaagttca tttctctttt ttggttgaga 44160 tggggctcag aggaggaagg acttgttcaa agacacacag ggagtgtttc agtgtgggac 44220 ggaggtttat ggagaaaggg tgaccatcca aggcttggac aaagatcatg acttcgacca 44280 gcaagcctca actctgtaga cttggtgggg gccaggccct cccaaacaca cctgacaggt 44340 gtctgtggtc ttggggacat tgtcgctccc cttcctgctg atgctctgct gtccctctcc 44400 catgaagcgt atctcttcgc cgtcccccat ccttgctgag agaggatggg ttctcttctg 44460 accaatactg aagatcttta gtaaagttct cttttttttc attttctgaa agtccctctc 44520 ttgagaaatc aggacaagtg agtcagggcc aggacaaaaa acagtgtggg acgagtgtgg 44580 tggctcacgc ctgtaatccc agcactttgg gaggccaagg tggcggatca cttgaggtca 44640 tgagtttgag actagcctgg ccaacatggt gaaacctcgt ctctacaaaa tacaaaaatt 44700 agccaggcgt ggtggtgcat gcctgtaatc ccagctattc gggaggctga ggcaggagaa 44760 tcacatgaac ccaggaggcg gaggttgcag cgagctgaaa ttgggccact gcactctggc 44820 ctcttggcaa cagagccaga ctacctctca aaacaaaaac aaaaacaaac gacaaacagt 44880 gtagactttg tgtttttctc aaaagcactg tcaagccagt gcccgcagca gtgggcctag 44940 acacctccag tcttgcctca gggtcagttt ccagcctccc tggacacttc ccccaggtat 45000 gtgtactttt tgattgtcct aaatccagag tctgtggcct gacctggttt gtcacagctc 45060 tcagtccctc cccatcccga atcccaggga gccgcaggtg tgtgcagaag aggcacacca 45120 cactcaatac atcttgcatc ctcgctggac ccaatccatt ggcttggtga tgtacagact 45180 gagcctcatt atagccgttc gttcctgttg acctttccag atcaatctgc cagcttggct 45240 tctccgagtt tcgcttgtca gcatttctcc aatcccatca tgtactttgg acctctttgt 45300 tgggtggctt gctttatctg aaattttcag atttgacttc aggtctctcc tttgtccctt 45360 aatatggctt aatggtggac cctgtcaggg gtagagaaaa tattgaggag ccctgacttt 45420 gaggtgcaca agttagaggg ttagacaagt ccagccacaa ccagcccaag ctgcagtgta 45480 gggaggcctg tccagctgct ccacggttga gggtggagca tacaggaagg cttccttctt 45540 gctgcagccc aggtgttctg gctgccctag ctgcctggct ttggtagaag aaagaaaggc 45600 tctgtctctg acttgtcaac taatggcact atgagattgc acataattaa cctgggtctg 45660 ctcttccaaa agccttgggc ctctgactgc aacatggagt ctgggtatca ctccccatcc 45720 ctgcgccact cacctgctct ggcgctaggc gtgtgcctaa tcacttaatt tctctgtgct 45780 gcctcttagg tatcacttcc cctgatccca aatacttacc aggtgtggga tgacacctga 45840 ctagttactc cttggaggta tctgcttctc accggggact ccgaaaccaa acgaaaagca 45900 aggccaagcc cagcctaaag gacgcttcct acatgacttc aggcttgcgg gggctggagc 45960 gtgggggtgg caatggagtt ggggggggct cagggagggg atgtggaagt gctttgcttt 46020 gcaaactcta gagaaccgtg taaataggag tgattattct gtcccttccc tttctttcca 46080 acaggaatca gcatcccaca gcccatgttc agctatgaag aatggaaact gaggctccgg 46140 gaggggtata gggaggagcc agcagggtct tgagttcata ttagtgccct ttcctccata 46200 ggcacatctg tgttttcttt tattttattt tgaatttaat tttttttttt tttggcagag 46260 tcttgctctg tcgcccaggc tggagtgcag tggcgcggtc tcagttcact gcaatctccg 46320 cctcctgggt tcaagtgatt ctcctgcctc agcctcccga gtagctggga ttacaggtgt 46380 acaccaccac acccagctga tttttgcaat tttagtagag acagggtttc acagtgttgg 46440 ccaggcttgt cttgaaatcc tgacctcaag tgatctgcta gcctcggcct cccaaagtgc 46500 tggtattata ggtgtgagcc actgcgctcg gccacatctg tgttttaaat gagaggaaag 46560 gggataatgt gcattttgtg gaagcttggg ccgtttgtgt ctaggactct tatgatcttc 46620 ataagttttc ccccagggag gacactgttc cacttaggga gtcaggaccc ccagtcctta 46680 caagattcag cctctcaaaa tggagacagc agttccaggc ctgggctggg ttctgttcac 46740 actaggagag ggcaagtgag tggtgtttgg gatgtgggga agtattatga aaacagagat 46800 gctccaattc ctagtgatag gaaaccatta agctacttgg catcttaaaa ccaagagcgg 46860 ttcaagttct gagattgtta acacacctta caacaccgcc gccgttatta ggaagaagct 46920 ctgtttgatg acgtcccaca ctgtgggtac ctttatgaac aggaatttgc tttttcaaat 46980 cccagagaag taagattaaa gttggctgtt ctccatcctt gaaaaatttg gttttagggt 47040 gaattcaaga atgactgacc atacagaatg gggagcaaac ttgggaagaa agaaggcaca 47100 gttcagagct ctcccaatag tcacccctga actgcacccg gaccatcagt tatctctgtg 47160 ggtagagctc aggaatctaa aatccatttt aaaattaaag tatatcgggg ctgggcgcgg 47220 tggctcatgc ctgtaatccc agcactttgg gaggccgagg tgggaggatc acgaggtcag 47280 gagtttgaga ccagcctggc cacatggtga aaccccgtct ctactaacaa tacaaaaatt 47340 agccaggcat ggtggcagac acctgtagtc ccagctattc ggaaggctga gtcagaagaa 47400 ttgcttgaac ctgggaggca gaggttgcag taagccaaga ttgtgccact gcactccagc 47460 ctgggcaaca gagggagact ctgtctcaaa aaaaaaaaaa aaaaaattaa agtatgtcat 47520 acatactgtt acaggcacag accttaagtg tacagcccaa tgaaatttta cacatctata 47580 cagctatata actaccacct atatcaagac acattccagg aactcagact ccatcatacc 47640 cctcctcagc agaggtaaca gacccacacc tctcctgctc cggtggtaat taaccactat 47700 tctaactttt ctatcaatta gttttgccca ttcttgagct tcacacagat atacattgtc 47760 aggcatgatg actcatgcct gtaatctcag cactttggga ggccgagacg ggagtatcac 47820 ttgagcccag gagttggaga ctactctgga caacatagtg agacccccga ctctacaaaa 47880 aaaataaatt agctggtcat ggtggtgcgt gcctgtagtc ttagctattt gagacgctga 47940 gagaggagaa tctcttgagc ctgggaggtt gaggctgaag tgagccgtga ttgcaccact 48000 gcactgcagc ctaggtgaca gagtgagatt ctgcctcaaa aaagaaaaaa tatggccggg 48060 cgcggtggct caagcctgta atcccagcac tttgggaggc caaggcgggc ggatcacgag 48120 gtcaggagat ggagaccatc ctggctaaca cggtgaaacc ctgtctctac taaaaataca 48180 aaaaaagaaa gaaaaaaaaa ttagccaggc atggtggcgg gctcttgtag tcccagttac 48240 ttgggaggct gaggcaagag aatggtgtga acccgggagg cagagcttgc agtgagccga 48300 gatcgcacca ttgcactcca gcctgggcga cagagtaaga ctctgtctca aaaaaaaaaa 48360 ggaaaaagaa aaaatatata tacattgtgt actttttggc atctggttta ttttgctcaa 48420 tatcacatct gcgaaattaa tctacactgt gtgtatgaaa ggttggttct ttttgttgtg 48480 atgcagtatt ccgtcgtgtg actacgggac aatttgctta tccgtattcc tatcggtggg 48540 catttgggct gttaccaggt tctggctgtt atgaataaag ttgctatgga tattcttgta 48600 cactacttct ggtgagcgta tgcactcatt tcgcttatgt aaatatcttg ggtggaatta 48660 cctgatcata aggtaggtgt gttggctttg taatgtgctg acttggttat gctgaattcc 48720 cttttttgtg tatttctggt tagagcggaa catgagggtg tctcttcagg gaatctggag 48780 ggtggaaggg aagcaggagt cggtttctgg ctcacacatg ttgtgactga actgctggta 48840 cacctggttg gcatggagct ggcttctcct ttggcgttgc ctactgttgg ggcaggtgtg 48900 tatgtggtta gctccatgca atgaacccgg gcttctgcaa aatacattaa caacgacaga 48960 gacaacaaaa gctgatgtgg atttaaaggc ttcagttcan nnnnnnnnnn nnnnnnnnnn 49020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49080 nnnnnnnnnn nnnnnnnnnc cagcagtggt tctcaactga gcatagtttt gcctcagagg 49140 ggacatttgg taatgtctgc agacattttt tgattgtcac agcccagccg agaaggtact 49200 actagtatct ttttggtaga ggctagagag gctgctaaac atctaacaat gcacaggaca 49260 ggcctctgta acaaaaaagt atccagtcaa aaatgtccac agtgttgaga ggtttaggta 49320 agtaggcgct aaaacataag gagactgtgc ctgagagcaa gaaggagtaa ttggaaagtg 49380 ctggtgtgat tagctctggg ttttagaaag ctcattttgg ctgcttgtag acagtgcatc 49440 agaggtggag gagggtggta agactggagg cagggaaagt aatttgggag ccactgaaat 49500 gatccaggtg aaaaacggtc agcaggtgac taggaaagtg gcagaggcaa tggggatggg 49560 tggctggatg agatggtgaa gaaagcacta taactaacta atgtgtggat gatgggcagg 49620 aggggtgaag gatgaccaga gtcctgcctt gcaggtctag ttggaaggtg atggtttctc 49680 ctgagaaagt gaccacaaaa agtgaagcag gtttgtgcgt gtgtgtgtgt gtgtgtgtgt 49740 gtgtgtgttg agttcagtct gagatgtgtt ggactcacaa tgtccatggg acatccaagt 49800 ggagaagcat cttgggtgac catatgtgtg agtctgcagc tcagaaacag gcctggggct 49860 ggagatgaag acttgggaat gatctgcgta tatatttggt agcttgagcc acaagagtag 49920 atgacataac ccgtggtggg tgtgcagaat taggagagac gtgcaccaag aagccaggtg 49980 atccccaata tttaaccatc tggaagaata agaggagcct gccaacagaa attgggaggg 50040 aatggccaca aaggctactg agaagggaag cagttcttaa gaagggggaa gtgaagaggt 50100 atcactactg cagaggtcaa gtaggataag aactgaagaa tgtctgttgg gtttggcaat 50160 ggggtagtca gtgggcacct gggcaaaagc agttttggtg gagcaatagg gataacagaa 50220 acaagactgc tatggtaaga ggaggaagag ggtgttgagg aagtggccag cgagtctaca 50280 ccacttgctg gaggagcttg gctttggtgc aaagcagaga agccagctca ctcattgact 50340 taacctccaa gaaacacaaa atcatccata tcctggctca aattccagca ctaccaggag 50400 atggttggcc cctagaaatg ccatcccact tctcctctgc ttatcctatc ctatctgtca 50460 gtctgttgag cccaggctaa gcgctacctc ctcaagcaag ccttctctgc ctgccgtcac 50520 actttaagtg atcctgacaa cactgaaaat gtgtgtctct tccattcatg ttagttctac 50580 acttctgagt atctcctcaa tatattgcct tgttttacta atatgctcgt tctgtttgcc 50640 ttatttatca gctaccttaa acctccctgc aactagagat tctctttaag tatttgttga 50700 ataaatgaat gaatcaatcg atgatccaga gcctggtaga ggcttgtgtc catggtggat 50760 gaggctcaga aaatacctgt agaatcgaaa taaatgcatg tgtgctctga tctaaactca 50820 gctaaacttt ctccaggggg taaagttcaa gttgattagt caattgatta attaattcat 50880 tatgtaatgg aaaaactcct tctatgacct gggcagagtt ataggcagtg aacaagacag 50940 acaaggtcct tgttgtcatg aagtttgctt tctgaaggag agagataata aacaagaaac 51000 cagtaagaaa gcaagattat atcattttgg taaatgttct tgtggaaata aatgtgatga 51060 tgtgtaacaa aagtaccaaa taggagagtg gggtgggtgg gcttctttta gaaagagttc 51120 tcggagaagg cttatctgag gaggtggcct tttaaccagt acaaatgctt tagcttggcc 51180 agtggagctg ggaccaggat gacaagggtc acttgtcatg ccagtgagtt tgagcttgta 51240 gacaagagcc tgatcatgaa agactttgca gatggtggta atgggtttgg gttaattgct 51300 actatgtggg aagactttga atgggaagca tggggacaat ggcctgtgat acatgttatc 51360 aaatatggtc gcaggggcta gtgaggtggc agcagagata gggagaagta gacggactgg 51420 ggaaggtaga agatggggca ggggaggcaa ttactgcaaa gacatattcc ttctaagctc 51480 actgagtgtt catggtctct gggagcagag gttcctggag gggaaagagg ataatgtcac 51540 ttcctgagga agcgggaaga acccatctga gacgtgggga ctgtgctggt tcgtttctaa 51600 ggggccttcc agatctcaca tgccaatcgt cttggtctat gtcaattgtt ggggcatcca 51660 aatggggaac tgttgtccag gccgatttca cagaacaacc gcccagtcca tatctcccga 51720 gccattcacc cttgcagtgg cgttagctct ttcaccagct tttatctgcc ccgtggggat 51780 gttggccaag cccagttaac aagcagttga tcagccccag agatcaggtc cctggagtct 51840 gtcacttttc tgagggtggg gagagaatcc tggagcagaa catgtaacta gaagggccac 51900 ctggcttcct atggtctgag ggagagaatg gtgggatctc tggcctgaat caaacctccc 51960 tttctcagtg tccatcttac ctctctgctg taccttcgtt attttccagc agctcctcag 52020 cccgttcctg tgggaccctt ctctgccaat ccctacaccc actgtaaatt tcaccgtggg 52080 agggagatgg gccttgaggg ctgtattagt cttctattct gcataacaaa ttgcctcaaa 52140 tttagcagct tcaaacaact catgtttatt agctcatcgt gagttcatca gcagtgtggg 52200 cccagcatgg ctaggttttc tgctcagggt ctcacaaggc taaaatcaag atgttgtctg 52260 ggctgtgtgc tcatctggag tttagggttc tcttccaggc tcacgtggtt gtggcagaat 52320 tctgttccct ggagttgcag ggctgaggtc ctgttttctt gctgactgtc agatgagggc 52380 tgctctcagg tcctcgaggc tgcccacatt gcttgccacg tgcgtggtct tttccatcct 52440 tgaagccagt gatggagaat ttcccttgga ttgaatcacc cacatggttg gactctctga 52500 cttcaggaag agagccctgt ctcttttatg ggatcacctg attagatcat acccatagag 52560 ggcagttcct tttccttaaa gtcaactgtg gcatgtaaca tcacacaacc acaggagtaa 52620 aatccatcat atttacagtc ccagggatta tgcacagtgc accaggggac aactgaattc 52680 tgcctgtcaa aagggccaag caggacttta ttggtgaaga acagtggaat gtcattcttg 52740 gttcttccag aaaaaaatca ctcagtaaag ttagaggttc tcttgccttt tgggaagtca 52800 tcaaagaatc tcatggaggg tttggacctt caccctagaa acatcacacc atgttttcta 52860 taattgcagg gttcatggtc ccttgaagcc tattcatagt ttccaggttg aaaagctctg 52920 ctgcagggtg tggggaggga tgcaggtgga ggtgagggct gaatagtgtg agctgcatat 52980 ctggagctgt ggtggttttt ttagtcttta agctgtcatg tgttgggggt tgggcatggg 53040 aggggcatcc caagagctcc ttggtattga caccatctcc aaggtgatct ctgctctgcc 53100 tggtgcacac atgtttttct cctgttgcaa cagcccactc ttgtagaaga gcagacccct 53160 cagtaccagg tctgaccctg gacagcttgt accaggagct acagcacact cccccacaag 53220 cctaaagttg ggatgagccc cccgagaatt agatcagaaa agattaaatg cagaggtgat 53280 ctgtcaggtc ccctttggaa gtgctggtat ggagaggatt gactgagtct gtttaggaac 53340 ctccaagctc tgtagtaact ttagggctag aaaggaggat gcctaagatt caggatcctg 53400 cagtgatgag tcaacatttc ttggggaagg aggcagggct gaggattaaa cggagatgat 53460 gggtatcgtt ctcttgctca aaggcactgg accccaaggc ctccagctct tcgctcccat 53520 ttgaaattca agtcctgagc acaccacagt tgtgatgcag ggaaagaatg tgcttatcag 53580 agagcctggg caagtgggcc ccttgtgagt accgttcaac ctcatttatg tcattggcac 53640 caaaagtaga catcagtctc ttgaaagttt gattaatgct ggtcacactc aaagaccctg 53700 ggtagcattc atttactaag caattactaa ataccagttt ctgtgctaaa tgctgcatca 53760 gtcagggctc ttaatggcag gcagcagaaa ctctccttgg ctgatctaag tagaaaaatc 53820 caggactgaa aggaaacgga gtagctcatg aaattgcagg aagggccgga aaaccagaca 53880 tggagccaaa gtcaggctgc agaacaggtc tagggaggat cccactgctg ctgagaccta 53940 gaccttgtgt ctggcaccca ggatgttgta gggctcagac cctggatcaa tgtatcctgc 54000 agtgcctctg tgggtactgc aactccagga actcaatctt gtcaacgcca ccgccagaga 54060 gaggccttct tggcctccat ctttttggtc actagctcca gattcaaaat cttgaataga 54120 tgcttcttct ctttgataga gcccagtcat atgcgttagc tgcaaaggaa gctgaaaatc 54180 tattaggaac ttttgtcttc aaaaatgaga ggcctgtcct ccaccaagat ccataggaaa 54240 tggaatccaa gaaaccacag gaaggggtga ggtgactggg cagctcacag catgcatgct 54300 acatgtgaat tatctcattc atttctcaca ctacccagtg aggtaggtat tgtcatccct 54360 acttcataaa tgatgatatg aggtacagaa agtttaagga acttgcccag gacacgacac 54420 gcagctatta agtgctagac ccagtcaatt tgagtctgac ttggactgtc tgactccaga 54480 agccaccctc tcagacactg ctgtatactt ccagtgaatg ttgatgaaat tttcagggtt 54540 gctaagctgt ggatttcaga tcctggattg tatgacctaa aagagagact tccctaggag 54600 tgagggtccc tgaacagtca actggtttcc aagaatgggc tccctctcat caccttatga 54660 cagtaatcct ctgtccaaca gccaaagagg tcctgtgggg agggcttgca gatgggagtg 54720 cgcagagccc agctcaaagc tcctgactag gctcttgttg agtattcctt tgattcctgc 54780 ttctgtcttt ttaaatcaat ggagacaggg gagggttatc tccatcctcg gctcaagatg 54840 aaatgcatcg ttcctcgttt ttctcattcc ttcccaatgt gtgtactgtt aactttagtt 54900 atgaaggaaa ttacagtgtc ctgtgcatat accaaggctg tccaacctcc acacctttgc 54960 tcaagctgtt ccttctactt gaaatgcctg tttccttccc ttctaattgc atctttccat 55020 ccaggtagga atcagctcct tggttcatgg agccttttct gctctgtttt actatgcatg 55080 gacttccttc tgaattagca gaggatgttt cctagcttgg tcttaaccct tctccttttg 55140 tttgacctca atttactcat cttacaaatt aggttgtaag ctaattgaat acaggatcta 55200 tgcttcactc tgattttatc tccacctgga tagcatcatt tttgacacac aagcaggcat 55260 atgggagggg agagaagttt ggtgccagaa agaactggat ttgaattcta accctgttgt 55320 ttacgtgagt acgttactta accattaatt acttcaatgt atatttatta agtacctact 55380 atgtgccggg cactgtacta agcaccaagg atacaatggt gagtaaagag atgcagcctt 55440 caccatcacg aaggaagaca gatgttaatc cattaaccaa gtaatctcac aagaaaagta 55500 aaatgactaa ctgataagga caagcccctg gagctacaag agggtgtata cagggcatcg 55560 atccaataag ggcagtgttg cggggagatc aggagccaca cagagcctgg gttgtctcac 55620 ttggaaaatg gggtatcaac cacctacctc actaggtttt taaaatcagg ttaaatgagg 55680 taatacttgc catgaacagt attttgttga ttgatgattg attgaaacgg agtctcactc 55740 tctcgcccaa gctggagtgc agtggtgcaa tctcagctca ctgcaacctc tacttcctgg 55800 gttcaagtga ttctcctgcc tcagactccc aagtagctgg gattacaggc agccacccct 55860 atgcctgact aatttttgta tttttagtag agacaaggct ttgcaatgtt gaccaggctg 55920 gtctcaacct cctgacctca aaagatccac ccacctcagc ctcccaaagt gctgggatca 55980 caggcatgag ccactgcatc cagccacttg ccatgcatgg catttaaaaa tgttcagtaa 56040 atgttaccat aatgaaggct ggtaggttgg ccaactgagt ggtctgattc agaaggaaag 56100 aagttagaca tacgtgaaca tttcctgtac ttgaagatcc tcaggacagt gactcctaga 56160 cccatcttcc atcacagtca gctgggaagc ttttaaaaaa atgcagacat ctgaccttca 56220 cgctagacct attagccaag cagaagtttc tgggcagggc atctgcatat ttttaaaaat 56280 ctttaataag gcagcctcaa aattacagat tcagcacgca tttaccataa ccactgaaga 56340 aatgcaaagt tataaaaaga agataaacaa caatctgtct cctgctttct tccctctcct 56400 cccctgcttc tggaggcaac aaggtcaact atttggtgtg attcctttta gcattccctc 56460 catcaatggt cacataagga tgctcacaga taagcaccta tgcgggggtt ttttttttcc 56520 ttgtaaaact attcacatac taaatacttt cctcagtatc ttgccttttt tcacttcatg 56580 tcacagaaac atctcttcag gtttatagat acaggtccag ctcttctttt catagccata 56640 taacattctg tagaatagag aggacacatt ttactcagtg tccgattgat ggatatcaat 56700 attgttttca tttctacaaa tagtcaagga ataacataac tctgtaaaag ttttattact 56760 tataggcgca tttatgccta aaggatagtc tcaaaagagt gaaactgatc aaatgtgcat 56820 ttttttattt taataggtat ggacagattt gttctcaaaa tgtttgtggc agttcaaaac 56880 accagtaaaa caggggagat atgtattttg gaaaagcacc caaggcgatt ctgaagtgta 56940 gcccaggata agaaccattg cccagagctg ttccagatgg cccctgggtt cctgaagtgg 57000 gtatcgggag agaaatcttc actgaatgaa tgagtgggct ccccagggaa gtgatgaaat 57060 ggtccttatc agccttgcta tctccctctg acagaggcaa actctctctc cctgggggaa 57120 gttcctccaa ggcctctata taagaagtct ttgtgagagg aagcaaagaa ggacctgggc 57180 tttgggaaga tctaaagacc caggaaggtc tctgggtggg tgagtgcttt ctctgctgtg 57240 gtggagctgg tgacagttta ttctcccagg aggtccctgg ctgtggctga cagtttctgg 57300 agggctggca ggcgtctacc tgtggctttc aggttatgag gatgtcagca ggggcagcct 57360 tcatcctctg ccttgcacat tccttctgcg ggatgtgaaa gtgctccttg gctggggaaa 57420 ggagatggtg gagacatgga ggagggtgtg ggtggcttct tgaactctga ggaggggaca 57480 taccttctaa gtcctatgtg ttcctaggaa agccaataat cattgcttct cccgcctttt 57540 ttatgtcata gactctgagg gacccattaa gtacaaacaa ataagcgtaa tagtcccttc 57600 tttacttccg ggcctgaagg aaagccagcc tcagccaccc ctcagggttt gctgcgttct 57660 gtttagaaag aggtccttgc gtcctggatc ctggagcatc aggagctggg cttggcatga 57720 gcttttctgg cccatcctga tttctattca ggccttcttt ttctccacct cactcccacg 57780 gtcccctaat ggtgtgattg tgatgtgtgt gcatgtgtgt ctgtgtgtgt caatgacaaa 57840 ctgtgttctc cgttgcagga taaagccaag atgaaactcc ccttacttct ggctcttcta 57900 tttggggcag tttctgctct tcatctaagt aagtgttttt tgccttcagt ctttctttct 57960 ctgttttttc cctttctatg gtagatgggg tcagagttac acacccaccc ccttctttga 58020 tcgtcttcta tttctgaatt tctgtgtgct taaagggatg gggactctat ggccaggagt 58080 tgaaaggatt tctcaaggcg tctgttatgt ctgtggtctt ggttctactg tgacattccc 58140 aattttgtcc tttctccatt atgcttactt tgagcttact gagtgccttc tctcctttaa 58200 ctctcttagc atcgccatga agtaggtggt attgtatacc catttcacag aaatacagct 58260 ggtggatgat ggaaccagta cccaagccca tgactgcccg actctaagtc catgctctta 58320 accaccttga ccttgtcagg cagcttgggt tcccctcata gagactgggt tccaggttcc 58380 ccttcccagg cagagttgag cactctgatg cccagggcaa ggtgtgagct gtctgtggtt 58440 ctggggagga acaaggggag atgtgaagga aggacactta gctatcctcc ctgccagggt 58500 ctgagacttc cacctttgag acccctttgg gtgctaagac gctgcctgag gatgaggaga 58560 caccagagca ggagatggag gagacccctt gcagggagct ggaggaagag gaggagtggg 58620 gctctggaag tgaagatgcc tccaagaaag atggggctgt tgagtctatc tcagtgccag 58680 atatggtgga caaaaacctt acgtgtcctg aggaagagga cacagtaaaa gtggtgggca 58740 tccctgggtg ccagacctgc cgctacctcc tggtgagaag tcttcagacg tttagtcaag 58800 cttgggtgag tggcctatgg ctgaggctga ggtgggagca tggaacgggt gtgggatatg 58860 cccccagcat tgctatcact ggctcttttt cccattgagg gccctggggg tgtcagtaga 58920 acctgagcct cagagaggtg ttggggtaag aggggagggc cacctacaaa cagaagttgc 58980 attttggtct ccaaccttca aatggttgtg gcaggggagg gagggaatga attgtgggga 59040 ctcaagaccc atgtgaattc atgtaggaag gatgctccat tctttgtctt ttatcctgcc 59100 ctgtagttta cttgccggag gtgctacagg ggcaacctgg tttccatcca caacttcaat 59160 attaattatc gaatccagtg ttctgtcagc gcgctcaacc agggtcaagt ctggattgga 59220 ggcaggatca caggctcggt aagagaagtg tgaacactaa atggggtgca cctgctgatc 59280 tcagccagca ctcagcttgc atcagatttg tctgtttttc tcctgtataa tctccagaag 59340 aaccagggat agatggacac ccacagacaa cactgagggg gctgcctggg cattcaggga 59400 agagctaagg atttagaatc aggaggtttg ggtccaagtt cctttccatc tctcactatc 59460 tatgtaactt aagttagctg ggcatggtgg tgcatgtctg taatcctagc tacttgggag 59520 gctgaggcag gagagtcact ggaacctggg agacagaggt tgcggtgagc cgagatggag 59580 ccattgcact ccagcctggg caacaagagc gaaactccgc ctcaaaaata aataaataaa 59640 taaataaaat aaaaaaaaaa ttaaaacaag accatgagtt tgtttcctca tctctaggat 59700 gagttggcaa cccttgttct accttttgtt agggctggaa ggacaagcct gtcactggga 59760 tgcatagaat ctgatggtga taattgccgt ggatcagcat ttcagatgac taggacagtt 59820 cccatcatgg tccagcaggg aagggcccat tgcccggtgg gcagcagaaa gagctggcag 59880 atacggggcc aggtctgctt ctctgccttc cctctgcccc atcccttctt cccctcttgc 59940 tttctccagg gtcgctgcag acgctttcag tgggttgacg gcagccgctg gaactttgca 60000 tactgggctg ctcaccagcc ctggtcccgc ggtggtcact gcgtggccct gtgtacccga 60060 ggtgaggtgg ggctggggat gaacgatgga aaggtctggg agatgggaag tgccccaagg 60120 aggagatgct acaaagagcc tgaccctttg tgggagaggc ttcctgggtc ttttatatac 60180 tctgactcca cagcagtgtg tgggtgggaa aagaggccct cctgtgggtt gagttgggat 60240 ggacaagagg ctgaaagtcc ctttctgttc tgccttcaca ggaggccact ggcgtcgagc 60300 ccactgcctc agaagacttc ctttcatctg ttcctactga gctggtccca gccagcagtt 60360 cagagctgcc ctctcctggg cagctgcctc ccctcctctg cttgccatcc ctccctccac 60420 ctccctgcaa taaaatgggt tttactgaaa tggatttatt ttctcctctg atcgcggatc 60480 cactctgctt agccctcatt gaaacttctt ccttatcatc tctccccaca ccacaacttt 60540 catagaagtg tcagaagcta ctactccttg aggaggagga tggagggtgg agttgggtct 60600 atggagcctt ttggagatgg aggaatgggc tcagctagtt ctcttcatag aacacctgat 60660 tactgggcac ctgcatagtg ctgccaggac ctttcaaggt tgtaggtaga ctcccaatgg 60720 cccagtttgc atctctgtaa ccaaaggcct tttctctctc tctctccaac cccagaactg 60780 tggttggttt tatatgtaag gaagttaaca tgtccctggg aacagtccac aacattcagg 60840 aatgaatgta taagtaccgc aatccccggc ccctcaagtg gaataaatct aacatgtatt 60900 gggcaccatt tcccagtggc ctgctgtggt agttggcctt attccatgca tttttatggg 60960 ctgccttccc ttcctcaact gcattctctg ctccttccta ctctctgcaa ctcccaaata 61020 aacacttgta cgcaactccc tctctcagga tctccttctg gggaaacctg atataagaca 61080 gcttgccatg cgtcagactc tgaatgaggc ctgggaatac aagacatagt cctctggcac 61140 ttgggatata tggttatttg taacataggc acaaaaacat ctactagttg ttatcgctta 61200 ttgagcaccc acaacatacc ccctgctgtg gcaggcacct tgcctagatg acctcatgtg 61260 atcaataatt atgagcccta ttttacagaa ccaggctcag agaagttagg atctgtcaaa 61320 agacttgccc aagactgaac ctctaaatgc aactcatatt gaaattcaac tctgctccaa 61380 agcatgttac tttaaccctt gtgcttttac agctggctac tctcccctta tggtcacacg 61440 gggatgaagc acggggggag gaaagccaga ctgtctcact cttgggttca tcttgggaca 61500 caggacacca gcccagctgg aggtgaggga gctttaatca gaggggaggg aggaaggcat 61560 tctcaacccc ttctgtacta gggaggtcag cagaagaaaa taattcaatg ttctaaagcc 61620 atttttttct ccagcattcc tccaattcat agatcttcat atgggattag gggctcagag 61680 aggggtgaaa caagaactct atttttttgg agtgtggtat agagaaggga tgctacttct 61740 ctaaggtcac atagtaagtt gagaaagaga gagaaatcaa actcaggttc atttcaacta 61800 ttgttccaca agaatctgtt gatttcaaag atggtggact atgggttcat ccctgtggtg 61860 agtgctgtga ggatgcagct gaggtggaac tttcactcct tgccctcttg gactttatat 61920 tctggtgtgg aaaggcattg cttcccttat ttcaatatta acaacaaagg gtaataatat 61980 ttcccattta ttaagcattt actaggtgtc aggtactgtg ctaaatgtta ggtgaacttt 62040 gtcttgttcc tcataaatct ctgccgctgt gggtgtgtac tttgacagaa gtttgacttc 62100 cagtccacag agatcttctt tgggggagta atatcaagaa ggggcacgaa ggaagctgca 62160 gggctcctag tcccatcctg tatctcgacc taggcatgtt tacattggtg cattcactgt 62220 gaagtttccc tgagcagtcc actctatagt gtgctttata ggagcacatt gtacatccat 62280 tgaaaaattt ttcttggccg ggcacggtgg ctcatgtctg taatcccagc actttgggag 62340 gccgagacag gcggatcacc tgaggtcggg agtttgagac ctgcctgacc aacatggaga 62400 aaccccgtct ctactaaaaa tacaaaaaaa ttagccgggt gtggtggcac atgcctgtaa 62460 tcccagctac tcaggaggtt gaggctggag aatcgcttga acctgggagg cgaaggttgc 62520 agtgagccga gatcgtgcca ttgcactcca gcctgggcaa caagagcgaa actccgtctc 62580 aaaagaaaga aagagatttt ttctttttct taaaaagtaa aaatcatgaa ataaggggac 62640 tgggctaata ttccaaaata tgggtttgtg tgtgaatttt cctctccagt aagatactaa 62700 ctaagctctg tgaaactgtt tatctatggt tctttatcat tgaatccttg gagttcctta 62760 cactgtgcag agcacagagt aggggctcaa tcaacagtgc actcattgct ttttcataga 62820 caagggccac cctcactcaa ctcatgtgcc aggcatagtt ctgagagctt tgcttaagct 62880 gatnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 62940 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnggtcact aatttggtat 63000 agagttatgt tcattgattt cattttattt tgtttctgat ttttaaagat tgttttactt 63060 gttttcttcc tattattatt ttattttatt tgtaaaacat ttacatatca gacatttaca 63120 ttttcccaaa ggtaaaactg tgaaacaaga tatattcaaa gaagtttact ttccctctct 63180 gtttcttgta ccccttttcc tcttctttag gtaaccattt ttattttttt aaatataaac 63240 attgtgtagg tgtatataca tgtattagtc tgttttcatg ctgctgataa agacctatct 63300 gagactggga agaaaaagag gtttaattgg acttacagtt ccacatggct ggcaaggcct 63360 cagaatcatg gcaggaggtg aaaggcactt cttacacggt ggtggcaaga gaaaaatgag 63420 gaagaatcaa aagtggaaac ccctgataaa cccatcagat ctcgtgagat ttattcacta 63480 tcacaagaat agcgtgggaa agactggccc ccatgattca gttaccctcc cccactgggt 63540 cccacccaca atacgtggga attctgggag atataattca acgtgagatt tgggtgggga 63600 cacagccaaa ccatatcaat acatttccct ctcttttaga taaaaggtag tatactgtat 63660 acactattct gcagagtttt tttttttttt gatgtaactc tatcctgagg gtgctctgta 63720 gcagggacct ctcatgcctt ttaaccactg cctgggtctc cattacatgg ctgcagcata 63780 gttgccacag cattcctgta ctgatgacta tttggattgt ttccagtctt ttgctattac 63840 cagtagtgtt acaaagagga tctggctaca tgttcagggt ggggaggggc agatgtgtag 63900 cctgtcagga gggtattgca gtaatccatg actgagttaa tggtagttta aagctaggat 63960 gagtcagtgg ggttggagag aagtgggcac atttgaatga tatgtaggag gtgaatgatc 64020 agcattattg atgagtttga ggtggggcat gtggggaaag gattcgagga tgactcccag 64080 gtttctgttg ggacagtgga tggatagtgg ctcctcccct ttttccaatc ttccttggcc 64140 cttcgctgac ttctgttggg ttggcctaca gagagcttct ttttcctctc tgttcgccca 64200 ggttcctcca ctttggcggt ggccctctgc tcgacggtgc cttcgctggc cctgacatcc 64260 ctgctgtgcc tgggcttcgc cctctgtgcc tcagtcccca tcctccctct ccagtacctc 64320 accttcatcc tgcaagtgat cagccgctcc ttcctctatg ggagcaacgc ggccttcctc 64380 acccttgcgt aagtggcctt ggggcgggct ctgtggagac ggacacactg gggcaaagag 64440 aagctggagg taaagaaatt gggaggcaag gcggggcctg gaggcagtca ggtgcgggag 64500 actgggtttg ggggcaggtg tggagggggt gagaccagag gtggtgggaa ggatagaaca 64560 ttcatgcact tgagccttta catctgcggt gccctctccc tctgttttct acctggtgaa 64620 ctcgtattca tcctctgagg cccacttctg tttcagttct ccagggaaga aatggaaaag 64680 tgtcttccct tctttgtgcc cttagtactc tagtcttact tcctttgcta gtgcgtgcat 64740 tgtctggcat gccatccatt tacatgcctg tcttttcttt cctggtgcag cctgcatgag 64800 ggtcctgtct gtttttccag ggccccgcat gtgccttctt ctgggttctg tgggtcaaat 64860 gtctgagcag agctgaagag ggaaaggcca gacaggtgtg gttggagggc aggcctagga 64920 caggggagct ggggacaagc ggccgacagc ccccagaggc caggcttctg cttggaggga 64980 gggtccctga agctcactgg aacccctctg gtttctctcc ccagtttccc ttcagagcac 65040 tttggcaagc tctttgggct ggtgatggcc ttgtcggctg tggtgtctct gctccagttc 65100 cccatcttca ccctcatcaa aggctccctt cagaatgacc cattttacgt gagtactggg 65160 aggatgggga tccctggcag gaggcctggg ccttaggcct tggctgcccc aaatctggct 65220 gtgatggcct gggtatgtag catggtgcag cttcccaaag ggtctgtgtt attcaagtat 65280 ttggggcaaa agtatttgtg tgtgtgggga aacagacatt ctggagtagg gtggggaatt 65340 ctcacgaaac ttcaagcaaa atcctgagac ctcaaaggtg tttcctgctt gtggtgagtg 65400 caggcccacc ctggcctctc ccctaggccc acacagggtt tccacagttg gccccaggga 65460 caggacctct gtgctttcac ctctgtgtcc ttacacctgg agggatgctc tgaggtcctg 65520 ctctaggagg tggtcgtgag tctcctgctc tttgcagaaa ctgaggctca aagaggttac 65580 ttacgtgttc agaggcacca gctaaggagc aaaagtcaac tttgaattct gtgttttgac 65640 tactgcacag ctctatttgc ctcatttttt atttttaaag cagcaaatct tagaatagga 65700 gtttaaatcc atcacttgga gaaaagaaag actaaatgtt ttttgttttt gttttggaga 65760 cacgatcttg ctttgtcacc caggctggag tgcagtggca caatctcggc tcactgcagc 65820 ctcgatctcc tggactcaag cgatcctctc atctcagcct cctgagtagc tgacactaca 65880 ggcatgtgcc accatgccaa gcttatttta ttttattttt ttgatagaca ctggggtttc 65940 gctatgttgc ctgggctggt tttgaattcc tggcctcaag cgatccaccc gtctctgcct 66000 tccaaaatgc tgtgattaca ggcgtgaacc actgtgcatg gccaaaagag taaacttgaa 66060 atctgaggcg aatgacttga ttgtgacatc aggtgaccta gtaatcagct gtgtattcta 66120 gctggtgcct ctaccagctt cccatgtgac cttgaacatg tcattgaatg ctcgctaggc 66180 ctctgtttct ttatctgtga aatgggcttg atattcctcc tctaccccaa ccgatagtgc 66240 agaatgaaaa gtaactgaaa gtccttcctc cagggcacca tagtgtctgg gtgaaaagta 66300 gaatataaac tcggtagact tctggtccct tcattggtca tggaatggac cagtgcttgc 66360 ttcattgagc aacagttctg ttgttcagaa ttcctggatt tcacctcact tctgctctcc 66420 ctgcaggtga atgtgatgtt catgcttgcc attcttctga cattcttcca cccctttctg 66480 gtatatcggg aatgccgtac ttggaaagaa agtccctctg caattgcata gttcagaagc 66540 cctcactttt cagccccgag gatggttttg ttcatcttcc accacctttg aggacctcgt 66600 gtcccaaaag actttgccta tcccagcaaa acacacacac acacacacac acacacaaaa 66660 taaagacaca caaggacgtc tgcgcagcaa gaaaagaatc tcagttgcca agcagattga 66720 tatcacacag actcaaagca aaggcatgtg gaacttcttt atttcaaaac agaagtgtct 66780 ccttgcactt agccttggca gacccttgac tccaggggag atgacctggg ggaggaagtg 66840 tgtcaactat ttctttaggc ctgtttggct ccgaagccta tatgtgcctg gatcctctgc 66900 cacgggttaa attttcaggt gaagagtgag gttgtcatgg cctcagctat gcttcctggc 66960 tctccctcaa gagtgcagcc ttggctagag aactcacagc tctgggaaaa agaggagcag 67020 acagggttcc ctgggcccag tctcagccca gccactgatg ctggatgacc ttggcctgac 67080 cctggtctgg tctcagaatc acttttccca tctgtaaaat tgagatgaat tttggtgttg 67140 aaagttcttc ctggagcaga tgtcctagaa ggttttagga atagtgacag agtcaggcca 67200 ccccaagggc catgggagcc agctgacctg cttgaccgaa ggatttctga cagactatct 67260 ttggggatgt tttcaagaag ggatataagt tatttacttt gggcatttaa aagaaaattt 67320 ctctcgggaa taattttata gaaaaataaa gcttctgtgt ctaaggcaac tactgtttcc 67380 atctctctag gctttgggcc ggggctgtgt gtgtgtgtgt gtgtgtgttt gtgtgtatgt 67440 gtatgtttct gaggaggccc taccctggca tgagagggta gggaatctgg ctacacatct 67500 agtgtggcag ctggacccag aggtggggca ggaaccctga ctatgattca ccccgctggt 67560 cctgggatgt gggcccagag acttcctccc ccaggaaccc ctctgcttcc tcttcctctc 67620 cacatcctta actaacttta gcagaaccct actcctcact acacaccccc agctagaagc 67680 gctggatgga atcagaaatt cctagtttga gtttcaattc tgcccctcag cagctgggca 67740 agccccttaa ccactctgag tcactagttc cccacctgca aagtgcagtt aatcatttct 67800 atctctgatg gcgattgtga gaatgtaaag tcattgcaac tgcctagcac atggtaggag 67860 cacatgaggg tttgctcctg tgtttactca tgacccttgg ggaggacggg ggcaaagagg 67920 gagaagttga gggtgcagga ggagagatgg caggtgggtg ggatgggaga atctggggca 67980 cacctgctgt ctcattccca ccttgctagg agagggacta ggaaagaaca gtgggaggca 68040 gggggatggg ggtggaaggc agggggtggc aggcaggttc atccatccat tcattcaaca 68100 aatgtttatt gagcacctgc cacgtgtcag gccctgtcct gggtgctggg gctataaaga 68160 tgcagaaggg tctgaaaccc agctcttcct tcttcctgtg gatgtcgggg tgtaatttcc 68220 aggggccagg agcctgggtc tgagggcgga caccaaagtt ctagtggtgt ctattagcag 68280 cgtttaaatc taatggatgg atttggtctt gttaccctgc tcaaaagctt tcagcagctc 68340 cccactgtcc acaggacaaa aatccagatg ctagcctggc attcaaggct gtcactagtg 68400 tgatctcaac ctctcccctt ccctctttac ctcctaccaa cagcggggca gagcccaccc 68460 ctgtggacca agattcccag tctctgggtc tgtgtgtgca ccagttcctc tgcgtgggtg 68520 gctcaccctg cctcagcttg tgaaatccat ctggtctgct gggatcctgc tcaaaatgtc 68580 atcttctcca aaaatcatta ctcaggcttt ccagcatgtc tgagtccctg gcacttggtc 68640 acacccttcc tggtgactgg catttgcctc cacatcatga ccctcccacc ccttgcctgg 68700 gcagcatact ccaggaggca aggtctgttc tcgcctggct ctaattaatc tgtgcttacc 68760 atccacatgg taccagctaa ttcttgttga atgaatgatc gttgaatgag tggattcttg 68820 ttttggcctc agaaccaatt agaaggagcc agaaaaacac atgggggtgg gggaggtgca 68880 gtgtggtgca gtggaaaaaa acccttctgg aaatctcagc tctgtcactt actttgtcag 68940 ctctgtgact ttggatggac cacttctttg tcagtatggt gggagaaata gacatgcctc 69000 tctgggctgt tgtaaggatt acaaattagg tcgagtgctt ggcatgtggt gggttgaaca 69060 gatcacagct agcattacag atgatatatt aaagccaaaa aaagatgcct aatgtccacc 69120 agttggtgaa cggacaaagg aaatgtacca tatttgggat attatttggc aatcaaaaaa 69180 agtactgaca cctgctacaa cacggatgaa tcttgaaaac attagactaa gtgaaagaag 69240 ccagacacaa gaaactgcta atgattccat ttaaatatga aatatcgggc cagggtgcag 69300 tggctcatgc ctgtaatccc agcactttgg gatgccaagg tgggcagatc acttgaggcc 69360 aggagttcgt gaccagcctg gccaacatgg cgaaaccccg tctctactaa aaattagccg 69420 agtgtagtgg catgcacctg taatcccagc tacttggttg gctgaggcac aagaattggt 69480 tgagcctggc aggtggaggt tgcagtgagc caagatcgtg ccactgcact ccagcctgga 69540 tgacacagtg aggttccgtc tcaaaaaaaa aaaaaaaaaa ggaaaaagaa aaaaagaaat 69600 ttccagaata ggccaatctg tagaggcaga aagtagattc atgattgggt aggcctgggt 69660 gtggaggcca tgggtagtga tggctaatgg ggaaggggtt tcttttgggg tgatgaaaat 69720 gggtggactt atggtatgtt aattatacct caataaaact gttatttaaa ggaagaaaag 69780 atgcctggat tccccaggaa gtgtacagta gacttctgtg agaatcagaa atgatttctg 69840 gggaagatgg gcgagaggag agtaagtggg agaagtgacc acgtgcgcaa ctctcatcgt 69900 tctgccctga gagccttcct cctgcaactt tatttattta tttattttga aacaggttct 69960 cactctgtta ccctggctgg agtgcagtgg tgtgatctca gctcactgca gcctcgacct 70020 gccaggctca agcaatcctc ctgtttgagc tcctgagtag ctgggactac aggcgcatgc 70080 caccacatct ggctaatctt ttatttattt atttatttat ttatagagat tggggagtct 70140 cactctgttg ctcaggctgg tgtcaaacgc ctggactcaa gtgatcctcc caccttggcc 70200 tcccaaagtg ttgggattat gggtgtgagc cactgtacct ggcacctcct gcaacttctt 70260 cctcaagtgg aaccaatgag gaagcaagca actcagagct ttcacaagtt ttgatttcaa 70320 tcagcaacgg gcttccaatg caacccttct ctcctgtaac cagcctcagt agagaggaac 70380 tggaggtgaa ttggccccca tcacaccccc acagtgccaa gctgggccct tccatcaggg 70440 ggagaacaca tgccgtgtaa gggacagcca acagcataaa ataggaattg tgtgatgatc 70500 ccttttaagc ctattcagcc cagggaagtg catatgatca gccccatttc atagatgaag 70560 aaagtcaggt tcacccatta gcacattgtg gggctggtat ttaaaccagg tctgtctggc 70620 tcccaaggtc acattcattt agacattacc tttactttac atttcttctt cttttcttct 70680 tcttcttctt cttcttcttc ttcttcttct tcttcttctt cttcttcttc ttcttcttct 70740 tcttcttctt cttcttcttc ttcttcttct tcctcttctt cctcttcttc ctcttcttcc 70800 tcttcttcct cttcttttct tcttcctctt cttcctcttc ttcctcttct tcttcttctt 70860 cttcttcttc ttcttcctct tcttctttct tcttcttctt cttttttttt tgaggtgggg 70920 tcttgctcta ttgcccaggt tgaatgcagc atcatcatac ctaaatgcag ccttgaactc 70980 ctggccttaa gcaatccccc tgcctcggcc tccaaaagtg ccaagatttc aggcatgagc 71040 caccatgccc agcctgcatt tattctcttg taagaaagat atcatttaaa acagacgaga 71100 aaataaagag ggacatgaaa aagacgcatc accattaatt ggaccactca gagataatca 71160 tggttaacat gttggtatgt tccctcccgt catttgactg gatgtatgtg ataatttaaa 71220 tgatctcata agcttttcct tatgtaatca aatagtagcc aaaaacatga ttttaaatgg 71280 ctgctcacaa ccccatctcg tggttctgcc acgccttgtt tatccccatc caccccctac 71340 tccctttccc cttccctgcc tgtgtggggg tcctagatga cggtgagcca gagggcagcc 71400 ttggtcagca gattggagag tgcaaataat aaaaacactc agaaggcgag ctgttgtcaa 71460 gtgggcttat cacaaaagag caccttggga tattccagag aatgacctca tacccgctaa 71520 tcactatcca taatctggtg ctaactgtac tttagctgaa ggtgctggca ggtcctgccc 71580 aggtgctgct aagaacactt ctattctgtg agaatcagag atgatttcta gggaaaatgg 71640 gcgagaggga gtaagcagga gaaacaaccc acaggcacag ctctcatctt tctgccctga 71700 gagccttcct cctgccacgt ggttttgttt gtttgtttgt ttgtttgttt cagatagggt 71760 ctcactctgt cacccaggct ggagtgtagt ggcaagatca tggctcactg aagcctcgac 71820 ctcccaggct caagcagtcc tccccaaatt caaagcttgg agtgatggtc ccagtggtta 71880 tgtctaggag ccctttttcc tgccagcccc tcaggggatt gatgactctc aaatgcttca 71940 ggtgtgacat gggcacagca gtgagtcatt cctctgacat tctttgggaa gaacattttc 72000 catccaggct tccaggcata agatccagtc ctctggtgat aaggagttca cagacaggac 72060 aatgtctgag tgtatcttaa acccaggacc atggcttgtg ttcacaccag accctccagg 72120 gattttgagg tgttttgttt gtttgtttgt ttgtttgttt gttttttgag acagagtctc 72180 tctctgtcgc caggctggag tgcagtggca cgatctcagc tcactgcaac cttcgcctcc 72240 cggttcaagc gattctcctg tctcagcctc ctgagtagct gggactacag gtgtgcacca 72300 ccacacccgg ctaatttttg tatttttaat agagactgtg tttcaccatg ttggacagga 72360 tggtcttgat ctcttgacct cgtgatcctc ccgcctcggc ctcccaaaat actgggatta 72420 caggcatgag ccaccgtggc ccgcccaatt ttgagttttt atgttctaat cccaaacatc 72480 tgctcacagg cccctcagca tattctttcc tgggtccagt gtcacctccc aggcctgcag 72540 gctggctaga gcagtagggt gtgtgggaaa gctctgggct ttgcaggcac tgatcagctg 72600 tgtgacctta accaccctga acctcagttt cctcacctgt aatggaaata ggtaccacgg 72660 cagtttgttg caaggactag agagtaacct tgggaataaa aggtagcagc agcttgggct 72720 ctggagatgg actgtccaag accaacttcc agttcctccc cacacaagct ctggcactta 72780 gattcctggt acctccgctg cttcatctgt aaaatggagt aacaatagga atactttata 72840 gagttgtaag gattgagtgg ctggatgaac gtcaagcact tcaaagggga cctggcatgt 72900 agtgagtgat caatataaac cacctggctt gtagcaggtg tgctgtgtgt ggctgcaggt 72960 gttattagta acatctgtgt gcccttcaga gcgtgcacca cacttcacac cttgtggagt 73020 ctggaatgcc actattatag ttcaggatag aaaacctccc tgcaagcact cgctttagct 73080 tgtctccacc gaacaaaaca acacaagttc tttattactt ggaatgggaa aacttcaaag 73140 gcaaaaaaaa aaaaagactt tcgagttacc ccaaatctta agccaaagtc aatgaaaaat 73200 atcaatcttc atattcaatt tttgcgatac ttttgtctcc ccagcagtca atggagagaa 73260 tccaagcaca cagaaatgtc aattaccagg ggcagggcta tgaattcctt tcagagccct 73320 gggctgggga agagtgcagg cagacagatc tgggtcctgt tatcacgttc ttagattggg 73380 tgtccttgta ggagtcatga agcatcttag tgcctttgtt tgctacctat aatgcctacc 73440 tcagagagta ataaggataa gtaaggctct acgtgaaaag tgctcggccc tggcacatag 73500 taggtccttc attaatggca gctactaatt tttattacat acgcaaaatc acattacagg 73560 tcaagtacgc tacatgacag tgaaacagtt tttttgtttg tttgttttga gacagagtct 73620 cgctctgtca cccaggctgg agtgcagtgg cacgatcttg gctcaccgca acttctgcct 73680 tcaagcaatt ctcctgtctc agcctcccga gtagctggga ttacaggcat gtgccaccac 73740 gccagctaat tttttttggt atttttagta gagacggggt ttcaccatat tggccagact 73800 ggtctcaaac tcctgacctt gtgatctgcc caactcagac tcccaaagtg ctgggattac 73860 tggcatgagc caccgcacct ggctgtgaaa cagttttatt gtgtttctgt ggaatgtgtc 73920 ctacccaacc tatagctaac tcctatagtt ccctcagttc tcagctcaga tatcccttcc 73980 tttctgtact gttacctagt actggttttc atagcaccag gtacctctct ggcatagagc 74040 ttgtcacagt tgcagtttaa tgtaccatca taggatttta aaaatattca gttgtgtctt 74100 ccattaggct ttcatttggg aactccacgc aggcagcagc tgtatatttt gtattgccta 74160 ctgtatcctg agaactttgt accctactta gcacagaatg gaggctcagt aaatactgga 74220 catgagagag agagagagag agagaggaga gggagagaga gagagagaga ttcaacctac 74280 aatcccagct ctgagcttct agttccctga tggtgaggac tgtgatgtgt ctcacacggt 74340 aatgagcact tatgcagaag aggctcagaa aatttctcct catggccaac ggaagactta 74400 gagttctttt ccaagctcca ccgtttgctg gcatgcaaaa tttggactat cacttaagtt 74460 ttccaagcct tgctttttct atccctaaca taggacaata ttcagcattg ttgtttgttt 74520 gttgggggca ccatgtttca ggcacttagt agattattgt accaccacat ttcaattggt 74580 cctcctcaag ccctgcaaca tctgtgaggt ggtcatcctt aacaactcac agatgagcaa 74640 caggagactg gggggatgag ggaactgcca aggaggtcca gcttatgggc agcagagcca 74700 agaatggaac cagggtcttt tattttttta tttttttatt tttatttttt aaccagggtc 74760 ttttaacatc cgaggaccac attctttgtg ctttccaaat catcacctgc cccatgcaac 74820 ttacagggta agttacatta aacaacgtat gtaaatggct ttgtgctagt tattcaccac 74880 cacaggggaa gtgagtcacg gacaagagtg cagccgctcc attcggatcc tggctctgac 74940 acttacctgg aaaatgactt aaccattccc aggatcagct gtttgtctgt aatttaggta 75000 gtttaatggc acttgtgtcc tagagttgtt tagaaggttg aataatatgg agcacttaac 75060 atacttagca cctagaaaca cttcctaaat attagttgct gctgttgtta tcgttattaa 75120 aatttctgcc taagatctca tttcagggag cccaactcaa tctttgacaa gcttaaacaa 75180 aaattgcttt tcttcattta ttcacttaca cagcaaacat gaattgagcc tgtactgtgt 75240 ttccagaact gtgcaggacc agagaggcac aggtgaagga agcaaggctc tggctctact 75300 ggggaaacag caagaagatt gctacaatga ggtgggaaga gggctggact agagagaagc 75360 cctgattagt gtccttgcta cctttctctg ggagagccaa ggcaggcttc ctggaagagg 75420 tgatccttgg ctgaaacttc gatgaagaaa aggaaagagc gcagtggtta gggaggaaag 75480 ggcattctgg gcagatgaaa tgacatgtga caaaatatgg gtgatcannn nnnnnnnnnn 75540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 75600 nnnnnnnnnn nnnnnnnnnn nnnnnnngca ctccagcctg ggtcactgag tgagagaccc 75660 tgtctcaaaa aaattaaaaa aaaaaagtcc agaagaacat ttgggtctca ctctgtggcc 75720 caggctggag tatagtggca caatcatagc tcactgcaca ttcaaactcc tggcctcaag 75780 tgatcctcct gccttagcct tgaaataagc ttggattaca gatgagccac cacacccagc 75840 cagaccatta ttcataatag ccaaaatgtg aaaacaaccc aaatctccat caactgacaa 75900 atggataaat agaatggtgg ttgatccata caatggagta tttactcagc aataaaaaga 75960 agtcctgata catgctacaa ggatgaacct cgaaaacatt atgctaagtg aaagcagcca 76020 atcacaaaag gctacatatt acaagattcc atttaaatga aatgttcaga ataggtaaat 76080 ctaactttta tcacaggcaa agctatgaca ggaaatagat gagtggttgc ctagtgcttg 76140 ggggcagagg tgggggtgag gcgagtgagt actgctaatg gtacagagtt acttttgggg 76200 ataaagaaac tgttctgaaa tggactctgg tgatggttgc actactctga acatactaaa 76260 actgttaaat tatatacttg aaatgggtga cttgtgaggc atggaaatta tatcttaata 76320 aagctgtttt acatatttta catatttaaa aatgcaggtg gagggatgag ccctctaaag 76380 agaagcagga gtttgaggag gttctaaata ttgtgtggtg ggtactgagg catataaatt 76440 tgtcagacct catcaaaatg tatgatgtaa tcttcaagaa agttgatttt aaagaaacac 76500 caccagcacc aggtggagaa ggcaggaaga agttacacaa ggggtaggcc aagagtggtg 76560 gctcatgtct ataatcccag cactgcggga ggccgagctg ggtgggtgac ttgaggtcag 76620 gtgttcgaga ccagcctggc caacatggtg aaagcccgtc tctactaaaa atacaaaaat 76680 tagccagacg tgctcgcgtg aacccagggg gagaaggttg cagtgagcga agatcatgcc 76740 aatgcactcc agcctgggtg acagagtgag actctgtctc aaaaaaaaaa aaaaaagtta 76800 cataggggac agtggcaggt gtcaagggca ggcagggtct ctcctatctc caggataaac 76860 tcatagggga cttagatgcc atgtgggtcc ctaatagccc tccacttggt tcttgcagcc 76920 actcttatgt gtatcatttc atgtcaggcc tcttcttccc aacccaccca gccatcccag 76980 cctggctgcc aaccccacct cctccagccc ctgtcacccc ataattgggg ccaggaggca 77040 tgggagagtc gccatctctc ggtgccatct gttgcatctt tacagataac catggctgga 77100 tgcggcagat cctggggtgg agcagccgct gttcagagca gtgatcaaga cctccccatc 77160 tccacccctc aaggaatcgg ttttcttcca tagccacatc aggtgctgtg caggaaggag 77220 ttgaaacgag aagccaggag caacgagaag gacactaaca tttattaagc actgcagact 77280 ctcacagcac tcccacggaa tcgatattat tatccccatt ctgaagacca ggcaactgaa 77340 gctcaatgtt taaggaactc accgaagtca ccaactgata aaagtgatgg aagctgggat 77400 tcaaatccaa gctaaacttc cttccaagct tactccacaa cacagaggtt ggggaaaggg 77460 gataaaaaga gaggggagcc caattccatt tccacccagc tcctgaggcg gagcttgtca 77520 gcacagctct ctccttccca gaataggaag atacccatca gaggcaagtc ctagacacca 77580 gcagtggtaa ctccctgccc caaggcagct gcagacagcc tatggctgta gttactgctc 77640 ccaaagagtg ttagaattcc cactcccagc ttcggggcca ctcacacaag gtgattgaag 77700 tggaaaccag agactctcca caatgccctc ctagagtaaa tgaggctatg taactttgtc 77760 caaatgagta atttgaaaac ctgggggctc ccagctcctg aaaagggaag gatgtggggc 77820 cctttatatt catactccac tttgtgcagc tctcccttgt cttatgatag ccctattaag 77880 aaattcctct cccagcacgt ctccttcaaa gagctctaga cctgaggctg tcagaggctt 77940 aggactctgc ctattagtcc cagggtctgg atgaccagca ggacacctgg cattcagtga 78000 ccactggatt agataaatga aacagtgggc agagtgccac ccaatctccc cctgaagttt 78060 gaagaggtcg agaagtgagg ctgtccaact gctgaccctg ctttctgtcc acctggccac 78120 ctaacctttt ctggcttcca cctgcccctt tgccatccct ccccccagcc cacccagccc 78180 attttcaggc atacctgggc acgtgctgga atagaagccc tcgttcttca gaatgatcaa 78240 cagggagccc cagcccagga gtacagcaga gaagaagagg ttctccagca cagccgtgca 78300 ggccatccac cagcgcctcc ggtacgcctg ttgcagcgtg ggggccatgc tggccccgag 78360 cctgcacaga aacagagcgc tgggtgaagg gccccccagt ggccccaggg aagggtcctg 78420 catcatggtg gcacccgaga cctctcgggc cagcccgcga ggagcccctc atggaggccc 78480 catagagccc tgggcttccc agccggtgcc aaggagctgg ctccgcgcgc actagcagtg 78540 ccagaggtgc acgcggcacg gggctcccgc tgagccacta tcggaaacaa ggaaggtcct 78600 gtctgcgcgc tgcagcttcc tagcaggctg ccgggttctc tcacccaggc cagggcgctc 78660 agggccgggc tgctggggag aaagtccgca tctgcccagg tccccagagg acagcaaggg 78720 gcagagcgcg ctctgaagca ccgcgggccc atgtccggac tctcgcgcca ggaaagaccc 78780 ctagaagctg gcaggaagaa gggcaagttc aaggctaccc tacgacccca tcttccagtt 78840 gcccctccaa gacctctcct tccctctggg gccgggcgac agcaagccct ccccctttcc 78900 gtatcaggtg acccacgacc ctacagtctc tcgggccaag ccaacagctg ccacgtggag 78960 ggagacccag gacgggctct cctcggttcc ctcctccccc gcgcgcccct cactcactcc 79020 gcagggctcg gggcaccagg ctttgcacct cggaacccgc ttgcccccct ccagccccgg 79080 gagggggctc ggacttcggc aggaagtctg gcggctgctg actttataag ggcagcggtg 79140 gcggatgggc tggcgggcgg gtgtgtttac caaagggagg gaaagagccc cagctccccc 79200 cgccgcggcc gctgcagcct cggcgggagg agagggaacg cgggcagcgc gggggcgggg 79260 agcgacaact gggatgagac cgaggaaagc ggagaggaga agggcaagaa agacccagag 79320 agaggggagg aagtaccagt cacttcttcc agggggactc ggtattctca tctgtgaaac 79380 ggggctttgg gttcaagcgc tccaggaggt ccgctggaac tctggcaaac gcgcagctct 79440 aagcagagga agtgcagcga gcggggaccc gggaggaaga gaagagtcgg aggggtcaga 79500 gaaaagaaaa gggaaggacg cgcttggcga gatgggacac tgtgccgcgg gaccgcgggc 79560 gcaagtaacg gtctttcctt gggaagcctg gcagtgtcgg cgggagccgg cctcggtgtc 79620 tctcagccga cgcatagccg gagaccctac gcgcgccccc tccccgccca cgctgctcac 79680 ctccggtcac cggcaaatga gcagccagca gctgcggacg cctccgggag cgcaacgctt 79740 tcgcggcgcg tccggagtcc cgtgggccca gccctgagcc gcgccggcgc tggggtcttc 79800 tctgcgtgca ggacccggcc gccacggagc ttcagcctga cagcccggtg gcctcgcctc 79860 cgctgtctcc tcggaagaag cgggggaact gggaacccgc cgggcgccag aggtctgcga 79920 agctgggctt ggatgaagtg gatctgcgga gttgatagtt gtatttacac gcgtccggag 79980 ctgcgccccg aggtgggggc gggggctccc ttcttttccc ctccccttag gtcgagtttc 80040 acgcgcacgt gactcgcccg ctggtcccgg acactctccc tctggcacag ccccagcacc 80100 tacatttcca ccctggaccc ccatcttctc ccccaagccc ccagactaac atcaggcagc 80160 gccctctgta tccttgttca aaacaaagtg cgattcggct gaagccgact gaccgcgatt 80220 cagggccgcc ttgggtgggg ttttgaactg tgcagctgga agcagtgttt tccgagaggc 80280 agagtggcac gggtttcttt ggagttagtc agatcgaggt ctgagtcttg actttttaac 80340 tgactaccct gggttaccta gggcaagtta cctctctgag cctcagcttc ctcctcttta 80400 aattcggtta aaatggaacc tacctaactg cccaaaggaa tcgcgattgt gatgcaggta 80460 aaatgctaag catagcattt ggcatagtaa gcataatgtt aattgttgct gctgtcatta 80520 tttcagaaga cctggtgatc ggatgcttcc agatcaacaa ttgattgact ccaggtaaat 80580 ctctcagcct ccctgagcct cagtatcctc atctgtaaaa tagactacta tggtgtggag 80640 taatgagaag taatctcatt acatgtgagt ttaattgtgt gttaagagtg ctgctaatgc 80700 atgctgagct taatacctag gtgatgggtt gataggtgca ataaaccacc atggcataca 80760 tttacctacg taacaaacct gcacattctg cacatgtacc ccagaactta aaataaaaat 80820 aaaatttttt taaaaaaaga gtgatactgg tggccaggtg tggtggttca tgcctgtaat 80880 cccagaactt tgggaggcca aggcaggagg atcgcttgag ctcaggagtt cgagaccaac 80940 ctggacaaca tggtgaaacc ccgtctctac aaaaaagaaa aaaaaatagc caggcatggt 81000 ggtgtgcacc tgcagtctca gctacccagc aggctgaagt gggaggatca ctgagctgga 81060 gagatggagg ctgcagtgag ccaagatcat gccactacac tccagcctgg gtgacagagt 81120 aagactctgt ctcaaaaaca aaacaagaat gactacagaa agctccaaga aggcctcaga 81180 taaaagggaa cccctgaaca gatgagccac caagccaaga gaggaactaa tggctaccat 81240 agacagggca ctttccaaaa taaaaatact gttattaatt cctcaagaca tcatggtccc 81300 atttaaacct catagctttt cacagaggga gaaactgcag gcttgaagct ggagcaaggt 81360 tagaggtagg atgcagagtc aggtcggcct ggcatttaag tacggctcct tccattcctc 81420 ccagaaggag aatggcaaga gcaaaggctt agctgtggga atggcacaag gagttctcgg 81480 tggccaaagc acatgtcagg ctctgatggt ttaacttctt aaaatgcaat actgcctccc 81540 agaacttcca gatcaaggtc aaactcctca gctctacaca gggggaccta gagtcaactt 81600 tctaagctag gagagtcatg gatccctttg agaatacaaa agacagtggg cgcggtggca 81660 gtggctcatg cctgtaatcc caacattttg ggaggctgag gcaggaggat cacttgagcc 81720 caggagttca agacctgctt ggtcaacata gtgagacccc tatttctaca aaaaattcag 81780 ctgagcatgg tggcatgtgc ctgtagtctc agttactggg gaggctgaag taggatgatc 81840 cctgagcctg ggaggtccag gaagctggag tgagccgaca tctcgccact gcactccagc 81900 ctgggtgaca gagaccctgt ctcaaaaaaa aaaaaaaaaa gaagaaatat gttattgatc 81960 tactcttgac aaaaatgctt gtgtgaacat ggacacacac actcatcaac attcacattt 82020 caaggttttc atggaccctt tccatgaggc tctagtggtc catggacccc catggctgga 82080 acacttgctc ttcctcatct caacccacat ttccatggag ttggactgtc tgctgcatga 82140 ggacacaggc ctcatttggt gtgttcattc actgctgtgt atcccagcac ccagaacagc 82200 acctcaccta aggggcactc agcacatgtg cagtgaagag tcagtcagct ggtttcacac 82260 ctcccagtct ttgcacctgc tattccttct tgtgggaatg acagatttcc ttcatttctt 82320 tttttttttt ttttgacaga ttccagctct gttgcccgag ttggagtaca gtggcacgat 82380 ctcagctcac tgcaacctct gcctcccagg ttcaagcaat tctcatgcct cagcctccca 82440 agtagctggg attacaggtg cacaccacca cctgtgagct gatatttttt tcttttcttt 82500 tcttttttcc tgagacagag tctcactctg ttgcccaggc tggagtgcag tggcgtgatc 82560 tcggctcact gcaagctcca cctcccgggt tcaagtgatt ctcctgcctc agcctcccaa 82620 gtagctgaga ctacaggcgc gcaccaccat gcctggctaa tttttgtatt ttttagtaga 82680 ggcggggttt caccatattg gacaggctgg tctcgaactc ctgacctcgt gatccgccca 82740 cgttggcctc ccaaggtgct gagattacag gtgtgagcca ctgcactcgg ccattttttg 82800 tattttttta gtagagatgg ggtttcacca tgttggccag gctggtcttg aactcttggc 82860 ctcacgtgat ccacccacct tggccaccca aagtgttggg attacaggca tgaaccactg 82920 cgctcagcct ccttcttcat ttctaatgta ctcatccttc acaactcagc tcaagtttca 82980 cttctctctg gaagctctac tctaggctgg attcagggcc ttgtccacat acccaccaaa 83040 tactctgctt acctctatgg aagtccccac actgatctag aataatcagc ttagttttct 83100 gcccccatcc cgccccatga gatgtacatc ttgtgggggc aggaaccacc acgtggtagg 83160 tgatttgtgt gcctgctgcc tatcacaggg cctggcgcct aataagcttg cggccaacat 83220 ttgttgaata aatgaaaagg gaatggtggg aaaggaagct gaaaaggtag gctaaaatca 83280 gtttggaatt acctctggga ggccaaggac tttcagtctt gcagggtagg taacaggaaa 83340 ctcctggatt ttgttttctt ttggttttgt ttgtttttaa tgaagggtag cgttatcgtc 83400 aggtttttgt gtttaattaa tggagcatat attggaaagg acagagacct taaagcagtt 83460 aggagaccac cataatagtt cacattttgc agccataaaa aggaatgagg ccaggcatgg 83520 tggctcactc ctgtaatctt atcacttcgg gaggttgagg caggcggatc acctgaggtc 83580 aggagtttga gaccagcctc accaacatgg agaaacccca tctctactaa aaatacaaaa 83640 ttatccaggc gtggtggtac atgcctgtaa tcccagctac tcaggaggct gaggcaggag 83700 aatagcttga atctgggagg cagaggttgc ggtgagccga gatcgtgcca ttgcattgca 83760 ggtacatgga tgaagctgga agccatcatc ctcagcaaac taacacagga acagaaaacc 83820 aaacaccgca tgttctcact cataagtagg agctgaacat tgaaaacaca tggacacaga 83880 ggggaacatc acacactagg gcccgttggg gagtgggggt tggggggtaa ggggagggaa 83940 cttagaggac gggacaatag gtgcagcaaa ccaccatgac acacgtatac atatgtgaca 84000 aacctgcaca ttctgcacat ggatcctgtt ttgttttaag aagaaataaa gaaaaaacca 84060 agaagaaaca aacaaacaaa aataattccc atttaaaaca ataaaaaata ggccaggcat 84120 ggtgactcag gtctataatc ccaacacttt gggaggccaa cgcgggcaga tctcttgagc 84180 ccaggagttc aaggccagcc tgggcaacat ggcaaaaccc tgtctctaca aaaaatataa 84240 aacaaacaaa caaaatagcc aggagtggtg gtgcatgcct gtcatcccag ctactcaggt 84300 ggctgaggtg ggagaatcac ttaagcctgg gaggcggagg tagcagtgag ctgagatcgt 84360 gccactgcac tccacctgga gcaacagagc aagattttgt ctctaaataa ataaataaaa 84420 taataaaaaa cagagaagag gaaagacacc tgagatatat ttccatatct gaatcaatag 84480 gatttatcaa cgttctcctc tacccccaaa actaattcct tcctaaactc tgttctcctg 84540 acactactca taggttaagt ataacagcat tatcacattg gctgtcatgt gggctcctgg 84600 ctagaggctg cttcacagct taatggacaa gagcactgag acagggtggg tctaaatcct 84660 ggctctgcag ctgattattt gtgtgatttt gtccaaatca ctccatctca tgagcctcac 84720 tcttctagtc tgttaagtgc tgaaaataaa agtatccaat tcaattcatt atttaatgaa 84780 ttatttagcc taacaaatag ctattataaa tatttaggct gggcacagtg gctcacgcct 84840 gtaatcccag cactttggga ggccaaggtg ggcagatcac ctgagtcagg agtttgagac 84900 cagcctgacc aacatggtga aaccccgtct ctactaaaaa tacaaaaatt agctgggtgt 84960 ggtggcatgt gcctgtaatc ccagctactc aggaggctga ggcaggagaa cgcttgaacc 85020 caggagacag aggctgcagt gagccaagat cgtgccactg cactctagcc tgagcaacag 85080 agcaagactc tgtctcaaaa aaaaaaaaaa aatctctgca tgaagaatgt acataaaatg 85140 gtgcagccat ttcggaaaac agtttggcag gtcctcaaat agttaaacat agagttacca 85200 ctatagccca gcaattccac tcctaaatat actacaccca agagaattga gaatatttgt 85260 taacacaaaa atgtgtatac aagtatttat agctgtatta ttcattacag ctaaaaagtg 85320 caaacatccc agcagtccat cagctgatga acggagaaac aaaatgtggt atacccatac 85380 aatgtcatat tatttggcca taaaaaggaa gtactgatac atgctacaac atggatgaac 85440 cttgataatg ttattctaag tgaaagaaac cagacacaaa agaccacata ttgtatgact 85500 gcatttatat gaagtgccca gaataggcaa atccacagag acagaaagta gattagtggt 85560 tgccagagac tggagggagg agataatggg aaatgtggaa tgactgctaa tggtatgggg 85620 tttcttcttg gggtaatgaa aatgttgtac aattagataa tggtgatcat tgtaaaactt 85680 tgtgaatata caacatgctg aattttatac tttattatat tttatttttt ttgagacaag 85740 gtctcgctct gtcacccagg ctggagtgca gtggcacgat ctcagctcac tgcaatctct 85800 ctgcctccca ggctcaagca atcctcctgc ctcagcctcc tgagtacctg acactacagc 85860 atgtgctacc atgcctggac aatttttgca tttttagtag agacagggtt tcgctatgtt 85920 gcccaggctg attttgaact cctggactca agtgctccgc ccacctcagc ctcccaaagt 85980 gctaggatta caggtgtaag ccaccactcc cggcctaaat tgtattcttt aaaagactga 86040 attgtatggt gtgcgaatta tatctcaatt taaaaaaaac aaaacaaaac aaaaaaaaaa 86100 cctttgcgtg tgtcaggcac tagggattcg atgctgaata agacacagac cctaccctca 86160 gagaacacag agcccagcag gagagagtca cagatgaatc aagtgttaca tcatctatag 86220 gaagcgccat ggaagaaaga catggtgcca tgagaacata cgcttagaga agggaatttc 86280 atctagactg gggctcaggg aggaatcttt cagggtgatg cttgtgctca gagttttcca 86340 tgtcagaatc agtagaattt atcaatcctc cagaggagga aacagcaaat gaaaaatctt 86400 acaacaggag gatgcggaga cattccgaga gctgatcaag ggctggtgtg aacaaagcac 86460 ataggatgca gagcctgtgg tgtgaggttg cagctggaaa ggtaaaacac taattacatt 86520 ggatcttctg agacaataaa gagtatgcaa taatctcaaa cgaccgaaac tgaccttcct 86580 cctccctaac ttgcttgctt ccactgttgc ccgtatcata aaagcaccac cctcttctac 86640 ccagtggctt aagacacgaa actcaagtca tcccaggctt tctccccacc tcactctcca 86700 catccagcct atcagcgagc ttgtgggtct taccacgtaa agacttctca tctccagcta 86760 ctaccatccc ccaagcccag atcaccatca gctcaggcct ggactcctgc aacctttcta 86820 accgggtctt cccaatccta cccccgcaac atgaccccaa tagcccatca gaatggacta 86880 atcgagatgt agatttgatc aggccacatc ccttgaaagg cttcctgtga ccctcgggga 86940 aatgcacaaa ctcccaatga tggcccctga gtcctgtgcc atctgggtct gccctctgcc 87000 ctctgtgtct ttgccatggt aacctccttc acacccatta atactccatg ctctctccta 87060 cctcaagttc ttcctgggct ggaacattct ctgcactagc ctagccaact aaccctttag 87120 atcttttgtt tgtttgtttg tttgtttgtt tgtttttgag acagtcttgc tctgttgcca 87180 ggctggagtg caatggtgca atctatctcg gctcactgca acctctgcct gccgggttca 87240 agcaattctt ctgccttagc gtcctgagta gctgagacta taggcaccta ccatcacgcc 87300 cggctaattt ttgtattttc agtggaggtg ggttttcacc atgttggcca ggctggtctc 87360 gaactcctgg cctcaaatga ccaccctcct cggcctccta aagtgctggg attacaagca 87420 tgagccactg tgcccaggca acacttcaga tcttaatgat catttccttt aagtgcctga 87480 cctcttgtag taactagcct gactccagca atgaatcctt ttgcaatgta acctatataa 87540 catctgagtt tccctttgat aaaactcatc atatatttgt tcctctgaca gttcagaggg 87600 caagggcctt tgcccacctt cctcaccact atcctctcac cacttaacac agaactcacc 87660 acccaccatg cctcctgcct gacaaattcc taaccatcct tcaaatctca ctcacctatt 87720 accttctggg aggcagtctt ccctgagcac caagacaatg ggacacattc ctttatacac 87780 cctgctgaac atctcttttt tgaggggcgg gtagagatga gtgtctcact atgctgccca 87840 ggctgacctc aaactcctgg cctcaagcga tcctcctgcc ttggcctccc aaaatgctgg 87900 gattacaggc atgagccact gtacctgacc gcaactgggt tagnnnnnnn nnnnnnnnnn 87960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 88020 nnnnnnnnnn nnnnnnnnnn nnncctgggc aacaaagacc cttcctctac aaaaaaaaaa 88080 aaaaaaaaaa aaaaaaaaaa aaattatttt aaattagcca gacatggtag tgcatgcctg 88140 tagtccaagc tacttgggag gctgaaggga gaggatcact tgagcccagg aggttgaggc 88200 tgcagtgagc cgtgatcgta ccactgtact ccagcttggg caacagagtg agacctcatc 88260 cctaaaaata aagaagaaaa tatggcaatt tgactgtaca tctctaatgg gatatatcct 88320 aaggatgaga aaggaataag gaaggacaga aaaaaggaaa caaagaagta gcaacagtat 88380 ttagcaattg tattgttatc aagtaacatc aatattggta aaaccagtaa ttatatttaa 88440 aatactatat atgtgtatgt acatttacat atgcatatgt taggaaccaa gtttatcaga 88500 ggaagagaaa gggctacaaa tgtaaaatca aggaaataaa aatttgaata aaaatatcag 88560 tattaagtat ttatgatatt tttcttataa aaaaattata tatatgttaa ctctatccaa 88620 aacccaaaag cagtgacaac ccaggagcaa taaaaaacct cagcatccag actgtagtct 88680 ctaccatttc caattaaaga aacccagggc tagttgggaa aaatgacaat ttcatgtcta 88740 gggcaagaaa cacacctagt gaaatggacc tgaacattta attgtgttag aaagtaagga 88800 aactctctag aaataatgtg atttcatcta aaagacacag attctgggct ggtaaagttt 88860 tcaatggcca aaggtgagac aatttgagca tcaagaagaa tcatgacaga acagattaaa 88920 acatgtcaaa tatattttaa aatgaaatat tataaaagaa acaattagta gccatccctg 88980 aaggtcacta gggcaccaac tcatatttca aactggtaaa taaatgtgta agccaagcat 89040 ttatttctgg gtaacaaaat agtaaggaat gtttttcttt ctagaagaat tctagtgatt 89100 aaaagtagaa gatagaaata gaaaatcatc cttttggcca ggggcagtgg ctgtaatccc 89160 agaactttgg gaggccaagg caggtggatc actggagatc aggagtttga gaccagcctg 89220 gccaacatgg tgaaacccca tctctactaa aaatacaaaa attagccagg tagtgggtgc 89280 ctgtaatgcc agctactcgg gatgctgagg caggagaatc gcttgaacct gggaggcgga 89340 ggtcgcagta ggctgagatt acgccactgc actccagcct aggcaacaca gtgagactct 89400 gtctcaaaaa aaaaaaaaga aagaaaagaa aatcatcctt ttgcgatcct aatgaaataa 89460 tgggcctagg cattgatcat taatggctcc taaaatcact aagtatatgg ttgatgggaa 89520 actttatagt ggatggatca gactcgcaat gtctaaacca gttgatcaat cttaacatcg 89580 taacaagaca acagacacca ggggctgctg acaggagaac agaggaaacc catagctcta 89640 ccactgagtt attcacggca aaaaaaaaaa aaaaaaaatt aaactgcgtt tcctccaagc 89700 ttctaatcct gttgtttaca ggaaataccc aaggaaagga atacttttaa atgacacatt 89760 aaaacaacgc caaatccaaa atatggggaa atgacccagt ttcttcaaca aataaacaag 89820 aaaaggtagg ggggaggact gttctagatt ttaaaagcta tagaagacac agcaaccaaa 89880 tacactgcat ggaccaggca tggatcctaa ttggaacaaa ccaactgtaa aaggatgtat 89940 ttgaaatgat tggaggaatt tgaacagtga ctgcacagta gatgatatga agaaattatt 90000 gttatttttt aggtgtgatc atgattttat ggtgatgttt aagtaaaaga ggccttattt 90060 gttagagata catgtacggg tatagagaaa tatttacgga tgaaatgata cgatgtctga 90120 gatttgcttt gaaaactcta gcaggtgtgg gagaagcagg tgcatgggtg ggggaaggga 90180 tagatgaaat aagtatgcaa aatgttagtc tacttttgtc cctcctgacc cagcaggtta 90240 aaatacctca gcatacctct actcctccaa ccaggtccaa ggatcaggcc aaaactccct 90300 gatgtggtaa acagcctgac cccttcttac ctctctctct ccagccactt ccctaagatt 90360 ccccagtgct ctgtgcccta gccagcccga ctcatctgcc cagattcctc aatgtttcac 90420 tctctcattc accattttga ccccactgtg ctcctgggcc actctccaag gccccgcctc 90480 ttcatctcct ccctccttac tcatccttca ggtcttggct taggtgccat tccctccagg 90540 aagccttccc tgacaccaat cccatcctca cctagaacag attatgtgcg cttctttgtg 90600 ccccccatgg ccccctgtgg gtttgcttca cggattataa ctgcctgact acctgccttt 90660 ctccaccctc tagactgaga acaccttgag aaaaagaaca catctatctt gtctgtcatt 90720 gaatccctgg tgtctggcac catggctgac acataactat cactcagtga ctagtgtttt 90780 aatgaatgaa tgagtgcaac tagacagggt taagaacaaa agagaagacc aggcatggtg 90840 cttcacgcct gttatcccag cattttggga ggctgaggcg ggcagatcac ccgaggtcag 90900 gagttcaaga ccagcctgac atggtgaaac cccgtctcta ctaaaaagac aaaaattagt 90960 gggggatggt ggcacacgcc tgtaatccca gctacttggg aggctgaggc aggagaatct 91020 cttgaaccca ggaggtgcag gctgcaatga tctgagatca caccactgca ctccagcctg 91080 ggcaacagag taagactcta tcacaaaaaa aaaaaaaaaa aaaaaaaaaa gagcgagaga 91140 agatgtcatg gggtaaatga agacctccct tcctggttcc ctgaccagcc cctgccctcc 91200 cccgcatctc acctgtcttt cttgcttcct tctggtactt ctgtttaagc cggtccatga 91260 gcaggccatt ccagggggca cacagcactc cgaactgagt gaaggcaaag gcatttgtgt 91320 aggtgctgac tgcagaagga agagagaggt tggttgatga gaagtttcca aaactccctt 91380 ccaggcaggg actctcccac cttacccttg tctgcatgtc cctcctcccc acaccatcag 91440 accctcctct ggtgtgtaca gccctgctgg gaggctctgt gttcccagct gggacatgca 91500 gatgggctac ctcccagccc taccacatac ctcgtgccat gtccccaccg gccatgttgg 91560 tcagcaagga gttgagagtg ccaatgaaga ggtagtgcca caactgtatc acagacagcc 91620 acaccaggtg ccaggcaaag cgccgagaga aagcgtagct ccagaaggag cggagttcct 91680 gcttctgccc tgcccctggg gtctctaatg gggagaggag gatctgggcg tgaattacga 91740 ggaaagtgga caggtaggat ggggagtgtg gaggcttcaa tggaacattt cagatccggg 91800 cccaccttct acccttggct cccagaactc acccggcgtg ctctgcacct gcctcggact 91860 cacacatccc cacctccctg ctttgtcatg ctggccctac caccttggat gaccctctgt 91920 gttcttctct attgaaatcc gatctgtctc tcacagcctg gtcaatgaca cttcttgcag 91980 taataccttc ctgatctttc tcagcgagaa aggtgaaagg aacgacaagg agaggagaaa 92040 gtcagaaggg agaggagaat gagtgtggat actctgttct aacctgctcc tcagcacctc 92100 cctttctttt gataccagta tcctgagttt ctttgggaaa tcttcctcta ccctaatctt 92160 catggtccag atgggaccat gaattcagtg ttctgttcct ctctcaaggt taaccaatga 92220 gatggttcct cctaacaagg cagaccggcc atgagtttag attaggatgg acttaatcta 92280 aaataggtcc tacaccctgg caagttcaat gtcctccctt gattttggaa gcttcccaga 92340 accctattct tctttcttta aaataaataa ataaatacat gttttggatc caattgtcag 92400 atggtaaaaa taaaaacaaa aaaatcaatt ttattctgta tatttaagat atacaatatg 92460 aggtcatagg atacatatag ctactaagat ggttactaca gttaagcaaa ttaacatatc 92520 catcatctca catttctacc tgttttgtga caagagcagt taaaatctac ttgtttagga 92580 aagtcccaaa cacaatgcag tttgatgacc tacagtcttc gtgctgtgca ttagatctct 92640 aggcctgttc atcctgctca tctgctcctt tttgtccttc gacctgcatc tcccatctcc 92700 tctcccaccc cgttcttatt tctactgtag ctagctgcgg tttgtgatgt gtgtaaccaa 92760 agacgcagaa cagagaggaa ggaaaggaag cagtgataga gttgggacaa taagagaggg 92820 cggacccagg agacctggag aaatgggggc actgtaccag acttagtgca atggcatcac 92880 agaagagggc agaaccgagg agtgggggga agggaaggca acccatggca ggcgggcttc 92940 aaggggtggg gaagtgatag gatgcgaaat agagaaaaga gggacagaaa agagacgaaa 93000 gccctggacc ctccattaag tgagagggtt gggaagatgc ctaaggccct ttttctgtcc 93060 tgcctttcct gattctgggt ccctggggga gctctggagg tgaggggcca ggaaaggcac 93120 aaggagaggc ttgggtctgg aggagagatg ggttagccag cagggctcac cttccttcgc 93180 tgaaaggaac tcctttgact gtagctccct gttttcatgc tcagctgttt ccttctcttc 93240 ctttgtggtg ccattcccag ggcacaggct atggaaacaa aagccccacc agcaaggcca 93300 aggactgtga gccgaacctg agactcagac tggagggaat agcatggtga atcccacatt 93360 ccaccgcact ttggaatcac cttttagcca ctctgatgcc caggttgcag accagaccag 93420 ttaaatcaga atgtctggag gtgagagcca ggcttccttt tctaagatct ctatgtgaat 93480 ctagtgattc taataagcag caaagtttag gaagcatgaa aagagtaggg caggcccagg 93540 ttcaaatccc agctctgcct cttcctagca acagaaagat ggctcagact taacccttct 93600 gagcctcatt ttttgcattt agaaaatgga gataaggata tctcagagga ttattgtgag 93660 gatgaaatca gagagcacat ggggtctgac aattagtaag tgagcagcaa aggaatgccc 93720 ttcctctact ccttgtggca aatgactgca aaaatgatca catttcttca cctcctctgt 93780 atttccccca atttgaatga gactgcagct ctatttcccc atgccctgaa tctgggccag 93840 ccttgtgaac tgcttcagcc aaaagaatgc agcagaagtg gctgtgccaa ttccaagctt 93900 aaatctcaag aacgcttgtg catttctgca ctctttcaga accctgaaat cacggtgtga 93960 atgagcccac gctggcttgc tggaggatga cagccacgtg acccaggcat ccctgtcact 94020 ccaaacctat gtgagtgagg ccatcctagc atagccagcc cccatgtaat cctccaaatg 94080 atcagatgta tgaatgagcc ctgtcaaaat catctacatc tggccctgat cagcggaact 94140 agccagctac ccacagactt gtgaaaaata ataaatgctt aacattttag gctgctgagt 94200 tttgagatag tttgttatgc agcaatagct aacagatgca ctgctccagt cctcctcctc 94260 tcctgtgata ggtttgcttt accctgtcca tcccacccta gggccaatga ggggctctgg 94320 cccacaatca ccagatagtc cttacccata gctgtagttg gggggcagtg ggtatgggat 94380 gtgcccccgg ggcatcagga ggaaagtgcg tgctacatgc caggtactgc agacagagat 94440 gaagatgaag gaggccctga ggctgatgcc tttttcataa agaagctgca gaaggagaag 94500 gaaaaagtca gtgtcacacc cacgttcata gcagcactat tcacaatagc caaaggatgg 94560 aagcaaacta agggtccatc agcagatgaa cagctaaaca taatgtgatc tatacacaca 94620 atggaatatt attcagcctt aaaaaaggaa agaggcaacc atgctggctc acacctacaa 94680 tcccagcact ttgggacgca cgaggatcac ctgagcccag ttcaagacca gccttgacaa 94740 catagtgaga ccctcacccc ttctctagaa aatttttatt taattagctg ggtgtggtgg 94800 catacacctg tagtgccagc tactcaggag gctgagtggg aggatttctt gagcccagaa 94860 gtttgaggct gcagtgagtc atgactgggc cactgcaccc cagcctggac aatgaaacat 94920 gaccttgcct ccaaataaaa aaaaaaaagg aaaggaaaga aattctgaca catgctgcaa 94980 catggatgaa ctttaagagc actatgcagg gccaagctca gtggttcctg cctgtaattc 95040 tagtgcttta gaagaccaag acaggaggat tgcttgagtc caggagcttg agaccagcct 95100 gggaaacagc aagacctcat ctctactaaa aataaataaa taaatcagct gggcgtgatg 95160 gtgcacgcct gtaattccag ctacttggga ggctgaggtg agaggatatg attacatgat 95220 tacatgcctg taatcccagt actttgggag gctgaggcaa gcagatcacc tgaggccagg 95280 agttccagac cagcctggcc aacatggtga aaccccgtct ctactgaaaa tacaaaaatt 95340 agtggggcat ggtggcacgc acctgtaatc ccagctactc gggtggttaa ggcaggagaa 95400 tcgcttgaac ccgggaggcg gaggttgcag tgagccaaga tcctgccacc gcactccagc 95460 ctgggcaaca gagcgagact ctgtctcaaa aaaaaaaaaa ggttaagata gtaaatttta 95520 tgttatgtat attttattgc atacaaaaac atcagcagaa gaggcagggg ctggaaccct 95580 gttttctaag gagtcctagt acaagccatc acctactatc ctgtaagctg attagggaca 95640 cctggtacac acatgccccc acccacccca agacacaccc ggcagtagag gagtcctcat 95700 acgacccatc cccacagccg gtggagcctc ctcgtgtggc tccccagaga tcttctagcc 95760 cagtgccttt tttcccccaa cgacagcaaa ggccttttgt tcaaagaaaa ttttacacaa 95820 aaattcatct tacaaaacac accaatgggg agcttgccag tcatctccct ctttattctc 95880 cttggtgact ggtatgacat caaagagaat ccctaagttc ctcaacagct cagtttgaaa 95940 accaccgacc tagcccaacc tcctcccatt ttacagagag tgacgttgag gtccagagag 96000 gtgcagtgaa ttgctcaata aattgacaga gtaagcagca gcaaagtcag attaaactaa 96060 gaattcctgt tcctgctccc tttccccttc caactctaga gagacaggag agaggctggg 96120 catggtggct catgcctgta atttcagcac tttgggaggt caaggaaggc ggattacttg 96180 aggtcaagag ttcaagacca gcctggccaa catggcgaaa ccccatctct actaaaaata 96240 caaaaataag ctgactgtgg tggcacgcct atagtcccag ctactcagga ggctgaggca 96300 ggagaattgc ttaaacccac taggcagaga ttgcagtgag ccaagatccc accaatgcac 96360 tccagcctgg gagacaaagt gagactccaa ctcaataata aaaaaaaaaa aaaaagagag 96420 aggaaagaaa gatgaggcag ccatctgggt tctccagggg aaggagggag aacccagaaa 96480 gtgactctta tgccaggagt agaaaggctt gagtgcctca ggggctcagt ctctgcataa 96540 ccctccaaac ctccaaagct tatgggacta agctagactc atgtctgggt ggtgactgcc 96600 agagatcctc ttctctgccc ccataacctg caggcagtgc caactgcctg tgacctaaca 96660 ctaagcccag agagaagtcc caggttggat ggcttgagat ccacactctt cccttccttt 96720 cactcagcca tctgtggtgt gctggcttta gtcctccagc ttgctgcctc ataattgaag 96780 catggttgcc acaactccag ctatcacatc ctcacaccac aacattcaat gaggaagact 96840 ttgtttttac tctgctttca ccttgcgtca gggaagaaaa gtccccttga atcttccact 96900 atatacactc cctttatctc attaaaaagg actggatcat atgctgacct ccacctatca 96960 ctagcaacgg gtaaatggat tgccatggtt ggctttaatc aatcaggatt catcccctgg 97020 gctaagcggg tcactgccca gataaaactg ttcgcaatga ataagacaga atggttgttg 97080 attgacctct aatagccttg gcaacagttc atcccctgat accccaacat cagccactgg 97140 gacagctgga caagcctctg tgtctgcccc tgctgtaccc actagccact tgccaccttc 97200 ttgtccaaac tagaagctca cagcagcaaa cgccccactc taaaggtccc ccagcctcta 97260 cccaacactg gcccaagcac attatgacca ctgccacaaa agcttgggca agtctgaaga 97320 aggggcttag cggttacaag ctcaggctct agaaccgaca agcctgggtt caagttccag 97380 tatcatggct actagctgca gaaccttcaa caagcttttt aacctcagag actcaaatgc 97440 ttcatctgta aaatgggggt aacacagtac ctacctcacc gagttgatgg agacaaataa 97500 tgcaggttca caagacaagt gtctggcata tacaagtgcc cagtgaatgt aggctgttgc 97560 tatatttacc ttaataataa ggaagactgc cgaggaagag tcaaatgctc cattgtacag 97620 