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Publication numberUS20050239060 A1
Publication typeApplication
Application numberUS 10/122,851
Publication dateOct 27, 2005
Filing dateApr 12, 2002
Priority dateJan 21, 2000
Also published asUS6569662, US20030104529
Publication number10122851, 122851, US 2005/0239060 A1, US 2005/239060 A1, US 20050239060 A1, US 20050239060A1, US 2005239060 A1, US 2005239060A1, US-A1-20050239060, US-A1-2005239060, US2005/0239060A1, US2005/239060A1, US20050239060 A1, US20050239060A1, US2005239060 A1, US2005239060A1
InventorsY. Tang, Chenghua Liu, Vinod Asundi, Jie Zhang, Feiyan Ren, Rui-Hong Chen, Qing Zhao, Tom Wehrman, Aidong Xue, Yonghong Yang, Jian-Rui Wang, Ping Zhou, Yunqing Ma, Dunrui Wang, Zhiwei Wang, Radoje Drmanac
Original AssigneeTang Y T, Chenghua Liu, Vinod Asundi, Jie Zhang, Feiyan Ren, Rui-Hong Chen, Zhao Qing A, Tom Wehrman, Xue Aidong J, Yonghong Yang, Jian-Rui Wang, Ping Zhou, Yunqing Ma, Dunrui Wang, Zhiwei Wang, Drmanac Radoje T
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Expression vector comprising nucleotide sequences coding protein factor for treatment, diagnosis and prevention of proliferative and differentiative disorders
US 20050239060 A1
Abstract
The present invention provides novel nucleic acids, novel polypeptide sequences encoded by these nucleic acids and uses thereof.
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Claims(28)
1. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-877, 879-1104, a mature protein coding portion of SEQ ID NO: 1-877, 879-1104, an active domain of SEQ ID NO:1-877, 879-1104, and complementary sequences thereof.
2. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide hybridizes to the polynucleotide of claim 1 under stringent hybridization conditions.
3. An isolated polynucleotide encoding a polypeptide with biological activity, wherein said polynucleotide has greater than about 90% sequence identity with the polynucleotide of claim 1.
4. The polynucleotide of claim 1 wherein said polynucleotide is DNA.
5. An isolated polynucleotide of claim 1 wherein said polynucleotide comprises the complementary sequences.
6. A vector comprising the polynucleotide of claim 1.
7. An expression vector comprising the polynucleotide of claim 1.
8. A host cell genetically engineered to comprise the polynucleotide of claim 1.
9. A host cell genetically engineered to comprise the polynucleotide of claim 1 operatively associated with a regulatory sequence that modulates expression of the polynucleotide in the host cell.
10. An isolated polypeptide, wherein the polypeptide is selected from the group consisting of:
(a) a polypeptide encoded by any one of the polynucleotides of claim 1; and
(b) a polypeptide encoded by a polynucleotide hybridizing under stringent conditions with any one of SEQ ID NO: 1-877, 879-1104.
11. A composition comprising the polypeptide of claim 10 and a carrier.
12. An antibody directed against the polypeptide of claim 10.
13. A method for detecting the polynucleotide of claim 1 in a sample, comprising:
a) contacting the sample with a compound that binds to and forms a complex with the polynucleotide of claim 1 for a period sufficient to form the complex; and
b) detecting the complex, so that if a complex is detected, the polynucleotide of claim 1 is detected.
14. A method for detecting the polynucleotide of claim 1 in a sample, comprising:
a) contacting the sample under stringent hybridization conditions with nucleic acid primers that anneal to the polynucleotide of claim 1 under such conditions;
b) amplifying a product comprising at least a portion of the polynucleotide of claim 1; and
c) detecting said product and thereby the polynucleotide of claim 1 in the sample.
15. The method of claim 14, wherein the polynucleotide is an RNA molecule and the method further comprises reverse transcribing an annealed RNA molecule into a cDNA polynucleotide.
16. A method for detecting the polypeptide of claim 10 in a sample, comprising:
a) contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex; and
b) detecting formation of the complex, so that if a complex formation is detected, the polypeptide of claim 10 is detected.
17. A method for identifying a compound that binds to the polypeptide of claim 10, comprising:
a) contacting the compound with the polypeptide of claim 10 under conditions sufficient to form a polypeptide/compound complex; and
b) detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.
18. A method for identifying a compound that binds to the polypeptide of claim 10, comprising:
a) contacting the compound with the polypeptide of claim 10, in a cell, under conditions sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and
b) detecting the complex by detecting reporter gene sequence expression, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide of claim 10 is identified.
19. A method of producing the polypeptide of claim 10, comprising,
a) culturing a host cell comprising a polynucleotide sequence selected from the group consisting of a polynucleotide sequence of SEQ ID NO: 1-877, 879-1104, a mature protein coding portion of SEQ ID NO: 1-877, 879-1104, an active domain of SEQ ID NO: 1-877, 879-1104, complementary sequences thereof and a polynucleotide sequence hybridizing under stringent conditions to SEQ ID, NO: 1-877, 879-1104, under conditions sufficient to express the polypeptide in said cell; and
b) isolating the polypeptide from the cell culture or cells of step (a).
20. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of any one of the polypeptides from the Sequence Listing, the mature protein portion thereof, or the active domain thereof.
21. The polypeptide of claim 20 wherein the polypeptide is provided on a polypeptide array.
22. A collection of polynucleotides, wherein the collection comprising the sequence information of at least one of SEQ ID NO: 1-877, 879-1104.
23. The collection of claim 22, wherein the collection is provided on a nucleic acid array.
24. The collection of claim 23, wherein the array detects full-matches to any one of the polynucleotides in the collection.
25. The collection of claim 23, wherein the array detects mismatches to any one of the polynucleotides in the collection.
26. The collection of claim 22, wherein the collection is provided in a computer-readable format.
27. A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.
28. A method of treatment comprising administering to a mammalian subject in need thereof a therapeutic amount of a composition comprising an antibody that specifically binds to a polypeptide of claim 10 or 20 and a pharmaceutically acceptable carrier.
Description
1. CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. application Ser. No. 09/552,317, filed Apr. 25, 2000, which in turn is a continuation-in-part application of U.S. application Ser. No. 09/488,725, filed Jan. 21, 2000, both of which are incorporated herein by reference in their entirety.

2. BACKGROUND OF THE INVENTION

2.1 Technical Field

The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods.

2.2 Background

Technology aimed at the discovery of protein factors (including e.g., cytokines, such as lymphokines, interferons, CSFs, chemokines, and interleukins) has matured rapidly over the past decade. The now routine hybridization cloning and expression cloning techniques clone novel polynucleotides “directly” in the sense that they rely on information directly related to the discovered protein (i.e., partial DNA/amino acid sequence of the protein in the case of hybridization cloning; activity of the protein in the case of expression cloning). More recent “indirect” cloning techniques such as signal sequence cloning, which isolates DNA sequences based on the presence of a now well-recognized secretory leader sequence motif, as well as various PCR-based or low stringency hybridization-based cloning techniques, have advanced the state of the art by making available large numbers of DNA/amino acid sequences for proteins that are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity.

Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences.

3. SUMMARY OF THE INVENTION

The compositions of the present invention include novel isolated polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies.

The compositions of the present invention additionally include vectors, including expression vectors, containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.

The present invention relates to a collection or library of at least one novel nucleic acid sequence assembled from expressed sequence tags (ESTs) isolated mainly by sequencing by hybridization (SBH), and in some cases, sequences obtained from one or more public databases. The invention relates also to the proteins encoded by such polynucleotides, along with therapeutic, diagnostic and research utilities for these polynucleotides and proteins. These nucleic acid sequences are designated as SEQ ID NO: 1-1104 and are provided in the Sequence Listing. In the nucleic acids provided in the Sequence Listing, A is adenosine; C is cytosine; G is guanosine; T is thymine; and N is any of the four bases. In the amino acids provided in the Sequence Listing, * corresponds to the stop codon.

The nucleic acid sequences of the present invention also include, nucleic acid sequences that hybridize to the complement of SEQ ID NO: 1-1104 under stringent hybridization conditions; nucleic acid sequences which are allelic variants or species homologues of any of the nucleic acid sequences recited above, or nucleic acid sequences that encode a peptide comprising a specific domain or truncation of the peptides encoded by SEQ ID NO: 1-1104. A polynucleotide comprising a nucleotide sequence having at least 90% identity to an identifying sequence of SEQ ID NO: 1-1104 or a degenerate variant or fragment thereof. The identifying sequence can be 100 base pairs in length.

The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NO: 1-1104. The sequence information can be a segment of any one of SEQ ID NO: 1-1104 that uniquely identifies or represents the sequence information of SEQ ID NO: 1-1104.

A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information is provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.

This invention also includes the reverse or direct complement of any of the nucleic acid sequences recited above; cloning or expression vectors containing the nucleic acid sequences; and host cells or organisms transformed with these expression vectors. Nucleic acid sequences (or their reverse or direct complements) according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology, such as use as hybridization probes, use as primers for PCR, use in an array, use in computer-readable media, use in sequencing full-length genes, use for chromosome and gene mapping, use in the recombinant production of protein, and use in the generation of anti-sense DNA or RNA, their chemical analogs and the like.

In a preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-1104 or novel segments or parts of the nucleic acids of the invention are used as primers in expression assays that are well known in the art. In a particularly preferred embodiment, the nucleic acid sequences of SEQ ID NO: 1-1104 or novel segments or parts of the nucleic acids provided herein are used in diagnostics for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.

The isolated polynucleotides of the invention include, but are not limited to, a polynucleotide comprising any one of the nucleotide sequences set forth in the SEQ ID NO: 1-1104; a polynucleotide comprising any of the full length protein coding sequences of the SEQ ID NO: 1-1104; and a polynucleotide comprising any of the nucleotide sequences of the mature protein coding sequences of the SEQ ID NO: 1-1104. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any one of the nucleotide sequences set forth in the SEQ ID NO: 1-1104; (b) a nucleotide sequence encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotides recited above; (d) a polynucleotide which encodes a species homolog (e.g. orthologs) of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of any of the polypeptides comprising an amino acid sequence set forth in the Sequence Listing.

The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising any of the amino acid sequences set forth in the Sequence Listing; or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in the SEQ ID NO: 1-1104; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of any of the polypeptide sequences in the Sequence Listing, and “substantial equivalents” thereof (e.g., with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid sequence identity) that preferably retain biological activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.

The invention also provides compositions comprising a polypeptide of the invention. Polypeptide compositions of the invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

The invention also provides host cells transformed or transfected with a polynucleotide of the invention.

The invention also relates to methods for producing a polypeptide of the invention comprising growing a culture of the host cells of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the polypeptide from the culture or from the host cells. Preferred embodiments include those in which the protein produced by such process is a mature form of the protein.

Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of anti-sense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.

In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.

The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.

Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a polypeptide of the present invention and a pharmaceutically acceptable carrier.

In particular, the polypeptides and polynucleotides of the invention can be utilized, for example, in methods for the prevention and/or treatment of disorders involving aberrant protein expression or biological activity.

The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample. Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions. The invention provides a method for detecting the polynucleotides of the invention in a sample, comprising contacting the sample with a compound that binds to and forms a complex with the polynucleotide of interest for a period sufficient to form the complex and under conditions sufficient to form a complex and detecting the complex such that if a complex is detected, the polynucleotide of interest is detected. The invention also provides a method for detecting the polypeptides of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting the formation of the complex such that if a complex is formed, the polypeptide is detected.

The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.

The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention. The invention provides a method for identifying a compound that binds to the polypeptides of the invention comprising contacting the compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a reporter gene sequence in the cell; and detecting the complex by detecting the reporter gene sequence expression such that if expression of the reporter gene is detected the compound the binds to a polypeptide of the invention is identified.

The methods of the invention also provides methods for treatment which involve the administration of the polynucleotides or polypeptides of the invention to individuals exhibiting symptoms or tendencies. In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising administering compounds and other substances that modulate the overall activity of the target gene products. Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity.

The polypeptides of the present invention and the polynucleotides encoding them are also useful for the same functions known to one of skill in the art as the polypeptides and polynucleotides to which they have the closest homology (set forth in Table 1). If no homology is set forth for a sequence, then the polypeptides and polynucleotides of the present invention are useful for a variety of applications, as described herein, including use in arrays for detection.

4. DETAILED DESCRIPTION OF THE INVENTION

4.1 Definitions

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

The term “active” refers to those forms of the polypeptide which retain the biologic and/or immunologic activities of any naturally occurring polypeptide. According to the invention, the terms “biologically active” or “biological activity” refer to a protein or peptide having structural, regulatory or biochemical functions of a naturally occurring molecule. Likewise “immunologically active” or “immunological activity” refers to the capability of the natural, recombinant or synthetic polypeptide to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.

The term “activated cells” as used in this application are those cells which are engaged in extracellular or intracellular membrane trafficking, including the export of secretory or enzymatic molecules as part of a normal or disease process.

The terms “complementary” or “complementarity” refer to the natural binding of polynucleotides by base pairing. For example, the sequence 5′-AGT-3′ binds to the complementary sequence 3′-TCA-5′. Complementarity between two single-stranded molecules may be “partial” such that only some of the nucleic acids bind or it may be “complete” such that total complementarity exists between the single stranded molecules. The degree of complementarity between the nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.

The term “embryonic stem cells (ES)” refers to a cell that can give rise to many differentiated cell types in an embryo or an adult, including the germ cells. The term “germ line stem cells (GSCs)” refers to stem cells derived from primordial stem cells that provide a steady and continuous source of germ cells for the production of gametes. The term “primordial germ cells (PGCs)” refers to a small population of cells set aside from other cell lineages particularly from the yolk sac, mesenteries, or gonadal ridges during embryogenesis that have the potential to differentiate into germ cells and other cells. PGCs are the source from which GSCs and ES cells are derived The PGCs, the GSCs and the ES cells are capable of self-renewal. Thus these cells not only populate the germ line and give rise to a plurality of terminally differentiated cells that comprise the adult specialized organs, but are able to regenerate themselves.

The term “expression modulating fragment,” EMF, means a series of nucleotides which modulates the expression of an operably linked ORF or another EMF.

As used herein, a sequence is said to “modulate the expression of an operably linked sequence” when the expression of the sequence is altered by the presence of the EMF. EMFs include, but are not limited to, promoters, and promoter modulating sequences (inducible elements). One class of EMFs are nucleic acid fragments which induce the expression of an operably linked ORF in response to a specific regulatory factor or physiological event.

The terms “nucleotide sequence” or “nucleic acid” or “polynucleotide” or “oligonculeotide” are used interchangeably and refer to a heteropolymer of nucleotides or the sequence of these nucleotides. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA) or to any DNA-like or RNA-like material. Generally, nucleic acid segments provided by this invention may be assembled from fragments of the genome and short oligonucleotide linkers, or from a series of oligonucleotides, or from individual nucleotides, to provide a synthetic nucleic acid which is capable of being expressed in a recombinant transcriptional unit comprising regulatory elements derived from a microbial or viral operon, or a eukaryotic gene.

The terms “oligonucleotide fragment” or a “polynucleotide fragment”, “portion,” or “segment” or “probe” or “primer” are used interchangeable and refer to a sequence of nucleotide residues which are at least about 5 nucleotides, more preferably at least about 7 nucleotides, more preferably at least about 9 nucleotides, more preferably at least about 11 nucleotides and most preferably at least about 17 nucleotides. The fragment is preferably less than about 500 nucleotides, preferably less than about 200 nucleotides, more preferably less than about 100 nucleotides, more preferably less than about 50 nucleotides and most preferably less than 30 nucleotides. Preferably the probe is from about 6 nucleotides to about 200 nucleotides, preferably from about 15 to about 50 nucleotides, more preferably from about 17 to 30 nucleotides and most preferably from about 20 to 25 nucleotides. Preferably the fragments can be used in polymerase chain reaction (PCR), various hybridization procedures or microarray procedures to identify or amplify identical or related parts of mRNA or DNA molecules. A fragment or segment may uniquely identify each polynucleotide sequence of the present invention. Preferably the fragment comprises a sequence substantially similar to any one of SEQ ID NOs: 1-1104.

Probes may, for example, be used to determine whether specific mRNA molecules are present in a cell or tissue or to isolate similar nucleic acid sequences from chromosomal DNA as described by Walsh et al. (Walsh, P. S. et al., 1992, PCR Methods Appl 1:241-250). They may be labeled by nick translation, Klenow fill-in reaction, PCR, or other methods well known in the art. Probes of the present invention, their preparation and/or labeling are elaborated in Sambrook, J. et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; or Ausubel, F. M. et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., both of which are incorporated herein by reference in their entirety.

The nucleic acid sequences of the present invention also include the sequence information from the nucleic acid sequences of SEQ ID NOs: 1-1104. The sequence information can be a segment of any one of SEQ ID NOs: 1-1104 that uniquely identifies or represents the sequence information of that sequence of SEQ ID NO: 1-1104. One such segment can be a twenty-mer nucleic acid sequence because the probability that a twenty-mer is fully matched in the human genome is 1 in 300. In the human genome, there are three billion base pairs in one set of chromosomes. Because 420 possible twenty-mers exist, there are 300 times more twenty-mers than there are base pairs in a set of human chromosome. Using the same analysis, the probability for a seventeen-mer to be fully matched in the human genome is approximately 1 in 5. When these segments are used in arrays for expression studies, fifteen-mer segments can be used. The probability that the fifteen-mer is fully matched in the expressed sequences is also approximately one in five because expressed sequences comprise less than approximately 5% of the entire genome sequence.

Similarly, when using sequence information for detecting a single mismatch, a segment can be a twenty-five mer. The probability that the twenty-five mer would appear in a human genome with a single mismatch is calculated by multiplying the probability for a full match (1÷425) times the increased probability for mismatch at each nucleotide position (3×25). The probability that an eighteen mer with a single mismatch can be detected in an array for expression studies is approximately one in five. The probability that a twenty-mer with a single mismatch can be detected in a human genome is approximately one in five.

The term “open reading frame,” ORF, means a series of nicleotide triplets coding for amino acids without any termination codons and is a sequence translatable into protein.

The terms “operably linked” or “operably associated” refer to functionally related nucleic acid sequences. For example, a promoter is operably associated or operably linked with a coding sequence if the promoter controls the transcription of the coding sequence. While operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements e.g. repressor genes are not contiguously linked to the coding sequence but still control transcription/translation of the coding sequence.

The term “pluripotent” refers to the capability of a cell to differentiate into a number of differentiated cell types that are present in an adult organism. A pluripotent cell is restricted in its differentiation capability in comparison to a totipotent cell.

The terms “polypeptide” or “peptide” or “amino acid sequence” refer to an oligopeptide, peptide, polypeptide or protein sequence or fragment thereof and to naturally occurring or synthetic molecules. A polypeptide “fragment,” “portion,” or “segment” is a stretch of amino acid residues of at least about 5 amino acids, preferably at least about 7 amino acids, more preferably at least about 9 amino acids and most preferably at least about 17 or more amino acids. The peptide preferably is not greater than about 200 amino acids, more preferably less than 150 amino acids and most preferably less than 100 amino acids. Preferably the peptide is from about 5 to about 200 amino acids. To be active, any polypeptide must have sufficient length to display biological and/or immunological activity.

The term “naturally occurring polypeptide” refers to polypeptides produced by cells that have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including, but not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.

The term “translated protein coding portion” means a sequence which encodes for the full length protein which may include any leader sequence or any processing sequence.

The term “mature protein coding sequence” means a sequence which encodes a peptide or protein without a signal or leader sequence. The peptide may have been produced by processing in the cell which removes any leader/signal sequence. The peptide may be produced synthetically or the protein may have been produced using a polynucleotide only encoding for the mature protein coding sequence.

The term “derivative” refers to polypeptides chemically modified by such techniques as ubiquitination, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) and insertion or substitution by chemical synthesis of amino acids such as ornithine, which do not normally occur in human proteins.

The term “variant” (or “analog”) refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using, e.g., recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added or deleted without abolishing activities of interest, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology (conserved regions) or by replacing amino acids with consensus sequence.

Alternatively, recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the “redundancy” in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.

Preferably, amino acid “substitutions” are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. “Conservative” amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. “Insertions” or “deletions” are preferably in the range of about 1 to 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

Alternatively, where alteration of function is desired, insertions, deletions or non-conservative alterations can be engineered to produce altered polypeptides. Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention. For example, such alterations may change polypeptide characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate. Further, such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression. For example, cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges.

The terms “purified” or “substantially purified” as used herein denotes that the indicated nucleic acid or polypeptide is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like. In one embodiment, the polynucleotide or polypeptide is purified such that it constitutes at least 95% by weight, more preferably at least 99% by weight, of the indicated biological macromolecules present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000 daltons, can be present).

The term “isolated” as used herein refers to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) present with the nucleic acid or polypeptide in its natural source. In one embodiment, the nucleic acid or polypeptide is found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same. The terms “isolated” and “purified” do not encompass nucleic acids or polypeptides present in their natural source.

The term “recombinant,” when used herein to refer to a polypeptide or protein, means that a polypeptide or protein is derived from recombinant (e.g., microbial, insect, or mammalian) expression systems. “Microbial” refers to recombinant polypeptides or proteins made in bacterial or fungal (e.g., yeast) expression systems. As a product, “recombinant microbial” defines a polypeptide or protein essentially free of native endogenous substances and unaccompanied by associated native glycosylation. Polypeptides or proteins expressed in most bacterial cultures, e.g., E. coli, will be free of glycosylation modifications; polypeptides or proteins expressed in yeast will have a glycosylation pattern in general different from those expressed in mammalian cells.

The term “recombinant expression vehicle or vector” refers to a plasmid or phage or virus or vector, for expressing a polypeptide from a DNA (RNA) sequence. An expression vehicle can comprise a transcriptional unit comprising an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an amino terminal methionine residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

The term “recombinant expression system” means host cells which have stably integrated a recombinant transcriptional unit into chromosomal DNA or carry the recombinant transcriptional unit extrachromosomally. Recombinant expression systems as defined herein will express heterologous polypeptides or proteins upon induction of the regulatory elements linked to the DNA segment or synthetic gene to be expressed. This term also means host cells which have stably integrated a recombinant genetic element or elements having a regulatory role in gene expression, for example, promoters or enhancers. Recombinant expression systems as defined herein will express polypeptides or proteins endogenous to the cell upon induction of the regulatory elements linked to the endogenous DNA segment or gene to be expressed. The cells can be prokaryotic or eukaryotic.

The term “secreted” includes a protein that is transported across or through a membrane, including transport as a result of signal sequences in its amino acid sequence when it is expressed in a suitable host cell. “Secreted” proteins include without limitation proteins secreted wholly (e.g., soluble proteins) or partially (e.g., receptors) from the cell in which they are expressed. “Secreted” proteins also include without limitation proteins that are transported across the membrane of the endoplasmic reticulum. “Secreted” proteins are also intended to include proteins containing non-typical signal sequences (e.g. Interleukin-1 Beta, see Krasney, P. A. and Young, P. R. (1992) Cytokine 4(2):134-143) and factors released from damaged cells (e.g. Interleukin-1 Receptor Antagonist, see Arend, W. P. et. al. (1998) Annu. Rev. Immunol. 16:27-55).

Where desired, an expression vector may be designed to contain a “signal or leader sequence” which will direct the polypeptide through the membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous protein sources by recombinant DNA techniques.

The term “stringent” is used to refer to conditions that are commonly understood in the art as stringent. Stringent conditions can include highly stringent conditions (i.e., hybridization to filter-bound DNA in 0.5 M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and moderately stringent conditions (i.e., washing in 0.2×SSC/0.1% SDS at 42° C.). Other exemplary hybridization conditions are described herein in the examples.

In instances of hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for 14-base oligonucleotides), 48° C. (for 17-base oligos), 55° C. (for 20-base oligonucleotides), and 60° C. (for 23-base oligonucleotides).

As used herein, “substantially equivalent” can refer both to nucleotide and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions, deletions, or additions, the net effect of which does not result in an adverse functional dissimilarity between the reference and subject sequences. Typically, such a substantially equivalent sequence varies from one of those listed herein by no more than about 35% (i.e., the number of individual residue substitutions, additions, and/or deletions in a substantially equivalent sequence, as compared to the corresponding reference sequence, divided by the total number of residues in the substantially equivalent sequence is about 0.35 or less). Such a sequence is said to have 65% sequence identity to the listed sequence. In one embodiment, a substantially equivalent, e.g., mutant, sequence of the invention varies from a listed sequence by no more than 30% (70% sequence identity); in a variation of this embodiment, by no more than 25% (75% sequence identity); and in a further variation of this embodiment, by no more than 20% (80% sequence identity) and in a further variation of this embodiment, by no more than 10% (90% sequence identity) and in a further variation of this embodiment, by no more that 5% (95% sequence identity). Substantially equivalent, e.g., mutant, amino acid sequences according to the invention preferably have at least 80% sequence identity with a listed amino acid sequence, more preferably at least 90% sequence identity. Substantially equivalent nucleotide sequences of the invention can have lower percent sequence identities, taking into account, for example, the redundancy or degeneracy of the genetic code. Preferably, nucleotide sequence has at least about 65% identity, more preferably at least about 75% identity, and most preferably at least about 95% identity. For the purposes of the present invention, sequences having substantially equivalent biological activity and substantially equivalent expression characteristics are considered substantially equivalent. For the purposes of determining equivalence, truncation of the mature sequence (e.g., via a mutation which creates a spurious stop codon) should be disregarded. Sequence identity may be determined, e.g., using the Jotun Hein method (Hein, J. (1990) Methods Enzymol. 183:626-645). Identity between sequences can also be determined by other methods known in the art, e.g. by varying hybridization conditions.

The term “totipotent” refers to the capability of a cell to differentiate into all of the cell types of an adult organism.

The term “transformation” means introducing DNA into a suitable host cell so that the DNA is replicable, either as an extrachromosomal element, or by chromosomal integration. The term “transfection” refers to the taking up of an expression vector by a suitable host cell, whether or not any coding sequences are in fact expressed. The term “infection” refers to the introduction of nucleic acids into a suitable host cell by use of a virus or viral vector.