agtgatgatg gtcgaacggt gttggccaaa taggttccca atctggggat gataggacta 97680 gcctggatca cttatttatt catgaaacag atacttcctg agcacccagc atgtggcaga 97740 ccctccttat acccaaactc accctccacc gctagagctc ccacctcagc ttgggccaac 97800 cccatctgag gcagccaatt atagaaaagg gtctctcctt ccctccacct tcccgccacc 97860 ctgccgagtg cctgggatta gggaaggctc ccacctgcag gttggtgatg agaaacagga 97920 ttcccccaat ggtgagcatt ggcatggcca ggaagagcag cacggctgag cctggaccat 97980 caaagtcaga ggtagggtgg tgtcatagtg caacccaaac atggagcccc aaactctgct 98040 cccacctgct ccaaattccc aacaatcctg gtatccaggc cccaattcta gccagcgttc 98100 cagcgtcctt caagggtttt taggataccg gccaaggctt ccccagatat ctctgtggaa 98160 gtcttctgag ccaccttctt cccaaccaaa gttggtcctc agtctgtggc aggccaggaa 98220 gtctacagac agaggcagag ctctaagtga agccacctct ctcttccctc agtaaaccac 98280 aagctgcctc tccctttcat ccttgacact cctggaaaag aagaccctgg actcaggtcc 98340 ctggctcaac cctctagccc attccctaat tcatggtatt ggccttgagc ttcaatcatc 98400 tgtttaatgg gaacaacagt tcctgctctt cctgtctcag gtgctatgag aactgagtga 98460 gaaaaggacc atggtctttt ctttgttcac taaactctga gcacttcttt ggtgccaggc 98520 attgtgcttg gcactggaaa tgcaagatga atcagatagt ccttgccctt aaatagactg 98580 acatgcaaac aaatggttat aacaggtctg gtaagtgtga gaccacagca aaaaagctca 98640 agagctgggc taggggaacc cttgacaaat tcttcctccc caaaccagac ttctgcccac 98700 cattattctg gccacaacct atgcctgtcc tattatttgc taaaatgttt taagttgact 98760 cacttttatc caaaaagtat ctatttttaa aggacacttt atatcactac tgtagatgaa 98820 aacactggca ttacttgtca tgaatagaaa gtaactgtca aaataaatac aatgaaagga 98880 aaacaatgtt attcaattgt agctggatgc atttgacctt agaatgttca aagcctaaga 98940 cctgctcttc ccatcagtgt taaaatcaca ctggccccac atgaagacat tctttcatga 99000 aatcagaagg actgaaagag aaataaaaag ggaatagctg ttctaccagg tgatttgatg 99060 tttgttagtg tagttcacgt agtatgcgtg tgcccctaac atcctcttaa ctaccgtgct 99120 ataccttaag aagcactgcc aagagctaat tttagagtat tcacacagtt taccattcaa 99180 tttctgtctt tataaaatgt acatctctcc tactactaaa ggttggagac tcctttcaca 99240 atagagtcct tatgggctca atgctttttt caaaactgaa aagccctata ttatggagga 99300 agaggaggat tgttgctcag acgatttgca ggcacgagtc aaacattacc cagccaccac 99360 ctccacattc agttgcttaa aaatcattta caggctttta gagtagatga tgctggtttg 99420 ataaggagag tggtttgaaa taattggttt gaggtgctgg gccatctcat gagatctgtg 99480 tgaacaaaga cactcagcct ctgtgtttgc ccagcatgag tgcagacaat ctcatgatgc 99540 tgtcagcttt agcatagctt acacacacaa gagtaatgta ctttctttcc taaaccaaaa 99600 attgagccac gggtctaaca ctaggaagga atattgggag gcatctcgtg gccaccataa 99660 ccaaggcaat gacagaaaga agagtgaggg atcaggaggc ctgcacatca ggcccacctc 99720 ccacttgctt tctctgtggc catggacatg tctttgcaag gggtcctgct gtggcttcag 99780 tttctcctct gtgtaatggg tggaagggtg gtggaaaata aaccagattg gagttccaga 99840 cttaacagac tggtgaaatt ttaaaacaaa gattttgagt acaatagggt tgtcaacttt 99900 taccctgcta agtaaggata tttgcaaaat ggtcattcat ataatcattt cattaaaaag 99960 agaaagagaa cattttaaca cataggagaa ggatgtaaag gttttttgtt gttgttttgt 100020 tttttagggt tttttgtttg ttttttggta gagtctcact ttgtcaccca ggctggagtg 100080 caatggtgtg atcttggctc actgcaacct ctgcctcctg ggttcaggca attctcctgc 100140 ctcagcctcc tgagtggctg ggattacagg cgcgcaccac catccagcta atttttgtat 100200 tttttttagt agagaaggga tttcaccatg ttggccaggc tggtcttgaa ctcctgacct 100260 cagatgatcc acccgcctca gcctcccaaa gtgctgggat tacaggagtg agccactgca 100320 cctggccaga tgtaaagttt tgaataaatt ctactctctg aagtaatccc tctccatcat 100380 ccttgctttt cacattttct caataaactg ttttcacaga ccagcaatag ctcaagatcc 100440 ttccaggatt ctttcaagct gcagatctct gaataaccat gtggtctgta tatcttgcct 100500 atagccctct gctcacacct gccccagcca ccaggtgcct ctgagcttgc atccctccca 100560 cccacctgac agcactcacc tgcagaggtg aaggctatga tgagtgtggc ggtggtgtag 100620 aaaaatctga aacacataac aggaaaagca gaatattgtc aaggagggag aaacctggga 100680 gaaaaaacat gattctgctc agccagccca caagtgtagg acttgaccgc accctcagcc 100740 tgggatgcaa cgggcactga tgcctctgag ccccaggctc aaaaccaggc gcaagaagcc 100800 gcgatgagat tgagatgtgg tcctgacctc atggacagtg cattttgctc attctgaggc 100860 ccaaggctag catggaaagt cttggacaat gagctcagct gacgatgtga ttggcttggg 100920 acttagccag gacagaatgg gcaaagcgaa ggtcctccca cctggaagcc ccaacagccc 100980 aaccccttgg agaaaggggt tagtgcctgg tctgcaaatc aaggccttga gttctaactc 101040 ctcctcactc tgtgaccttg ggcaaggcgc tgtccttctc tgggcctcag gagccttttc 101100 tataaaaaga aatgatcgga ctgatctagc tcagagtgct atgatttcag gactacagtc 101160 ccaaggttat caggctccct tagcatttgg gggtcttgta aggcatggag taaaaaaaaa 101220 aaagcaatat cctaaggctg gagaagaggg aggggacaaa ggaaggggag gaaaggggag 101280 gtagcaggga gccaaggacc aagaaggact gaggtacagt cattctgcat ccaaaggctt 101340 aaattgtaag ggactggctt tactctggct gtttccggaa aggcaggccc agccagccct 101400 cccgtctctc tctctgacag ccaatctcac atgtgcctcc ctgggagcac ctgctctgag 101460 ctgtatcagc ccccagcagg ccgctgatta ccactgagcc tggccacaga gcacgagatt 101520 aggatgcagc aacacactgt gtgtgagatc acgtcccgaa ccttctgact catctgcaca 101580 ggaaaccccc ccagtctccc ctccagtcag aagggacctg aaattccacc agtggcaata 101640 ccaaagaaac ttcctattag ctaagcccct agggagtgat tggctgttgg ggcggggagg 101700 gggggcggtg aggaggatga ggatgaagcc tgggcaacct ggatgtgagg ctgtgcaggg 101760 gatgaggaca aggatccttg gggtgaagga agagaagagc aattttaggt tttgctaatt 101820 ttgtaaccct ggctccaagc cagcccttac aggaagtcac cctggcctcc ggctcaattc 101880 agcacgtgat agggaagcca catttatgca gagcagggaa cgaggtaagg aaatggaagt 101940 ggggctgtgg tgaagtgggc aagtctagag agagtcccgc tgcctggggc tgttcctaac 102000 agctgctggg agcgagctgc aggtgtggtg cctggcaggg tggccgggct gtctgactct 102060 ggatttcact ccaagctagg ctgctgcctg aaggattcct cttacccacc tttgcctggg 102120 ctggcctttg ggacttacat ggctatgagg cgtgccacgg tggtcttgaa ccggtcaaag 102180 atgtagccag tggggaatgt catgaagttg ttcatgaagg accccagggt gaagatgagt 102240 gagaacctct catcctgggc tttgcagtct ggagtagaaa aaaggtctcc catgcatccc 102300 agccttcctg ccaaatgagc acacaggctg ggctcccctc cacctcagac agcttgtcgg 102360 tcgcaaactt gtcccttaag ctgagttgaa atgtggctgc ccctaattac ccctcaggag 102420 ctggtgcctc cctcccaggc acttcccaga tcaagtgggg tgagagctgc tgacccttcc 102480 tctcatcata gaaagagggg tgggcagggg gcagagtcct tcctgctcct tgccaccacg 102540 tgggagccag acttaacttc cttagaaaag tcatccctgc ccttaccagc ctgccctgtg 102600 gcattgccaa tcggcccagc atctggtcca cacagatcct taaagtaatc ttcattcttg 102660 aagacaaaca ctagtgaagg ccagccaaag aggacgccag caaagcccag gcattccagc 102720 agcccagtca gcagtgtggc cacgtgcagg ggcaggccct ggcccgccat gagcagaagt 102780 ggagtggatc ttcaaatccc actttgtcct cctggacgga tcacaggcgc cgtaagcctg 102840 gcgtttgagc acttggaaaa ttcctctggc aagccaagcc cttcctttcc cgtagctctc 102900 tggttgtttc aggcctgggc aaaaaccatc agcgggtgat tctctggatc ctgtagaata 102960 aagatagagg ctgctggaag aggaggcctg cgggaaaggg aaaggtagac tagagttatt 103020 tgtgaggtgc attaagaggc aggatgatca tggccgctgg cagcaaatgt ggggaataaa 103080 tactccaata catcatctta ggcactgcat ttgagtaacc acgtggcaag tagagaggca 103140 ggtcttgatg gccacctgga gtcacaggtg agataaacga cttacccaat agctccccgg 103200 gagcaggtgg agaagcggag ctcctgcgct cgaattctga ataccgtccc ctaaaataat 103260 gacagcaact caaccaggtg cagaggcagg gagatttcat acagaagaca caaactcccg 103320 ctgccaagtt ggtgttatct tcagtttact gacaatgaaa caaaagctcc catggatttc 103380 aggaacttgc ccaaggtcac agggctagtt tagtcacgac gcaggccatt ctactgccag 103440 aaataccccc aactcccatg accctcgcct aggactcgca aacctggtcc ccgccgccct 103500 tcctcgcatc aacttctacc aggaaagcct ccgggggccg ctccccgcca gcctccgcac 103560 cccgctccag cctgcggcct gccctccccg cagaggagcc cgaggggcca ggccgcgctc 103620 ggcgccccat ggcgcccgaa aggggaccct tcgccctacc cgcctgctcc gcgccggggc 103680 tctccgcgcc ctttccgcac gggccaggtt cgcattcgcg cctctcgcag cccctcccag 103740 tcccctgctc gcctccgccc cctcctgccc gcccggaagg ggctggggca gacctcccac 103800 tctccatcac ttccttcttc ttttcccttg ctcacagcct cccgcgccct ttttacctct 103860 ccctcttgaa acttctccct ctagaacccc ctagaacccc agcggtgtct ttccctccct 103920 cctcgctgcc tttcagcctc ccagccccct tgcctctgcc tcccctaacc aagttagttg 103980 aatgctgtta ctcgctcagg cccacctagg gaaaatgtca cacccagcac ccagaggaca 104040 cacagacagc acatgagggc atagggacac acacactcta tttgtgcatt ttgccttgac 104100 cgctgggttg gcagggaaca tatttttcct atttgctcac cagcttaacc gtctctccca 104160 gtttcacact cccagagctg ccaaaaaaat cccaaccaca gaatcaggaa gccaagaacc 104220 aggactgagg gcttttcaga aaccatcccc tggaggactg ccccatattt tcactcccaa 104280 aaacccctta gatgactccc tgcctcaccc ccgcccccca ggttctgaaa gagccttccc 104340 gccagactgc attgattaac cattcattgc cccatttttt attaatcaaa gacatatata 104400 attgctcatc ggagcttgtg atcagcgtga ggccttacta agcagctgcc ttactatcct 104460 tccagcccag agcacgtgag ctgacgtctt ctttggcctg tgtggccgtt tccttgccaa 104520 aagctcagtt tggggagagc ttcttgcgta ttagatgcag tctgcagact cccaacccca 104580 gctacctgga tcccctgagg gcccaggaac tccagctatt ccaagcccac tcctcttttt 104640 tttaagagga agaaatagag gttacgatag gggacagcca gaactgagga ttttccagct 104700 caccaccaaa gcacaaaaga taaaagtctg caaccaccct agtgacttga ctgaatggag 104760 gaagggtggc tggggtcctg taccccaagc tactcactag ttatacaacc tgaggcaagc 104820 tctttggctg ccccacctgt aagacgagga caatagtacc ttaattatag gaattgtcat 104880 aaaagaagta taagatgggt gtatgaggtc cctgcatggc gcaggtgcta taggcagatt 104940 gtagggtagt agattttcta gtctgcagtt atgtagacag agccagagaa gcagctctgg 105000 ggaggaattt caaaggaact tgcccacggt cattctacaa agctgcagta ccttcccaac 105060 tctgaaacgt atgctctcat caccccgtct taacaaacat ttggacatta gagaaaacaa 105120 gtcttttctt aaaataacat tatttatggg agaaaatcca caaaaatata gcatcccagg 105180 acaaacaggg cttaagatgc aagattttct attttactgc aagacacaaa gactctgaaa 105240 ttaatgcatg ccctatcttc tgctctggca tacattttag tctcctgggg ggatcagtaa 105300 gtgtggaagt agcaagggag aaacagaaaa aagtcaaagt aaagagacag attttagaat 105360 gttaatctgc aggagcctgc cagaaagatc tagctcatgg gctatctgta catccaggac 105420 tgaagcacgg gacacggggc aggtcgtcca gggttctgtc caccttatct tgttacctct 105480 cttgactctt agagcctcca ctccacatct cccatcaatg tctgcagaag acgtggcctc 105540 cactaacaca agtcttactg aactgatggg acaggaaatt agaatatcct ctgaaccatt 105600 cccatgttct ttggttcgaa ttccagcagc tagaaaaggc agatgctatt ctgatcactc 105660 tcctgcgtgg ctccaatgag gattaatgag taacatcaga gagagaagtg attataataa 105720 ggtctgacgg tgcacccgat gtcttcatcc ttttctcttc gcctccttcc tcatcatctc 105780 acaccttttt ttttttaatt gactgattgg ttcaacaaat acatgtggta cctcaggctc 105840 tgtgccaagt gccgggattc gtagagaaga gattcagtgc ctgctctcaa ggggctcatt 105900 ctcttgtggg agagacagac aaagaaaccc aagatttctg gagtgtggga atggtcttcc 105960 aggcagatgc tagcacagca cattgaaagg cacggaacct caacaaaaca ataacattta 106020 ggaaccagct agagcacagg gtggtgaaga aagtggaaag atttgaggcc agcgtcgcca 106080 tctaagtgag ggcattaaga attcagccca catcaatcaa tcatgtccta ttgatttcac 106140 cccttaatat ctctcctatc tatccgtggc cactgctcta tgcagacact catcatctct 106200 cacagaggca tcatctgctt ccaagccatc gccattctcc tgcaagagtt tatttccatg 106260 gttcccactg gatggcttca cttaactgct caaaaccctt ctgaggtcca gtcaactggc 106320 tggtaaggac cagtccaggg tctggggatg ccagccatga gacattgctt tgaggggaag 106380 agggagcata gaactggatc tcctgcatcc tactgcccaa gtaccaatgc tggaggtggt 106440 tttccttccc atcatcagca agtctggata tccaggatcc accctatgga tgtttttatg 106500 gacagagtgg gaagatggat atgtttaggt tagggaaaga gggtttgcca aagagggcag 106560 tataagtgag ctgcactcca tcattcccct ggcacaaaca atggctagta tcctctagtc 106620 ctcaagagca ccaccttcca atgcagtccc tgcctgtcca cagacctctc tcctcaaact 106680 tcctctgaac aacctcagcg agggcaattg ccactctctg ggcagagtcc agatattctc 106740 tcctaccctc tgacatcact ttctaaattt gtatatgtag ataaactctg agccattcac 106800 ataaagggct ttgatttcgg atacgccaaa cacataaaca aacaaacata agctttcctt 106860 tcacaatggg ctcatgtaaa ttaaaatgtt tggttttcca cctacggtct tgaaaggggt 106920 ttctacagcc tgttttggaa gtcagaaagc aaaaggtaaa tgcaaacatc atttcacctg 106980 cagagaaaat tctaatcctc ttgaggcagt gccaaaaata atacaagcac actgctatcg 107040 agccaattac tggtatctct gagcttccgt ctcctcatct ataaaattgg aatcgagctg 107100 tatggattaa agataatgta tgaaaactgc ccaattagta cactattaat aaatagcagc 107160 tactgttgtt aacaaatatt attgacttac tggaaaacaa agaggaaata aagtcacatt 107220 tagggagaat ttcaaagtgt tcctaaccta aaaaagaaat aaattagggg gaaaaccact 107280 aagtaatggg tgagctcagt ttaccttgct taagaagtcc caccctagag aactgatctc 107340 tagatgacac ccaaatgcac tcagtacaac cccccaagac tgtctgggct taaggcaggg 107400 gcttggattg tcctgtaagc tgtgggaagt ctgttcatga gccacagtag acaggaaggg 107460 gatggagtct tagagctggc tcttcagggt atctcctagt gtgttcaaag cagttctcag 107520 gagggtgggg aactactaca tagccaagta aatatgaggc ctccttgctc tggggagacc 107580 tttctcttta acagaggtga atctgaaagg atacccaaag aggcactgga gggtgggggc 107640 cactctggcc cctcagagca gccagctcag cttcagtgga tgctagaggc agcagaggat 107700 cagcctggat cagcctccct ttcaccatgc agaaaacaga gctcccccac cagaccagat 107760 actggaagca ctgggccagg cctaagagaa agcagagccc caagccccac cacaccaggg 107820 cttatgaggc tactgctacc caccctccca agccccagct ccacttctat gttcatcaag 107880 caactgttta ctggtaactg cacttcccga tagactttgc tagaaaggaa tgcctcagtg 107940 cactgacaat atctaaacct gcaactctaa ggactaggct ggggaacact gtgtcaacat 108000 ggaggcacgt cctacccctg agaagaaaaa taaggaatat caataatacc tgctgggcac 108060 tgagcactga ctatgtatct gattcgaagc gctttttgct taatctgtca ctgaatctca 108120 cgataggtgt tgttattagc atctattatc tgggccaact gaggcctaga gggacgaagc 108180 aactccccca acatcaccag gtagcagtgt caggactggg atagaaacct gatgctctga 108240 ctgaaactaa tgcttttttt tttttttttt tttttttttt tgagacagca tctcactctg 108300 tcaccaaggc tggagtgaaa tggtgtgatc tcagttcact gcagcctcca cttcccaggt 108360 tcaagtgatt ctcctgcctc agcctcccga gtagctggga ttacaggcgt gcaccaccat 108420 gcccagctaa tttttttgca ttttttgtag agatggggtt tcgccatgtt ggccagactg 108480 gtctcaaact cctgggctca agtgttctgc ctgccttggc cccacaaagt gctaggatta 108540 caggcgtgag ccaccatgcc cagccagctg atgctcttaa tctgtgccct acccagcctt 108600 cctgggaggc ttcccaagag ctacacagag catgagttct ggaatcgggt tgatgggggt 108660 accagttatt actaatagga atgaagatgg gtaattcttt cagacagcac ccttgattaa 108720 aacaagagag tagtgctgcc tctctgtgat tctgtgtctc cctgccctgc tcacacagac 108780 accacaccca cccacacgca tgatcatgaa aagaggaaat ggatccagga gaaggagacg 108840 actcctgagt gaaaacaacg gggtttttca cattgagagc tttgcccaac accccaaaga 108900 tgaaaagagc aggaaactgc tggggccgat tgaacactgg acttttgttg tggaaaaagg 108960 caaagggaag ccggaagaga ctggaacagt ttccatggtg ctggaggatg gggaagtggg 109020 tagggattag ctggagggag aaggagaagc tggggtggga ggggaacctc cacttgccag 109080 gagagcacat gtaggatggg aaccccagat gatactcaag gcatggcatt agaccagaag 109140 caagtctgtg gtgaaattag ggaaggctcc actgcggact gtagacagag cactggacaa 109200 ggaagtggga gacccagggt ccagtcctgg ctctggagcc ccctgggtgg gctgccccgg 109260 gcacctttct ctcttggggc ctccattcct acctctgtga agcgagtgct gaacctctct 109320 tagccctgac ttgctgaaat gctgggactc tgtacagagg ctgacattaa gcagggatct 109380 gtcgtggggt gctgcaatgt tcctccagat gctgcacggg agagggcaga aaaggcctat 109440 atggtgagtc cgccctggga gcctctgctt ggaagctgaa gtggcctgag agtgactcag 109500 aaaccacgga agttcccggg gctgatgggt tcttatagat tgtacatgca gctctcctcg 109560 tgggctgcaa aaccgcaaga tgggctgtga ccactctcaa ggaaagagcc ctatctgcaa 109620 aaagcattct gccctccagg tcttaaagca aacacagact caatccttat tccttttaag 109680 acaaaattgc ctcaggggca tcagggaggc agcaggcctc aaatgtgtgc ctttctagaa 109740 ttctcaatga aagcaccctt ttgggtatta ataatgacaa cagtaatgac agtcatttac 109800 tgagtgctgc ttttgggaca ggcattgggc taagagctat atgtaatata tattattatt 109860 tgatgcccac agccacccca taaggaggcg gaggtactat cattatgcca actttaaaga 109920 tgaagaaact gaggcctcaa gagatgaagt aacttggcca agtcactcag ccagtaaatg 109980 ggaagagata gacttcccag tatccagagc ccatgttttc accattatgc tgaagtacct 110040 cttttcctgt gccaatgtga tctgcctcca ggaatcctgt cttgatgttc ccttccccat 110100 acagaagtcc tctctgtgtc ctcttcagcc tgatagtata tcttttcata ccattctttg 110160 gacatctctg ttatactact ccaatggtgt tccctcccct acccctccct gggagcttag 110220 ttgttgtgat taagtatagg ggaaatgacc cacactaaac aaactcataa gagactgatt 110280 gataaacctg aaatgcaatt tattaattaa cactgagaaa tgaaaccacc cagcagatgg 110340 gaatcctaag gctgactggt cagcacaatc tctttcagga aggacaggct tttgggaaag 110400 gaaatcaata ccagaaggtt ctttgttgag tacaaagtca gagggaaggg agttgatgga 110460 ttgacacata ggtgaagctt gacatacctc tataaagcct ccatcctgcc aaggatcaga 110520 atatccaagg cagggagcca tctgggtgtc ctctcctttg gacagtgctg gatttttctg 110580 gatcctatga agatcttacc tttctggctg catttatcat gattgtggaa ggctttttgt 110640 ttccttgttt gcttagatta atttctgcgt atttaataga actgaaaggc aatttcccat 110700 tgagacccac tgaagaggaa taatcaatac atactagttg tgttgccctt tgcagagaat 110760 tcacttctgt gttgtcactg tatcctcatg cttccttata atggagggac agagatggta 110820 aaaacatgga cttggaagcc agaccgtctg ggtttgaatc ctggctctgt tacttataag 110880 ctctgcaacc tcgggcagat tacctaagtc agtttcccct tctctgaatt ggggatataa 110940 tagcacccac ctcaacatct gtcaagagga ttcaatgagg gaatacacat aaagtgctca 111000 gaacagtgtc tgccatctgg taagcagtcn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 111060 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 111120 nnnnnnnnnc taatttttgt atttttggta gagacagggt tttgccatgt tggccaagct 111180 ggtctcaaac tcccgacctc aggtgatcca cccgcctcgg cctcccaaag tgctgggatt 111240 acaggcatga gccaccacgc caggccccac acacctttta aacaaccaga tttcattcat 111300 cgggaagtgc ctgtggggct ggtgtggaca tgtgggtgaa ggtggcactg ggagaagtta 111360 ggattctcca tgacctctgt tactcatatt cccacactcc tcaaattagc ctgagtctcg 111420 aggacagtct gatggctggg caaaccctgc ggcaaaccat tccccagccc tgccctctca 111480 accagagtcc ttccgataca tgattctggg cagctgttgt tacccgtgtc ctccatgttc 111540 ttccagagat atccatgcat gcatcggcat atgtgtataa ttattatatc tatatttcat 111600 cccacaagct tttgacatca atagtagcat attattaaat tgttctgtac attattttat 111660 taacttggta tctctggtac tgctcaatat aagaacatat agacctggcc aggcacagtg 111720 gctcacatct gtaatcccag cattttggga ggctgagatg ggtggatcac ttgaggtcag 111780 gagttcgaaa ccagcctggc catcatggtg aaacccccat ttctactaaa aatacaaaaa 111840 ttagccaggt gtggtggcag gcgcctgtaa tcccagctac ttgggaggct gaggcaggag 111900 aattgcttga acctgggagg cagatgttgc agtaagccga gatcacgcca ctgcactcca 111960 gggtaggcaa caaagcgaga ctctgtctca ataaaaaaaa aaaaggaatg tatagacctt 112020 ctttattctt tttgatggct gtaggtggat gttctaaaat ttgtgtaacc aatctcctat 112080 tgataatatt taagttatgt cttcagcatc atatgaaact tacaaacaag gttgcattga 112140 ctatccatct gtaaatgtct ttttgaacat ttctagaata attgcaggat aaactcctaa 112200 aatgagaatt tctgggtcaa agaggatatg cattttacat ttaatagata tttgtcaaat 112260 tgtcttccaa agtggtcgta ccaattaaca ccccgacctg taatgaatga gagtgccttt 112320 tttccccaca ccctggagag atgaaaaatt tatgggccca ctttggagtg catggtggag 112380 gaagctgttg gccgttatat aaccctcgtc attaataagc ctgggggtgg ggggggagaa 112440 agagaggtta gttagtgggt gcaaacatac aattagatag aagtaataag ttctaatgtt 112500 cgatagcaga ggagggtgac tatagttaac aacaatgtat tgtatatttc aaaatagcta 112560 gaatggagga cttaaaatat tccaacacat agaaataata aatgcttgtc tgcggccatg 112620 ccaccctgaa tgtgccagat cttgtttgtt cttggaagct aagcagggtt gaacctggtt 112680 agtatttgga tgggagaaat gataaatgct tgaggtgata gatatcctaa ataccctgtc 112740 gaacattata cattctatat atgtaacaaa atatcacacg tatcccataa atatgtacaa 112800 atataatgta tcagtaaaga gagggctggg cacggtggct cacatctgta atcccagcaa 112860 tttggcaggc cagggtggga ggatagcttg aggccaggag ttcaagatca gcctgggcaa 112920 catagcgaga ctctgtctcc acaaaaaata aaaataaaaa cgaattagcc aggcgtggtg 112980 atgcatgctt atagtcccag ctacttggga ggctgatgca ggaggattgc ttgagcccag 113040 gagtttgagg ctgcagtgag cctacgactg caccactgca ctctccagcc taggcaacag 113100 aggaagacca tgtttctaaa agaaataaat taaataaaat aaataaaaat aaaaagactg 113160 aaaagcagag tggtaagaga aaggactttg gggctcaaca gtactagcct tgaaccctgg 113220 ctgttactta cccatcgtgt gataagcaaa tgccttaacc cctgtgtgcc tcactttctt 113280 aacatataaa atagaagtaa aaatcatacc cacttcaagg gtcattataa aaagccaata 113340 gagataatgt atataaagct tctggaataa tgcctggcac acagtaggag tttaataact 113400 ggaaattcat tgttgtagtg ggcagccttc tgaatctgtg tcctctttgt ccactaatgg 113460 ctttgatctg gatttggctc aggcaagacc tggggaaggg cagagactga gggcaactgg 113520 aggtataggg tggtctgagc ttccccagca gagtgaggct gggaaaggtc tgggagacag 113580 accaggcagg tgctgataag accggaatgg gaggctggag cataaggcag ttcagttttt 113640 cccaaagggg ggtgtacaaa acgatctcgt atgactcctt tatactgtta atgttttcat 113700 tttatcgcgc actgaaaaac aaaaccaaca tatttaatga atgattccaa ggggattctt 113760 gcttttacaa aaaatgctaa agtaggcatt cacatgttta aaaattgagt tgatttaaat 113820 tttaaaatta ctaagtcata gtacataatg tgtgagccac agctatcccc aaaatcatga 113880 tagcgataca ttaatgactg aagttcttta aacatcaaca tacaatgcca attccagaat 113940 tcagctcaaa ttctgcaatt acacaggctg gggttgaaac ccagcttttt tgctaactgt 114000 gtaaaattag gcaggagggc taacctcgct gaatctcaga tgtctagtct gtaaattgaa 114060 gataatgttt gtttttatct cacagagttg ttgtgaagat tcaataaaat cacaacatgt 114120 gaggatgatc tggctgtgac acctgtcacc ccactgatct ccagagttga ttcggctgat 114180 caggctggct gggcaggtgt cccctttctc cctcaccact ccgcatgcat tcctcccgaa 114240 actgcacact tggtcaaaga ggaagacctt tcctgataga ggaggaccat tcttcagtca 114300 agggtatatg agcacctgtt ctgtcctgcc agaatctccg aaggagctct cagtaaaatc 114360 acaagatttt attgtgcatg gtagcatgag cctgtaatcc cagctactca ggaggccgag 114420 ctaggaggac tgcttgagcc caggagtttg agaccagcct gcgcaacata gtgagaccct 114480 gtctcaaaaa aaagaaagaa agaaagaaaa gaataataat agtaataaat cacctgtgca 114540 acgtgctcac ttctctcttt ggaatgtagt aagtgtacct aataaatgtg atcattgtaa 114600 tcatcacagt gagcacaggc taaagcatct tgactttatt ctataagcaa taaaagagga 114660 tttgttttta cagaactcat tatgttgtga aaataatttt ccaacattaa caaagaacat 114720 tcttcaagta aaaggaaaac cacccatcat tctcccaacc ttcaataatt ttcaattttg 114780 catattctcc agactttgtc aacatgaata cttactttac atggtcgcaa tcagtgttca 114840 tgcaaattct tttatcctga cttttataaa caaatatgat gttataaacc ggtctccatg 114900 tttctgcata ttctttataa ttatcatttt gtggctgcat aatattgcat tgactatgtt 114960 aactgcagtt ttcttaacca tttcactgtc tggggaaatg gaggataatg ccagggtcat 115020 gcctggagct tttttttgtc tattgcatta tattcttaag atcaaatccc agcagtgaga 115080 ttagtcagtc aaaaagtaat aatattttca aggctcttgt tatattttac tagattgttt 115140 tccagagttt tgcacactgc tcccagagat gtaggaacac agacgtcatc caaccttgcc 115200 agtgctgggt gatggtgttt ataaacttct gctaatttaa taagtatgaa atgctatcct 115260 cacacggctt tcatttctat ctctttgatc attaacaggt tgaactattt tccaagtatt 115320 tgtttactct ctgcataccc tcttgggtga agtagtcatc cacttccttc acctgtttat 115380 ctgttgaagt cttgaggctt gttttataaa tgtgagcgag cacttcagag tcaatagaca 115440 ttaattgctt ccagccagat ttggccactg aggctcctga gcaggggaat gcatgatcaa 115500 aactacaccc tggacagatt aaattaattg gagaaaatgg gctgagaggc agagatatgt 115560 gtcactggcc tactgtgttt gatcctatag tgggggcctg aactggggca acggcctgag 115620 tcccccacta ccagtagcag gaggctccat gtgtccccca tattagagct tgcggcactt 115680 ccatttgccc cacctcctac aataccccac atacatgtac tcactctccc ttgcaaatct 115740 agtggcttca acccacagaa tttaagggga aaggaattgt tctgtcctgt tcacttactg 115800 cagaaatgag aaaagcgttg ttcacatggg atcacctaat gaagggatgc catccccaac 115860 ggtgcctata aggaaatggg ggagggttgg agagttgtgc aaaatgcaac agggaatcat 115920 cagagtctct tgccccatga tagagggttc tcaaattaag agagtctaca gcaactaatc 115980 tcacggccac tctaggcagg gcttcccaat gcttccccaa ccccacctcc atcctagact 116040 ttacccactc tgctgaacac agatgttacc catagcacct tgcaccatga ttgtttgatt 116100 agcacctccc acagtagact gtgtttctga taggtcagca acatttgctg agcacctact 116160 ctgcagggct gtgccaggtg cacaaaataa acaaagccaa agacaacatg gaccctgaac 116220 tcagcaagtt cagagtcaag tgggataggg aggctctctt cactggaagg taactccaag 116280 aaacaatggg actcaacttt ctaaccaaga gaactccagg gagctaaaat tctgacttct 116340 ggttaagact ggtgtggagc ttcattaaag aagaaaagat tcacccagac ttgagttcat 116400 agcctggctt tgcagctttt aagtcatgta acctttgatg aagttatgtg acctctccac 116460 ccagctgccc ctaacacctt gcaggggcag ggctggagtg caaagggagg cactggtacc 116520 acagcctggg aggcaccacc ccactagtgc aagccgggca acctctgccc ccaaggcatc 116580 cctagcctcc caactgcaag catcaatctt gcacttggaa aggaacctca cctttgaaat 116640 ctaggttcaa atttagaatg atccagctcc ttgaagttct atacagaaat acagccagca 116700 gccaggcccg gtggctcacg cctgtaatcc cagcactttg ggaggctgag gtgggtggat 116760 cacttgagga cagaagttcg agaccagcct gaccaacatg gtgaaacccc gtttctacta 116820 aaaatacaaa attagccagg catggtggta cacgcctgta atcccagcta cttgggaggc 116880 tgaggcagga gaattgcttg aacccaggag gcagaggttg aagtgagcca agatcgtgcc 116940 attgcactcc agcctgggca acaagagcga aactccatct caaaaaaaaa caaaacacac 117000 gcagcttccc tccacttccc aaccacagct ccatctcaga caacaagggg cctcatgtcc 117060 atgcacatga atatccaacc aacatgtcta aggcccaacc acaccctctc caaacatctg 117120 ccccttggcc accctttggc catgggttca tgcactggca gaaaggtagt tcagagaaga 117180 agcccacaaa gggccgggaa gtccacttgg gctttttgag attccagggt ccaggataac 117240 ctaagtgtgg tctagaagag agatgcagct tctgggaggc acattccttg gtcttaggga 117300 cttcttgccc ccatggaggg aaactggcta gatgagggcc aaagcagagc cctctaaagc 117360 acagggctca gggaaggact ctttttgacc agatctaaga gcagcactac ctctctgagc 117420 ctgtttctcc atctgtaaga aggggacatt aatagactct ccccgctaga gttactctac 117480 atcagccagc acacgtaagt tcatgacatg aagcaagggc ttaatatata cccgttgtac 117540 tataaataat aggccaggcg tggtggctca cacttgtaac cccagcactt tgggaggccg 117600 aggaggatgg atcacaaggt caggagtttg agatcagcct ggtgaaacct catctctacc 117660 aaaaatacaa aaattggccg ggtgtggtgg cgtgcacctg tagtcccagc tacttgggag 117720 gctgaggcag gagaattgct tgaacccggg aggcagagtt tgcagtgagc caagatctca 117780 ccattgcatt ccagcctggg tgacagagca agactgcatc tcaaaataaa taaatacaca 117840 catacataca tacatacata catacataca tacatacata catacaatac atggacaggg 117900 accctaaaaa tgagacaggg aaagagaaaa acatgttctg acaaccttgc cctttatact 117960 aatttaggtt ttcttgcctg ttttagaaag ggcctggaca ggagccctgt tcccctcagg 118020 ccaggcagaa caaggtgtgg aactcactgt ggaagggttc tgggtgacaa gtgcagcccc 118080 gtccctccac ctcccagcac agtaggcagc acgtgtctcc attgactggc tcaggagcag 118140 gcctggtgac cagtgggaga gctgaggagc ccagggtggg gtctgaagga atccctagaa 118200 aatctgattt tcccccaggg cccacatcac gtgcccagag ctgggaaagt ggaggcagca 118260 tgggatctag ctgagaggct ccatttttgg tagcttctag tttgggagtc acagagacac 118320 ctggatgata cgaagatgta gctttgcagg actctctaga acatggagtc caagatattc 118380 ccttcaatga tgggacactg aagcccacag aggagaggtc tgtcccagtt actcagccat 118440 tcggaggcag agaccaggct agaactcagg acttttaatt tggaccagga ttccttttac 118500 cacagtgggc agccctagca agtgccaggt agggtggaac tgtgaaggtc atccgagggg 118560 tagtacacgt gggtaggaag tcatatctaa gaactgaccc ccagacctgg ctctgccact 118620 cactccttat gagaccacag gtgctgggtg cagtggttca cacctgcaat cccagcactt 118680 tgggaggcca aggcaggcag attgcttgat tccaggagtt cgagaccagc ctgggaaaca 118740 tagtgagacc cccacctcta ccaaaattag ccaggcgtgg tggtgtctgc ctgtagtccc 118800 agctacttgg gaggctgagg tgggaggact gcttgagcct gggaggcgga ggttgcggtg 118860 agccaggatc atgccactgc acaccagcct ggatgacaga gtgagacaga atgacacact 118920 gtctcaaaat aaataaataa atgacagcag atcatcattt ttctttctgc ctctagactg 118980 caatgcctat ttctccaggt agtcactagg ataaaagtaa aaataatatt atcagcattt 119040 accaaataca gggtcagcta ctctgttatg ttctttcatg ctttgtttct tttaagcctc 119100 aaacaactct atgagctggg aacaagtatc gtccttcttc ctcccatctt atttatttat 119160 ttatctatgt atttatctat ctattcattt atttatttat tttgagacaa ggtccttcta 119220 agtcaccagg gatggcctca aacttgagct aggaactagt gtcaccaccc cccaatttct 119280 tttattgatt gattgattga ttgattgact ggttaatttt gaggcagggg tctcgctaag 119340 tcgacagggc tggtcttgaa ctcctagtct taagcaatcc gcccgcctca gcctcccaaa 119400 ttgctgggat taccaacacg agccaccatg cccagcccct cccatcttct gaataggaaa 119460 actggggttt gaaaaggtaa gcgacttgcc caaggtcccc tccctagcta gagagcttca 119520 gagccagggc acaaacccat caaagcctgt gctctcgccc attgagccac cggacctcgt 119580 acactaaccg ccaagtgttc tacacagtga aggtgacaaa gaggtgaagg gaagagccag 119640 ggaggttctg tggactcact cggtgggtat gcccagaggg aagggggatc ttgggtggca 119700 cattgagagt agctgcgctg ttagtaagtg agaactcgga agtccagact catccagtct 119760 gtgccaataa acccctcctt ctacctggtc tcctttccaa agccagctgt tctccagaca 119820 atggggtggg cgggggcggg tgtcctcctc cttctcaggg aaaatccgac gctgagccca 119880 tctccagaga tcttggcttc ccgtggggct gcagatccac ctagagccac cagagggcgg 119940 gccagcactg cggccaaggc ttgaagaccc agcacaccaa agcccggcca agcctccagc 120000 ccagtgtcca agagtccagc cagaggccga gtcctcgatc tcaaaatgtc taactgcaga 120060 agcccaactc atgttcaggc atgatgtgtc tgattctact gggacaatca ttgccaccaa 120120 agaattactg ccaaaatagt aacgacatta gctacctacc acccctccac acaccaacac 120180 acctcatttt accaagcact ttctcatgcc tggggtgcct ggagacttaa tgcagcctcg 120240 cgatgaccgg gtagcctcac cgtacagatg aggaaactga ggcacaagga aggggagtac 120300 attgtctagg gtcacctgga gaactctgat ctccagactc aaatttccaa ttcgtccccc 120360 ctccccccaa ccctaaccgg agctaggtgg ggtggggaca gcaaatgtgg atggggggag 120420 gtaagagggg tcagagtgct ctacagagaa gaccaaatgc attgtggcac ctactgtaaa 120480 atgagaccag ccagccaccc acccaccagc cagccaccta aaagtcttca gtgggcacct 120540 gctggaaaca cgacaatgga tgacacgagt tccctgccct caaaaagctg gtagtctagt 120600 tgggggtgga gggggtgagt cagcagataa ttatgggaaa ccgtgacacc tgtataaggg 120660 gcggggatga gcagaggggc tgcgactgcc tggagccagg gattcccgga cggggcttcc 120720 ctttcctcgc agctcgtccc aggaggagga gctccccccc agcttcgggg ttccgcctgc 120780 cttgggggcc cggggtcccc tcccacccct ccccgaagag cgcgggcccc gggaaccgat 120840 gacagcacac ctgagtcagc ccgccgccca cccgcccctc agcgtctgtc tccgcatctt 120900 gtgatatttc gctccccggg agccagcccc actgcgctcc ggaggcagct cggcaaacaa 120960 acccagcgac agattgtgcc gcggctcatt ccggggaagg acgccaaacc ccaccctgct 121020 acccccaaca ctccctcccc gccgccgcct ccaggccctc cccccaggcg caggccctag 121080 tcggggtggg tcctggggaa acgcagggtc ctgtcctgcc tcctggaaat agggggagcc 121140 ctgggtgagg gaagacggga gccccagaga cttttctttc tgtttctacc tgatccgaaa 121200 acgagagggg cgggaaagga aatttagggg cacagagagg agctgggggg ccgagaaggt 121260 ccgaaaatgg aaccagcagg gggcacccga gagccgaggt gcccacgggc cgggagcctg 121320 ggaatgaaac tggggaagag ggggagagaa agggaggcag agacaccgag acacacagag 121380 acgagagaca gagacgcagg gagccccgcg gggaggagga gagagacgaa gacacagaga 121440 gacagtgaga aagacagaag accgggcagg gaaacagacg agtagagaca gaaaaggtcc 121500 gagagagagt gagggaggga gggaacagag agacagagac cacgaaatat gagtaagagt 121560 cgggggagaa aaccagagaa atcgaatgag aacgcgagaa gaacgagaga ccgtggaggg 121620 agcagagaat gaatgggaag aataagacca acatttatca agagccgact gtatgccagg 121680 cactgcattg gaccctggca cggataggaa aggaggagcc gcggcgcggg cagcggggcg 121740 aggggcttct gtgctcgcgg gagcggcagc ccagggggct cagcagcccc ggcaccgccg 121800 cacctgcggc tccagcagcc ccaaccccgc cagcgctgcc tggccaccgt acccgaagcg 121860 gctcccccga gggccccgag cctatcctac gccggggcgg ctccgcggac gcgccgggcc 121920 gagtcaagac ctgggtcaac cgccctgcag cctttgtagg gaagtgccta ggtgatgggt 121980 ctgctgatac cgcctgtgac caggccatga agggccagag gggctccagt gagaccataa 122040 tccgcccctc tttaaaaggg ggtagaggaa gttcacgcga agccaacagt cttctcccca 122100 gctttgggtc ctctcctgca cccccgcggg agataaggtc tcccctcccg gacacatcat 122160 acatacacaa aaaaacgcac acactcgcac gcgcgcccat ctcgcacccg cttgtaaatg 122220 cactaagggg catacacaca ccgggcacat atttctttcc acccatcccc aagatcgcaa 122280 gcgcaaaacc tcgcacagcc tcacgtttcc caccagctca gacatgcacg ctggcggact 122340 ttcagcggct cacccgtgtg cacactcacg tgcccccccc cccgcttccc caagcccgta 122400 caaagggtaa cgggcaagca tcctgagtca cacctgcaca agcatccttg cgcgcacgtg 122460 cacgctcata tgcactcgat cttgcacgca caaactcttg catatactat tcttatagtc 122520 gcacactggg cttgaggtct gggagtggaa ggaaaagtgg aatcttggag ctgtcccagg 122580 ggacagaaat gctggaggct gggacactgg cgcgagggac gcggctgggg gcgggggagg 122640 gggtgaccca gaagctcatc ttctcctgga aagttgggag gggggaacag gacaagtcca 122700 cggcgttcct ctaaactacc gcattccccc aagaagggat ttctctagaa gagtggcgcc 122760 gcgaggacga tcgaacacag tcctccgggt cgcttaagcg ggggggaggg gggcggggtg 122820 gagggggtta gaaagccgct cccgcctcct agtggtcgag aaagggttaa gtcggcaagc 122880 cagcaaacga gggaggagcc agcgagtgcg ggaaggagtg ggggtggttg ggaagagctt 122940 cctcgctgtc cccactctcc ctcggctagc agcctgggca cacggacaga cggactgacg 123000 gactctcgag cggacagcgc agctagcggg gcgcgggcgc tgggcgtcga cggccagccc 123060 cagccttccc cgccccgtcg cgccccgccc cgtcccgtcg gggccgatgg ctcctcccga 123120 ggcccgcagc ccgggcggcg cagggtagag cgccgcggcc cggccacgca gcccggggac 123180 tcccgggccc tcccggagcc ccgcggggtc cccgccgtgc atccggcggg ctcagggagc 123240 gagtgggagc gccctccccc cgctgccccc tcccccgagc atcgagacaa gatgctgccc 123300 gggctcaggc gcctgctgca aggtaagaac gccagcggcg ggagagcgga gggcatcctg 123360 gggagagaag cagggcgtcc cctctttcag ggattgaggg tggggcagtt ggggaggtgg 123420 ggtaacctgg ggaaggggaa aagctcagcg ctggggccgc gcccccgccg ccagggctgt 123480 tctcagcagg agggcacttg gctgggagcc cgcgggcgcg tgcgaggagc tcgtgaccga 123540 ggtgggacgc agggggcagg tggacccggc ccggagcggg gagggaggct caggttccgc 123600 tgtccccgct ccacctgctc cgggggacgc tgaggactcg ggccggctgg ggaagcgccg 123660 actcagcaac tcctcctgcc cggtgcctca gcactttctg gccacctggg aagacaggag 123720 atgtgggtag ggggctgtct ggggaggtag gaggcgcaga gggaaatcca agtggccctc 123780 tctggtagga gagatggagg gcgctagaaa gaggatagtt ctactgattg agtgacagat 123840 aagggtgtgg gccagagact gggggtgggg tggggagggg tcagggggag agggatagga 123900 aggagaactc aaagatggag aaagtggtga gggaagctca aaggaggagg gagatggagc 123960 gggggagggg gagaaggaat aaaggttaga tgggaaaagc gtggagggaa gtgggaccca 124020 ggtgaagacc aaggaagagg gaaggagagg aaagaccaga tcaggggagg gatgggaaga 124080 agactatgga cagggaccca gaatcctggg atggaggtag cgggaaagag aatcaggact 124140 gggaccctgg ggactggaat ggaaaaggag aatggaaaga tcagaaacca gagaaggatg 124200 gggatggtga ctagagaagg ggtatcagga accggcgaag agggttggag acagggaacc 124260 atggatggga gaggggctgg agaggaggga agaggaggag gaagagaaag gctgagagag 124320 agggactggg gattgggggt gctgcccagg gatgagacaa agaggcttct ggtaaccact 124380 tccacgtggg aagccctcca ttcccaaagc gcctgcctgc cacatttctt ctctcaggga 124440 gtggctggtg ggccagatgg ggggtgcttt gagctcaggg ccctgggggt ggctgtgagg 124500 gacagagggt gaggactttg gaaggggagt gacagcctcc gagggtgggc aaacaggctg 124560 gctcctgtgc tgccatttat ttatccggcc cggacgttgg attctgcagc cgctgccgcc 124620 accacggtgg ctgcttattt tggggtgtta cattctggca gagtgagaag ctgtttgcag 124680 cagctctaaa cctccgtcac ccgcgtcagt gcctccccag gcccctgcgt cactggcatc 124740 accaccacct ccatcccact cctcagctcc cacctcctca gcccctgccc cctcagcatc 124800 tgcccgcagg ccccagccct tccctgaagc agcccgttgg gtgtggagcc cttgcttctc 124860 gtctgggacc ctgtgcccct ccttccagag cgagaggcct ctgctgcctt tccagggagc 124920 atcctttcct gggaccactc tgcaccagcg actctgccct gtgggtgggt agcctggatc 124980 ctgcccccta ctttgggtcc agttttcttc tcctcaagtt ccttcttcta caggggcctc 125040 cggcccaaag agtggcctgt gggctgagaa ctttgtttct gagccttggt actccaaggt 125100 ttgatagcca gagtcctgga cagtggtccc tcagtgaaca gatacttttg gctctggaca 125160 cttcagcctt ccgggatcaa taccatgttc tggcctctct tggctccctc ccctggtcag 125220 ttctggccat atattctgga caggggtcat ctcttcttga ctcccacatg taatcactac 125280 tctagaacaa ccgcaactgg aagcctagga ggtgaaagtt gcagagagag ctggagtccc 125340 ttccttgcct tgaccctgaa tagccaaaca gactcagcat tgtggctggc ccagccctag 125400 gcacctgggt gcaatttctc tcctgtcttt acctcaaggg cagtgtctca cacattcagg 125460 cgtggtttct gcggaggatg tggccacctc ttaaagaaag atcagagtgt ctctctgaca 125520 tgggcttgat gtccctcttt tccaatctgg gttccacctt gtactagctg catgacctga 125580 ggccactgtg tcatgtttct ggggctccct tccttcatct gcaaattggg ggccacaata 125640 ttgacctcca ggggattatg tgtgttgtgt tcaatgtata aagaagttaa cctgtacaaa 125700 tgcagtgcct aggacaaaat aggtgcttct tggtttcctc ctaccctgct gtactctccc 125760 ctgcagctct agccatcccc tgctgacttt agaggagggg gtgagcagag agggtggggg 125820 aggctgctac aaagggcttt cctctgtcca tgaagtagtg gagggatgaa atgaaggctt 125880 ctgagaaaga caatgaaggc gagctgtaga gacctggtca ggaggcctgg ggtgctcaga 125940 aactcacact tcccctcccc agccctcaat ggtgttacct atgatgtgag gggtcggctc 126000 taggtggcca ccgaggtatc cccctttcca gctctgatac tctgtgcatc ttgccccagt 126060 ctccaccggg aattcacaaa atgaaggcca ggagtggagc cgtggtcctc gggagagaca 126120 ggaggcctgg gcctggaggg aaggagtggt ggtgctgagg aggagtgaga acagggggtg 126180 gggaagggac gtggcaagaa agaaaagggc acacactggg cagggcaggg actgagggcg 126240 ggggagagag ggaaaggcac agctctctag tcccccaacc ccccagtccc accacctctg 126300 ccctggagtg ctcgctccag ccccagcagg cctggggcag tgaagcccag agccccctcc 126360 cctcccctcc tccttgcctc cagtgagagc cgctgcgtga attatggatg agctccttgg 126420 gttacagctg ctttgcacgg cagtggcaag ggccagaaat ggcaacagag tcactgttat 126480 gcagcagctg ttatggagga gcccccagca ccgggtcgct cttcagagag cctgcaggga 126540 ccactatcat gggctggggg aggtgagccc tggttggggg agacatggga acaagatgga 126600 aggagagtgg ggaaagagaa gagaagtagt ctaatgtggg caggtgggga gcaggagagt 126660 ctagggagag aaagaggagt aggcaccctt gccagctcct gcagagttta ccctcaaggc 126720 cggaaggaac cctgatgcca ggggaatggg ccttgcctct gagattgcac atccttccct 126780 ctgtctctcc tggggcagcg gtcagtccgg aggctggggg aaagctctgt aatcctccag 126840 gggctagcgg ccatcagggc tcacactctg gtgagcttgt ggataagggg taggattaag 126900 ggatcagaga aggatttggc ttcttttggt gtcaagtcct tagggaagtg gagatcagag 126960 ggtgactctg acaggaaggg aagtgccctg gctgggcatc aagagacttt tctggccctt 127020 tccctgccaa cactttgctg tgtgaccttg ggtaagtcgc ttgctctctc tgagctccag 127080 tcatcacctc agtagaactg atgcttgaac cagaggaatc gaggggacct ttgcggcttt 127140 gaaatctcca gttctaagcc ccaaacctca accctcatga aacccactca gggtccccac 127200 tgtgcttcca cactccacct ctgcctggtt cagatgaggg gtaagagaca ttgctcctcc 127260 accccacgtg ggtctaagaa actcgggagg agaaagtaat cgtgaaacgc cgcacggggg 127320 aggggtgaga agggccgaga aacgcggagg tggtgtgaac gaatggaaca gcagccgctg 127380 tgtcactgag tattacatca cacccagcct acacacgcac ggggcccggc gctcacacac 127440 acgcggagga cagccagcac gcaccgacgc agcaccgacg cagcgccagg aggggccggg 127500 gacactcacg gtggggccca aaagcgagga gcagcacact gggagtgtgg atcttccacc 127560 ccgcacctgt gtgctccccc ctctggagga ggaacaccag ggcagctggg atgccagcgc 127620 cacactcggg gcctgtcagt cccatgcgtg cacacctggc tgagcagcac tgcatttggt 127680 gagcacctgg ctcacgccac tacccaaaat cacagataca tacacacatt cacgcacacg 127740 gcaacctcag gagcgtgaca caacacacac aaaaccacca ctaagcaagt gcaatttgca 127800 gccttggaga ccccacactc aaaatcacca acccctcagt ctctcccagg gtctctgaac 127860 cccaaggagc cccaggatgt cagagtgcag aaacaagtct tcctcccctc tgccttcaaa 127920 agcctaggac gttgcttgaa gcagaaggtg ttcagtcact gtgtgcccag ggaatgactg 127980 cctggctttg ggggtgcagg ctcccttttt ccccaggcaa aactgccaga agaaaatccc 128040 aggagtcacc tggaaatcat aagaaagtgt agaggtcaag ctagttccgg cctagaactt 128100 tatcagctat agtgacggca aaggccaggg atgatgggag gccctgcacc cctattaaaa 128160 tatgagtaca gacacctgca ctccactctc tagcccccag gctctctggg cctgcttttc 128220 catcagtatc ataataagga tggatcatat ccaaccttca aaagttactt tgggggaaaa 128280 aaaaaaaaaa gctttggctg gatgcggtag cttatgcctg aaatcccaac actttgggag 128340 gccaaggtgg gaggattgtt tgaggccagg agtttgagac cagactgagc aacatagcaa 128400 gaccccatgc ctacaatttt tttttttttt tttttttttt tttgatacag agtctcgctg 128460 tgtcacccag gctggagtgc agtggtgcga tctcggctca ctgcaagctc cgcctcccgg 128520 gttcacacca ttatcgtgtc tcagcctccc aagtagctgg gactacaggc gcccgccacc 128580 atgcccggct aaattttttt tttgtatttt tagtagagac ggggtttcac cgtgttagcc 128640 aggatggtct cgatctcctg acctcgtgat ccacccgcct cagactccca aagtgctggg 128700 attacaggcg tgagccaccg cgcccggcca aaaattttta aaaaattagc tgggtgcagt 128760 ggcacgggcc tgtggtccca gctcctcagg aagctgaggc aggaggattg cttgagccca 128820 agtgatccaa gctgcaataa gctgtgatcg taccactgca ctccagcctg ggcgatggag 128880 caagaccctg tctccaaaag aaaaaaagaa agaagttttt aagtaactgc gaatgaggag 128940 agcctggggt gtaaaatgca gattcccagg ctgtcccccc aggaattctg catagttcct 129000 aggactggct ggtggcctca cttagagacc cgacccttaa ggcccctccc ggcacaaaga 129060 ggctctgact ctgcaagggc gaaaagtaca ggaaagtaag ggcactgggc accagtgggc 129120 tggcaagacc agaccccaga gtgagtccat ttcacacggg cctcagatct ccaaagggtc 129180 ccaagttact tccagtcatt ctccaatggg gtgactttgc cccccagggg acatttggca 129240 atgtctggag acattttggt tgtcacaact ggaggcaggg tgctgctggc atctagtggg 129300 tagaagacag agatgctgct aaatgcctta tatagggctg cccccacaac gaggaactat 129360 ccggcccaac tgtcaatact gaggcagaga aaccctgacg ttagtctttt gacattaatc 129420 tctagacaag gtcaaacatg caatagtgaa aacaggaatg aagagatgat cattcttcaa 129480 ccaatttgca gtgctttcta caatggcctt ttggcattat tttttaatat atgagaagcc 129540 tcagaaagtg gaagtggcca ggccacttga ggctataacg ttgtcccctg agcccccaga 129600 catgggagca ccagggctct aggcctttat ttttattttc tattttttcc cctgaaacag 129660 ggtcttgttg tgttgcccag gctggagtgc aagggtgtga tcgttgctca ctacagcctc 129720 aaactcctgg gttcaagcga tcctcctgcc tcagcctccc aagtagttgg gactacaggc 129780 acatgccacc atgcctggct aatttttttt tttttcttgt aaagacaggg atctccctta 129840 tgttgcccag gatagtctca aactcctggc ctcaagcaat cctcctgcct tggcctccca 129900 aagtgctggg attacaggtg tgagccacca tattcagccg ggtctaggcc ttttaccaag 129960 ttggggggct ggcccccagc tggcactcct gccctggaag cccacctagt aagttctgct 130020 tcccctcccc acagctcccg cctcggcctg cctcctgctg atgctcctgg ccctgcccct 130080 ggcggccccc agctgcccca tgctctgcac ctgctactca tccccgccca ccgtgagctg 130140 ccaggccaac aacttctcct ctgtgccgct gtccctgcca cccagcactc agcgactctt 130200 cctgcagaac aacctcatcc gcacgctgcg gccaggcacc tttgggtcca acctgctcac 130260 cctgtggctc ttctccaaca acctctccac catctacccg ggcactttcc gccacttgca 130320 agccctggag gagctggacc tcggtgacaa ccggcacctg cgctcgctgg agcccgacac 130380 cttccagggc ctggagcggc tgcagtcgct gcatttgtac cgctgccagc tcagcagcct 130440 gcccggcaac atcttccgag gcctggtcag cctgcagtac ctctacctcc aggagaacag 130500 cctgctccac ctacaggtga gcctgccctg cccccaccct cagccccttt ctggtttcct 130560 ctctctgtgg gcccctctgc tccccgaccc tggcgtgcgt ccctcctctc tccccaggcc 130620 acccttcctg cctcagcatc tccatttctc tctgtctatg tctcttttct ctcttacatt 130680 ctccaggggc tttacttttt cccttctgcc tctctacctg tttaggtccc ttgctgttcc 130740 tctctctctc tctccctcta actccacaac cttcacctct ctgcctctgc ctgtctgtct 130800 gtctatccct ttccatccat cactgcctct ctcactaact tgcctccccc atctgtcttc 130860 tgcctcttct gtctgtctcc cttcacacac ccactccgca tacaccccca tgtctgtctg 130920 cgtgtgtgta tctgtctctt tctgtgatct cacgtgtttg ccttcagggc actctgcctt 130980 cccccagggt cccctgccca aaggcctttg cagctgtttt tctcacccac cctcaagtct 131040 gcccacatca cggtgaagta gagagagaag gcagagccac agccactggc atcccacaga 131100 aagttgcgct tctctccaat tcactgggca atgggacggg agaagcccac accccttcta 131160 gattcccatt ttccaaacct gtcatctcaa tgcaggggaa gaaagaaaag ggtaaatctc 131220 tgttatgcag ctggagaatg gatgctctga aaatggaagg aataccagta attgttattc 131280 attgttatta ttattgatct aattattgtt tattgttgtt atgctgactg tttgacacgc 131340 aaatcatccc actccatttc cccaggaagc aataacacac cctccaaacc accctgagag 131400 aaaatcttcc cttggctaca gagcctccgg ctggaagggg gtgaaaatat ccaaattctg 131460 ccctctccct acttgaacct ggaacgtgct tcctctgcct catccagggc tagtgcctaa 131520 ctagttatca atctgctagt tggaaaatca ggtcagtgct gatgatgcta atgataataa 131580 caatagccat aacaacctaa caaacatact gagcacccac tacgagctag atgctaagaa 131640 tacagtagtg aacagaacag accaaacccc ctgccttcac agagatacca ttcccatgag 131700 gagggaaaga agtaaaatgc acggtatatt ggaaaaatat gtcttatatt attcttattg 131760 ttgcctaaat agtgacagta atagcagtag ccgccaccac ttagtgggta cacagggtca 131820 gccacagtgc caagcacttt ataggtatcc actctgccat ttacaagcgt gtgacatttt 131880 ttttttttac ctcctcagac ctcagttttc tcatctgtac aatggggtag caagagcacc 131940 catctcctag ggattttgaa agcattaaat gcatgaataa tttgtaaagc acttagaata 132000 gtgcttggca tacggtaagt gctatataaa tgcttgttaa aatactattt taaaaaaaga 132060 aacgagcctt atttaacatt ggtttcagtg aagtggccca acttggactc catcctgaag 132120 atgtgggtca acttcaagga ttatactaag gtcatgagtg agtcccagaa attgcacctc 132180 acagtttatg aagtgcactc agccacctca tctcatttct acagcccagt tgggagatta 132240 ttttcacctc cttgttaaca atggagaagc tgaggctggg ggccctgaag accctataga 132300 gatatagtca cctccaatca taaatctttt caaccattgt cggtgtgacc ggaggcttat 132360 gtcttctcac catcatgttg agcctcacaa caacctggtg atagggacag ttaggggcac 132420 tagggacatg gaatgaatgt tcctgaggcc acacacccag gaagagctgg cgcttgaacc 132480 tcatggtctg gctacaaggg gacagtactc tggagtacaa ttgagcaggc tcatttttga 132540 aagcacacag tttggactca gcaagaccta ggttcaaatc ctggctccta tatatatgac 132600 tttggacaaa ttacttaacc tctctcagtc tccatttcct catctctaaa atggcaatca 132660 ggatagtact taataataat cttttttttt tgagacgacg tcccactcta tcgcccaggc 132720 tggagtgcag tagtgcgatc tcggctcact gcaacctctg cctcccaggc tcaagtgatt 132780 ttcctgcctc agcctcctga gtaactaaga ttacaggcat gtgtcactac acccagctat 132840 tttttgtatt tttagtagag aagggtttca ccatgttggc caggctggtc ttgaactcct 132900 gacctcaggt gatccactca cctcggcctc ccaaagtgct gggattacag gtgtgagcca 132960 ccatgcccag ccaataataa tccttattta agaagttttg taaggattaa aatgtaaggc 133020 atttagcaca aggattaaaa tgtaaggcat ttagcacata tgggcactat aataataatt 133080 actactacta ctactactaa tactgagatc aaatactact acaaattgat catgcattta 133140 atgctttcaa aatctcctta tcaatatata ttagttattt aggaggaatt tggagtcaga 133200 gggcctgagc ttgaatcccc gatctactat tttctgactt atttaacttt aagcaggttg 133260 ctaaccctct ctgaacctca cttactttat ctgcaaactg ggaataatga aaataatacc 133320 ttccaccaag aatggctgta aataggaaac gagttagtgt atagaaagcc catagttcag 133380 gctggtgtgg tggcccatgt ctgcaatccc agcacttcgg gaggccaagg tgggtggatc 133440 acttgaagtc aggagttcga gaccagcctg gccaatatgg tgaaaccctg tctctactaa 133500 aaatacaaaa attaggcagg cggggtggca ggtgtctgta atcccagcca ctagggaggc 133560 taaggcagga gaatcacttg aacctgggag gtggaggttg cagtgagctg agatcgtgct 133620 actatactcc agcctgggtg acagagcaag actctgtctc aaaaaaggaa aaaaaaaaaa 133680 aaaagcccat agttcagtgc tgaagaaatc atgttattat gaccccatcc tccattgact 133740 ctcaggccaa caacagcaat caggacctga ggtcagcaaa ggcttgggca gaggggacct 133800 caggtggaca ttggggtctt ctgaaatggg aagtgtttgt tctctacgcc cctggcatga 133860 atggtaccag gcatcatggg aaggaagcaa cttcacacct ggccttttat agaggagatg 133920 gaaaacacag cctctgcctg tgaactgcct ggtagggctg ggctgggaga tgccacaggc 133980 aggtgaggaa acatgggctg gggtgagatc cgcagggtgc aggtgtgacc caagatggag 134040 ccaggcctgc cccaaagggg agctttggag gaaactccac cagaggacca cagcttttca 134100 gaatggggaa gggccaggca ctgtgccagg tgagttcatt catcaacaga tatttactga 134160 gtatctacca catgccaggc aatgttccag gtgccaggga ttcaggagag aacagaaaca 134220 gtggccctgt tctcccagag catattccct actcaagtgt agccagatga taaagacact 134280 tgttttcttt cttttttttt tttgagacga agtctcgctc tcttgctcag gctggagtgc 134340 agtggcacga tctcggctca ctgcaacctc tgcctcccag gttcaagcga ttctcctgcc 134400 tcagcctccc aagtagctgg gattacaggc atgtgctacc atgcctggct aatttttgta 134460 tttttagtag agacggggtt tcaccatgtc ggccaggctg gtcttgaact cctgaccaca 134520 ggtgatctgc ccaccttggc ctcccaaagt gttggattac aggtgtgagc caccgcaccc 134580 gccgacactt gttttctctt tcagtcatta cagtggcctg catggttttt gtttgttttg 134640 ttttgttttg tttttgtttt tgagacagtc tcactatgtc acccagctgg agtgcagtgg 134700 cgcaatcttg gctcactgca gcctcacctc ctggggtcaa acaattcccc catcttagcc 134760 tccccagtag ctggaactac agacatgtgc caccatgtcc agctaatttt tctattttat 134820 agagacgggg tttcaccatg ttgcccaggc tggtctcaaa ctcctgaact taagcaatcc 134880 acccgcctcg gcctcccaaa gtgctgggat tacaggcatg agccaccgta cacagctggc 134940 ctgaatggtt taaaaatagt ctttatgctc aagcagatca gatctcagtt tgaattccag 135000 ccacacctct aatttgctct atggctttgt gcaagttatt taaccactct gagcctcgat 135060 ggacccatct gtgaaatggg gataacctgt accttggcga gcaggggttg tgaggattaa 135120 aggagatact actgagctca cagcccaatg tctggtacaa agtgagtatc caatgaatgg 135180 tagctatcca ttaacaccag ggaggacacc aactgaagct cagcaaaata aaagcacagt 135240 ccaaggtcac ccagctagta aggaacatga cctagaattg gcccaggtct gtctgactcc 135300 agagtgcagt tgttcagagg tctctggagt tggaagccac gttccactgc atattagctg 135360 ttggacccta ggcgagtcac ttcacttctc tgaggctcca tctcgtaatc tctgaaatgg 135420 agataataat agtatccacc tcatagggtt gtgacaatta agttactata taggatctgt 135480 gtagcacaga gcttggcaca tggtaagagc tcaatcagtt acctgcttga caatgctgac 135540 gccgatgatg acgatgatac ccatcctaga ctgatgagct ctgtaagcgg gggtgcctgg 135600 cacagagtag acactcggta cagctctgtg gaatgaatga ggcacatccc agaactcacc 135660 aattcataaa aatcagatgc agatgggatc ttaaagatca cctatcctaa gtcccttgtt 135720 tcacagatga aaagacccag gcccagagag gtgcttggag ctgcgcaagg tcacacagcc 135780 aagcagctca tttgattagt gtcagagcca agagctggga gtttggaggg aggcaaggtt 135840 aagaacagga tgctgtcagg gaagcaggca gggatgctgt gttaagattc caaatggatg 135900 cagagagctg tgaaccggcc agtggggagg caagggaaat gtggtttttg aaatggaaga 135960 ggatgacttt agcagaggct ctcagcccag agggagggga gatagggagg ggagataggg 136020 aggggcgggg ggagggctag ggctgtgaaa gtcaagagct tattaatgca tagagaacgg 136080 ttttaacagt ggagagagga aggaccggat ttgaaagcta cattcaagga agtggcaacg 136140 ggatttggca acagcttgga tggggggagg aggcaatgga ccccaaggca gaggctcaga 136200 gaagggaggg gcaggacttt ttgcagagaa acaaaaggag aggagaggag gttagaatca 136260 agaaattctg tgggccaaaa cctggggctg tgggtcaaag gcacctgaat tccctaggat 136320 ctctggaact ttggtctact cttctgacct cccgaggtcc cccaaaatgt ggattacccc 136380 tgctcactct cccccaaccc ccggcccctt atcgatcctc tgaccataca tctctgggtg 136440 tgtcctactc ttgctgacac ttcataaaaa gaggaacccc atttaggtgt tttgagtggc 136500 agggattcca agcctacccc ctggatgggc ctggaagaga acaagagcac caggccatgg 136560 tgagtcaggc tgaggccagg gaggtgcaag gagccagctg gaggcctgag ccaggatttg 136620 gggtggtggc agcagggggc ggagaatggt ggtgtcagag gcagccgaga aggttgaggg 136680 ggacggatct caatgtggcc aagaggaggg ctcttggcac gctcagttcc tgtagcgaag 136740 agggcggaag ccagatggga gggggcgaga acaggcagga gcacaggaag gtggaggctg 136800 tgggtgtagg ctgggagtca atgccctccc ccaacctgag gcctccgacc aggctcctgg 136860 gtggcaggca tggggaggaa agcgtctccc caggcagtga gggagggaga gccacagtca 136920 gggaacaggc cccctgggtg aactggcctg agcagagtgg atgctcctgt tctgagaccc 136980 agacctcctg gaacctgctg accacagtga tgccctgcac aagaggggag gacctcaagg 137040 cagtgaggtc agggagctga agtcctgctt ccctctctgg caagccctta tctctttgag 137100 ccccagtgct ctcctctaaa aaagtgagct gggctgatgg gtgccaaggc attagctccc 137160 aagtcagctg atcatcagaa tcccctggtg agctggttat aatgcagagt ccaggaatcc 137220 ccactggccg tgggccacac acacccgccg ccccccgctg ttaattctga accatagttc 137280 caaggtcctt tctgcactaa tgtggcctga ttaggtgact ccctagcacc aggcaggtgg 137340 gacagcgcct ctaaggggag tagtaatgca atgtggcttc cttcctctcc tcccctgccg 137400 cctctggggg tggagctgat gcccctcacc ccaataccca gcctagtagc agtactttgg 137460 ttcccccagg gagctcctct tttaaagaaa agggacagga cccaattgtt actgagcccc 137520 tattgtcata gtagccacca tttattgatg gttgactatg cacctgccag atactgtacc 137580 cttaacagca tttatcatcc aaccctcctt tagcctgctg agggggttat acataataag 137640 gaatattgta catactgagg aacctgagac tccatgaggt taaaacttgc ctaaaataac 137700 acagctaggg aaaaggcaag ctggattttg aactagggct ctaagtgctg agcctgtggg 137760 cttcataatt ggaccaaatc cctgtgtgct gggcacgtgt ccagcacttc cctcatatga 137820 tctttatgtg aaccatcctc tggaatcctc agaacaaacc caggaagtag gtatactcat 137880 ccccatttta cagatgagga aacaggcaca gagagatgac tggcttggcc aagttaagaa 137940 taatggctaa caaacaaaaa caaaaacaaa aattaaaaaa aaaaaaagaa taatggctaa 138000 ctcatggaac tcatagaact ccacaaggaa aggtgttcta agcaccttca tacatgctgc 138060 ttcatttaat ctctacatta tacagatgag gaaactgagt cacagatatc ctgagtgact 138120 tgcccacggt ggcatcagtt aatgacagat ccaagatttg aaatcagaaa ggctggctcc 138180 ccagtctcca tacttcacca aaccagaagt tctgaaactc aaactgtggt cctgccaatg 138240 gccacactgg cttccctggg gaacctgtag acatggggat tcccaggctc caccccaaac 138300 ctcctgaatt agaaactctg ccccccgccc caccccgctc agagatccgc aggggatcct 138360 aatacacccg aaagtttagg aaccactgac ctcaccaata ccactttttc cacagcaaat 138420 aggttagagg aggcagaatc caaatccagg atgctatgaa tcaaaaggtc aaccctttct 138480 cttctgccac ggtgcacccc cttccctccc ccggccaagg ccccagcggg gtctgcaccc 138540 tgcctcaggc ccattctctt cttctgtgcc ccactccacc ccacccagga tgacttgttc 138600 gcggacctgg ccaacctgag ccacctcttc ctccacggga accgcctgcg gctgctcaca 138660 gagcacgtgt ttcgcggcct gggcagcctg gaccggctgc tgctgcacgg gaaccggctg 138720 cagggcgtgc accgcgcggc cttccgcggc ctcagccgcc tcaccatcct ctacctgttc 138780 aacaacagcc tggcctcgct gcccggcgag gcgctcgccg acctgccctc gctcgagttc 138840 ctgcggctca acgctaaccc ctgggcgtgc gactgccgcg cgcggccgct ctgggcctgg 138900 ttccagcgcg cgcgcgtgtc cagctccgac gtgacctgcg ccaccccccc ggagcgccag 138960 ggccgagacc tgcgcgcgct ccgcgaggcc gacttccagg cgtgtccgcc cgcggcaccc 139020 acgcggccgg gcagccgcgc ccgcggcaac agctcctcca accacctgta cggggtggcc 139080 gaggccgggg cgcccccagc cgatccctcc accctctacc gagatctgcc tgccgaagac 139140 tcgcgggggc gccagggcgg ggacgcgcct actgaggacg actactgggg gggctacggg 139200 ggtgaggacc agcgagggga gcagatgtgc cccggcgctg cctgccaggc gcccccggac 139260 tcccgaggcc ctgcgctctc ggccgggctc cccagccctc tgctttgcct cctgctcctg 139320 gtgccccacc acctctgact gcggtgctga gatcgaagag gccagtgtcc gatccccgct 139380 tcccgtccac ccggggctgc ggctccggcc ccagtcgccc caccttccct ggccttgctg 139440 cctccctttc ccctcccagc tcctctcctc cccggggagc aggccgcctc tccttgcctg 139500 ccccctgggc tgtcctgact tgtggcagcc ccaagagggc gtgtgtggtg gctcagccct 139560 gccctcccca gttctggcca ttaactcttc cccatcccaa ggctggggtg gggcccccca 139620 ggcagccgct gacccgcact cctaagggcc cacagcggac accagagggg cttttgtctg 139680 cagagcgtct tccaccagca gagcctttgg aagctccccc agggagcccc acccaggacc 139740 ctttggggga tgcctcagtc agggccaggc tgaccctgac ccctgcttac cctagtcccc 139800 tcaacctcct gacactggag gaatactttt ctcctaagtc taccctggac actttttagg 139860 gcacctggag agaactttcc tctccactgt ggcccctgcg tggtgaagat caaaagaagt 139920 tgtttgggaa aaaaaattta ttaaaaaatt ctattatttt atctactgta agatttgttg 139980 acttgggacc ccgaaagcgg gatgaggtct cagaatgtaa ggattgcagg gccaggaggg 140040 ttggagaagg ggagccgtcc cccgccatca aagagcttcc tggtggctgg aggtggtgtg 140100 cgctcccccg ccatgaggag gagctgaagc cctgcattct aggtgaggcg cagtgtggca 140160 gccaagagtg ggtgctggtg gcacctcttc tcttcatttg tccaggggaa gagctgcagc 140220 caaccctgag tggtctggcg cctgaggaac taagcctggg gaagacctgc tgtctggtta 140280 acagccctct tccagaccct gttccttcag gaaacaagag cagttctcct gcaaggagga 140340 gtcacataca cactcctggt cacagacagc cccaacatgg ctttgggtaa atgtgaacaa 140400 ggcactgctc cctcagggaa acacagcccc atgccagagc aaacacctta gcaaacagag 140460 accaaggctg ggtttccgcg tacacttgcc tccttggcta agtgcccttg tgcagtgcac 140520 agcgtacaca cctgcacaca gcaaccctgt gggtatgtgg tctctctctc agctcctgtg 140580 aggtagaagc catcagggat gaaccaggtc agagaagcag gtttccaaac aggctagaag 140640 agggaccgag gaactcgggt gatcagaggg acaggaatcc caaattggga tgcattactg 140700 gcttgaggta caatcagaac cttcatcttt ctggtgtgtg gaagagaggc tggggactgg 140760 gaagagctca ggctaagaag gacttgggtt gggatttagg ggtgagtctc atcagactga 140820 gcacttggag agaagtttgg tagtttgaat ttggagctaa gaatctagct tgggcagggt 140880 gtggtcgctt gcacctgtaa tcccagctaa ttgggaggct gacgtgggag gatcacttga 140940 ggccaagaat ttgagactag cctggacaac atatcgagac tgagtctctt aaaaatgttt 141000 ttttaagaat ctagtttgga gtggggtgtg atgtctcaac gtctgtaatc ccagcactct 141060 gggaggctga ggtggacaga tcacttgagg tcaggagttc aagaccagcc tggccaacat 141120 ggcagaaacc ccgtctctac taaaaattca aaaaaattag ccaggcgtga cggcgggtgc 141180 ctatagtccc aggtactcag gaggctgagg cacaagaatc actccagcct gggtgacaga 141240 gactctgtct aaaaaaaaaa aaaatctagc ttgggaggtg ggaatagaaa gatagagggg 141300 gcctagatgc tagggcttga ggaagcaggc tgaggttctg tgattctggc tagggaggtc 141360 aaatgatctt gagaagaaga gaagaaagga gaagaaatca gcatctaagc ctgaggcagg 141420 tagactccgg ttaagggtgt ggggtgggct gggggagagt gagagcagct ggtcagaaac 141480 ccagggagct cggagtctgg ggtcttgcag gggcttgtgt caggctggct gtgaggaggt 141540 taatgggttg gattggaggg acagccagac aagagctctg gtggaggagg ggctgctggg 141600 gcctgggcag ggggagggga gctgctggta aattagaggc aggctgtcca ggtcatagaa 141660 ttatcattgt gaaatattca tgggccatcg gtccagatgc tatttcagaa cagtgaaagc 141720 aagaggagtg tgtgagcctc aggaagaagc ctgaagcaaa gccactctcc accaaccccc 141780 acccctccca ccaccagccc agacagaccc acggacgccc atcacgtgca cacccacact 141840 cccgagctct cacacacact cgcaccaagc agagccatgt agcacgtgca agcacaccaa 141900 ccacccacgg gtcccacaaa caggcaggtg tcccctaaat tctgacatgc acactgacat 141960 gcacacccac tcaatcagga cccagcagag atcacctcca gcgatctcac atgcgcagac 142020 ccccaaactc tccaaacaac ccagattcac caccttgacc cacacaccct gagataggag 142080 ggatgttcaa ggccatccag cccaaccccc accaatgctc tgatggggaa actgaggcca 142140 tagaaaggaa gggatttgtc tgagattcct ctatcccctg aaaaaagcaa aattcattca 142200 cctcccacat tctgagtgta cccccattct gcattttcgt ctgccagaca cccagcctag 142260 ttgtaattaa ctcctccctt tctctaattt cctgcatcta ttcagttacc cagtccccca 142320 cccagccaca gtctatccct tccttcccat tctccccacc acctccctgc tccagctact 142380 cattacctca tgcctggaat ataaaagaaa actgcgataa cctcctcgct ggtttcctac 142440 atggaatctc tccctccctc ccacccagcc ataccgtggt gaccagattc atctgatcaa 142500 aatttgcata tgttatgatg tcactcagga gcctgtaatg gcttcctaat gcctataggg 142560 taaaggtaaa acaccttagc agagcatcaa agatccctca gagtctggta ccaactgctt 142620 ttctagcctt ttctctcaca atctcatccc aaaccttcac tccagctaga acgtttgtat 142680 catactggcc accagttatc atgtatgtga aacccaccaa ccgactttga gtgcccccct 142740 aaaatttctc agtctctcct gaagtaggaa acctcttccc cctcctcaga tctcagactc 142800 cagagccctt tcccaaggcc aagactgcac ctctctgacc atatacaggg gttcttcaaa 142860 gcagcagaca gaggctcagg ctctggctcc ctccaagcag acggctgccc ccgactggcc 142920 accttgggaa gcacagccag gtttcagtcg tctagaacag agaatgagca tctaaccgcc 142980 tggggagagg actaggacac cagatgataa ggtttataag cccttaagcc tctaaggttc 143040 ttacacccag agtagggggg ggacggttct cagccctgtt tccctagctg cgggctccca 143100 attttcgatc cctaatccga gaggaactcc tctccaatga aatacagact tgggactctc 143160 aggacactgt ggaagggaaa tttcccaaca gactctgaga gtccaggagg ccagggatag 143220 accaggtggc aggcccaagg tccagctggg gtcaggtttc tatatgaatt tttaatgctt 143280 ccagatagac ttgtcagatg ttctgaaaac tgagcatctc ctttcacctc tgtacatgat 143340 gcccttctcc aaccccattg cccctgcagg agggcaggcc tgggacagat attcagtggc 143400 ctctggagaa acggttttgg gacagtagaa gggtaaatga cctagttatg ttcccactag 143460 taagctgtgt gaccttgggc aagttactta acctctctga acattagagt tctgtgggtt 143520 tgtttttgtt ttgtaagctg gggacaatag tgccagccta aatcaatttg ttgtggggac 143580 tcagtgcaat agcccatggc aaagtgacct acatgcttgc tgttattatt ctctttcctc 143640 aagttctgcc tccctcttcc agcttttctt ccaaccccaa agatgtctct ggctattgct 143700 tcgaaggtag gaactttggt tggttctccc ctttctcttc aggcccaaac tccccacctc 143760 aagatccttt ggcctttgta gaaacttcag gtgaggaggt ggcagagaaa taagaaagtg 143820 tgcaaggctg gtggagtgag agaggaggat agatggcgaa gccctagcag aggggaggga 143880 agtgggcagt ggagagagg 143899 16 215980 DNA Mus sp. modified_base (1001)..