As used herein, an “uptake modulating fragment,” UMF, means a series of nucleotides which mediate the uptake of a linked DNA fragment into a cell. UMFs can be readily identified using known UMFs as a target sequence or target motif with the computer-based systems described below. The presence and activity of a UMF can be confirmed by attaching the suspected UMF to a marker sequence. The resulting nucleic acid molecule is then incubated with an appropriate host under appropriate conditions and the uptake of the marker sequence is determined. As described above, a UMF will increase the frequency of uptake of a linked marker sequence.

Each of the above terms is meant to encompass all that is described for each, unless the context dictates otherwise.

4.2 Nucleic Acids of the Invention

Nucleotide sequences of the invention are set forth in the Sequence Listing.

The isolated polynucleotides of the invention include a polynucleotide comprising the nucleotide sequences of the SEQ ID NO: 1-1104; a polynucleotide encoding any one of the peptide sequences of SEQ ID NO: 1-1104; and a polynucleotide comprising the nucleotide sequence encoding the mature protein coding sequence of the polynucleotides of any one of SEQ ID NO: 1-1104. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent conditions to (a) the complement of any of the nucleotides sequences of the SEQ ID NO: 1-1104; (b) nucleotide sequences encoding any one of the amino acid sequences set forth in the Sequence Listing; (c) a polynucleotide which is an allelic variant of any polynucleotide recited above; (d) a polynucleotide which encodes a species homolog of any of the proteins recited above; or (e) a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptides of SEQ ID NO: 1-1104. Domains of interest may depend on the nature of the encoded polypeptide; e.g., domains in receptor-like polypeptides include ligand-binding, extracellular, transmembrane, or cytoplasmic domains, or combinations thereof; domains in immunoglobulin-like proteins include the variable immunoglobulin-like domains; domains in enzyme-like polypeptides include catalytic and substrate binding domains; and domains in ligand polypeptides include receptor-binding domains.

The polynucleotides of the invention include naturally occurring or wholly or partially synthetic DNA, e.g., cDNA and genomic DNA, and RNA, e.g., mRNA. The polynucleotides may include all of the coding region of the cDNA or may represent a portion of the coding region of the cDNA.

The present invention also provides genes corresponding to the cDNA sequences disclosed herein. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. Further 5′ and 3′ sequence can be obtained using methods known in the art. For example, full length cDNA or genomic DNA that corresponds to any of the polynucleotides of the SEQ ID NO: 1-1104 can be obtained by screening appropriate cDNA or genomic DNA libraries under suitable hybridization conditions using any of the polynucleotides of the SEQ ID NO: 1-1104 or a portion thereof as a probe. Alternatively, the polynucleotides of the SEQ ID NO: 1-1104 may be used as the basis for suitable primer(s) that allow identification and/or amplification of genes in appropriate genomic DNA or cDNA libraries.

The nucleic acid sequences of the invention can be assembled from ESTs and sequences (including cDNA and genomic sequences) obtained from one or more public databases, such as dbEST, gbpri, and UniGene. The EST sequences can provide identifying sequence information, representative fragment or segment information, or novel segment information for the full-length gene.

The polynucleotides of the invention also provide polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides recited above. Polynucleotides according to the invention can have, e.g., at least about 65%, at least about 70%, at least about 75%, at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide recited above.

Included within the scope of the nucleic acid sequences of the invention are nucleic acid sequence fragments that hybridize under stringent conditions to any of the nucleotide sequences of the SEQ ID NO: 1-1104, or complements thereof, which fragment is greater than about 5 nucleotides, preferably 7 nucleotides, more preferably greater than 9 nucleotides and most preferably greater than 17 nucleotides. Fragments of, e.g. 15, 17, or 20 nucleotides or more that are selective for (i.e. specifically hybridize to any one of the polynucleotides of the invention) are contemplated. Probes capable of specifically hybridizing to a polynucleotide can differentiate polynucleotide sequences of the invention from other polynucleotide sequences in the same family of genes or can differentiate human genes from genes of other species, and are preferably based on unique nucleotide sequences.

The sequences falling within the scope of the present invention are not limited to these specific sequences, but also include allelic and species variations thereof. Allelic and species variations can be routinely determined by comparing the sequence provided in SEQ ID NO: 1-1104, a representative fragment thereof, or a nucleotide sequence at least 90% identical, preferably 95% identical, to SEQ ID NOs: 1-1104 with a sequence from another isolate of the same species. Furthermore, to accommodate codon variability, the invention includes nucleic acid molecules coding for the same amino acid sequences as do the specific ORFs disclosed herein. In other words, in the coding region of an ORF, substitution of one codon for another codon that encodes the same amino acid is expressly contemplated.

The nearest neighbor result for the nucleic acids of the present invention, including SEQ ID NOs: 1-1104, can be obtained by searching a database using an algorithm or a program. Preferably, a BLAST which stands for Basic Local Alignment Search Tool is used to search for local sequence alignments (Altshul, S. F. J. Mol. Evol. 36 290-300 (1993) and Altschul S. F. et al. J. Mol. Biol. 21:403-410 (1990)). Alternatively a FASTA version 3 search against Genpept, using Fastxy algorithm.

Species homologs (or orthologs) of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.

The invention also encompasses allelic variants of the disclosed polynucleotides or proteins; that is, naturally-occurring alternative forms of the isolated polynucleotide which also encode proteins which are identical, homologous or related to that encoded by the polynucleotides.

The nucleic acid sequences of the invention are further directed to sequences which encode variants of the described nucleic acids. These amino acid sequence variants may be prepared by methods known in the art by introducing appropriate nucleotide changes into a native or variant polynucleotide. There are two variables in the construction of amino acid sequence variants: the location of the mutation and the nature of the mutation. Nucleic acids encoding the amino acid sequence variants are preferably constructed by mutating the polynucleotide to encode an amino acid sequence that does not occur in nature. These nucleic acid alterations can be made at sites that differ in the nucleic acids from different species (variable positions) or in highly conserved regions (constant regions). Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site. Amino acid sequence deletions generally range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous. Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues. Examples of terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells and sequences such as FLAG or poly-histidine sequences useful for purifying the expressed protein.

In a preferred method, polynucleotides encoding the novel amino acid sequences are changed via site-directed mutagenesis. This method uses oligonucleotide sequences to alter a polynucleotide to encode the desired amino acid variant, as well as sufficient adjacent nucleotides on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed. In general, the techniques of site-directed mutagenesis are well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183 (1983). A versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller and Smith, Nucleic Acids Res. 10:6487-6500 (1982). PCR may also be used to create amino acid sequence variants of the novel nucleic acids. When small amounts of template DNA are used as starting material, primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant. PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the polypeptide at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasmid and this gives a polynucleotide encoding the desired amino acid variant.

A further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315 (1985); and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be used in the practice of the invention for the cloning and expression of these novel nucleic acids. Such DNA sequences include those which are capable of hybridizing to the appropriate novel nucleic acid sequence under stringent conditions.

Polynucleotides encoding preferred polypeptide truncations of the invention can be used to generate polynucleotides encoding chimeric or fusion proteins comprising one or more domains of the invention and heterologous protein sequences.

The polynucleotides of the invention additionally include the complement of any of the polynucleotides recited above. The polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides of the desired sequence identities.

In accordance with the invention, polynucleotide sequences comprising the mature protein coding sequences corresponding to any one of SEQ ID NO: 1-1104, or functional equivalents thereof, may be used to generate recombinant DNA molecules that direct the expression of that nucleic acid, or a functional equivalent thereof, in appropriate host cells. Also included are the cDNA inserts of any of the clones identified herein.

A polynucleotide according to the invention can be joined to any of a variety of other nucleotide sequences by well-established recombinant DNA techniques (see Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Useful nucleotide sequences for joining to polynucleotides include an assortment of vectors, e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.

The present invention further provides recombinant constructs comprising a nucleic acid having any of the nucleotide sequences of the SEQ ID NOs: 1-1104 or a fragment thereof or any other polynucleotides of the invention. In one embodiment, the recombinant constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a nucleic acid having any of the nucleotide sequences of the SEQ ID NOs: 1-1104 or a fragment thereof is inserted, in a forward or reverse orientation. In the case of a vector comprising one of the ORFs of the present invention, the vector may further comprise regulatory sequences, including for example, a promoter, operably linked to the ORF. Large numbers of suitable vectors and promoters are known to those of skill in the art and are commercially available for generating the recombinant constructs of the present invention. The following vectors are provided by way of example. Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLneo, pSV2cat, pOG44, PXTI, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).

The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein “operably linked” means that the isolated polynucleotide of the invention and an expression control sequence are situated within a vector or cell in such a way that the protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, and trc. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an amino terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product. Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotech, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced or derepressed by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Polynucleotides of the invention can also be used to induce immune responses. For example, as described in Fan et al., Nat. Biotech. 17:870-872 (1999), incorporated herein by reference, nucleic acid sequences encoding a polypeptide may be used to generate antibodies against the encoded polypeptide following topical administration of naked plasmid DNA or following injection, and preferably intramuscular injection of the DNA. The nucleic acid sequences are preferably inserted in a recombinant expression vector and may be in the form of naked DNA.

4.3 Hosts

The present invention further provides host cells genetically engineered to contain the polynucleotides of the invention. For example, such host cells may contain nucleic acids of the invention introduced into the host cell using known transformation, transfection or infection methods. The present invention still further provides host cells genetically engineered to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell.

Knowledge of nucleic acid sequences allows for modification of cells to permit, or increase, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the polypeptide at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the encoding sequences. See, for example, PCT International Publication No. WO94/12650, PCT International Publication No. WO92/20808, and PCT International Publication No. WO91/09955. 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 carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.

The host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate transfection, DEAE, dextran mediated transfection, or electroporation (Davis, L. et al., Basic Methods in Molecular Biology (1986)). The host cells containing one of the polynucleotides of the invention, can be used in conventional manners to produce the gene product encoded by the isolated fragment (in the case of an ORF) or can be used to produce a heterologous protein under the control of the EMF.

Any host/vector system can be used to express one or more of the ORFs of the present invention. These include, but are not limited to, eukaryotic hosts such as HeLa cells, Cv-1 cell, COS cells, 293 cells, and Sf9 cells, as well as prokaryotic host such as E. coli and B. subtilis, The most preferred cells are those which do not normally express the particular polypeptide or protein or which expresses the polypeptide or protein at low natural level. Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989), the disclosure of which is hereby incorporated by reference.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 (1981). Other cell lines capable of expressing a compatible vector are, for example, the C127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements. Recombinant polypeptides and proteins produced in bacterial culture are usually isolated by initial extraction from cell pellets, followed by one or more salting-out, aqueous ion exchange or size exclusion chromatography steps. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Alternatively, it may be possible to produce the protein in lower eukaryotes such as yeast or insects or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain the functional protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequence include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the host cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT1US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

4.4 Polypeptides of the Invention

The isolated polypeptides of the invention include, but are not limited to, a polypeptide comprising: the amino acid sequences set forth as any one of SEQ ID NO: 1-1104 or an amino acid sequence encoded by any one of the nucleotide sequences SEQ ID NOs: 1-1104 or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides preferably with biological or immunological activity that are encoded by: (a) a polynucleotide having any one of the nucleotide sequences set forth in the SEQ ID NOs: 1-1104 or (b) polynucleotides encoding any one of the amino acid sequences set forth as SEQ ID NO: 1-1104 or (c) polynucleotides that hybridize to the complement of the polynucleotides of either (a) or (b) under stringent hybridization conditions. The invention also provides biologically active or immunologically active variants of any of the amino acid sequences set forth as SEQ ID NO: 1-1104 or the corresponding full length or mature protein; and “substantial equivalents” thereof (e.g., with at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, typically at least about 95%, more typically at least about 98%, or most typically at least about 99% amino acid identity) that retain biological activity. Polypeptides encoded by allelic variants may have a similar, increased, or decreased activity compared to polypeptides comprising SEQ ID NO: 1-104.

Fragments of the proteins of the present invention which are capable of exhibiting biological activity are also encompassed by the present invention. Fragments of the protein may be in linear form or they may be cyclized using known methods, for example, as described in H. U. Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S. McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of protein binding sites.

The present invention also provides both full-length and mature forms (for example, without a signal sequence or precursor sequence) of the disclosed proteins. The protein coding sequence is identified in the sequence listing by translation of the disclosed nucleotide sequences. The mature form of such protein may be obtained by expression of a full-length polynucleotide in a suitable mammalian cell or other host cell. The sequence of the mature form of the protein is also determinable from the amino acid sequence of the full-length form. Where proteins of the present invention are membrane bound, soluble forms of the proteins are also provided. In such forms, part or all of the regions causing the proteins to be membrane bound are deleted so that the proteins are fully secreted from the cell in which it is expressed.

Protein compositions of the present invention may further comprise an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.

The present invention further provides isolated polypeptides encoded by the nucleic acid fragments of the present invention or by degenerate variants of the nucleic acid fragments of the present invention. By “degenerate variant” is intended nucleotide fragments which differ from a nucleic acid fragment of the present invention (e.g., an ORF) by nucleotide sequence but, due to the degeneracy of the genetic code, encode an identical polypeptide sequence. Preferred nucleic acid fragments of the present invention are the ORFs that encode proteins.