(1100) a, t, c, g, other or unknown 16 ttgggggtat aaacccagaa gtgggattac tgcaccatac aataatcctc taacttcaag 60 caatttttcc acaatggttg tatcatttta cattcccact ggctacgaga agggttccca 120 cttctacaca tcttcaccac catttctgtt tttgtttttg agtaacagct gcctaatgac 180 tgtgaagtgg tatcttatct cagtgttgat ttgcatttct ctgatcatta atgtgggaag 240 gcatcgtttc atatgtttat tggctgtttg tgtatcatct tctttggcga tgttgattca 300 agttatttgc ttgttttttt aattggagtt ttaaaaaatt gttgttgagt tgtgggagtt 360 cttcattagc tctgcatatt aataccctga tgaaaatgat taacaagtat ttgcttccat 420 tttgggggct tccattctgg gctgttttta ttcttttgat actcttttga ttctcaacag 480 tttaatctga ctaaaattca gtttatttct tcttttaatg gccatgctat tgacacatcc 540 cgtaatcact gccaaatcca gtcatgaaga gtttctttca agagatttat agttttagct 600 ctttaagttt gtcatgtctg tttcacttaa ttttgtatag tgtacaaaag tctaacttca 660 ttcttttcta tatggcttgc tactagtata cgaagagcta aatttctctt tccttgagtc 720 tcaacctctg atgtgtagca atttcttcag aggaaaacat ggtgggaagt tccttaaaca 780 taggatgctc catggaggtg aaatagttca tcctacaggg aagcttgtta aacacaggaa 840 gtacatactc agcagctcta gtaagtgagt gaaactgact ggaggcacta ggtccctcct 900 tccctacgca tatagaagct gtaaggattg ggaagagata ctgtcaggtc agctcagctg 960 ctgcccggaa gaagctcaga cccactggcc tggctccaag nnnnnnnnnn nnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn atcactcttt actcaggcca cctacacgct gtttatagcc 1140 tgcctttgtc tctttggcta tacttcctgt ttatgtctat gcctcccctc tttctttttc 1200 tttctcttct cttctcatct catctcatct ttcttcaggg gggagcctgg tctagaactc 1260 acaaagattt gactgtctct gtctccttgc actaattaaa aaatctttta caagcatctt 1320 ttagcaattc ttacagggaa attttggaat gttaaactct gattgttagc gggctgaaga 1380 taacaatagc tctgatgata aattgcttgc caggcaagtg tgaaaatctg agtttgatcc 1440 aaaaagccgg gtacagaggc caaagagtcc ataatcctag taggggcagg aatcagggat 1500 gggtgggtcc ctggggtttc ctggtttgtc agcgtagccc aattgggaat agccaggttt 1560 cagtgaacga tgctttctgc aagctgagag aggtccttgt tcaatctctg tgacccaact 1620 ggagggagaa gagagccagc tctccagaag tggtcctctc aactttgtgc atgcatgtcc 1680 atgttcacac agggaatgga taatgcttaa aaggaagacc ggcagggggt tggtaatgca 1740 cctcctttgg tgacatgctt tcctcttgtt catgctgctc caggtgtggt cggcagcacc 1800 aaaaaccagg tgtatgtttg taatcccagt attctctggt cgtcagtagg aaatgaaaag 1860 cgaggtcatc ttcgtataga gttagcaaac tctaagccag cctcggctac atgagacttt 1920 gtctcaaaac aaaggaaaaa tcaaggagga cggctcccga gcactgtcac ctgaagctga 1980 cctctggcct ccacatgcat gtgcgcaaac acatgtcctg cacaaacaca cagacacccg 2040 catctgctcc ccgacaaaag aacctgaaac cagtatactt tgagaatttc ccattcatag 2100 ttaccattgt gtgttccttg tgnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnggtggtgc ctttctctta cccagtctag aagggctgga ggcagggtgg atggggcact 2280 ttgaactccc acctaggcaa aaaacccagt gatctctggg ccagtgtgtt gtttgcaagg 2340 gaataaggta gagagccgcg gaggaagaca ttgggggttc tatgagtatg tgaaggggtg 2400 cacacaccac acacacacat tttttttgtt ttaaatttac aaacattaaa ataggctgta 2460 atgtggctca gtgggtagaa aaacctgctg tctaagcctg gtacgagttc aatccctgac 2520 aagctggaag gagacaacca accacaactg ctagcagcca gaagcactgc ttgctaacac 2580 tcaagagagc ctggagtgga agacactgga tccccagcag gcaagcctgc aagaagatgt 2640 gccttgccta gacaacggca gaacaaacat caaggctggc agagctgtcc aggactgttc 2700 atattaatca tgtatagata agagggaatg gcacagacag aacaattcaa cacacggggt 2760 atgaaaggaa aggaacaagg cacacaaagg acaaagaacc tagcatacaa gaaagcctaa 2820 gcagagagtg gcacttccca gaagggagtc ataaaataga ctgaattcat taaaacaaga 2880 gccaaagata aacggctcaa aaaactcacg gaaaacaggt caaaataacg tcacccatct 2940 gacagttgat actgtcaact taaccgtatc tagaactcca gcaggcacat ctccaggcat 3000 gcccctgaag gggtctttgg actaggttaa ctgacgtggg agtgacacca tctatggacc 3060 gaagcctcag acagaataaa aaggagccag tgagctgagc gtcagtgctc attgcttctg 3120 gcttcctgtc tgtggctgca gcgagacacg gtgcttcctg ctttagctgc catgacagac 3180 cacaccctca aaccgtgaac caaaataacc tcctctctac attgctttta ccaggcattt 3240 ggtcacacca atgagaaagg ttaactaata cagcactcaa tacttaaaaa cataaacacc 3300 aaccttgttt gcatgtgtga gactttgaag ctcacgggcc agttatgccc aatgccaggt 3360 ctgctggcta agggtgagag tgcacaccta taatcccagc tgctgtggaa tcagcaaaag 3420 cgctacagat ggaaggcagc cagggcagct gagactgact caaactgata gaggtgggag 3480 gcatagagaa aaccagatta atagagtgtt ccccactatg caagaagccc tgggtttcag 3540 gacgagagaa ctaagaatac agaagtctac tgtgtagaag cactgctagg tcacacagaa 3600 acatcactca agtgtctctg gatgctacac ggagggcgtg tgaagtattg cttcctgatg 3660 atctgtatct actacagcac tgctgtttta gtatgcgctc ctccactaca gctcctcacc 3720 acaccaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnaat taatcaaaga 3840 aaacacacac caccagttag agaaagttaa tcaggccgaa tggcggcttt cccctgtatc 3900 caggctaccg tcaggacggc tcactgccac tggcaactct gcctgaacaa agcccgcagc 3960 caacgtgggc ttcaggggct ctaaacactg caatcaaagg ttgtgtgtgg gggtgggggt 4020 gctgctgcta ttcaaggatt cccaaagctt agatgtattc atcatactca caggaaagcg 4080 tgttcaaccc atcactcatg agcagtcggt accggggtga cctattccct gtagaaatgg 4140 gacggatgtt ctggaaaagt tgacagaaaa gttgattcat taggcaggct ctttgcccaa 4200 gccctgaggg taagcaaagc taactggcag gagactaggt ttgccattaa tctgagacaa 4260 gatgaaccac ttgcccatcc tcctgacacc taaatactaa tgaaagaaca atggattgag 4320 ctggcattat taaaaacgat agaaacagaa gtatcaatag tcatgtgttc tttctcccat 4380 atgtcaaaac aatgtgtaag atggcatcga acacatgcag aaactgttta gggaacatgc 4440 tgaaaatatg aagtaaaatt aaaattggaa agaaagacaa tttgcctaaa gcagctcaga 4500 gctggagaag ggaccgaggc agagataaca gcaacgtgtg gacatacgga tctggggcag 4560 agcagtcacg gactcagccg gaaagggtgg ggcagcctct gaaggaagtt aaggtaaata 4620 gagccacaag gtgattggcc caggagtggt gccaccttca cctcctgcct caaagtctga 4680 aggaatgatc ctggagtctc ccatctattg atatatgaaa ttcacagtat gttttagaac 4740 ccactgaatg atgggtagat taactaaaag aaatttaagc ggggtggtgc aggtctttta 4800 atcccagcac ttgggaggca gaggcaggtg gatctctgtg agttcgaggc cagcctggtt 4860 ccaggacagc cagagataca tagagaaacc ctgactcgaa aaaacaaaat taaaagctca 4920 tcaaaacaac aacaacaaca aaaaaaacaa aaaaacaaaa caaaacaaca aaacacccta 4980 tagtacctgt tggtgagttt gagtgagtga gtgagtgtgt gttagagaga ggggcgggga 5040 aagtgtgttc tggaaatggg agaaagagaa tgtgcatgtg tgtttctggg atgtagacaa 5100 aactacatgt cttccatcaa atgcaatgtt taattatcta tgagttgaac catcttcatt 5160 ctgctaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 5220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnaac aaaaataaac 5280 caaaccagta aacaaaatcc tgtaagataa agcctaagac aagacacttc ctggggctgg 5340 ggagttgctt agaccataag gagttcataa tccaggcgtg agagcccgag ttcaggtccc 5400 tgggcttcca agtcaggagg agaccaagga atcaacaagt ctcgactttg gtctctagtc 5460 ccatgcacac acacgcgtgt aaatacgtag atgttcactc acacacagaa gactgcacct 5520 ggctctctca catctcagcc aacatataaa gcctgcatta tcagaacatt ctaggttcta 5580 gtttcagtca actcttacac agaatggcca tcatactccg tctacaactt ctcctgatct 5640 acccacgtgt cattgcttca gtattaacaa aacccagaat aaccagctgc gtagatcctc 5700 cctgatgccc cagtcattgt cttactgaga ctactaagtc acaaggtagc actctggatc 5760 caaaaagcaa tatccaattg agagttacaa cctataagga ggagtttacc ttcattatag 5820 ggcactggat tcccaatctt taatccaacg tcttcagcag atttcataac ttccaagtcc 5880 atcaaaacaa ctactttcct acaaagacag acacaagtta gaattaagaa ctctgcagcc 5940 tttcagatga gttactaaga agcttacttt agtagttgtc tggctaaaac tgtatccttt 6000 accaaccttt tctcattctg gactaacttg agaagtatta attcctaagt aaatacttca 6060 cttattcttt ccccacatct ccaatgtttt tgtctttaat ttattatagg gcaattcatt 6120 tcctatctag ttccctgatt aaaacagtag accttgctgc atgccattat cctcatggag 6180 gcactgatac aatttagatt attaaataca aaaccctaaa acacaaaaag atgatttttt 6240 tttaaaacaa gattttaaaa aaagcatgtg ctacgcttcc ttctgccact aagcctacac 6300 atggtcctct gactgaattt ttcccctcat tctgcttcat ctaatatgtg cttttcaaac 6360 ctggaattga accagggact tattcatgct aggcaaatgc tctaccatag agctataccc 6420 ctccaactcc catctcaaat atcatttcca aagacatttt cttggtctct tatttagatc 6480 aggtttcttt gtcctcctgc agctatgact tcattccttc agaacactcg tcttagcttt 6540 aagttctgta ttaattagtg attgttttca ttctctctgc tagaatgcac tttcaataaa 6600 ggcaggtagc cagccacagt gcttaattaa gcaacagccc aacgatgtca ttcactacat 6660 actgggacaa gatgcctaac atcatctgca gataaagacg aactactggt gtcaggagac 6720 agctaagggg tccagggctt gggcacgctg agtgtgagca ctggagtccg ggtgcccaga 6780 aacgcacata aatgcaatat ggatgtggca atctacctct aattccttct ttaagacagt 6840 ggctctccag agcaagctgg ctagcaagac aagccatatc agtgagctct gggcttgacc 6900 aagaccctgc ctccaggtgt aactcccaag caaaaggatg atggctcaca aatctcaggc 6960 tatcatgttc atgtacaaaa tgtcaaccgg catacacaca tgcacacaca tgaaaactgg 7020 gagaaaataa gaagaattgc aaccaaaaaa tgtaatttga ggacacataa ttgcaggcgg 7080 ggagtggggg gatgacagaa ggtgaactga gtggaccgag ggaaagctgt gctagcggca 7140 atgagaagaa gggtggggca gtctgagcaa gggttcagca atcaccacgc tttactgtct 7200 gcacagcctg gctgtagaat gctgggcttt atcacacaga attattcagt atgtgctatc 7260 tttacagtaa agttattcta tcaggctatg ctacttcaat agaacaagcc tgaaaaagtg 7320 gtctgctgct gagaacctga caaagatgac ctgttagaac tgtctgccaa gtgtggaatt 7380 ccagcactgg ggaccaggag ctcgagggtc accccagatg cagggagtta gaggccagtc 7440 ttggcaacat aacatcatgc ttcagaaatt aaaaacaaaa nnnnnnnnnn nnnnnnnnnn 7500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 7560 nnnnnnnnnn nnnnnnnnnn catgagatag ttaataaact gaagaaagcc atacaaggag 7620 taaagtagat agttgcaagc atgaagaaag acaaaccact tgagcttttc ttttgtcgta 7680 aggaggaaac cagacaggtc cagagagatg gctcagagat taagagcact gactgctctt 7740 ccgaaggtcc tgagttcaaa tcccagtaac cacatggtgg ctcacaacca tctgtacagc 7800 tacggtgtac tcatatacat taaataaata aataaataaa taaataaata aatcttaaaa 7860 gaaaaaaaaa aaaaacctaa ccaatcagcc aggcgatggt gacacatgtc tttaatccca 7920 gcacttggga ggcagagaca ggtggatttc tgagttcgag gccagcctgt tcttcagagt 7980 gagttccagg acagccaggg tgatacagag aaaccctgtc tcaaaaaaca aacaaacaaa 8040 caaacaaaca aacaaaaaag gaggaagcca gacaggatgc actttatacg tgaatggaat 8100 tgacaaaaga caagttctat aagtgttagg gaaaggggga ggacaacggg ggttcatgtc 8160 tgtggtggaa cacgtattag aaggctctgg gtatcctgtt tccgacaaac aggcactccc 8220 aatcacacag gccactggat gtctcaggca gagaaagatg tgatagattg actttttaac 8280 aatcacagac tgtgtggaaa atatttgtaa ggttgtcatt gtcacccagg atagagctga 8340 tggttattca aacgaggatg ggacaacaga aatgggagag agggatgtga gaaccatttt 8400 gaaccagggt gatttactgc gcacgtgtat agggtctaca gggagtggga tatgtagagg 8460 aggcctatgt tcctaacttt ggtaatgagc ttattacagt tactatgcac agcctggaag 8520 atactggaaa aggtgcaggc taggctagaa aggtactaac tgagggtttg acagcccctt 8580 ggatgtcagg atgcagcaag cctacctctg tatgtagtca atcccttctc aggctatggg 8640 tcctgcagat catccgtctc tgtatccatt attcccagtc catcctctga gtggctccct 8700 cttatccagt ttaacaaaat gctgactgca agctcccaag cccagggctc tggctccttt 8760 actccttgtt attgtacttt accctgtttg cttgggatag agtgtgccct ttataaacat 8820 ttgtgaaagg gggaatgaag aagaataann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8940 nnnnnnnnag agagctcaat ggttaggagc actggatgct cttccaaagg tctccagttc 9000 aattcccagc atcaccatgg cagctcacaa ctgtctgcaa ttccagttcc aggggattca 9060 acactcagaa acataagtgt aggcaatcta cgtaacataa aaataaataa atgagctgga 9120 aaagaaaaca tgtttcaaaa tatacaagta atggggctgg aggagatgtc tcaatgggta 9180 agatcattgg ctgctctttt ggaggttctg ggttcaattc ccaccaccca catgacagct 9240 cacaactgtc tgtaactttg gtcctgtggg agctgatgcc ctcttctggt gtgcagacat 9300 acatgtagac aaaacacctg catacataaa ataagttttt aaaaaagtta cacatacacc 9360 cgtgtgtaat ataacacaca ctggcttaac ttcctcagca ctgactgttc accatacgga 9420 ttcccatgag gttttggttg cattctatca ccgaaaaaaa aaaaaaaaaa ttagaagaaa 9480 gtatatacat ataaacctct ccctaaaata aagttttctt ttctaaaagt acatccttat 9540 ttttttattt tttttttttt ttaagaaatg ggaacaacag ttctgctcac actgtatttc 9600 tagcatgtaa catcttgcaa gtacttaacc gtattctata tcagctcaac acacttacta 9660 ccgaagactc aagatcacaa aaaaaaaaaa aggacccaga ctggataatt aaacgtttct 9720 tttgttgtag taagcgacct cttccttaga agatactaca gtaatgctga agaaatgaca 9780 catctactgt aatctgttct ctgggattcc aacttgtttc ctctgctact cctcccttgg 9840 cggcaatgtt cgtctgcatc cggctgagct cctcgctgcc ttgttaaacc tccttcctga 9900 acttccgacc tgtagttccc gctctacagt gcaagcgagt ggataaggaa gcgcatacct 9960 gccgtctttc agggtgttga cgatgaactt gtggacctgg cagacacagt tgctggccag 10020 ctgccctccc tcgaccaggg tgttcagctg cgtggccagc atgaacgctg caaaagcaga 10080 gagagagggg ctcagtctcc aagcctttcc ttaacccgaa agctcatcac aaggagaacc 10140 attaaataca gctgtttaaa actcctccgc cctgcagaga ggaaagcagc atcaatccgc 10200 cccatgtaaa agtctgaggc tcttcctaaa tggtatctgt ttctcacagt ctccaaatca 10260 tttttactgt aattctagtt tctggggaaa gacctttctc ggtctttagc cccgtgacta 10320 gagacaacag gcaaatattc cagaaaggcc cccattttct ttttaaagct tctannnnnn 10380 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 10440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnngcacat cttgtgaagt gtccacatct 10500 ttcggtccct cgaatttggg tttcttctgg gacgtggtag catgtgactg tcactccagt 10560 gcttggagca gcagaggggt caggaactcc aggctggcat tagctgcaga gctggagcag 10620 gtcctggaga acagaaactt tggttgcagc attaatgaac tagaagaatt tttttgtctt 10680 ctgttaaata taaatacctc cattatcttc tcataaacag tgttgccttt ttatttaagt 10740 ttttaaggat caggcacaga gactccatgc cagactacca ctcaaccact gagctacacc 10800 cccaacttgc ctttctgcta ttttttaaat tgtatcagtg gccaccaaac atggggagag 10860 gtcagggggc tacgtggagg aattgtttct ctcctaccaa gtgggcccca ggtttcaaat 10920 tcaggtgacc tggcttggca gcaagcacct ttacccctaa gccatctcat tggcttcatc 10980 ttttaatggc cccttcccct gctctgaggc aggctctccc tatatagccc tggctggcct 11040 caggctcgca ggtccaccag tgagcaccag gtttctgctt gtccttacct ccccagcact 11100 gtggttataa gcatgtgcca ctgtgtcaaa ctcagtcact aagctttgcc aagccatagc 11160 ccagcccttg agtttactgt ttgtctgtgt ggtgatttgt caaaccactt ttgttccact 11220 gaggtatttt gtcaagtttg acaaaattag ttgagtatgt aggtcttttt ttctggaatc 11280 ttctgttata gcttagtctg gtcttgaact catgatcttg cctcaacctc acgattattg 11340 aggattattg agatggacag gctgtgtgac catgctcggc tgtgtgtttt agcatgcatt 11400 agtcatttga aaaacgttgg ctcatgacac tttacaggtc ttccatgttt gatatgtttt 11460 atttaatcca aagtaattcc agcaccagag gctgagacag gaggatctca aggtcaacct 11520 agagatgcat agcaggcggg gccccactcg gttaggttaa tatcatcact gacttcagga 11580 gaaaagtctt aagtattggg gactaaaagc aggaggatct gaagttcaag gtcatcttta 11640 ggaacttagc agacttgagg ccagcttggg cgctgtggga ccctgttttt aaaccagaaa 11700 acaaattgaa aggaaaaaaa aaaaaagctg gaggaagtga atgtgagtgt tcacatagtc 11760 ctgtttccac aagaaaacag ggttactttt ggcaacaaat aggtgctttc tttgaaggct 11820 ggcatttttg tgacttgtca ttggagaaat gatttaatta agacttttct actgagtgcc 11880 tctgaagagg ctcttttaaa tttagtttaa ttttatctca ttgttagtgt ggtgtgcttg 11940 tgcacacaga aggcagcttt ctagagtctt ttcactctct cctccacagc tcctggagtc 12000 aaactcaggc cctggctagg caagctctta ggacagtgtt agctgtagct tattaagttt 12060 ttaagaattt ttataagact ctgtttttct ttctcaggtc atgatacagc aggaaaatac 12120 atccataaag cccatcctgc aggtcattgt aagtaccggc atgtgtgttt agcataatga 12180 agatggttca cttatagtta attaaacatt ggattggatg gaagacatgt agttttggtt 12240 acttcccaga aacacaaatg cacattcttn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 12360 nnnnnnnnng aattcagagc tgatatgtag tactaactcc tactcaatga atcctttgtt 12420 cttctattcc ttcattacat tactgttaat agtggtaact atgtaccaaa gagtcaaata 12480 actcttggac catccaaggc agaaggaagg ctggcaaaaa tgtatgatga tctgggatgg 12540 gaatgtactt cagtttgtac aggaggccct tggttcattc catttctggc aatgcataga 12600 cctgtaggat ctcagcactg gtggggggtg gggggtgagg gtgaaggggc gggaggttaa 12660 aggcagaata gtcataaatt caaagtctgg gtcctggaaa gaggactaaa cgattaagag 12720 ctttagctgt tcttctagag aacctggtgt gatccccagc acatggtgcc tcacgactgt 12780 ccgaaactct gattctaggg ggatctgaaa accctcttct gccctctgta gatacagaac 12840 acacatggtg cacatacata catgcaaccc aaacaaccca tatacataaa atattttttt 12900 ttcaaaaaga cattcaaatt cttcctcggc tatatagtgt ttaccaaacc tcaaaaacaa 12960 aacaaaacaa aacaaaacaa agaatcatta atgttttgcc ttcatgtatg tctgcccacc 13020 acggacatgc ctggtaccca gggagattaa aagaagacat tagctcccct ggaatggaga 13080 taggtatgat ctaccacttg ggtgctggga acctgggtcc cctgcaaaag cagtaaatct 13140 ttttaacccc taagctgtct ctcccaacgc ctaaagattc ttgtaacaca gcatgatgag 13200 cactggcaag catagcatgg taatctgact tcagggcgcc agattttgag cttaatgctt 13260 gattattaga agtaacgtac tagatttaat gcctggagct tcaagcaaca aaattaactg 13320 aagaataaaa ataaaaaccc tgccagccat gatggtaatc ccagaacttg agaggcagag 13380 gcaggtgatc tctgtgtttt gcaaggccag ccacaatcta catagcacgt tgcagtannn 13440 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnncga ataaatctac acatgtaaaa 13560 agaaattcaa agaaacaaat gccaaataaa tacacatatt gtaataaaga gataattgtc 13620 taaaaaactc aaggctttaa atggtaagat atcatattct tggatgaaaa gatctaatgt 13680 caaaatatat caatttaatg caattatgta tattcaggag atctctggtt ggcttttgaa 13740 cttgatagca ctcttataat tcacatagaa gaaaaaaaac catgaaaact gccaaacatt 13800 attagaatac tccacagatg gtattttggc agcacataca tcgaagggct gtgaaagatg 13860 tgtagatcat ccacgccttg ctagggagag ggcgggtgtg tgtggggggt atagctgttt 13920 gggaaaataa cctggtaatt cctcattagt taaatcatag tcagaacctg gactagcaac 13980 ttctctctaa aatacattca ccctcagcat ctgcattgcc aggaaaccac tcctagcagg 14040 atctgtacgt ggatcaaggt agtagcatct gcatttaatt gacattctcc taaatgcttt 14100 aaattatctc tagattactt atagtagcca agatgatgca aattatgtta cactgtatta 14160 tctggggcgt aacaagaaaa tgtctctact caggttcatt caggtgcagt acttcccctg 14220 aatacttctg aatacacgga tcaagaagcc acagaaagag ggctaaccat atacaagcat 14280 atagtacact aataaccatg tacaaccata tagtacacta atattcagtg cattactcaa 14340 aatgcaaaca gatggaaaca atccaacagc ctgtaagctg aaaaacaaga taagcaaaat 14400 gtgctgggcc tagaggccca ggtctataat tccaactaag gtcgaggcag gaggatctca 14460 agttcaaggc cagcctagac aacttagcaa gaccttgtct caaaacaaaa agtaaagagg 14520 ctgaggatat agctcagtat agagcatctg cttagcatgt gcactgacag ccgtatcaca 14580 gaggaaaaaa aaaaataagc aaaatgtgat ctgtctgcac aacaggatat cacagccccc 14640 taccacaggg gaacgacaca gtaacacaac aaaaacttag ccctgaaaat actatggtaa 14700 ataaagaagt gtcactgagg atcaggaaat gcatgactcc atttacatta tatagaaatg 14760 agaagatcag tgagcctcta ggactcaaga gatttgggat tggcagctaa agggtactgg 14820 gtttctttat gggggtaaga aaacattcta aacttaactg tgagaatgac tactcaacaa 14880 tgtcaagtgt tcaaaaatca tacttttttt tttttttggt ttttcaagac agggtttctc 14940 tgtgcagtcc tggaactcac tctgtagacc aggctggcct cgaattcaga gattcacctg 15000 cctctgcctc ccaagtgctg ggattacagg catgcgccac cattgtccgg ctcaaaatca 15060 tacttttaaa aattgcccag tgactcatga atacaatcag aggcgggaga ggacagtggc 15120 aaactcagga taccagtgtc ttttatgtct gctgcccaac tatcaatttc ccatagttac 15180 cagagaactt tttggtttgt ttcatcttat ttgttgcttt tggtagaatc tcaatatagt 15240 aagatacaag gctggcctca tactatatag ctgaggacga ctttgaactt ctaatcctcc 15300 tgcttccatc tcccaagtgg tgggattaca ggggtgtacc gctatgccca gcaagcacaa 15360 agccatttga accacacccc agccttttca gagaaacctg tacaagcctt agtgccttag 15420 catattaagg caacaaaaga cataatgcgt ggctaccata gagtgtttgc ctaccatgtg 15480 tgaggctcta ggctaaatgt ccagcactta taaaaaagag ttaaaaacac tcatgactca 15540 aggatgacta tgcagtcttg tgtacaaagc cccgcattca atccccagca ccgtgcacat 15600 caggcaggct ctgtagagga cccagcttaa ggtcatcctt aggtaagtta gaggccttag 15660 atggctacat tagatgagac cctttctcat aaacagaata aataatttaa agctcctgat 15720 caaacactat gccttcccat cacactcaga ataaagcact ctactggccc tttaaggact 15780 gcccatctgg aagagaaacc taagttacat tccttgcttg tgtcatatgt gataacaaac 15840 tcactggaaa tacgaaaata cagtcttaag cttggtcaga aagcttcccc agcaacatga 15900 tntcagagga cataatgcag aaagtggaca aatgcaaann nnnnnnnnnn nnnnnnnnnn 15960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 16020 nnnnnnnnnn nnnnnnnnaa tcagaggaca tctttcagga gttagttctt tcctccctct 16080 atagctttca gggatcaaac tcaagtgtgt actgagcgct tatgcccagt gcgccatcgc 16140 accaggcctg cttctttgtt ttttatgggt ctgaatcaat tagcaccatt acaacaatgt 16200 tgacaatcag caagtacctt tctctacctg gctagtaaga gaagtaagtg cctttggtgt 16260 gtgaacgcag tttctcttgt gaagtgcatg gacttgatct ttgctcacaa cgttttttag 16320 gtccttaagt tgcttgggtt ttatgggaaa ggctcttggg ttttttgaaa agattttact 16380 acaacttgat ataatcatta tttttaatcc tttaaatagt atgacttatt ttaacagatt 16440 aatattgaac tgttctttca ttcttacata ataaatcctg ccttaaaaaa taatcctctt 16500 agcttccttt ctctattttc aaatttgttt tatatttttg catgattttg aacatttata 16560 aaagtaggca gacaacacag tagaaccaag tccccatata gctgtgcaca tagcttcaga 16620 ttattgcctg ataataggtc ctgttttgtc tctgttttct cacagggatg tgttattgtg 16680 tgtgtgtaca catacataca tatatgtatg tatgtatgta tgtatgtatg taatgaactc 16740 cctttaacaa aacaagtact gggctgggaa gacagcacag ttagttatgt gtttaaccgc 16800 acaagcatga caaccagagt tgagatcccc accaaccgca taaaaagctg ggcatagtgg 16860 cattgacctg tagccctggt gctggatgaa agctggggag gcaggtagat cggcagagct 16920 tactggcaac aaatctgccc agtaggtaag ctctgggctc agacatccta tataggaaaa 16980 agatgaaggg cgaggcgcag cggcacacac ctttcgtggt agtgcttgag aggcaggggc 17040 aggccagtct ctgtgaccag cagcctggcc tacatgtcaa gttgcaggac agccagagcc 17100 accacctact gagactgtct cagaaataag ttttttaaaa aattgagatg aaggagctgg 17160 taagatggct tagaaggtaa aggcacttat cactaagcct gaagccccga gtttgaccct 17220 ggaccccaca ctgtagaacc aactcctcca agttcttctc agacctccag cagagcacaa 17280 gtgtatgcag acacacacac taagtaagtg aatgtaaaaa acatgacgta gtggcactgg 17340 cctttaaacc cagcattggg aggaagaagc gggtggatct cttgagtttg agaccagctt 17400 agcctacata aggaatttca aggcagccag ggctacctag aaagtagctg tttatgaatg 17460 aatgaataga aggaaggaag aaagagagac agacttaaaa aatatatgct ggagagtaac 17520 agaagaggac accggcttgc tggtgtcttg acctctggct tgtacacata cacatgtgta 17580 gtgcatacac ccacatacaa ttgtactcag acacacacaa acatgtactc attcatatac 17640 tgcacacctc aacactcaga aaatgaaaaa acaggtacca tttacacctc cgtgttcggt 17700 ttccaaccac tcatatgtat gggttgtaaa tgcttatatc tgtatgtgtc tgtatatttg 17760 tgtatacatt caaagttgag tcaggatcca acgtaaactt ggatagtagt gggttgatgg 17820 tctggaagcc tgctcgcagc tgtctttttc tcctcgtacc ttttcccctg tttgtttcta 17880 cgacagcagg tcatttgtct ctaagtgtta gtttcccatc ctctctcttt tgctgatggt 17940 agccttgtag tagtcacctg tgttctctgt aaaatggctt tgccgtgtta tttcaatatg 18000 ctatcatcct catcttgcta tatttcattc aatatatgta tatattacaa gatagattaa 18060 aattatttta attttatgct tatgaatgtt ttgcctaagt atattgcacc ttgtgtgtct 18120 agtgtccaca gaactcagaa gaaagtgtca catattctgg aactggaatt gcaggtggtt 18180 gtaagccacc atgtgggacc tggaaaccaa atccaggcgc cannnnnnnn nnnnnnnnnn 18240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 18300 nnnnnnnnnn nnnnnnnnnn nncctttggt caaagatcct cagtttcgac tttgattacc 18360 cagacttcct gtttctctca tggaacagtt tccccctgag atttactagt ggaagaaagg 18420 cactcaaaaa gcagggagcc ctcgtacaaa tgagacttcc tagctatata attaggccga 18480 gatgcacaca tccacagtca ttacccttct tcagagcctt tgtcatgtca agtgtatttc 18540 gcccatgtga actttagaac tggcttgttg tgtttcataa aaagtgtagt tgggctcttg 18600 attgggattg tgttaaattt atagattcat ttagggagag ctgacaactt tacagtatta 18660 aatgttttca tccgcggaaa aggttgtctt gccacttact tgggctttct tttatgccct 18720 taagtaaagg tttatagttt tctttatatc agtcttgcac atttcctgtt agatttattt 18780 ttgcttgtaa ggtttccttg gttggtattg tgtataaaca ttttctccca attacatacc 18840 ataattgatg gttaggagta taaataaaag gtagaatttt aaaattgctg tatgaatgac 18900 tgctttgcta taattcaaca tcttttcatt tttatgccaa tctacttgac atgtttaggt 18960 taaatgatga tatctgtaca gagtaatcct ctgatccagt atttgcacat cttactttct 19020 aacgtccata gcatagatac acatcttata ctgttgagta catatatatt taaggtattt 19080 accatagtct tataatatgc agcgtgcttt ggttcaagac agttgccctg tgttcctcaa 19140 cattaacatt tttttcatca caaatacaca ttgaccttta tcaaattttt aaaactatct 19200 tgagagaaat gaccattttt cttaatctgt taatgtaaaa tttttataaa aatagttata 19260 aataatatta gcctacatat ttcttctgtt ctctttttca actcttagaa tcagagtatg 19320 gtagtctcag actaaaccag gagcttccta tctgtttctc tgttcttaag tcacttatat 19380 aatgtaagga tgctgtgtat atctgccagc taggccttat atacaaaagg cacccatcac 19440 aaccttctaa aacagtctta ccacttagag accatgttca aacatatggg cctttgaggt 19500 aattgccaca ttcaagctat aatattgtta tctaagggaa tatcttcact tctagcagat 19560 gcctaaaaat atctaaaggt aaacactggt aattgctgtg tttgttgatg ctgctcttcc 19620 tcctcctcct cctcctcttc ctcctcttct tcctcctcct cttcttcctc ctcctgcttc 19680 tccttttctt catcctcctt tcttttctta tttttgaggc atgatttcac catgtagccc 19740 taggtaaccc gtaacttact atgtatgtag accaggctag cctctgtctc ctgagtgctc 19800 atattaaagg tgtgtatcac catatccagc aacacttgct ttgagatggt tagaggaaaa 19860 aaaaatatac gtaaataaag atggatgcca attactaaat tgttacttcc agtcaaactt 19920 tgtacctagt ctaaggccaa aatagggatt ttttttctac tttgcaagtt ggctccatta 19980 agaggctttt cttctcttgg tctcactaga taggaaggag agagaggagg gaaggagaga 20040 aagcggttga ggagtgggag gtagtgtgac cgagaatacc cagtaggctc atatatttaa 20100 atatttggtc cctagttgat agaactgttt agaaagatta ggaagcatgt cttaggggct 20160 ttgaggtttc aaaatttaat gctagaccca gtctttcaag ggagggggcg gtctgtctct 20220 ctctgcctgc tgcatgcaga gctctcagct actactctag tgtcaagcct gtgtgcttcc 20280 tgcctcaatg atcataaatt aactgtaagc aagcctccaa ttaaatgctt tcttttatag 20340 ttaccgtgat catggtgtct cttcacagaa atagtaacct gtggtgattt taatatgcct 20400 ggaccaggga gtggcacttt taggaggaat ggccttgtta agaggaagtg tgtctctgtg 20460 ggggtgggca atgagaccct cgtcctaacc atgtgagaac cactcttctc ctattggcct 20520 tcagatgaag atgtagaact ctcagatcca cctgcaccat gtctgcctgg aagctgcctt 20580 tgttcccacc ttgctgcccc aattaaatgt tgtacttata agaattgttt ttggggggct 20640 ggagagatgg ctcagcagtt aagagtactg actgctcttc cagaggtcct gagttcaatt 20700 cccagcaacc acatggtggc tcacaaccat ctgtaatggg atctgatgcc accttctggt 20760 gtgtcagaag acaggacagt atacccacat acattaaata aataaataaa taaataaata 20820 aattcttttt aaaaaagaat tgctttggtc atggtgtctg ttcacagcag taaaacccta 20880 acataaccct gactaagaca acaagtgagg aaaggtgttg tgtgacactc tggatctctg 20940 gaagctcacc tcagcatgaa gcttgtcgaa gcgnnnnnnn nnnnnnnnnn nnnnnnnnnn 21000 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 21060 nnnnnnnnnn nnnatctaag tacactgtac tgtcttcaga cacaccagaa gagggtgtca 21120 gatctcatga cagaggttgt gaactcagac ctttggaaga gcaatcagtg ctcttaactg 21180 ctgagcatct ctccagccca aaataattct tactagtaac atggaacaat caagttttat 21240 tatatgatac atattaatca acttataagt acatgattat gcacatttat catatcgtgc 21300 aaccatcact gctgtcgttt tgttttgttt tgttcttttg aggcccggtt tctgtgttgt 21360 tctggaactc actctgtaga ccaggctggt cttgaactca atgatctgcc tgcctctgcc 21420 tcccaagtgc tgaaaacaaa tgtgtgcacc accacctctg gctatcactg ctgtcttttt 21480 ttttttttta acagttattt atttcgtgca tgcatgtgtg tataagcatg taacgtatgc 21540 catggtatgc atgtggaggt cagaggacaa ctttcaggag ttagttcttt cctcccactg 21600 tgggttctag gaaccaagct caggttgtta gacttgcatg gcaagtgcct ttaccacaga 21660 gccatcctgc tggccctact ataggtcctt atataaaaag atcatatgcc gggcaaaaac 21720 caaacaaaaa ataaacctca aaaaacaaaa ggaccatata atattgtggg ggagtggatg 21780 aagtcctgaa cgaatgtgtt ctgttgacat gtctgtactt cagacccatg ggaattggca 21840 aagccttcct ctggtcctgt gaggatgctg atagtctgtc taaaaactag agatcacagc 21900 tttctcctct ggatgactgt aaccccagat tgttcctctt cagagactgt ccaccaagct 21960 accctgccta cttaagctgt acacaatgaa tgagctgagt ttccaggtta cagcacagta 22020 gacactgtcc atcagtgaga gcacagccta gcctaacagt acacatgtct gctttcttca 22080 cgtttccaga accaagcctt gctggataga gcatatttgt ctgtttggct tatttcactt 22140 gataaaaagt tttcaaggag ggccaggtgt ggtggcacac gcctttagtc ccagcactcg 22200 ggaggcagag gcaggcaaat ttctgagttc gatgccagcc tggtctacaa agtgagttcc 22260 aggacagcca gggctataca gagaaaccct gtctcaaaaa accaaaaaaa accaaaacaa 22320 aacaaacaaa caaacaaaca aaaagccaaa aatccaaccc cccccaaaaa aaaaaccaaa 22380 ccaaaaacca aaaaacaaca acaacaaaaa gtttttgagg tttaatttat tgcatgtcac 22440 agaatttcac tgtttaaaaa aatggctgaa taatatttca ctatccattc acgtatttgt 22500 aggcattcat gtgtgtagtg gtttaaataa aaatagcccc cataggcttc tacagttgaa 22560 tgcttagtca ttgagtagca gtactagaga gggaattgaa ggtgtggcct tattggagta 22620 ggagtggcct tgttgcagga attgtgtcac tttgaggtcc cagcaacaag gttgctctga 22680 tcacatccaa agacattcta ggtctatgtg atctggctgg aattcagaca tgcccttaat 22740 acacaccttt aatcccaaac aatgaaggta aagttagttt ataaaaagaa gcacccatgt 22800 ttgaaagtga cgtttaatta agagtgatga attagagaaa gatctgctgt cacagagcag 22860 agaggaaaga gaggcagcat aagagggagc atggcagagg gagagggagg aggggttttc 22920 accagggcat ttgtacagag acaggttgca gagctagaac aggtgaagac agaacaagcc 22980 agagaatgag aaggagccag gagattagga cagattgcca atgttaatag gctaagcaga 23040 gcattttagt cagaaactga gagaagtcaa attgaatcag ttagcttgga aaggagtttg 23100 agcagcaaca gctgagttaa actagccaac agaatccaga aagaactaga aaagatgagc 23160 ttactcagca gcaaatctca gaggctaaaa acatcttaga cctagattag actgcatgga 23220 ggctagacgc ttccagggct aggcctaggt tagcagacgg agagagtaat aagccttgga 23280 gacaacagtt aatacagaag actatgtaca gacatggata tgaacctctc agccacttct 23340 ccagcgtcat gcctgtctgc attgttagga gtcatctagg aaaggctaag ggcaggcaag 23400 caacttttcc agagatggtc cactgttttt tgcatggctt ttgagaggcg agctctgaga 23460 gggaaggttc caagagactt catcccagga ttgctgctta attacgacat gccttttctt 23520 gtcactgtta tttagtataa tgactcctga gctttagccc atcctattgg gcatatttcc 23580 tgcagatcaa cataaagatg aactttcaca aattaatgct gtttagatga ataaatgatt 23640 ttataaaatt cctgatttga tttaaataat tttaggaaga aagctttagg agatagttta 23700 gttggtttgc cagaaagatg taataacgtc agaatcaaga atagaatgtg gctgggcagt 23760 ggtggcagat gcctttaatc ctagcacttc ggaggcagag ataggcggat ttctgagttc 23820 gaggacagcc tggtctacag agtgagttcc aggacagcca gggctacaca gagaaaccct 23880 gtcttgaaaa acaaaacaaa aagaaaagta agtaaaggct gcataataaa gaatacaatg 23940 agctttcaca actacaccaa aaagagacat gcttgggaca aatttgtgat caaggaaaaa 24000 tattcattct agatcaggtc caaggatgaa gccacaagtg tgtgatatga tgaacaagac 24060 catggataaa ctgttgtttt gagcttaaag aataaaacac tgctttgaaa ttaactatca 24120 acattctact gtaactttcc tttttataaa ttttatctat gagataattt tctaaagaac 24180 ttgtgtctat aaaggtatag aaggacagag agaaagaaat aaggtgtggc atctgggctc 24240 tgctccatcc acccaaataa atatgtgtgt gtgtgtatgt atgtatgtat gtttatctat 24300 atgtatgtat atacatacat gtgtaggtag gtatatgtgt atgtatataa gtatgcatga 24360 acacttggga agttgatgag acaagtgaga ggttgggccc ccnnnnnnnn nnnnnnnnnn 24420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 24480 nnnnnnnnnn nnnnnnnnnn nngaattcac tctgtaaacc atgctggcct tgaactcaga 24540 gaaccgtgtg cctctgcctc taaagtgctg ggattaaagc atgtaccacc acaacccagc 24600 tagtttaaat gtttcttatt tttttgttta tgggtctttt acctgtatgt atgtgtgtgc 24660 accatgtgga tgcatggtgc ccttagagtc cagaagaggg tatcagatcc cctggaactg 24720 gagtgacaga gggttgtgag ctgggacttg aacctaggac ttctaaaaga gcagcaggtg 24780 ctcttaatag ctgagcctta tctccaggcc gtcccatgga tttggggggc tttgtttcat 24840 tttattttgt tttgagacag ggtgtgtagc tcatgcttga atttactatg aagccctgac 24900 tcccctcaaa gtaaagatcc tcctgcctct gtctacagct gctaggattc gaggtcttgt 24960 accacatgct cagcacagcc atgattcata acaataaaaa aagaaagaga gacctaaatg 25020 gccttagaga taaataaatt attttttttt taaagattta tttatttatt tattacatgt 25080 aatgcacact gtagctgtct tcagaccccc cagaagaggg agtcagatct cattacagat 25140 ggttgtgagc caccatgtgg ttgctgggat ttgaacttcg gaccttcgga agagcagtcg 25200 ggtgctctta cccactgagc catctcacca gccccgagat aaataaatta taatgtatgc 25260 gtaaggtggg atcatctcag tctccgggaa tcttgcctgt tactccttcg ctctcccttc 25320 tattcatgct tgggtaactg gccctggctg attgatgaga gctgatttcc ccactgccct 25380 gtggcaggga ccactgcgcc cacagggctc cctcaggatc ctcagtacag agctgcacag 25440 ctgggtggaa gtagagggct gcatatataa cacgatctca actttatttc tttaaataaa 25500 aattttattt aaattttata cagctctata taaacgaagg aactattgaa ggttcagcaa 25560 ggacctgcca acggttgtca agggtaatgg cgatgtagtg attttttttc ccccttccat 25620 tttacttcca tactttctac attaccccac aactggcaag tattatttta aaatgaaagt 25680 aaatagtgac agatgacttt gaaggaaaat tgaatcggta aaaagaaagc tgagagacca 25740 cccgggaagc ccaggctaaa tgtaatctgg gtcaggcctc ccaggcctgg ggtctcaaga 25800 tggtcagctg agggaccctg gtgaccctct tgggccagca gggacgggga ggagccggaa 25860 gctgagtacc caaagtgctc ctctgggctc caagggcctg cacagagact gtgtgggaat 25920 caaaggatac aggcatgagg actgaggcct gacgaaccca gctatcattc gtcctagaac 25980 aggaggcaga gctccaagag tccaaccaag aggcaggaag ctttgggacc cgagatgggc 26040 gatgggatta gaaaggcatg tttgcaaata ctttcaaatt tacgatgcac actcactgga 26100 aaccccaccc ctgggtgtcc cttccctgcc tcttgccaca cccaatagct gacatcactg 26160 gagaaagtcc caagaccagg ctggctggag ctcctgatag gttccaccct cctgcagagg 26220 gccctcgaag actagcttgc tcgcccacac cgccagatgt ctgtgtcttt ctctcttttg 26280 cctcccaccc tcgtctcttc ctccaacctc agtggagggt cccctgcttc ctggggaaag 26340 tagaacttgc cagtgctcac tgtaatgtcg tccctgtagg tgtcatggtc ccccattact 26400 gggagcaggt atgcctcaga tctccctcta ttcgctgccc tttcaggctg tctcagtttc 26460 tctctgacag ttcctctcct cctgaatcct gcttgttggc atgcgaacag gctcaatatc 26520 ttccatctca aaaaacaaac actgggaagg tgttgagaga cagagagcat gggtaatggg 26580 tgccccagct tggctgggaa ggggtaactt acaatgctct actgcccagt agggtagctg 26640 cagttgtcaa ttaattgtaa atttcaaaat agctagtaga gaggatttta gatgttccca 26700 atcccaacac aaagaaatga taaacattca aggcgatggg tatgctaatt gctctgatct 26760 gatcaccgca cattgtatac atgtttttga aatgtcaggc tgtaccccat aaatatgtac 26820 aattaccgtg cagtgattca agataaaaac tataatttta aaaagctaaa aacagaagga 26880 aatagctgcc cttgaccccc ccacccccac aaggtccttc ctgtttgtcc agccacttaa 26940 tgtcagagct tcctgtggga gggtggtttt ggtgtacaca gacactcctt cctccctcct 27000 tccccataag aggagtcacc cctgtcccac gatgccatgc agggccacat gcgtgatatt 27060 aaccagtaag atgtgagcag ggatgatacc tgtctcttat aacaaacgga aaaaaaacca 27120 caccaaacca aaaacaaaca aacaaacaaa caaacaaaaa cagggttggt ctgtccctgt 27180 gtcttttccc acataaagtt aagcacacaa agtagccacc atttatttat ttgtcccctc 27240 ccccacccct ccccgagaca atgtttctct gtataacagc cctagctgtc ttggaactca 27300 ttttgtagac caggctggcc tggaactcac agagacacag agattcacct gcctctgcct 27360 cccaaatgca gggattaaaa gcatgagcca cgaactaacc agtaccccag agctcttgac 27420 tctagctgca tacgtatcaa aagatgacct agttggccat cactggaaag agaggcccat 27480 tggacacgca aactgtatat gcctcagtac aggggaacgc cagggccaaa aaaatgggaa 27540 tgggtgggta gggaagtggg ggggagggta tggnnnnnnn nnnnnnnnnn nnnnnnnnnn 27600 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 27660 nnnnnnnnnn nnnggcagga tcctgtgttc atgtgcaaca ctcgatgcaa gctgtgtagt 27720 gtttggttct gagcacctga aggggaccaa gcaggctgat gcccaggcca cgggttcttt 27780 ctggccccac tgcccactcc caccctctgg catccccatg atgaacatgg ccacagatca 27840 cactactctg ctcctctccc agatccacgg agccataggg tccccagatt catctctgca 27900 gctaacaagc tgggcagtgt cacctccctc aaggttcctt tcctgctctg agcagcagtg 27960 tctcccacag tgagacactc atgtccactg gaagatattg tagccattaa attcctgtgc 28020 taaaataact agggggactt gtcaatcact acactcttag ccccggactt ctgactcata 28080 gagggtggtg acagctcagg gacctgcatt ctaccaaata gccatgtgtc cctgatggag 28140 gaactgcccc tggacaacct ctgcagcaac tgaaccctct gtggtctcct agttcttctg 28200 gacaggtgtg accccagtac ctagtgccag gtgagagagt gctagggcca cactaagggg 28260 tgacaggaca aggttggagc tggtagatgt ttgggccacc aaagagaaca ggtcagtagt 28320 aaaagccatc atggcctgag ccagcctgcg agtctcctct gcagttggga cactcttgca 28380 gtgtcctggg gacctcttga gggtagcatg gtcaccaaaa tcctacaagg acagatcaga 28440 agtcagtgag gtcaagggaa cagctctagg ttctctgtgt ccctcacgga cctttttttt 28500 tttttttttt tttttaagat ttatttattt attatatgta agtacactgt agctgtcttc 28560 agacagctcc agaagagggc atcagatttc gttacggatg gttgtgagcc accatgtggt 28620 tgctgggatt tgaactcagg accttcggaa gagcagtcgg tgctcttaac cactgagcca 28680 tctctccagc cccctctcag tcctgatgcg acagggcagc aaaggccttg tcccagatct 28740 gaggagagtc atgctgaagt ccttcctacc ccaccccttc cgaacccctg aacatcagcc 28800 ccataactac tgactccccc acccccattc ccttgcttcc actgatccgg tcctcctctt 28860 ccctctggcc ccacccattc ttccccagcc ccacctgatt gtacctggtt gtccaacttg 28920 aagagggcag gcaggggcag cttctgctgg gcctgctcac tcactggctg tagaaatgag 28980 aaaggagatg aagaaaaggc ccttcccatg ggtccccatc ttgccaagac ataggtgagt 29040 ccctttggct cttcccccta aacctctcac ttttgagtac ctgctggccc gggagatcca 29100 cggcgctcac cggagagaac tgttgagaaa agggagaaca gagaactcag cgttcctccc 29160 tctccaccct tctggcctct cccagatttg cccccgcccc ccagcatctc cttcagcctg 29220 actgaccact tcccactcag acctcagctc tgcctcaccg tgaaacaggg accttgcagg 29280 caggacaagc tgagtacgag gagcccccgg agcagtgcca tgttcctgta tccagaacag 29340 ggagtgttag ttcctacctc acgctcgaag gccaagcagt agactgctat ccatgggttc 29400 cttgaccgca ccaggctgcg gaacctggac tcaaaacata gcagctgtgg acctcactca 29460 ctctgagagg tgggatttcc ataagctttt tttttcacct gtacatttag tcttcattct 29520 tttcgtctta cactgtggat cagtcctggg ttcaaattta aagccctcat cttgcaagag 29580 gaccttgcgc atctcccttc atgcctttgg ctttaccctg tcttggtaat tcatggcaga 29640 agttcttcct gctcccatgt agatgttgag gacccaaata agaatctctg taaatactga 29700 gcatgatgcc tggcccccac cctagcaaag ccacctgacc tgttgttcat ttcatccagc 29760 ctttctcagg ctgccctggt cctacccaaa ggctctgaga gctaatctgg gctggcaggg 29820 cagccagaaa cttctttgtt gaccaatgaa tgactggccc agacaccttt ggacttacgg 29880 gaactacaag cctcatccca cttctgctcc aagttctgat ccagggtgct tcggggaagc 29940 ccagctggcg gaagggggga ggctctcagc ctagagagcc ttcctttcca tcctcagccc 30000 cctacccagg ccttatttca ggcaccagct cttctaaaag gtccttctgt tatccctaga 30060 cctccacaac tgtgttcaag aaccttcagc cagggcctca tctccaatct ggatatatga 30120 tttttctcgc caagagtagg cctccaggtt ttggagttct agaggtttct cctggagctg 30180 cctggacctc tgctcctcac caccccagga cgctgtgaag ctgcaggctc cctgaataaa 30240 ttcatccaga ccccttgcca aggtgccagc tgtctacttc ctctgctgcc caagcagcag 30300 gctgcaccac ccctccatcc tacctcttca ggcttcttag cgcagcacac gcagcacacg 30360 gtgttctcct ggaccagctt gctccccacg ctcccccagt gcagccagca gggcctagct 30420 ctctcctccc acaggacctt tgctctcagc caacccccgt tcagcttgtg ttcagtgctg 30480 gtaaatattg acctgtacat ccggttaaac attgatatgg gggccagaag accctttccc 30540 atcaaggcta cccagaaccc tgcctgagcc tggagaaggg gtttacagga gcagataagt 30600 gaggaggttg ggcctggcaa gccttctaat gatccctcaa cataggggat tatccacagt 30660 cagtgaggct cagagaggct gtgtggcctg tgtaagggcg cagagtgggc tccagagtca 30720 cagccaaagt cccaccacca ccaccaccac caccaccacc accaccacca ctaccaccac 30780 caccaccacc accaccacca ccaccaccac caccaccact accaccacca ccaccaccac 30840 caccaccacc accaccacca ccaccaccac cacctcatct acccatacta anttgaggct 30900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30960 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tctatgaggg ttacatttta 31020 gaacatctct ccttttttcc tttttgagac aatcttacta tatttaggct ccccttgaac 31080 gtgtgatcct tctgcctctg cctcccaagt gctgaggtta caggcatgca cagtcacatc 31140 tgcctacaca atgtcttagc agcccttagc agcaccaggg gtcaggaagc cctcaactgt 31200 ccctttagct ggcttctctt gtgaagggct atgtcttctt ccccttccta gcatggagac 31260 ggctcttagc cccagagcct tccttccttc aggttaaaca gcaccagttt ttggtgggac 31320 ctcccatttc cctctatctc cctaagcaac gaccttttct gctctgactc tcatctggca 31380 cttggaccac aagacaaaac tgcagcctgg gctgtgtgtc ctcgcacatc attcctgtgc 31440 ccccctggag tcaggtctag gggaggaaga cagggttcac gactcagaaa agaccactgg 31500 ctgtcctagt gtgccctcac ccatcctata gcacgcacat gctgatgtgc cccctccgct 31560 ccatcaccat cctctcatgt acacgtgccc tccctcgcca gacacatgca tcactaactt 31620 ttctgacttc ccagaaaaat atctgatctg agaagttagg agtctgccat catcagctat 31680 ggtccttaaa attaagtcag acaatccatg ggacatgaag ggcaacaacg agaagactcc 31740 tcgttccttg ttcactctgc ttttggcagc accaccagca ggaaccaacc tggctctccc 31800 taatccctca tctatagcag gtctcccggt gggaatttta gggacctctg tgttctcatc 31860 caggggcact gccactcagc tgctcaggga gagacccctt agaacaacaa agaaatcaat 31920 gcagatttag gcttcttgtt tcccttccca gcccctccca tcacaggcaa cagcctccct 31980 tggctgagcc tcaggaggct gatttatcag agaggtgctc agagaggcac ctctggtccc 32040 tctgggtagg tagcaactga gacaggagga gatggtcacc ctgggcatcc tctaccagga 32100 agtaaatgag atacccttgc agatgggacc cctgaagttc ctcccggggg cgggggtggt 32160 ggtggtggga gtctaagtca cagatctttg ttaccacgtg gttagactga ggactgaatc 32220 tgaggtggga aatctgatgt gcatggggaa acacagaggt ccaatgctgg ccaagagcta 32280 caagcaggga caggtgctag ggggatgtct gaatgttcca ccccaagcca caggaataac 32340 ggaaatggag actctaaagg gcagaaagtg agggtgtgca gcaggggctg cacaggacac 32400 atgcaaggcc ctggctgcaa taactgggtt ggggaggcag tcattggcta gccaggggca 32460 ccaggacagt gatgccatcc tgtccaaagg gcagtgtcca agccagattt ctaggctcca 32520 gggggaggag ggtccgggga gaggggtcaa gattctcccc ctctgagtca aggttggcct 32580 tcccatgtgc cccaaatcag gaggcacaga aactgggatg ttgtggtctc acatccaagc 32640 tgagaagaca agtgggagcc agtacatgtg tttcagatta aacccagtcg gagacaaaca 32700 tgttgctcct cctcctccca gagccaagct gccttcaagc cacatggcag tgaatatgcg 32760 gacagtgcag gggaggacac ctctctctcc actggctcaa ggacagtttc aaggggttca 32820 ggctggctgg ctcatggcta cgccgctcac cccctggaca gtttggggtt tttccctcct 32880 gaaatcttgg aatctgaatc agcctgagat accccataat tgtacctccc aacaccccca 32940 gaaaggtcag ccctgcagaa cagaactctt tggtccccac ccatccccct cagccctgga 33000 ggctgaactg atgggcagct aaggtccaga cagtggctgg ctcttggaaa gcctgtctct 33060 ttcctttgac tcagaccact ccctgccgtg gcttacatca ggaggtgcaa gggctgcagg 33120 agggcagcca gaccccacaa accagctagg ctaaatggtg cttattgttc gcaagaggcc 33180 atgacctcat ttgtctccca gctcttttgg taagagagaa tgagaggaag ctggacagag 33240 aacctagcag gcctcaggca gcccactgct ccttgctgta agggaaccag caccgatggt 33300 tctgaaaagc agcgatccga atggagtcag gctgagctgc aggaagctca ccttccttgc 33360 tcactgctgg tggaagcaac ttcaggaaga gcccagccta tgggactata gctcctccgg 33420 ggtactgctg agtccagccc cagagcttag ctccctgctt cccaccaccc accaccacat 33480 cctttcccaa caccattcaa aaccccagtc cagcctctcc tactggtcta cagtgagcgg 33540 ctaatagagt cctgggcctc tgtcccccca attctctctc ccctctcatc tgttcacctt 33600 ggttcctaaa ctgcaggggc tactataacc ctacctccac ttccttgcac ccctcttttc 33660 tgctctctgg ggtgcccctg ccactcccag tccctctagc cagggagcct cttccatatc 33720 tgtcttcccc aggctagacc aggcgctgcc ttacctgtgg ttgcggcagc ttctctcaca 33780 gcctgcactc tgaggggctc caggaagcag tgaggggagt agctgcctct caaccagcgt 33840 ccagcaggct tcagattaca gctactcttt tcttaaagtg acctgactcc atttggaatc 33900 tgtgattgca tcattgtctg gtgttaactt taacccactg ctgcccttcc gccatgtggc 33960 tccaagacca cacgttggcc accctcctct cccaccacat ctcccttgga tctttatctc 34020 tcttcattgg gaccttcatt gggacatgat ggctaacttc aggggcactt gggccagcct 34080 ggggtaggtc atgagtctga acttgaacat ctgaaaggat tggctgagag gcaggctgca 34140 tggagagact gtgagccagc cggtatggag atgctgggtt cttccaggcg cttggctctg 34200 gctcactgca ggtgggagca aggtgattct tctcccctcc tcacctggaa aatgaaggaa 34260 tgggactgta cctgacagct ctgaaggttc caaaggacag tggggtgggg actagagagt 34320 tggcccagtg cttatgagca ctggctgctc tcgcagagga cctgagttct gttcccagct 34380 cacatagcaa ggactcgaaa ctgcttggaa ctccagctcc agagaatctg acgctatctg 34440 ctnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnatgtcagg catggtggtg 34560 catgccttta atcctagaac tcaggaggca aagcagatgg acatctgaat ttgaggcccg 34620 cattgtctac atagcaagtt ctaggctagc caggtacatc ataagaacct ttctcaaaaa 34680 ataaataggg ccagttggca aaatttagct tgccctccta acacaagaac ccaaagtcaa 34740 ccccagcaac catgtaaaaa gaagcaaggt gtggtggcac ttgcttgtga tccagcattg 34800 tcaaggtgga gacagacgga tccatggggc tcactggcca gccagctagc tggtctactt 34860 agtatgctcc cagccagtga gagactgaaa aataaataaa taaataaggg gttaggaaga 34920 ggtaacatgg tggctcagtg agaaaagata cttgtcatac aagcctagca accctcaatt 34980 ttcagtggcc actaaaggtg gaaggagaga accaactcca aagaattgtc tcctgacagt 35040 tttatgctgt ggtacacaca cacgcacaca cacacttgtt acatgcatat gcatacaatt 35100 aataatttaa aatgttatgt gtatgggtgt tttgcctaca tgcatatctt tctgtgcacc 35160 acatgtgtgc aatgcctgtg aaggctagaa gaggacatca gatcccctcg gagttacaca 35220 gggttgttag ctaccatgtg gattctggga acaaaaccat gggttttcca aaagaggctc 35280 ttaatcactg agccatctct ccatcccctc aatagtatat atttctgggg ctggagagat 35340 ggctcagagg ttaagagtat tgactgctct tccagaggtt ctgagttcaa ttcccagcaa 35400 ccacatggtg gctcacaacc atctgtaatg gaatctgatg ccctcttctg ctgtgtctga 35460 agacagctgc agtgtactca tatacataaa gtaaataaat aaaccttttt tttttttgtt 35520 ttgttttttt tgtttttcga gacagggaga cagggtttct ctgtatagcc ctggctgtcc 35580 tgaaactcgc tctgtagacc aggctggcct tgaactcaga aatccgcctg cctctgcctc 35640 ccaagtgctg ggattcaggg tttgcgccac cgccaccacc tccaaggctg ctgctgcggc 35700 caccaccacc ccaccaccac actacctgac tatttaactt ttaaaggcag ccatctcatg 35760 gaaaatgaca cctagcattg tcctctggtc cctacatgac cccatgtgca aacacatacc 35820 tgcataaaca cacataaata cataagtaaa cttagtctgg ttgttttgga aatgtgctat 35880 ggtttggatt gtgtcccccc aagggaaaaa ttgggtccca gagtagtact attggaagag 35940 agtagaactg ttagggttta ggcctggtgg gaagtggcca tggctagaga gacatgccaa 36000 ccaaggggaa tctctggctt catcttttcc ctttgctttc aggtcctaag acagtcacaa 36060 ggctgcttca ccacatgccc agaagcaagg ggagcagtca tggctgggac cgctaatgca 36120 gctgttgatt tccccagata tttgtagtag taagagacag gtgaggaacc ccacagcaag 36180 tgttagtaat tgtgtgtgga ggtgccctcc ggggacgggg gccctcctgg ggcaggacgt 36240 tcctcttcct catccacctg cactccgaga acaggaaatg gtgactttgg cagagcttaa 36300 gcagagcccg ttcatgttac aagtatgtaa attcataagg accagtttct ctccatatga 36360 aacagcttca aacaggagaa ggaagaagca aacattaagg aaaagctctt ttattgcaga 36420 ggctacactg aagctaccgg ccgccttcct ggaatgtata atcagcttcc ctctgggggt 36480 tctgtagagc actgagacat taagtactac tggggtccag gattctgcct atgaagagga 36540 gggcccccgt gtccgtgtcc ctcagaacaa agaggaaagg ttggttaagg tgatagtcta 36600 gcgggaaggt gaggcggacg ggctggaggc ctgggctggg gctgcttcct gccccctctt 36660 cattccactc gaaagcagcc ctgtgttcca cttgggtgag cttcacgggt ttgccagtaa 36720 tcttgctgaa gtcgggtgat tcaaacaacg actgtagctc tgtggagatt cagagattcc 36780 attaacacca cacacacaca cacacacaca cacacacaca cactccctgt ttgtgtaggc 36840 tgattttcaa gaaagcaagc tagaagtgga gtacctcaca gtgacttgtg agctatgagg 36900 cactctgtga caggctcagt gacctacctg agaacttata gccaagatgg ctgaagccag 36960 acctggcctg agagaatgtt ttgggctgtt ataggacaca tagagataca cacacacaca 37020 acacacacac acaccaagga ctgagtctaa tgggaggtgg ttcttcattc ccctcccctg 37080 taatggtgtc acatgttccc tgagccaccc tacaaagaaa gccacaggac tcagttctgt 37140 cagcaaggtg gcaggctcca agactcagcc ccgagcgcaa agtggccttg caaacatact 37200 catgtcctgc agagacttgg taagttcgcc ttcgaagctc agcttcagct tggggacagt 37260 cagcacagct tggatagtct tcagttctcg gtcgatgtca tgaatgaact cagaggtgag 37320 gctctcttct atcatggtca agttctgggt cacggtcagg ggcaggaaga agatgatgct 37380 catacttcct gtcaagggca gctgggcaat ctaacccaac agagatgcgc acaggttagt 37440 tgtgagccag aaaaaacaaa acaaacaaac aaaaaaacac caacagctgc cttcccctct 37500 gctgtaacgg ggccccagcc ttgtgctccc cagcctcagc ctgggctgta ggctactggt 37560 tactggcagt ccttccatga gtagggagtt ttcttctcag cctaaaaccc acagaagttt 37620 aatgaacaca cgtttgtttg tggttccgct acggtttcta ttgtgataaa acatgactga 37680 aagcaacttg gagaggaaag ggtttatttc atctgacaat tcgcagggtg tcttctcatc 37740 actaagggga ctcagggcag gaactgaagc ggaagccgtg gaggaacgct gctttctggc 37800 ttgctccccg tggcttctta gcctgctttt ttatgctatc cagaaccact tgcccaggag 37860 tgacactgcc cattgtgggc tgggcccccc cacatcaatc actaatctag aaaatgaccc 37920 acgggtttgc ccagaggcca gtctggtggg ggcattttat caattgagtt tcacccttcc 37980 aaatgactct aacttgtgtc aagttgacca cacgaatcag ggcctggttc ttaggagctg 38040 aagtggaatg tcccccagag actgcctgcc agcactgctg accatttgct ttgtatagag 38100 cattgaacca gaaatgaaca ataaaatgga tcctttgaac agatgtgttg atcctagggc 38160 ctgtggacac agcgactggg cttcccagag cccccatgga atcannnnnn nnnnnnnnnn 38220 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 38280 nnnnnnnnnn nnnnnnnnnn nnnntggcct catcaggtat cagagagaga gagagagaga 38340 gagagagaga gagagagaga ggataaaagg ttagcccagt ggtggtggca cataccttta 38400 attccaacac ttgagaggca gaggcagggg gagctctgtg ggccagtttg gtttacagag 38460 taagtttcag aatagccagg gctacacaga gaaaccctgt cttgaagaga aacacacaca 38520 cacacacaca cacacacaca cacacacaaa taagatcttt aagaagaaaa gaaaggatag 38580 tggggaaaca tctgagcaga ggaagaaatg gggtgcgcag gacacccacc ctcagaggag 38640 gccctcactg gaggtgtctg cacaggagaa cacttgcact cagcttgccc tagggcgtca 38700 gaactcagaa ttcagtttca aagcactgac aggagcagtg actggggacc ccaggttgaa 38760 tccccccttt atctaaaatg agtaagaacc aaaaaaacaa aagtgtttgg gatttggaat 38820 ctgggttatt tgcatctaca aaagaggtct tggggaggaa acccagttct acccctggaa 38880 ttcatcagtt tcctataccg ctgactacac aggggctgaa ggtaatctca atgttttcat 38940 aactggtgtg gtgctacttg ctcatgatcc caacacttgg aaggtaggtc agaagttcaa 39000 gagcagtctt gactactcag tgaatttgag gctagcctgg gctacatgaa aactcataaa 39060 acaataaaag aaaagaaaag gtggcgagtg aggtggccca tcaggtaaaa gtgccaacca 39120 cctcgcctga aagcctccac acggaagggg aaagccagct tctacatgtg gtcctctgcc 39180 ctccgcatgc accatggctc gtgcaccccc acacccaccc acccacccac ccacatgaca 39240 taaatacttg taatgattag tttctgaaga acaatatttt cgttgatctt gtttagggaa 39300 caaagttcgt gcacattgac ctgtcgaccg tgtagtacgg gatccgctcc aggaagctaa 39360 agattttggg atgcttcatg ttgcagctac tctgatgaac agtgttgctt tctgggccag 39420 gtaatggtgg catatacctt tgatcccagc acttgggagg cagaagcatg tagatctctg 39480 tgagttcgag atcagcctgg tctacagagt gagttccagg atatccaagg ctatacagaa 39540 aaacccctgt ctctaaaaat cactaattta aaaaaaaatt cctttctaaa cctatataac 39600 aaatgttttg taggctgcct taacaaagcc caatggccat tcagagaagg ctcaaaagag 39660 aaagtttagg ggactctaca agcatcctca ggaaggccac agaaagcaga gcctgggcca 39720 gtgagacttt gcagtgggca aggttcagct ctttatgtag gaagaagaga gtcaacagtc 39780 agagtccagc tttccataaa acctgtgcag ggcctctagg caaagccctg tgttaggggc 39840 aaaggcattt gcagtctaag cccggtgaca tgagctcaat ccttggaacc caggtggaag 39900 gagtgtgctg actccacaaa tttgtcctct gatctataca tgtatgcacg tgcacgcaca 39960 ctcacataca tgtccacatg cacacatgca tatacatgcg catgcgtgca cccacacaca 40020 gggtcaaaag cagcaagaga tgccctgtga aaaacgtctc attcagtctc ccatcatcca 40080 gtgccacact ctgagcacag gtggtactga tatcgttcct gattgatcga tcagttgatt 40140 tgagaccccg cctcactatg tagcccaggc tggcctggaa ctcacagtga tcctcttgct 40200 tctgcaagat gagcccatca tgcccaccat gttattgaag caataccatg ctctataaag 40260 caaacctagg caggcaggat ggtggactcc tgtaatctca ggacttgaaa agtagaaggg 40320 agatgaggag ttcacatcaa tctcccgtat gcgttggagg ctggagtggc tgttccctgg 40380 gcgcttctgc cagcacctga ccaatgcaga tgcagatgct cacagccaac catcagactg 40440 agctcgggac cccagtgagg gtgctggggg gaggactgga ggagctgaga cgggattgca 40500 agcccatagg aagaacaatg tcagctggcc aaaccaccca gagctcccag ggactagacc 40560 acgaaccgag gactgcacat gaagggatcc atggctccag atgcatatgc agcagaggac 40620 agccttgtct gacagcatgg gaggggaggc cattggtcct gtggaggttt gatgccccag 40680 tgttggagga tgctggagcg gtggggcagg agtgggtggg taggtgggga gcaccttcat 40740 agaggcaaaa gggatggggg agaaggcaga tgggatgggg gggttgtgga ggggtaagaa 40800 agaaaaaaga tgtctctgaa agtaaaaagt acttgtcact aagcatgagg atatgagtca 40860 acccccaagc cccacagggt ggaaggagag aaatgagtcc cacaagttat tttctgatct 40920 atacgtgcaa tccatggcat acgcagaaac gcaaagacag acaatgagtt gggtgtggtg 40980 gtgcacatgt aattccatca ttcaggagac agaagcagca gagttgttgg aaatctaagg 41040 ccaacctaaa gacctacacc caaagaagga caaactataa ggaaaaaggt ggtcgaccaa 41100 tgtaacatta aagttagaaa tctctcttca cactgtgtag atactgtaca aggaagagaa 41160 aaggcagcca catcaaaaca gtgtaaatca acgagaaaac cagaaacaac tcaagagaag 41220 gctgcagggg cctgaattct gttctcagaa cctgcatcaa gccaagagaa tcaaaactgt 41280 ctgtaactcc agctccctgg gatccaacac ccatttctgg cctccatcag catcactcac 41340 aggtgtgcac acatacacat caataaaaat caaaaccagg gatgaagggg tagggaggtg 41400 catgtggatc tgggaggagc tgagaggcac tgggtgaata caataaaaaa tttggtgcat 41460 ggtggtgcac gcctttaatc ccagcacttg ggaggcagag gcaggcgaat ttctgagttc 41520 aagaccagcc tggtctacag agttagttcc aggacagcca ggtctacaca gagaaaccct 41580 gtctcaaaaa aacaaaacag ccgggcggtg gtggcacacg cctttaatcc cagcacttgg 41640 gaggcagagg caggtggatt tctgagttcg aggccagcct ggtctacaaa gtgagttcca 41700 ggacagccag ggctacacag agaaaccctg tcttgaaata aataagcatt tgttgctgtt 41760 acagaaaact ccagcccagt ttccagcaca cacagggtga ctcacaacat cataactcca 41820 cttccagggg atccaatgcc ttcttctgac ctctgtgggc accaggattg catacagtgc 41880 acagacatgc acataggcaa aacactcaca aaataaaata aatctagcaa aaaaaatttt 41940 aactaataat ttaaagaaaa aaataaggaa gccgggggtg gtgtcgcacg cctttaatcc 42000 tagcacttgg gagacagagg caggcggatt tctgagttcg aggccagcct ggtctacaaa 42060 agtgagttcc aggacagcca gggctacaca gagaaaccct gtcttgaaat aaataaataa 42120 ataaaaaata aggccaagta attcttggaa gaatcccaag gggacactaa gtgtatataa 42180 aggcgttcca tagggctagg aatgaggctt agcgagagca acttcgctgg tgtatgaaag 42240 tccctcagct gcatgtggta cctttaatct aggctctccc gaagcagagg cagaaggatt 42300 tctgtgagtt caaggccagc ctggtgtaca tagctagttc caggacagaa agggcgatat 42360 aatagaaaca tcntacctag agcccngcca aanaaagggg agacctgaga ccagagagat 42420 gactcagtgg ctaagagcat tgactgctct tccagaagtc ntgagttcaa ttcccagcta 42480 aaaatttatt taaatgttta ttacttgtat tattatttaa atttaaataa ataagtaaat 42540 gggagcctag gtttgagtcc ccaaatcacc aagaaaaaat gttatcattg ctaataatca 42600 aattaagagc ataagaactt ctttttaaag aattcttatt tattttatgt atgtaagaac 42660 actgtagctg tcttcagaca caccagaaga gggcattgga tcccattaca gatggttgtg 42720 agccaccatg tagttgctgg gaattgacct caggacctct agaagagcag tctgtgctct 42780 taagtactga gccatctcta cagctcttat caggttgata aaatttaatc tcgtggagcg 42840 ctgagaccaa gaactaaagc tgggagattg aaaaatgcag accaccaagg ccctgctcat 42900 ttctccagtt ctgatcagct cccgtaccag gggtctaacc aggcctgtgt ctgcttccct 42960 gagtagacca gaggccccat ctaaacagcc tgcctgcagc agctcctctc tctaggtgga 43020 cagatgggaa tttcagacca atgtcatttc ccaggacatc aacacagcag ccaaatttat 43080 tggtgctgtg gctgccacag ttggtgtggc aggatcaggg gctggcattg gcacagtgct 43140 tgattattgg ctatgccagg aaccagtctc tcaagcagca gctcttctcc tatgccatgc 43200 tggggtttgc cctgtctgag gccatgggac tcttctgttt gatggtcgcc ttcctcatcc 43260 tcttcgccat gtgaggctcc ctggggtcac ccagccgtcc ctgctgcctt gactccatgc 43320 cagtcctggt gctggagtct actgagattt accattaaac agcaacgttt ctctaaaata 43380 ctattaatta attaattaat cacgtgacaa ccccagcgtc catatgggtg tggaaaatga 43440 ggaactctac ccatcataca tggcgactat gaagaacaat gtgacagaaa atgctaacat 43500 catgtgtgac cgcatgcatc agccctgact gctaaaagtg gacaagcccg aagcgaaagc 43560 ccaatgttct acttctaaat gcatgcacca aacgcctccc acaggaccag aggtgcagct 43620 ctgatagggt ccttgcctgg catgcatgaa gctgtggaca cgaggcatta ttcgcaagaa 43680 cattctagct gtctggaggg ccctcaatcc actgtgttcg cgctgttcca gcaccagtgc 43740 ctcctggggc tgcacctgaa aaaggggact gcttaagagg gctcctacca agcctactgc 43800 cacagatgca tgatgggaaa gccttctgga agcaactggc tgccaaaggc tctggacaag 43860 agatcaccct ctactggaaa ggtggtttca gtctaggttc tgtgggattc caggaaatta 43920 gacaacactg gcagtccaac agacagacga tctaaacttc caaggcacag ctggtagaac 43980 ttgctgcgga accagacaac aaggtacgag ctactcccat acaacataca aaaaagcaga 44040 gagagagtca gagacagaga cacacagaga gagacagaga gagagtctaa agagagtcag 44100 ggtctcagga ctgagggtat agtctactgt agagcatttc cccagcatat acaagaccct 44160 ggattcaatc tgaacacagg aaaaaagggg gggggggact tcgattatct caaattctcc 44220 tttttgtgac acacccctaa agtcactgcc tacttccctc accgccatga agtaaagagc 44280 tgtttgcgct tatgtctaca cagtctcggc tcccacttcc tcctcccctc tgcttctgtg 44340 ctcatctcct ctgaaaccac tgcagcaagt gacttgtgtt gactgccaca cggaaactct 44400 cctcagtagc aggcagcaga gcagagctct gtcttctcgg agcttcttct ctcttgtcgc 44460 cattttctcc cacccttaag taccctatct tctctgtctc tgcttgttga tccttggacc 44520 cttttccttt ctatgaacaa aatatctcct taaaggatct cttctagttc agggtccccc 44580 cgcccccact gtggagaaaa cccagggcct tgcacatgct cagcaggagc tccatccagt 44640 ctctagctcc atgacttaaa gcatctctgt gctgtcaaat atacacttcc agcccttacc 44700 aaaatattca gtcaactcct tgccattcaa aatggatgac ctcaaagcca gagtcagcgg 44760 tgctatgact cccagatcca tccacttggt agcccaggaa tgaactcann nnnnnnnnnn 44820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 44880 nnnnnnnnnn nnnnnnnnnn nnnnnnnngg ttactgggat ttgaactcag gacctttgga 44940 agagcagcca gtgttcttaa ctgctgagcc atctctccag ccaccaccac caccaccacc 45000 accaccacca ccaccaccac caccccacca ccaccccgcc ccacctgctc attcctgatt 45060 ggttggttag tttagtctgt gagacaggag ctgtcccttt tctatagtgg aaggtgaata 45120 agaaactcct gaaagtgaag gcctacaaaa cagccacact tatttgttgg aaaatactgt 45180 aaatgtgaca tgtaaataca tgctaaaata attcgttaag tcagtgaaca accttaaaac 45240 acagtctgta gcctgaatta cagacacgac acgagccatg acagaggctg aaataagacg 45300 cctttgcaag gagaagggca gaagcttcca tccttgctag caaatctttg ttccaagctt 45360 tatcagattt tattgctttc tctttctgtt tttctttatc atatttgttt atttgttggg 45420 ggaaagccta atcttcatag cccatgtatg tgagcacttc agcatatgtg tgtgaacacc 45480 aacagcacac gtgtatgagc accacagcat aggtgtgtga gtaccacagc acatgtgtgt 45540 gagtacgata gcacgtgtat agagttcaga ggagaactga gagagtccgt cttttcctcc 45600 tactgtgtag gtctcagggg tggaacttgg gctcagcctt ggtggcaagc tcctttatcc 45660 acagagtcat cctgccagcc cagctttctc tttttctctc tgttatgtct atccactctg 45720 ttcaaggcta actcactgac tctgagttat cagaactgct tgtgagagca ggagtaactt 45780 tggacatctg tgctggtagg aacaccatcc ccactcggct tggatgacga aggggaaaaa 45840 aagcatcacc aaggagttcc accacctcaa ccagcaaata tttacctcct atacatggat 45900 aggtggggtg ggtgagcctt gtgatttatc gttaggatct catgggagtg attacagctg 45960 gtctactcca tgaccaaaat ggtgacggtg gctgaccaaa aagaaacagc tacacctggc 46020 tctagttttc tttctttctt ttttcttttt cttttacccc acggtactaa ggattgaacc 46080 caggaatgca agagctctgc caagtgagct acattcccag atctgttttt ccatttcttt 46140 ctttcctttt agattttatt tcgatttatt tgtctatgtt tgtgtgtatg tgtgtatgct 46200 tatgtgtacg tatcagtatg ccatgggtat acagaaacct gagaaggcca gaagagagtg 46260 tctgggttac aggagtttcg agctgtcctg tgggttctca aatgcagcag caccaccacc 46320 aatcaccccc acccccaccc agccttcgag ttcaattctc atcatcacaa aaacacacac 46380 acagacaagg gcctgcaaga tggctcagca ggtaacgaag ctcgtgtcat aagcctgaga 46440 acctgagttc actgtctgga acccgcgtaa agggggaagg gaagaatcaa ctctatgatg 46500 ttgtcctctg ctctccccat gtgtgccatg gaatgaacag ccctcccaaa cacacatcaa 46560 gaataaataa aactaaaatt agcttagtaa cttttatgtt gaaagtggtt tttacatgcg 46620 tgggcaacaa taacaccgag agtagaaagg caagcatgta tgtcactgaa cagcattgaa 46680 gaaaaaacaa acacatttcc tgtacatcgt tctgggagtc tgagttaggg tttctatgct 46740 gggataaaaa caccctgaca aaaattaacc tggggaggaa gctgtttatt tcagctttta 46800 tgtctacaac atgacctgtc acccagggaa gtcagggcag aaattcaaac aggtcagaag 46860 cctggaggta gaagctgata cagaagccat ggagctgctt ctggcttgct ctagcacaca 46920 ggagtactag cctaggggct gtactgccca cagtgggcta ggctctccca cagcaatcat 46980 aaatttagaa aatgcactac aggtttgcac acaggccaat ctggtagggc cattttctca 47040 attgaggttc cttcttccaa aaggacttta gcttgcatta tgttgacata aaaactagcc 47100 agcatattgg gattatagat attctcataa aaaaaagaca tttagattcc cacataacac 47160 catattcaga aattaactca atgtgaacca gaagctctga aagtaagagt taaaactatg 47220 aaaaattctt acaaccatcc ataacaaaaa tctgatgccc tcttctggag tgtctgaaga 47280 cagctacagt gtacacacat ataaataaat aaataaatat ttaaaaaaat atatgaaaaa 47340 tcaggctggt gagatggctc agtgggtaag agcacccgac tgctctttcg aaagtccaga 47400 gttcaaatcc cagcaaccac atggtggctc agaaccatcc gtaataagat ctgactccct 47460 cttctggagt gtctgaagac agctacagtg tacttacata taataaataa ataaatctta 47520 aaaaaaaaaa aaactatgaa gaactatgaa ctacaagaag tcaggaatag ggctgggggt 47580 gtaacccaac agaaaaacac ttgcctggcc tgcgtttggt ctctagcacc accaacgtag 47640 aaagagaaca gcagaggatg agggcatcct gacttgagtc aagtgacaag tgataatcct 47700 cgagacacca aaatcacaat gataaaagag atcaacaagt tgggctttat ctgaataaag 47760 agctgtgtcg ttaaatacca cgcaggaagt gaagaggagc tgagtctggt aacacaggcc 47820 tgaaatccaa gctactgggt ggactgaggg aggacaacag ctagctcaag gcccacctgg 47880 acgccagagt taactcagag agcagcttgg gtagctttaa tgagactctg cccaggccag 47940 tgcacaggag agatggctca gtggttaaga gcattactcc tcttgcaaag gacctgagtt 48000 caattcccag cacccacgtg ggcacttaca atcatccata actttagttt caggggatcc 48060 aatgcccttt tcacagtacc aggcatgtac acagtgcaat tacatacata catgcatgca 48120 tgcatacata cacaggcaaa acttacataa aatactaagc agataaatct taaaagaagc 48180 cgggcgtggt ggcgcatgct tttaatccca gcacttggga ggcagaggca ggtgtatttc 48240 tgagttcgag gtcagcatgg tctacagagt gagttccagg acagccagga ctacacagag 48300 aaaccctgtc ttgaagaaaa taaaaaaaaa aaagaaaaaa atcttaaaag aaaaggagag 48360 gactggagag atggctccac agttaagaac acttgttctg aggtctacag agtgagttcc 48420 aggacagcca ggactataca gagaaaccct gtttcgaaaa accaaaacca aaacaacaac 48480 aacaacaaca acaaaaccac ttgttcttac agaggacttt ggtttgattc tcagaatcca 48540 catgatggtt cacaaccatc agttgcaggg atccaaggtc ctgtcttctg tgggcaccag 48600 gcatatatgt ggtgtacata catgtataca ctcatataca taaaataaaa agttttaaaa 48660 aggaggctgg gtttgtagcg cagaggtaga ggtaaaaaga ctctagcttg tttaatgttg 48720 acatgaaaaa aaaaagacat ttagattcct gcatcacacc atatccaaaa attaactcaa 48780 tgtgaatcat aagctctgaa agtaagaata agcctagtat gcactgtaag gctctgggtt 48840 cactccccag cactgcaaaa gatcatgaaa ccagaaatgc agatcctctg aaccacagca 48900 tgggaatgta actcagccga tgcagtgctc acctgtcgta tacagagcac aggataaatt 48960 gattgtggtg gtgcatacct ataagctcac tacgtggaaa gtagaggcag gacgaccaaa 49020 ggttcagtga catccttggt cacatagaga atttgaggcc agtctggtct gctggtctat 49080 ttggaatgct gtctcaataa ataaaagaaa gaaagaaaaa gaaaagaaga agtcctatga 49140 ttgtcttaac ctctgacctc tgtgttcatc aagtctcctc ctcaggaact cactggtcat 49200 cttgtgaaaa cctaccccag agtctctgtt cagaggaccc aggctccagc tgtggttacc 49260 acataggatt tttatactag aaaaataaaa tgaataagta tgtatttttt aaaaaggtgc 49320 agagctggat atggtggtgt ctagttatag catccagaac tgagacagga tagccatgag 49380 gttgagaaca gctagactat acggtctcaa caaacaaaag taagggatct gagtagatga 49440 ggttttaatt tttttctttg tgtttgttac ctaacgtgta tggttgtttt gaatacatgc 49500 atgtctgtgt atcacttgtg tgcctgaaac ccaaggaagc cagaggaggg catcgggtcc 49560 cccggaagta ttattacaga aggttgtgag cagccatgtg ggtgctggga atcaaatctg 49620 aaagagccac ctcgggctgg agagatggct cagtggttaa gagcactcaa tggctgctct 49680 tccagaggtt tggagatcaa atcccagcaa ctacatggtg gctcacaacc atatgtaatg 49740 ggatccgatg ccctcttctg gtgtgtctga agacagctaa agtgtactca aataaataga 49800 tcaaaaaaga aaaaagaaac agccacctct ccactctccc tttttaaaat cctcttgcct 49860 ctgtccctta atgttaataa cacaggtata tgatactatg ccttgtttat gaatagaaaa 49920 tacacgtgct aaagcaagtg tgaaccttaa atacattatg ctgagtaaaa ggagtgagtt 49980 gcacacaaga cttttctgct caagagtatc tgtatgaagt attgaacatg tgaactctga 50040 aatcgggagc tgaggaagat atggggagtt ctaatggcta caacatttct ttttggaatg 50100 atgaggatgt tctagaactc aaaaatggtg ataactcagc atatatacta aaactcattg 50160 aattgtacac tttaaatgaa tgcaataaaa cttgtctcag taatgtggtt tagaagatgt 50220 acagacatgt gtgtgtgtgt gttaaaacat ttcttggcat ggcaataaaa atacagtttt 50280 agccaggtgg ttgtggctca aaaaataatg ataataacaa taataaaaat aatgaaaaca 50340 gaggctggag agatggctca gcggttaaga acactgactg ctcttccaga ggtcctgagt 50400 tcagttccca gtaaccacat ggtggttcac agacatctgt aatgggatct gatgccctct 50460 tctgatgtgt gtctggaaac agctacagtg aaagtcattg caaggacttt acaatagtga 50520 ccatgataac attgaagcta gacttgctac tactgctgag tgtgtctgct ggctctttct 50580 aaggagtaat gttagctttt tgtcctaaat ttgtttcctt cctttcctct ctccctctgc 50640 tgttttttct tacccctctt ttactttgct ttcccctctc atctcctctc ttaacagagt 50700 tgtcctatgc agcccaaatg ccatcttcct gcctcagcct ccccagtgtt gaaaaatact 50760 ctttccacag gttatgttag gagactggag tctgctcagt cggggaggga gcctgggtca 50820 agttctgagc tcaattcctt ttctttcttt ctttctctct ttctttcttt ctttctctct 50880 ttctttcttt ctttctttct ttctttcttt ctttctttct ttctttcttt taagacaggg 50940 tttctttgta taccctggct gtcctggaac tcactttgta gctggcctgg aattcagaaa 51000 tctgcctgcc tctgcctccc aagtgctggg attaaaggtg tgcaccacca ctgcccagcc 51060 ctgggctcaa ttcttaacat tgtggagaga aaagtattgt agctgttctg gccacctgga 51120 attactttgt ttctgatctt ttgctgcagt caaatccttc tcatccatct ttcctcgtca 51180 ggctataata tagactctcc ttgcaatact tggaaatgct ctacagtcag ctacatcctc 51240 agtcctgctc ctatattttt tcctaagctt ccttctaagg tctttattgg tttatgattt 51300 acacagaaca tttttttttc ttgtctatag catgcgttag agtgatcgtt gccagataga 51360 ggaaagagaa atgagagaan nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 51420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt 51480 cagctactga ttcctcctcc tccctcctcc ttcctccctc ctccccagcc tcatgctctg 51540 ctcatcttgg acttctgcgc atgtcctcag cccagacctt ctgctcttgc ttctcctctc 51600 cccagcagcc ccccagttct cttcctgaaa cttctgaggt actctccatc acctcctttg 51660 gctcctgctc tgattggtgt cacctgctgg ataggcttgc tcctgactcc actgttcgtg 51720 tctcaattag ggaccctcac cctctgatat accacacatt tccctagtgt ctccacctcc 51780 cacccccacc ctatacgcac atacacactt agctgcatca ggatcctaca ccagggactt 51840 cttacccttc taatcctccc caccggacac tgcccaggga cactggggct ccagagggct 51900 attgccacac ggacacacag gagatctcat caaggagatg tgcctacccc agagggtagc 51960 tctcaccatt cacaagcaca ccacttctgc ctccagcttc tactctctcg caggaagtag 52020 ccagcccggt gccaagtatc cccaactaca tccccaaaat tctcagacac tgccagcctc 52080 cagctgtcag cctggccccg gctggcgggc gcctgctcct ggcatagcga ctagggtgta 52140 attagaaacc cgctagctcc ctaattgcca gttctgagct gtccttgtta ccggctgccc 52200 gaggcacaca tagaggaaaa ggctgagagc tgagccaggc tggcatggag gtagccctag 52260 tagacctaga gaggactggc atgtggccag ggaccaaacg tggcacagag agggctcagt 52320 gcaatctgcc ccgtgggtgc ctcccagcca catccatttg cccagaactg tgacgtcaaa 52380 ccagcccggc ccattcattc tttattcagg tggcataaaa atcactacaa aaactttaca 52440 aaagagtctt gggagctaaa gggtcccttc cttgcctcag tccccaagat tcctggcagg 52500 ggaggacaag agagagaaga aggaggaaga ctcctggcag tgttggcatc tccaaatacc 52560 agaggggtga cttgggtgac aggacacagg ttggggacct gaatgtcttc agcaagggac 52620 actcttgtag ggtaggtcag cctccaacca tgaagtataa caccaaggcc agtctaagct 52680 tgggagacca acacttgtct ctccttttcc cacccagggt gtctggaata tgtctaaaga 52740 tggcctctcc agcctctgct tacaaatgtg gagggaccct aagttaggga cttgcctaac 52800 ctacctctag ccaaaactgt gtccacaagt gccagcccac aaaagatcac cccctgagcc 52860 ccttgggaag aaatgaagat tccccatgcc tgccttcctc caggccccac cccacctgct 52920 gcaagagaac agcttctaca ctggtgatgg tccttccggt cccaccctat cccacaaagc 52980 tggttagaaa gagtcacagg agctgagagg ctgatccagg tggggactca ggatgctgct 53040 gcccagggcc cctcctcact tgggggagct gaactggggg tagtcttcct ccatgcgggg 53100 tgcaagtttc aagtcaggac caaaggtctt gcctccatgg aagtcagctt tgtcattctg 53160 gcctatgagc ctgttgtcag gggaatctcg ctgttcctgg agctggggca gcgcgctggg 53220 gttagggttc ctcacactgc ccacaaagag gggcacgcct atggtgtcct ccatgatgaa 53280 gaagaggaag ggtcggttca cagtgaagga ggagagggac attcgattca tggctacgct 53340 ggtagctgcg gctgcctcca caccagcctc gctgagctcc atggtagact gatgttgcac 53400 gctagacacc accagattct gctcagagat cccacgaagg tctgggccct ggaacaattc 53460 ctgcaggcct gcccagaaca gcagatgact ggtcagtgct gccccaaggc tatgtggatc 53520 tgtctagcat cctggctaaa gggaacactt gaacccagcg gttgattgga atctgttaga 53580 cctcagtcta gacaacactt ctagaaacct tttttttttt tttttttttt ttttaaatca 53640 ggatctgcgc taggtacagg acagaaagtc tagaggagca tatcaaatgc tcccatccag 53700 gaagcagggc cacctctggc tcaggcacac tggcagctcc cgtactctgc ccagaccacc 53760 taggggcacc ctatccccaa gctccttacc cagttggctg agggtggcca ccaggtccag 53820 ctgctgttgc agatggagtt taggcagcca caccttggtg ggcctctcct gcagcgaggg 53880 atggtacaga gtatcccagg tcaggttggc tagtacctcg gacacgttcc actcaaaata 53940 agtgggcatc acgaccacaa agctcatgtt gttcttaaag gggaaatgag ccacctacag 54000 ataagaaaag gagagaacat gaggaccaga cagcacctgg acctgtctgg agtctgggcc 54060 aaaattactt ctgtactttt gagacaagag ccagaaattc agggttagca tgctttcact 54120 taactggtga agtggaataa taccacttac ccctttgcaa ggtgacatgg gaccaaatga 54180 gataatgctt ttacacctct ctgtgtgcac acataagcat atatgtttgt atcggtgtga 54240 gtgtgtttgc tcatgggtat atggagtcag aagtaggtaa acatcagtcg tcttcctaca 54300 ttgctctcca cttttttttt tttttttttg gtgttgccat ctttttgttg ttgttatttc 54360 aagacaggct ttctctgtgt agccctggct gtcctggaac tcactctgta aatcaggctg 54420 gcctcgaact tgcagagacc cacctgcctc tgcctcctga gtgctgggat ctaagatgtg 54480 tgtaactaca catagctccc tcttttttgg acacagggtc tcatggatcc caagctggct 54540 ttgaaatgac tgtttggggc tggagagatg gctcagcggc taagaacact gactgctctt 54600 ccaaaggtcc tgagttcaaa tcccagcaac cacatggtgg ctcacaacca tccgtaacaa 54660 gatctgactc cctcttctgg agtgtctgaa gacagctaga gtgtacttac atgtaataaa 54720 taaattaatc ttttttaaaa agagaaagaa atgatggcta catacttctc tctcgtctct 54780 ctgccccaag tgctgggatt acagagctgt acaacaagcc caagtttgtt gtgttttaga 54840 catgctaatg tatcccaggc tgtcctcaga ctctctatgt aattcagaac gaccttgaac 54900 ttcttttaag gtttattttt atcttatgtg tatgggtatt ttgcctgagc atttgtctgt 54960 gtaccgtgtc cttgcagtac cctcacagtc cagaggaggg caccatttcc ccctgaactg 55020 gttgtgagct gcatggtggg tgctgggaat caaaccctgg tcctctgcaa gagaagccag 55080 taagtactct taactgctga gccacttctc caccttgagc ttttcttcct cctatctcga 55140 tctaaaagta ctagggatgg cggatgtgcg ttcatgtgcc tggtttatgt gttgctaagg 55200 gttgaacaaa gggctttgtg catgccaggc aagcactcaa caactgagct acacatcccg 55260 acagactttg actcttctag tagtagtgtc tccactacag cctgagttct ctatctgctg 55320 tcagcaagct gtacaaacaa gctatgggcc ttcctgtcct tgcctctcag ttctctccgc 55380 aggtggggct actggctttc aaaatgaccc atagaggagc cacagcaaac agtaggaagc 55440 ttgcccctcg tctttcaccc tctcccagag agtcagctat aattcgagtt tttttttcct 55500 ctctctctct ttaaacagga tctggttatg tggccctaac tatcttcaac ttcagtcttc 55560 ctgcttcaac cttctgagtg ctgggattat ggtgtaagcc accacactca gctcacacaa 55620 cctttttttt tttttttttt tttaaagaat ccatgcagtt aggacagcat ggaaatgacc 55680 aggctcaggc ctccctgggt accagcataa tgcctgcagg cgggtcctct gccagtgggg 55740 ggatggaaag atggagccag aggatctttc ctctctgaac ctcaatgtcc cacagtgaga 55800 cactcatgtc cactgggaga tactgtagta ttcaaggaag aagcaacagg aaggtgagag 55860 ctaagtggag ctgagcaggc tcgtatcctc tcaccacggg ctacagagaa gtctggctgc 55920 cccctccaca tggctcctcc ctgcagaact ggcaatgctg ggcccggctt gcccagtcaa 55980 actaaccaac agaatggatg agcatgtgtg gtgccacaca cctgggaccc cagcactcag 56040 acagctgggg cagaagggtc atgagtccaa agcgaacttg tgtaacattg tcagaccctc 56100 gaacaaacaa aactagcccg tcctgttatc tcagccacag atgatgggcc caaggatcag 56160 tactctagcc aaggagtcac ggttaggcta gaagcaaggg aagccttagc tgagacagct 56220 tggcacggag cttcatccaa tcagaatgtt cagagcaata agctttgaaa cccgacttcc 56280 atctatgaag cactgtgtgg gaactcctct cttcccttac gagcagggcc ctggtcctct 56340 tgggctccgc taaaacccca gcacagagaa cagttacctg gcacgtgaca aaaactcaat 56400 atattttctt tgaggagatg aacctcaaag aagctgtgtc ctggatagac acagcataat 56460 aaacccttca ggagctacct acccagggac cagactttac ctcccagtac caggcctcgt 56520 ttgccagcca aaggcaaagt ccagactgac ctgtatctca ggttgctcca gcaggaacca 56580 tcgaagagga tatgacaccg cgtgcatcat gtccaccgac actgtgaacc gctcatccag 56640 gtggaagaaa tctttctggg tgaggctcgg gtcaaacttg gtcctccaga aacctgcagc 56700 caggcagagg gcaggagcca tgtaacataa aatcagcctn ctgcctgtct tgcctagaac 56760 ctatnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 56820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnaaccaa ggcaggtctt 56880 ggaaaaagga atcttaaatt agaagatgcc ttgataagat tggcatgtag gtatgtctca 56940 ctaatgattg atgtggaaag tcacgaggga tggtgtcacc ctgggcagat ggcctggggt 57000 atataaaaac acaggctgaa caaaccacaa agcagtagtc ctcaatggct tctgctttag 57060 tttctgtctc aggttcctac cttgacttcc ctcagtgaag gcatgtcaca tgagagttgt 57120 aagaggaaat aaaccctttc ctccccacat agtttttggt tatgatgtta tatgtcaaca 57180 acagaaacta taactaatat agttggtttt ctttttttgt ttgttttgtt ttgttttgag 57240 acagggtttc tctgtatggc cctggctgtc ctggaactca ctttgtagac caggctggcc 57300 tcgaactcag aaatccacct gcctctgcct ctgcctccca agtggtggga ttaaaggcat 57360 gcgccaccat tgcctggctg gttttctttt ttttttaata catttataat gcattttaga 57420 tttaaaaaaa aaaatggcca tggcatataa tataaaaaga agtgcttaca aatcaccatg 57480 tgcccttgcc ataaattatg taaaaatttc catatggaca tcagtctcaa gcttacaatc 57540 tcagcactca tgagcctgag gcagaggcag gaggatggtg agctcaaggc cagcttagtc 57600 tacataacaa gatcctgtcc aaataataac aacagtaata atttcataca tagaactaga 57660 aggggccact gcaaagacag tatgacaaaa ccactggccc tgcctaattg tattttaaat 57720 aactgtcctc ctctctgtaa ttttcagttt ctaattttta cataactacc atgtattctt 57780 tttgtaattt taattagttt tttaataata gaaacaagct aagtgctaag aatattttca 57840 tatgaacatt ttcaaggcac ttgatacata cctcagattt gccctccagg tgagcagtac 57900 caattacgtg ccaccagcaa tgttagcttc cttttttccc taccatctga ttctgtttca 57960 gtctattcgt agttctgatc ttgttatatc cctttttatt gtttccctgg gttccaacac 58020 ctcccagttg agtgttctca ttgaatttca ttagcagctg tttcattaat ggcacagaag 58080 aaggattaca gtgttaacta ggatagactt tgacaaagaa ctatgagaac atatcttatt 58140 atctttgcat aaattctttt taatcaaagt tcctcaaaag cctctctctg ttcccatctc 58200 agggagtagg tctggccact gatgagtgtc caggccacag tacaggtgtg cgtggttctg 58260 tccctgtggg aagggcacat ctgtgttgta acaggattcc tgtcttaaca agccttgctc 58320 aggctctaag tggtcctgag ctagctaact gcccttggct ttcccttgat taccagataa 58380 ctattcactc ttctcatttt gcagagcact taccaggtag ctatgtcctg gaagtacgaa 58440 tgagtccttc tattgttttt cttttactta aatcccattt gaaatgcgcc agggacactt 58500 caatccaagg tacacttttg ctaaagaatc actcattttt atatgcaaaa tgtcacctat 58560 taactgcagc tgatatggta catacatatt ctctcttcct attatccact aataggtgac 58620 taatgcgaaa tattgagtaa tttttaaaaa tcaatactca attttttaga aataattaga 58680 gagacattca actctgacac cagcacccta ctcagttcct gagccttcct ctgccggagg 58740 agaatctata aataactcac gaagctgaca ttactcactg tgttgcagtc atttttttct 58800 gagaaaattt tagcaactgt tctaatagag cctgccagtt atcagtagtt gagaatgcaa 58860 gtcaactttt aattatgcag acgctgatta ttcagacgac aaattgttgg tgcctgcacg 58920 gctccttcct gctgcctacc tttaaccgtt ctcagtgctc attagcacat gttccagaag 58980 gtaggctttg gaggggcgga caggcactca aaccagctaa gcacttagag aagctctgat 59040 gaaagatgtt aatgcagttt gtagaattat tgactaaaat tgagtcattt ggattccctg 59100 tgaattgtat ttacatgccc tgtccctgtc ccccatagca acagataata ggattgtctg 59160 cagagagaca acatagttct tatatttaat tttttccttt gtcgaacatt ttcacatgat 59220 ggttcgtggt gtttcctttg ttcattacat ttgtatccag actagttact tctgataagc 59280 ggttagttag gattcctggc acgcggacag tgacaccaca gttgtctgat cgtttcccac 59340 ttttttacaa aaccgtttgc ctttaagagt cagtgttttg cacatttcac ccagattatt 59400 ggaaatatta tttccctcct gcttaaaccg aagctgtgat cataatttaa gcctttctag 59460 gtagccgatc ttacatgtat catacctatt tctggcatat gtttgtctat tacaaagacc 59520 tcgtaggtat gcagttagaa gcctctagtt aaatgaaatg ttgcgtgtgt gatgaacctg 59580 gagtggggat ggccttttgt gtgccccaag gctgttgtgt ttcacacagt tgttttctgc 59640 ctcctctggt ctatcactat cctgccactg ccagaaaacc ctgctgtgtg ttccccgcgt 59700 ggaggatctc tgcttctgaa cttctttggc ctgagaaact ccataaccaa atcagttagc 59760 attttgttta aagagcaggt aggctgttag agcttgggtc ttacatgtct cccaggtcca 59820 cttgccagcg ccttgaccac tgttaacttt tgttaaccaa ctcatctttt gctgcctgtt 59880 ttttgggggg tttttttggt tttgtttaag ccaagatcag ttatatggcc caggctgagc 59940 ctctcttccc agcctctcaa atgttagaat tacaagcatg catccctcag catacctttc 60000 ctttgctttt tttaaaatag agttttgcca tagcaacaga aatctaacct aactaagcat 60060 agccgtgcac atggtatgag gaactcacat atgtgtgaat ggaagttcat agagaccggc 60120 atcactgcct agaggcccct ttcttccttc cttgcagttg tcgtgctagc tgactgtact 60180 acaaaagagg ttgtctgagg cataagacta ccttcaataa aacatgcaca gacagtttgc 60240 ttctctgaga tttcagagca gtgactacct tcaataaaac atggacagac ggtttgctta 60300 cctgagactg cagagcagtt tccaaaaatt ttagacaaag ggtaggatga agaaggctgc 60360 ggggttttgc acacacttaa ggtgcgtaag taaataaact gagctacact gacaggatgc 60420 tcgttctagt agccaaccaa agagcagttg aaccaaagca cctagacttc aaacatcgtg 60480 gggagataat cttaggagtg ctatgcttct gcgtcctaca agtattatga aactgtctag 60540 aaagcacccc actggtaatc cctttttgat tatttttttt ataaattcta gtcttggggt 60600 tttgagtggc acacagacat aatggttagg cttcggtgtg tgctcattca ctttgcttcc 60660 tggggaccag agtttgcgat gagtcatgtt ccatctgatt tctgtcggat ccggctgcag 60720 agccatgact cagatgggct tcaggcccag ctgctcagtt catcttctgg ggaatagatg 60780 acaaggacgg gacaaatgtc ctgacgcaca tttccttctg ttcttgcact tccagggtct 60840 aacgagagca tcattaccaa cagcaggcag atacgccttg ccacaggcat cttccctgtt 60900 gtcagcctcc tgaaccactc ctgcaggccc aacaccagtg tgtccttcac tggcactgtc 60960 gccaccgtcc gggcagcaca gaggatcgca aaaggacagg agattctgca ctgctatggt 61020 gagccagcct ttctttccac taccctgctg tgcctcacac ctcacatgaa aaggataagg 61080 ggacaggaat cagcagatat gggcccagtg cctctactca tcctctgagt ctttcctgga 61140 aagggcaatg catccttggg ccaataaaaa aggtcttctg gctgtaataa aaaagcccgt 61200 tgagggcagt gagccatatc cctccatgcc ttgtagacag cctatcctga aaatgagcga 61260 ggagcacttt cttggcttct ttcttcctgc cccagcagct tggaaacgta tccactttca 61320 cccgtgtttt gttgtttttt ctgagatgat agggcagagt acccaacctc atataggcta 61380 ggctagtgtc tatcactgag ccaggacccc aacccagcac caccatgcca gtcacgtgat 61440 gactaggcca gcccctcggt agagtaggca ttgactctct tggtgtgact aggaactgtg 61500 ggtaatctct ctccagggcc tcacgagagc cggatgggcg ttgctgagag gcagcagagg 61560 ctgagttctc agtacttctt tgactgccgc tgtggggcct gtcacgctga gacactgaga 61620 gcagctgcag ctcccagatg ggaagccttc tgttgtaaga cttgcagagc gctcatgcag 61680 gtaaatctct gctgttccca ggggcagggc tccagctaaa ggttgtcagt cgccaggaga 61740 accattcctg cttcccttct tgtaactcct ccctacatgt cgcccggtcc tgcagaaaac 61800 acaggttgta tttcctaata ttttccctat aagtgacaca aaatcttaaa ttacacaaag 61860 ggaccaaaaa aaaaaaaaaa aaaaaagccc tagaaattta cttgctcaaa taagtcatca 61920 aaagttgtgc atcaggccta gcacttgggt actggtaacc ctagcactca ggaggctgag 61980 gaagaaggat ctcaagtcgg aggccagtct caagtgacac cccatctaag agatcaccat 62040 tccaaggagc tatttcagag atggtttaat ctggggaccc agattgtgga ttttctgtct 62100 gttcaattcc atctctctgt gctggcctca tcagacacac tctgtagtaa ctgtgggaaa 62160 atccgaccca catagttttc cctcagcctt tgacccagag ggaagagcca cagtggagag 62220 catgagagca gacccttggg tgctactgcc aggtaatggt gtagacactg gagtcttcaa 62280 cattcatgcc ccaatgcaaa atggtctcca caccagagca tggcattctc attagaaata 62340 agtaaatgga attggctgtg ttgaaaattg taaagccaag ggtcaagaat gaagccttcc 62400 ccagcatgtt ttgttttgtt ttgtgtttta ggcagcgtct ctctgtgtag ccttggctcc 62460 tgccctctgc tacctctccc aggtgtgcca ccatgctggg cctaagcgcc ctgtgcatta 62520 gtgctccctc gatcctgctc actcttgaga cagtcttcct tctactctgt atccccagat 62580 aacctagagt tcacttcaga gcccaggctg gcctcaaact tgagatcctc gtgtcccagc 62640 ttctcaaatg cagtgatatt tacaggccta cacctggctt tccctgatag attcctagta 62700 agatgattat cctttgagcc atatctctct tctgcttctt cctctcttcc tgcagggttg 62760 atctagaatt tattctaaag ctgactggcc tcagaattgc catccttctg cctttagnnn 62820 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 62880 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnaca tagtgtcaac tttcaaattc 62940 tgccttaaga gttctttgtt tatgggaatt tatgggaatg ttccacagaa cccatccagc 63000 ggagttctgg ctgttgtttt ttaatcttta ttcatcttgc gtgtgtgtgt gtgtgtgtgt 63060 gtgtgtatgc gcgcgtgctc aacttgcaaa attgcaaaat tcagtctcct ctttccaccc 63120 tgtaggtcct ggggatcaga ctctgttagg cttggtggta ggtgctttac tgagccatct 63180 tacaggcccc ccatggacaa ctttttcttt gaaaacctgt ttctggcttg ggtgtgatag 63240 ctcacacctg tgaccctacc accactcatg aggaagaggt aggaggacta acagaattgg 63300 aagccagcct ggactacaca gtgagtaaag gctatctata tactcaccac atggcaagac 63360 cccgttttaa aacactgggc aaggtgaaac aaaagtcaat taatttcaca taaagtcaat 63420 agcttcatta acggcctagt tatctttaaa actgtatgca ggttagtact tggtttcaat 63480 tttattactt tttctctgga acatttaaaa gtactttagg ggctggagag tcagttaaga 63540 acagtggctg ctgccaaagg actggagttc actcccaagc acccaggtgg caatcacaac 63600 tgtctgtcat ctaattctag gggatctgac accctcacag actcacaggc agtggaacac 63660 caatgtacat aaaataataa ttaaaaaaat gaaataaaat accaggcaag gtggcacacg 63720 cctttaaccc cagcactcag gaggcagagg caggcagatt tctgaattcg aaggcagcct 63780 ggtctacaga gtgagttcca ggacagccag ggctatacag agaaaccctg tctcaaaaaa 63840 aaaaaaaaaa aaggacttta aattgggctg gagagatgga ttaaaagcat tggctgctct 63900 tcccagaggt cctgggttca attcccagca ctcaaatggt ggctcacaac tgtctatatc 63960 aacgcaatct aacaccctct tcaggcatgc aggttacatg tagacaaaac atccatatgc 64020 ataaaataca taagtaaatg agtcttttaa tgtatactag aagctgggtg gtggtgcatg 64080 cctttaatcc cagcacttgg gaggcagagg caggtggatc tctgagttgg aggccagcct 64140 ggtctgagta aatagagcct tgtacttcta cttatcacta cagttacatt ttataacttt 64200 gggccctagt gcttccattt tccactgttt gcttaaccac tggggcctga agcttttgtg 64260 ctgacacttt tgttcgctaa tcatcaggca accaatggtc tctacactcc atcaccatca 64320 acacaaacaa aacaaaacac aacactacgg atcctggcat ggtggaacat ctttagcccc 64380 agtacgtggg cttgagttca aggccggcct ggtctacata gcaagttcta ggatagtagg 64440 gatagtcttt aaaacaaaac actattttat ttatgaacaa aacatgtaaa gaaagaaaaa 64500 aaactgcaaa tttatctatg aatgaagtct aagtaatact tcaatattgg aaatagcttt 64560 ctaaaatatt tttatttaaa gaaaactcag caaattattc aaacaacctt ataaacgttc 64620 gttataaaag taaagaatta tttgcaattg ccttaagggt ccaaggtggc agcctcttaa 64680 aattcagaac aatccaagct tcacattcca gttcaacatt tctacagccc taacgtattc 64740 aaatacctcc attctgacaa ctgtttcccc tcttcttttc ttctaagctg cttagatgtc 64800 tgtcccaggc ttttcatgat tttagtcatt cacacaacta gcaaacatta tctagggact 64860 aaaacttgcc agatactggg atatcaccct aaagggggac tgaaagtagc tgcaggctac 64920 agtctctaca atctcctgaa tgaaatacaa agtagctaat atttaccaaa taaacatgta 64980 cacctgtgat gattgctagc tgtactagca gaagctaaac actaaatcta gaaactcagt 65040 cctccaacta gccccttgct cggcttcagc ctcattttta caaacaaggg aaagagtttg 65100 gaatgttgcc caaagccata cataagtgaa caaaaaggag ttggagtctc caaatgcatg 65160 gatttgggct agttactttg ccaaccaact cagtaacaac tgagctgaac aggaacactg 65220 tggtagcaaa agaaactgga actatcaatg gcctctagag caaaaatata tttaaaaaga 65280 aaaaaacaaa caaggcctgg caaggagact gtgagaagag tgtgctgact gaaattgact 65340 agttcagcca acaaaagact attccagggc tggtgagatg gctcagtggg taagagcacc 65400 cgactgctct tccgaaggtc aggagttcaa atcccagcaa ccacatggtg gctcacaacc 65460 atccgtaaca agatctgact ccctcttctg gagtgtatga agacagctac agtgtactta 65520 catataatca ataaataaat ctttaaaaaa aaaaaagact attccagtgg ggatggaaaa 65580 gttaagtgtg gagttaaaat atacttcaac tggtgatgga ctaggtgtcc agagtcgggc 65640 aaaaggatgc tctgtggtag aggtgcctgc tgtgtaagcc cagctacctg agctcaatcc 65700 acagaatcca cagcggagtg ggaagagaaa caacgtccca gagttgtcct ctggcatccg 65760 acgcacattc gccatcccca agatgtcata catatgtgta catactacac actggcgcac 65820 gcgcacacac actctttttt aaaattcaga cttagaggga cataaaggat ttgctctgat 65880 atatgttcaa ttgaaaatga ctttgaagat agagggcaga tcgaaggaag ctcagcagga 65940 aagaattaat aacatgcagg tgaagggcta taaactagtc tgcagagggc cttggctcga 66000 caaaaaaatc tatggggttt gccggtaaaa taaggaaaaa gttgtcaaca tgaaacacag 66060 aacactagca agagaggagt gttagcagaa agaagccaac aagctcaaac aattaggtcg 66120 gctgaaaaat tttaaaatgt cttctgattt ggctactggg aagccactgg tgacttcggt 66180 cagcgttttc tctctcgtga ccagagagat gtctagtagc aataatgagt taggaggatg 66240 taaaagaagt aaaacagccg aaaacaagtc caaaaagttt ggggtgatgg agaaagggag 66300 gaaacagagg ccgccgaaga tagacagcgg catgtttatt tgtcttgttt tcttagatgt 66360 aaacaaacta aaaaaactcg tgagttcttc tgccagtacc gggttgcctc cagcatcctc 66420 tgatggtctt agagaccccg ggatgctccc ccgcggccgt ataatttcct ccctgacgct 66480 ctcccgatcg acagcggctc cctccccggg tcctctttgc accgctccaa ggccgcgctg 66540 ctagggccat cgagcccgct cagggtcgtc tccttacctc gatggccccc tcgctcaggt 66600 gtcccaccat ggctgcaccg ctaactcccg cgctcgcgct cttgcaccgc ctgagcttct 66660 ctgccggggt cccgcgggct gctcaacgat tggctagagc aactgtgcgt gccgatccgc 66720 ccccagcgtg agcgcggtgc gaggggcggg cctagacgcc gatagccacc gcattggcta 66780 ccgcgcggca ggcagagcac gtgactcttc cgaggccggg ttcgaggcct agtggcggga 66840 tggcgggacg tgagggcggg gcgctgggtc gcagtgcgcc tgtgtcagcg cggtgctact 66900 gagttgttcc cccgccagct gtcggaactt tgcccgccca gtcctttggc ggacagacag 66960 aatggcaacc cagggaacag tcggagctct cccctggtaa ctgctgctaa atatagtcaa 67020 agcagtgacc tgggtacttc ttcacgcagt gcgtgcccgg cgccggtgcc aggcccagag 67080 cttggcactg tgggataaac aaggtaaatc agactcagtc tccgccctct tgagttccac 67140 ctgagagttg tggccgcaag gaacccagcc tcaaggatgg tagacgcgat atgggccaca 67200 catgtggagc tccagagtgg gggtcaaaaa tcaatcaggc tttcgagagg cgatgcggtt 67260 tgaactgagt taaagtgtgt gtagaaattt gtcaggtgga ttccagtgag gatagtgatg 67320 ttcctaaaag cccaaatggc ctatgcaaaa gtattggaga gcctggcgtg ctggctggct 67380 ctgatctgtt tgtaatccca gcctttggga tgtagaagca gcaaaagttc aaggtcaccc 67440 ttaacaccgt tgagttcgag gtcaacctga actaaatgag accctgaaaa atcaaaattt 67500 gggacccagg cgtggtggca ttcgaggtaa aagcaggcag atctctgagt tcgaagccag 67560 ccaggctaac ataagatccg gtctcaaaaa aaaaaagtaa taaaaataaa aagggagaga 67620 ggctatatga actgaaagaa agacctggag atcaaaacag aaaactgagc cgtctaagaa 67680 atgaaaatat ttaacttcat agttgctgga gtaagaagtc tggaaaactt tgggcaacta 67740 aggtaaacag gtctagaaag actggaatag tagccatcta ctggtatttt gatctctgtt 67800 tgtacaacca caacctacta tagtttctca aacagttcca aagaatatgt ctgggtgaat 67860 tggtaccaca ccacagatta actctccttc agcatatcaa cagctataga aaaccccaga 67920 agaaatgatt ttggttgcgt gtcacttggt aggatgaaat ctcgattttc tagaactatg 67980 cattaataga aagctgaatc ttcatgttct gactttacag agctgcggca gcatggatct 68040 accggtggat gaatggaagt cctacctact taagaagtgg gcttcactcc cgaagtctgt 68100 gcaggacaca atttctacag cagagacttt gagcgacatc ttccttcctt cttcttccct 68160 tcttcagtaa gtgaatggaa acttcaggga aattttggtc tggaaaatgt tctgccttgt 68220 catttggtct gaatatctct tttttatagg agagagtagc tttatattct ttatagtatg 68280 gggcatttag cagttactgt tggttttcac gtttctccct agtctgtgat tactagaatg 68340 ggtaggcact aactgctttc ctcttttggc atgtgttata cttaaggaat gtagtatctt 68400 gctgtcgtcc cagtgctgtc actcatagga tctggtgcag gttgtgtagc tgcccctaga 68460 agctcattca gtcctaatgg ggagaaagaa ccctggcact tggttagttg agacccanaa 68520 cttctcaagt tctnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnntttagtt 68640 tccaagtgcc actttactgc aatgtgtcac cacatccaga gttctgtgtt tgtttatttg 68700 tctgtttttg agacagggtt tctctgtgta gccctggctg tcctggaact cactctgtag 68760 accaggctac cctcaaattc actgagatct gcctgcctct gcctccagag tgctgctgta 68820 cactaccacc acccatccag ctttctatat tggttttcct atggcgtttt aaaatagcca 68880 ttatacgtgt gtttatcatc taaagtcctg gtcccaaaag gagatgagag gggctgctaa 68940 ggtgaaaagg attacaaacg cttatcaatt ctgttcaaaa attaaacctc agagtgggat 69000 tagctgcttc tttcattaga attgctatca gaattcactc aggccttgtt tgcgtgtgtt 69060 attgaagaag tctcttcctc atcaggtcag tgactcctta gctcaagtac atgcaatatg 69120 cagtattgat aactgttctc gcttaggaat aaaaatagaa ctgacttcca cagggaaatg 69180 atgtgctgag ctgtagcaac gaatcttgca caaactctgt cagcagggac cagctagtct 69240 ctcgcctgca ggaccttcag caacaggtct gcatggccca gaagcttctc cgaaccggta 69300 aaccaggtga gattggctcc ctcgccctag gcctcagccc ttcccttgtt tattttggta 69360 tcaccttgcc ttactgagca gtcctcaata aatgactgag gacttgaatt taattatccc 69420 agcaccagcc acaagatggc tatgtaggcc agtgagacca gactgtgacc agctgttact 69480 ctggtgccct tgaaagtctt cctgatggtt taagctgtgt ctgctgcgcc agatagttct 69540 agcagctcga gcaccagaaa ggctgtctga cttccatggg ctttgtgtgg ctccagaggt 69600 ccaatgccat catctgattc ccagcttaag gacctaagct ccgagaaggt tgctctgccc 69660 tcagcagcag cagcaagtcc tgagtgctgc ctgggctcgt ggtgtgactc aggagtagag 69720 ctcggtagct agcctgagct gagagctgag agaaagaaag gactcctctc ttttcagaaa 69780 gggatttgca gaactcgatg ttagaccctg acatggtagg aatctgtttt gactattcta 69840 gcctagattc tgaagttgac ctttagccta gagtcaagaa aactaatgat tacaggagga 69900 atgtagagtt ggttgttaaa tgttggttgg aaaatggatg ttagaagccc agggtaaatg 69960 tgaggaagcc tcatctaaca cctcttttac tgaaagagaa aacataagca accaacagct 70020 tccctggaat gcccggctgt tgactccgtg agataaagag gcattttcac tttgacctaa 70080 ccgatagaga ccttgcaacg tggtctctcg tgtccaggac tagatctgta tctgttgtga 70140 ggcatttttc ctttgaatcc atagagcaag ccattcagca gttgttgcgg tgccgggagg 70200 ctgctgagag cttcttgtca gcagagcaca ccgtactggg ggaaattgaa gatggcctgg 70260 cccaggccca tgctacctta ggtatgctac cttaggtata gccggagttc tccttccctg 70320 ccgtgtgttc agtgcggccc ttgccttgtc tgtttggttc tctcttgcca tctgaattga 70380 cgctcttctc cctcccattc tgcattcctt gcccccagag ccttaggcta atggtgtttc 70440 ttttccggaa tgagacattt ctcttctcac agggaactgg ctaaagtctg ctgcccatgt 70500 acagaagagt ctccaggtgg ttgaaactcg ccatgggcca tccagtgttg aaattggcca 70560 tgagctcttc aaactggccc aagtcctatt caatgggtag gcctttcttt ttcctagtgt 70620 ttggccaggg cacacagtgc tctgtgtttt cctaggtgct tctgtgtatg gctttttgct 70680 acagtgcttt aaagcatgtt gaaactcttt tatttcctct ttaggacaga tatttgccct 70740 ctgcttcact gatagacttt aagctttgaa ttccttcctg aggatgtgga gaaagccatt 70800 aggtctgcat ggagcttccc agggaggatt tggaggcagc ctcacccgcc tctagcattc 70860 ctgtctgctt aatcacacct cccttggctg cctcagtccc tgctctctca actccagggc 70920 tcggcccttt ccctggtttg cctcttattc cttttaaagc agtggttttc aactagaagg 70980 gattgcaaat ggcatttggc agtgtttaga gacagttttg attgttatgg ctgccagcat 71040 ctagtaaagg ctaaacctac agtgcacagg accgcctcca cagtggagag acccaagtta 71100 gctatgtgaa ggctgagaat ccctgctttg gagattaaaa aaggaagctg agggaaccac 71160 tcagttggaa gcacccttgg tggcatgcac aaggccctgg ttctgtccct agctctgcac 71220 aaaaaataga atacaaggaa gagtaaccct aatgagctgg tccctcaccc agtgtgccac 71280 tgaggtcact tgaagggaag tctagcccca atttagtatt ttttgtggct gccatacctc 71340 cagccttgat caaatctcat ggtatacatt ggtaagaaaa agggtttgaa acatagacct 71400 gatactcgga catggaaaca gtatgtttgg tcagagagag cgaaggacct gatagacgag 71460 ggcaatatca gagagagggc atcagtcggg ttagacacga gcattccaca gtgagcagct 71520 ctggataagc ttttataaat gctggttaag gttttgaatt tgcccaattt tgtcaggatc 71580 ccagagtcta tcacaaacat acacagtttt ctcaaatctg ctttgcagta tgcccgtgaa 71640 tgtctcttat ctatactttc agatggtaag accctgaggg cagaggaact cagacccttt 71700 gtgccccctg taagaccctg ggggatgcag tggacccgac tttgtgttct ctgcacagaa 71760 aggagtccac tttcgttgag actaaggaag ggaactgaca agcttccctt tctggcttca 71820 ggttggcagt gcctgaagct ctgagtgcca tctggaaggc agaaaggatc ctgttggtgc 71880 actgtggccc tgagagtgag gaggtccggg agctccggga aatgaggtcc tgcttactgg 71940 actcgtcatt cgtccctgtg gggcccttgg tgtagagcaa tcatcctcac cctcaagaag 72000 gagctctggt gatgactgag atgttctgtt ggcttggagc tctcatcaga gaggacggga 72060 ccttcccacc tgacctgagc ctagtgtctg gcacagagag cacttgaaaa cagattgaga 72120 cactcacctg ccatgctggc tgctgcttgc aagagctaac tgccctctga tggaaacccc 72180 atgcccagaa aagactaaat ccagtatcta aaggctgctt taaagggttg tcactgcagc 72240 cgggcttggt ggcacacgcc tttaatccca gcactcggga ggcaggcgga tttctgagtt 72300 caaggccagc ctggtctaca aagtgagttc taggacagcc agggctacag agaaaccctg 72360 tcttgaaaaa ccaaaaaaat aaaaaataaa aataagtaaa aaataaataa ataaataaat 72420 aaagggttgt cactgatctg caggcagctc atgctagcct aggcttttgg ctcgatttca 72480 tctcactaaa cgatgaatct gtttccctgg aacattccta tggtttctag tagtaatgaa 72540 gtgctgtgtt ccactccagt gagaacttca attcttagtc ttgtattata attgaaaaat 72600 aatatatagc aagaaatcag tatgactgct tacctcaaga gacatacaat tccacttaca 72660 atatcctgct tccttaaatt tttcattaag actggtgata tataatttgt gaatggagaa 72720 ataaatacgt cttactgttg gcagtttctt cctgggatgg caactctgta ttggtttcct 72780 accagtgtcc taattcttac tcagtggctt tcattgagtg ttcttggcac tcactgtcca 72840 agcactgatg caaggcaacc ctgtagcatg acttcatagc acaggcctcc ttgttagcac 72900 acctgaaagc agaccactct ggctgtttca cttgcagaca gaatcttact ctgtaagcca 72960 gtctagcctc aaacaacatc ctcctgcctc agccttccaa gttctaggtt tataggaaaa 73020 ggccaccttg cccagcttga gactgcttct tactgccatg tctcttcagg ctcacacatg 73080 aagtccaggg cactccagga ggagccgtga gtctgtctgc agggcactcc agggggagcc 73140 atgagtctgt ctgcagggca ctccaggagg agccgtgagt ctgtctgcag ggcactccag 73200 gggaagccgt gagtctgtct gcagggcact ccagggggag ccatgagtct gtctgcaggg 73260 cactccaggg ggagccatga gtctgtctgc aaggcattcc aagagcagcc atgggcgtca 73320 ctcattggta gactgtgagg ctacatctcc agatgccccg agtgctgtgg ttgtgagcac 73380 tgctgctcat ggtttccaac tgagacagag ggaaggactt tgcccctttc cctaaggatg 73440 ggtagtaata gtccagacca caagggacag atagctatgg ggttttctga ctcatcctta 73500 gtacattatt gctgatgacc agtttgtttg gatgagttag tgggaaagaa gacccaagtc 73560 catacactct gctttttaga acttgctcat cctagccatg cccaaggagc agccgttgac 73620 tgtcatggca ttacagtgag gaaataaaca gtcctgaagg tgcctggcag cagcttttca 73680 agaagctggt gttaaaagac agtattcaaa catctgcgga ctgggaactg ggcagcattt 73740 gagtctcctg ctgtctgtta atttaccctg acaaggaggt gacttgaaag gtttgttttg 73800 tttggggtag agctttttca ggaaaaaagt ttagtcctac agacaactct atagttattc 73860 tagtccaaac tcatgccttg tgttttattc ctaaaagccc tgtcacactt tgtaaaatag 73920 gtgctcttcc tcaaaggata tatttaacgt tttatatatc aggccttatt ctgtgcatgg 73980 aagctttttt tagatgcttt gtaagatggc tcagtggtta agagcatgta ctgctcttct 74040 ggaagtcctg ggtttgattc tcagcagcta acaccagctg ttattccagt tcctgggatc 74100 tgatgccctc ttctggccta tgtgagcact gcatgtgcgt agtgcacaga caaatgcagg 74160 caaagcactc atacataaaa ctaaattcaa aaaactcttt cattgtctca tgtgacctag 74220 cttgagaata cctgtgctta tattataatc tagtatgagc cagccacggt agcaacacac 74280 ctattatctc agcactcaga agattgagac tagatggtca agagctagag tctgggttac 74340 aaaacacctg tctcaaaagt aaaagggctg aaaaagtgtc tcagcagcta agagcacaca 74400 ctgcttctcc agagggcctc atttcagttc ctaataccca caccgagtga ctcaaccacc 74460 tgtaactcca ggtccatgag atccaacacc tctggtcgtc tgcataagct cctacactca 74520 attatacaga gagagagaga gagagagaga gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 74580 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 74640 nnnnnnnnnn ntctagagtg tttcaggttt tttttgtttt tttttttttt gagacaaggt 74700 ctctctatta tgctgcctgg aactttctat gtagaccagg ctggactcaa acttatagtg 74760 atccactact tctgcctctc agtactggta ttgaaggcat gtgtcaccac accccactac 74820 ttcaagatct tagatttcca aagaagccgt agcctagaaa aggttaataa gtactgattt 74880 aaaacagaaa gaaatcaggt acacttagag ctgtagaatg tcagcatgtg acatttgtga 74940 caagttgtca aaactttgct cttaattcta aagagagaag ctgtcaaaag acttgaactg 75000 gggctgtagc caacttggtc gagcccttgc atgaagctgt gtgtttactc cccagcactg 75060 tggggtttga attgatttga acccagtaga ttcgtatatt tgaatgttta cctcatgggg 75120 aatgacatat tacaaggtgt ggccttgttg gaggaattgt caatttgggg gtgagctttg 75180 aggtctctct gctcaagctc tgcccagggt agaaagggag cctcctcctg gctgtctaca 75240 gaggacatag tctcctggct gccttcagat caagatgtag aactcttggc tcctccagca 75300 ccaagtctgc ctgcacaatg ccatgcttcc taccatgatg ataatgaact gaacctctga 75360 aactgtaagc cagccccaat taaatgtttg tctttataag agttgccttg gtcatggtgt 75420 ctcttcataa caataaaagc ctaactaaaa cacattcctg ctgggcagtg gtggtgcacg 75480 cctttaatcc cagcacttgg gaggcagagg caggaggatt tctgagttcg aggccagcct 75540 ggtctacaga gtgagttcca gaacagccag ggctacacag agaaaccctg tctcaaaaaa 75600 aaaacaaaaa caaacaagca aacaaatgcc agcatttggg aggtagagtt aagaagattg 75660 ggagtacaaa gtcgtctcag ctagtatgtt tgaggccagc atggaccaca tgagacgttc 75720 tcaaaacgaa agaaacgaat gaatagataa acatttgagt gtccagtttt ttcctttctt 75780 tcttgctttg tttttggcgg tgctgaggat taaacccagg accttgttca tactaggcaa 75840 gcattctcca ctgaggaaca ccctggcgag tgcctagtct gtctgtctgc ctgcctgcct 75900 gcctgcctgc ctgcttgtta tgtgtatgag tggtaacctg catgtctgtc tgtataccac 75960 agacatgcct ggtatctgca gaggccagaa gaggatgttg gatcgcctgg aactgggatt 76020 acaaatggtt gtaagctgcc atgtaggtat tcagaattga acctggtgct ctgaaagagc 76080 agccagtgct cttgttgttg gttttattgg gggcaggagg tagttatttg gttggttggt 76140 tggttggttg gttggttggt tttcttgaga cagggtttct ctatgtagcc ttggctgtcc 76200 tggaacttgc tctgtaggct caaactcaga gatctgcctg cccctgcctc ccgagtgctg 76260 ggataaagtc atgtgccacc aactccagac aagcagccag tactcttaac cactgagcca 76320 tcattccagc ccttctttgt gttttgagat ggtcacaaag tacaactcag actgagctct 76380 tgatcaccct ccctcagcct cctgactgct gggggttaca ggtgtgtcac tgtcctcaat 76440 tctgagtgtc agatcttgaa aacccattct cgtgaccttg atccttaaaa caaaccctgg 76500 gagaatgagt tctgataact atttctcact cctcttcaag aaaaggaaag ccagagaaag 76560 gggggggggc aagccccaga aacattgata acttgcccaa agttacacag caaaattcag 76620 acagcctgca catctcagtg gccatctgtg ccatatccac cctgcccttc tctgacctcc 76680 ccacctccat ccctacagac cttgcagttg agatcagagt ccaagccgta tcgtaagatg 76740 gccttaggat ctgacatcat ggggactctc acggtcctgt cctcgtccaa atgaaaatcc 76800 tggagggtcg tctttctcga gtcaaacttg gttacccact gccctggaag gaaacagatg 76860 gagcatcctg agccactgtc cccagaaagg ccacaggtcc acgctgtgcg tccactggcc 76920 aaagcaacct gagctgtcag cagcaagaac acaggagccg ctgggtccca gcatgtgtgg 76980 cacagaccat aggctccatg caccacgggt tctggctatc ctcctgtagt aaactcagaa 77040 ataagtgggt gttctctctc tgacttggat caccacgctc cttctgttta aagtggcctt 77100 taatatgctg gtgtgtggta cgtgcctgct ctcctgtccc ctggggactt ggagtaggaa 77160 gccccagggc tttcctctaa gttaggatcc actcttgcta ctactccata agatggtcac 77220 aaagcaacgt aaaatggaaa ttaatcaaac cattcctgcc acaagaataa aacagatctc 77280 aggggaggcc tgtggaaggg tctcctgagg ccttaccact gtctagaagg aagttgacag 77340 cagttcttga gcaggggtgc gactccagga gttgggggct gctctgagag caggacagca 77400 tgtattgtag agtgtctggg agggagctgt gttatcctta ccgtgaagat gaggacacgg 77460 gctcatgggg gcagagccag gattaaacct ggtctgattc aaaaagccag agatctgtgc 77520 ccagccccac gcagccattt cactggtcaa ctaattcaga aacacttggt ctgatatgct 77580 catatgctac aagcactgtg gccttcagat ctccctctgg cctggtacct gcattcaggt 77640 tcaccaccat caccacacac acacacacac acacacacac acactcggcc agagacaagt 77700 ggggaagccc tcacccttga agtaagccac gccaaggaga aggatgctga gggcactggg 77760 catttccctc gtggaccggg caatcttccc tttcatctgg gcctgcaccc agttgttaat 77820 ctcctgaagg tctactcgag ggttgcccgt gaggatccgg ggcctggtcc cataggactt 77880 ctccagaggg gcaacaaagc tggatttgac tcgaagttct tgagaggaaa cagatcaaag 77940 atgagagctg aatcagcacc ctcactttga aagcatgcca gaccccagct tcctgctcag 78000 catcttcctt tgacttgctg gggcatctgc cggcttgccc agaccctggc tagggaacag 78060 tggattccac cgtttgcatt ccccgtccca ggccctcctg ctgtctccca gagcccactt 78120 cctctttctg ttcctctgtg gtctcactgg ctctttcctg cccaccagtg ccaggcctcg 78180 cctgagcaca cacagcctat tgtttagaca tcatggaagc atacagacaa cccaggccaa 78240 tgaagcaact tcacgccagg cataatgggg cgtgcctgcc cttcagaagc agaggcagct 78300 ttatgagttg ggggaccagc tgagactcta tagactttga gaggtggggt gggggtgggg 78360 ctactgactc ctctcaaaca caattctgga agcactcttg aggttcttct caggggcagt 78420 aacagaggca aggagctcct tgtaggtgct gtggatgtca gggttggtga tcaggtcgta 78480 gtagagagcc cggtgaatga cagactctgt tcgatgttca gctcctgcca gagagaaaag 78540 gatgccaagc ttcataactg cccgtgaggc ccgcatcagg atagggacgt tagacatcaa 78600 tccttttgtc ctctgagagc ccgaggaggc cgatattgca gatgttttag ctggacaaga 78660 tcttcagggc gtggaaagaa ataatgaccg ccttgctagg aagagctcta agacagggca 78720 aggttatcag agctacagag agaagagtgg gatgtggtcc tgaagttctc ccatcgtaac 78780 ctaccctgtt ctgaggagga gccagctctg ctcacggcag ctgtacccct agaacctggt 78840 taaatgacta aaacacgata ggaggccact taaggaacca aggtcgagtg ccacttacaa 78900 agtggtaggg attgtgtgtg tggcccccac cgcccctttc ctgttcctct gacggcggca 78960 gcatggaaac tctgagtggg ggaaattcag gtccacctgc agccttcttc agttgacact 79020 cacccagaga aagggcagag agggccgtgg ccacgctgag tggagacagc aggacgttgc 79080 ccgttgggct ggcactggat ctcaggcggt acagatcgta gccgaagttg gagacagctg 79140 ctgccagctt gttcacaggg accttgaaga aggggtcctc ctcctccacg ggctcgcccg 79200 tgctgtccgg gactggggag ccctgggtta gaatacaagg accagtaggg aggcacagtg 79260 agtacatcac ctcctggttg ggttggtcct ctagtccctg gggccatgag tctgaggtca 79320 gaatgagtgt gtgctctctg actccacaac ctgtgtgctg ggaggtgggg agtgggaagg 79380 gcaacacaaa agggcttgcc agacctgaac tgtggtctga gaacctgaag cctggcccac 79440 tttaaaataa aacttgtagg gctggggaga tagcacagta gataaagtac cagcatgcaa 79500 gttcaaggac ctgggttcag tccccagagc tgggcacggg ggtgcatgct tataatccca 79560 acactgggga ggcagagatg ggcaggtcct ggggctcatt ggccaatcag cctgaactaa 79620 tcagcgtatt ccatctcagt gaggggtcct gtttcagagg gcctgaggaa tgactctggg 79680 ttgactacta gcctactctg tgtctgtttc tgtctgtctg tctgtctgtc tgtctgtctc 79740 cacccctctc tgtccccttc cctctgcagg gaacttcctc accaccacca acccccaaag 79800 aaacccaccc tcagaccagt cttccctatt cagcttgctg gctggtccta gtctgcctag 79860 gttctgctgt gacgccctcc ctgtctttcc tgacaagcca tcccctctga ctagacccga 79920 gaggaatttg tcgttttctg acctgttttc agtgtcagcc tcttccttat gagactttct 79980 gcttttttgt tttgttccca gggctttgga tcaaagctgg gctcttacat acgttaggca 80040 aatgcttggc cacccagctg tacctcccgt ccctgttgct tttcggtttg gaggactttt 80100 tttttagttt tctgtttggt ttttggtttt gattttttgt tgtttgtttg tttgttttga 80160 gacaggattt cgctatgtga ctctagctgt cctgggactc actatgtaga ccaggctggc 80220 cttagattca gagatccacc tgcctctgcc tcctaagtgc tgggattttt agattttaat 80280 ctgtacctac caacctcaaa ggaagtgtcc atggatagag ttcagtacta catcatgtgt 80340 gtaacatgtg tgagggcctg ggcttcaccc ccaacagaga agggggagtt agtaggtgag 80400 gaataagtga ctggctagtg gcaagacagt attgtctaag gtcactaagc cttaagccac 80460 acttaaagcc cacaatccag gtctaatatg cccatctgcc ttgtccttgt gtgacatgac 80520 cccaccccta cttcctccgt atagtggcag ctcctctgga tcctgaaagg agagggaaga 80580 tattcttgtc tcgatgttaa agtaaccaag gcctagaaga gtgaaggcca aagcccaccc 80640 tggatccagg gctgcctccc tgcactgtct cttctgctgt cccacctacc caccctactg 80700 acctcagagc tgctggggac gttctggctg ctgccgtgcc cgagcagggc tccagtccag 80760 aggagtagca ccagggcctg catcccggaa ctacaagaga aacaagagag caaacgactc 80820 ccctcaccca caccctcccc tgccactgca cattgcacac tgcacaggga caagagtcag 80880 gcagagtgag cccttcccct ccctccagct ctcagcccca agtggaccct tgacttgagg 80940 tcttccgtcc ctgacctgcc cctgcacttc tccttgagct gtgcccccat gttggttcct 81000 atcaggagac ccaccttccc atctaagctc cagcacaggg aagacccagc agcaggctct 81060 tcaggcccca agacaatgct ctagcacaaa cacacaccaa ggcttttccg tggaggcaca 81120 cggccagctc ctttggtagg atttggaacc ctgtctcagg atgggagcag agcccaggtc 81180 atagacttac agaacatctg gtctggtcct gctatccacc aatagttctc tgaccaaagc 81240 ctatgttaaa gacacacaca cactttcttc taggtaggtt cttgtgtatg tagcccaggc 81300 tagccttgaa gttgcagcta ccctacttca tttgcctcct gagtactaca atgccaggtg 81360 tgagccatca tgcctgactt gactcccttc ttctatttca agagcaattc ttagttaaga 81420 gggtatgaac cagggccaca ctgccnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 81540 nnnnntctta tttttagctt gtttgttttt cttacttgag acctggggag gggaggtgta 81600 tgtgtcttcc atttgcttct tctacctaat aaagtttctt ggtgatgttt gggggggggg 81660 gagggggtag gactcaagaa gggattctcc cataagctgt tccgtttggg tagtactatg 81720 taaggaagtt acaggtgggc agagctcggc tctgcctgac tggcgtgctc tgaggtaaag 81780 gtgagatggt gcaagatttg ggccctcagg agttggctct gttggccctg taccttctgg 81840 tctgtgggta aggatgacca gtaggtgaga gatgagggaa ccagaacaga aggtgaaagt 81900 tagtggggcg gagccccaga ctagtcaggt gggggtaaac tagatgactt tctggaaccc 81960 caaggggctc ggagactagt ggtgttggag aagacctcta atgtgttgta aggcctctga 82020 actcagtagc cgaacttgat gccagaaagc cccaaactgc taaacccaag caggagcggg 82080 acgccatccg ttccatggct tcacccgagg tggccccatg gctgcgccaa tcaatgagca 82140 gccgagagat aggggcgtgg acaagccagg aaaagttaca gcacgctgga aagataatac 82200 aggccaggaa gccccaggca cagcagggtg gaaaagctag atcccgattc tgccggaggg 82260 gggccccttc gaggtcccgg gcaccgggtg ccaggatcag agaaactgac tgaaacctag 82320 ctgacctgcc cagaccatgg catcctgggg actccttgtg gctggcgctt ccttcacggc 82380 gtttcgggga ctgcactggg ggctgcagct gctgcccacc ccgaaatctg ttcgggaccg 82440 ctggatgtgg cggaacattt tcgtttcgct gatacacagc ctactctctg gagtaggggc 82500 gctggtcggg tgcggaactt ggggactgac aaagcactga ggggcggggg tggaaaagag 82560 ggcctggaag actgaagttg gaaccttttg gaatggaact ggtttgggtt gtggatgggt 82620 gggagtaccc agtgggagaa tggatctagg tctgggagaa attgacctta gctctttgtc 82680 ttctccaggc tgtggcagtt tcctcaaatg gtcaccgacc caattaatga tcacccaccg 82740 tgggcacggg tcctagtagc agtgtcagtg ggtgagtgta cagaaaaggc tgaatcggga 82800 aaggccttgt tggaccggga attctaggtt cctcccccat ctttggaatg gagcagatgt 82860 tgctggaggt ttgctgtgag gaattaagga cctgagaaaa gtgggacttg agatatctag 82920 gctgtgcatc agctctgagc gaggagcctc atagtcttct ccggtgcctt caggttattt 82980 cgctgcagat ggagttgata tgctgtggaa ccagacattg gcccaggcct gggaccttct 83040 ctgtcaccat ttggcggtaa gactctgaag ggagaggcca ggtagtaagg gagcatgtcc 83100 aactcaaggg cccaacctct ctcttcagtg ttctgtcctc tgacttttcc acaaagcccc 83160 ctgaaaacct atcctctcag acttggattg agttggaggg aggttttgac tggctagcca 83220 ctcctgggca ctgcccaagg agtttggttc tccccacaaa cctccagctg atcataaaaa 83280 aaaaaaaaaa aaagccagga atgaaagcta gggtatgcta tgcaaatagt gtggcttggg 83340 gtaagagaac ctctggtcca gggctgctca tgccccctag ataagggtca gcagaaaggt 83400 caggattgga ggcagtccta aaaaatgctt gggtaatata aagtgaataa ataaaaaata 83460 aataaatact aatttttaaa aagctgatac ctggaaggat gaggcagaga gtagaaaaaa 83520 catgcgtggg tgtccctagg ataaggagct gggacttgtt gggcacaggt catgcaaagc 83580 ctgaaccttg aaccttgcct gcaggtagtg agctgcctca gcaccgctgt tgtgtctggc 83640 cactatgtgg gcttctctat ggtatccctg cttctggagc tgaactccat ctgtttgcat 83700 ctacggaagc tactgctgct ctcccataag gccccatcct tggccttcag agtaagcagt 83760 tgggccagcc tggccaccct ggtcctcttc cgccttctgc ctctgggatg gatgagtctg 83820 tggttgtccc ggcagcacta ccagctgtct cttgctctgg ttctgctttg tgtggctggg 83880 ctggtcaccg tgggcagcat aagcatctcc acagggatcc gaattctgac caaggatatc 83940 ttgcagtctc agccctaccc gtttatcctc atgcacaagg aaaccaagac acgtgagcct 84000 gttgccagga acacttccac tctcagtctg aaaggtgtgg aagttttctc ttctgtcagc 84060 ccccagggag gtggggctgg gaagaggaga tggtagccca ctgcatagtc tactatgtag 84120 caaggactag actgtatcat cagagagaga gagagagaga gagagagaga gagagagaga 84180 gagagagaga gaacattgta tgagatctcc attacagtca ggaaatcagg agatctaaat 84240 aactttaaaa gtcccacagt ctttacatat tcttaaaatt tcaatctctt taaaatatcc 84300 atctctttta aaattcaaag tctttttaca attaaaagtc tcaactgtgg gctccactaa 84360 aacagtttct tccttcaaga gggaaaatat cagggcacag tcacaatcaa aagcaaaagt 84420 caatctccaa ccgtccaatg tctgggatac aactcacgat cttctgggct cctccaaggg 84480 cttgggtcac ttctccagcc aggccctttg tagcacacgc gtcatcctct aggctccaga 84540 tacctgtact ccactgctgc tgctgctctt ggtggtcatc tcatggtact ggcatctcca 84600 aaacgctgca tgaccccttc agtcctgggc cttcaagaga gaagactaga gcctggcaaa 84660 gtggcacatg ctgataatgc tagcacttgg gaatgacaag cagaaggatc agaagttcaa 84720 ggccagcctg ggctacaaga gactctgttt caacaaacaa acaaacaaac aaaccaaaga 84780 agagaaagaa aaaactggac atgacagccg gaacattatc tgacattcat aaggtcctga 84840 gttcaatgcc aagttggcag tgcctagttt gataagggtc tagccactct ggtaatacca 84900 tggctgactg aacaccttac ccagcaactt gctgatagac tctgccttcc agcaaaaggg 84960 aggagcttcg ctgaggagag aacattgaac cctattgtat atgaataaat tgctgtgcaa 85020 atgatttcat cagtctcttg tgaatgtgat tgctttgagt catttttctt ggctccagtg 85080 ttatcctggt ctgcagtgtg gtgtggagtt gtggaagctt tgagttggga gggtttcctg 85140 ttaaggtttc tctggctctt ttctttcctc ccggtttttg ttttgtttgc ctggtggggt 85200 tctctggtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt tagaagttgg 85260 cggggggtgg agggggctgg agagatggct cagcggttaa gagcgccaac tgctcttcca 85320 aaggtcctga gttcaaatcc caacaaccac atggtggctc acaaccatcc gtaacaaaaa 85380 aatctgatgc cctcttctgg agtgtctgaa aacagctaca gtgtgcttac atataataaa 85440 taaataaata ttaaaaaaaa aagaagttgg catggatgat gtagtgaaga ctggcattag 85500 atatctctgg atccccctgc ctctacctct tagacactgt gagtatggaa gtgtaccacc 85560 gcaccaggcc aggctagaac attctctgat ctacaaatac ctagagtatt attcctctat 85620 gatcagaaaa cagacccagg gggccacaga aatgtcttag taggtaaaaa cacttgcttt 85680 caggcctgat aacctgcggt tttttgtttg ttctgggggg cgggagaggc tggctggctg 85740 gctggcctgg aattcacaga gatccacctg cctctgcctc ctgagtgtca ggtaccagga 85800 tcacaggtgt gtgccaccac acttggccta actgcctgag tttgagcatc agtactcaca 85860 tggtactgag gatagaatag actctcacca gctcttctga cttccacatg tgccctgcag 85920 catgggctct ccttccccaa aggaaaaata aatgtaagaa ttaaaaaaaa aaaaaaaaag 85980 caaacccagg tcttgtgtga tggctcagca tcaaagctac ctcccgccac agctgaccac 86040 ctggtgataa cttatagcct tgttatgctc tcctttgacc tccacgggca tgctgtacac 86100 gtatgtgtgc ccacacaaac acacaatcaa gaaataaatg cagccaggcg aggtggcaca 86160 cccctttaat cccagcactt gggaggcaga ggcaggtgga attctgagtt cgaggccaac 86220 ctggtctaca aagtgagttc caggacagcc agagctacac agagaaaccc tgtttcgaaa 86280 taaccaaaaa aaaaccactt taaatattat ttttattttg ttttgtttat cctggaactt 86340 ggtctgcaga ccaggctggc cttgaactca cagagatcca actgcttctg cttcccaagc 86400 acattaaagg atgtcccacc actgcctggc taaagattta ttttttcttt ctttttgttt 86460 tgttttgttt tgttttttct aaaaaatttt tttaaaaaga accatccctc ctagcactca 86520 ggagactctg aagtcagggc cagccaggtc tactgagtga gctctagggc agccagggct 86580 ccacaaagaa accctctctc aacaaacaaa caaaagagaa cagacccaac cagacctgag 86640 gacacacact tgtaatctaa gcccttgaga ggctgagaag ttcaaggcta gccacaagtg 86700 tgtggtgcat tcaagagcag cctgggtggg ctacagaaaa agaaagaggg agagagagaa 86760 tggttaatga agatgactct ggaaaagtga aactcaagag aaagcccctc agatttgctt 86820 aagacgagtt gagggtggag aaccgccaaa gcggacgagc cagacagaga ctgccaacaa 86880 agttcaatcg gttcaggtac attacttcca aaacgccatt gccacatcag gatgcttcaa 86940 tcagccaaac caacgcagcg actattgact tctgcatttc agagacttcc gtctctgtcc 87000 agggcaatgt cactttagct ttcctttgca gaaaggaaaa gtccctgcct ctgatgtggt 87060 agatcctcac acaccttctg ccagatccag acactggtat gactcagcct cggggagctc 87120 tatctacaga gataagggta caaggcgtgt gtgtttaaag tatgtgttta aaagtacaaa 87180 gtgagagtcc ctggaaaggg ctccctgccc tcaccatcac cgaaagcaca aaccttaggg 87240 taatatctga cattcctgga aatgtatgta tgtattcatt atgtagccct gactgtcctg 87300 gaatggggta taaaccagga tggcttcaca tctcagagac ccatttgcct ctgcctccca 87360 agaactaaga ttagaggcat gcactaccat acttggctca tgatttactt aactttattt 87420 tatgttcacg aatgttagcc tgcatgtatg tgtgtgcacc atgtgcatgc ctggtgcccc 87480 agaggccaga agaaggtgtt ggttggattt cctggagatg aagtcccaaa caactgtaag 87540 cagtccaatg tgtgtgctgg agatgaaact tggttcatcc acaagagcag tatgtgctct 87600 taactgtgga ggcatatctc cagcctcaga tttcccagtt aatgtttgct ttcgcaccca 87660 ggcccatctg cgcatgcgct ggagacctcc tttaccgcct tgagcctcat tggccaattg 87720 tggctgggag acttgcagat cccaagtggt acaagagaag aataaactgg tgtgctatga 87780 actcacctct tctctgtagc cattggctga gcatactttg cctcaaccta ccgcccttcc 87840 ttcccctaat cctaaatctt tgccctctcc aaatgtgctc ctcccccgca gtaatccagt 87900 ggtcgctggg gctctagaga gatggggggg gggggagcaa cgggtacagc ttaaggcagc 87960 tgcagcagaa cttttttgct gtatattgag tcttaaaaat tcatataaac tttgtgttct 88020 gtttctaaat ataaccccat ctgtttcaac acaaaatgca acaacaaaat gtttcaaatt 88080 gctatttgga ataattaaaa aatttcaata cttgatttaa aaatgcttta actttttaaa 88140 taaattttaa atgttattat ttttaaaaag ttacaagttt aaaaaaaaga aagatagaaa 88200 tcacataatg aaattaacca tacgcaagtg aggctcggtg cactggtaca cagttacagt 88260 agccatttgg agtggaggcc atggcgcttc acattgaatt ttatactttc tttatagaat 88320 attttttatg cacctatcta ctactgataa caaaacaccc atgagagagt tagaattaga 88380 catcaattag ctttgatcct ctgtcataac tcgtgtccac tccctgcctt agtcctacct 88440 catccctgtc ctcttttcta catcttatac tgaatccaca cactcagttg tttacacaaa 88500 cacatacatc actgtccann nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 88560 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnag 88620 tgcctggctc tgttttgctg ttgttgtttt tgtttgtgtg tgtgtttaga tttggtttgg 88680 tttggtttga ttttgttttt ttttagagaa tcttactatg tagctcaggc tgtccttgaa 88740 ctcacagaga tctcttgtct ctgccttcca agtgctgaga ttaaaggtat acaccacctt 88800 acctggcccc tttcatctat ctatctatct atctatctat ctatctatct atctatcatc 88860 tatctatcta tctaaaattt atctgtgtgt gtctgtgtgt acattcccca gagcctgtgt 88920 ggtagtcaat aagtaaccct cagaagttgg ctttctctaa tccttggatc aaacttgaat 88980 tgttaggctt ggtagcaagc atgtttaccc actgagccat ttatgacccc atggcccagc 89040 atcttccatg ggttctgggg acacaaatgt gtactttgat gtttacagga caagcgctta 89100 accaaccaag tcattttccc agccccatcc tgactcccat taagtgttct ttcccccaac 89160 ccaggaccaa atctagagga gtgtccatgc ccaacaaaca ctctgccaag cctctcccct 89220 tactgctctt ctcccttccc ttccttcatt tcttcgttcc ttcttttctt tctttttgaa 89280 acaggtcttt tctctgcatc ccaagctagc cttgaacttg tgatgtagct caggctggct 89340 ttgaactcac agctgtcctc ctacttcagc ttcccaaaca ctgggattat agacctatgc 89400 taccacacct ggctcatttt tcaaataaat aaaaagaaaa tcaaaaagtt cctagaacag 89460 tcacaggatt cacaaaaact ttggaaggag actaaaaatg gatttttaaa aaatgcttga 89520 agcacaaaga gttgttgaaa gaagagagaa gaggaaaagt tagcttagta ggtagaagtc 89580 aatcaagcct cacaccctga gttcaattcc tgaccctatg gtagaaggag aagagcaatg 89640 ccggaaacat tatcctctga cctccagacc cgctgtggca cgtgcatgca cacacacaag 89700 caggagccct tggagggaag tcctagaaat gaatcttact gaagcaggtc tgccaggccc 89760 tgtgctcagc cattttattt ttcctttgtg tacccgacac gcttccattc tcaaagttgt 89820 gagtctgaga ggaagtactc actgtgtccc cagtgagctt ctgtcttacc ctgggtcact 89880 tagatggggt cacttagtgg tagccttggt gtggagaaag agaacacagg tccgagtagc 89940 cagtagacct gagtctttat atctgcaaag ggtgttgggg cataatcaaa tctccccccc 90000 tccccggggt cctgatacca ggttgtatta agtgtatgtg catggtcctt ccaagtcttg 90060 acacatcatc caactccaag tggctttctc atttttcctt gccagtagcc tcttggtgag 90120 gaaatggctg aggaaaacag agttgcagaa agacagggcc atggcctggc tgcaggcttt 90180 ctctgagtct gaagagggtc agcgactctg agaaatgaag ctatttctga gtgagagggg 90240 ccaaagaagg aacacggcag agggagagcc cccgaggaga tggagacaga agccggagag 90300 ggaccctgtg cgaggctgga ggggaggaag aggggggagg agtgagaccc actgtcatct 90360 gttgggcaga gaggggctac attcatctgc agtatggtgt agaggggaca gagagtgatg 90420 gtaacaggaa aaatttgggg ttgagggggg cagcctgtag ggctgggccc cagcagtgta 90480 cagctaggta gagtacacag taactcccag aattctctgg ctccactaaa tccctgttcc 90540 gctccgtgca gagtaaaacc cacacagggt ggatttcagt ctcctttgca cccccctcca 90600 ccccccctcc acccccagct ctggtcacag ccagtcagag ttgggggtgg ggcagatctt 90660 gtaaaagagg ctggtgagga catcggaaag tctgtaccct ccactagcaa agtgccagac 90720 gctccgtgac actttaaatg cctcagataa aacagtgaga gactctcctg gtggcaggca 90780 agatgatggg tcagggacct cagcgcctct gaggctcaga caccaggata aagaataaaa 90840 acaccacgga gacccctgtg accccctcgt gcagagggag aatgccaatg tggcccagct 90900 agctgctgga gcgcaagcct caaggcctgt gctagttatg agtctactgc tgctgccttg 90960 tcccaataaa cccctttccg cccaggatta gtggacacgc cttgctcaag ccgagtccct 91020 gatctgccac cttatcacac acatacaaaa atcccttgag gatggttacc atcctgggac 91080 aaagcctcat tctctgctta acccaagtga cacctatatg gcagatccct gtgtccttct 91140 cctgatgata acaacccttg caatccaata gaggggaact cgggtttctg tcagcttcct 91200 ttatgctgat agaaatgtac tctgcatgtg gggagcctgc cttgctcacc ctgagacccc 91260 atggggctgg ctggggcttt gcacatcatt gggactcaga gatgttgact acatgaacgt 91320 cccacacttg gttgcacaag gcagaatgac aggatgttat gcctggtgtg tgagtgtgtg 91380 tgtgtctctg tgtgtgtaaa acgcctctct ctggagccct cctgtctgtc tgcctcttgt 91440 tcaatggctg cacaattgtc ctttctcttt ccaaggacct ctgtatgggt gtgtccttca 91500 ttcagtgcct ttcctctgtg ggtttgtcct gctagccccc tgtcactgag aaagtcttct 91560 gtctgtcctt gggttgtctg gctagaacac agacatcatt gtcttttttt tttttttttt 91620 ttttttttaa agatttattt atatgtaagt acactgtagc tgtcttcaga cactccagaa 91680 gagggagtca gatctcgtta ggatggttgt gagccaccat gtggttgctg ggatttgaac 91740 tccagacctt cggaagagca gtcgggtgct cttactcact gagccatctc accagccccg 91800 acatcattgt cttgcccacg actgctctcc agaatggtgg gcaggaggat gtgacccccc 91860 acccccaggc accgggacac aacatcttct acacgtgtag gtcttgtgca ctggctttgc 91920 tttcttcttc caagcaggtc tcccaggaaa tggcacttac agagattgaa gagtttaata 91980 catgtctcgc tgcctctctt ttcgggaacc ccccagaggg agcagcagaa accagggctg 92040 gcaggggctc taagctgcct gggcaaagga gcagggggta gcatggagcc ttagccaatt 92100 tggaaagcac tgtgacccaa gcacattttg cagcagtaat gtcaaattct gccgttcagg 92160 catgccattg atgtgcacgc tgccacacag aaaccagtga cacaaaggca cagccttctc 92220 caccctcctg gtgcttagga actaacggct ctaatgagaa atgagagctg aaaggagaga 92280 gacgggggcg ggccacagca gcgcaggctg gcactgcgtg ttggaggagg ctgacccact 92340 tctcgtagag gtaaggggcc cactgaaatg tcacttaaat tagccaccac tcccaacact 92400 agatctcctt tgtccccata cctcagcccc acgcttcttt ctttttttct tctttttctt 92460 ctcctctggg gcagcctcaa gcccagcacc cactttttag agctgtaaac caccctggtc 92520 ctagaagccc tcttacgtta ggggatgaca ggaggtagag atcaggaagg agggagggag 92580 gggaggagga aaggaaaagg gaggggagag agggaaagag atcgagagag catgcattca 92640 tcacaaagag ccctcttttc tggctttttg actgcactgt gagttattta gccaacaata 92700 gatgtttatg tattttttta gaacccgtat ttattaacag cctgaaagga gagagacgga 92760 gatttatata ggaagtgcag tgagttaagg ggggcaatta agagagcaga aagagatacg 92820 gaacacagac ttgtaaaggg ttttgtaaca tccaatcaaa ggtgcttcag gtattttcca 92880 aggaagcaga aggtaaaaaa aaaaaaaaat tgtcccatta gaagctgaca ctggatggag 92940 caatggccca ggcggaactc ctgcttgaaa gaaggtgaga agggagggac acagaccagg 93000 atccgatgag ccagagtgtg gccatagctg ggtcatgagg cccagggttg gaaggacccc 93060 actaaagtgt gcactggcct ttccttgaca aaggatgcac ctatagctag gcgtggtggc 93120 aagtggttgt tattctagta cttaggaggc tgaggcagga ggatcaccat gagtgtatgc 93180 ccagcctgga ctgcatagca acacccagtt tcaaaataac aacaaaagga agtgggggtg 93240 gggagggcaa catttggaat gtaaataaat aaaacatttt ttttaaaaaa agaaaggggc 93300 tagtgagtta gttcagcggt taagagcgct gactgctctt ccgaaggttc tgagttcaaa 93360 tcccaacaac cacatggtgg ctcacaacca tccataagga gatctacgcc ctcttctggt 93420 gtgtttaaag tcagctacaa tgtacttaca tataataata aataaattct ggagtgaggg 93480 ggccagagca agtagaggtc ctgagtttaa ttcccagcaa ccacatgatg gctcacaacc 93540 atctgtacaa ttacagtgca ctcatataca taaaataaat aaataaatct ttaaaaaaag 93600 aagaaagagg gtggggcagg ggagggaaga agaagaaagg taagaagcta aataaaaggc 93660 acagagatga gcttcatgtg gaaacacagg cctgtagtcc tggcactcag gtggggttgg 93720 ggggggctac agtgagagta tcatgagttc aaggtcaact tgggtgagac cttgtctcaa 93780 aaaatacata ngcnaaaaaa aaaaaaaaaa acatagccag gcatgatggt atacatttat 93840 agtcccagca cttagaggac tgaggcaggg cagaaagaaa aggaattcaa gatcaggctg 93900 agctgtatgc agtcctgatc ctatcccctc cccccccccc ccagagacag acagacagac 93960 agacagacag agagaaacac aaagaaaggg gccttcagat ggctcagcaa ttaaaggcgc 94020 ttgctattca gaccccatga cctgagctca aagcctggga cccaaggtag aaggcaagag 94080 ccaactccac agagctgttc tatgatctct atatgaatgc tggggcatgt gcctacacta 94140 tgttgtgcac acatgcacag attagaaaaa gaggaggaag aaaaacataa gattgtttca 94200 agaaaagaaa ggctggcttc ttccacgtca gtgtgagagg agggtctggc ccctttgtag 94260 ccaggtcctt cccagtccag tgggggctga actgaggcag cggaggaggc aataacggag 94320 ctttcccaac gcagtgtcca gcaaactcaa ctctacagcc tgtcctgatc cacagagaag 94380 ccttcctggc tccctcacca atgcgggggc attggctccc aggctcctgg gccccccccc 94440 acacctgtgg agtgctaggt gatttgctaa tgttgggcaa catttgccca cgtggggttc 94500 ttggctcttt ggtaatagac atgcctagca ggagggcgga gcttggaggg gggagtcctg 94560 gggttgcccg tggctccctg cagctggggt gtctggccag ctgaagaagg agccatggca 94620 cgcaaatggg agagcatgga acagaggctg tggatgctaa gcaatatggg aggcagtcta 94680 agcttggaag cagcaggtgt ctgggaacgg gcctgtggcc caggcagatt tccagtgagc 94740 actccagttt tttggcacaa ggaacaagct ggctgagccc aagaggcaag tggtgataat 94800 gaaacccgca gttgaggaac agcgggtaag ggtgccatgg gagcccatgt gctcatgaag 94860 aggctggggt gtgaagaaga gcccatgcag ggaagccaca catcccctcg agttccaggc 94920 agaggcagag tccctgagtg gggctccctg ggtctcccct tacctaacca gtctcccggc 94980 accccagcaa acaaaatccc atccataatt tgaggtttat agagacctca aaggctgagc 95040 tactgtgtgc cactaaccat cagcctaacc ctcccccact gtcttctcta gctgcccctc 95100 tttcttctga gactgtgata gtggcgggga cgggttggga gtgtgtgtga agccctctcc 95160 gactctccaa ccccagctga gccccttgtt ctgcagctca gtaacacagt aacacaggct 95220 cagttctaca ctggttgaga acactcacgg ctctctcagc tccttagaga gcctgttttc 95280 tcattttcct gtccccaaag cctagacaat ggctggtcca tttgtaagct tatctgagga 95340 tgccaggggc caccccatgt ctccactagg ctggcaatgt tctctgtcac tgtagtacag 95400 aagactgcct ggtgggaggt gagataagga aagggatggt ctcccctggg gttcccacac 95460 agtgctgagc ggaaaatggc agaatgggct gggaggtaac tctgttgcta gagtacttgc 95520 ctagcatgtg caaggaaggg cctgggttcc atccccagca ctacagaacc caggcgtggt 95580 ggttcatgct ggtattctca acattcagga ggtacagtca ggaagagcag aagttcaagg 95640 ccatcctcag ctacatagct agcttgagac cagcctgggc tatgtgagac tttgtctcca 95700 acaaacaaca acaaagcagc agaaggccaa ctggcaagag gagtattacg taaagtaaat 95760 ccatctcaaa aagcaagtag catgtatctt ctttcatttt tttttacatt ctataaaggc 95820 ctatcaagtc atgtatatat gcatgtatgt ttgtatgata tgaaagtagg gggctggata 95880 gatggctcag cagttgagag cacttggatg ctctttcaaa gaacctgggt tcaattccta 95940 gcacccacat ggcagctcac aactgtctgt aattccagtc tcaggggatc tggcaccctc 96000 acacagatat ccacgcacat aaacaccaat gcacataaaa taaataattt ttaaaaaaag 96060 aaattggaag taaaactctc taaggagaca aaagggactg aggggaagtg ggaggggcat 96120 gaagggggag ggcataggtg tgtggtgtgt ttaacatgca gaatacactt ctataaaagc 96180 tttggggttc attatgcaat gtatacatgt gtgggtgcaa gatgtaagct gtgcatatgt 96240 gtgggggcca aaggtctcct cctcaatccc tctctgcctt attttcattt aaattataat 96300 tattactatt agtgtgtggt gtgatgtgtg tgggtgtgtt aagccctcac ggcaatcaga 96360 ggatgtctgt ggtctgagga tgctctctta ccatgttcgt gtgggttctg tggatggaac 96420 tctggtagtc aggtttgcaa agctagtgtc tttatctgcc gagccacctt gctggccttc 96480 aaccttattt tttgcattga acatggaact tcctgagttg cctggacagc aagtccccaa 96540 gaccctcctg ttcctgcctc ccccntgtcn nnntcacang aggacacacn gcttantggg 96600 tntccggatt gctgcncacc tccccgccnc ccnagcctcc tgcctccccg cccctcgccc 96660 ccgctggncc ctcccccccc cccccccccc cccccccccc cttccccccc ccccnnnnnn 96720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 96780 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnncagaca cacacactca aattaaatat 96840 agctctaact gctgttaaat tcacactcct tcacatcccc acgctaggac tctaaggagg 96900 caccagcaag gcccaggtcc agcttgactt agagcaaagc atcctccccc ctccacacaa 96960 tggaaacgga cggaaagggg catggaagca gaaccagaca acagcagcct agccaagccc 97020 aggactctgc tccttccccc catgcctgcc gtgcaactgg ggaggcaaag ccccagccgg 97080 tgctttctga ccgcttagcg gaagacaagg ggagcctgtg attatgattt ctgctgattt 97140 gcaatgaaac actaatgcag tgggcttttc attaagccag atttattcaa tctaaagatt 97200 ttatttcctt tatgtagaaa gtgcatcttt atatgttgtt ggaggagcag agatgtgata 97260 aaaagaaatt tctcttatga actaatagca ctgatacata gtggtagcta tgcctaggcc 97320 tctctctctc tctctctctc tgtctcctgt gcatgtgtgt gtgtgtgtgt gtgtgtgtgt 97380 atgaatgcac acaaagtagc ccccccccat attatttctt ctgtgggatc tccagactca 97440 gcaaatggtg gtgactggga agtctggcca tgcaattctt gccttttctc ttgccagccc 97500 aatccctttg cattcaaacc cgggctgctt gctgtggcca gccctttcac ctggagtcct 97560 tcctcctcct tacctgtctt cccatccttt gcagacaatt atcctcaata actagccaat 97620 tacccttaag gacaattata ctcttccatc agcaaacacg ggtgttcttt ccttgagtct 97680 tttgatgaag tcgatattaa agagatgctt tatttacata aagtcaaata gctccctttt 97740 agaagggttt gggttcgatg tcaaagtttt aaaatcttaa ctagaggatg ggtgtagagg 97800 gcttttggct agggtagaaa agagatggag atacttattc tgatgttgct ttaaaaggta 97860 ggatgcccag agaaggtgga aggatggggg agggagggtc cctcctcaag ctaatgaatc 97920 taaaagcagg gatgagctgg gcgctaggag tggaaccagt cagaagtgtc tgcctttgac 97980 tgaccacagc tcctgccctc ccctccccca gtctctctgt gaaccgccag cattaggagc 98040 taatcgcttc agaaagccag attggaatgt gttgctcacc ctccactgct cagaaaacct 98100 ttattccagg caaggactga cccaaaccga tcatggcatc tgccaatcag gaggccaaag 98160 gtgccggcag ggcgggacct agctgtgcag aaacagctcc gttatggcgc gcagaaaaag 98220 ctggggggaa aggctaccgt tttatctctt ggcagatggc ttctctcttt gatgctttgg 98280 gccttacctg ttactgcctg cacttgactt gacctaggca aaaatagcag cgagatacag 98340 gttctcgaag ttagaaggaa aaaaaaaaag ccccaaacca caacacaacc cggaagtgtg 98400 cccccgctgt gtttctaaag agctgttttc ttcccaagct ctacagcgtg gtggctctaa 98460 tcggaaattt ctttttaatc atagcaggag tcccaattag cgtgttgggt aatctttcaa 98520 gtagagtggg agttccgtgg ccacagagag cagaggcaat attcagcata aagccctaga 98580 gaaagaggtg ttgtgggcct gtgcacacat gtgtgtcaac gcacatgtgg cttgtggagg 98640 ctggcttccc actctcaaga tgaggtgtgt gcaccccagg ccttttgatt ctcaaagctt 98700 tattaggacc agagggactg tgtgtgtgga ggggtgttgc tcacagtgca gaaacccaaa 98760 cctggcttct ccaggagccc acatgccaac aaacaggctg cacactcttg ctagtacatc 98820 ccctaaaggt atggggatga gggaccaagt gctttgcaag acagcaggca cagagttctg 98880 ggacgctcct gtaccccaga ctcagccgcc acccagggcc agctctgatc tggcttgacc 98940 tactttcttc tgttgttgtt tttggaagtg ctgatgtcaa tgcagaattc agcagagtgg 99000 ctgagtgaga aaaaagagga gagggaggaa aagggggggg ggacgggacg ggccgaggcc 99060 aacaggaaag ggcaggcaac aagacaatga ccacaaggtc cctgtaacta cactaactgc 99120 ttacctttcc tgacccccag ggcttagcca atatagctga gacccagtct tggtgctgtg 99180 gcttcaggct aagtaaacag ggaagagttg gacatgggtc tccattctct ctcctcatcc 99240 aacaagggga ggaggcagtg gccaggcagc catgcccacc gatgccatcc ttctgggagg 99300 agccagacat ttcaggcacc tctccttccc tgggtgccta gaggtgctgt gtctgcatcc 99360 atctgccatg cctgccatct gagagaggcc actgggactt ggtagagagg ttctccacac 99420 atgctggcct ggaggaaatt ggtctttagg gacactgaag gcagtttcct ctgttcagtg 99480 gctccttgga aacccacgtg acagagctcg catgacaact tgccggctct caactcccat 99540 tcttagctgc ctcaagcact gtaaggttta ggagagcccc agatgtaagt atggatggga 99600 agaccctcca gggagtcatt gcctaccctt ctgaactcta acatggtcca gcttttccat 99660 tccacaattg aggagacgcc agacctggca ggggagcaag cctttgtttc tgacccattt 99720 gcaaacccca gccactgagg aacttgcata caagaaactg cctctgggcc tctcctggac 99780 tgagccctgc ctcccagggg acaactgggc aacagatcct tccaggtggc tgcagtgaca 99840 gatccatgct tttatgacat agaaaggcct cagtctcagg atttcacaca ctgtatttcc 99900 ctcatcctgg ggaccaggga aggcgagcat cttctgctcc ccccaaacaa gtgtgggaat 99960 gattaaaatc attttttttt tctgctccat gaactcatac agttttcaga taccgaggag 100020 acaaagccct cctgtgctga aattagaccc cgaaaaatag gttagctgac aattacttgt 100080 ttctaagtgg agtgtgatgt agtggcagga gcgcaggatg ggctgccagg gctgcagtct 100140 cccccccccc aaacttactg tctcttaacc tctcgagtcc ctgggtttct tgccgggatg 100200 ataattctcc ccatctccct cctctggtgg gctggtggaa agcgtaatga atcaacgctt 100260 gaagcacgct gaagaggcca gactcgggat gccatgtaag tacacagcat cgccagccac 100320 ctctcaagtc tacacggagc tgatttattt acctcccgtg aaagagacaa caatcatcat 100380 atttacactt catgccgcag cttcctgcgt ggcacggcag caccccctcc ctctccgctg 100440 ctgaggactc catcaagcac gctgccttgc caggatgaca gcagcccact ctcagcctct 100500 ccctggcctc cttacagatc atgacctcct gccccgtgag gtctgtcacc cgaaaaccac 100560 ggtacaccgg gggctgcagc ctctctatgg gggaggctga ggaaatgaat tccgtaggta 100620 aaaggcttcc taggaaatca gacgctgcta gtaattaagg agcgaagcat aggtgcgtga 100680 aaggtaaatg gatgttattt aaatgttgcg tcatttaaag agtgtcctgg tgcttcagtt 100740 ccttgttacc atgcagggct gtggacgggt ggcaattagg ctggcacggg tagagctcac 100800 ctgctgagct gagggagggt ggggacacac cttccggtaa ttgctgctgg gcagctctgg 100860 gtctccccac ccccgccccc gccctcactc cccacccccc acttctttcc tgacagctct 100920 ttcatttgca gcagcttaca gggcttgttg cccttaccca gaaaatcacg ttggaagaaa 100980 tataagaaaa agaggaatga aagagaaagc cagaaaagtt catattaggt tcggatctgc 101040 ggccaaacct ggccgagaga atccatgacg gtccgcgcgc atataaccct gtggcaacag 101100 ggcccggcac aacagggccc gccacaagag cttcttgagt tgccacctgc caggagacag 101160 gatgaatgaa tggatcatct gtccttagag cacaagccag gcctgattct ccaatattga 101220 tgtgtgaggg agatgtcaac agaggttccc taaagaatga tgcttctatt tccatgctaa 101280 tcctggggcg tcagcttcag tcggaacagc cggaccgtta ccttagctct gctgttctcc 101340 tgtctgtaac ccgcagaggg aagggcgggg tcacccagca ttgccactcc ccccaccctc 101400 acgtggtcca gacccctctt gggttgatct gctcctgaaa aacagtgttg gctcaagttt 101460 gcctctgaag gtatgtcacc gctggctcag ccagcttatc tccccggtgc tttcaagatc 101520 aaaacaccca aacgaaagaa aaactttgtt tcaagagcag agtgtggtgc caactctgat 101580 caaagtgttt ttcagcatga caactcactg cccgtgacaa ccagtacttg gctgttgtgg 101640 ctcagagtga gatgcggagg gaagtggatg acaacagctg tatccaggtc caaacagagt 101700 agattcacgg ctggcagaaa atggctgaga gccttgggct gcatccctcc tcccctcctg 101760 cctctctctc ttttcaaggt ggtttttgga aatgtccttc ctgtgggttg tgtgcctttt 101820 ccatgtagga cctggggcct gtgcagatgg ccctgtgttc ctggtgctgc tgttgagatg 101880 tgaacgagtg ataggaaccc aggcactaaa cacacaatgt ggttgtatct gactagaagc 101940 aaggcaagag caggaggcat ttgagggtaa aggagtgtaa ggactgtgta aagagatgag 102000 ggttctatct gggaggcagg agtcccaatg ccagcaaata caatggactc tcctggtcga 102060 cccaaccaga gagaattcaa gatggcagag ggacaggctg tctgagtttc ctatggctgc 102120 accgataaat ggtcataagc agagtagagg aaaaccacag acagaaattc atgccattga 102180 gactagaaat ctagctcaag gttgtgtgtg gcagggttgg ttcctgggtg ttcaaccttt 102240 tcacactgtg acatgatgct gtggtctgca gatgtgttgg gctgcatcca tagctaccct 102300 gggacacatt catggaccgc aggttacaca tgctatttaa aaactccaag ggaagggcta 102360 gagaaatggc ctggtagtta agtatgcttg ctgatcttcc agaagacctg agctctgttc 102420 ctagcagcca tgttgggcag cttacaacta actatgactt ctgagctcca aagctctctt 102480 ctaatacata catacataca tacatacata catacataca tacatacata cgtacacaca 102540 cacacacaca cacacacaca cacacacact ttaaagaaaa aaattctggg ttggagaggt 102600 ggctcagcaa ttaagagcac tgactgctct tacagaggtg ctgagttcaa ctctcaacca 102660 catggtggct cacaaccatc tgtaatggga tctgatgccc tcttctggtg tgcgctgaag 102720 acagatacaa tgtactcata tacattaaat aaataaataa gaaagaaaga aagaaagaaa 102780 gaaagaaaga aagaaagtgt aaacgaggaa aattcctaat taaaaaagaa agaaagaaag 102840 aaagaaagaa agaaagaaag aaggaaagga attctgaggg agaatctgcc ccttttccta 102900 acttccaggg ctataggcaa cctgtggcct ggggaagctg tagacaacct gtggcctggg 102960 gaagctgtag acaacctgtg gcctggggaa gctgtagaca acctgtggcc tggggaagct 103020 gtagacaacc tgtggcctgg ggaagctgta gacaacctgt ggcctgtggc agcatcatgt 103080 caacgcctca ccctctgtgc ccaatttcct gttctctaag gacacatgcc atcaaatgca 103140 taggacactc tacatcaaga tgatcttgtc tcaagatgtt taacaaaatt acatctgcaa 103200 agacctatct ttacatgtga ggtcactcca caggttctag acatattttt gaggagccac 103260 catccaactc actatgtgac agagtcatct agagatttgt gtccaggaca gactggctgt 103320 atctgctctg agagtcccct gcctgcccgt gggaactccc cagtggtcct taagggccct 103380 gaggactttg gatctgcaaa gccacatctt ccaaaaccat tttcctcttt tggagagcta 103440 ctctaccctg aaaccctttt ctctgaggtg gcttttagag aggcaggtct cagcagggca 103500 ctgtgcccac aagaagtccc ggggagaagg gacccaaggg ccagtgctga actatcgctg 103560 agactgagaa cattgtgtct cacctaaaat cggtggtcgc aaggaccaag caggctctat 103620 aaatgtctta ctgcctttat tccttttcct ccgctccatc ttactcctca tttttgtttg 103680 tttgtgtgtt tgtttgtttt cttctgagat gtagcccagg ctggccttca gctcactatg 103740 taactaagga tgactttaaa cttctgatcc tttcttccct ccacttccag agtcctgggg 103800 caggtgtgtg ccaccgtacc ccagctttat ttgagactat gattcaggct ccatacttca 103860 tgcatattag gtaagcatgc taccaacttg gctatattcc cagcctttct ttctttcttc 103920 tttgagacaa tgtctttttt tttttaatta tatgagtaca ctgtatctgt tttcagacac 103980 accagaagaa ggcattggat cctattagag atggttgtga gccaccatgt ggttgttggg 104040 atttgaactc aggacctctg gaagagcagt cagtgctttt aaccgctgag ccatctcgcc 104100 agtccttgag acaatgtctt gctatatggc acatattggc ctcaaactca gaatccttcc 104160 gcttcagcct cctaaatact gggattacat gtgagccatg gtgtttggct tctagccttt 104220 cttccttccc tttcccttcc cttttccctt ccctttccct tttccctttc ctttccttcc 104280 cttcccttcc cttcccttcc cttcccctcc cctcccttcc cttcccttcc cttcccttcc 104340 cttcctttcc ctctctctct ctctccccct ctttcttttc tttcagagag tttctctgtg 104400 taatcctggc tgtcttggaa cttgctctgt agaccaggct ggcttgnnnn nnnnnnnnnn 104460 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 104520 nnnnnnnnnn nnnnnnnnnn nnnnnnaaga agaagaagaa gaagaagaag acaacaacga 104580 cgacaacacc ggcgccgctg cctccactgc catccacctg agacaggact caaatccaga 104640 ccaattttta aaaccagtgt ttcaagccgg tacactgaag tagtagtccc acttgggatt 104700 atagctcctt actttgtttt gctttgacgt tttgtgacat ggtgtgatgt agtcttggct 104760 gtcctagaac tcaatgtgta aattaggctg gccttgaact tgcttctgcc tcctgctggg 104820 atgatagact gatggtgtaa aactccactt aggaggcaga ggtgggagga tcagaaattc 104880 aaagtcatcc ttggctatgt tgtgagtttg aggaccaacc ttggctacat gatatcctat 104940 ctcaaaaaga aataaatgta ttgccgggca tggtggcaca cgcctttaat cccagcactt 105000 gggaggcaga ggcaggcaga ttttctgagt tcgaagccag cctggtctac agagtgagtt 105060 ccaggacagc cagggctaca cagagaaacc ctgtctccaa aaacaaaaaa caaacaaaaa 105120 agtgaacccc aacagtactg ccggacagtc tggtgtcttt cctaagtctc ctttcaactc 105180 tgtttaccca ggtgtaccca caaggtgtgt gagcagctct atacccagag gtgatacggt 105240 tgtttgaatg agagaaaagt ttcccatcag ctcgggtgtg tgaatactcg gtccccagtt 105300 ggcagtattg gctggagagg tgatggggag gtgtagcctt ccggtggaga tgggctttgg 105360 gagtttaaag cttcctccac gaagaccact gggctgatct tgttaccaac agaattggat 105420 tggcctgctc ttggctccgg cctcagttta tctaaaattt acatgttacc tgatcaaaaa 105480 ctgtttcctc ccccacccct ctccctgtct gtattccctg ccctctagtg gtgctggctg 105540 tatactacac tggtgatctt gactgtattt cagtttacct cttgttctct ctctgctgac 105600 tctagagatg tcctttcatg gctgggacct ggctcagaaa tttctaaggc actgagcctt 105660 cctccatctg aactttagga aacttcttgg cttaaaggtg tatttctgac ttagtatgca 105720 atgagacctt ggagcctgca ctttgttaag caccctgggt ggtggtggtg gtggtggtgg 105780 tggtggtggc agtagtagaa ctctgatgaa cagttagtta ttcaaggccc atctagaaaa 105840 agaaaggctt tgggtgcaca tggctataac tcagtggcga gagacgtgct tcccatgaat 105900 aataatgatg acgatgaaaa taattctctg tgactgttct ccccacttcc ctctctctca 105960 ccctagctct tatctaccga atccctgcac aagcacccat ggggtttaca gaatctgggg 106020 cggaacgtta gtcacttccc ttcgcctact tcagtattgt gtttccagaa gtacccattt 106080 tggctagtca ctgaggaaaa cggcagctgc ctgtgggcca ccagcccatg ccaagtgagg 106140 tcagcaagaa agaagctgac agcaaatgtg ccaactgtgg gtctgctgga tttctactgt 106200 gctaagtggt ttcaagaagt ttcttcttaa ccccctacaa gaaaccacaa attttattat 106260 ctacactgtt ttgtagatga agaaaacacc attccgaagc tcactgccag tcagcactgg 106320 aactggaatt tgtttggcta attcagtggt tctcaaccag ggtggatttg gactccccag 106380 gggatatttg gggacacttc tggtcgtcat aattgggatc atgtgctact ggcacctagg 106440 gtagaggcca gggtggtact gaccttccta ctatgaacag ggcagccatg tacataaatt 106500 ctctcgatca aaacatcaac agtgctaagg ttgagaaatc tcaggctgaa accctgtcat 106560 ttggccttga ggtgggtggg aggaggttag agagtggaat aaaatcagaa gggccaccac 106620 agaggcctcg agtgaggagg gaacagggct cctatgctag ggataatgga gaatagggca 106680 gcttgttgaa acttttcttt cttccaagct tggctagagc cctgctcaat ttcccccaac 106740 tctgccaagt cagtcccggg acttcgcact aagtttgtcc tggagtgacc ctgactccag 106800 cttggagctg gggcaacaca tttacctggg tcttcccagg agtgggttaa aagtcaaaga 106860 taagtggcat ctgagaagtt aaaagtgggg tgagtgggta aaggcaggag gaccccatca 106920 atcagctgac ctaggaagga agagagcaac tgaagcagca aagagctggt ccaggagact 106980 ggattctgac ccactaggct tatcttccac agcctttctt gtttaggctt gggctcagtt 107040 tccctgcatc atccgcagga gcccctggag cacccacatt cagccggccg ccaggacagg 107100 ctccccagca gtggcctccc cactaactga cagtggtgac aggaaatatc tcccattcca 107160 atctcctcag aagtctgaat aagtaaggga cagatgttgg ggagaggcgt cactcttggg 107220 ttgatgaaga aaagatcatg agaagcatac attttacccg ctatggttgg ggttccatta 107280 ccacccatgg cggggttgag ggggaagggc agaaaaaagg agatggagaa ggacagacac 107340 gtaagaagga ggatgtggtt agggctgatt cgtcccctgg ggtcgaacaa tcagctgtta 107400 ctggggcgga aggcaggaag tctccttaaa gacacaatat tctgaacgtt gaactcagga 107460 tttgaagcaa gcccagcagt caccttagtg gagcccatac ttaatttaac acagaagcgg 107520 ctatctcagg cttccctctc atttctttgt ctcagatgct ctcacttaga aagcttagat 107580 gctcttagaa atgactcaaa agtcaagaac cccaggccac aagtttctct ttgggggtgg 107640 ggagggagtg aagaggtggt cccagtcttg tccctttaaa taagcaattc agcagctttt 107700 gccaagtcat tgggttcatt tcggtttttg cccatccccc gcctttcaga ctctgattgg 107760 cccctaggga aggagccgcc tcttcattgg tctccacctt tgaaatcact tccctaagta 107820 ggcctgagtc agagaagcgt ttcggagggc gggactgaat gggtgttaat cttagaaccg 107880 ggtttctggt tgatactact ttggtaaaga tcttccccta atttttaaaa agacgcttcc 107940 tctctaaaag tgagggcgaa tcctttgtta agaacgtgcc ccttgagaag ccgtgggctc 108000 ttcagcgact aagacgagac attcactaga aaagatttca ctaaacccac gagggataga 108060 ctagacctcc agtgaagatt gggcctgtgc gggtgacatt tgtccctata ccccgaagac 108120 ctcgagctag ctctccagtg aagactgggg ccgtgcgagt gacagtggtc cctatacccc 108180 gaaaaaaaaa aagtcctatt tgtggaaaaa aaaaaagact tcgggtgttc tgctgcatcg 108240 gtggctggct tccatcttta gttctactca ctcctgttgc ttcgcgtgct ccaccttcgc 108300 ttagctcagg cctcctgtga atcagttttg aggctaaaag aagttccaag aaggaggggc 108360 tgtagccctt taaggacttc cccgcgaccg agtcagagat cagtttaaaa atgccaactc 108420 acagagcgcg ctgcattctg ggaagctgag tgtcaccgta agaacttcat tgaccggaat 108480 gcactgcaaa aatacacgcc tatacttcct tctgctcttt aaactgtagt ttgacgtaaa 108540 gctggtctaa gcaagtcgcc taggccgagg gttagccaca ccttttcagc cattggccag 108600 ttggttagtt ggtaggcgtg gcttagagaa gctcctccag gcaagggggt ggcctccttg 108660 ccaatcagag cccagacgcc tgaatgggcg ggagtaagca gaggtgctgg cgcccccgag 108720 tgggtgtggt cacgttgccc agcaatgggc ggtgattggc cctgggtggt tcattcgcag 108780 ctcgtgcgtc acgacgccgc cagctgatcg gagactggag ccggtgtgtg ctgggcgctg 108840 ggaagagaca gagcggtcgg ccgtgcggac aggtcgcagt gattttgctc ctctgtccac 108900 agcaaccccc gcacccagca tcaggtgggt gtgatctggg gacccggtca tcccgggggg 108960 aaccgcggta accgggtgat ggggaaagta gggtcctgac ggccacaccc tgcccttctg 109020 ggggagggga gagggggcgg cggggacagg ggcgctcttg ggagaggagc ctggactctc 109080 ccgagtagtg tgtctggacg tttaaagaga gagtcccgga caggagtcgt ggcagaaggt 109140 ttggagaagt aactggggag gaatatgaga ggccagaggg ccgggggcgt ctaaccccga 109200 cgccctttgg tttgaggatg cccgagctga ccatttagcc tagggaggat ctggacgagc 109260 gaggggtgcg gaggtgcatt gcctctaccg gcgctgactg ggtcagggcc agttcaagtc 109320 cctggcaggg aaggggtcgc tgggcggtcc ggcccctcct ctcgttccct cccggggatg 109380 ttatgtaagg ggggagggga aaggagtagg gggcggcggt gcggaggcct tatgcaaccc 109440 aaaggttagg gtttcaccgc gggttgggcg gaggttgggg ggggcggaca ggaggagtgc 109500 ctggaaactc tacccgcacc ccccctccca gcctaactgg ctgtcttgga cagagagaag 109560 gtcacctttg cacctccccc ctagtatgtc cggtagagag gcccctagcc cgggcttggc 109620 ctgactgcct gggaagccgg ctggctgggt ggggcgcctg ggttagtcat cgctgggctc 109680 cctctctccc cacctcctgg ccaactcttg gcccctcccc acggcctccg gttaggctaa 109740 cgttcccacc tccctctggc cctagtttca gtctccaact catttggcct gtcaccctgg 109800 ctgttagagt aggctagaag ctgtcatggt gccagagagt tgatggagca gctggtcaga 109860 gggtcagtgc cctgggccca ccccgccccg cagccaaggg cacctgcttg gcacaaactc 109920 tcagcagcca gtgaaccctg tggcctgaac agagctatcc tgggcagaga gaagtggaca 109980 gagactgatc acctaggaga aggaagatcc gacaaagttt atacttccca agaggctttt 110040 ggaatttgaa agttgcccac cctagtgtaa tctttccact ctctgaaaat agaaatccca 110100 aggcaaagtc tccttggccc ttctatctgg cagtggccat gtccttggac tgactgtgca 110160 gaaccaccct ctcgggctcc cagccctcta gcctgccacg cccccagccc cctccctgag 110220 ccatgctgta gggccccggc ttttactgct gattcatgcg ttggaactgt gggggcgggg 110280 cttggaactt ggaacaaagt tcagacgtgg aggggccggc agacagcctg gaattcatac 110340 cagatgtacc cggaatgtgc aagcggaatg cctggcatct ctagtcctga ggaagctgcc 110400 cagccaccct acccatacct ccctcccctc ctgcctttgg tcagctgtcc tccctcagac 110460 tcctgagagc ccctgctgac cttccaactc tagtgcccct cccatttcta accctacaca 110520 aaccctcctt gctgctgaat tccctaagaa caagtcattt gagttgatca cagagctcat 110580 atttctgaag tacatttttt tttttaactt gggacttggg ttctacaccc tgccctttga 110640 atgccgaaga tgctgggctc cttagcaggt tgccaagagt tgccagctcc tagtctgtaa 110700 aggggcacaa agcaagtgca tttagaagcc tcttgcttct tattcaagaa cccctcatta 110760 gaaggtactg aaagtcagct agagccaggt ttggatggcc tctgggtcgc tggccctgtc 110820 acccagcttt cctgtttttt tttttcctcc ccttcctttt aggaacctgt gcctcccaca 110880 ccctcacctg gctgagccgc agtagttctt cagtggcaag ctttatgtcc tgacccagct 110940 aaagctgcca gttgaagaac tgttgccctc tgcccctggc ttcgtggagg aagaggagaa 111000 gcagcagctt tgcctatcat ccggaaggtg acagaactgg ggtgggaagg tctggacagc 111060 tggggtgatg gctttatggg agggaaaccc tggtcctctg gggagccctt acccccactg 111120 gcccagtgaa agatttaggt taaaggcact gtctataaat tggggaatag gtgactccac 111180 ctccccaaga ttagttgatg tctgtgtggc agtgggaaga aatagaagga aaagtctgtc 111240 tgtttactga gacttccttg taggcctgcc tttcttatct tcatcatcac catgccaaca 111300 cacacacaca cacacacaca cacacacaca catacacaca catacacaca cacacacaca 111360 cacacacttt cctttccatg aggtccaaaa gtaaatgtac tcaggaaggg ggacattgaa 111420 actccgttct aagtagtcat ttgtgtattt actttttttg tttatttgtt tgattgactt 111480 tcgagacagg gtttctctgt atagccctgg ctgtcctgga actcactttg tagactaggc 111540 tggcctcgaa ctcagaaatc tgcctgcctc tgcctcccaa gtgctgggat taaaggcgtg 111600 tgccaccacc gcccggctgt atttacattt ctttatttat ttttagtctg gcccagattt 111660 tgggtttagg ggtacttacc cttacacctg tggatttttc cacctgtata atggggaatc 111720 ccatagataa gtaggcagga gggcattaaa agtccaccag tggtgactca gagcctgggc 111780 tcttcttctt ctcgtggatg gaaacgaaac agctcttcac atgaactgtt gtccttcccc 111840 caccccctga ctactcaccc agctcagggg gattaggatg gaaggaaagg ctatggttaa 111900 gtcccaggca agctcgtggg aggctagtcc tctactggct tctcaccatg catgggtggt 111960 ccaaggcttt ccctccacct aaagcaaaac tgtagctctt ggttgggttc tagcaaccac 112020 tgccatttat tttctgcctt tgctttccag gatagtgaga ctctgctcaa tactgtgcag 112080 gcaagaaatt gtcaggggag atgggttgta tgatatgagt cccttctgct gcctctagct 112140 cctgattcat tctcacgtat gggcttggtc tctgattgtg gttcaccttt ggcccagtct 112200 tcctaacaga agatgggttc agggggtaca ggaggctgtt tgttgtattt gacaggagga 112260 ggagttctag cctgttcccc atttgtgaga aactgaaagt cataggggag actagatcat 112320 ctaatccagc cccactgcag tctaagctga gggataggat gtgtaaggga ctgtagcaga 112380 cgggctgggg aggctgagtc ggctcacaca ttgcgacaaa gattgccctt ccctcgacct 112440 cgcttgcttt ctttcctcct cccttccctg gccacagtgt gtccctccag cactgggtac 112500 atggctctgc tgtcctcatc caacatggag cctcagaggt gagaaagggc agcctggaag 112560 caacagaggc aggcacaaga cagtggagga cctggcctgg aaccacaagg gcctatccgg 112620 acattggtca gagaggcacg tagaagcctg gagaacacca ggaaagagag cagccagcca 112680 gcctcagtga aagacacgtg cttccagcca tctcctctca ggacctgcct tcctgggaga 112740 tgaagggcct ccaggaagta tggtcccatc tctaccctgc agtttctata aacagcctca 112800 aggagcatga gccacctctg aaaggaaata cacagcaaat tcaaaaagag attcaaatgt 112860 gtaacactgt gggaaaacat atctatgact ggggttgtag ctcagttggt aggtttgctt 112920 aacatgcacc aagccctggt tctgtcttct gcattgcata aaactgaaca ggttggccca 112980 ggtctgcaat cccggcactc tggaggtggt ggcaaaggag cctacattca aggtaatcct 113040 ctgctataca atgagttctg agccagcctg ggctatatga gactgtctca aaaaataaaa 113100 caaaataaaa taaagcattg gttagtaatt caaagaaagc agatgtggct gaaaccgttt 113160 tccctgatca taatacaaca agcaaatgaa agccagaaga aggctcctgt gccttgtgtg 113220 tggcagtacc aaccattgtg agagatgcct ttggacctgg tagtttgctg tcttagaaat 113280 gtatcctaaa ataaggattt ggttataaaa tgttcatctc agggttgtaa tagagaaaaa 113340 tggaacgcag ctgtttgttt ggaagtccat tccttttctg ctgtcatgaa aatgtatagc 113400 tagggcttgc ctaagtaaat tatattcatc tgatggtggt gttctgtgca gccatccaaa 113460 gtcttacaga agaaaaattg agtggaaata taaatattga atactaaaaa gattataaaa 113520 gtatgagttt gtgactgttt ttaaaatatg aacacatact tgtaatatat ttttttaaaa 113580 accatccaat tgagtggaaa tataaatact gaatactaaa aagattatga aaagtatgag 113640 tttgtgactg tttttaaaat atgaatgcat acttgtaata tattttttaa aaaaacactg 113700 aaagtggatt caaaatgtta agaatggttg tttttgtatg gtgggatagt acaattgtga 113760 attttcccct tgttttttct gtctttctaa tttttaaata ttgtgcattg ctttcatatg 113820 ttaaataaaa tacaaaagac aaataaatgt tttaaaattt ttactctttt atgagtgttt 113880 