A variety of methodologies known in the art can be utilized to obtain any one of the isolated polypeptides or proteins of the present invention. At the simplest level, the amino acid sequence can be synthesized using commercially available peptide synthesizers. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with proteins may possess biological properties in common therewith, including protein activity. This technique is particularly useful in producing small peptides and fragments of larger polypeptides. Fragments are useful, for example, in generating antibodies against the native polypeptide. Thus, they may be employed as biologically active or immunological substitutes for natural, purified proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

The polypeptides and proteins of the present invention can alternatively be purified from cells which have been altered to express the desired polypeptide or protein. As used herein, a cell is said to be altered to express a desired polypeptide or protein when the cell, through genetic manipulation, is made to produce a polypeptide or protein which it normally does not produce or which the cell normally produces at a lower level. One skilled in the art can readily adapt procedures for introducing and expressing either recombinant or synthetic sequences into eukaryotic or prokaryotic cells in order to generate a cell which produces one of the polypeptides or proteins of the present invention.

The invention also relates to methods for producing a polypeptide comprising growing a culture of host cells of the invention in a suitable culture medium, and purifying the protein from the cells or the culture in which the cells are grown. For example, the methods of the invention include a process for producing a polypeptide in which a host cell containing a suitable expression vector that includes a polynucleotide of the invention is cultured under conditions that allow expression of the encoded polypeptide. The polypeptide can be recovered from the culture, conveniently from the culture medium, or from a lysate prepared from the host cells and further purified. Preferred embodiments include those in which the protein produced by such process is a full length or mature form of the protein.

In an alternative method, the polypeptide or protein is purified from bacterial cells which naturally produce the polypeptide or protein. One skilled in the art can readily follow known methods for isolating polypeptides and proteins in order to obtain one of the isolated polypeptides or proteins of the present invention. These include, but are not limited to, immunochromatography, HPLC, size-exclusion chromatography, ion-exchange chromatography, and immuno-affinity chromatography. See, e.g., Scopes, Protein Purification: Principles and Practice, Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel et al., Current Protocols in Molecular Biology. Polypeptide fragments that retain biological/immunological activity include fragments comprising greater than about 100 amino acids, or greater than about 200 amino acids, and fragments that encode specific protein domains.

The purified polypeptides can be used in in vitro binding assays which are well known in the art to identify molecules which bind to the polypeptides. These molecules include but are not limited to, for e.g., small molecules, molecules from combinatorial libraries, antibodies or other proteins. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.

In addition, the peptides of the invention or molecules capable of binding to the peptides may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for SEQ ID NO: 1-1104.

The protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component of the milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the protein.

The proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified proteins but into which modification are naturally provided or deliberately engineered. For example, modifications, in the peptide or DNA sequence, can be made by those skilled in the art using known techniques. Modifications of interest in the protein sequences may include the alteration, substitution, replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more of the cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Techniques for such alteration, substitution, replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration, substitution, replacement, insertion or deletion retains the desired activity of the protein. Regions of the protein that are important for the protein function can be determined by various methods known in the art including the alanine-scanning method which involved systematic substitution of single or strings of amino acids with alanine, followed by testing the resulting alanine-containing variant for biological activity. This type of analysis determines the importance of the substituted amino acid(s) in biological activity. Regions of the protein that are important for protein function may be determined by the eMATRIX program.

Other fragments and derivatives of the sequences of proteins which would be expected to retain protein activity in whole or in part and are useful for screening or other immunological methodologies may also be easily made by those skilled in the art given the disclosures herein. Such modifications are encompassed by the present invention.

The protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987), incorporated herein by reference. As used herein, an insect cell capable of expressing a polynucleotide of the present invention is “transformed.

The protein of the invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the protein may also include an affinity column containing agents which will bind to the protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl™ or Cibacrom blue 3GA Sepharose™; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

Alternatively, the protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag. Kits for expression and purification of such fusion proteins are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The protein can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. One such epitope (“FLAG® ”) is commercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an “isolated protein.”

The polypeptides of the invention include analogs (variants). This embraces fragments, as well as peptides in which one or more amino acids has been deleted, inserted, or substituted. Also, analogs of the polypeptides of the invention embrace fusions of the polypeptides or modifications of the polypeptides of the invention, wherein the polypeptide or analog is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such analogs may exhibit improved properties such as activity and/or stability. Examples of moieties which may be fused to the polypeptide or an analog include, for example, targeting moieties which provide for the delivery of polypeptide to pancreatic cells, e.g., antibodies to pancreatic cells, antibodies to immune cells such as T-cells, monocytes, dendritic cells, granulocytes, etc., as well as receptor and ligands expressed on pancreatic or immune cells. Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment, for example, immunosuppressive drugs such as cyclosporin, SK506, azathioprine, CD3 antibodies and steroids. Also, polypeptides may be fused to immune modulators, and other cytokines such as alpha or beta interferon.

4.4.1 Determining Polypeptide and Polynucleotide Identity and Similarity

Preferred identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in computer programs including, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, BLASTX, FASTA (Altschul, S. F. et al., J. Molec. Biol. 215:403-410 (1990), PSI-BLAST (Altschul S. F. et al., Nucleic Acids Res. vol. 25, pp. 3389-3402, herein incorporated by reference), eMatrix software (Wu et al., J. Comp. Biol., vol. 6, pp. 219-235 (1999), herein incorporated by reference), eMotif software (Nevill-Manning et al, ISMB-97, vol 4, pp. 202-209, herein incorporated by reference) and the Kyte-Doolittle hydrophobocity prediction algorithm (J. Mol. Biol, 157, pp. 105-31 (1982), incorporated herein by reference). The BLAST programs are publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul, S., et al. NCB NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).

4.5 Gene Therapy

Mutations in the polynucleotides of the invention gene may result in loss of normal function of the encoded protein. The invention thus provides gene therapy to restore normal activity of the polypeptides of the invention; or to treat disease states involving polypeptides of the invention. Delivery of a functional gene encoding polypeptides of the invention 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, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Introduction of any one of the nucleotides of the present invention or a gene encoding the polypeptides of the present invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes. Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of polypeptides of the invention will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of polypeptides of the invention.

Other methods inhibiting expression of a protein include the introduction of antisense molecules to the nucleic acids of the present invention, their complements, or their translated RNA sequences, by methods known in the art. Further, the polypeptides of the present invention can be inhibited by using targeted deletion methods, or the insertion of a negative regulatory element such as a silencer, which is tissue specific.

The present invention still further provides cells genetically engineered in vivo to express the polynucleotides of the invention, wherein such polynucleotides are in operative association with a regulatory sequence heterologous to the host cell which drives expression of the polynucleotides in the cell. These methods can be used to increase or decrease the expression of the polynucleotides of the present invention.

Knowledge of DNA sequences provided by the invention allows for modification of cells to permit, increase, or decrease, expression of endogenous polypeptide. Cells can be modified (e.g., by homologous recombination) to provide increased polypeptide expression by replacing, in whole or in part, the naturally occurring promoter with all or part of a heterologous promoter so that the cells express the protein at higher levels. The heterologous promoter is inserted in such a manner that it is operatively linked to the desired protein 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 carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the desired protein coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the desired protein coding sequences in the cells.

In another embodiment of the present invention, cells and tissues may be engineered to express an endogenous gene comprising the polynucleotides of the invention under the control of inducible regulatory elements, in which case the regulatory sequences of the endogenous gene may be replaced by homologous recombination. As described herein, gene targeting can be used to replace a gene's existing regulatory region with a regulatory sequence isolated from a different gene or a novel regulatory sequence synthesized by genetic engineering methods. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of said sequences. Alternatively, sequences which affect the structure or stability of the RNA or protein produced may be replaced, removed, added, or otherwise modified by targeting. These sequences include polyadenylation signals, mRNA stability elements, splice sites, leader sequences for enhancing or modifying transport or secretion properties of the protein, or other sequences which alter or improve the function or stability of protein or RNA molecules.

The targeting event may be a simple insertion of the regulatory sequence, placing the gene under the control of the new regulatory sequence, e.g., inserting a new promoter or enhancer or both upstream of a gene. Alternatively, the targeting event may be a simple deletion of a regulatory element, such as the deletion of a tissue-specific negative regulatory element. Alternatively, the targeting event may replace an existing element; for example, a tissue-specific enhancer can be replaced by an enhancer that has broader or different cell-type specificity than the naturally occurring elements. Here, the naturally occurring sequences are deleted and new sequences are added. In all cases, the identification of the targeting event may be facilitated by the use of one or more selectable marker genes that are contiguous with the targeting DNA, allowing for the selection of cells in which the exogenous DNA has integrated into the cell genome. The identification of the targeting event may also be facilitated by the use of one or more marker genes exhibiting the property of negative selection, such that the negatively selectable marker is linked to the exogenous DNA, but configured such that the negatively selectable marker flanks the targeting sequence, and such that a correct homologous recombination event with sequences in the host cell genome does not result in the stable integration of the negatively selectable marker. Markers useful for this purpose include the Herpes Simplex Virus thymidine kinase (TK) gene or the bacterial xanthine-guanine phosphoribosyl-transferase (gpt) gene.

The gene targeting or gene activation techniques which can be used in accordance with this aspect of the invention are more particularly described in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; International Application No. PCT/US92/09627 (WO93/09222) by Selden et al.; and International Application No. PCT/US90/06436 (WO91/06667) by Skoultchi et al., each of which is incorporated by reference herein in its entirety.

4.6 Transgenic Animals

In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122, incorporated herein by reference.

Transgenic animals can be prepared wherein all or part of a promoter of the polynucleotides of the invention is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression. The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

The polynucleotides of the present invention also make possible the development, through, e.g., homologous recombination or knock out strategies, of animals that fail to express polypeptides of the invention or that express a variant polypeptide. Such animals are useful as models for studying the in vivo activities of polypeptide as well as for studying modulators of the polypeptides of the invention.

In preferred methods to determine biological functions of the polypeptides of the invention in vivo, one or more genes provided by the invention are either over expressed or inactivated in the germ line of animals using homologous recombination [Capecchi, Science 244:1288-1292 (1989)]. Animals in which the gene is over expressed, under the regulatory control of exogenous or endogenous promoter elements, are known as transgenic animals. Animals in which an endogenous gene has been inactivated by homologous recombination are referred to as “knockout” animals. Knockout animals, preferably non-human mammals, can be prepared as described in U.S. Pat. No. 5,557,032, incorporated herein by reference. Transgenic animals are useful to determine the roles polypeptides of the invention play in biological processes, and preferably in disease states. Transgenic animals are useful as model systems to identify compounds that modulate lipid metabolism. Transgenic animals, preferably non-human mammals, are produced using methods as described in U.S. Pat. No. 5,489,743 and PCT Publication No. WO94/28122, incorporated herein by reference.

Transgenic animals can be prepared wherein all or part of the polynucleotides of the invention promoter is either activated or inactivated to alter the level of expression of the polypeptides of the invention. Inactivation can be carried out using homologous recombination methods described above. Activation can be achieved by supplementing or even replacing the homologous promoter to provide for increased protein expression.

The homologous promoter can be supplemented by insertion of one or more heterologous enhancer elements known to confer promoter activation in a particular tissue.

4.7 Uses and Biological Activity

The polynucleotides and proteins of the present invention are expected to exhibit one or more of the uses or biological activities (including those associated with assays cited herein) identified herein. Uses or activities described for proteins of the present invention may be provided by administration or use of such proteins or of polynucleotides encoding such proteins (such as, for example, in gene therapies or vectors suitable for introduction of DNA). The mechanism underlying the particular condition or pathology will dictate whether the polypeptides of the invention, the polynucleotides of the invention or modulators (activators or inhibitors) thereof would be beneficial to the subject in need of treatment. Thus, “therapeutic compositions of the invention” include compositions comprising isolated polynucleotides (including recombinant DNA molecules, cloned genes and degenerate variants thereof) or polypeptides of the invention (including full length protein, mature protein and truncations or domains thereof), or compounds and other substances that modulate the overall activity of the target gene products, either at the level of target gene/protein expression or target protein activity. Such modulators include polypeptides, analogs, (variants), including fragments and fusion proteins, antibodies and other binding proteins; chemical compounds that directly or indirectly activate or inhibit the polypeptides of the invention (identified, e.g., via drug screening assays as described herein); antisense polynucleotides and polynucleotides suitable for triple helix formation; and in particular antibodies or other binding partners that specifically recognize one or more epitopes of the polypeptides of the invention.

The polypeptides of the present invention may likewise be involved in cellular activation or in one of the other physiological pathways described herein.

4.7.1 Research Uses and Utilities

The polynucleotides provided by the present invention can be used by the research community for various purposes. The polynucleotides can be used to express recombinant protein for analysis, characterization or therapeutic use; as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in disease states); as molecular weight markers on gels; as chromosome markers or tags (when labeled) to identify chromosomes or to map related gene positions; to compare with endogenous DNA sequences in patients to identify potential genetic disorders; as probes to hybridize and thus discover novel, related DNA sequences; as a source of information to derive PCR primers for genetic fingerprinting; as a probe to subtract-out” known sequences in the process of discovering other novel polynucleotides; for selecting and making oligomers for attachment to a “gene chip” or other support, including for examination of expression patterns; to raise anti-protein antibodies using DNA immunization techniques; and as an antigen to raise anti-DNA antibodies or elicit another immune response. Where the polynucleotide encodes a protein which binds or potentially binds to another protein (such as, for example, in a receptor-ligand interaction), the polynucleotide can also be used in interaction trap assays (such as, for example, that described in Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotides encoding the other protein with which binding occurs or to identify inhibitors of the binding interaction.