tgcctgtgag tggcaggaat ccaacattgt cttctgaaag cagctagcgc taacttctga 113940 gccgtctctt cactccctct gtaattttta aaaaatatat ttgtatgtta tattatgtgt 114000 ctttgtgcac cagagtgtgg gtgcacattc tgcagaggcc agaagagggc atcagattcc 114060 ctggagctgc acgctgtttg gatcttctga tgtggatgct cagaatcgca ctcaggtcct 114120 ctagaagagc agcaaatgct cctagccact aaagccatct ctccctctag tcctcattgt 114180 catgtttaga ttttggagaa tttgcttgta ggaggatggg ctacaccaag tgccaggtga 114240 aagaaaatgt ttgcttggga tacctattgc ttcttgagtg tgtgtgtgca tgcttgtgtg 114300 tgtgtgtgta tgtgtgtgtg tacactggag ctaggaatca tatccagggg ccttttcaag 114360 ctccaccaca ctaaagtcaa ttctatgaac ttcattaatt gtctgaatcc acctactctc 114420 tacacacagg aagcattcct ctgactttct gactgtcagc cagctaagga ggtgtggctt 114480 agaataagaa agaagggaaa tgctcaaaac ctgtcactct ntggggnnnn nnnnnnnnnn 114540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 114600 nnnnnnnnnn nnnnnnnnnn nnnnnncccc tattttacca agcattgcag gataaataag 114660 aacatagctt aaagaattct agagaaaaaa aaaaaagtcc actaatgttc aatgttttaa 114720 ttaattttta atgggaaaaa gtaatatcaa agaaatccat gtctacggca tattaatgtg 114780 gttaatgcaa acaggacaga ttatcttggc caaattaaac cacagtaaca cgttttgaac 114840 taaaagtcat ctatttatac gagatgaaat gtgaattagc gtgcgcgcgc aggtgcacac 114900 atgcacacat acaaacagag tctttttatt cttaagccta ccaaataact ctttaagcag 114960 taaataattt taagctctaa aatttaaaaa atagtgaaac cccttcaggc tatgacagaa 115020 tgctgctttt gccattcttt ctgataaaag tcccaaaggg tgtcataatc tgtatccttc 115080 ttagaaaagt aaggagcaca tcctatgagc tggtgacctg agttttacac ccaagtcaca 115140 cgtcagcaca cagcaatgtc ctggaccatt tgtgaggagc cccacgctgg tcctgagcaa 115200 caactcactt ggactggtac ggggtgaggc tggcgatggg caccactttg gactgtgtcc 115260 cacctgaagg ctgcagcagg ccagttccgg caggttttcc aaatggcttt gaggcaccat 115320 aagccttagc tgcagtggaa cctatgcaaa cacacaggaa aagggcagtc attcccgttt 115380 tatttctcga agtgaatgta caggcaccat ggcacgcatc tggaggtcag atgataatct 115440 caaaaaaatc caacttttgt gagacaggct cccttcattg tcttcccacc acacttgcca 115500 ggttaggggc acaagctccc aaggagtatc tgctctccta agggcactgc catcagagac 115560 actctgacta ctacacctgg cttttaggtg ggttctgagg atttgaacgc agatcatcat 115620 ggttgtatag caaattttta ttcaccaagc cagacaaccg ttttttaact ttttttaaaa 115680 aaagatttat ttttatgagt acagtgtggc tgtcttcaga cacaccagaa gaagacatca 115740 gatcccatta cagatggttg tgagccacca tgtggttgct gggaattgaa ctcaggacct 115800 ttggaagagc agtcagtgct cttaaccgct gagccatctc tccagccctt atttaacttt 115860 taaaagttta aaattaattc tcattatgca cgtgccacag ggaagtgtaa cataagcaca 115920 tgtgtttgaa gtatatatgg ggatgtagtt atgtgacggc cagaggacaa ctctgtgaag 115980 ttgattcttt cctttcactg tgggtcctcg gagcgtcaat agggtcagca ggcattttac 116040 cagctgagct atcttgtggg ccaccaaact tatttttaaa aagctagaag ttggttaaag 116100 agaaggaatt catgtaattg aaatagaaaa gcagagttga agagggagga gaggaggtga 116160 gtatgatatg gtaagaaaaa ccaagaaaaa acctctacaa gtaaatctag tcgtgttcca 116220 cagatcacta tgttcaagga tggtgtccca gaaaagggtc ctaaaacaaa gcccaggaag 116280 taatcaaaat gatcccatcc agacctgcta cagagaagct ggggggaagg agggagaggc 116340 cctgtaacct caagtccccg ggcagcacag cagagctgtg cccaatgatc tccgtgatac 116400 aggacaacag gatatctgag aaggagtccc agtgagcgtg tagcagaagc cagaggcctc 116460 gaacaaaacc aacaacttca ccatgatgaa catcatgaag atgtgtggac aaaagggtgc 116520 actctgggac acagtgtcac acactacagc ttccacctgg gacaaaaagt cacacactac 116580 agcttccacc tgggacacaa tgtcacacac tacagcttcc acagtcagat tatttttctt 116640 ctagcggaga ggctgcaagg gtggagggca ggtacaaggg aacaggggtg agtgggatcg 116700 gggtgcatga tgtgaaactt acaaagaacc aataagttag gaaagagaga tgaagacata 116760 gaagccatgg ccaacaaccc cagcactctg gaggcaggca ggagggtctc catgccaaag 116820 gtaatgaatg ctatgtctaa agagatgata aagtcccatg agagttacaa agtcagagga 116880 gaaacttgga cagaaaagcc aaatgtaaca aatgcatggt aggtggggaa aggtggggat 116940 ttaaaagaaa tttcacaaaa aggcacagaa tgtaaggtga aagcgagcga ctaccagata 117000 aagcatcaac taagaggagt caggaacagt gtattttaac tacctttata aaaaaaatac 117060 tcgggctggt gagatggctc agcggttaag agcaccgact gctcttccaa tggtcctgag 117120 ttcaaatccc aacaaccaca tggtggctca caaccatcca taacaaaatc tgatacggtc 117180 ttctggagtg tctgaaggca gctacaagtg tacttacata taataagtaa ataaataaat 117240 aagtattaaa aaatatgcaa taattggaat tattctttgc aattctacta tgaatggagt 117300 ttttgttcct gttgtttgtt ttccgagcct gtttcatgta atccaaactg gcctggaacc 117360 tgctaaatag tggaagatga ccttgaactt ctgattcccc tgtctccacc tcccaagtgc 117420 tggcctaggc tactctgcct gactcaggct cagtgaattt tcaaacactg cttcaacctt 117480 gacccctaac ttccatctct tgggtcccat cttacccatt cccagactcc cattctgtgg 117540 ctggggcttg ctggctggtg gtgtcgctgc agaggctggg gaagggactg cttgctgctg 117600 ctgctgctgc tgctgctgct gctgctgccc atatcctgga gatcaaaaca ggccaattca 117660 gtgcaaaccc agtcaagaga tttcaacacc agcagtaaat accttaggaa aacccacctt 117720 tggctcttag aggagcagac tgcaccacgc tgcacaccct gtgtctgcaa agggctcact 117780 tttttgtctg cgcatagtaa agactggttc catttccctt tcaactgttt agaattgaca 117840 gctttcagtt ttaatgaggt tctccctgaa gccttgccca tttctccttc aagaacctgc 117900 agtaaagtca ctgggctcaa tctgtgatac taccactatt tccatagcaa caaatcgatg 117960 tcatcaacag gacggtgtgt tcgtggcagg atccattaga agagaaagca gggtggtaag 118020 gaaaacctaa aagcagttca attgtctctc aagtgctttg gcttcaaagg aaaaggagac 118080 gtttacaaag ggttctaccc tttcccccaa acaaagcaac cttattttgc aactgacagc 118140 tggtggaaaa tatgagttga aagagtctca ggtaccgtga agtgctagtg aactgtcaca 118200 gcagggagag atgagaacag agttcagaag cagccgtgtc aaggcaacag ggagataaaa 118260 aggaggggga ccgagcacgc ttctaaagca gcaaataaga cgcgccaccc tgtcggagtg 118320 tgtatttgcc cttcaccctt tatacaaatg tttaccaagt tgaagaatgt taacattgta 118380 aattgcctac cactatttct aaaataacga gtttatgggg tttgatttta cttctgtgat 118440 tggctctgaa ggctgaagcc agcctcgttt aggctgctca cctggagtgc ttgtagatga 118500 caaaggcagc taaaaaaaaa aaaatgtccc agagctcctg aaacactaaa actggtgcgc 118560 acaggcagga agtctccctt gccactgagg ctctcccttc tccaactgta agttctaact 118620 cctgctgggc ctcctgaggt cccaactcac cgggcatgct acatgcccat ggaattcatt 118680 tttggcaaaa ttactcttaa gtagctcaag gaatgagacg taattgtgtt gctggagaca 118740 aatacattca gcacaaagtt gagaagatta aatagaattg attatgcttt agttcatcct 118800 acagagagaa aaagtgagac acctatttac ataggagagg ggcccaggct actgacaatg 118860 agcccttcct ttacagctca gccttcccat tatcactcaa ctcagacacc cagcccagtt 118920 cagtctatgt gaagcatttt taaaatcagc aagagaaagg tgagttgctc agtagtacac 118980 tgaaggaaag tagaaatgag cacacagtga cctgctgcat aagacacagt ttaaaaggtg 119040 acctatcttc cagcgaagtt cttgccttct ttaaaaagaa tgtggtattt gctggtatgt 119100 gcacctcacg aaggtgcttg cagctatgtg gagggcagag accatcctga gggaatcggg 119160 tctccttcca ccagagtcct agggatggca caaaggcacc gggcttgaca gatgccttta 119220 cctgctgagc cctttcccca gcctctcctt ttctacagtc tccaaattac ctgaggtagg 119280 agcctcattc tcagaaccat ttctgctgcg catgtctgac tgaactggat atggtcactc 119340 gactcaattt ctataaaaag ataactgagg agccggcttg gttggtagag aaaacataaa 119400 gacccgagtt ttgttcccac atggggcgtg cgtgctagag aggtgaggac ggactaggca 119460 gggagcagag gtaggcagat cccaggcctc aatagccagg cagcccaacc tgagctctaa 119520 gttcagtaag ggagcctgta accacagaag acactgtgcc gtgtaaccac ggcaagcggc 119580 ccgctgcagt ggcatctgct catagccaca gctacccagg aggctgagcc acaagactca 119640 ttgaagctgg aggtcaaggc cagcataggt accataggta gacccccatc tcaaagttga 119700 acagtaaaca tatattttac tacaattaaa aaaacaaggc cgggtgtggt ggcgcatacc 119760 tttaatccca gcactcggga ggcagaggca ggcggatttc tgagttcgag gccagcctgg 119820 tctacaaagt gagttccagg acagccaggg ctacacagag aaaccctgtc tcgaaaaacc 119880 aaaaaaaaaa aaaaaaaaaa aaaaaaccaa aaacccaaca catgtaccta ttctaacata 119940 aaattttcat tttttataaa aattacagtt ataattttta gttgaacata atacaatgat 120000 aagtctccaa cttgataatc tgaggctggg ggtgtagctc atttagtaga gtacctgcct 120060 agcagtcgct aagtccgggt agtcccttgc ctgtatccca gtgcttgggg aacaggcaga 120120 aagaggacca gaagttcaag gtgctcctcc tcttcaggta atgaggagtc tgagccagcc 120180 tgggatgcgt gagacacacc agtaacagca actactgcct ggagtgctca ctctggacca 120240 ggaacaagag ttaagtgcgt tactcccgta actgtctgca caatgatgga gacagtacgt 120300 catctttaac atctttggct gagaagagaa aacctggtat tcctcagctc gttctggcta 120360 agttcatgta cttcatcatc atgcagttaa tttgtaaaca accaatcctg gcagcaataa 120420 ctctaattat atagaacata agatgtggta attaggaaaa gctactaatc cacttaatag 120480 agtaaccttt atctcttgca aatctggtac aagagacagt cccaaatcaa atgatggcaa 120540 gaattccaga gcattgtaaa tagcagcaat tgcccttcaa ttaacgcatt gtaacgcagc 120600 agctgcccac aagacctcaa atcaatcagt ctatagctaa ggaaaaatct ttctaaagcc 120660 aaaaccattc tacaaagcag taacgtaggc tccgtttata ataacctgtt ttgggccacc 120720 tgcaaatcaa gctatcccag gaagccagat cgtaattctt aggctctgct ggctacacac 120780 tggtcccaag ccatgaggga actagattac agcaggctcc gccctcggtg acctgctcat 120840 agctatcatt cttacagtct attatggcaa gtgagctctg ggcagagaaa aattcacaaa 120900 caaacccacc aacttcccaa gcaagcattt tcttaacaaa cacaaagaat aaataaatag 120960 agcctgccat ggtagtgcac acctgtaatc tcagcatgtg ggaggcagag gcaggcagat 121020 ctctgccagg agttccatgc cagcctggtc tagacagttg caagatcaac aacgctatat 121080 ggtgaggccc tgtctcaact cccaaaccac tgaaaacaag taagacgata tggatcaatg 121140 caatatttct cagttcctac tgacagaaaa tggacacaat taggctgggt ataattccaa 121200 tatgaataat aagtatatta tacagtacct agtttaagtc tgagcaagat attatcgcca 121260 acagcacaga tacaggacac acacacagct gcaagcgtga aggatttaaa ggcacccatc 121320 cctacagtat atgcagcatt gactgtctag ttttatttcg cacactttga atcatccatc 121380 catttttgct caatatggca gcagtaataa aatgtatatt tgcattttga tgcatggtgg 121440 aattcttact agcctgggct gtgtttgctc actaactcca gtggacttct gacgtaagag 121500 gcgctggact agcttccaga ggatgtaaat ctaactttgg ttctcggcct cccctgaagc 121560 ctttgctgtg gtgaaaggtg ctgtttctga agccacgaca gtcccatggt ggtttgagta 121620 acaatactcc tggcttggta acaatgccaa aaaataccaa aaaaaccaaa accaaaacca 121680 aaaaaagcac agagctcaca tctgagccaa aaaaaccgac actccctatt ttttgaagaa 121740 ctcacagaaa tcaagaagaa aaacaagcaa acaaacagca cataacaggc taacaacaac 121800 aacaagtcca cttcagaggc caaaaaccaa aaggcaaagg ggctcttaaa catttacaag 121860 gacagacctc actcagaaca caagctatag ccatgatact gctcttcatc catcaattct 121920 taaagacaca gaagatgggc tgctggcatg actgggaaga gaccagtgtg ctcactcatt 121980 gctggggtac gctgtaacaa gcagaggaga atgtctaact gtgtctgcca atcacaggca 122040 cattcccagt taacccagca atcacttctg gcaaaaaaag cccactctgc ccgcacactg 122100 gtgtacccag gaggtaattc actgcggctt ctggtggatc ttttttcctt cttgcagtgc 122160 ttgggttcaa attaggatca agcacacttt tatagtcaga gcacggagac aaaaggatct 122220 aaccacagag ttggttaaat aaacaacaga acagccacac cagaactgct ggggggcggg 122280 gggagggggg ggagggaagg gggagaagct agaatcccag cattcaggat gcagaggttg 122340 gtaaacaagt ttcaggctca gtcggggtat aaggtaagac tttatttcac aaaaataaat 122400 aattttttta aagaggacag aaaaaagaca gcgtagagaa ctagctatct tttatgtaaa 122460 ccatgtgggg tacaggagca gcatttgcat ttggttactt ggcgaatagc cctggtagga 122520 taaagaaacc ttaaactgtg ttatctataa agggcagcag tgggtgcaca atgcagtggg 122580 taggagccca cccattgcac gccatagttt tgattacaaa gtcacgcagc tctactcaaa 122640 aattaaaaca aacattaatg cttgttaaag aaacagggct gcagagatga tggctcagtg 122700 gttaagagca cagcgcccat atggaggctc tcagccatct gcttctccaa ttccagggga 122760 agctaacgcc ctcttttggc cttcaagagc actgcatgca catggtacgc ttacctacat 122820 gcccgcaaac attcaaagaa aaatacaaac tgctataaaa cccatatatg accatcttaa 122880 gattctttca tttttttgag acagggtttc tctgtgtagc cctggctgtt ccagaacttg 122940 ctctgcagac caggctggcc tccaacccag agatctgcct gcctctgcct ccacagtgct 123000 ggaattaaag gtatttaaca cacacattat acatatcttc cttcttcttc ttcttttttt 123060 aaagcgtttt gttgtttttt gttaagtttc aattaaaaaa ctacatagtt ttataggcaa 123120 acataattaa aaatgccaat gtgaaataaa taatatatac atatataaca ttctgtaata 123180 gattcactca cacaacttat atacttaaat acaattttca caataatgaa aagctttgaa 123240 atgaagactt ctggatacat tagaaacgta ccctgaaaat cgcaaatgac ggttttcatt 123300 tctttgtgtc agacattagt gtgagtgtct aaacttgcat aaaggctctc ttctctatca 123360 cttcctacct attgcagtgg ttctcgctaa acctggaacc tggggctcct gttccttggc 123420 tggactacaa ggcagcaagt cccagcaatc ctcctgtctc acctttcttg gaaccaatgt 123480 tataagtgtg tgtgggcact agccttgtta catggctgct gggactagaa ctctggtctt 123540 caatattagg catcaagagc tcttaactgc taagccatct ttctaccctg attagaattt 123600 cttgaagcaa aagaaactca cagatggtca gagtttacac acacacacac acacacacac 123660 acatacacac acacacacac tcacacacca aggcttagtg accactgtga aaagggaagt 123720 gcggtgagga actgtaaaaa taaggtgtca ggaaagctct gcacaaaatg gtgtcctctg 123780 gacaagccag ggcctctgcc ctcatcagct cttagtaact atggttgcct acagcaaacc 123840 atgccaggga ccactctaac atggagctgg gatgggctcg agaggccctg ttattaaagg 123900 agaagctgta gagagttgat ttgatggatt ctaaagtagg gggaatcgat ttcctttatc 123960 gatatgggtc agccatgctc tagtgagtgg ccccacaccc acccatgagt atgtgtggac 124020 agcacacacc ggacctggca agtcaataaa acaaaaacaa aaacaaacaa ataaacgttc 124080 tggtccacca tagtggctgc tcgtggttgg gagctgtccc gcacttatgg caggaagaca 124140 gtggctgcaa agaggacaaa agtctctgga actgatcaac tctagagtct gcttgttatg 124200 agaactggga agtacccgct gggacagaag cagactctga aggtgatcag gacagagatc 124260 acagaaggag agactggtta tcggaggaaa tctgaaacat aactcgacgc atactggtcc 124320 aaactggtgc ccatcactac aacagcagta attgaattgg gcacaacatt cagaaaacag 124380 aaaaagacta cagagtacgc accctggcta tcatcaaccc aggcgattct ggcactattg 124440 gaagcaagcc agactggaga aaaggaaaca aaaagttatt taacaaaact tcccagagca 124500 tgttaaaaaa aaaaaaaaga aagccagaca tgggggtgca cgtttttaac cctagcactc 124560 aggaggcaga ggcaggtgag gcaggaggat ccatgagttc gaggccagcc tggtctgtac 124620 agtgggttcc aggaaagcca ggcaacaaag aaaccctgtc tcaaaaatca ctgactgggg 124680 agagaggaag tggatccaag agcaagagag agagagcaga gagagtgggg gtttggatgt 124740 cagcgttatt aaatgacagc agaaaagatg ggcccgacca atgacatccc agaaatggca 124800 aagatgaaaa aataaacaca agttctaaat atcattttaa taatggctgt gtgtctgctg 124860 gctcattctt gttatccaaa caaactaaag caggggtggc cgggatttac ggccagccag 124920 gactagagtg agacactgct ttaaaaaagc aatagatgca cgctaaccat taatcagcgt 124980 aactggagtt tgagggaggg agtgggcccg ggaagcctgt gcctataaac ccaacctgcg 125040 aggcctgaag cccgaaggtt gaactgagaa catctcaaga caaagcacag gcacaatctc 125100 ttacaaacag tttaaacaca ataccagcaa taaattgtca gctttatgac agataggctg 125160 acaggcatac cacaaagatc gggaagaaga aacgggattc ttgcacaaca ttttacaaat 125220 cgacacagct gagcctagtg acaggccgtg ataagctcaa aacatgcaca ctgcaaacac 125280 cacagcatgg ccagagtgac agggtcacag caatacagca acagagacag taagaaacat 125340 tggaaaaggg aagaaggcaa agagcaccac gagagaaaag ttaacccgcg attccatatg 125400 agggcccagc atccctctcc cacctgacag agaaaaccag gaggacgcag ctgaaacact 125460 gtacaactat aaatactcct tcctagtgta gacagagttt accaaagggt atcgtaatct 125520 gaagcacaaa cataacactg taagttacca gaagtttaga cacgttgaga atttaataga 125580 agttgagaca tcaaacacac gtttgatcct aggggaatta aattattggt aacagaaaag 125640 ctctttacaa agtctccaca tttataaacc aaatacactt gtatcagaat tgttgtctga 125700 gccagaggcg gcggcacaat ggtggctgag gcagcaggat caagtctgga gaagcccctg 125760 ggtccaggga gcactagtga attcaagtaa acattaaaca ttaaataaga aagacggatt 125820 ctacaggagc ctagacaccc tgtaaggtca gtactactaa gataccaaaa ccaaactgac 125880 tcagggggct gatgagatgg ctcagtggct aagggcatgc actgctcttg cagaggacct 125940 gagttcagtt cccagaaccc gtatcaggca gttcgcccac ctgtaatgta ccagagaccc 126000 taaacatttc tatcctccaa gcacagacac acataaacat aattaaaaat aaatcttaaa 126060 aaaaaatctc tcgtggacag acagcaaaaa atactgaacg aaatctaatc aggtagactc 126120 agcaaagcaa atccaaatat gcgaaaagga taatacaatg caaatccaga cttatcccag 126180 aaaccccagg tcgctttagc atccaaaaaa ttcaatcatc ataattcacc gtattagcta 126240 accgaaacag aaaaggcatt tgattattaa taaatacagg gaaagcattt gacaacattg 126300 accattcact cttaataaaa atgttaccaa acaggagaaa gaagtaaaat ctcctcaacc 126360 cgatgaacag gaacacaaac tggctgggca cagtggcaca tcctgaaacc ccagcaccca 126420 gatagctgcc agactgggct ggcatgagaa ccaagggtaa gaggcaccca aacacttaat 126480 gatccaaggt gctgcctccc ccccccccct gtgtcactta aatccgtaat taaatttctg 126540 ggaggagggg tttgacacag ggtctcactc tgtggcacag gctagcctga atggagtcaa 126600 tgctggcctc aaaactcttc tgcctcagtt ccagagtgag gggaaacaat catgagccac 126660 ctcacccagt tttaatgcca tttaacagta caaggttaaa cgctgcctga aaagaacaag 126720 acaaagaaag atcacacaga caaacatcac acaggtgctg tttgagacta ggtctctagt 126780 gatgtaggct ggtctcaaac ttgcacaaga aaagaatact tttgccatca agatcaaggt 126840 tgctatcacc catggtggca tttcggaagc agaggcagga agatcagtag tacaaggtca 126900 tcctcagcta ccatgggtgt gaggccagcc aaggcagcac gtgagccagc tgtggaagca 126960 cacatctgta accccaccac tcaggaggct gaagcaggaa gttcaagcca cacaagagcc 127020 acttaaaaat aaataaaaac ataaataagg ttggagagag ggnnnnnnnn nnnnnnnnnn 127080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 127140 nnnnnnnnnn nnnnnnnnnn nnggaaaaca cccattagac tgctgaagag actgagaaat 127200 tgtcatcttg aagggttgaa atcactgagg aatggctaat gacaaaactg cttaaggaga 127260 attttattaa aaaacaaaac tccttctgga ggagtactgt gctcccttca gcattaacta 127320 ctgtgccagg gaacccactg ccagcagaca cccaacttca aggataatca agcttgctac 127380 gtaacatggc tgcttacgca gacaccacac acacttcctg tatgctgggc atcacctcta 127440 gcttacttat aatatcaaac ataagtacca tgtgagcgct acagggcctg gccaatgaac 127500 agcgatggga aaaataacct gcacatggtc agtaatgggc aatccactcc ccagacactt 127560 ctaacctcag ctgcagactt gtgggtactg agaacggacg tgactaagtc aattcacaga 127620 agcagagccc tgtgcctgtg ccagggaacg gaggacgctg ctcaatgggc agagcttcag 127680 ggcgggaaga gatggaaagt ggagctaggg cccgatgacg tgaatatggc taatgccata 127740 ttgtgtgtgt gtgtgtgtgt gtgtgtgtat gtatgtgtat gtggtgtgta tgtatgtgtg 127800 tgcatgtgtg tgtgtgtgta tgtatgtatg tgtgtgtatc caaaatggtt ataaaaatcc 127860 catacaatgg ctatgcttat gtgtctttta ccacaagtaa aaaattttaa gtaatcttag 127920 gaacacattt ctaaaatttg gaatacttgt tggggaagct atctgagccc cagcatgaat 127980 caggaaatgg gtaggagagg caggagagat ggcttagtgg ttaacacaca catggtggcc 128040 ctttcctggc accaactagg tcactcacaa ccaggatctg acagcctctc ctggcctcct 128100 caggcaccag gcaggaaagc ggccccacat ccctgcaggt aaaacatttg tactgaaact 128160 aaataaaaac ggagcagaag ctgggcctgg aagtccttta cctccagcac cgggaagcag 128220 gtggatattt tgtgagttcc gggtctacat agtaaaaact tgtcgccaag taaaacaaaa 128280 caaaaactgg gggctggtga gatggctcag tgggtaagag cacccgactg ctcttccaaa 128340 ggtccaaagt tcaaatccca gcaaccacat ggtggctcac aaccatccgc aacaagatcc 128400 tcttctggag tgtctgaaga cagcaacagt gtacttacat atattaataa ataaatcttt 128460 aaaaaaaata cctttaaaaa aacaaaacaa aaactcccac taaaataatt ggaaaagtcc 128520 agggaaagcc acactcgatg gcgcaagcgt gtgatcacag tattctgtag gtaaggcaag 128580 aggatcacag cgggacggag gccagcctca gctacacagg ccggtctggg ctacagtgtg 128640 agaccccggt ctcaaaacaa aacaaaacaa aaaaagcgtt cacattatca tatactcaag 128700 gccacaaaac accttcttct tcccaatcct tgaatgctat cgaatttggt tacttttttt 128760 tggtaatttt ttgtttgctt ttcaaggcag ggtttctctg tgtagccctg gctatcctgg 128820 aactcactct gtagaccagg ctggcctcta actcacagag atgtaattct ttttcaaagc 128880 tggattttga tttgggggtg tgtgggtgat accacaggga cacttgggcc ccaaaccaaa 128940 ccaaagaaaa aacacccccc ccccaaagta tgaacatcaa ctgtatataa aactaacagt 129000 tcatataagc taagtagcct gcaggtacat ggttatggaa acagctttat catacagact 129060 tctttcagta gatgccattt gagaaaaaaa aaaaaacaaa acattctttg gattttcaac 129120 atgtaaacga aaatatgtta aatcttaaaa aaccttaaag cagacgccac tgctctttgg 129180 ggtcagggaa gcacggctgc aggcccccag agcagcacat tccctgaggg aagccttgtg 129240 cgtcctcacc agcaccaaga acagccaact ataataagct cataaaaaat cctgaaaggc 129300 tgcccaagcc tgaaaatctg atatgaaaac agagggcaag acagataaaa ggcaaactat 129360 actttaaaag tcaattccag ctatctgtgt ggaaggactt ctcgggacgg actatctgct 129420 aagaccttgt gggtgattta aacagcgtgg agggcagaaa aagaaaggaa agtgcaggaa 129480 acaatggcaa aagctcctcc tccgtggcct ctacacgcat ccaagcgata tggagggagg 129540 agggaaggac actatacaca ggtaccaatc cccacgaact agaaaacaca gcttttacta 129600 actagtctat tttttttttt accttagcta gtgtctttct tatgtttggc ataatttctg 129660 ctctgcatta aataaaccta gtgtataaga aggcaaatga gtaaaggtaa agtcagttaa 129720 tgttcatttg ttttgactgt gtggtgtgca tgccaggcgc attcgtggag gtcagaggac 129780 aaccgcatgg agcaggtctt ctccttccac caccccaggg gtcgcagggg tcccagggat 129840 ggaactcggg tggtcagatt tgcttggcaa gggcttcact cactgaggcc ccccaagggt 129900 cccaattcac ctgtttactc taatgtatat atttttgaga cagggtcttg ctatgtagtc 129960 caggctagct ttgaactcac tgtggacact agctggcctg gaggtctgag tgaacctcct 130020 gtttccaccc aagtgctggg acaacagaca gcaaccatca aggcaaaatg aagtcttccc 130080 ttcacaccaa agtggcttcc tatgaccttc tcctgacaac cccaaacagc aagtgcccgg 130140 atatgcactt gcgtttctct ttttttactt caagctttga ctccactttc ctaaaaggtg 130200 tttactaggc tgaagcacac ttcaggagac accctgtagc tctggagtga ccggaaacac 130260 accagctctg atgcaaaaac aaaaaacagc gatggggatg gggcaaaggt ggcttgtgct 130320 tctgtgcaat ggcattccct gacgcattac ctcacaccag atacatacag aaaagacaaa 130380 ggtaagtcct cactcagtgc gcgcatccac cacacccaaa gcaagcattc aacagctcag 130440 tggcataagg ccagcattat cacagcgcgg acaaacacaa agccaacctt ctctttgagt 130500 tccccaaggt acatagtgtt ctgtccaagg atggcctctg cagactcctg gaaacacgtc 130560 acccactggt tctcttgaaa atctgcaata tttgcctaaa aaacacaact ctattatttc 130620 agcgaataaa taacaaaagg aagacacccc aaggacgagg gaagattatc ttttcctact 130680 taaagtaact tccaagggag aaatgacatt gccgccacac acagcccccg actctttggt 130740 taacggttcc cccttatgag gaagtattca cttctggtca ccccgtgttt cacttttagt 130800 acagtatcta atgaattaca ggagccactc aataccttta tcataaagtg ggctttgtgt 130860 taggtgattc tgaccaagcg taggctatgc aaatgctctg agcacactta ggggaggctg 130920 ggccgtgcta gccctgctac atgcaggtaa tgctacgcca gtcttacgcc acaggaagaa 130980 gcttaaaatg tggtcgaact caatctgcta gaggtgtgag tttagacctc gggagacggg 131040 ccaagtaaac ttccgcagat gtcggagcat cagacagagc tgtcccctcc tgatgcaaaa 131100 ggcttcgcac ggcaagtttt taatgagcct ccatggtaat agtgggttcc tctctctcct 131160 cctcccttat caatatgctt gacatctttt agttttttga gacagcaaat aacgtagccc 131220 aatctgcccg catacttact gggcagccac agctggccct gaactcctcc tgcgtctcct 131280 ttaccccgta acaagtgctg gggttacaga catatgccgc catgttcagt gtaagtgacc 131340 gctgcccagc agggcaaagt cttccttatt tacaaagcag cagccaagcc ctgcagccca 131400 ggcctatctg atttcctcag cacaccccca agggtctcac cgatggcagt cagtccatga 131460 acaccgtagg cttctcacaa tccacactac tcaccgataa gatcatgcgg tatttgaaat 131520 tgggaaattc ccggtcacac ttctcacagc ggtacaaccc attctgctgg tcaatcactt 131580 tcttattgca gtcctgggtt gggcaggcct ggtacataca gttctctttg cggagaaaca 131640 ccaccgctgc cacagtgctg aaatagtccg cctgcaggcg aaaggaagac cgccatcagc 131700 aagcaacaca ggtctggaac aggcaactca aagctgctct tccttgcaag tggtgagcgc 131760 gtgtacatcc tagctcccac gctcacgagc gatatgcaga atcactaact ctggtttcag 131820 aaatcacacg tgctacacgc agaacccaaa gaagtaacaa accggcactc acgcacctca 131880 ctttttccat tttggagacg gcggctcact agctagcctt gaactcagag tttggtctgc 131940 ctctgtctct atagagtgcc tggctaccac acctgggcac tttgttttcg agacagggtt 132000 tctccggaac tcactctgta gaccaggctg gcctcaaact cagaaatcca cctgcctctg 132060 cctcccaagc gctaggatta aaggcatgtg ccatcagcgc ctgacccaat tctttttatt 132120 tatagttatt atttgttaca tatgtgtatc agtgtgtatg acgtccatat gtatatgact 132180 gtgtgcagtg tgcacatgtg tgcaggtcag aggacaactc tcaggagtca gttctctcct 132240 cctactgtgg cgtttgggga actcaggttc caagatagca ggaaaagtgc ctttaacagc 132300 tgagttatct cgacactgac atgtaatgat tcagtgtgca cacagtggtt ttgcatgtag 132360 tcatataaac cacagcgtgc atgtgcaggc taggaaacaa cctgtgggag ttggtgatct 132420 cccctcacca tgtgagtgag ggagaggaac tcaggctgtc aggctcggtg gcagtgcctt 132480 tactcactga gttcccttgc tggccaagca ttatttataa gatggtgatg tctaccctta 132540 tctttaggga tcagtaagtt tttccccaag acaagccaga aaaacctttt aggcccaatg 132600 agccattcag tcagtctcta ctgccactcc tcaacctgtc tgtggggcag gacaagccac 132660 agacaacctg aagacggaag gtgagccaat aaaatcttac tgacagtaac agccagccag 132720 ctcacaggct tgacaggcaa ttcttggact gaatgcgttt aagagaatgc agaattaccc 132780 atgactaaga tcttctaaat ggaaaaatgt ctggttaagt aacccagcaa ggagctaagt 132840 cacgcaagcg gtggatacct gctttgcctc tgaaccctgc acaggttttg gtttatgttc 132900 aatcatgtca agtacctaca aaatccagat ctagcctgaa ttcaaagata ggctacctac 132960 cctgcccccg gacccccacc cggggtctca ctgtgtagtc ctggctgtcg tagcgctctc 133020 tatacagacc agctgttatc aaattcagag acccacttgc ctcccaagtg ttgggattaa 133080 aggcatttgc cactatgcct ggctctcact ggttgtacca gaggagcaaa acaatgtggc 133140 catttaaaga gacgaagcta gaaaccagtt tagacagctt tggggctgtg ggtgtagctc 133200 agtggaagag cttgcttagc atgcacaagc tgttggtttt aaccctcagc gtgacagaac 133260 cgaatacata agaaggctta gaggaggggg tggtggtgga gagatgggtt agaggttaag 133320 agctggttgc ttaatttccc agtccccaca tggtggctca caacatccat aactgcagtt 133380 ccaggggatc tgatgtcctc ttctgacctc cttggggaca tgcgactcat ttggctcaca 133440 tgcaggatgc ctcgggccac tatgctggct caggggtgaa ggtgcttgct gccaagctgg 133500 gtggatttga gtttggtccc tgggacccac aaggaagagt ttgacttcta tacactgagg 133560 tggaacatgc atgctctacc cgcaaattaa aaacttaaaa tttaaagagg aagctgtaga 133620 gaaatagctt ttaggaggat gcctaaggaa cttctctgcg ttttcaggtg agattcagac 133680 tcaaagccca atttaaaagt ttgagtgctg tcacgtgttc tgtatgccca gttctggctg 133740 tccgttgtct gtctttagtt taagagagca actgggtgag aagtaactga gagtctagcc 133800 gatgtttaat tctcaagatg tcctgtgata gcattataag ttgctgtgga tgacagtgat 133860 ggatcgagca cacagtgaaa tgagacagtg aggaagaaaa cctatattgt atacgaggac 133920 aataatggag cagtggcgag cgatattttg ggaatacaat agaaataaat gattcaaata 133980 tccaagagaa cgcaggttag accaaggtag gaagaggatc actgggctgg agagatggct 134040 cagtggttaa gagcactgac tgctcttctg aaggtcatga gttcaaatcc cagcaaccac 134100 gtggtggctc acaaccatcc gtaataatat ctgatgccct cttctggagt gtctgaagac 134160 ggctacagtg tacttacata taataaataa ataaatcttt aattaaaaaa aaaaaaaaaa 134220 aggaagagga ccactgagca cacattgaaa tggaagatgc aactgaaatg caataccaga 134280 gccagtctgt ggcatggcag caggaattac agtctgttca aaacccagca cggacctgag 134340 gctacattat gtccctctac actggccgtg actgaaaagc agcaacgtgg taccctggag 134400 caggcctgag gtcccaggta aggacacagc cctgacctgg cactaggaaa caggtgtaaa 134460 aactcaagcc cctgccgttc tctgggtgtc tggagcgagg ggtgcgaggt accttgtctc 134520 cctggcccag gttctcagat ttagcctcat gcaaagtttt ccagttggtg ttgccccctc 134580 cggcccctcc actcctgtgg tcagagatgg aaacaccatc taaggcttgt ccttctgagt 134640 caaacctagg ggagaaagaa acaaacgtac tgctgacagg aacttcacat ccttctcaaa 134700 tgtgctgtga atgcaccagc cccgagctgc gccccctcgg ctctcaccta ggtctcagca 134760 cacctagtcc actcaagaaa tgcaatgccg ttgctccttc catctcgtgt ccttcaatgc 134820 ccctcctcct ccacccgcaa ggtcagacaa actgccaaag taccacagac cctttcccta 134880 aactgggctc acattgacag cagccatgaa caaaggcagc aacagcaagc cacactggca 134940 cccctccccc caccctcggc ctcaagcctt caccccaatc accaaaatga accagatgaa 135000 aagggacatt tttgtttcat gagtctgcca aaaatgtttt catgggagaa aatcttttaa 135060 gcccaatcag ttatagaact caatcagaag gcattatctg ctgtgttaga gatttgcatt 135120 ctcagtgaga atttgtttat ggaaatatca aggttaaatt catttatatg aaattattat 135180 taaaattgac tatacttctt gaccatctat tggctaatct gaaggaaaag aggccaggga 135240 gaaggtagca aggacaggct agtggcagag gacagtgagc ctgagatgaa aacacctcaa 135300 ctacacgatt ggtggtaact aaagctgccc ccacacagga ctgactccct aaggagactc 135360 ccaacagagt agcggtggcc caaggcaagc agtcacatcc gtcagaggac aaggtccata 135420 aatccgtctc accactacta gccgctcaag tttaacttca cacaggcatc ggcaaccaaa 135480 gacggctcat caggagtgaa cagagccagg agacaggcag cctctcttat agtaaaaggt 135540 ccactcttag agccaagatc taaagatcgc taatccttgc ctgggtaggg ggggtatact 135600 gaacttgtag atcaatgaca gtattttgtt cgggggacag acatgcctgt ttgtgatttc 135660 tgtgatcaca gttactgaca gtgaaatatg aacaccttaa tgtagacaca caattaacct 135720 ggggttgtgc tcagagtctg ggagaaaaac caaccaacaa accagaccaa tgccatggct 135780 gtctctggtc agacttctgt cctacaatgc aacgctagct atcagcacag ttacacagcc 135840 agacccacac cactgaggat gccttaagtg acagacaccc cagatggatg ctctaagtag 135900 taaatattgt tattggttta catcagactc aaaacaaaaa tgaggagcgt gaaatatgag 135960 ctgtttttct tgagtctctt aaattctacg acacagctgg aaaccacaca tgcccaccct 136020 ggtactacag ctattcaatt tccttaagct tgggctaggt aaagtatttt ctttgaccac 136080 ctcaatcctt ctacacaaca ccctcaggat gaagtgctct tccaaggcag agtttaatgc 136140 cttaaaaagc tagatatggt gtggaggttc taacaagctc tcctcatctc ctgagtgctt 136200 tgggaatgac aatgtacagg caacaaagag tccattgttc tcactgtact ttaccgagtc 136260 cagtgacaac acaacagaag atgtgaccag ctccagagac tcagcagagt aagaaagtac 136320 attctacccc cacatcagct gcccacagtg caaactgaag atgctctcag cactgttctt 136380 cagcgcgcag ctgttaaagc cttgccgtac ctggcgggat caaggtcagc acttagattc 136440 taattgcttt ctctggctct ctatgttcat caggggatcg tgggtttgtt ggtttaagag 136500 ggctttcagg ccaccatctc agaagacata atgcctcgaa gaagtatagg aaccactcca 136560 ctcaatagca aaggcttggc aaaacagctt ggcagcagtc cacatgctga cagtgtccag 136620 ctctggttta ccagagcccg agcacagata acccctgagc tgagttgcac aaaacctacc 136680 agccacgaag cttataggcc tctgggatgt caggattcac aatgacagtg ctggatgaga 136740 ggaccgagag gctccgtcca ccgaagtcag agactcgggc tcctttgatg gccatcacgg 136800 gctgccgaga gccgtcaaac ttgtcagcct gcaaatcaaa cgcacgcaca catcactgac 136860 aagaagaatg tacaggatgg ttttatggag aagagtcagt caatgtctgt ggactctaag 136920 acagacctcc cactcgggaa ggagcattgc actacaagaa gctgcaataa ccgatcatct 136980 cacacagcga aggtcttcaa atacttactg gatagcacaa ctccctgagc taaagcccca 137040 ccctcaggac tcggctcagt gcaaggactc acatcttctc cccacagagt tgtggtcacc 137100 accttccctg acatgtccat caaatagata tttctcttag caacttctct gttgttcgac 137160 ttcactgtga ttttaatcga atcttcatag ctcttgcaga ttccaatgat gtctgaaaca 137220 aagaacactt tgtaagagct cccatcaagg ctcctcttta aaacacggca gggacgaaag 137280 gcaagcacgc tagtcacgca tcaccccaaa cactggagac aaaaatcacc actctttgcc 137340 ctcaaccctg gacgcaccta ctagtgcgtc tttagccttg ctctctaggt caccgatccc 137400 tgtgaaatca aactgaactg tgggtaagtg atggccatct tcacagggaa ggacagaagt 137460 ctcattattg aaggtcatct catagtcatt tttaacagcg gagaactgtt tgttagcgat 137520 cttcagggcg ccctttgaga agtaatacac ctgcaaaaga ggccaagtca gggcaagcta 137580 ctcagtccat caaagcccgc cccacaatgc agccttccaa actgtgctca tggctctcac 137640 agttcagcct gtcagctctg actcaacacc gtcttcttac cttgttcact tcaataaggg 137700 gaaagaactt gtccacttgc tcattgaaag cagtagctct gatttcaccc tgcagaagca 137760 agggagagca cattagaact gcgctgctag gccctcgctc tcaacagcga gagcaggcca 137820 ttttgacagt tagacaccat cctcggagca acagtccaag tgtaaagaga ccctcacagg 137880 gctgcagaca ttgcagaatc ccacatatac agttacattt tcaagctatc agctctggta 137940 aaacaaaagc agctcagcca cccgagccta cagttgtaag ttaacacata aaacgaggga 138000 gcctcgagat gatctggaag acaaaggtgt ttcctgtgcc aacctggcca ccctagttta 138060 atcccggaac cctcgggatg gagaagacaa tcgccccacc aagttattct ccagcctttg 138120 cacacgtgtg catgcataca cacgaaaata attcttacat cttatcaaaa catttttaga 138180 aggaatagct taatattccg ataatgaaac ctaatattct gggtcccgag tgatgggctg 138240 gaagcccaca ggaaggcgtg ctgaactcca atgattcagc actcttcctc acgcccgcaa 138300 cctaagtgac ggcttacttc acagtcagga gctggtgcca aaaatgttcc aacaagtcat 138360 gctttcaagc tgactgcact gttacttttc tgtcaggcaa tatgttacag atggataaga 138420 aacttaacta aatggctaaa accttaccca gacaagctgc aggaaattag tttctgatac 138480 tgaatctgcc atcacagtta agatatctgc tgtgctacac cgtgacaaga ggaagaagag 138540 ggggagagag ccccaaagct ttgcccttcc cggtatcatg gcttatttta cgtgtgtggg 138600 tgttttacca gcatgtatgt gtgagcatca ctgagtgtct gatgcctgag gtgaccagga 138660 gaggtactgg atcactgaca cgggagtcac agatggttat gtgctactct gttggtgctg 138720 agaattgagc ccaagtcctc tcaaagaaca gcgagtgctc ttaaatgctg agcagtctct 138780 ctggcccctg cattttgctt taagtaaagc ctagtgagta taaatgatca gaagccctcc 138840 ccgcagacta gttaaagctg agtagctgct gctccttctg ctggaggcaa acccgccctg 138900 ctcggcaggt atcaccccag ggcttcaact gtgcccctag caatgtaatt agagcgctgc 138960 agtctctgca gacggagact atttacagtc caccaatctg tattgctaca gacgcgtccc 139020 tttaggagca ctttatctca tgtctgaccc tgtgccccag atagcatcaa aggtctccaa 139080 cagaaggaaa gccacaatga ccgccatgtt cctcggagct gagccccacc cgnnnnnnnn 139140 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 139200 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntttttatt aattatgtat agatatagat 139260 atctctatat atgaatgagt atcctgtagt tatcttcaga cacaccagaa gagggcatca 139320 aatcccattt acaggtggtg gtgagccatc atgtaattgc tgggaattaa actcaggacc 139380 tctagaagag caatcagtgc tcttaacccc tgagccccct cccttttttt tttttttttt 139440 ggcagcttat ttttaattgt tttaaatcac gtatatgttt ctgtatgtag ctatgttcac 139500 ataagcgcag gcactcatag aggtcagaga tgtaagatgc ctttggagct ggacttaaac 139560 atggttgtga gccatctgat atgagcacca agtggacttg ggtcctctgg accgccttgc 139620 tgccttccac ctagaattac agtatatgca attagctgct cagccacccc ctcagtcctc 139680 cttagtactt tcaacatagt gtaaatcgga gagcactcct tggtgcaaag attctgtgtg 139740 ttctatcatt ctctacaccc aggcttcatt gggacgtgtg atgccagcga tgattcattg 139800 gcgtgaagta tgagttagag tcagacaatc agtgctctct gctctgagtg ggtctccctg 139860 cccctgtgtc tctgagacag gggaggattc tcctgttgga tcacatagtg catagagccc 139920 tgtgtgcaca gcgctctcaa ccatttgttg ccatttcaaa tacagtgact tcagagtctt 139980 ccttgaaata acagatttct ccattttgtt tgttcccttc tttcgtcaca aagacctgag 140040 gatgagatgt ttttgaagga actttctagt agttactcgg tggaaaagga caatgatgct 140100 cccctcttct acagagaaga aggaaacagg aaattccaag aaaaggagta cacagatgct 140160 gcagtgctgt actctaaggt aacgtgcgtc caaccagcag ttgaacaagg cggaagatag 140220 aggcaaagtg cagactcata accagtccat tgttttgctt tggttagttg ggttggttgg 140280 tttgtttgtt tgtttgtttc ttcttcttta ttagtcggat tggatttggg aaaaatgtag 140340 tagaattcta ttgttgatta tattcacaaa caaaagactg tttatatgtc ctgaattttg 140400 gcaagattct tcccatattc cttcagaccc atagcagcag caagcttagt ggccctttgc 140460 caggtattct cactgagctg tacagcattt gtctgaagaa ctgatgaaca ttttagctta 140520 tgctcctatg ttagtaaata gctaggtttt tagctagcca ccttgctagc ttacaactag 140580 aatgcctctg gctgagtttg gtggcacttg cctttagttc cagcactcag gaggcagaag 140640 caggtgaatc tctgagttca aggcagcctg gtctacataa caagttccag cctggccagg 140700 gatatgtagt aagactctgt ctcaataaag taaacagcca gaatgactta aatattgcta 140760 aaaaagaaag aaagaaagaa agaaagaaag aaagaaagaa agaaagaaag aaagaaagaa 140820 agaaagaaga aaatgagtgg atttgccata gcctagtcaa actagatttt cttggttata 140880 tgttaacata ctactattaa caacaacaaa tactttacac taaccatgac aagctatatt 140940 tttaaattat tattttatat gtatggtgtg ggaagctgtt gcagagtggc agttggctac 141000 tgctggccac cacacataca taggcagtga aggttctttt gccaagacaa gttaaccaat 141060 cagatgtgag acacgcctct cctaggccta tgtaagcagc accagttctg ggctcagggt 141120 ctcttcgcct ctacaatcaa gctctcccaa taaacgtgtg cagaaggatc ctgttgcagc 141180 gtcgttcttc ctggccagtt gagcgcgcac aagagtatgg gttttcacct acatttatgt 141240 gaactgcatg tgcacttggt acccagggag cccagaagag ggcatcatat cccctggaac 141300 tagagtcaca ggttatgtat gggttctagg aatcaaaccc atgtcctctg gaagaacagc 141360 cagtgttctg gatttaactg ctgagtcatc tctccagccc caccatgatg gatttaactg 141420 ctgagtcatc tctccagccc caccaagctg gatttaactg ctgagtcatc tctccagccc 141480 caccacgctg gatttaactg ctgagtcatc tctccagccc caccacgctg gatttaactg 141540 ctgagtcatc tctccagccc caccacgctg gatttggaag cagaactgag gctttgaaca 141600 ctgtgttatt ctaactcttg cttccttgga accctgagaa aattccttct attggctttt 141660 cagagatctg tatgggctta aaacaagaat atgtccaact cttgatctct gattttatat 141720 attaaaagaa tagagtgagc ctaggagttt ggcacactcc tatgttccaa gcttcaggaa 141780 gggaagaagg tcaggagttg aaggctagtc agtcttagct ttgtgacaag tttgaggcta 141840 acgtgagctc tatgagaccc tgtctaggag aggagaaaga aaggggtaaa gtgaggtgtg 141900 gctccacgct accacttgcc taacacgcat gacgcccaag gtttccgttc ccagccctgg 141960 ggttcagagt attgtgcaca ccctgtatct ttaaaagtca atagtcaacc cttggaatta 142020 actttccgaa aaatattaaa gctacatcat ccactctaca aacttgtaaa agcccttctt 142080 tgtaatggtg taagaaagta tttggcttca gttttatggc ctttgcatcc taaatgttac 142140 cccatggaaa gttgcttaat gaaaacaggt aaaaataaga caggagggac tgcaaagatg 142200 ctcaggctca cgagtgctta ctgcccttgc agagaccatg gcaggcagtg ggtctcccta 142260 ctgcagggaa tccaaaaccc tcttttggcc tctgcaggca accacattaa cacatgcaca 142320 catacacata attttaaaaa taaaataaat cttttaaaat gagctctaga acgagtttga 142380 catcagtcta ggctacacaa gaccttgtct caagaagaaa gaaatgaagg tttgctgtgg 142440 atggagaggg agatgcactt cctattccat gaagctgcta ttttggtgat tatggtactt 142500 tgcaatttta tagagagctg ctattttctt tcttttaaag taatgcttgt tgttttgact 142560 gtaaaagtaa taaatgttac tttggaaaat atagagaagt ataaagagta aaaaaaaaag 142620 tcataaccag tgaagtaccg ttaacatttc tgcttctatc cggccagcca gagtttttcc 142680 tgtgagaatg tgtttttctt ttacaaaatt gggataatgc tgcacttact gttttgtagc 142740 ccactctttc ccttcacgat ttattgtacc cattttctca cagtattaaa ttttcagctc 142800 caagtaattt tccatagcta actctgtgtt ctgttacaga gaagaaatgt acttaattta 142860 agatctaata ttggcataat atttggcatt atgatgctat aataagcatc tttctgtata 142920 aatattttta tgtacagcca gtgttttgtt tgggatgaat tagcacaaga gtaaagtgtg 142980 gggtcaagcc tctgagtgac gctgaattgt tctcaggaaa ggactagtta acatccattc 143040 tgaaagaatg tgggaatgct catttcttaa gcaacactgg ttattattac atactattat 143100 tttgttagta ttatacattt atttaggggg ataggaaata tgctcatata gttcaatttc 143160 aagagttgca aaaatatatg gtgtggtttc ttttcctgtc ccctagttta gtctcacttc 143220 cagccccata atctaccttg ttaaatatac tgtatataag gcatatgtag gtgaataaaa 143280 aacagcctag tatggtggcg ttaacctcgg gttacttgag aactgctctt gcagagaaca 143340 tgactttggc tccgaaagcc ttcctggaat tccagctcca agggatgcag tgcctctggc 143400 atccttgggt acgtcactca tgtgcacaca tacacatttg gtttttaatc ttaggaactc 143460 caagtgggcc aatgagatgg ctccatgtat aaaggcagtt atgcaaaggt ctggatgaca 143520 tgagttcagt cttcagattc tgcaagataa caggagagga ccaacccctg cgagttgtcc 143580 tctgacctca gtacacatgt catggtacgt gtgtagtatg cacatgcaca gaagtcccag 143640 cactcgggag gcagaggcag gaggatctct gagttttagg ccagcctggt ctacaaaacg 143700 atttacagtt atataaagaa actctgtttt gaaaaacaaa acaggggttg gggatttagc 143760 tcagtggtag agcgcttgcc tagcaagcgc aaggccctgg gttcagtcct caactctgga 143820 agagagagag agagagggag agggaaaggg agagggagag ggaggagagg gaaaggaagg 143880 aaggaaggaa ggaaggaagg aaggaaggaa gaaagaaaga aagaaagaaa gggaaagaaa 143940 gaaagaaaga aagagagaaa gaaagaaagg gaaagaaaga aaggggaagg aagaaagaaa 144000 gaaagacaaa gcaaagcaaa actaaataaa atacatacaa tatattaaat tttaagactt 144060 gagggcatag atcagtgtta taatgcttac ctagcatgcg taaaactctt ggcttctaaa 144120 cctagcaccc taccgcaaaa tatttgctct gtcttgctaa attatattgc tagttgtcag 144180 actactgtgt actttcacta gcaacataat gagaatgttc actatcccac tcctctgtca 144240 agtaatctgt tcttggtttt atttttcttt gccaaattga tgggtgaaca agtatttcag 144300 ctagcctaga acacacagag aactctcttt atgaactcaa gtttcttatc cttttatgat 144360 ctccagaggt ttgtttttgt gggtttatta gtgttttttg tttggttggt tgtttgtttg 144420 tttggttggt tggttggttt tgctttactt tttcttattc atttttttat ttctttattt 144480 ttttgttttt aatttaatgg actggttcca tgtggccaag gatagcctca actttgtagc 144540 agaaactggc tttgaacttc tggtcttcct tcatctacct cccaagtgat gggattaagg 144600 cacgtgccac cacatctaac aatatctggg tttctttatt ggagtttgaa agggattcct 144660 ccagcattac tttgactctt catagtttct tccagagtta ttacactttc atttgttaca 144720 ttaagagttt gatccagggc tggagagatg gctcagtggc taagagcacc aactgctctt 144780 ccagaggtcc tgagttcaat tcccagcaac cacatggtgg ctcacaatcg tctgtaatgg 144840 gatctgatgc cctcttctgg tgtgtctgaa gacagctaca gtgtaatcat ataaataaaa 144900 taaataattc tttaaaaaaa aagagtttga tccatttaca ctggacttct tgaggcagca 144960 ggatcataaa ttcaaggtga gcctgggtga actggcagaa gtggcagaag ctgtgtctca 145020 cactgctgta ttcatttcct cattgatctt cagaggtttg ctaacgggaa gtaagtggaa 145080 cagaaggttc agtattcttt ttttcccaat tctattcagt ctttagtagt agatccctca 145140 ttatctgaga tgcagagtcc cctttattcc tgtgaccatc tcgttgtttt tcagggagtg 145200 tctcattcaa ggcctaacac tgaggacatt tcactgtgct atgccaatcg ctctgcagcg 145260 ctcttccatc tgggtcagta tgaagtgagt attgaagaac ctggtgtcct gcctgtggct 145320 gcagtggaaa atgagctcct ctctgttctt ctgcacacat tgaaatcaac tagcttgcaa 145380 acactgacat ccacccagac ccattctctc ttctgactca tgtcacctct cataggtgac 145440 cacaaacaat atgtagttga caagaagtag ctatgtcatt gtccacagtg catggatttg 145500 ttccaatagg ccagcacttc tgtgtccata tcagctagat gtgctgctga tagtatttta 145560 gattccaaaa tgtgtccaga tattacctcc ttcgtttgtt tcttctaaat aagccaggca 145620 caaagacttg aaagatggct tgatggctct tccagaggct gggtttgatt cccagcccca 145680 acatagcagc tcacaatagg ctataacgtc atttccaagg ggtctgactt cctgttctgg 145740 cctctacagg cagaaagcac agacatacat gcaggcaaaa cacataaaca taattgaaag 145800 aagatattaa ataatagccc acgcttgagc ttattcctct gatgatacag ctctcctgaa 145860 gtcatcatgg gcagtgtaaa agtaaaggtg ccccgccctg cccaggggca tcagtgaggt 145920 agctgactgt gagtggcttt cttctcatcc cctaactgct gcaacatcat caactgtgga 145980 gcattatatc cgtggatttt aagttaggaa atgacaaaga ttagatctat ggccaggcac 146040 tagtgcacgc ctttaatccc agcactcagg aagcagaagt aggtggatct ctgtcagttt 146100 gagtttacag agagtgtcta ggcagccagg gctatataga gaaattctat ctggaaagaa 146160 taaacaaatc agatctgtat ttcaggaaga tgcctatgag ctgtttgaca tgtgtgatag 146220 aggtccttaa ggacaggaaa gtattccaca tgtgtgcatt ttacagaaat tggttataca 146280 ctggagttaa ggactgttgg aatagttgaa tgttcacact cagtgttctt caaatcaaat 146340 agaagaacaa gaatctatct gggcatggtg atgcacaact gtattcctaa catgtagaag 146400 actgaggcat gctatgtgtt tgtggctaac ctgggctaca tagtcaatat tggacagtca 146460 gagctgctac taaaacctaa aaaacaaaaa tctaataatc tgggatttta tattttcctt 146520 tttttaaaaa aagagtgggc agaatgtctc tgattttgtt cagatggcca cagaacctag 146580 aaaaactgct gctgctgctg ctgctgcata gcacacagct aatatttgac tatacgtata 146640 taaattttgt tgtatacttt agctgtgctg tcaactttgg aaaaaaagta tcccagttta 146700 tcattttaaa ttggcactgt acagaaatta acagccatat tagtctagac acattaaact 146760 tcatttttcc atttatacag aagcaatgta ctgtattaaa tattcagtct tatctacagg 146820 ggtttgatta cagaaactat caaagtattc tctaaatgat gaaaaaagat tcaagaatct 146880 gactgtagat ccaaaggaca agtggagaaa aacttaggaa gaattttccc tttatcccct 146940 ccctatattg atcatctctt ttacttctaa taatagtggc catttattga acatacccag 147000 gagttccttt catcacttta gatatataat ttatctcatc ctaaaatgac ctgttgatga 147060 gtcatctctc tttcagatga gaaacaaaga cattgaaaaa tctaacttgc ctgcataaga 147120 tcacacctag cctcttactc actccatgaa tattcctttt ttttttttcc tttgagacag 147180 aatctcacta tgtagttctg gttgtcctag aactcaatat atagaccagg ctagcctcaa 147240 actcacagag atctgatagc ctctgcctgc cgagtgctag ggttaaatgg atgtgtcacc 147300 aagcccagca aaatttacct tcttaatttt ctaagacgtt tctcctctta aaaaatggaa 147360 ctattgagcc agtcatggtg agacaggctt aatctttaat ctcagcactt aggaggcaaa 147420 gacaggccta tgggttcggg gcagcctgat ctatagagag agttctatgg gttaaagttt 147480 agggttaaag ttttgagaca aaactttgtg tcaaaaacaa acaaacaaag ccagactgct 147540 taataagaca aatcagacat aatattataa acaagtatta gtgtcactca attaaaaagt 147600 cactcaggag gtgagatcaa tccccagcat aatgagggga ggaggggaga gaaggaaatg 147660 aatgggaggg gaggaggaag gaagagagaa aaaggaaaga tctcaaagca gaacacagga 147720 tgtaatttaa ggcctaagct ctcgactgaa gttgtccact tttaatgacc cttttcatgc 147780 tcatggtttt ctgtcttcgg tacatagtga agagtggaaa ccaagggtca tcctggaatt 147840 tccttttgtt ttcaggcatg tcttaaagac atagtggaag caggtatgca tgggtatcct 147900 gaaagactgc agcccaagat gatggtgcgt aagacagaat gcctggtgaa cctggggaga 147960 ctccaggagg caagacagac catcagtgat ctcgaaagca gcctcactgc caagccaacc 148020 ctggtgcttt cctcttacca gattctgcaa aggaatgtcc agcatctgaa aataaagatc 148080 caagaaaagg agactctccc agaacccatc cctgcagctc tcaccaatgc cttcgaggat 148140 atagccctgg gggaagagaa cacacagatt tctggggcct ccctctctgt cagcttatgc 148200 acacaccctt tgaaaggccg ccatctagtt gccacaaaag acattctccc aggagaactg 148260 ctggtgaagg aagatgcttt tgtaagtgtc cttatcccag gagaaatgcc acgacctcat 148320 cattgccttg agaacaagtg ggataccaga gttaccagtg gagacctcta ctgtcaccga 148380 tgtctgaagc acactttggc cacagtacct tgtggcagct gcagctatgc caagtattgc 148440 agccaggaat gtatgcagca ggcatgggac ctctaccata gcacagagtg ttctcttggg 148500 gggctgctcc tcacactcgg ggtcttctgc catgttgccc tgagaatgac tcttttagcc 148560 agatttgaag atgttgatag agttgtaagg atgctttgtg acgaggttgg tagcacagac 148620 acctgtttac ctgaaagcaa gaatctggtc aaggcatttg attacacaag tcagggagag 148680 agtgaagaga agagcaagat aggtgaaccc ccaattcctg gatgcaatgt caatggaaag 148740 tatggaagta attataatgc tatcttcagc cttttgcccc atactgaaaa gcatagccca 148800 gaacacagat tcatctgtgc catcagtgtc tccgcactgt gcagacaact caaagctgac 148860 agcgtgcagg cccaaacctt aaagtcccct aagctgaaag cagtgacccc agggctgtgt 148920 gcagatttga ctgtttgggg agcagccatg ctgcgacaca tgctacagct gcagtgtaat 148980 gcccaggcaa taacatccat atgtcacaca ggtaagtcag aaatggtttt tacttacatt 149040 attggtattt caagagctaa tgtttaagga gaaaaacact ataaaggaag cctggcatca 149100 aataaatcag tgacctaaaa ggaaaacaca gccgtcttat atatcattat gctattgaga 149160 agctttgagc acatttctgt gaacccagag cttgggaggt ggagatagga tgattaggag 149220 tctaagacag ctttagctat acagcacgtt tgaggtcagc ctgaactaca tgagaacttg 149280 tctcttaaaa acttgagcca gagccaggtg gtggtggcac atgcatttaa ttctagtact 149340 caagaggcaa aggcaggcag atccctgaat ccagcctcgt ctatatagtg agatccccac 149400 caggctacat agtaagatcc tgtttcaaat aaataaatat aacaaaaaca gcaataataa 149460 caatagcaac aaattaattt tttagatgta tttatttatt ttatgtatga gtacaccatt 149520 gcttttttca gacacaccag aagagggcat tggatcccat tacagatggt tgtgagccac 149580 catgtatgtg gttgctggga attgaactca acacctctgg aagagcagtc ggtgctctta 149640 gccactgagc catctctcca gtccattaat taaaaattta aaactagagt atttttaaac 149700 atttattcat tttgtgtgtg gtatacatac tataatacag gttcataagt caattctctt 149760 ctaccatgtg tgtcttggag atcaaactca ggttcttagg catgggagca agtatttact 149820 tcctgaacca tctccctagc catttctagt attcttttct tttgtcttga aagatttatt 149880 tattatatgt aagtacactg tagctgcctt caaataccgg aaaggggaat caggtcttgt 149940 tagagatgat tgtgagtcac catgtggttg ctgggatttg aactcaggcc ctccagaaga 150000 gcagtcagtg ctcttaactg ctgagctatc tccagcccca tttttagtat tcttattaag 150060 tggtttccat tttatccaaa gatgccttta agggcctggg aagatggctc agtgggcatt 150120 gaacttggtg tgtgagcatg aagaccagag ttcagatccc tagcacccag gcagatgctg 150180 aatgatggtg gcctgcctga gattccagga caacggagac agacaggggc cctagctaac 150240 catactacac actagctgag ctgtgtgctc aagagagcag ccctggctta ctgtgcagga 150300 tggagagtga tcatctccac aggcaagcac acacctgagc acacagacat gcacaaagga 150360 aagaaaagtc cttttaaggt ggtggtggtg ttgttgtttg ggggtctttt gttttgtttt 150420 tttctccccc tccctcattg tgatggcaca ttcctttaat ctcacatctg ggacaaagag 150480 gccggaggat ctttgtgaac tggaggtcag cctgttctac atagcaagcc catttcagcc 150540 aggacgacat agatataccc tgtctcaaac agacaaaaat tatttatttt atatatttga 150600 atgttttgcc tgcatgtatg tctatgcaca ttatgtctgg tgcccatgaa agccagaaga 150660 gggcatcaga tctctcagaa ctggaatttc agacacttat caagtactgc ctgagtgcta 150720 ggaatcaaac caaggtcttc tggaagagca gcaagtagtc tttttttttt ttaatatttt 150780 tttattacat attttcctca attacatttc caatgctatc ccaaaagtcc cccataccct 150840 cccccccccc ccccgagcag caagtattct tcattgctgg gccatctccc catctccttt 150900 tctagttaat taagctgaaa gggagggagg tagatgttgc ccaaacttag gatttattga 150960 cagattaata ctctgttagc ctaactacac tatagaagct tattctttag actttcacat 151020 tacactgtcc agattttgcc atcctttttg ngtgtatatg tctacagatc ttaattcagc 151080 tgccaattta tacagtgttt ataggtattc tttgtgacgt ggatctttta cccatcttaa 151140 agcagtagga tttgaaagct gacatttatg tggcctatgg tcctgttaaa tcacatttca 151200 agttagtctc tgtggtacac attttggggt ctatctgcgg ttccgcatct cacacttttc 151260 cctctcaggg tgtccagaag ctgctgcaca ctgggctgga aggatgaagt ggagtccaga 151320 gtgagtggaa ttctgcagca tcccggtcca gctgggagtg aatgctgggg tcaggaggag 151380 atgggtgaga gggccttctc caagggcctt cttagtgtta cagctctagg caaaggcctt 151440 ctctgacaat cttagcctgt gcatagtttt ttattcgaga tgagcttgta tgcatacact 151500 ttattggcag taaatcagag gttatccact cttagggaag gagataggaa tacccaaggt 151560 ggacagaggt cattggctga aggataacgt actgagatgc tcattagcac ggggaggcat 151620 cccaggaatc tcaggtgctt gctgactggg tttcttaggg ggttgagagg gtagcagtga 151680 tttcaccaag gttatgtatg gcagagggta taggggtttc aggctcccca gacaaagaag 151740 gagaaggaga agccctgctg ataagggaag tcccccattt tgagccactt cagaaggcta 151800 tcaagacact gatagacttt gtccttaatt agcagggccc aacaagtgtc tgttttcttt 151860 tctgccttca ttggctcttt gagccactgc tacaaaatat ccaaatctgg gccaggaagc 151920 tggctcggct agtaaaggtg tttgcctcta agcctgaagg cctgagtttt cacttgattt 151980 ggtttggttt ttgttttttt gagacaaggt ttctcagcat agccctgggt attctggaac 152040 tcactctgta gatcaggctc aacttgaatt cagagatctg cctgcttcta catcccgagt 152100 gcttagatta aagttgtgcg ccaacactgc ccacctaaaa aaaatatgag gggctggtga 152160 gatggctcag tgggtaagag cacccgactg ctcttccgaa ggtccgaagt tcaaatccca 152220 gcaaccacat ggtggctcac aaccacctgt gatgagatct gatgccctct tctggtgcat 152280 ctgaagacag ctacgggtgt acttacatat aataataaat aaatcttaaa aaaaaaaaaa 152340 aagaacacta ttcacaaggt acaacaacag tgtactagga aactttaaaa tagcccatca 152400 ttcattctag gggaaaaatc tttttaaact ttagtgtgta agaaagagag aggggctgta 152460 gaaaggccac agcacgtgta tccaggttag gagtcaactt ttcagaagcg gagtctcccc 152520 ttctacctgt ttttgaggca gtctcttgtt tctgccctac actttgtaca tgaacttcaa 152580 gatggttgtt ctatttctgc ctctcatgtt gccctatgca tgctgagctt actgatgcca 152640 gccaccacat aagcatggca ccagcactga gccaagggca ggcatctggc tcacatggag 152700 agttacccat caagccatct tgctagcccc agaaaatgta tttttgacag gtgtggtggt 152760 gcacatattt aatcccagca ctcaggaggc agaggcaggc agatctctgt gtctgaagcc 152820 agcccagttt acaaatcaag tcccagaata gccaaggcta catagagaaa ccctgttttg 152880 aaaaacaaac atccttctgt gttccagtca caatgactgc tgtaacaata atatgaggat 152940 ttgggcgtgt caaaaatcac aagtagcaag gtgtaatggg caggccttta gtctcagcac 153000 ttgggaggca gaggcaggag gatctctgtg agtttagcac agccagggct gttacacaga 153060 gaaaccctgt ctcaaaaaaa ccaagcaaaa atagaattac aagttaacca gggtattggt 153120 gttaatatga aatagcagaa ctcaagatag ctaatgaaac aaaggattct attttataaa 153180 ggagacattc catactgaaa tatatgcaga gcctgatgct tgcctagcaa ctgaactaca 153240 cnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 153300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngcctgggca agagtaagca 153360 aaccgtcagg ctcagaggca ggatggcagt gatgtgtttt ggggcacaag gccccctttc 153420 attaaagcac caaatcttgt actaaaaaca gccctctgct ctcgatctgc agagatacga 153480 gagaggcaca catagcttcc gggcccttgc cctctgctct cccggtcagt tcctggactc 153540 ctgggagaag cttgaagctc aagaactggc cgctgttgcc tttgtgctga cgccagaacc 153600 agcagctatt cagcagctgc ggctcttggg caagcttgga agtagctcgt tccttctcct 153660 tctgcagggg aaagacaagg agatggcact gagctgggta gccaggatgt gggaagtaaa 153720 ttgtgtctat gtgggtgagg gaagtgccgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt 153780 gtgtgtgtgt gtgtgacaga gacagaaagg gagagtgcat gttcttgtgt gttcctgtga 153840 gtcagtgctc cctttggctc ctcctgccaa aaagcatgct gtgttctcag agtcaagctt 153900 ggccaggctc gcccacccca ccaaagccca ggatctgcca ccccattaag aatgcggggt 153960 taggaaataa aaaacagggt tcttgtccaa gagaattttt attattattt tttctctctt 154020 aattggattc agcatttctt actcctcagt atcctctctg gtagggaatt caggtctgtc 154080 tatgcagagc acaagagact gtgcttgcca gaaacccttg ggccaacagc cctattccct 154140 gactgggctt gcctgcaggc tgccttctgg gctgacccct gagtctggcc ctctgacctc 154200 tgcccgtcct ggggtcatcc aggaggagca ggaccactgt gatacagggt tcctgagatg 154260 gctactgaaa caccctcatc tgctaaggcc actagatttt tttcccactg ccagactact 154320 cagggccctc agtaggtcac tggccaaaga gcctagatgt taaaactgca gctaaagcct 154380 ctcctgaggc cagagctcag agcctccctg gcctgcacaa agtgctaaga aggacattgt 154440 ccatccaacc cattggacta acactgtaga cgctgccttg tctgccagct tgaaaacccc 154500 agaaacctct tccccagctc cgcctagctt gcctccagca ccctgacatc cacttctctc 154560 gctaatgtct gcagcttcta cagtaggggt gacggggtgc tccggggtgc cagacaggcg 154620 tgtgttgcat ataaaaacga ggtgatgttc taagtatcta agaatgttgg tcccctgaag 154680 tgattcttgc tgcttctctt ccttcccgac tcttcccact gacacctttg cccccagcaa 154740 gcccagggac tattgttgct ggctggggtt ctgaacaaaa ttgccgcagt tttttgtttt 154800 tttttttctt gctagaagtt accgatacag tccttaattg agcaaatata ggttcccgta 154860 tagtttataa acatgataag acatacagtt tggtaaggag tgggttgggg gacctgtgca 154920 tttatatatt tatatatata tatatattat gtgggtgtgt gggcagagtg aggatatata 154980 taagtggaca taggtataaa actgcacctt ctgtgtgact ctcattgcga gtacagttct 155040 aaatgtcatc cactggcgat ctctcctttg gtgattggtt cttggacccc agcaggtctg 155100 ccgggggctg cctgagacgt caggaatgag aggcattacc cccatggata gggactgagg 155160 gtggcatagg gttggacagg gcaggttaac taagtgatct cagacaagag ccaagagtgc 155220 tctgagattg ctggttgccc cagctggctc tgggagagcc ttgttctgag tcctgctcct 155280 tccaaaacca gcagggtcct tcagcccttc tctccaaatg accaggcttc cgcagagccc 155340 agcttcttca aggggcgcat gtcccgacac cactaatgac tcactttgcg tgcctttgac 155400 cactgtgctg gagtggatac ggtccagagg cgctcggtca ggacagccga gtgagacgtg 155460 atacccttcc cgtctacggc tgtacatttt gggcttataa tccaccagga agggtccaga 155520 cggtggctga aggcttcagc agccttttcc tgaaacccag cagatcttcc acttaggaaa 155580 aaaaaaagaa agaaagaaag aaaagaagaa aaaaattctg ttccttgctg gacatgtggc 155640 agtgctgggt gacggagccc tgggtcctca gcggagagtg actgccagcc ccagtatcca 155700 ggccaggagt ggggccccaa gggccgtgcc tgaggatgcc tgctgcaccc cactgggggc 155760 acggatgggg gtcctgcgag cacacttgcc cttcctcttg ggtcgtgcgg tgggcatgat 155820 gtcaaagctg aacttgtgct gatagtcggg ggcatagtcc tgcagttcgg taagctcttt 155880 cccagagctc accttagaga tctggttccg gttcctgtgg ctggtgcagt tcttgcctgc 155940 cttcttgtaa cctgacctgg agccaggcgg atggccatgt gggtggcctt tgtccctgga 156000 ggccccatgg gacggatggt gctccttgcg ggcagccctg tcagaggtgg taagcgtgtg 156060 agacttgatc tggtgaggag acactggtcc tgtgcagttc cggaagtcct ccaccctcag 156120 cagcttcaga tcctggcctt gccgcagctc gggggtcgcg caggggacag cagagctaga 156180 gccacggaac cttcgcagcc attcccacag ggaacgtgcc cggcagccac agtcccaagc 156240 attcccattg aggcgaagga actccaaggc caccaggggg gccagacagt caccctgcag 156300 ctcagtgagg ctgttgttga agagaaagag ggtggttagc ctgtggaggt catggaaagc 156360 cttgtggtga acccactgta gctggttctc atgcagcagc aaccggtcca ggttcaccag 156420 gccccggaag atgccttggc ccaggctcca tagcttgtta ccatggagaa acaagtgact 156480 gagattgacc aggtccacaa agatgtcatc ttggaggtac tcgatatggt tgtcctgcaa 156540 gtagagatac tgcaggctgt gcaggccacc aaagatgcct gcgggcaggg cgctcagtcc 156600 acacttatag aggtagaggg cgtgaagctt caccaggcct tggaaggtct cgggtgccag 156660 cgttcgcagc tgtcggttgt ctccaaggtc tagctcctcc agatgcacaa agccctcgaa 156720 ggtgttggga gcaatgaaag tgatgttgtt ggagtagatc cagagggtga ccatggcggg 156780 gctgaagtgg ccctgctgga ggaaggtgat gcgattgttc tgcaggaaga tgcgctcact 156840 gtcctctggg atgccctccg ggatggcagc aaagttgtgt gcctggcagc tgacagtcat 156900 gggcgcaggg tagcacacac agtctcgagg acaaccacca cccagaggta gctctccagc 156960 gagcagcaac agcagcaatt ccacacagca ccctggtggg gagagacaga acagcagtga 157020 ggggctgccc agaggaggtg gagatagatg gggaacagag ggtggaatgg gggactggca 157080 aatgactctg ttggctcaca gaggttctgt cctctgtatt gtatgcaggg gtcccttgga 157140 cagggcattt ggggccaagg cccacattat ctcctcacct ctttagctct gtccctaaag 157200 tctctaattc catccgaaca cttcttcaga ctgtcagccc caccgcaggg tggaacacac 157260 ttgtgaacac aggcgagtcg gcctccggct ctgggtccgg ctctgccact cgctcactgt 157320 tagctgcctt agcaaggaat gactctaaca aagcaaatct ggagtcctga atgatcactt 157380 tatttaaata attcctcaaa ataaagaaag cattgagtcc atggtaccaa agcatgcctc 157440 aataagcgcc tctttcacac tgtggtacaa aaacttcaaa cctacaactc ctccatggct 157500 gtttcctcat gagttaaaca cagttcacag ggctgtgtgt acagacaagg cacatttctg 157560 tgaggggctg tgagtgacac cagggctgac gcacgaggct tcccttgggg ttcacagtac 157620 tgccagatcg aggctgcatg ctcctctccc ccattcacac ccccccctcc tgtgctggag 157680 attgccaggc tgtggctgta aaaccgggcc ttgcctcttg actgtccaga gcatttcctc 157740 tgtagcttcc ctctaattgg gcattaatta ggcattcgtt aatggatcct taaaataatt 157800 attttcggat gtgtccagcc tgtggtcggg taataggcct atgctcatta tggaagccgc 157860 ctcattatgg acgattgtca ttacctgcct ttttccaggg tcacagctgc cccaagtggc 157920 ccagcaggcg cgtcaggaag atggggacag gctccaggcc tacgggcgcc caccctgaag 157980 ggccaggcag ccacgaccta tgtcgcctca gttggcctct tgccccttct tttccagctt 158040 gttcagctgg gactcttggg agagccaggg cccctggggg aatatgagct gagctgaatc 158100 ttcttgctgc tagctgtgct cagagcaagt ggaaggagca gggaccttct gaccaggctt 158160 cccacttggg gtcccaggcc cagggactgc ccaggccccg gcagagtagg ttgccacctt 158220 gacttctgac gccccccccc cattcccaac agaaacagca tcgtaagttg acagctccca 158280 gctgttggga gttatgggct cccagagagt ggcagctgct tctcgtccct gtaatcaccc 158340 ggcttcagct agaatgtttc tagcacataa aaatcatcgc atataattta gtttttgcat 158400 aattgggttc agttgtgatt tcagagcaat tatgacctca gcagcagggg tggcacagca 158460 taggacccct ttctgggccg ggccatgccc tgcaggccct ctgcagtgct ttctgcccac 158520 cggcccttag acagcatgca ggctataacc atctctgcct caatttcctg ctccaaaacg 158580 tgtctagatg ttactctgtc gatcttcctc ctcagcatcc tgggtgtggc ctccagcctg 158640 ccagcctctg tcctagggat cctgtgctgc agagggaggc acagtcggag ggaggggagc 158700 ctgccctgtg ccaccagcac tcacactggc tgcacagtcc acagacccac agctccaacc 158760 tccctgcttg gcttgcaccc tctcttccag gaaggccatt cttgccagaa cctttcccaa 158820 cggtcccctg ggaaagcctg gactctaggt tcaaggacat tcatgatgct tgccccacat 158880 tttatgctgg atgagacaca gcagagcctt cttcactggg gggtcctgtg aaaatgaaag 158940 cttttcttcc ccggcctgca gctgcaggca ggtaggggtt gcagtgggct tatcactaat 159000 accattcgac atttgtacag ctcatcggag tttacagagg gcttttgttg taccctcaac 159060 ttcccctgtg ttctcccacc tactgtggct gctctgtctc tgtgcacccc aaaagaatct 159120 ggaagtccct tggggagatt acccccctta cataggggcc tccaaggaat acaggctaca 159180 gctctattta ggaaaaaaaa aatcaaactg aaccaaacct caggtgtgga cttagtaacc 159240 agtttataaa cataccgtgg caagtggagg aggcaggcgg cagaacggca tgaaggtagg 159300 actggggttt tccctctaaa agggcaatgg ggggacaaag ggactctagc caagacctga 159360 tgcagaacca ggctcagttc ccctgttatc tcaaggctat catcactagg aggcctaagg 159420 caatggacca caaggacctt gtccttgtag gacagtcact tcctgcagtg aagtgctctt 159480 ctggaagcat actaatagga tgtaggctca ggacagctgg tctctgtcct ttagtatttt 159540 tccacatgcc agggatgtta accttccaag cctccatctc ttctaatggg gggggggtgt 159600 tgaggggctc agccactctg catccatgtc tttgaaagcc agtggtatta ctccaggacc 159660 ctgagcaagc tgtcctagtc agccctggcc acttctggac tccttgcctg agtcagtagg 159720 tgccaatcct aggattgtca ccagcaggtt tcttcctagg gaggcaagca ctgtatcacc 159780 atggcgcctt ctatgccccc tctatgaggc ccttgggagc cccgccccac tgattgcctg 159840 attaatgtac caacaatgag gatggagcct ttgccatgca ttttaacatt gcaaattagc 159900 aggaatccaa gtctctgtgg aggggccctg cacctcttct gccagactca tcaagcgcct 159960 cttgggcagg gctgccttct acttgagggg gcggaaggga gaagacccag ttccactctc 160020 cttcccctcc aggaggtgcc cttcatcgtg ttctgcttcg ttactctcaa gcctccggcc 160080 tcccacgcac gtgagctccc aaggggctct acagcctccg tcattccttc ttccattcat 160140 acttgccccc tagtctagga gagccatgga agacagtgtg ggaagggctt gacaatgagc 160200 atcatgcccc atttgcatat gcggtggcaa taccctggtg ggtaccagga gagtataggg 160260 gaaattaaga gaggggccta aggaaagcct ctgctatccc tgggctacca gtcagcattg 160320 cttggtcact gatcccctct gtaacaccag cccttctgca acctgccaga gtttttgacc 160380 tttgaactag ggctgagaag ggtctgctct gttcagctgc cttggctggg aggggaatct 160440 gctcagacct cagcacacac tcaacagaag gcatgcaagc aagggagcta gcagtggcct 160500 tgggtcagct ggcaagcccc aaactcttcc tgccaagctg agcatgaaaa gccacctcac 160560 catggtccca tgggaccaga cctggtagga taggtggcaa ggctaaggca gcggaatagc 160620 atgtgcaaag gcactggggt gggaaagggc ctgtgcttct caccccctct aatggtgcag 160680 agcctccaag gaatactgta acctcagctc agctgggctc gggtggccag agagcttggc 160740 accagaacca gcatcaacag ggcctgtctg ctaaacccag acctcacaag ccagtttagt 160800 aggggccctg tagcaccctg gccaccagaa ctaacgagga agatctgacg ctgggaatat 160860 gtctttaatg aaaagccctt ccggaagcca catttgcaca gaagaaaatg aggtgcccag 160920 agcatcagtg ggctggttgc agctggagaa cacagcaggg ggacaggtcc taccaagcta 160980 ccctgccttc aggctggggc tctagccagc tccctgatgc ctggagtagg taaagcagcc 161040 tcgaaatggg ctgggtcagc tttttcaggc tccaaagggt caggacagct gctgcagctt 161100 agcacccaag ggggctgccc ctctacccct aagtagaggc atccccatgg cccctgggca 161160 ggtcagtggg tctctctgaa gctttgtagg ctgcttcttg gccatgtagc caatctctct 161220 ggccttcagt cctccctccc tgcccccagc ctggccagct gctcttctct gagcaatcga 161280 tgttaaccga atgctctctt gctgtgggga tggcggcctc aggccaggcc agctgcactc 161340 ctggggctgc tggcgcctca ggccacttgg cacttgtgcc acttgtgttc taaacacagg 161400 ctccttcctg gctcggccct gaagacaaga aagctggcca gggaacagct gggctcccat 161460 ctcagcctcc actgctgtgc agagcggccg gcagcctcct atccatggct gtgagtagaa 161520 cagggctgtg gagccagagg cctaagttga atcctggctg ctccttttaa tgcttgcagg 161580 agcctcgttt tcctcacctg caaaatgggg cagtcattgg aagctcagcc agtcctccag 161640 cccacagata ctggtaccca cctgcccttc ccacatctcc atctgtctaa ctgaaaccat 161700 ccaaaccaag ctcttctctt cctctccggc ctcctccgtt cacaacttct ccatcttggg 161760 taaagatggt ccctttacat cagctgcctg ggaggaacac ctcagaatca ccgtggctca 161820 ctctggggaa attctgctgg ctagatttta gaatgtatcc agtatctatc cacttatcat 161880 actcgctatt gctaccattc acccagtagc ctcctggatg ccctcccccc ccccccgcaa 161940 gccctgcctc ctcaccccta cacctccttc aacaggaact agggtagtcc agggaaagtg 162000 agtcaggaag ggctgctcct tagtctgcat cctccagagt gcccatctaa ctagaagctg 162060 ccccaggtct ttctcccagc ccagaggccc tgcctctccc tggctacaca gaccttgctg 162120 ccgtccctcc cacatgccag ccctcagcct ccctcctgcc tttgtccatg ctgttccatc 162180 tacctggacc ggttcccagt gtgtctgcag ggctgcttcc cagagaagcc actcttgagc 162240 agcgatttgc aaggagcttc tttcctggga cattttactc tacagcacgc gacctcttgg 162300 acagcacgac ataagtcact tgtttccttt atgtccgctg ccactttgtt ctgatgagtg 162360 tgctccctgc acacagtagg tgctcattaa cagttctggg ggagggaatt accttctcaa 162420 gtctctggag aattgaatga tgacactcag gaagccctag gctcaagcct ggggcctgga 162480 tcatagtagg tgctcgataa atgttggttg taattagtcc tgggagactc agagccttca 162540 ggagaacaga cacctgaact tggctcacat caagactcct taggcccatc aaggaagtga 162600 ccgtttgttg gatgagcact ctgagaagga cccaatacca gtcctttcct gggcaagggg 162660 aaatagactg ggactgggga gtttcccaag gtatgggatt ttcagcacta aactggaata 162720 atgctaaaga aaaaaaaaag ttagtcactc taaaaggggc caggaccaaa acctttcaaa 162780 cagaaatgtc tgggtttatg aagagaggaa gccagatatg gtggggcaca tctttaatcc 162840 aggtgcttgg gaggcaaaga cagatggagc tctgtgagtt tgaggccagc ctattctaca 162900 aagtgagttc taggacagcc aaggctacat agagaaaccc acttgacttg ccacccaaat 162960 taaaaatctt agtgggggag cagtcaagga ggaaacacac acacacacac acacacacac 163020 acacacacac acgttagtat aatatcatac tatggctctg tgcctgcagt ccaggaatga 163080 gggctgaact cagagtgtta gtgtgtgcta gtggatattt gagctctgta tttatgtgca 163140 tgtctgtgta gatgtgtacc tgaggtgttt atgtgtacac aggtgttggc ctgttgcata 163200 tggatggaga catggttgtg tttgctaggc atttgcatgt gtctgagttc atgcacataa 163260 actcacatct acctctggag actgagagtg acaacccagg gcccttttat cctgcagcac 163320 cccaggccca gcaccccgac ccagcatccc aggcccagca ctccagaccc agcaccccag 163380 gcccagcatc ccacgcccag catcccaggc ccagcacccc agacccagca tcccaggccc 163440 agcaccccag acccagcacc ccaggcccag catcccaggc ccagcacccc gacccagcat 163500 cccaggccca gcaccccagg tccaagcact caatgcccag caccccgacc cagcatccca 163560 ggcccagcac cccaggccca gcatccaagg cccagcatcc cagggacagc accccaggcc 163620 cagcatccca ggcccagcat cccagggaca gcaccccagg cccagcatcc caggtccagc 163680 atcccaggga cagcacccca ggcccagtat cccagggaca gcaccccagg cccagtatcc 163740 cagggacagc accccaggcc cagtatccca gggacagcac cccaggcaca gtatcccaca 163800 tggaggcagc acatactgaa gatagggaat gtctctgagg cctcttatct tggtccttac 163860 cctcattgct ttcagcacct gctctcctca cactcggaat caaacaccct gtgcaggttc 163920 tcccagtacc aggattcccc tcagctgagg aatgggtagc taccattttg gcttttgtct 163980 gtctggggtt ggcagcccca tgctaattgg actgacagtt tctcctgaga gcaatttggg 164040 cagcacatcc tgcccattag gcctaacctt gcctgcaggg gtgtgctgta ggggcaggga 164100 tggagcctac cctgtatagc tctgtattga ggcactcccc caagctatga cccatgccag 164160 tgggagtcat ttcacctagg caactccaga tgggcacaaa aatctctcca ataagggtag 164220 gtatgggaat aggtaaggag agcatagtga gcctggctgg gcacctgaga cctgagcagc 164280 ctgcacggga gattgtgtca ctgtggttcc agactgccaa gacatcttgg ctttcacccc 164340 aactcaggat ggtccagaat ccagagctct taagagagca gatgctgaga ggcacttaac 164400 ccagggctaa gacccttcct tggacggttt cttggctttc tactctgtcc tctgtcccag 164460 tctgtcatcc ccatctgtgc ctaacagctc tctgtggaaa acatgaggcg tatgagctct 164520 ctacttctcc cagcatccca tgcccgcacc ccagctcact gtgtgccctc atgttactca 164580 aatcttctgc taggtttgag ggccccaggt tgaggctgtg ggtttccctc catctgtccc 164640 tcccttttac caccaccact aatcctcttc ctcctcttcc tcctcttcct cctcctcctc 164700 tttctncncn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 164760 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnna caaaggacgc 164820 aaattgaact tacagaggaa acacaaatga gctgtaatga cagaaaagga atactatttc 164880 ccttgtgatc agaaagctat agattaaacc ccagcccttt ttgcctttgt ttttcatatt 164940 tagtggttct ggggactgaa cccaaggaag gacagctgct tggaatgttg gagacctctc 165000 aggcattgct tatgctaagc tgacaaaggc tttctgaatg catatggtag tatctattca 165060 tcttaagatg cctataacca tgggtggagc aattctctaa gagtctgtct gaacttctta 165120 agcatgtgct caaaaacaca cataaagatt gtttactgaa gcagtatttg caacagtgaa 165180 acaaaaccaa acaacaacaa aaagtactta atagctcatc agtaggaaaa agaacaacaa 165240 attgggacac atttatacta ttctcgttct atcacacgac tattaagaag attaaggtgt 165300 gaggatgaaa agcccctgca gaacaacgca cacaatgaaa ttgaatagag aataaagaaa 165360 aaaatatata gtgtatttat gtaaaacata cataaacaca taaaaagaat agaaaactct 165420 ttgactgtat atctttgaaa agaaccagca gagtgctatg gtttataaat gaaaagttcc 165480 cccaaaattc ctgagttgga ggttcaatgc agagttcata ctcttaggaa gtaactaaat 165540 caggaatgct ctgatctaat caatggattg accaactgat agattaataa tctgaaggca 165600 acacttccag gaggcagaaa caaggtgagg cctaactgga ggaagtttgt caccagaggc 165660 atgtccttgg agaggctacc tcatccttga tccttccctt tctccgcttc ctggctgtaa 165720 aagtatttat ttgactgatg taactgtcat cttggaggct tactggctcc atcagctaac 165780 ctaggcctag ccctggaagc ttctagcttc catacaatct aatccaagcc tagaatgttc 165840 cagcctttag gacttgctgc tgagatcacc gtttcctgtt ctttctgaac tctagctggc 165900 tgattcagtc cccctgttcc gggctcaaac tcctctcccc gatgatttta ttcacaatct 165960 gtcttttctc ttggcctctg aattgctctg cttggtctca aactaactct agcaatcttt 166020 tctaatctct tgtctccttc acactctctt gcttgttctg tctttactgt gtctagtttg 166080 ttctctcttc catccttctc tgtaaagctc tcccggtaaa cctgcctcct cctccccctc 166140 tgtgccgctc tactctccct ctcagctcta ctgcactgct ctccacagct ctcctgtatc 166200 ctgtgctgca ctctcttctc cggtaccacc tgtgtctccc ttacgtagct tccctttcct 166260 ctctcttctc ctgagggttg ggcagatcct atcctgtcaa acctttctct gattcttcac 166320 tttgtctgcc actcaattag acatcacttt caagcaggag tgcctcctct acaaaccaac 166380 tttaccttca ttgtttcaaa ttaaaggtga gtactaaggg tgtgtctctt tttcagccag 166440 tgagagtaaa gatgtgtgct aataaggctg agccaactct agctagaaat agtttctttt 166500 tctccataaa taacagaatc ttagggttca caatacgatc aaatatcctg agacagctgg 166560 ctgccatgtg gtgaagagaa tgcagagagt taaggatgtg ctcttgaggt ttcagatggg 166620 aataagcatt ttcttgggag ttggattaga gtcattcctg tgacactgtg acaaaggact 166680 cgactacatt ttgccatgcc ttgagactgt ggagggctga gcgatggatt aatacactgg 166740 agtgaatttt aaggcagcca aggctgtggt tggactgtta ctagctacgt ttagctggat 166800 ttatattaag aattgggaac aaaaaagcag agtagaaagg acagttttgg cagaaggagg 166860 tttcttcata tacaacttag tttttatagt tagcttttta tgttgctatg accaaaatac 166920 cttatggaaa ctgaagaata aaaattttta ctgaactctt cttcacccca gaacccgacc 166980 cctcccatct agagattgtt cccggaacac tcctgaactc ttcaccccag aatgctttcc 167040 tgaactcctc accctagagt tcgaaccctc ccaactaaaa actgttccaa gaacattttt 167100 gagataaggg cctcctaaaa caacctcaaa atgaaccggg tacattgcca aataatagga 167160 catgacccct tagttacgta gattcccttg gcagaacccc ttgtcccttg acagaacccc 167220 ctagtgatgt aaacttgtac tttccctgcc cagctctccc cccttgagtt ttactatata 167280 agcctatgaa aaatttggct ggtcgtcgat tctcctctac accactaggt gcatgagttt 167340 cgaccccaga gctctggtct atgttccatg tgctttcttg ctgttgttct attaaatctt 167400 gccttctaca ttttgagtac ggtctcagtg tcttcttggg tccgcggctg tcccggggct 167460 tgagtgcttg agtgagggtc tcccttcggg ggtctttcat tttggtgcat tggccgggaa 167520 acagcgcgac cacccagagg tcctagaccc acttagaggt aaggttcttt gttctgtttt 167580 ggtctgatgt ttgtgttctg tttctaagtt tggtgcgatc gcagtttcgg ttttgcggat 167640 gctcagtgag accgcgctcc gagagggaac gcggggtgga taaggataga cgtgtccagg 167700 tgtccaccgt ccgttcaccc tgggagacgt cccaggaaaa acaggggagg accagggacg 167760 cctggtggac ccctttggag gccaagagac catttggggt tgcgagatcg tgggtttgag 167820 tcccacctcg tgcccagttg cgagatcgtg ggttcgagtc ccacctcgcg ttttgttgcg 167880 agaccgtggg ttcaagtccc acctcgcgtt tggtcacgag atcgtgggtt cgagtcccac 167940 ctcgtgcaga gggtctcaat cggccggcct tagaaaggcc atctgattct ttgagttgct 168000 tgtggtcgac gcagagtcgc cgccgtttct ggtttctttt ttgtcttagt ctcgtgtccg 168060 ctcttgttgt gtctactgtt tttctagaaa tgggacaatc tgtgtccact cccctttctc 168120 tgactctgga gcattggaag gaggtgcggg tcagagccca caaccagtcg gtggaagtca 168180 gaaagggtcc gtggcagacc ttttgcgcct ccgagtggcc aacgtttaga gtaggctggc 168240 cacctgaggg tgcttttgac ttgtcactaa tcgctgccgt caggcgaatt gtttttcagg 168300 aggaaggggg tcaccctgat cagatcccct acattgtgac ctggcagaat ctcgtccaat 168360 tcccacctcc gtgggtcaag ccttggaccc caaactcttc gaaactgacg gtcgcggttg 168420 cccagtctga tgcagccgga aagtctggcc catcagcacc ccccaagatc tatccagaga 168480 ttgacgacct cctctggata gactcccaac ctccccctta ccccctgccc caacagccac 168540 ctgcagctgc cccaccacag ggaccaatag cgagaggggc tcagggaccg gcgggggaga 168600 ctcggagtcg ccgaggccga agccccgggg aggaaggggg gccagactca acagttgcct 168660 tgccactcag agcacatgtg agagggccag caccaggacc taatgatctc attcctttac 168720 agtactggcc tttttcctct tctgatttat ataattgaaa aactaaccac cctcccttct 168780 cagagaaccc ctctggactt actgggctcc ttgagtcact tatgttctcc catcaaccca 168840 cttgggatga ttgtcagcag cttttgcagg ttctttttac aacagaagaa agaaaaagaa 168900 tcctcataga ggcgagaaaa aatgttctgg gagaggacgg cacacccact gccctcccta 168960 acctcgtgga cgaggctttc cccttgaacc gccccaactg ggactacaac accgcggaag 169020 gtaggggacg cctccttgtc tatcgccgga ctctagtggc aggtctcaga ggagccgcta 169080 gacggcccac caatttggct aaggtaagag aggtcttgca ggggcagact gaaccaccct 169140 cagtcttcct tgagcgtcta atggaggcat ataggagata cacccctttt gaccccttgt 169200 cagaggggca gagagccgct gtagccatgg ccttcattgg tcagtccgct cccgacatta 169260 agaaaaagct gcaaaggctg gaggggctcc aagatcatac gctccaagat ttagtaaaag 169320 aagcagaaaa agtctatcat aagagggaaa cagaagaaga gaggcaggag agagagaaga 169380 aagaaataga ggagagggaa aatagacggg atcgccgtca ggagagaaat ctgagtaaaa 169440 ttttggccgc agttgtgaat gatagacagt caggaaaagg taaaataggg ctcctgggca 169500 acagggcagt gaaaccgcaa ggtggcaaaa agataccact ggaaaaagac caatgcgcct 169560 attgcaaaga gaaaggacac tgggctagag attgccctaa aaagcgggag cgatccaagg 169620 tcctaaccct agaagatgat tagggaagtc ggggctcaga ccccctccct gagcctaggg 169680 taactttgtc cgtggagggg actcccgtca acttcctgat agacaccgga gcagaacatt 169740 cagtactcac taacccccta ggcaagctag gctccaaaaa gaccatggta attggagcca 169800 ctggtagtaa attttacccc tggacgacca aacgagctct tcagatagac aaaaatatag 169860 tgacccactc ctttctggtg atacctgagt gccctgctcc cctcttgggg cgcgatctgc 169920 taaccaaact aaaggctcaa gtccaattta cttcagaagg cccacaagta agctggggaa 169980 aggcccctgt tgcctgcctt gtcctcaaca cagaaaaaga gtaccggttg catgaagaac 170040 aacccaaaaa tgcagtctct tcaggttggc taactgcgtt ccccaatgtc tgggcagaac 170100 aagcaggaat ggggttggct aaacaagtgc ctccggttgt ggtagaactt aaagctgatg 170160 ccacccccat ttcggtaaaa caatacccca tgagcaagga agctagaaaa ggcatccggc 170220 ctcatatcca gaggttgctg ggccaaggag ttttagtggc ctgtcagtcc ccctggaata 170280 caccacttct gccggttcaa aaaccaggga ccaatgacta tcgcccggta caagacctcc 170340 gggaggttaa caaaagggtc ctggacattc accccacagt cccgaacccg tacaatttat 170400 taagctctct cccacctgag agaacatggt atacagtcct agacttaaaa gatgccttct 170460 tttgcctgcg tttgcaccct aagagtcagc tcctgtttgc ttttaaatgg agggacccag 170520 agggcggaca gactggtcaa ctaacttgga ctaggctacc acaggggttc aaaaattccc 170580 ccaccctgtt tgacgaggcc ctccatcggg atcttgcgcc ttttcgcgct cgaaaccctc 170640 agcttaccct actacagtat gtagatgatc tcttggtcgc ggcggcctcg aaggagctgt 170700 gtcaccaggg aactgagagg ctcctcacag aactgagtga cttggggtat cgagtttcgg 170760 ctaaaaaggc acaaatctgt caaactgagg taaccttcct ggggtatacc ctccgagggg 170820 gcaaaagatg gctcacagag gcccggaaaa agactgttat gatgatccca tcgccaacta 170880 ccccacggca ggtacgtgag tttctgggga ctgctggctt ttgtagactc tggattccag 170940 gctttgcaac cctagcagca cctctatatc ctttgactaa ggaaggggtt cctttcaagt 171000 ggaaagaaga acaccaaaga gcttttgagg ctatcaagtc gtctctaatg actgccccca 171060 cgctagcatt accagacttg actaagcctt tcgtcctata tgtggacgag agagcgggtg 171120 tagccagggg agtattgaca caagcactgg gaccctgaaa aagacctgta gcctatttgt 171180 caaaaaaatt agatcctgtt gctagtggat ggcccacatg tctgaaagct attgcagcag 171240 tagccctgct gatcaaagat gctgacaaac tgacaatggg acagcaggtg accgttgtag 171300 cccctcatgc cttagaaagt atcgtgcgac agccacctga cagataagat gacaaatgcc 171360 cgaatgacac actatcagag cctgctgcta aatgagcgtg taacctttgc gccccctgcc 171420 atcctcaacc cagctaccct tctccctcta acaaatgatt ccgtcccagt acatcaatgt 171480 atggacatcc tcgctgaaga aactgggacc agaagtgacc tgactgacca accctggcct 171540 agagctccca gttggtacac ggacggcagc agtttcctga tagaggggaa gcaaaaggct 171600 ggagctgcgg tggtagacgg gaaaaaggta atttgggcaa gcgctttgcc tgaaggaaca 171660 tcggcacaaa aggctgaact tatagcgctt atacaagccc tccgagaggc taaaggtaag 171720 atcgttaata tctacactga cagccgatat gcttttgcta ccgcacacat ccatggggcc 171780 atctacaggc agcgagggct attgacctcg gctggtaaag acattaaaaa caaagaaaaa 171840 attctggccc tgttagaagc catacatgca cctaaaaagg tagccatcat ccactgcccc 171900 ggccacccaa aaaggagaaa acttggtggc caagggcaac cgaatggcag acttagtggc 171960 aaaacaagtt gctcaagggg ccatgatctt aactgaaaaa ggtgatccgc ccaaaagccc 172020 tgaggatggg aggtataaca taaaagagct atggtagacc agtgatcccc tcccatactt 172080 tttttgaaag aaaaatagaa ttaactcccg aagaaggaat aaaatttgta aaaggactac 172140 accaattcac ccacctggga gttgaaaaaa tgatgagact aattaaaaat tcccgatacc 172200 aagtccccaa cctgaagtca gtggctcaaa agattataga ctcctgcaaa ccatgtgcat 172260 tcactaatgc aactaaagcc tacagagaac ctggaaagag acaacgggga gaccatcctg 172320 gagtgtattg ggaggtagac tttactgaag ttaaacctga aatgtatggt aacaagtatc 172380 tgttagtatt tgtagacacc ttttcaggat gggttgaggc atttcccact aaaacagaga 172440 ctgcccagat tgtggccaag aagatccttg aagaaatcct gccaagattt gaaatcccta 172500 aggtaatcgg gtccgacaat ggaccagcct ttgttgccca ggtaagtcag ggcttggcca 172560 ctcagttggg catcgattgg aaattacact gtgcttaccg ccctcaaagc tcaggacagg 172620 tagagaagat aaataggacc ttaaaagaga ccttgactaa attagccatt gagaccggca 172680 gaaaagactg ggtggctctc cttcctcttg cgctcaaaca cccctggtcg tttcgggctc 172740 actccttttg aagttctgta tggaggacct ccccccttaa tggaagctgg tggaacatta 172800 gtttccgact ctgaccctgt cttaccctcc tctttgctta ttcatttaaa ggccctaaaa 172860 gtgattagga cccagatttg ggaccaactg aaagcagcct ataccccagg gaccaccgca 172920 gtaccccacg ggttccgagt tggagacaaa gtcttggtca gacggcatcg aaccggtagc 172980 cttgagccac ggtggaaggg accctatttg gtgttactga caacccctac tgcggtaaaa 173040 gttgacggaa tcgcctcctg gatccacgcc tcccacgtca agagggccgc cagtcaagat 173100 gaagaaaacc acgacgacaa ttggacagtg gcagtcactg acaatcctct taagcttcgt 173160 ctgcgccgca ggcgccactc tagacctagg gaaccttaac cctcatgctc caattcaaca 173220 gtcctgggag gtgcttaatg aaaaggaaaa cattgtatgg gcaaccactg cagtccatcc 173280 cctctggatt tggtggcctg atctcacgcc tgacatctgt aagttagcgg caggatcccc 173340 caattgggac ctctcagatc atactgatct tagcaaccca ccccctgagg agcggtgtgt 173400 cccaaatggg atagggagca catatgggtg ttcggggcag ttctaccgag ctaatcttag 173460 agctgcacat ttttatgttt gccctggtca gggtcagagc aaaaggcttc aacaaaaatg 173520 cgggggggca tcagattact tttgtggtaa atggacatgt gaaacgacag gagatgctta 173580 ctggaagccc tcctctaaat gggacctaat cacggtaaaa cgaggtagtg gctatgataa 173640 gtcaaacgaa ggagaaagaa acccctataa atatcaagag agtgggtgcg cttttaaaaa 173700 cagagcaccc tcaggaccat gcaaagataa atactgtaac cccctacgta taaggttcac 173760 cgagaacgga aaacaacacc gtctaagttg gcttaaagga aataggtggg gttggcgagt 173820 atacattcca ctaagagatc ctgggttcat tttcacgatc agattgacag tgagagaccc 173880 ggcagtgaca ctcgtagggc ccaacaaggt ccttataaaa caggggcccc ccagtcgtac 173940 tggctccccc aaaggtcccg actgtaccag ctccaccaac tccacagccc aacacagtgg 174000 taccctccct aggaactaat actctcctca taaagcctac cttggcttcc ccaccgcccc 174060 taggaacaga ggaccgtctg gtcagtctag tccaaggagc ttttttagtt ctaaatagaa 174120 ctaaccctaa tatgactcaa tcatgctggt tatgctatgc ctctagcccc ccttattata 174180 aaggaatagc tcagatcagg acttataata ctacttcaga tcattctcaa tgcctttggg 174240 gaaaaaacag aaagttgact ctagcagcag tttcaggaag agggctttgt ctgggccggg 174300 tacctcagga taaagggcac ctctgtaatc agacccagaa catccagtct agcaaaagcg 174360 gtcagtatct ggtgcctccc ctagacacag tgtgggcttg caataccggt ctcactcctt 174420 gtgtgtctat gtctgttttt aatagttcca aagatttctg cattttggtt cagcttattc 174480 ccagactctt gtatcatgat aatagttctt ttttagataa atttgaacat cgggtccgct 174540 gaaaaagaga acccgttacc ttaactttgg cagttctatt aggattggga gtagcagctg 174600 gagtaggtac aggaaccgct gccttaatta agaccccccc aatactatga agaactacgt 174660 gcagttatgg atattgatct tagaactata gaacagtcta taaccaaatt agaagaatct 174720 ttaacttccc tgtccgaagt ggtgctgcaa aatagaaggg aattagactt attattcctt 174780 aaaaaaagag gactctgtgc tgccttaaaa gaagaatgtt gtttttatgt tgaccattca 174840 ggagtaatca aagattctat ggctaaactt agagaacgcc tagatatacg taaaagagaa 174900 agaaaaagcc aacaaagatg gtttgaaagc tggtttaata agtccccttg gctcaccact 174960 ctcctctcca ctatagcagg acctttaatt acacttatgc ttttgcttac ttttgggccc 175020 tgcatcctta ataagttagt agcttttatt agaaaaagga taaacgcagt ccaggttatg 175080 gtactaaggc aacaatatcg ggtccttcag gaggttgaaa actcgctcta agattagagc 175140 tatctcctaa aagaagtggg gaatgaagaa taaaaatttt tactgaactc ttcttcaccc 175200 cagaacccga cccctcccat ctagagattg ttcccggaac actcctgaac tcttcacccc 175260 agaatgcatt cctgaactcc tcaccctaga gttcgaaccc tcccaactaa aaactgttcc 175320 tagaacattt ttgagataag ggcctcctaa aacaaccgca aaatgaaccg ggtacattgc 175380 caaataatag gacatgaccc cttagttacg tagattccct tggcagaacc ccttgtcccc 175440 tgacagaacc ccctagtgat gtaaacttgt actttccctg cccagctctc cccccttgag 175500 ttttactata taagcctgta aaaaatttgg ctggtcgtcg attctcctct acaccactag 175560 gtgcatgagt ttcgacccca gagctctggt ctatgttcca tgtgctttct tgctgttgtt 175620 ctattaaatc ttgccttcta cattttgagt acggtctcag tgtcttcttg ggtccgcggc 175680 tgtcccgggg cttgagtgct tgagtgaggg tctcccttcg ggggtctttc aaaactactt 175740 cagaggaaaa atgtattctg cctcatgggt tcagggggtt tccctcagca aattcaggga 175800 agacaagatg gaacagctca acctgctggc aggagggtgt gggaaaggac aagtgttcat 175860 tgtgtggtgg acaggaaaca gagagctgcc tacagtctta caggcctacc accactgacc 175920 tacctctgtc cgtcaggccc tacatcttaa aggatctaca gtttattaaa agaacactac 175980 cagataggaa ccaagtatca aaccaccagt ttgtagggga taaaaataca aggaacacat 176040 ctcaatagga gtgtgttcca ggatgtggac aaggagaaca cagttgttta aaagcttaac 176100 gctggccagg agagctgcac acctttaatt ccatcactcg taagagggaa gcaggttcat 176160 ctctgtgagt tcaaggcaag cctgggctat acaattctag attagccaga gctacatcgt 176220 aggagcctgt ttcaaaacaa acaaaaccaa accataaaaa agcatttctg aggctttggg 176280 tttaatcccc atgacctcaa atagccaaac agctctcctc agtccaaacc aaactgcaaa 176340 attggagcta gtgagatggc tcaacatatg aaagtccttc ccaaaaatat tgacaactgt 176400 agcttatctc tggggacaca cataatggga gaggaccaat ttctacaagt taccctctga 176460 cctccacaca tatgcctccc acaaataaga aaatatatat aataaaaaga aagaagtcta 176520 cagctgcaca tggtcatgca tgcctataat ccagcactcc agaggctgag gcaggaggat 176580 tattagtttg agatcgcata gcaagcagta ggctagacag ggctacatag tgtaaacctg 176640 ccttaaaaca caaaaatcaa ttaagcaaca ataacagtaa caaccacaac aaaaacccaa 176700 aagagtactt tgtagtaagg acaataccaa aaatgttcct ttaaggacag ttctggaatc 176760 agcaatagcc ttccgagtgc tcagggatgt ataaatactt agaaaacttc ccctggagaa 176820 atgagcacca gggtacactg ctctcagagc tgcccagaaa gttgtttatc ctggattcat 176880 ttcagccttc ctaactgctc aggcattcag aggtcacttc tgtagtagcc aatgtctaaa 176940 aaggctaaac tactgctcag catggctgtg gtacttggca ttatcatttt gtgactggtt 177000 ttgtagttat gcagaattca agagttatag catcatgaaa gtttccacca agttcctgat 177060 ccagtcacct cttaaaggtt ggatgcacca agtgcctttg gggtgataaa ttatattcaa 177120 ataatggtat tccaccctaa tccccaaaga cttctggcca tctcataatg taaaatgctg 177180 agccatcgca ccagcccatg gccttgaact cttgatggtc ctgtctcagc ctgtgtttgg 177240 attataaatc tgttggtgag gtattccttt gctgataata caagcaaatt cttcaagctt 177300 ccatcctaga ctgaagacca gcagctctcc aggagtcctc aatgcagact ggcccagctg 177360 ggacattgag cctcatggac tcagccgcta ctagattcgc aacctattca gacaagccac 177420 tgttggacta cccagacaat actatgtaag ccaatcccat tttaatacac atattcatct 177480 gggtgtgtgg cacacacctc tactcccagc acgcaagagg cagaggcagg cagatctctg 177540 atttcgaggc ctggtctata gagtgaattc caggccagcc agggctacac agagaaaacc 177600 tgtttcaaca aaaccaaaac cgtaaattca ttctatcagc tctatttcct tagagaattc 177660 taatacatgt gggtaccagg ggttgaactc aaagtcttca tgtttacgta gcaagtttcc 177720 ttctgctagc ctagtgaagc tgaggcaggt acagccggtt ctttactgct ccttgcaaat 177780 ggtcctcctg agctttcctt tgagagccta caaagaactc tttttttctt taggtctcca 177840 ggttttggtc ttaagaggtt ctggacttgg atctgtagct gtcatatcac agacattcaa 177900 catctggcaa atgtcttgac aaaggagatc acttgtgttt gctgcagtgt cccttctggc 177960 tgtgagattt tgctcctcac cactgcagga ctgcagatct attctgcctt tttagttgac 178020 ttttcattcc tgagaactgg ggaaaactga ctttgtattt gggctttgaa tttgtccatt 178080 tgtcaatcca tcacaccaga cctaaccaac tgccaagagt tctgctgact tttgttttct 178140 ctagggtggt cactttgctg ggctcatcct catccttggc ctgcagttta tccccaggaa 178200 agaaaatggc taacgactgc taagaagcag tctttccttc cagaaatttt agtctatcta 178260 gaccttgctg cagtctgaag tctttaaaat gtgtttgtta tggtagaata ttttgagttg 178320 ccttaggagt attgcttgct gtcacctatc atattctatc aggaagcaga cgtcccattt 178380 accaaatgtg aagaaatatg gcatcaatac ccactgcaaa aagtgtaaat aaataataaa 178440 aaaatagatt tattacagag tgcaagggaa aagaaaaaaa tcagccagtt tcagaattgt 178500 aactggacaa atgttggtac agttcatgaa gaggttctac aaaatggctg ggggtgggaa 178560 cataatgagt tagtttgctt ttttttttct ctttccttcc ctttcctttc cttacaaggt 178620 ctcatgtagt ctatggtctc aaactcacca ctgtaaatca ccttgaactt ctgatccttc 178680 tgcacgctgg caatgtaagc atgtgccacc aggcctggct cacacatttg gtttttcaat 178740 acagaatagc tctgtgatga ttaacttcaa tcatcaactt gacataacca agaatcgtct 178800 gaggaagagt ctcagtgact gggtgggcta agggcatgct cataagggat tatcctgatt 178860 gttaattgac atggaaagat caagtccatt gtgagcagca acacgccctg aacagaagtc 178920 ttctgaagta taagaggaga aagcttgatg agagcaagca ggcaagcaag ccaggatcca 178980 cgtgtttatt ctctgtctgt tcttgaccgt agatgtgatg gctgtcttgg cttcctggga 179040 aacatgaact gcaccctgga attgcaaggc aaacaaacct tttcctcttc caagttgctt 179100 tatgctaaga tattttatcg cagcaataga aatgaaactt agaacaggcc cataactgcc 179160 agctttggaa ctgaacctaa ggctgttata attcactagg atagggacca ctggaagtga 179220 atctgatttt gatggtaaaa tcatgtgttt gtttctggat atgatagatt tatcaatttg 179280 agactcagaa aagaagttag gacttgaatt ccgttttaga gacattccag agaaaactga 179340 tgtcattgtt ctgaatgtaa gtgcctcagc tgaaaataca aagagtacag ggaagaaagc 179400 ccaggctaga atctgaagga actcctctat tttttgtttg cttgtttgtt tggttggttt 179460 tttgagacag ggtttctctg tgtagccctg actgtcctgg aactcacttt gtagaccagg 179520 ctggcctcga actaagaaat ctgcctgcct ctgcttccca agtgctggga ttaaaggcgt 179580 gtgccaccac accaggctag gaactcgtct attacacatt aacacccctc tttaattaac 179640 tgttcctgcc aatgtaccaa atagtcaatt gattcctgtt tatttaccac atgtttctgt 179700 tagtaaacca gaataactta tctagccaaa gtctgcctat tagccatatt ttcatcagtt 179760 cccaaccatt tttggaattc tgtgagggga atccacagat gctgtagacc gctttagaca 179820 tttttcagct tttttcaagt tgcaggtcat gattcagtgg gtcatgaaat taatttagtg 179880 ggttctgatt agcatttcaa aatgaggcaa gcagagggca tattgtcaca gcacagcaca 179940 tgcggtaagc agccacacac tcttgcttgg aggcttagtc agtttctggc tctaaacgcc 180000 ccaggtttgt ttctctatcc taggcctctc tcttaaattc caaacatagt tagacattac 180060 cattggggca cgtgcaactc aaacacggag tgtgactcct ttccccatct gcggttccca 180120 gatttggcaa tgtcaccctc ctcccttctc cctagggtca gttttacctc tcacactcca 180180 caacacaaca cctctcatct caagaattgc cattagggct ggtgagatgg ctcagaggtt 180240 aagagcaccg actgctcttc tgaaggttct gagttcaaat cccagcaacc acatggtggc 180300 tcacaaccat ctgtaatggg atctgattac ctcttctggt gtgtctgaag acagctacag 180360 tgtactcaca tatattaaat aaataaatct aaaaaaaaaa aaaaaaagaa ttgccattaa 180420 atgtacctca gagtccaaat gcttcttcct cccctgacta cactcacgct ggcctgagtc 180480 cattttctta ttgaggttac tgcttctctg cttctaccct ggctccttct gctgcctatc 180540 cttgacacag cagacaagca gttctttaaa gcagggctca ggaccagtga gactgatcgg 180600 ctctggtggc acttcctgcc atgactgatg atctaaggtt aagcctagaa cccacgaggt 180660 agaagcaaag gacctactct ccaaagccgt cctctgacca ccatgtgtaa actgcacatg 180720 tacatgcatg cacatggtac acacacatac acagaagtaa aaagagattt aaattgaaaa 180780 tcattaaaaa gaaaaatcag ggctcagcaa actttccgtg tagaaaacta gagtacttag 180840 gctttgaaag ccaagaagtg gatattaatt atagttattc attatagcag agatttctaa 180900 aaccttttga caaaactaaa aaatataaca gagtgtattt tttttgtaat gtaagtttac 180960 taatggcagc agtgggatta gtttcttttt tagattattg ttattatttt tattaattat 181020 tagtgttttt gtgtttattc atattccaca gcatgtgtgt ggaattggat ttctgcttcc 181080 acctttgtgt gggtcctaga gattgaactc aagtcatcaa gcttgcacag taggtggtca 181140 ggcttacaca gtaggtggtc aggcttgtat ctttggaagg caagcatttt acttcctgtg 181200 ccagctcact ggccttcttt gtttaaaaaa agaaaaaaaa agtccttttt tgtttaatta 181260 gggttcatgg ccagtgctct ttatcttaaa atcaactgca aacttttatc tggtaaaaag 181320 ccatccttag ctgtggtcct aggagaaaaa catacagttg gatggcttta tcctgcaggc 181380 ttagtttgat catctctctt tgaagatata atcagctcac atcacactca agcctctgcc 181440 aacgagtttt ctacttctgt tcaacaaact acccaagctg agcagctcca aacaacagcc 181500 agttatgatc ctcacagtcc ggtgggtcag aagcctaagc gggcgtggct acctcgctgc 181560 tattgcctga ccctgctcgg tgcatccaca ttcacatcct ttcctggtga gtgtggttct 181620 ttgactggtt ttgttccaat ttttagtata tgtgctgctg aaacaatctt tttgcctctg 181680 cctccagact gcagggatta atgttcttga ctgccacaga gcactaatat ttactgaaca 181740 tgtgatcatg tggtgctcag cactcttgca cccaaggctc ggggaacatg gaggaagagg 181800 gggtggaaag attccaagaa ccagaggaag aagaaagtca gaggtgagac tgcatctcct 181860 agaaatgtca gggacatttc tagacctctg aagtctcaag aacaaggcct gaaagtctta 181920 tttatatagg ttaacctgaa aggggaaaaa attcttacag gggtccaacg ttagacaaag 181980 aactctaagc aactaaggaa tgttgggggg ggggtagtct tccccaggga acactcctct 182040 acccttcaag ccccacccaa gctggttatc caaaacaaac tggtcagtcc tgaagccata 182100 tacgcacaag taacatcata tggatgggca gattgcattt aggaatacac acatacacac 182160 acacaactta aaaagagagg ccatgaattt aagagagagc aaagcaaagt gggaaggggt 182220 acatgggaag gttggaggca gnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 182280 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 182340 nagagagaga gcctattatg tcgttggttg cttctaatca ttagaaaacc actctcttag 182400 gctgagtcag aactagccta gctgagacac tgtccaccca ctgtcccaga gcaaggccat 182460 cgctgtccca gatttgcctt tgggggccct tgaaatgaaa gtcaccagca ggctccggaa 182520 gctgcctcat gctaatcagt tgaggttgtg aaaatagccc tgcagtggtt cctgggcctg 182580 cagctgggcc agagccacta aggggagtct ggtcctttgg agcagagtta acagtcatca 182640 gtgctttttt ttttttttta aatgttccct gctttaggct cagtgctgtg cgctacttct 182700 aatccttgca ataggctgca agacaggcaa gaatatcatc cctgttttgc cctcaggcaa 182760 attctgaagt ctggcaaatg aaagatgtgg gatttgaaca cagacttgtt tggccaaaga 182820 attctcactc tacttctgcc tgtgccacct tcctctcatg cacggggagg ggaggggagc 182880 ccacctccca tgctcagggg ctaggaagtg gggagaagat ggatgtcctc aaagcagggt 182940 gagaatgaag tagaagccag cttcaaatct aaactaagca atgttttatt tccatttccc 183000 tgaacataaa gttcagttac atttggttta aaaaaaaaaa tccctacaca actggttctt 183060 gagaaatgtc aagtgctaca attcagtgga tgtggatgaa acaatcaaaa tgttgaacac 183120 ccccaaacag atacaaacct tcatcaaagt ctcttccaaa ggctgggtct gaaaagagcg 183180 actcatgttc cagcccagtt ggctccttct catgtgagct ccgacttcca aagactgctt 183240 gcaccaggag gaaatataat agatgtcctt tttaaggggg gtggggtctg tctgacaacc 183300 tcccacagtg actgtggata cagcccagtt agtagagttc tcgcctagca agcgtgcggc 183360 cctgggcttg agatctagca ccctaaggca tagtggtaca tgcccatgac cacagcactt 183420 gggacgtaga ggcagaagga tcagttcaag gtcagatgag gggtggggag gcattccttt 183480 aatgccagca tttgagaggc agaaacagat gaatttgtga gttcaaggcc agcctggtct 183540 acagactgag ttccaagaca gccaaggcta cacagagaaa ccctgtcttg tcaggaaaaa 183600 agatagtggg agagaattca aggttatctt ggactgcata agactttgat tccaaaataa 183660 acaaaaatgg agcatgaatg cttgcaactg tggacaatat tgggttcata catattctgt 183720 tttgtcacct acataccaat tatacaaatc agattcagct gggcccactg gtgcatgttt 183780 gttcccagca tctgggaggt agagatgggc agagctctat gactttaagg ctagcctggt 183840 ctacaaagta agttctagga cagccaaccc tacagagaga tacactactt ctaaatcaat 183900 caatcaatcc atcagtcaat catgggctgg agagatagct cagtgatcaa tagtgccatt 183960 cttccagagg acctgggttt gattcccagc acccacatgg cagctcacag atgtctgtaa 184020 ctccaatccc aagggacatg acaacttcta ctggtctctt tggtcaacag gcatgcacgc 184080 agcatacaat atatatatgg gtaaaatgct atatatataa aaatcagatt cacaaatcaa 184140 gtacagaaag agatcaacta taatgaaaca accataacac atactgtttt aaaagctacc 184200 tttcctgtct gacatctgac ttcttgcgtg acccagtgcc tccacaatga gacaagcaga 184260 tggtgtagtc cctgtgaccc aggattaggc aaccactgcc cctgggaagc cattcctagt 184320 aacaggatca aatcccacag tgctccactg taccccgcca gaggacgtga agcctccctt 184380 ccccggctgt ctgtgcagca ccgcccagat gcttggtgtc actgagtgac catcggagcc 184440 caactgagga gtgcctcagt gctcctcagg gcagtgtgca attgaaactt gcatgtgatt 184500 tctggaattt gtcatttgat atttccagac tccagctgac cttggataac agcacagagg 184560 gcagaactgc agaggaaaag gggcactact gtgactgtta tcccctgcat gattatagaa 184620 gggtctgtgc tttctcttac aaatcattgc ggcctctctt cacttccctc ctttgaagtc 184680 gaaaaaaata gatgcatcct cacagtacag gatggcaggt acgaggcggg ttccgggact 184740 gaggcaggac tcatcatgca gcctctttcc ctcaactaca ccgccgcccc tgcagagttc 184800 cctctgatca aatcagtttc aggcctggaa agaaacggcc actcaggctg gggatgtggt 184860 tccattagag gcctgcatgc acgaagctct ggatttgatc ttcagcacgg gcataagccc 184920 agtatggtag tatctgttta gcatggtgtg gaggtatacc tatctctgca cagggagacc 184980 agaagttcag agttatcctt gcacttacag taagttcaaa gctagcttgg gcaacatgaa 185040 gttttgtctt aacaaaacga aacaagaggg gccggggaga tgcttcgctg tgctgagtca 185100 tttgctgcca agtttgatga cctgagtttg gtcccttgag cttatggtgg aaggagagaa 185160 atgacgcttg aactccacgc acatgccaca gcccacgcat gaatgtgtat acacacacac 185220 actaagtgga taaatgttaa aaataaataa atcaaaggaa tggccactca aaatctacca 185280 tcgttgggaa gggaggggaa aaggcaggcg agggagatag ataaccctga tatgaacacg 185340 gaaagagcca gtgtgccacc aaagctgccc agtgtgccac caaagctgcc cagtgtgcca 185400 ccaaagctgc ccagtgtgcc accaaagctg cccagacttg attacagatt tggccaggga 185460 cacaggaggc cagcaggagc agccaggttc cacctcagag gtggagccac aaacctggaa 185520 atgaaacgtc tttccctttc ttcagaccac agcagtgaca gctgtcctgc agagtctgga 185580 gggctggcag ggctcatcca ctctagtgtg cctgtggcca gaacaggcct cagtcacagg 185640 tgcttttcca aggtcttagt gtctaattaa ggttagcagc caaattggag agagaagggt 185700 gctggacttt actctgctgt aaggactttg ggcattgttc cattccgtga tcaaatacca 185760 ctggctctgc caaccaccat gtcagtgggt cttcagaggt agaagaactc atcctttttt 185820 gagaggtttg gtctggtcct tgtctaatgc aaaatgcctg gggcaccagg ttaatgtcaa 185880 ctcaaaggca agtgctggtc cagcatgtgt ggaatcctaa gttcaatacc catcagagcc 185940 ccaagcccta gaggacagac atgctttaaa aaaaagtcat gctttaaaaa aattctgttg 186000 agggggctgg agaaatagct cagcagttaa gagcactagc tgctctttca gaggacccag 186060 attccgttcc taacattttc atggtggctc aaagctgtct ataattcaag tcctagaggg 186120 gaatctgttg ccctctctgg ttttctcagg caccaagaac acatgtggtg caaacataca 186180 cgcaggcaaa acactcatac atatgaaaca ttttttataa acctgctcgg atgtggtggc 186240 tcatgccagc gatgctctca gcactcagat ggcagaggca gggggatttt gtgttgagcc 186300 cagcctgagc tatagaatga gatgctgtct caaaaagaaa aaaaaaacaa aaaaaaacaa 186360 aaaacaaaaa caaatctgtg ggcttaatca ttcctagcca gaaggagctg gctccagcaa 186420 caatggatcc ctagagctct gctttgcccc ctggtctgga gagcttgctc tagaaaggaa 186480 ttctccatag accacatttc tattttgggg accactctgg gcctgcacat ggaaaaatgg 186540 agagttgagg tttacatggt catttttttt gttgttacag taatgagagc tttgaaatga 186600 tcacaaaagg aaaataggaa aatatgcctc ctaaaaagag ccgaggcaaa ttattatagc 186660 aaccgaatcc taaaaggaat gttcaagtaa aaaaaaaaaa atatggctca tgcgaagttg 186720 ttatggcaac tgacatttaa aagtaacagg atttgggcaa gtttctttct ccaccctctc 186780 tgcaaagtct tgcaggaact tcatgctaaa attatgcttt aattttaatt agccaaatag 186840 gtcataaata tagcttattc ccaaatcctt agaatttcta ccctgcaagg agctgaacta 186900 ctaatgagga aatatttcca caaaaaccca cttaccatta agaaccccca tccgatattt 186960 ttctaatata atttatcaat ttaaacacat tgcataatgt gccactctgt agcattccat 187020 taaaatgata aatagcaata gtggtgaggg gtggggggca aaaaccagga gattaaacat 187080 atccaaagca gtgtagctat atttaaatac ctcaatccat tgtagaggaa aacacactgt 187140 tctcatccgc agatacagtc tagactcaga gcagcatatc cttgactgta agggtattaa 187200 taggacagac gaaggggggc aataagaaat gacaggaaac ttcagaagaa ataaaatttc 187260 tattaggctt tgttataaga ttacatcaaa gcagttcata tgttttaatc tggggaggaa 187320 aaaaagcaac tacttggggt ttgcgcctgg gggctgcctc tgtgtactga accagacagt 187380 ttgcataatg aacaattttc attcaatcag gatctcagca gagatagctc ctactcaaag 187440 gaacccggca caggctcata gtttttatct cccagctcca cctgctggag aaaccttgta 187500 ttgcagggag agaaagcagt cgggaggcat tgtcctagtg gctgtgtacc taaagttaca 187560 gacctgactt taaacagttt ctctctggag gttgaaaggg gctctgtaag ataccagagt 187620 ggattgctct caaagactct cggactcctg ttacaggcaa gtaaggtcct agcagatggt 187680 agcatggatc tccggccctt ctcactgctt tcttgaatca gggatttaga aattgctatt 187740 tgcataccag gaggactgaa gtttggctcc cggtgaccag aggacaaggt cattgtttaa 187800 aaccacccaa actcatttcc gacttggttg gtcaaatttt caagtttccc agcagtctaa 187860 ggattcataa aataaggcag aggcagagaa acggagggtg tgtgtgtgtg tgtgtgtgtg 187920 tacccaaaat ggaactgcat tttcatgcac aatagaaaac ttaaagactg aaccaatcat 187980 tttggaaaac tggcacagct gacattggct agaggaagga acggccaggg cgagccagct 188040 gcaccaagac ccagggctga ggcctaatcc gcctttatcc gagggtttag tgaggctccc 188100 gccgctcacc aatcccggct ggagccgcag aagagctctc ttcacttggc tcagtcccag 188160 cacagtcgca ctatgctctc ccgtggggag gccgctccgg gagggggagc gacatcaagc 188220 tttgtgaaac tgttttcgaa aacctgggat gatcatttaa atgtttaaaa tatgcacatg 188280 gtaattcaaa actaattacc ctgagcacat ttgaaacatt tatgccatca tcttggatcc 188340 tgcctactga ttgtgcgctg cagctcactc tggtgtttct ataaactgct tcagcgattt 188400 taacttccag gctaaatcag gcagccacag gcgctgcctc cagccctggg ttggtggaga 188460 gacccccatc cctgacttcc aggcgaggag gcggcccgtt tctccagaga gccgtttgtc 188520 agggtcttgt agttctggct gccgaattat tgctcttatc cgtgttcata attctcatct 188580 gcattattta atttaggcta gaatgacctc tttccctccc gagtcttcct ccctcattcc 188640 catttcctct tcttcaattc gtggccccca ttttctgatt ggtccaaata tatagacaaa 188700 tatccttgat cgtcccaccc cacttggcta catcttcatc tgggagccaa tgtggtgagt 188760 tttctgggtt tgcaaggtgg tcaggtccac cagtcatcct aaggtgtgtg agagaggtag 188820 accaacatga gcggcgcaca gccgccatca ctgagagagc acgtgccctg cagctcaggc 188880 acaggcatgc acacaccggc agacatgtgc acatgcgctt tccccagcaa accctgcttg 188940 cagagtaatt aggcctaggc agttcctgaa gcaaattcat ttcccccttt tccagaataa 189000 aatgagttct cttcctttgg gggtgctaaa ccagcatgcc agtggctaga agcctgagat 189060 gggtgatgtg gctgaaacca tttctgcagc caagcctgtg ggcagaagct aaccttgggc 189120 tggggagctg cagtcggaag aggcacaatt ctgggatcaa gaaatgagca ctggtttata 189180 ggtacactcc cagaaataga cagatgaggg ctgcctcctt attagcgctt tgaagatgcc 189240 catggcgggt ttttagacat ttaggaatat aaaagtaggt tggattccca cagtcagctg 189300 aagtttgaca gagtgatatt accgggttta actagagcca ttaagagact cttcattatc 189360 ccacaccacc gccacccaag ttatcacatg agccataatg caagagaatt ttcattccat 189420 caacaagaga gggagccggt ctatctttgt ccaaaggaaa tgagcagccc agcgtgaagc 189480 ttgtgaggaa ttgagtgtac aacactccaa taacatcccc tgcaggattg cctctgcgat 189540 ttagtcggtg aagcaggggt aactgcgctc gagcagtctg cctgtgtacc tggcttgcaa 189600 gaacaccagc tcgaggaaca ccaaaaaggc cgattaatga caaaggacac tcatagaggc 189660 ccgaattcca cagggcttaa gtattaagcc ccaaagaaat caaggtctag gccattctcc 189720 tggcgctcag caatctcatt tattatttct ctacaaagat ccaacactca atttcccagg 189780 tatcccctgt atctgactca cattctcctg ctcagtaagc catcctggtt tgaaacgggc 189840 ctcccctcct cctgcctatg catgctttgc gtcttcacaa cgacagctgg taatttgcaa 189900 gaccccctcc actggactct ctcaccccac atacttggaa ctactccttg gaactacttg 189960 tttatcaagt gttctgttgg tgagccttct cttgcattaa agctgtgaga aggaaccaca 190020 gtttctattt cctttacatt tcttgtagcg tctcacatgg gagacaccca ggttagatat 190080 actgagggtc ctggtagttt tagagttgga gttagatgac ccagcaacat gccttccccc 190140 accacgcacc aagcaaaaat tgcacccacc cttccctcag atgttcctgg catcttataa 190200 ctcgcccaaa gccagattta ttgctcctgc tgtaaagtgt atcttctcta agcctcactt 190260 aaaagctacc acttggcaga agatcaagtc agaagtgcag gctagcaggt gacggtgagg 190320 acagggcggg atggggcggg tagggtggag cgaataattg aagctccaag agttaccagc 190380 tcaatattta acctaactgg taatttgctg tgacaattac gccatgaagg gaacgctgcg 190440 actatgcaag aatgttgctc tctaattaag agggctctgc atttcctagt cacccgcact 190500 ttaataacac acagaatgag ccttggctcc gggagctaaa ggttccatta ggagcacggg 190560 cagcatatgg ctgtgcacat aggccgtgag tgatgcagcc cagttaagcc cgctaacacc 190620 ttcaattcgt cctcagatag agcccagaga gcgcggctca ggccctcacg ccacgagccc 190680 catttgactg acaggcatct tcccggaaag cctgcgcgtg cctacactgc aaatggacct 190740 gcttcccaca gcccggcttt caaccaggaa ggcttggcgt gggtctgatc cttcaagagt 190800 aactttaata aggattttct cacagaaaga aaagtccatg ggaacaaatc ctcctcttaa 190860 gagcgtgaga caggaatggg gacacaagcc aacaccccaa ttgctaggct aactctgata 190920 tgagacaaaa gaatattaat atcttggcta tgaaggagga tggtgccatc ttctgaattg 190980 atgggagttt tgaggcatgg ctaagctggg caaaccattt tctttttttt ctcttcttaa 191040 ttagtggttc atttatggag ggcttgctgc ccggagagcc catcagaaga gagctcgctt 191100 tatggagatg tagcttataa aactactcag attttaaaca aacagtgcag gaggccagag 191160 gtagaagtgg tgggggtggg gtggggcaag agaacaattg catctgcaga aggctagccc 191220 tgcaccccaa gcctatgttt agggttgatc agcttcccga ggcaagccca gaagcctcta 191280 aaattttagg ccaatagaaa tgacctctgc accacggctg actgaagcta taaataagcc 191340 tcgagttgag cagtggtgtc aacggagaga gcagaggaaa gtccaatcag agcttcattt 191400 ttttttttta aagtccactt gcttgggact cacctgaagg cagggcattg agtagagcct 191460 tggctccctg cagcgagagg ctccagtttt cccaggcacc agcccatcgg ttggttacct 191520 aaccaccgaa agggaactgc acagcacaca agttaaatat aggctgggtt atctgcattt 191580 tacaagctct gagcaagcta tctgaagaag ctgtcatttt taatgacggc acaaacttcc 191640 aattaccgac tgggtaatcc actagggagc aggtagtttt ggaagaacag ttcaccatta 191700 ttaaaagttt acacaatcac ttttgagttg actataagta tttcacacga ggcaggtggg 191760 attagggact ttttgggtgg tttactcgag gctgcaacca acaatgagtg ttttctcaag 191820 aattatacat tgagatttgt caactgctgg ggagtagtgg agggtcctgg taatgcagaa 191880 aggttatgaa atggccaggt aaggttgggt gcttccaagt ctcaaatata ctcctaaggc 191940 cagctccaag tcataagctc aaacaagtct tcaaggggcc tggagagtta agacaaataa 192000 ggatcactta ggctacccac ggacaagcac ttctcataca aggaccggct acctccaaca 192060 ccatcttccc aacatggctt ctatgttgct tcaacaacca gggcagggtg aattaggggt 192120 gggtctctcc aatgtggact caaatcatga ctacagcntg gggttttttt tttttttttt 192180 tttttttttt tntttggttt ttcgagacag ggtttctcca tatagccctg gctgtcctgg 192240 aactcacttt gtagaccagg ctggcctcgg actcagaaac ccgcctgcct ctgcctctgc 192300 ctcctgagtg ctggaattaa aggtgtatgc taccacgccc ggccgagtcc gtcttgataa 192360 tgaagttccc agtgacctgg atgtcaactg aagttggatt ttactgtgat gactactgag 192420 tccggctcag aattttgggg ggacaaggta ccttgattta actgggcact acacgactgt 192480 aacccccaca ttgggagagg cagaggcaga ggcagaggca gaaagttggt tggaggctag 192540 gcaaggctac acagcaagaa gctgtctcaa aaccaaagac atctttcttg atccaaatcc 192600 tgtcggaggg tgtgaggcct tgggggccag aacaaggtgg tcaaggaaga ccactgactc 192660 tgtcctttgc tccattactt aatcagaatc gccatcacag atatagctag gagattttaa 192720 gccttggtgg ctgcaatctg catttaagag ctaagtggga taaactcagg ggtgggccca 192780 atgcctccct ccccaccctc cctgcatccc tccatttacc tgtttccagg gatctgctta 192840 atttacctgc cagcctttgg tgggacacag gcttagtggc ttagcgctgc tcggggcacc 192900 agagaccctc acagaagcac ctgaatgtac tttcagcgct gcagagcacg cacggctcag 192960 gcccatcaga agaacccagg cttatgctaa ggagccagaa agtagaagca gctggcaaga 193020 gtgattcagc cccataaatt tacacatccg tacagccaaa cccacttgaa gtgatccaga 193080 gccactttta ttgaaataga aaagatgcct attctggagt gctaagtggt acaggagggt 193140 gggtatataa gagataatcc catgttgtct ttgatgtggt gctagggaga taacccagga 193200 cctcacgcct gcctgcaagg tagccaccaa gccacaccca caacctctat ttatacacac 193260 actaagtgtg gaggtatgga taaaaaaaaa tgtcccaaga cctcacgaat ctgcaaacat 193320 ggtgcctggt tggtggcacc gtttggggag gcagtggacc atttggtctt gcaggaggaa 193380 gttatgtcac tgggtatggg ctttgagagt ttgtagcttt gctccccttc cagttaactc 193440 tgctctcgta aggttcctgc caccatgttt cctctgccat tatggacacc tggtcctcta 193500 gaactgtaag ccacttactc tcaggtctct ttcagtcctg gagtcttatc atagcaatga 193560 aaagtaactt gtgtggcagc cagctaagca agggctgtgg ccgactgctt gggattatgg 193620 ttgtgtctgt ctgtctgtct gtcattccat ttatatagtc ctgagaattg aaccacttta 193680 ccactgacat gtctcagtcc tcttggtatc atatattcac ttaagacaag atctcattaa 193740 gtcattcaga ctggttttga gcttgcaatc ctcctgcctc tgcctcaagg cgataggatc 193800 cctagggtac tcgaccagac tgggagtagc aggttctgtt ctcttagctt tctacagtga 193860 ttgtggatta tttgtgtata aagatctgat ggcccgaccg actcccttcc ctttaagtga 193920 acatcaacag tatttagcat caacttaata aactcatttg gtaaagccat ctccccacct 193980 cttgaacaaa tgaaaatcaa acagcagtac ctgttctcct agagcagcgg ctctcagcct 194040 tccggccttt taatacagtt cctcgtgttg cggtgacccc cccccccccc agccgtagaa 194100 ttatttcatt gcttaaccag agttaactgg aagggttaat aataaaacca gtctgggaga 194160 ctaaggttac ccaaccacgc taggaaggag aggaaagggc cactcgcaca aacctgtctt 194220 tgagatgaag aacaatcaac ataacaggga cagagcagtc cttgtaacaa gtgcaaagga 194280 gagagagagg ctgagtttct acttctataa ataaaccctt ggcaggcgga tcactaaagg 194340 aacacaagtc aatataaacc tttagacatg gggctgccaa acttcacttt tcgacagtat 194400 attaattatg tagtcaatag ccatgggttt cattagcgta ttaaatacca cgatcaatat 194460 tatttatact tttcgaagac aagccactca gggaaaaaat ggtgggggga ggaggaggaa 194520 caatttgacc ctgtagttca aaaaaagtca gaacagcaca ctagagatta gcaagggttt 194580 aatggaaggc ataaaacact ggaaatatgg acagaaatca gatccctgcc ttcatttttc 194640 tgccttttac aaagagactg gagggaattc agaaactatt taaaataaag gcaaaatgat 194700 tagagcccct ccctcccctc agctgcttaa cactggggtt gtggtggacg caaaataagc 194760 attgagctct aagtgataga tgagaatcag aacaggaaca gtgtttttga ggcaaaatat 194820 gtccaagaga attcaaagaa ctgtgggcca gaatctactt aggcagtcct ctgggacccg 194880 aatccctcac aggcgttaac agtggaacca atttccaagg cagccctgct ggtgatctga 194940 tttttgagta gggaaatctg ttaaacatcg tcccacgagg gagcccagct ctttcactcc 195000 ccacgggttt ctacatgcag ctgtgctaga tctgctgaag tggccggtga ggaggtgtgg 195060 ggattggttc agcgacctca gaggacattc ttgttcacta gccctcgtgc actggggcga 195120 tgaccgaatg ctgtgagcag gagatatcaa aggccggcta ctggactgaa aactagatca 195180 ccatctctaa cctgcaattt gtcaatctca gacagcaatg aagactgtga ttttctagtc 195240 aacgctttgt aagcaaggtc agatagaggc tccataaaaa ttgttcaggg ttcaggcaga 195300 gaatcaagtg taactcaatc cctatctcct gagattaggg aagggaagga aggctgtgtc 195360 tactaaacca gtgagcctca agcaaagcct gtctgttctc agcaaggtga gccacccacc 195420 aaagatgcca acagctaagg gccagggatg tagtgcaggg tgctgtgata tcaacagctg 195480 ggagacagaa acaggaggat caggacttca aggtagtttg ggctataaaa tataagcttg 195540 aagctaccca cttgaagact gtccccaaca aaacaaacaa gctgggtatg gtggtcgatg 195600 cttgtatttc tagtgtatga gacgaaggaa gaaagctcaa gcccgagacc tgcttgggtt 195660 acatagggaa gatggtgcct caaaaacagg acagccgagg agcagacaga cagggcagac 195720 agggtgcatc gatctaaatc cacatacctg gatttaaagt aatatctggg agactggtct 195780 gtgagggccg ttccagagat ttaataaaga cccaccctga ctgagtatgg gcaacaccca 195840 tgggtggccc aggggtccag actgaataaa gggaaaatgg gaggaagttc agctggtagt 195900 gtttccaagt attaggacca cagcctggcc cctggcatgt gctggccagc tagtctagct 195960 ccagtatcaa gcttcaggcc agcggcaggg cactggacag ttcccacaca cgacacacac 196020 acacacagag cactagcatt cacctcctgg tctcttcttg acaacagata aaatgtaact 196080 ggctgccaca gtgagagtcc cctaccttcc tcaccgttaa ggatggtaca aactgtgagc 196140 cagcagcagc catttctcca gtaacttgct ttccacagat actgttatag cactaagaaa 196200 agcaactgaa acatggggtg ctgtgacccc ttggcaccac aaagccatgg caagctgaag 196260 tgcacacatc acaggccagg cctgaagatg ctgggggact gcaatgctgc ctggattctg 196320 gcagagatgt gcagcagatg ccaagaggtg ggctgcagca accagagata attaatatga 196380 ttaggaacac actgagcagg catgctcttg ccgaatgaaa agcctcgcag tgtaatgact 196440 gttttcttcc tcgatcacgg tctccacgtt tcagagttgg cttggtgtta ggctgccgcg 196500 taaacatcaa tccaaccccg aggggccaga tcatcggtgt tcctgggctc aatcgccttt 196560 ccttttgtgt tttcattcat ttaaagatgc attccagggt tgcaaacatt agtgagaatc 196620 atctccaggc ctcagtctaa tctctgagtc tgtaatgagt taacatcttt ccctagtgaa 196680 tatttattat gaaggctaat taattgcttt ccagttacaa gaatccttta cagtcaaaga 196740 aagtaggatc cacaaagata tactgtttat tcaaacaaag caaaggaaac aaagcttctt 196800 tcttaaattc tatttaacat agctttaata aaggtacaca ggtccgcctg gcaaccgaac 196860 ggtaactgat gcaaactgaa gccatgctct gtagcagcct ggatgtccca gtgccacctc 196920 tgtctgcagg ctttgtcgga tttactaaga ttctgttatc ttcaaacagg gattgtgtct 196980 caagtaactg accccactat gtggataatg aagtaaatta tgcaatttgg gggtttgctt 197040 ttccccaagg ggacagcaag ccagtgctta tcagccgtcc tcagaggaga caattctgat 197100 taatatcaga gtcatctgac tcagtctatt aaacctatca aaccctgaag gaaggatatt 197160 cagatattaa cgataggcct ttgattaata attctacctt gttgccattc taagcattaa 197220 caaccatgca gtaactctgc aaaacagacc ctttgattcc aggcagacgc accctctgaa 197280 cacctgggtt ctcccctact cttctccccc caggaggaac tcaagacaaa aaggtgccac 197340 cactggaaaa gcacactcca ggttacataa tttgcctcat tatccagagt ggggttaatg 197400 acttgtgaca taatttctgt ttgaagataa caaaatttca tgaaatccga caaagccgga 197460 aggcaggagg aggggactgc tgccacacta ccggtggctg agaactggag cggaaggttc 197520 acacacagcc ctctgagctc actgtctttg cttatcagtg agtcccaaga ggggcccaga 197580 tgggttgcca gcctccccta gaggatcttc attgtggagc tgtcccatgg ggcgggaagg 197640 aagccattct atttctgttc ttctctcttc cgttctggcc accagtggta cttgctccca 197700 tcacatgttc ttcctgatgt tcgcgatcag ccgtctgcca tagtctctga agtccacggg 197760 cttcacgtcc atcacagtgg ccttaattcg agattcatcc tttagaaaag agagaagctg 197820 tttgtgagtg gcagagcctg gcgtgcagcg gaagagagaa ctttctttgc ttcagtggct 197880 tcaatgagtc cagcaggaag aaggaaagtt tacaagtctc agagagaaag tgctgtgact 197940 tcctggagtt gggccagatc ctcttccaca gaccctttcc ccatcctagg tgccctgtgc 198000 tcagacctag catcctcccg gagaagcctc tgtctttcta tgggtgcagt gggggcccag 198060 agcagacagg taactcaccc taaagcatca ctttcatcta gaggagctct gtggtagtag 198120 ggactgaggc ttctgctcca gctctgggca aggttacttc tctgctcttc accattcctg 198180 tccatcccag gaagacagaa aatccctaca ctctcccttg atctacccga ctttctgaca 198240 ccagcctacc tatgttcatt taatacaaca actaaaatat ctattcacag gcactaagct 198300 ggtgataacg cagaatgcac aaactctgcg gctgcagggg agacggcaga gttcctcctc 198360 cacttgtctc cttgaactaa acagtgtctt tgaggcagaa cagggtgaca cctagggaca 198420 cacaagtcta gctgggggcc ttcatgcttc catgtgctta gtaattaatt actacatgca 198480 ccgctgttta caagtatggt taggagcccg actgcctggg ttggcctctc gcctctgcca 198540 ctccatggct ttaggttcag agtcattctc tgcatgcctc tgcctgtctc tccgttggta 198600 aagcttgcaa caacagctcc aacacagaaa gtgctgtgag ggtcgacagt ggatagatgg 198660 ctagatagat ggggcaggac ggactgtcca gtaagcaggg ttcatcatgg ctatgcagct 198720 ctggacatca ggattagttt aaacacttgt caggtggggc acttttacca gcacgtgcta 198780 tttgtttaat attctgagtt ttagaaccta aactgtggga aacaagagtc cacacataac 198840 annnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 198900 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ngccttgaac ttgcttctgc 198960 ctcctgctgg ggttacaggc ttgagccacc atgacagctt tagcaatagc tttgtaaatc 199020 cacagtgtca agctggatat gatggcacat gcttgcaata ctaacctcca aagattccct 199080 gaactcgagt tggtgaaata gtccaccagg taaaagagct tgctgcccaa gcctgaatct 199140 gattccctgg tcacatgctt taaggaaaga acttgccgaa gatgtcctct gagtgccacg 199200 tgtaccgatg catgcttgca gccacccaca cacccacaca agtgcactgt ctcacacagt 199260 gagaacagca agtgaacaaa caaacaagcc gggggggggg ggattgtgac cagaataatt 199320 gagggggggt gtaaagctct tggcaggtgg ctggctcctg gtaacactcc ataagtgggg 199380 aagttccaca tgtaaggtca tgtgatcgag tacatctggg cctccaacag tccttgnnga 199440 agaaacagat gcagtctgtc atatctaaac cattgttgtc gtatctctgg gtagtctttc 199500 ttttctcctt cctttctttt ttctctccct ttctctttta aaaaattatt tatttattat 199560 tatatctaag tacactgtag ctgtcttcaa acacaccaga agagggtgtc tgtcagatct 199620 cattatggat ggttgtgagc catcatgtgg ttgctgggat ttgaactcag gaccttcaga 199680 agaacagtca gtgctcttaa ccgctgagcc atctctccat cccccaaccc ctttctcttt 199740 tgagttaggt tttgtgtagc cctgggtggc gttaccttaa ctacactggc tttgaacttg 199800 caatgatact ctgcctgatc tgtcttaatc attttgagat agggactcac tacatagcct 199860 ttgctggtct ggaactaaca gagatctgcc tgtttctgcc ttgcaaatgc tgggaataaa 199920 gttatgtacc accacacctg gagtttaagg gttttttgtt tgtttgtttt tcgagacagg 199980 gtttctctgg gtagtcctgg ctgtcctgga actcgctctg tagaccaggc tggccttgaa 200040 ctcagaaatc cgcctgcctc tgcctcccaa gtgctgggat taaaggcgtg tgccaccacg 200100 cccagtttaa gggttttttt ttgtttgttt tttcctgaga cagggtttct ctgtgtagct 200160 ctggatgttc tggaactcac tctgtagagc aggttagcct tgaactcaga aatctgactg 200220 cctctgcctc ccaagtgctg ggattaaagg cgtgtgccat cactgcccag tgattttttt 200280 ttttttaatg tgtgtatttg tatgggtgtg tgggtgcttg tggaagccag gtgtcagatc 200340 cccagagcgg aagtgttctt aaccgctgaa ccatctctct tccctcttcc ctaactctga 200400 ttttaaaggc accaaactct taggtaggag actatacaca cacacacaca cacacacaca 200460 cacacccgta cacacccgta cacaccacat gaccatgcct gagcacacaa gtggttttat 200520 tgctggtctg gcctgtgtat gagctggaac caaaaccttt gtcgggagat ccgcagtctg 200580 cagtttgagc acaggctctc tggtttctgt tctctgtcct gtgtcgcatc ttgactagag 200640 gcagagaagc atctgcaagg ctgtgaccac gctggctggt gctctgccat ctacattttc 200700 aacaggaaat ctcaggagag tatttccttt taagaacgcc agacttttgt gcctgggcca 200760 cttctctact tcccagaaca ttgtgtgcca agtggcaagt tattaaccaa gtgctttgga 200820 aaattaaact ccttggtttg cagagtagca tgggagcatt gagagggtgt atgcctaaag 200880 gcctggttct gctgctggca gagctgacac ttggctaaag ggctggcatt tctgagatga 200940 gcctcactag atccgcgtct cagagtctgc aggagaaatc agagagggga gaaggtccag 201000 tggcctgttc aggatgatct tcctctgcat ttaagggcgg ctggtttgcc cacgtagccc 201060 cagaaccaaa cgagcctcgg acgaagcccc ctaaaggcag taggagagac tgagccttgg 201120 ctcttcagca ggggtgggga caagagcaag aggcgggatc tcgcccggcc ctttagagac 201180 acgtgcggtt gtttccgtgt ctgggagatc acatgacccg catcagctga cccgtcacgg 201240 tggagctcag cgctggtgct tcgcgctccc cgccctgctg cgccccggag cgcaggaccc 201300 tgcggagggg taagaaaacc cccaggcttt ctttcctttg tcgctggttc gcgcagtcac 201360 ctgcacccta ccccccgctc ctcgttcatc ccagtcttcc cggcctggca ccccggaagc 201420 cactgcgagg agggccgtgg ccaggctcag ccttgcgctg cccccaggcg gccaggacca 201480 aatggcccag gggagcagaa ggcggaaagt ggttcttaca gcagggtccg agggctggtc 201540 cccttcctca ggacctgaca tggaggagct gctccggagc gtggagagag atctgaacat 201600 tgatgcccgg cagctggccc tggcgccggg gggcactcat gtagtggccc tagtgtccac 201660 gcgttggctg gctagtctcc gggagcgccg actgggaccc tgtccccggg ctgagggcct 201720 gggtgaagca gaagtcagga ctttactgca acgttcggta cagaggctgc ccccaggctg 201780 gactcgagtg gaggtgcatg ggctgcggaa acggagactg tcctacccgc tgggtggagg 201840 cgtgcccttt gaggaggggt cctgtagccc tgaaactctc actcggttca tgcaggaggt 201900 ggctgcccag aattaccgga acctgtggcg ccatgcatac cacacttatg gacagcctta 201960 cagccacagc actgccccct cagctctacc tgccctagac tctatacgac aagctctcca 202020 gagggtgtat ggatgcacct tcttgccagt gggtgaatcc atcccatgtc tatcaaatgt 202080 cagggatggg ccctgcccct ctcggggcag ccctgcctgc cccagccttt tgcgagctga 202140 ggctttgctg gagtcgcccg agatgctcta tgtggtacac ccttatgtgc aattctccct 202200 gcatgatgta gttaccttca gccctgccaa gctgaccaac agccaagcca aggtgctctt 202260 tcttctcttc cgtgttctga gggccatgga tgcctgtcac cgccaggggc tggcctgtgg 202320 ggctctgtct ttgcaccaca ttgctgtaga cgagaagcta tgcagtgagc tccggctgga 202380 cctgagcgct tacgagatgc cttccgagga tgaaaaccag gagggctctg aagagaaaaa 202440 tgggacaggc attaagtctg aaaaagaggg ggaagggaga actgagtgtc ccacctgcca 202500 gaaagaactt cggggccttg tgctagactg ggtccatggc cgaatcagca acttccacta 202560 cctcatgcag ctgaatcggt tggcaggtcg acggcagggg gatcccaact atcacccagt 202620 gctgccctgg gtggtggact ttaccacacc ttatgggcgc ttccgagacc ttcgtaaatc 202680 caagttccga ctcaacaagg gagataagca attggacttc acctatgaga tgacccggca 202740 ggcatttgtt gcaggtggtg caggaagtgg ggagccaccc catgttcctc accacatctc 202800 tgacgtgctc tctgacatca cgtactatgt atacaaggcc cgtcgcacac cgcgctcggt 202860 gctctgtgga catgtccgag cgcagtggga accccacgag tatcctgcca ccatggagcg 202920 gatgcagacc tggacaccgg atgagtgcat acccgagttc tacacggacc cctctatctt 202980 ttgctctatc caccctgaca tgcccgacct ggatgtgccg gcctggtgca gttctaacca 203040 ggaatttgtg gctgcccatc gagccctcct ggagagctgg gaggtgtccc aagacctgca 203100 tcactggatt gatcttacct ttggctacaa actccagggc aaagaagctg tgaaggagaa 203160 gaatgtgtgt ctgcacctgg tggacgctca cacccatctg accagctatg gcgtggtaca 203220 gctatttgat cagccacacc cccaacgcct ggctggatct cctgccctgg cccctgaacc 203280 tccactcatc ccccggctgt tggtccagcc tattcgggag gccacaggcc aggaggacat 203340 ttcaggacaa cttataaatg gtgcgggcag gcttgtcgta gaggccactc catgtgagac 203400 tggctggact agagataggc ctgggacagg agaagatgat ttagaacagg ctacagaagc 203460 tctggattcc atctccctcc ccgggaaagc aggtgaccag ccaggctctt cctccagtca 203520 agcatcacct ggcctgttgt ctttttctgc accctcgggg tctcgaccag gccgtaggag 203580 caaagctgcc gggttggacc ctggggaggg tgaagagggc aagattgtcc ttccagaggg 203640 cttcagtccc atacaggcct tggaagagct ggagaaagtg ggtaacttcc tggccaaagg 203700 cctagggagc cagttggagg agcctgaaaa gcctcacgcc cagccacctg tgcacctgca 203760 gagcctcttc catcgagaca tgcaggtcct gggtgtcctg ttggctgaga tggtgtttgc 203820 caccagggtc cggatactgc agcctgatgc acctttgtgg gtacgctttg aggctgttcg 203880 gggtctctgc atacgccact ccaaggacat ccccgtgtct ctgcagcctg tgctagacac 203940 actcctacag ctgagcggac ccaaaagtcc catggtgtcg aagaagggca agctagaccc 204000 actgtttgag tataggccgg tttcccaggg attaccccca cccagcccag cccagctcct 204060 cagccccttc agctccgtgg tccccttccc tccatacttc ccagcactgc acaagttcat 204120 tcttttatat caggcccggc gtgtggagga tgaggtccag ggtcgggagc tggcgtttgc 204180 tctgtggcag cagctgggtg cggtgttaaa tgacatcact cccgagggct tagagatcct 204240 cctgcctttc gtgctgtcgc tcatgtctga ggagcacacg gctgtgtaca cagcctggta 204300 cctatttgaa cccgttgcca aggccctggg ccccaaaaat gccaacaagt acctcctgaa 204360 gcctctcatc ggtgcctatg agagcccctg ccgcctgcat ggccgcttct acctgtacac 204420 cgactgtttt gtggcccagt tggtggtgcg gctgggcttg caggccttcc tcacccacct 204480 gctgccccat gtcctccagg tactggctgg ggtggaggct tcccaggagg agggcaaagg 204540 cctggtcggg accactgagg atgaggaaag tgagctcccg gtgtccgggc ctggctcctg 204600 tgcctttggg gaagagattc agatggatgg gcagccggct gcttcctcag gactggggct 204660 cccagactac aggtcgggcg tcagcttcca tgaccaggcc gacctgccgg acacggagga 204720 cttccaagct ggactctacg tggctgaatc tccacagccc caggaggctg aggccgtgag 204780 cctgggccag ctgagtgata agagcagtac cagcgaagcc tcccagggcg aggagagggg 204840 tggggatgat ggcggtgccc ctgcggacaa gaacagcgtc aagtcagggg acagcagcca 204900 ggacttgaag cagagcgaag gctctgagga agaggaggag gaggaaggct gtgtggtgtt 204960 ggaggaggac caggaggatg aagtcacggg aacatccgag ctcactctgt ctgacacgat 205020 gctgtccatg gagacggtgg tggctcctgg tgatgggaga gacagagaag aggaagagga 205080 gccgctgaca gagcagacag aaggcaaaga acaaaagatc ctccttggtg agcccgtggg 205140 ctgagggggc atgggtcagg tgcttttcct tcaggctctc atatgctggg tgtgggtcca 205200 accagatcca ctgtagcacg cacagccaca gtcagacaca gtgcatggaa tgtggaagtg 205260 ctgtgtgtga gtggaaagtg gggcttagat ttagctttca ggagacagaa agctccttta 205320 aaagccatac cttgggctga ggctgggagt ggagttgagt ggtagagcac ttgtctggta 205380 tactcgaggg ccctgggtgt cttatctcta gccccagaag aagtattaag aaataaaagc 205440 aagtggtggt tgagatgtga atggagccag aactggccgg aacagtcggg tggaagtggg 205500 aagagtgttc cagacaggga acagtgtgtg tgtacctctg aggctctcat ggttccatca 205560 gagaggcagg gaaaggctaa aatggttttc ttaagagagt ccagaagggc tgggcttggt 205620 ggtgcaggcc tttaatccca gcactcagga ggcagaggca ggcggatttt tgagttcgag 205680 gccagcctgg tctacaaagt gagttccagg acagccaggg ctatacagag aaaccctgtc 205740 tcgaaaaaaa aaaaaaaaaa aaagagtgta gaagggtgga agccagggac aagtctgtac 205800 aagaaggaac ttgggagcat tgccgaaagg atgacctctc tgcaggtcct gcccgaggag 205860 ccagtttctg gggaccttga ccatggctag gtgaatggac ccaggatggg atggtcaggc 205920 ttgctagcag agccacagcc gagttggctg ggtggggtgg ggtggggtgg gaagggtgag 205980 ttatctgatg agctcaggac cttttcctgc cctgcagata cagcctgcaa gatggtccgc 206040 tggctgtctg ccaagcttgg ccccacagta gcctctcgcc atgtggcccg gaacctgctg 206100 cgcctgctga catcttgtta tgttggtaag gtctgtggtt agtgctggag accaggttcc 206160 ccagccaggc ttctgcccat ccttagccct ctctaggcga ctccttccct aacttcccag 206220 cactccctga gcagggcctg ggtctcaccc attaagctgg gttttcttgg gtaagtgggg 206280 aagagcccag tattgaatga atagaagcca ccccacagtc tcagaaggcc ggcttccctc 206340 ctgccctcca ctggcttctc aacgctgctg cccttccttg gtagggccca ctcgacagca 206400 gttcaccgtc agcagtgatg acacccctcc actgaatgcc ggcaacatct accagaagag 206460 gccagtccta ggtgacatcg tgtcggggcc tgtgctcagc tgcctcctcc acattgccta 206520 cctgtatgga gaacccgttc tcacctacca gtacctgccc tacatcagct acctggtcag 206580 tccctggttc gtcaaacccc ggcttggggg tgggggcaag gatccaagga ccagccccag 206640 gtcttggggg ttccaggagg tctgtggggt gacctgtccc tccctcatct attctgtggt 206700 tctaggtagc cccagggagc aactcaaacc ccagccgact gaacagccgc aaggaggccg 206760 ggctgctggc agcggtgaca ctgacgcaga aaatcatcgt atacctctct gacacgaccc 206820 tcatggacat tctgccccgc attagccacg aggtcttgct gcctgtgctt ggcttcctca 206880 cctccttcgt cacagggtag gcccctgctg cttgggagag ccacctggct gagggggccc 206940 ccaggaaggg ctaggaagct cagggagaag cagataccgg cctgagtcat ggttctgatg 207000 ttgggggtag tggcacaggt ctttcattcc agcacccaga ggagggcaag tttctgtgag 207060 tctgagacta gcctggtcta cagagagagc tccaggctat ctaaggctcc atagtaagac 207120 tctgacttaa gaaaagagtc gtggttcatt ctgggttgtg ggtgtggctt ggtgatggga 207180 cactttccca gcatgcagga ggagctatgc ttgagttcca gcccttcaga aaaacaaaaa 207240 tgggggctgg aaagaatagc tcagggttta agagcactgg ttgctcttcc agaggatcca 207300 ggttagattc ccagctgcca catggtagct cataaccatc cggcagttct atggaacctg 207360 ccaccctcct tcggtctctg tgggcactgc aaacatgtgc acagacatac atgcaggcag 207420 aaaaaacacc catacacata aaattagacc aaaaaagttc atgttctctc ctacctgtag 207480 ctctgactaa gctacactgc ttccctgtgc ctcagtttcc tcccctggtc tggactgatc 207540 agccttacat gcagctcctg ttatttgaag ttcctggtaa attggtcaag tccttcaggg 207600 aagggctggg aactcttgca ctttgattct aggttcccca gtggggccca ggcccggact 207660 gtcctatgcg tgaaaaccat cagtctcatc gccctcatct gcttgcgcat cgggcaggag 207720 atggtccagc agcacctgag tgagccagtg gccaccttct tccaagtctt ctctcatctg 207780 catgagcttc ggcagcaggt aggcaggcag cttctgggct gggtgggcca ggccaggcca 207840 ggccaggcca gggcagtgga cccactgaat ctgtggtctt cctacccgca ggatctgcca 207900 ctggatccta agggctgtac tgagggccag ctgccagagg cgaccttctc tgatgggcag 207960 cgacgaccag tggaccccac cctgctggaa gagctgcaga aggtgttcac cctggaaatg 208020 gcgtacacaa tctacgtacc tttctcctgc ctgttgggta ttgcccatca cgttcctttg 208080 cacagagttg gtgactacat ctcttccctg gggtgggccc cgatgctttc acctccagag 208140 tcagcaatgg aatcttttta tttttatttt gacatggggt ctcatttagc ccaggctgac 208200 ctttaactcc agctccttcc agcttccacc gtctcctgtt ggcattgtag tcatgtggca 208260 ttgctcaggc ttcttncatg ttcttatttt taaatgacct gtgtgtgtgt gtgatatctg 208320 tgtgagtgtg gaggtgacag aataacagtt ggggggtcag cagatgcctt gcctgatgag 208380 catctctcta gatccagttt ttggttttgt gggcttttat gtgtgtgttt gtttgtctgt 208440 ttttgtagac agggtctctc tgtgtagcct ggccatcctg gaactcattc agtagaccaa 208500 gctggccttg agctcacaga gattcacctg cctctgcctc ccagtgctgg gattaaaggc 208560 gtgtaccact cctgcctggc tttgtttttg ttaaccacca tcctcctgcc tcagcatctg 208620 cctcccctgt gctgggatta caggtgtgtg ctatcacacc cagctaacag tggatttaaa 208680 cgtaggaatt ttaggatcag agtgaccaga tttggtccta gggcccaatt tccacagtga 208740 ttatctatct tagttaggat ctctgttgaa aatcatggtg gaacatcatt accaaatgca 208800 acttggggag gaaaaggttt attttgtctg acaactctca ggtcaccaag ggaagtcagg 208860 gcaggaactc gaggcagaag ctgaagcaaa agccatggaa gaactctggc ttgttcctca 208920 tggcttgctc agtctggtgt accccctccc cacccacctc cccacaatgg tttctctgct 208980 tatgcctggc tgtcctagaa ctcactctgt agactaggct ggcctcaaac tcaagagatc 209040 cccctgcctc tgcatctcaa gtgctaagat taaaggcggg tgccatcacc cctgccccag 209100 gggtggcact acccactgta aattggtccc ttcccatatc agttgttaaa taagaaaact 209160 cctccatagg ccaatctggt gggggaattt tctcagttga gggtttctct tctcaaatga 209220 ctgtagctga tgccaaattg ataaaacaaa tctcaaacca ccaccaccaa caacaataaa 209280 accaaacaaa ccaaacaact aaccaagaca gtgacttata aagagaatct gaacattttc 209340 cagcaggaaa ggctcaggag ctggccattc aagtctgggg aacagaatgt aggggaatat 209400 gatggtctcc agaagctacc tgcaaaggaa tgaacagctt gctgggtttt gtggcttccc 209460 ttatgggatg ggcgctgtac tgggcttctc tctgagtagg atgggccacc ctgtagttgg 209520 gaatattttg ctcctacaga attgtaagtt cccagaggca ggacacatct gtcttattct 209580 tcattgtgtg tctgatgcta gaatggtgcc tggcatacac gtgtgtgtct ctatagagac 209640 agcactcatg tctacgtatc gataaaggaa gctgttttgg ggggaggaaa caggcttaca 209700 gacgagaact taataaccca gagtagccca gtcagtacct tgccttggct tctgttgttt 209760 ctaagctctg ggtagatagc taccttgcca tcttccctga tcttagaact ttccccactc 209820 ccctgtaggt gacatcatcc ggaaaatcat ccccaaccat gagttggtcg gggagctggc 209880 agggctctat ctggaaagca tgagcccgag ctctcgaaac ccagccagca tggaacccac 209940 catggctagt gccggccctg aatgggaccc tcagagtggg agctgtctcc aggacgatgg 210000 ccactcaggg acctttggga gtgtcctggt tggaaatcgc atccagatcc ctgactctca 210060 gccccagagt cctgggccac tgggctccct ctctggagtg ggtagtagcg gaggcctcag 210120 caacaggaat gaagacaacg ccctgaagcg ggagctgcct cggagtgccc atgggctgag 210180 cgggaactgg ctggcgtact ggcagtacga gatcggtgtg agccagcagg atgcccactt 210240 ccacttccac cagatccgcc tgcagagctt cccagggcac acgggggccg tcaaatgcgt 210300 ggccgccctg agcagtgaag acttctttct gagtggcagc aaggaccgga ctgtgcgcct 210360 ctggccgctg tacaactatg gggacgggac caatgagacg gcttcccgcc tcatctatgc 210420 ccagcaccgc aaaagcgtct tctacgtggg ccagcttgag gccccgcagt atgtggtgag 210480 ctgtgatggg gcagtgcacg tctgggaccc cttcacaggt gagcgggccc aggtgaggcc 210540 tgttcgacgg ctgctttact gtgccttagc caggcctctg ggaacgggac ctagtgcgaa 210600 acgtacaatg gcgtattttg acggggaaga ttcagtgagg caggaagaga agaagagtca 210660 ggacttagaa tctgtgggac ccaagtttga atccactccc ccaacttacc agcaatcggc 210720 tcagttgctg caggcgtctg ccttctacct gtaagaacca aaaatttaga agattccacg 210780 agtatggctt tggcttcttg tacgacgtca cctgtcgtcg ttgtaaagag aagtatcgag 210840 tggaggaggg tcagggcaga cggaggtcgc agctagttag agcatgctat gtgaagagag 210900 cagactgttc tggggctgga cccttgactt cactgtggaa gcagcaagat gagaaagccc 210960 tgagattgtg ttttctgagg gtcactgggg aatgggatgc aggtgtgggg tgagttggag 211020 tttgaagtag ccagggctct ttgatagcca ctaagtcccc agatgtgtcc tttttcagga 211080 aagacccttc gcacagtgga tccttcagac agccgggtgc ccctgacggc tgtggctgtc 211140 atgcctgccc cacacaccag catcaccatg gccagctccg actccactct gcgctttgtg 211200 gactgcagga agccaggctt gcaggtcagg aggggtgcag ttcctgggct actgggggtc 211260 tctaggtacc agtcaggaaa gacactcagg ggactccacc aggaacgctg cagtgacagg 211320 cagccctgtg tgggtggggc gctggcacgg atggggcttt tctcttccgg ggatggagtg 211380 ggagggtcag gcctactggt ttcgtgggcc tgaatggggt gagctgcagt agggtgggtg 211440 gcagtgatgg atggcgacgg gcacttgaac acaatctcct cctatagcat gagttccgac 211500 tgggtggagg gctgaaccct gggcttgttc gctcgttggc cgtcagcccc agtggccgga 211560 gtgttgtggc tggcttctcc tcgggcttca tggtgctcct agatacccgc acgggcctgg 211620 ttctacgagg ctggccagcc catgaagggg acattctaca gatcaaggtg actgactgcc 211680 tgaggtccta tcctttcatt tctacttagg gcctggtctg ggagaggaca ggtttatgct 211740 ggtgtccctt ataactactc ggggacattc agtggggtgg gaaaatggcc ctcgtaggcc 211800 agctcaggaa ccagctgcac aggaggcagg ctaggggcag gaatcagggc tagaactgac 211860 cctgatgctc cacagcgatg ttctaatgag taacccttgt ccatatttgt cttgcttgga 211920 ggatcagggg tcacgccctg tccgtgaccc agttcaggtt aaataaagcc aggaggctgt 211980 ttactgcctg gagaccactg agcagagtcc atgccccctg ctgggctgtc ctgatggggg 212040 gcaggaacag gcgcaggcct gcgcatcgtg ttcctgcctc ctatattcaa tcatagacct 212100 cagagctcag caggttctgg gaggggagaa atagggctgc ttgtgggagg atttctccct 212160 gcagtgggaa ctctcctccc ccgccgtcca gatggaggtg aaagacaggc actgttgctt 212220 acagggaagg caggctgccc ccagctctat ccaggacccc aggggaccct gggtctcagt 212280 gtctctaaat cccaacattc taagaaagtg tcaggatggc tctggggtca tcctgggtgt 212340 tagtcccagc tctcggagtc ttctctgagc accagttctt ttcctatggg aagtaaggac 212400 atgccaggtg ttctttgaga ggacctgagt ttggttctca gcactgtcta gctctggctc 212460 cagggggttc aacacccttt atggcttctg tggacacata ttcttatgtg gcctgcacgc 212520 acacacacga acacaaataa aaataaatgt taaaagaaga cagcggcacc ttgtacctca 212580 catgttagta cgattggatg tggcagtgcc tcacagaatc tgtgggactt tatttattta 212640 tttatttttt ggtcttaaaa ttttaaaaga ttggtttagg agtggtggta cacaccatta 212700 atcgcaacac tcaggagcag aggcaggtgg atctctatgg gtttgaggcc agcctggtct 212760 acagagcaag tttcagggca gccaaggtta cacagagaaa ctctttctca aaaaataaaa 212820 acaaaatatt taaaagattt acttacttct tatttgagct aggatctccc tattagccct 212880 ggctgtcctg gaactcactg tatagaccag gctggcacct taaactcacg aagatcctcc 212940 tgcttctgcc tcctaagtgc tgagattaaa gtagtgttat accatgcccc actattttct 213000 ttatagattt ggtgttttgc ctgcttgtgt atatatgcac taccttcatg cagtgctaat 213060 aggggtcaga ggcgagtatc agctcttcct ggaactagag ttatggaagg ttgggagtca 213120 ccatgctggg actgggtcat ttgcaagagt tacaagtact tctgagccat ctccagcccc 213180 ctagagtttt tttcccccct ggctgtcctg aagtagaatc tgttcttgtt ttgttttgtt 213240 tttcgagaca gggtttctct acataggcct ggctgtcctg gaactcactc tgtagaccag 213300 gctggcctcg aactcagaaa tccgcctgcc tctggctctc agaatactgg aattaaaggt 213360 gtgcgccacc acgcctggct cagaatctgt ttttaaatga gagtaatagt tacaggtttt 213420 ttgttttgtt tttttctttt ctttttttgt ttttgttttt tggctcattt gttttatttg 213480 ttttgagaca ggtctcactc tgaaccccta ggtggcctgg agcttgctat gtagaacaca 213540 ctgactttaa acttgttttc tgagtgctgg atttatgggc ttgtgctatt ttgcccagcc 213600 tctgatggtt gttaataaca atattattta gcttttcttt tggagatagg ctctcactgt 213660 atatcaccca gacagtggct ggtctggaaa tcactgtgta ggccaggctg accttgaatt 213720 cacagagatc tgcctcccga gttcagaaat taaaagcact ctgggatggt tttggagttt 213780 ggtgagtacc caagcctcca ttgatgctat ctgtccctcc cgctctctgc aggctgtaga 213840 gggcagcgtg ctcatcagct cctcttccga ccattccttg actgtttgga aggagctgga 213900 acagaagccc acgcaccact acaagtcagc gtccgaccca atccacacct ttgacctgta 213960 cggcagcgag gtggtcaccg gcactgtagc caacaagatt ggtgtctgtt ccctgcttga 214020 gccaccctct caggccacca caaagctcag ttccgagaac ttccgtggca cgctcactag 214080 tctggctttg ctgcccacga aacgccacct cctgctgggc tcggacaatg gcatcatccg 214140 cctcctggca tagggccagc caggagttgg ctgagggcag ggcgagatga catctctcag 214200 ggcccgctcc tcattcttga tctcgaagcc gattcttcta ggcaagcccc aggctctggc 214260 tacccacatg gcctgctgtc tgggattgca cagctcctga atctccaaag ccttgaagtg 214320 gcttcatgaa actcgggaga tactgttcct aaccagcaag aattggggca aggaaagcac 214380 tgtgatcccc attgctcccc agttctgcct tctggattca catggggaca gggcagctcc 214440 aggaaatgaa aggagttggg cctttgctca gccagcttcc tctagccacg ctctccttag 214500 ctctgtttct cccttgggta ggaaactgct cctgtctagg gttctgatgg tactgggact 214560 ccaggctcag gagggctggc caggacctac gactttcagg gcttggtctg gggttttagc 214620 attcattcag ccaggtcttc agtatgggac cagaaaaaag gggatgtgag aacagggcta 214680 gggaaggggt tatatgggcc cagctggtcc aggaatgaat ccatgccttg ccttggtacc 214740 cctaaccaca gcgtttgtgc cttcagccgg ggaggcagcc cttgggacca gcatccctag 214800 ggacaggagg cagcgggaat catctctgta tctcgggttc tgcccagggg atgggcagac 214860 tctgccatct cttgagtgtt cgtttggaga agcctgagat gtggcccctg ctgccttctc 214920 actagttgca gtctatgtaa ataaggtcaa taaattcttt ggaagagcca cggagctgag 214980 tgaggctgtg ttgtgttttg ctttgcctag gctgggctca ggcagctctg cctcagcctc 215040 ccaaggagct ggggaactgg tatatgtcac tgtatatgtc actgtgcctg gcttatggct 215100 tggcttggct ttttttcaga tggtctcaag tgcctcaggt tggccttgat cttgggatga 215160 ccttcctgct tgaaacagag tagtgggctt ataggcatga cccaccaggt ccaattttta 215220 ttttttaaag gcattgattt ttatacgtgt atggttgttt tgcccacttg tacatatgca 215280 caccatactt gtgtctggtc cctgcggagg tcagaagagg gcatcgggat cacctggaac 215340 cgaagttaat gaatggttat gagccacatc tcgatgctga agattgaacc tggatccttt 215400 gcaagagcag ccagtgttct tacccactga gccatctcta agccccacac ccagcttctt 215460 ttgatacaag gtctggtagc tcaaacttga tatgcagccg aggaggttga cctggtattc 215520 cctacctacc ctcttctctc taccttccaa gtgctgatat tatacatagg catggatagt 215580 catgcccacc agtttgcctt gatggcacca gagtcaggaa agtccaaacc tggtagttgc 215640 aaacacagca agagggtaga ggcagccatt gtcctctggc tgccttggat acagagcttc 215700 tgggttgggt ggccttgggt cagttttccg aatggttcac ccttggggaa agggaacact 215760 gctgaagagg tgggaccctg ggagggccgg cctccagctg ggtctctcca gccctcgcct 215820 tggaacctag gctggaggga gccaaccagg atcctggact tgctacagtt aggtgaacag 215880 gctcctgcag cctccccttc ccttgggtag ctgtggtggt ggtggtggtg gtggtggtgg 215940 tggtggtggt ggtggtggtg gtgggggggg gggngnngnt 215980 17 473 PRT Mus sp. 17 Met Lys Arg Ala Ser Ser Gly Gly Ser Arg Leu Leu Ala Trp Val Leu 1 5 10 15 Trp Leu Gln Ala Trp Arg Val Ala Thr Pro Cys Pro Gly Ala Cys Val 20 25 30 Cys Tyr Asn Glu Pro Lys Val Thr Thr Ser Cys Pro Gln Gln Gly Leu 35 40 45 Gln Ala Val Pro Thr Gly Ile Pro Ala Ser Ser Gln Arg Ile Phe Leu 50 55 60 His Gly Asn Arg Ile Ser His Val Pro Ala Ala Ser Phe Gln Ser Cys 65 70 75 80 Arg Asn Leu Thr Ile Leu Trp Leu His Ser Asn Ala Leu Ala Arg Ile 85 90 95 Asp Ala Ala Ala Phe Thr Gly Leu Thr Leu Leu Glu Gln Leu Asp Leu 100 105 110 Ser Asp Asn Ala Gln Leu His Val Val Asp Pro Thr Thr Phe His Gly 115 120 125 Leu Gly His Leu His Thr Leu His Leu Asp Arg Cys Gly Leu Arg Glu 130 135 140 Leu Gly Pro Gly Leu Phe Arg Gly Leu Ala Ala Leu Gln Tyr Leu Tyr 145 150 155 160 Leu Gln Asp Asn Asn Leu Gln Ala Leu Pro Asp Asn Thr Phe Arg Asp 165 170 175 Leu Gly Asn Leu Thr His Leu Phe Leu His Gly Asn Arg Ile Pro Ser 180 185 190 Val Pro Glu His Ala Phe Arg Gly Leu His Ser Leu Asp Arg Leu Leu 195 200 205 Leu His Gln Asn His Val Ala Arg Val His Pro His Ala Phe Arg Asp 210 215 220 Leu Gly Arg Leu Met Thr Leu Tyr Leu Phe Ala Asn Asn Leu Ser Met 225 230 235 240 Leu Pro Ala Glu Val Leu Met Pro Leu Arg Ser Leu Gln Tyr Leu Arg 245 250 255 Leu Asn Asp Asn Pro Trp Val Cys Asp Cys Arg Ala Arg Pro Leu Trp 260 265 270 Ala Trp Leu Gln Lys Phe Arg Gly Ser Ser Ser Glu Val Pro Cys Asn 275 280 285 Leu Pro Gln Arg Leu Ala Asp Arg Asp Leu Lys Arg Leu Ala Ala Ser 290 295 300 Asp Leu Glu Gly Cys Ala Val Ala Ser Gly Pro Phe Arg Pro Ile Gln 305 310 315 320 Thr Ser Gln Leu Thr Asp Glu Glu Leu Leu Ser Leu Pro Lys Cys Cys 325 330 335 Gln Pro Asp Ala Ala Asp Lys Ala Ser Val Leu Glu Pro Gly Arg Pro 340 345 350 Ala Ser Ala Gly Asn Ala Leu Lys Gly Arg Val Pro Pro Gly Asp Thr 355 360 365 Pro Pro Gly Asn Gly Ser Gly Pro Arg His Ile Asn Asp Ser Pro Phe 370 375 380 Gly Thr Leu Pro Ser Ser Ala Glu Pro Pro Leu Thr Ala Leu Arg Pro 385 390 395 400 Gly Gly Ser Glu Pro Pro Gly Leu Pro Thr Thr Gly Pro Arg Arg Arg 405 410 415 Pro Gly Cys Ser Arg Lys Asn Arg Thr Arg Ser His Cys Arg Leu Gly 420 425 430 Gln Ala Gly Ser Gly Ala Ser Gly Thr Gly Asp Ala Glu Gly Ser Gly 435 440 445 Ala Leu Pro Ala Leu Ala Cys Ser Leu Ala Pro Leu Gly Leu Ala Leu 450 455 460 Val Leu Trp Thr Val Leu Gly Pro Cys 465 470 18 283 PRT Artificial Sequence Description of Artificial Sequence Consensus sequence 18 Cys Pro Xaa Xaa Cys Xaa Cys Tyr Xaa Xaa Pro Xaa Xaa Thr Xaa Ser 1 5 10 15 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Pro Xaa Xaa 20 25 30 Xaa Xaa Arg Xaa Phe Leu Xaa Xaa Asn Xaa Ile Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Trp Xaa Xaa Ser 50 55 60 Asn Xaa Xaa Xaa Xaa Ile Xaa Xaa Xaa Xaa Phe Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Leu Glu Xaa Leu Asp Leu Xaa Asp Asn Xaa Xaa Leu Xaa Xaa Xaa Xaa 85 90 95 Pro Xaa Thr Phe Xaa Gly Leu Xaa Xaa Leu Xaa Xaa Leu Xaa Leu Xaa 100 105 110 Xaa Cys Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Phe Xaa Gly Leu Xaa 115 120 125 Xaa Leu Gln Tyr Leu Tyr Leu Gln Xaa Asn Xaa Xaa Xaa Xaa Leu Xaa 130 135 140 Asp Xaa Xaa Phe Xaa Asp Leu Xaa Asn Leu Xaa His Leu Phe Leu His 145 150 155 160 Gly Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Arg Gly Leu Xaa 165 170 175 Xaa Leu Asp Arg Leu Leu Leu His Xaa Asn Xaa Xaa Xaa Xaa Val His 180 185 190 Xaa Xaa Ala Phe Xaa Xaa Leu Xaa Arg Leu Xaa Xaa Leu Xaa Leu Phe 195 200 205 Xaa Asn Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa Leu Xaa Xaa Leu Xaa 210 215 220 Xaa Leu Xaa Xaa Leu Arg Leu Asn Xaa Asn Xaa Trp Xaa Cys Xaa Cys 225 230 235 240 Arg Xaa Arg Xaa Leu Trp Xaa Trp Xaa Xaa Xaa Xaa Arg Xaa Ser Ser 245 250 255 Ser Xaa Val Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Asp Leu 260 265 270 Xaa Xaa Leu Xaa Xaa Xaa Asp Xaa Xaa Xaa Cys 275 280 19 50 PRT Artificial Sequence Description of Artificial Sequence Consensus sequence 19 Asn Xaa Trp Xaa Cys Xaa Cys Arg Ala Arg Xaa Leu Trp Xaa Trp Xaa 1 5 10 15 Xaa Xaa Xaa Arg Xaa Ser Ser Ser Xaa Val Xaa Cys Xaa Xaa Pro Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Asp Leu Xaa Xaa Leu Xaa Xaa Xaa Asp Xaa Xaa 35 40 45 Xaa Cys 50