The polypeptides provided by the present invention can similarly be used in assays to determine biological activity, including in a panel of multiple proteins for high-throughput screening; to raise antibodies or to elicit another immune response; as a reagent (including the labeled reagent) in assays designed to quantitatively determine levels of the protein (or its receptor) in biological fluids; as markers for tissues in which the corresponding polypeptide is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state); and, of course, to isolate correlative receptors or ligands. Proteins involved in these binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developed into reagent grade or kit format for commercialization as research products.

Methods for performing the uses listed above are well known to those skilled in the art. References disclosing such methods include without limitation “Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds., 1989, and “Methods in Enzymology: Guide to Molecular Cloning Techniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

4.7.2 Nutritional Uses

Polynucleotides and polypeptides of the present invention can also be used as nutritional sources or supplements. Such uses include without limitation use as a protein or amino acid supplement, use as a carbon source, use as a nitrogen source and use as a source of carbohydrate. In such cases the polypeptide or polynucleotide of the invention can be added to the feed of a particular organism or can be administered as a separate solid or liquid preparation, such as in the form of powder, pills, solutions, suspensions or capsules. In the case of microorganisms, the polypeptide or polynucleotide of the invention can be added to the medium in or on which the microorganism is cultured.

4.7.3 Cytokine and Cell Proliferation/Differentiation Activity

A polypeptide of the present invention may exhibit activity relating to cytokine, cell proliferation (either inducing or inhibiting) or cell differentiation (either inducing or inhibiting) activity or may induce production of other cytokines in certain cell populations. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Many protein factors discovered to date, including all known cytokines, have exhibited activity in one or more factor-dependent cell proliferation assays, and hence the assays serve as a convenient confirmation of cytokine activity. The activity of therapeutic compositions of the present invention is evidenced by any one of a number of routine factor dependent cell proliferation assays for cell lines including, without limitation, 32D, DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+(preB M+), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2, TF-1, Mo7e, CMK, HUVEC, and Caco. Therapeutic compositions of the invention can be used in the following:

Assays for T-cell or thymocyte proliferation include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., I. Immunol. 149:3778-3783, 1992; Bowman et al., I. Immunol. 152:1756-1761, 1994.

Assays for cytokine production and/or proliferation of spleen cells, lymph node cells or thymocytes include, without limitation, those described in: Polyclonal T cell stimulation, Kruisbeek, A. M. and Shevach, E. M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Measurement of mouse and human interleukin-γ, Schreiber, R. D. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic and lymphopoietic cells include, without limitation, those described in: Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991; Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and human interleukin 6—Nordan, R. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al., Proc. Natl. Aced. Sci. U.S.A. 83:1857-1861, 1986; Measurement of human Interleukin 11—Bennett, F., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and human Interleukin 9—Ciarletta, A., Giannotti, J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology. J. E. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto. 1991.

Assays for T-cell clone responses to antigens (which will identify, among others, proteins that affect APC-T cell interactions as well as direct T-cell effects by measuring proliferation and cytokine production) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their cellular receptors; Chapter 7, Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad. Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun. 11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988.

4.7.4 Stem Cell Growth Factor Activity

A polypeptide of the present invention may exhibit stem cell growth factor activity and be involved in the proliferation, differentiation and survival of pluripotent and totipotent stem cells including primordial germ cells, embryonic stem cells, hematopoietic stem cells and/or germ line stem cells. Administration of the polypeptide of the invention to stem cells in vivo or ex vivo is expected to maintain and expand cell populations in a totipotential or pluripotential state which would be useful for re-engineering damaged or diseased tissues, transplantation, manufacture of bio-pharmaceuticals and the development of bio-sensors. The ability to produce large quantities of human cells has important working applications for the production of human proteins which currently must be obtained from non-human sources or donors, implantation of cells to treat diseases such as Parkinson's, Alzheimer's and other neurodegenerative diseases; tissues for grafting such as bone marrow, skin, cartilage, tendons, bone, muscle (including cardiac muscle), blood vessels, cornea, neural cells, gastrointestinal cells and others; and organs for transplantation such as kidney, liver, pancreas (including islet cells), heart and lung.

It is contemplated that multiple different exogenous growth factors and/or cytokines may be administered in combination with the polypeptide of the invention to achieve the desired effect, including any of the growth factors listed herein, other stem cell maintenance factors, and specifically including stem cell factor (SCF), leukemia inhibitory factor (LIF), Flt-3 ligand (Flt-3L), any of the interleukins, recombinant soluble IL-6 receptor fused to IL-6, macrophage inflammatory protein 1-alpha (MIP-1-alpha), G-CSF, GM-CSF, thrombopoietin (TPO), platelet factor 4 (PF4), platelet-derived growth factor (PDGF), neural growth factors and basic fibroblast growth factor (bFGF).

Since totipotent stem cells can give rise to virtually any mature cell type, expansion of these cells in culture will facilitate the production of large quantities of mature cells. Techniques for culturing stem cells are known in the art and administration of polypeptides of the invention, optionally with other growth factors and/or cytokines, is expected to enhance the survival and proliferation of the stem cell populations. This can be accomplished by direct administration of the polypeptide of the invention to the culture medium. Alternatively, stroma cells transfected with a polynucleotide that encodes for the polypeptide of the invention can be used as a feeder layer for the stem cell populations in culture or in vivo. Stromal support cells for feeder layers may include embryonic bone marrow fibroblasts, bone marrow stromal cells, fetal liver cells, or cultured embryonic fibroblasts (see U.S. Pat. No. 5,690,926).

Stem cells themselves can be transfected with a polynucleotide of the invention to induce autocrine expression of the polypeptide of the invention. This will allow for generation of undifferentiated totipotential/pluripotential stem cell lines that are useful as is or that can then be differentiated into the desired mature cell types. These stable cell lines can also serve as a source of undifferentiated totipotential/pluripotential mRNA to create cDNA libraries and templates for polymerase chain reaction experiments. These studies would allow for the isolation and identification of differentially expressed genes in stem cell populations that regulate stem cell proliferation and/or maintenance.

Expansion and maintenance of totipotent stem cell populations will be useful in the treatment of many pathological conditions. For example, polypeptides of the present invention may be used to manipulate stem cells in culture to give rise to neuroepithelial cells that can be used to augment or replace cells damaged by illness, autoimmune disease, accidental damage or genetic disorders. The polypeptide of the invention may be useful for inducing the proliferation of neural cells and for the regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders which involve degeneration, death or trauma to neural cells or nerve tissue. In addition, the expanded stem cell populations can also be genetically altered for gene therapy purposes and to decrease host rejection of replacement tissues after grafting or implantation.

Expression of the polypeptide of the invention and its effect on stem cells can also be manipulated to achieve controlled differentiation of the stem cells into more differentiated cell types. A broadly applicable method of obtaining pure populations of a specific differentiated cell type from undifferentiated stem cell populations involves the use of a cell-type specific promoter driving a selectable marker. The selectable marker allows only cells of the desired type to survive. For example, stem cells can be induced to differentiate into cardiomyocytes (Wobus et al., Differentiation, 48: 173-182, (1991); Klug et al., J. Clin. Invest., 98(1): 216-224, (1998)) or skeletal muscle cells (Browder, L. W. In: Principles of Tissue Engineering eds. Lanza et al., Academic Press (1997)). Alternatively, directed differentiation of stem cells can be accomplished by culturing the stem cells in the presence of a differentiation factor such as retinoic acid and an antagonist of the polypeptide of the invention which would inhibit the effects of endogenous stem cell factor activity and allow differentiation to proceed.

In vitro cultures of stem cells can be used to determine if the polypeptide of the invention exhibits stem cell growth factor activity. Stem cells are isolated from any one of various cell sources (including hematopoietic stem cells and embryonic stem cells) and cultured on a feeder layer, as described by Thompson et al. Proc. Natl. Acad. Sci, U.S.A., 92: 7844-7848 (1995), in the presence of the polypeptide of the invention alone or in combination with other growth factors or cytokines. The ability of the polypeptide of the invention to induce stem cells proliferation is determined by colony formation on semi-solid support e.g. as described by Bernstein et al., Blood, 77: 2316-2321 (1991).

4.7.5 Hematopoiesis Regulating Activity

A polypeptide of the present invention may be involved in regulation of hematopoiesis and, consequently, in the treatment of myeloid or lymphoid cell disorders. Even marginal biological activity in support of colony forming cells or of factor-dependent cell lines indicates involvement in regulating hematopoiesis, e.g. in supporting the growth and proliferation of erythroid progenitor cells alone or in combination with other cytokines, thereby indicating utility, for example, in treating various anemias or for use in conjunction with irradiation/chemotherapy to stimulate the production of erythroid precursors and/or erythroid cells; in supporting the growth and proliferation of myeloid cells such as granulocytes and monocytes/macrophages (i.e., traditional CSF activity) useful, for example, in conjunction with chemotherapy to prevent or treat consequent myelo-suppression; in supporting the growth and proliferation of megakaryocytes and consequently of platelets thereby allowing prevention or treatment of various platelet disorders such as thrombocytopenia, and generally for use in place of or complimentary to platelet transfusions; and/or in supporting the growth and proliferation of hematopoietic stem cells which are capable of maturing to any and all of the above-mentioned hematopoietic cells and therefore find therapeutic utility in various stem cell disorders (such as those usually treated with transplantation, including, without limitation, aplastic anemia and paroxysmal nocturnal hemoglobinuria), as well as in repopulating the stem cell compartment post irradiation/chemotherapy, either in-vivo or ex-vivo (i.e., in conjunction with bone marrow transplantation or with peripheral progenitor cell transplantation (homologous or heterologous)) as normal cells or genetically manipulated for gene therapy.

Therapeutic compositions of the invention can be used in the following:

Suitable assays for proliferation and differentiation of various hematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify, among others, proteins that influence embryonic differentiation hematopoiesis) include, without limitation, those described in: Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al., Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al., Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify, among others, proteins that regulate lympho-hematopoiesis) include, without limitation, those described in: Methylcellulose colony forming assays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y. 1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992; Primitive hematopoietic colony forming cells with high proliferative potential, McNiece, I. K. and Briddell, R. A. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39, Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., Experimental Hematology 22:353-359, 1994; Cobblestone area forming cell assay, Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long term bone marrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y. 1994; Long term culture initiating cell assay, Sutherland, H. J. In Culture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

4.7.6 Tissue Growth Activity

A polypeptide of the present invention also may be involved in bone, cartilage, tendon, ligament and/or nerve tissue growth or regeneration, as well as in wound healing and tissue repair and replacement, and in healing of burns, incisions and ulcers.

A polypeptide of the present invention which induces cartilage and/or bone growth in circumstances where bone is not normally formed, has application in the healing of bone fractures and cartilage damage or defects in humans and other animals. Compositions of a polypeptide, antibody, binding partner, or other modulator of the invention may have prophylactic use in closed as well as open fracture reduction and also in the improved fixation of artificial joints. De novo bone formation induced by an osteogenic agent contributes to the repair of congenital, trauma induced, or oncologic resection induced craniofacial defects, and also is useful in cosmetic plastic surgery.

A polypeptide of this invention may also be involved in attracting bone-forming cells, stimulating growth of bone-forming cells, or inducing differentiation of progenitors of bone-forming cells. Treatment of osteoporosis, osteoarthritis, bone degenerative disorders, or periodontal disease, such as through stimulation of bone and/or cartilage repair or by blocking inflammation or processes of tissue destruction (collagenase activity, osteoclast activity, etc.) mediated by inflammatory processes may also be possible using the composition of the invention.

Another category of tissue regeneration activity that may involve the polypeptide of the present invention is tendon/ligament formation. Induction of tendon/ligament-like tissue or other tissue formation in circumstances where such tissue is not normally formed, has application in the healing of tendon or ligament tears, deformities and other tendon or ligament defects in humans and other animals. Such a preparation employing a tendon/ligament-like tissue inducing protein may have prophylactic use in preventing damage to tendon or ligament tissue, as well as use in the improved fixation of tendon or ligament to bone or other tissues, and in repairing defects to tendon or ligament tissue. De novo tendon/ligament-like tissue formation induced by a composition of the present invention contributes to the repair of congenital, trauma induced, or other tendon or ligament defects of other origin, and is also useful in cosmetic plastic surgery for attachment or repair of tendons or ligaments. The compositions of the present invention may provide environment to attract tendon- or ligament-forming cells, stimulate growth of tendon- or ligament-forming cells, induce differentiation of progenitors of tendon- or ligament-forming cells, or induce growth of tendon/ligament cells or progenitors ex vivo for return in vivo to effect tissue repair. The compositions of the invention may also be useful in the treatment of tendinitis, carpal tunnel syndrome and other tendon or ligament defects. The compositions may also include an appropriate matrix and/or sequestering agent as a carrier as is well known in the art.