Claims (30)

What is claimed is:
1. An isolated nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising an LRRCT domain consisting of the amino acid sequence:
N X1 W X2 C X3 C R A R X4 L W X5 W X6 X7 X8 X9 R X10 S S S X11 V [SEQ ID NO. 19] X12 C X13 X14 P X15 X16 X17 X18 X19 X20 D L X21 X22 L X23 X24 X25 D X26 X27 X28 C
wherein X is any amino acid or a gap and the polypeptide does not comprise the amino acid sequence from residue 260 to 309 of SEQ ID NO: 5 (human NgR1) or SEQ ID NO: 17 (mouse NgR1).
2. The isolated nucleic acid according to claim 1, wherein X17 and X23 are independently selected from the group consisting of: arginine and lysine.
3. The isolated nucleic acid according to claim 2, wherein the amino acid sequence of the LRRCT domain is selected from the group consisting of residues #261-310 of SEQ ID NO:2 and residues 261-310 of SEQ ID NO: 2 with up to 10 conservative amino acid substitutions.
4. An isolated nucleic acid encoding the polypeptide of SEQ ID NO: 2.
5. An isolated nucleic acid encoding the polypeptide of SEQ ID NO: 4 (mouse NgR3) or SEQ ID NO: 14 (human NgR3).
6. The isolated nucleic acid according to claim 1, wherein the polypeptide comprises: (a) a NTLRRCT domain, and (b) less than a complete CTS domain, provided that a partial CTS domain, if present, consists of no more than the first 39 amino acids of the CTS domain.
7. The isolated nucleic acid to claim 1, wherein the polypeptide does not comprise an intact GPI domain.
8. An isolated nucleic acid consisting essentially of a nucleotide sequence complementary to a nucleotide sequence encoding a polypeptide selected from the group consisting of: a polypeptide consisting of residues 311-395 of SEQ ID NO: 2, a polypeptide consisting of residues 256-396 of SEQ ID NO:14 and a polypeptide consisting of residues 321-438 of SEQ ID NO: 4, wherein the nucleic acid is from 8 to 100 nucleotides in length.
9. A vector comprising the nucleic acid of any one of claims 1, 4 or 5.
10. A host cell comprising a vector according to claim 9.
11. A polypeptide comprising a LRRCT amino acid sequence:
N X1 W X2 C X3 C R A R X4 L W X5 W X6 X7 X8 X9 R X10 S S S X11 H V [SEQ ID NO. 19] X12 C X13 X14 P X15 X16 X17 X18 X19 X20 D L X21 X22 L X23 X24 X25 D X26 X27 X28 C
wherein X is any amino acid residue or a gap and the polypeptide does not comprise the amino acid sequence from residue 260 to 309 of SEQ ID NO: 5 (human NgR1) or SEQ ID NO: 17 (mouse NgR1).
12. The polypeptide according to claim 11, wherein X17 and X23 is selected from the group consisting of arginine and lysine.
13. The polypeptide according to claim 11, wherein X19 is glycine. [SEQ ID NO:11]
14. The polypeptide according to claim 11, wherein the amino acid sequence is selected from the group consisting of residues 261-310 of SEQ ID NO:2, residues 206-255 of SEQ ID NO: 14, residues 271-320 of SEQ ID NO:4 and amino acid sequences thereof comprising a conservative substitution.
15. A polypeptide comprising a NTLRRCT amino acid sequence:
C P X1 X2 C X3 C Y X4 X5 P X6 X7 T X8 S C X9 X10 X11 X12 X13 X14 X15 X16 P [SEQ ID NO: 18] X17 X18 X19 P X20 X21 X22 X23 R X24 F L X25 X26 N X27 I X28 X29 X30 X31 X32 X33 X34 F X35 X36 X37 X38 X39 X40 X41 X42 L W X43 X44 S N X45 X46 X47 X48 I X49 X50 X51 X52 F X53 X54 X55 X56 X57 L E X58 L D L X59 D N X60 X61 L X62 X63 X64 X65 P X66 T F X67 G L X68 X69 L X70 X71 L X72 L X73 X74 C X75 L X76 X77 L X78 X79 X80 X81 F X82 G L X83 X84 L Q Y L Y L Q X85 N X86 X87 X88 X89 L X90 D X91 X92 F X93 D L X94 N L X95 H L F L H G N X96 X97 X98 X99 X100 X101 X102 X103 X104 F R G L X105 X106 L D R L L L H X107 N X108 X109 X110 X111 V H X112 X113 A F X114 X115 L X116 R L X117 X118 L X119 L F X120 N X121 L X122 X123 L X124 X125 X126 X127 L X128 X129 L X130 X131 L X132 X133 L R L N X134 N X135 W X136 C X137 C R X138 R X139 L W X140 W X141 X142 X143 X144 R X145 S S S X146 V X147 C X148 X149 P X150 X151 X152 X153 X154 X155 D L X156 X157 L X158 X159 X160 D X161 X162 X163 C
wherein X is any amino acid residue or a gap and wherein the polypeptide is not the polypeptide of SEQ ID NO: 5 (human NgR1) or SEQ ID NO: 17 (mouse NgR1).
16. The polypeptide according to claim 15, wherein X6, X37 and X38 represents a gap.
17. A polypeptide comprising an amino sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:14.
18. The polypeptide according any one of claims 11, 15 or 17, wherein the polypeptide comprises: (a) an NTLRRCT domain, and (b) less than a complete CTS domain, provided that a partial CTS domain, if present, consists of no more than the first 39 amino acids of the CTS domain.
19. The polypeptide according to any one of claims 11, 15 or 17, wherein the polypeptide does not comprise an intact GPI domain.
20. The polypeptide according to any one of claims 11, 15 or 17, wherein the amino acid sequence of the polypeptide further comprises an amino acid sequence of a heterologous polypeptide.
21. The polypeptide according to claim 20, wherein the heterologous polypeptide is an Fc portion of an antibody.
22. A method of producing a polypeptide according to any one of claims 11, 15 or 17, comprising the steps of introducing an isolated nucleic acid according to any one of claims 1, 4, 5 or 8 or a vector according to claim 9 into a host cell, culturing said host cell under conditions suitable for expression of said polypeptide, and recovering said polypeptide.
23. An antibody that binds to a polypeptide of any one of claims 11, 15 or 17.
24. A composition comprising the polypeptide of claim 11, 15 or 17 and a pharmaceutically acceptable carrier.
25. A composition comprising the antibody of claim 23 and a pharmaceutically acceptable carrier.
26. A method of decreasing inhibition of axonal growth of a CNS neuron, comprising the step of contacting the neuron with an effective amount of the polypeptide of claim 11, 15 or 17.
27. A method of treating a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of the polypeptide of claim 11, 15 or 17.
28. A method of decreasing inhibition of axonal growth of a CNS neuron comprising the step of contacting the neuron with an effective amount of the antibody according to claim 23.
29. A method of treating a central nervous system disease, disorder or injury, comprising administering to a mammal an effective amount of the antibody according to claim 23.
30. A method for identifying a molecule that binds a polypeptide of claim 11, 15 or 17 comprising the steps of:
(a) providing a polypeptide of claim 11, 15 or 17;
(b) contacting the polypeptide with the candidate molecule; and
(c) detecting binding of the candidate molecule to the polypeptide.
US09/972,546 2000-10-06 2001-10-06 Nogo receptor homologs Abandoned US20030124704A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US09/972,546 US20030124704A1 (en) 2000-10-06 2001-10-06 Nogo receptor homologs
US10/735,256 US7173118B2 (en) 2000-10-06 2003-12-12 Nogo receptor homologs
US11/544,013 US7456255B2 (en) 2000-10-06 2006-10-06 Nogo receptor homologs that decrease inhibition of axonal elongation
US12/277,187 US20090175850A1 (en) 2000-10-06 2008-11-24 NOGO Receptor Homologs
US12/952,747 US20110129477A1 (en) 2000-10-06 2010-11-23 NOGO Receptor Homologs