The compositions of the present invention may also be useful for proliferation of neural cells and for regeneration of nerve and brain tissue, i.e. for the treatment of central and peripheral nervous system diseases and neuropathies, as well as mechanical and traumatic disorders, which involve degeneration, death or trauma to neural cells or nerve tissue. More specifically, a composition may be used in the treatment of diseases of the peripheral nervous system, such as peripheral nerve injuries, peripheral neuropathy and localized neuropathies, and central nervous system diseases, such as Alzheimer's, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome. Further conditions which may be treated in accordance with the present invention include mechanical and traumatic disorders, such as spinal cord disorders, head trauma and cerebrovascular diseases such as stroke. Peripheral neuropathies resulting from chemotherapy or other medical therapies may also be treatable using a composition of the invention.

Compositions of the invention may also be useful to promote better or faster closure of non-healing wounds, including without limitation pressure ulcers, ulcers associated with vascular insufficiency, surgical and traumatic wounds, and the like.

Compositions of the present invention may also be involved in the generation or regeneration of other tissues, such as organs (including, for example, pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac) and vascular (including vascular endothelium) tissue, or for promoting the growth of cells comprising such tissues. Part of the desired effects may be by inhibition or modulation of fibrotic scarring may allow normal tissue to regenerate. A polypeptide of the present invention may also exhibit angiogenic activity.

A composition of the present invention may also be useful for gut protection or regeneration and treatment of lung or liver fibrosis, reperfusion injury in various tissues, and conditions resulting from systemic cytokine damage.

A composition of the present invention may also be useful for promoting or inhibiting differentiation of tissues described above from precursor tissues or cells; or for inhibiting the growth of tissues described above.

Therapeutic compositions of the invention can be used in the following:

Assays for tissue generation activity include, without limitation, those described in: International Patent Publication No. WO95/16035 (bone, cartilage, tendon); International Patent Publication No. WO95/05846 (nerve, neuronal); International Patent Publication No. WO91/07491 (skin, endothelium).

Assays for wound healing activity include, without limitation, those described in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H. I. and Rovee, D. T., eds.), Year Book Medical Publishers, Inc., Chicago, as modified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

4.7.7 Immune Stimulating or Suppressing Activity

A polypeptide of the present invention may also exhibit immune stimulating or immune suppressing activity, including without limitation the activities for which assays are described herein. A polynucleotide of the invention can encode a polypeptide exhibiting such activities. A protein may be useful in the treatment of various immune deficiencies and disorders (including severe combined immunodeficiency (SCID)), e.g., in regulating (up or down) growth and proliferation of T and/or B lymphocytes, as well as effecting the cytolytic activity of NK cells and other cell populations. These immune deficiencies may be genetic or be caused by viral (e.g., HIV) as well as bacterial or fungal infections, or may result from autoimmune disorders. More specifically, infectious diseases causes by viral, bacterial, fungal or other infection may be treatable using a protein of the present invention, including infections by HIV, hepatitis viruses, herpes viruses, mycobacteria, Leishmania spp., malaria spp. and various fungal infections such as candidiasis. Of course, in this regard, proteins of the present invention may also be useful where a boost to the immune system generally may be desirable, i.e., in the treatment of cancer.

Autoimmune disorders which may be treated using a protein of the present invention include, for example, connective tissue disease, multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmune thyroiditis, insulin dependent diabetes mellitis, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory eye disease. Such a protein (or antagonists thereof, including antibodies) of the present invention may also to be useful in the treatment of allergic reactions and conditions (e.g., anaphylaxis, serum sickness, drug reactions, food allergies, insect venom allergies, mastocytosis, allergic rhinitis, hypersensitivity pneumonitis, urticaria, angioedema, eczema, atopic dermatitis, allergic contact dermatitis, erythema multiforme, Stevens-Johnson syndrome, allergic conjunctivitis, atopic keratoconjunctivitis, venereal keratoconjunctivitis, giant papillary conjunctivitis and contact allergies), such as asthma (particularly allergic asthma) or other respiratory problems. Other conditions, in which immune suppression is desired (including, for example, organ transplantation), may also be treatable using a protein (or antagonists thereof) of the present invention. The therapeutic effects of the polypeptides or antagonists thereof on allergic reactions can be evaluated by in vivo animals models such as the cumulative contact enhancement test (Lastbom et al., Toxicology 125: 59-66, 1998), skin prick test (Hoffmann et al., Allergy 54: 446-54, 1999), guinea pig skin sensitization test (Vohr et al., Arch. Toxocol. 73: 501-9), and murine local lymph node assay (Kimber et al., J. Toxicol. Environ. Health 53: 563-79).

Using the proteins of the invention it may also be possible to modulate immune responses, in a number of ways. Down regulation may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing T cell responses or by inducing specific tolerance in T cells, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process which requires continuous exposure of the T cells to the suppressive agent. Tolerance, which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon reexposure to specific antigen in the absence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (including without limitation B lymphocyte antigen functions (such as, for example, B7)), e.g., preventing high level lymphokine synthesis by activated T cells, will be useful in situations of tissue, skin and organ transplantation and in graft-versus-host disease (GVHD). For example, blockage of T cell function should result in reduced tissue destruction in tissue transplantation. Typically, in tissue transplants, rejection of the transplant is initiated through its recognition as foreign by T cells, followed by an immune reaction that destroys the transplant. The administration of a therapeutic composition of the invention may prevent cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, a lack of costimulation may also be sufficient to anergize the T cells, thereby inducing tolerance in a subject. Induction of long-term tolerance by B lymphocyte antigen-blocking reagents may avoid the necessity of repeated administration of these blocking reagents. To achieve sufficient immunosuppression or tolerance in a subject, it may also be necessary to block the function of a combination of B lymphocyte antigens.

The efficacy of particular therapeutic compositions in preventing organ transplant rejection or GVHD can be assessed using animal models that are predictive of efficacy in humans. Examples of appropriate systems which can be used include allogeneic cardiac grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which have been used to examine the immunosuppressive effects of CTLA4Ig fusion proteins in vivo as described in Lenschow et al., Science 257:789-792 (1992) and Turka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). In addition, murine models of GVHD (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 846-847) can be used to determine the effect of therapeutic compositions of the invention on the development of that disease.

Blocking antigen function may also be therapeutically useful for treating autoimmune diseases. Many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and which promote the production of cytokines and autoantibodies involved in the pathology of the diseases. Preventing the activation of autoreactive T cells may reduce or eliminate disease symptoms. Administration of reagents which block stimulation of T cells can be used to inhibit T cell activation and prevent production of autoantibodies or T cell-derived cytokines which may be involved in the disease process. Additionally, blocking reagents may induce antigen-specific tolerance of autoreactive T cells which could lead to long-term relief from the disease. The efficacy of blocking reagents in preventing or alleviating autoimmune disorders can be determined using a number of well-characterized animal models of human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic lupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes mellitus in NOD mice and BB rats, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (e.g., a B lymphocyte antigen function), as a means of up regulating immune responses, may also be useful in therapy. Upregulation of immune responses may be in the form of enhancing an existing immune response or eliciting an initial immune response. For example, enhancing an immune response may be useful in cases of viral infection, including systemic viral diseases such as influenza, the common cold, and encephalitis.

Alternatively, anti-viral immune responses may be enhanced in an infected patient by removing T cells from the patient, costimulating the T cells in vitro with viral antigen-pulsed APCs either expressing a peptide of the present invention or together with a stimulatory form of a soluble peptide of the present invention and reintroducing the in vitro activated T cells into the patient. Another method of enhancing anti-viral immune responses would be to isolate infected cells from a patient, transfect them with a nucleic acid encoding a protein of the present invention as described herein such that the cells express all or a portion of the protein on their surface, and reintroduce the transfected cells into the patient. The infected cells would now be capable of delivering a costimulatory signal to, and thereby activate, T cells in vivo.

A polypeptide of the present invention may provide the necessary stimulation signal to T cells to induce a T cell mediated immune response against the transfected tumor cells. In addition, tumor cells which lack MHC class I or MHC class II molecules, or which fail to reexpress sufficient mounts of MHC class I or MHC class II molecules, can be transfected with nucleic acid encoding all or a portion of (e.g., a cytoplasmic-domain truncated portion) of an MHC class I alpha chain protein and β2 microglobulin protein or an MHC class II alpha chain protein and an MHC class II beta chain protein to thereby express MHC class I or MHC class II proteins on the cell surface. Expression of the appropriate class I or class II MHC in conjunction with a peptide having the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3) induces a T cell mediated immune response against the transfected tumor cell. Optionally, a gene encoding an antisense construct which blocks expression of an MHC class II associated protein, such as the invariant chain, can also be cotransfected with a DNA encoding a peptide having the activity of a B lymphocyte antigen to promote presentation of tumor associated antigens and induce tumor specific immunity. Thus, the induction of a T cell mediated immune response in a human subject may be sufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, be measured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl. Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., I. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bowman et al., J. Virology 61:1992-1998; Bertagnolli et al., Cellular Immunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092, 1994.

Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol. 140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544, 1995; Inaba et al., Journal of Experimental Medicine 173:549-559, 1991; Macatonia et al., Journal of Immunology 154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine 182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993; Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca et al., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243, 1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai et al., Cytometry 14:891-897, 1993; Gorczyca et al., International Journal of Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica et al., Blood 84:111-117, 1994; Fine et al., Cellular Immunology 155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al., Proc. Nat. Acad. Sci. USA 88:7548-7551, 1991.

4.7.8 Activin/Inhibin Activity

A polypeptide of the present invention may also exhibit activin- or inhibin-related activities. A polynucleotide of the invention may encode a polypeptide exhibiting such characteristics. Inhibins are characterized by their ability to inhibit the release of follicle stimulating hormone (FSH), while activins and are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, a polypeptide of the present invention, alone or in heterodimers with a member of the inhibin family, may be useful as a contraceptive based on the ability of inhibins to decrease fertility in female mammals and decrease spermatogenesis in male mammals. Administration of sufficient amounts of other inhibins can induce infertility in these mammals. Alternatively, the polypeptide of the invention, as a homodimer or as a heterodimer with other protein subunits of the inhibin group, may be useful as a fertility inducing therapeutic, based upon the ability of activin molecules in stimulating FSH release from cells of the anterior pituitary. See, for example, U.S. Pat. No. 4,798,885. A polypeptide of the invention may also be useful for advancement of the onset of fertility in sexually immature mammals, so as to increase the lifetime reproductive performance of domestic animals such as, but not limited to, cows, sheep and pigs.

The activity of a polypeptide of the invention may, among other means, be measured by the following methods.

Assays for activin/inhibin activity include, without limitation, those described in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al., Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Mason et al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci. USA 83:3091-3095, 1986.

4.7.9 Chemotactic/Chemokinetic Activity

A polypeptide of the present invention may be involved in chemotactic or chemokinetic activity for mammalian cells, including, for example, monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Chemotactic and chemokinetic receptor activation can be used to mobilize or attract a desired cell population to a desired site of action. Chemotactic or chemokinetic compositions (e.g. proteins, antibodies, binding partners, or modulators of the invention) provide particular advantages in treatment of wounds and other trauma to tissues, as well as in treatment of localized infections. For example, attraction of lymphocytes, monocytes or neutrophils to tumors or sites of infection may result in improved immune responses against the tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cell population if it can stimulate, directly or indirectly, the directed orientation or movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate directed movement of cells. Whether a particular protein has chemotactic activity for a population of cells can be readily determined by employing such protein or peptide in any known assay for cell chemotaxis.

Therapeutic compositions of the invention can be used in the following:

Assays for chemotactic activity (which will identify proteins that induce or prevent chemotaxis) consist of assays that measure the ability of a protein to induce the migration of cells across a membrane as well as the ability of a protein to induce the adhesion of one cell population to another cell population. Suitable assays for movement and adhesion include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Marguiles, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measurement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376, 1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol. 25:1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994; Johnston et al. J. of Immunol. 153:1762-1768, 1994.

4.7.10 Hemostatic and Thrombolytic Activity

A polypeptide of the invention may also be involved in hemostatis or thrombolysis or thrombosis. A polynucleotide of the invention can encode a polypeptide exhibiting such attributes. Compositions may be useful in treatment of various coagulation disorders (including hereditary disorders, such as hemophilias) or to enhance coagulation and other hemostatic events in treating wounds resulting from trauma, surgery or other causes. A composition of the invention may also be useful for dissolving or inhibiting formation of thromboses and for treatment and prevention of conditions resulting therefrom (such as, for example, infarction of cardiac and central nervous system vessels (e.g., stroke).

Therapeutic compositions of the invention can be used in the following:

Assay for hemostatic and thrombolytic activity include, without limitation, those described in: Linet et al., J. Clin. Pharmacol. 26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987; Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins 35:467-474, 1988.

4.7.11 Cancer Diagnosis and Therapy

Polypeptides of the invention may be involved in cancer cell generation, proliferation or metastasis. Detection of the presence or amount of polynucleotides or polypeptides of the invention may be useful for the diagnosis and/or prognosis of one or more types of cancer. For example, the presence or increased expression of a polynucleotide/polypeptide of the invention may indicate a hereditary risk of cancer, a precancerous condition, or an ongoing malignancy. Conversely, a defect in the gene or absence of the polypeptide may be associated with a cancer condition. Identification of single nucleotide polymorphisms associated with cancer or a predisposition to cancer may also be useful for diagnosis or prognosis.