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US23836100P 2000-10-06 2000-10-06
US09/972,546 US20030124704A1 (en) 2000-10-06 2001-10-06 Nogo receptor homologs

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/735,256 Continuation US7173118B2 (en) 2000-10-06 2003-12-12 Nogo receptor homologs

Publications (1)

Publication Number Publication Date
US20030124704A1 true US20030124704A1 (en) 2003-07-03

Family

ID=22897532

Family Applications (5)

Application Number Title Priority Date Filing Date
US09/972,546 Abandoned US20030124704A1 (en) 2000-10-06 2001-10-06 Nogo receptor homologs
US10/735,256 Expired - Lifetime US7173118B2 (en) 2000-10-06 2003-12-12 Nogo receptor homologs
US11/544,013 Expired - Lifetime US7456255B2 (en) 2000-10-06 2006-10-06 Nogo receptor homologs that decrease inhibition of axonal elongation
US12/277,187 Abandoned US20090175850A1 (en) 2000-10-06 2008-11-24 NOGO Receptor Homologs
US12/952,747 Abandoned US20110129477A1 (en) 2000-10-06 2010-11-23 NOGO Receptor Homologs

Family Applications After (4)

Application Number Title Priority Date Filing Date
US10/735,256 Expired - Lifetime US7173118B2 (en) 2000-10-06 2003-12-12 Nogo receptor homologs
US11/544,013 Expired - Lifetime US7456255B2 (en) 2000-10-06 2006-10-06 Nogo receptor homologs that decrease inhibition of axonal elongation
US12/277,187 Abandoned US20090175850A1 (en) 2000-10-06 2008-11-24 NOGO Receptor Homologs
US12/952,747 Abandoned US20110129477A1 (en) 2000-10-06 2010-11-23 NOGO Receptor Homologs

Country Status (12)

Country Link
US (5) US20030124704A1 (en)
EP (1) EP1325130B1 (en)
JP (2) JP4465148B2 (en)
AT (1) ATE458815T1 (en)
AU (2) AU2002211539B2 (en)
CA (1) CA2424834A1 (en)
DE (1) DE60141409D1 (en)
DK (1) DK1325130T3 (en)
ES (1) ES2341608T3 (en)
NZ (1) NZ525422A (en)
PT (1) PT1325130E (en)
WO (1) WO2002029059A2 (en)

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030113326A1 (en) * 2001-12-03 2003-06-19 Children's Medical Center Corporation Reducing myelin-mediated inhibition of axon regeneration
US20040259092A1 (en) * 2001-08-27 2004-12-23 Carmen Barske Nogo receptor homologues and their use
US20050048520A1 (en) * 2000-10-06 2005-03-03 Yale University Nogo receptor homologs
US20050221420A1 (en) * 2001-10-22 2005-10-06 Carmen Barske Nogo receptor homologues and their use
US20050271655A1 (en) * 2002-08-10 2005-12-08 Biogen Idec Ma Inc. Nogo receptor antagonists
US7119165B2 (en) 2000-01-12 2006-10-10 Yale University Nogo receptor-mediated blockade of axonal growth
US20080274077A1 (en) * 2003-12-16 2008-11-06 Children's Medical Center Corporation Method for Treating Neurological Disorders
US20090054325A1 (en) * 2005-07-07 2009-02-26 Yale University Compositions and methods for suppressing axonal growth inhibition
US20090111753A1 (en) * 2004-10-01 2009-04-30 Yale University Nogo-A Polypeptide Fragments, Variant Nogo Receptor-1 Polypeptides, and Uses Thereof
US20110071088A1 (en) * 2003-12-16 2011-03-24 Childrens Medical Center Corporation Method for treating neurological disorders
US20110123535A1 (en) * 2006-05-15 2011-05-26 Biogen Idec Ma Inc. Use of Nogo Receptor-1 (NGR1) for Promoting Oligodendrocyte Survival
US8669345B2 (en) 2006-01-27 2014-03-11 Biogen Idec Ma Inc. Nogo receptor antagonists
US8992918B2 (en) 2008-03-13 2015-03-31 Yale University Reactivation of axon growth and recovery in chronic spinal cord injury
US9120111B2 (en) 2012-02-24 2015-09-01 Rain Bird Corporation Arc adjustable rotary sprinkler having full-circle operation and automatic matched precipitation
US9156043B2 (en) 2012-07-13 2015-10-13 Rain Bird Corporation Arc adjustable rotary sprinkler with automatic matched precipitation

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2004005510A1 (en) * 2002-07-05 2005-11-04 塩野義製薬株式会社 Novel Nogo receptor-like polypeptide and its DNA
SI1606409T1 (en) 2003-03-19 2011-01-31 Biogen Idec Inc Nogo receptor binding protein
AU2004227392A1 (en) 2003-04-04 2004-10-21 University Of Rochester Identification of novel nogo-receptors and methods related thereto
EA009643B1 (en) * 2003-04-16 2008-02-28 Йейл Юниверсити Treatment of conditions involving amyloid plaques
EA008253B1 (en) * 2003-08-07 2007-04-27 Байоджен Айдек Ма Инк. Nogo receptor antagonists
US20080045926A1 (en) * 2004-01-30 2008-02-21 Relton Jane K Treatment of Conditions Involving Dopaminergic Neuronal Degeneration Using Nogo Receptor Antagonists
EP1776136B1 (en) 2004-06-24 2012-10-03 Biogen Idec MA Inc. Treatment of conditions involving demyelination
EP2478917A1 (en) 2005-07-08 2012-07-25 Biogen Idec MA Inc. SP35 antibodies and uses thereof
AU2006282856A1 (en) * 2005-08-25 2007-03-01 Biogen Idec Ma Inc. Nogo receptor polypeptides and polypeptide fragments and uses thereof
CA2653456A1 (en) * 2006-05-24 2007-11-29 Richard Daneman Permeability of blood-brain barrier
US20100047232A1 (en) * 2006-11-14 2010-02-25 Jasvinder Atwal Modulators of neuronal regeneration
ES2437110T3 (en) * 2006-11-14 2014-01-08 Genentech, Inc. Neural Regeneration Modulators
WO2009009743A2 (en) * 2007-07-12 2009-01-15 Institute For Advance Study Sequence optimization for expression of a foreign gene
WO2009076359A2 (en) * 2007-12-11 2009-06-18 Genentech, Inc. Modulators of neuronal regeneration
CL2009001155A1 (en) * 2008-05-13 2010-06-04 Genentech Inc Isolated anti-pirb / lilrb antibody; polynucleotide that encodes it; vector; host cell; method of obtaining; pharmaceutical composition comprising it; kit; and its use to treat neurodegenerative diseases.
JP2011527572A (en) 2008-07-09 2011-11-04 バイオジェン・アイデック・エムエイ・インコーポレイテッド Composition comprising a LINGO antibody or fragment
EP2392208B1 (en) * 2010-06-07 2016-05-04 Helmholtz Zentrum München Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Fusion proteins comprising a DNA-binding domain of a Tal effector protein and a non-specific cleavage domain of a restriction nuclease and their use
JP2015518829A (en) 2012-05-14 2015-07-06 バイオジェン・エムエイ・インコーポレイテッドＢｉｏｇｅｎ ＭＡ Ｉｎｃ． LINGO-2 antagonist for treatment of conditions involving motor neurons
WO2016112270A1 (en) 2015-01-08 2016-07-14 Biogen Ma Inc. Lingo-1 antagonists and uses for treatment of demyelinating disorders

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5250414A (en) 1988-11-04 1993-10-05 Erziehungsdirektion Of The Canton Zurich Diagnostic methods using neurite growth regulatory factors
CA1341050C (en) 1988-11-04 2000-07-11 Martin E. Schwab Neurite growth regulatory factors
KR100196540B1 (en) 1988-11-04 1999-06-15 마틴 이. 샤브 Neurite growth regulatory factors
CA2117889A1 (en) 1993-02-11 1994-08-18 Martin E. Schwab A combination of neurotrophin and antibody directed toward myelin-associated neurite growth inhibitory protein promotes central nervous system regeneration
WO2000073452A2 (en) 1999-06-02 2000-12-07 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
US5858708A (en) 1996-08-12 1999-01-12 Bandman; Olga Polynucleotides encoding two novel human neuroendocrine-specific proteins
WO1999007891A1 (en) * 1997-08-05 1999-02-18 Human Genome Sciences, Inc. 90 human secreted proteins
WO2000070050A1 (en) 1999-05-14 2000-11-23 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
PT1490386E (en) 1998-03-10 2008-11-24 Genentech Inc Novel polypeptide and nucleic acids encoding the same
AU4682999A (en) 1998-06-16 2000-01-05 Human Genome Sciences, Inc. 94 human secreted proteins
PT1098972E (en) 1998-07-22 2010-12-14 Smithkline Beecham Ltd Protein similar to neuroendocrine-specific protein, and encoding cdna
WO2000031235A2 (en) * 1998-11-06 2000-06-02 Schwab Martin E Nucleotide and protein sequences of nogo genes and methods based thereon
JP3695642B2 (en) 1998-12-01 2005-09-14 ジェネンテック・インコーポレーテッド Promotion or inhibition of angiogenesis and cardiovascularization
DE69935085T2 (en) 1998-12-22 2007-08-23 Genentech, Inc., South San Francisco Method and composition for inhibiting neoplastic cell growth
KR20030002292A (en) 1999-03-08 2003-01-08 제넨테크, 인크. Secreted and Transmembrane Polypeptides and Nucleic Acids Encoding the Same
CA2362427A1 (en) 1999-03-08 2000-09-14 Genentech, Inc. Compositions and methods for the treatment of immune related diseases
WO2000058473A2 (en) 1999-03-31 2000-10-05 Curagen Corporation Nucleic acids including open reading frames encoding polypeptides; 'orfx'
WO2001009162A2 (en) 1999-07-30 2001-02-08 Millennium Pharmaceuticals, Inc. Secreted proteins and uses thereof
AU2575901A (en) * 1999-12-08 2001-06-18 Millennium Pharmaceuticals, Inc. Novel leucine rich repeat-containing molecules and uses therefor
US20020012965A1 (en) * 2000-01-12 2002-01-31 Strittmatter Stephen M. Nogo receptor-mediated blockade of axonal growth
US7119165B2 (en) 2000-01-12 2006-10-10 Yale University Nogo receptor-mediated blockade of axonal growth
US6436703B1 (en) * 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides
AU780798B2 (en) 2000-04-18 2005-04-21 Teijin Limited Oxygen concentrating apparatus
AU2002211539B2 (en) * 2000-10-06 2007-01-25 Biogen Idec Ma Inc. Nogo receptor homologs
AU2002331180A1 (en) 2001-08-27 2003-03-10 Novartis Pharma Gmbh Nogo receptor homologues and their use
US20050221420A1 (en) * 2001-10-22 2005-10-06 Carmen Barske Nogo receptor homologues and their use
KR20050062525A (en) * 2002-08-10 2005-06-23 예일 유니버시티 Nogo receptor antagonists

Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080219984A1 (en) * 2000-01-12 2008-09-11 Yale University Nogo receptor-mediated blockade of axonal growth
US20100278831A1 (en) * 2000-01-12 2010-11-04 Yale University Nogo Receptor-Mediated Blockade of Axonal Growth
US7119165B2 (en) 2000-01-12 2006-10-10 Yale University Nogo receptor-mediated blockade of axonal growth
US8394929B2 (en) 2000-01-12 2013-03-12 Yale University Nogo receptor-mediated blockade of axonal growth
US20050048520A1 (en) * 2000-10-06 2005-03-03 Yale University Nogo receptor homologs
US7456255B2 (en) 2000-10-06 2008-11-25 Yale University Nogo receptor homologs that decrease inhibition of axonal elongation
US20070104713A1 (en) * 2000-10-06 2007-05-10 Strittmatter Stephen M Nogo receptor homologs
US20090175850A1 (en) * 2000-10-06 2009-07-09 Strittmatter Stephen M NOGO Receptor Homologs
US20040259092A1 (en) * 2001-08-27 2004-12-23 Carmen Barske Nogo receptor homologues and their use
US20080118951A1 (en) * 2001-08-27 2008-05-22 Carmen Barske Nogo receptor homologues and their use
US20080090270A1 (en) * 2001-10-22 2008-04-17 Carmen Barske Nogo receptor homologues and their use
US20050221420A1 (en) * 2001-10-22 2005-10-06 Carmen Barske Nogo receptor homologues and their use
US20090136970A1 (en) * 2001-10-22 2009-05-28 Carmen Barske Nogo receptor homologues and their use
US7309485B2 (en) * 2001-12-03 2007-12-18 Children's Medical Center Corporation Reducing myelin-mediated inhibition of axon regeneration
US20030113326A1 (en) * 2001-12-03 2003-06-19 Children's Medical Center Corporation Reducing myelin-mediated inhibition of axon regeneration
US20050271655A1 (en) * 2002-08-10 2005-12-08 Biogen Idec Ma Inc. Nogo receptor antagonists
US7465705B2 (en) 2002-08-10 2008-12-16 Yale University Nogo receptor antagonists
US8030456B2 (en) 2002-08-10 2011-10-04 Yale University Nogo receptor antagonists
US20110071088A1 (en) * 2003-12-16 2011-03-24 Childrens Medical Center Corporation Method for treating neurological disorders
US20080274077A1 (en) * 2003-12-16 2008-11-06 Children's Medical Center Corporation Method for Treating Neurological Disorders
US8912144B2 (en) 2003-12-16 2014-12-16 Children's Medical Center Corporation Method for treating stroke via administration of NEP1-40 and inosine
US20090111753A1 (en) * 2004-10-01 2009-04-30 Yale University Nogo-A Polypeptide Fragments, Variant Nogo Receptor-1 Polypeptides, and Uses Thereof
WO2007008732A3 (en) * 2005-07-07 2009-05-28 Univ Yale Compositions and methods for suppressing axonal growth inhibition
US7893032B2 (en) 2005-07-07 2011-02-22 Yale University NgR variants and compositions thereof for suppressing axonal growth inhibition
US20090054325A1 (en) * 2005-07-07 2009-02-26 Yale University Compositions and methods for suppressing axonal growth inhibition
US8669345B2 (en) 2006-01-27 2014-03-11 Biogen Idec Ma Inc. Nogo receptor antagonists
US9228015B2 (en) 2006-01-27 2016-01-05 Biogen Idec Ma Inc. Nogo receptor antagonists and methods of increasing neurite outgrowth
US20110123535A1 (en) * 2006-05-15 2011-05-26 Biogen Idec Ma Inc. Use of Nogo Receptor-1 (NGR1) for Promoting Oligodendrocyte Survival
US8992918B2 (en) 2008-03-13 2015-03-31 Yale University Reactivation of axon growth and recovery in chronic spinal cord injury
US9120111B2 (en) 2012-02-24 2015-09-01 Rain Bird Corporation Arc adjustable rotary sprinkler having full-circle operation and automatic matched precipitation
US9156043B2 (en) 2012-07-13 2015-10-13 Rain Bird Corporation Arc adjustable rotary sprinkler with automatic matched precipitation

Also Published As

Publication number Publication date
DK1325130T3 (en) 2010-05-25
ATE458815T1 (en) 2010-03-15
US20070104713A1 (en) 2007-05-10
WO2002029059A3 (en) 2003-01-23
EP1325130B1 (en) 2010-02-24
WO2002029059A9 (en) 2003-05-15
DE60141409D1 (en) 2010-04-08
US7173118B2 (en) 2007-02-06
US20050048520A1 (en) 2005-03-03
US20110129477A1 (en) 2011-06-02
US7456255B2 (en) 2008-11-25
CA2424834A1 (en) 2002-04-11
JP2004528809A (en) 2004-09-24
AU2002211539B2 (en) 2007-01-25
NZ525422A (en) 2006-09-29
WO2002029059A2 (en) 2002-04-11
JP2009100767A (en) 2009-05-14
ES2341608T3 (en) 2010-06-23
US20090175850A1 (en) 2009-07-09
PT1325130E (en) 2010-05-31
EP1325130A2 (en) 2003-07-09
JP4465148B2 (en) 2010-05-19
AU1153902A (en) 2002-04-15

Similar Documents

Publication Publication Date Title
US20030124704A1 (en) Nogo receptor homologs
AU2017267184B2 (en) Method for assessing a prognosis and predicting the response of patients with malignant diseases to immunotherapy
RU2735551C2 (en) Compositions for modulating tau protein expression
AU2013358958B2 (en) Polynucleotides encoding rodent antibodies with human idiotypes and animals comprising same
KR101708544B1 (en) Methods and nucleic acids for analyses of cellular proliferative disorders
KR102291355B1 (en) Identification of patients in need of pd-l1 inhibitor cotherapy
US7507542B2 (en) Method for regulating immune function using the FOXP3 protein
ES2744098T3 (en) Compositions and their uses aimed at huntingtin
KR102046668B1 (en) Methods and nucleic acids for determining the prognosis of a cancer subject
JP2022506613A (en) Use of adeno-associated virus vectors to correct gene defects / expressed proteins in hair cells and sustentacular cells of the inner ear
CA2941594A1 (en) Genetic polymorphisms of the protein receptor c (procr) associated with myocardial infarction, methods of detection and uses thereof
KR102362647B1 (en) Method for obtaining globally activated monocytes
KR20220012230A (en) Methods and compositions for modulating splicing and translation
US20020119541A1 (en) Tumor suppressor CAR-1
US20020164645A1 (en) Assays for taste receptor cell specific ion channel
KR20190104400A (en) Human antibodies of transgenic rodent origin with multiple heavy chain immunoglobulin loci
JP2002017376A (en) Secretory protein or membrane protein
US20040171003A1 (en) Cancer-associated genes
CN113249470A (en) Method for in vitro diagnosis or prognosis of lung cancer
KR100877554B1 (en) A marker for predicting survival period of a hepatocellular carcinoma patient, a kit and microarray comprising the same and method for predicting survival period of a hepatocellular carcinoma patient using the marker
US20040002102A1 (en) BIVM (basic, immunoglobulin-like variable motif-containing) gene, transcriptional products, and uses thereof
JP2003259875A (en) Single base polymorphism (4) in human gene
US20030170683A1 (en) Formin-2 nucleic acids and polypeptides and uses thereof
KR20220008245A (en) Metastasis-specific markers for diagnosing prognosis and determining treatment stratagies of patient of Prostate adenocarcinoma
CN114053413A (en) Application of COL4A4 gene as acute ischemic stroke treatment target

Legal Events

Date Code Title Description
AS Assignment

Owner name: YALE UNIVERSITY, CONNECTICUT

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:STRITTMATTER, STEPHEN M.;REEL/FRAME:012347/0489

Effective date: 20011005

Owner name: BIOGEN, INC., MASSACHUSETTS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:CATE, RICHARD L.;SAH, DINAH W.Y.;REEL/FRAME:012347/0505

Effective date: 20011127

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: BIOGEN IDEC MA INC., MASSACHUSETTS

Free format text: CHANGE OF NAME;ASSIGNOR:BIOGEN, INC.;REEL/FRAME:019448/0817

Effective date: 20031113

AS Assignment

Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:YALE UNIVERSITY;REEL/FRAME:024107/0024

Effective date: 20100318

AS Assignment

Owner name: NIH-DEITR, MARYLAND

Free format text: CONFIRMATORY LICENSE;ASSIGNOR:YALE UNIVERSITY - NEW HAVEN, CT;REEL/FRAME:052875/0253

Effective date: 20200609