Cancer treatments promote tumor regression by inhibiting tumor cell proliferation, inhibiting angiogenesis (growth of new blood vessels that is necessary to support tumor growth) and/or prohibiting metastasis by reducing tumor cell motility or invasiveness. Therapeutic compositions of the invention may be effective in adult and pediatric oncology including in solid phase tumors/malignancies, locally advanced tumors, human soft tissue sarcomas, metastatic cancer, including lymphatic metastases, blood cell malignancies including multiple myeloma, acute and chronic leukemias, and lymphomas, head and neck cancers including mouth cancer, larynx cancer and thyroid cancer, lung cancers including small cell carcinoma and non-small cell cancers, breast cancers including small cell carcinoma and ductal carcinoma, gastrointestinal cancers including esophageal cancer, stomach cancer, colon cancer, colorectal cancer and polyps associated with colorectal neoplasia, pancreatic cancers, liver cancer, urologic cancers including bladder cancer and prostate cancer, malignancies of the female genital tract including ovarian carcinoma, uterine (including endometrial) cancers, and solid tumor in the ovarian follicle, kidney cancers including renal cell carcinoma, brain cancers including intrinsic brain tumors, neuroblastoma, astrocytic brain tumors, gliomas, metastatic tumor cell invasion in the central nervous system, bone cancers including osteomas, skin cancers including malignant melanoma, tumor progression of human skin keratinocytes, squamous cell carcinoma, basal cell carcinoma, hemangiopericytoma and Karposi's sarcoma.

Polypeptides, polynucleotides, or modulators of polypeptides of the invention (including inhibitors and stimulators of the biological activity of the polypeptide of the invention) may be administered to treat cancer. Therapeutic compositions can be administered in therapeutically effective dosages alone or in combination with adjuvant cancer therapy such as surgery, chemotherapy, radiotherapy, thermotherapy, and laser therapy, and may provide a beneficial effect, e.g. reducing tumor size, slowing rate of tumor growth, inhibiting metastasis, or otherwise improving overall clinical condition, without necessarily eradicating the cancer.

The composition can also be administered in therapeutically effective amounts as a portion of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of the polypeptide or modulator of the invention with one or more anti-cancer drugs in addition to a pharmaceutically acceptable carrier for delivery. The use of anti-cancer cocktails as a cancer treatment is routine. Anti-cancer drugs that are well known in the art and can be used as a treatment in combination with the polypeptide or modulator of the invention include: Actinomycin D, Aminoglutethimide, Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine, Mechlorethamine HCl (nitrogen mustard), Melphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate, Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

In addition, therapeutic compositions of the invention may be used for prophylactic treatment of cancer. There are hereditary conditions and/or environmental situations (e.g. exposure to carcinogens) known in the art that predispose an individual to developing cancers. Under these circumstances, it may be beneficial to treat these individuals with therapeutically effective doses of the polypeptide of the invention to reduce the risk of developing cancers.

In vitro models can be used to determine the effective doses of the polypeptide of the invention as a potential cancer treatment. These in vitro models include proliferation assays of cultured tumor cells, growth of cultured tumor cells in soft agar (see Freshney, (1987) Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described in Giovanella et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility and invasive potential of tumor cells in Boyden Chamber assays as described in Pilkington et al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays such as induction of vascularization of the chick chorioallantoic membrane or induction of vascular endothelial cell migration as described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999), respectively. Suitable tumor cells lines are available, e.g. from American Type Tissue Culture Collection catalogs.

4.7.12 Receptor/Ligand Activity

A polypeptide of the present invention may also demonstrate activity as receptor, receptor ligand or inhibitor or agonist of receptor/ligand interactions. A polynucleotide of the invention can encode a polypeptide exhibiting such characteristics. Examples of such receptors and ligands include, without limitation, cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, receptors involved in cell-cell interactions and their ligands (including without limitation, cellular adhesion molecules (such as selecting, integrins and their ligands) and receptor/ligand pairs involved in antigen presentation, antigen recognition and development of cellular and humoral immune responses. Receptors and ligands are also useful for screening of potential peptide or small molecule inhibitors of the relevant receptor/ligand interaction. A protein of the present invention (including, without limitation, fragments of receptors and ligands) may themselves be useful as inhibitors of receptor/ligand interactions.

The activity of a polypeptide of the invention may, among other means, be measured by the following methods:

Suitable assays for receptor-ligand activity include without limitation those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28, Measurement of Cellular Adhesion under static conditions 7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868, 1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein et al., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol. Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

By way of example, the polypeptides of the invention may be used as a receptor for a ligand(s) thereby transmitting the biological activity of that ligand(s). Ligands may be identified through binding assays, affinity chromatography, dihybrid screening assays, BIAcore assays, gel overlay assays, or other methods known in the art.

Studies characterizing drugs or proteins as agonist or antagonist or partial agonists or a partial antagonist require the use of other proteins as competing ligands. The polypeptides of the present invention or ligand(s) thereof may be labeled by being coupled to radioisotopes, calorimetric molecules or a toxin molecules by conventional methods. (“Guide to Protein Purification” Murray P. Deutscher (ed) Methods in Enzymology Vol. 182 (1990) Academic Press, Inc. San Diego). Examples of radioisotopes include, but are not limited to, tritium and carbon-14. Examples of calorimetric molecules include, but are not limited to, fluorescent molecules such as fluorescamine, or rhodamine or other colorimetric molecules. Examples of toxins include, but are not limited, to ricin.

4.7.13 Drug Screening

This invention is particularly useful for screening chemical compounds by using the novel polypeptides or binding fragments thereof in any of a variety of drug screening techniques. The polypeptides or fragments employed in such a test may either be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or a fragment thereof. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, the formation of complexes between polypeptides of the invention or fragments and the agent being tested or examine the diminution in complex formation between the novel polypeptides and an appropriate cell line, which are well known in the art.

Sources for test compounds that may be screened for ability to bind to or modulate (i.e., increase or decrease) the activity of polypeptides of the invention include (1) inorganic and organic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of either random or mimetic peptides, oligonucleotides or organic molecules.

Chemical libraries may be readily synthesized or purchased from a number of commercial sources, and may include structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.

The sources of natural product libraries are microorganisms (including bacteria and fungi), animals, plants or other vegetation, or marine organisms, and libraries of mixtures for screening may be created by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of the organisms themselves. Natural product libraries include polyketides, non-ribosomal peptides, and (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998).

Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds and can be readily prepared by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide 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, Curr. Opin. Biotechnol. 8:701-707 (1997). For reviews and examples of peptidomimetic libraries, see Al-Obeidi et al., Mol. Biotechnol, 9(3):205-23 (1998); Hruby et al., Curr Opin Chem Biol, 1(1):114-19 (1997); Dorner et al., Bioorg Med Chem, 4(5):709-15 (1996) (alkylated dipeptides).

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 bind a polypeptide of the invention. The molecules identified in the binding assay are then tested for antagonist or agonist activity in in vivo tissue culture or animal models that are well known in the art. In brief, the molecules are titrated into a plurality of cell cultures or animals and then tested for either cell/animal death or prolonged survival of the animal/cells.

The binding molecules thus identified may be complexed with toxins, e.g., ricin or cholera, or with other compounds that are toxic to cells such as radioisotopes. The toxin-binding molecule complex is then targeted to a tumor or other cell by the specificity of the binding molecule for a polypeptide of the invention. Alternatively, the binding molecules may be complexed with imaging agents for targeting and imaging purposes.

4.7.14 Assay for Receptor Activity

The invention also provides methods to detect specific binding of a polypeptide e.g. a ligand or a receptor. The art provides numerous assays particularly useful for identifying previously unknown binding partners for receptor polypeptides of the invention. For example, expression cloning using mammalian or bacterial cells, or dihybrid screening assays can be used to identify polynucleotides encoding binding partners. As another example, affinity chromatography with the appropriate immobilized polypeptide of the invention can be used to isolate polypeptides that recognize and bind polypeptides of the invention. There are a number of different libraries used for the identification of compounds, and in particular small molecules, that modulate (i.e., increase or decrease) biological activity of a polypeptide of the invention. Ligands for receptor polypeptides of the invention can also be identified by adding exogenous ligands, or cocktails of ligands to two cells populations that are genetically identical except for the expression of the receptor of the invention: one cell population expresses the receptor of the invention whereas the other does not. The response of the two cell populations to the addition of ligands(s) are then compared. Alternatively, an expression library can be co-expressed with the polypeptide of the invention in cells and assayed for an autocrine response to identify potential ligand(s). As still another example, BIAcore assays, gel overlay assays, or other methods known in the art can be used to identify binding partner polypeptides, including, (1) organic and inorganic chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides or organic molecules.

The role of downstream intracellular signaling molecules in the signaling cascade of the polypeptide of the invention can be determined. For example, a chimeric protein in which the cytoplasmic domain of the polypeptide of the invention is fused to the extracellular portion of a protein, whose ligand has been identified, is produced in a host cell. The cell is then incubated with the ligand specific for the extracellular portion of the chimeric protein, thereby activating the chimeric receptor. Known downstream proteins involved in intracellular signaling can then be assayed for expected modifications i.e. phosphorylation. Other methods known to those in the art can also be used to identify signaling molecules involved in receptor activity.

4.7.15 Anti-Inflammatory Activity

Compositions of the present invention may also exhibit anti-inflammatory activity. The anti-inflammatory activity may be achieved by providing a stimulus to cells involved in the inflammatory response, by inhibiting or promoting cell-cell interactions (such as, for example, cell adhesion), by inhibiting or promoting chemotaxis of cells involved in the inflammatory process, inhibiting or promoting cell extravasation, or by stimulating or suppressing production of other factors which more directly inhibit or promote an inflammatory response. Compositions with such activities can be used to treat inflammatory conditions including chronic or acute conditions), including without limitation intimation associated with infection (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine-induced lung injury, inflammatory bowel disease, Crohn's disease or resulting from over production of cytokines such as TNF or IL-1. Compositions of the invention may also be useful to treat anaphylaxis and hypersensitivity to an antigenic substance or material. Compositions of this invention may be utilized to prevent or treat conditions such as, but not limited to, sepsis, acute pancreatitis, endotoxin shock, cytokine induced shock, rheumatoid arthritis, chronic inflammatory arthritis, pancreatic cell damage from diabetes mellitus type 1, graft versus host disease, inflammatory bowel disease, inflamation associated with pulmonary disease, other autoimmune disease or inflammatory disease, an antiproliferative agent such as for acute or chronic mylegenous leukemia or in the prevention of premature labor secondary to intrauterine infections.

4.7.16 Leukemias

Leukemias and related disorders may be treated or prevented by administration of a therapeutic that promotes or inhibits function of the polynucleotides and/or polypeptides of the invention. Such leukemias and related disorders include but are not limited to acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronic leukemia, chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J.B. Lippincott Co., Philadelphia).

4.7.17 Nervous System Disorders

Nervous system disorders, involving cell types which can be tested for efficacy of intervention with compounds that modulate the activity of the polynucleotides and/or polypeptides of the invention, and which can be treated upon thus observing an indication of therapeutic utility, include but are not limited to nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention include but are not limited to the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems:

    • (i) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries;
    • (ii) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia;
    • (iii) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, syphilis;
    • (iv) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, or amyotrophic lateral sclerosis;
    • (v) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including but not limited to, vitamin B12 deficiency, folic acid deficiency, Wernicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration;
    • (vi) neurological lesions associated with systemic diseases including but not limited to diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis;
    • (vii) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and
    • (viii) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including but not limited to multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

Therapeutics which are useful according to the invention for treatment of a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons. For example, and not by way of limitation, therapeutics which elicit any of the following effects may be useful according to the invention:

    • (i) increased survival time of neurons in culture;
    • (ii) increased sprouting of neurons in culture or in vivo;
    • (iii) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or
    • (iv) decreased symptoms of neuron dysfunction in vivo.

Such effects may be measured by any method known in the art. In preferred, non-limiting embodiments, increased survival of neurons may be measured by the method set forth in Arakawa et al. (1990, J. Neurosci. 10:3507-3515); increased sprouting of neurons may be detected by methods set forth in Pestronk et al. (1980, Exp. Neurol. 70:65-82) or Brown et al. (1981, Ann. Rev. Neurosci. 4:17-42); increased production of neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.

In specific embodiments, motor neuron disorders that may be treated according to the invention include but are not limited to disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including but not limited to progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

4.7.18 Other Activities

A polypeptide of the invention may also exhibit one or more of the following additional activities or effects: inhibiting the growth, infection or function of, or killing, infectious agents, including, without limitation, bacteria, viruses, fungi and other parasites; effecting (suppressing or enhancing) bodily characteristics, including, without limitation, height, weight, hair color, eye color, skins fat to lean ratio or other tissue pigmentation, or organ or body part size or shape (such as, for example, breast augmentation or diminution, change in bone form or shape); effecting biorhythms or circadian cycles or rhythms; effecting the fertility of male or female subjects; effecting the metabolism, catabolism, anabolism, processing, utilization, storage or elimination of dietary fat, lipid, protein, carbohydrate, vitamins, minerals, co-factors or other nutritional factors or component(s); effecting behavioral characteristics, including, without limitation, appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) and violent behaviors; providing analgesic effects or other pain reducing effects; promoting differentiation and growth of embryonic stem cells in lineages other than hematopoietic lineages; hormonal or endocrine activity; in the case of enzymes, correcting deficiencies of the enzyme and treating deficiency-related diseases; treatment of hyperproliferative disorders (such as, for example, psoriasis); immunoglobulin-like activity (such as, for example, the ability to bind antigens or complement); and the ability to act as an antigen in a vaccine composition to raise an immune response against such protein or another material or entity which is cross-reactive with such protein.

4.7.19 Identification of Polymorphisms

The demonstration of polymorphisms makes possible the identification of such polymorphisms in human subjects and the pharmacogenetic use of this information for diagnosis and treatment. Such polymorphisms may be associated with, e.g., differential predisposition or susceptibility to various disease states (such as disorders involving inflammation or immune response) or a differential response to drug administration, and this genetic information can be used to tailor preventive or therapeutic treatment appropriately. For example, the existence of a polymorphism associated with a predisposition to inflammation or autoimmune disease makes possible the diagnosis of this condition in humans by identifying the presence of the polymorphism.

Polymorphisms can be identified in a variety of ways known in the art which all generally involve obtaining a sample from a patient, analyzing DNA from the sample, optionally involving isolation or amplification of the DNA, and identifying the presence of the polymorphism in the DNA. For example, PCR may be used to amplify an appropriate fragment of genomic DNA which may then be sequenced. Alternatively, the DNA may be subjected to allele-specific oligonucleotide hybridization (in which appropriate oligonucleotides are hybridized to the DNA under conditions permitting detection of a single base mismatch) or to a single nucleotide extension assay (in which an oligonucleotide that hybridizes immediately adjacent to the position of the polymorphism is extended with one or more labeled nucleotides). In addition, traditional restriction fragment length polymorphism analysis (using restriction enzymes that provide differential digestion of the genomic DNA depending on the presence or absence of the polymorphism) may be performed. Arrays with nucleotide sequences of the present invention can be used to detect polymorphisms. The array can comprise modified nucleotide sequences of the present invention in order to detect the nucleotide sequences of the present invention. In the alternative, any one of the nucleotide sequences of the present invention can be placed on the array to detect changes from those sequences.

Alternatively a polymorphism resulting in a change in the amino acid sequence could also be detected by detecting a corresponding change in amino acid sequence of the protein, e.g., by an antibody specific to the variant sequence.

4.7.20 Arthritis and Inflammation

The immunosuppressive effects of the compositions of the invention against rheumatoid arthritis is determined in an experimental animal model system. The experimental model system is adjuvant induced arthritis in rats, and the protocol is described by J. Holoshitz, et at., 1983, Science, 219:56, or by B. Waksman et al., 1963, Int. Arch. Allergy Appl. Immunol., 23:129. Induction of the disease can be caused by a single injection, generally intradermally, of a suspension of killed Mycobacterium tuberculosis in complete Freund's adjuvant (CFA). The route of injection can vary, but rats may be injected at the base of the tail with an adjuvant mixture. The polypeptide is administered in phosphate buffered solution (PBS) at a dose of about 1-5 mg/kg. The control consists of administering PBS only.

The procedure for testing the effects of the test compound would consist of intradermally injecting killed Mycobacterium tuberculosis in CFA followed by immediately administering the test compound and subsequent treatment every other day until day 24. At 14, 15, 18, 20, 22, and 24 days after injection of Mycobacterium CFA, an overall arthritis score may be obtained as described by J. Holoskitz above. An analysis of the data would reveal that the test compound would have a dramatic affect on the swelling of the joints as measured by a decrease of the arthritis score.

4.8 Therapeutic Methods

The compositions (including polypeptide fragments, analogs, variants and antibodies or other binding partners or modulators including antisense polynucleotides) of the invention have numerous applications in a variety of therapeutic methods. Examples of therapeutic applications include, but are not limited to, those exemplified herein.

4.8.1 EXAMPLE

One embodiment of the invention is the administration of an effective amount of the polypeptides or other composition of the invention to individuals affected by a disease or disorder that can be modulated by regulating the peptides of the invention. While the mode of administration is not particularly important, parenteral administration is preferred. An exemplary mode of administration is to deliver an intravenous bolus. The dosage of the polypeptides or other composition of the invention will normally be determined by the prescribing physician. It is to be expected that the dosage will vary according to the age, weight, condition and response of the individual patient. Typically, the amount of polypeptide administered per dose will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight, with the preferred dose being about 0.1 μg/kg to 10 mg/kg of patient body weight. For parenteral administration, polypeptides of the invention will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle. Such vehicles are well known in the art and examples include water, saline, Ringer's solution, dextrose solution, and solutions consisting of small amounts of the human serum albumin. The vehicle may contain minor amounts of additives that maintain the isotonicity and stability of the polypeptide or other active ingredient. The preparation of such solutions is within the skill of the art.

4.9 Pharmaceutical Formulations and Routes of Administration

A protein or other composition of the present invention (from whatever source derived, including without limitation from recombinant and non-recombinant sources and including antibodies and other binding partners of the polypeptides of the invention) may be administered to a patient in need, by itself, or in pharmaceutical compositions where it is mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a variety of disorders. Such a composition may optionally contain (in addition to protein or other active ingredient and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and erythropoietin. In further compositions, proteins of the invention may be combined with other agents beneficial to the treatment of the disease or disorder in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), insulin-like growth factor (IGF), as well as cytokines described herein.

The pharmaceutical composition may further contain other agents which either enhance the activity of the protein or other active ingredient or complement its activity or use in treatment. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with protein or other active ingredient of the invention, or to minimize side effects. Conversely, protein or other active ingredient of the present invention may be included in formulations of the particular clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the clotting factor, cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent (such as IL-1Ra, IL-1 Hy1, IL-1 Hy2, anti-TNF, corticosteroids, immunosuppressive agents). A protein of the present invention may be active in multimers (e.g., heterodimers or homodimers) or complexes with itself or other proteins. As a result, pharmaceutical compositions of the invention may comprise a protein of the invention in such multimeric or complexed form.

As an alternative to being included in a pharmaceutical composition of the invention including a first protein, a second protein or a therapeutic agent may be concurrently administered with the first protein (e.g., at the same time, or at differing times provided that therapeutic concentrations of the combination of agents is achieved at the treatment site). Techniques for formulation and administration of the compounds of the instant application may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, a therapeutically effective amount of protein or other active ingredient of the present invention is administered to a mammal having a condition to be treated. Protein or other active ingredient of the present invention may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, protein or other active ingredient of the present invention may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein or other active ingredient of the present invention in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

4.9.1 Routes of Administration

Suitable routes of administration may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Administration of protein or other active ingredient of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Intravenous administration to the patient is preferred.

Alternately, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into a arthritic joints or in fibrotic tissue, often in a depot or sustained release formulation. In order to prevent the scarring process frequently occurring as complication of glaucoma surgery, the compounds may be administered topically, for example, as eye drops. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a specific antibody, targeting, for example, arthritic or fibrotic tissue. The liposomes will be targeted to and taken up selectively by the afflicted tissue.

The polypeptides of the invention are administered by any route that delivers an effective dosage to the desired site of action. The determination of a suitable route of administration and an effective dosage for a particular indication is within the level of skill in the art. Preferably for wound treatment, one administers the therapeutic compound directly to the site. Suitable dosage ranges for the polypeptides of the invention can be extrapolated from these dosages or from similar studies in appropriate animal models. Dosages can then be adjusted as necessary by the clinician to provide maximal therapeutic benefit.

4.9.2 Compositions/Formulations

Pharmaceutical compositions for use in accordance with the present invention thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. These pharmaceutical compositions may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Proper formulation is dependent upon the route of administration chosen. When a therapeutically effective amount of protein or other active ingredient of the present invention is administered orally, protein or other active ingredient of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein or other active ingredient of the present invention, and preferably from about 25 to 90% protein or other active ingredient of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of protein or other active ingredient of the present invention, and preferably from about 1 to 50% protein or other active ingredient of the present invention.

When a therapeutically effective amount of protein or other active ingredient of the present invention is administered by intravenous, cutaneous or subcutaneous injection, protein or other active ingredient of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein or other active ingredient solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to protein or other active ingredient of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained from a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the invention is a co-solvent system comprising benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be the VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD:5W) consists of VPD diluted 1:1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. Naturally, the proportions of a co-solvent system may be varied considerably without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity nonpolar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose. Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein or other active ingredient stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. Many of the active ingredients of the invention may be provided as salts with pharmaceutically compatible counter ions. Such pharmaceutically acceptable base addition salts are those salts which retain the biological effectiveness and properties of the free acids and which are obtained by reaction with inorganic or organic bases such as sodium hydroxide, magnesium hydroxide, ammonia, trialkylamine, dialkylamine, monoalkylamine, dibasic amino acids, sodium acetate, potassium benzoate, triethanol amine and the like.

The pharmaceutical composition of the invention may be in the form of a complex of the protein(s) or other active ingredient(s) of present invention along with protein or peptide antigens. The protein and/or peptide antigen will deliver a stimulatory signal to both B and T lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) following presentation of the antigen by MHC proteins. MHC and structurally related proteins including those encoded by class I and class II MHC genes on host cells will serve to present the peptide antigen(s) to T lymphocytes. The antigen components could also be supplied as purified MHC-peptide complexes alone or with co-stimulatory molecules that can directly signal T cells. Alternatively antibodies able to bind surface immunoglobulin and other molecules on B cells as well as antibodies able to bind the TCR and other molecules on T cells can be combined with the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention may be in the form of a liposome in which protein of the present invention is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithins, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. Nos. 4,235,871; 4,501,728; 4,837,028; and 4,737,323, all of which are incorporated herein by reference.

The amount of protein or other active ingredient of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of protein or other active ingredient of the present invention with which to treat each individual patient. Initially, the attending physician will administer low doses of protein or other active ingredient of the present invention and observe the patient's response. Larger doses of protein or other active ingredient of the present invention may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method of the present invention should contain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg) of protein or other active ingredient of the present invention per kg body weight. For compositions of the present invention which are useful for bone, cartilage, tendon or ligament regeneration, the therapeutic method includes administering the composition topically, systematically, or locally as an implant or device. When administered, the therapeutic composition for use in this invention is, of course, in a pyrogen-free, physiologically acceptable form. Further, the composition may desirably be encapsulated or injected in a viscous form for delivery to the site of bone, cartilage or tissue damage. Topical administration may be suitable for wound healing and tissue repair. Therapeutically useful agents other than a protein or other active ingredient of the invention which may also optionally be included in the composition as described above, may alternatively or additionally, be administered simultaneously or sequentially with the composition in the methods of the invention. Preferably for bone and/or cartilage formation, the composition would include a matrix capable of delivering the protein-containing or other active ingredient-containing composition to the site of bone and/or cartilage damage, providing a structure for the developing bone and cartilage and optimally capable of being resorbed into the body. Such matrices may be formed of materials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The particular application of the compositions will define the appropriate formulation. Potential matrices for the compositions may be biodegradable and chemically defined calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydrides. Other potential materials are biodegradable and biologically well-defined, such as bone or dermal collagen. Further matrices are comprised of pure proteins or extracellular matrix components. Other potential matrices are nonbiodegradable and chemically defined, such as sintered hydroxyapatite, bioglass, aluminates, or other ceramics. Matrices may be comprised of combinations of any of the above mentioned types of material, such as polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be altered in composition, such as in calcium-aluminate-phosphate and processing to alter pore size, particle size, particle shape, and biodegradability. Presently preferred is a 50:50 (mole weight) copolymer of lactic acid and glycolic acid in the form of porous particles having diameters ranging from 150 to 800 microns. In some applications, it will be useful to utilize a sequestering agent, such as carboxymethyl cellulose or autologous blood clot, to prevent the protein compositions from disassociating from the matrix.

A preferred family of sequestering agents is cellulosic materials such as alkylcelluloses (including hydroxyalkylcelluloses), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, and carboxymethylcellulose, the most preferred being cationic salts of carboxymethylcellulose (CMC). Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). The amount of sequestering agent useful herein is 0.5-20 wt %, preferably 1-10 wt % based on total formulation weight, which represents the amount necessary to prevent desorption of the protein from the polymer matrix and to provide appropriate handling of the composition, yet not so much that the progenitor cells are prevented from infiltrating the matrix, thereby providing the protein the opportunity to assist the osteogenic activity of the progenitor cells. In further compositions, proteins or other active ingredients of the invention may be combined with other agents beneficial to the treatment of the bone and/or cartilage defect, wound, or tissue in question. These agents include various growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinary applications. Particularly domestic animals and thoroughbred horses, in addition to humans, are desired patients for such treatment with proteins or other active ingredients of the present invention. The dosage regimen of a protein-containing pharmaceutical composition to be used in tissue regeneration will be determined by the attending physician considering various factors which modify the action of the proteins, e.g., amount of tissue weight desired to be formed, the site of damage, the condition of the damaged tissue, the size of a wound, type of damaged tissue (e.g., bone), the patient's age, sex, and diet, the severity of any infection, time of administration and other clinical factors. The dosage may vary with the type of matrix used in the reconstitution and with inclusion of other proteins in the pharmaceutical composition. For example, the addition of other known growth factors, such as IGF I (insulin like growth factor I), to the final composition, may also effect the dosage. Progress can be monitored by periodic assessment of tissue/bone growth and/or repair, for example, X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for gene therapy. Such polynucleotides can be introduced either in vivo or ex vivo into cells for expression in a mammalian subject. Polynucleotides of the invention may also be administered by other known methods for introduction of nucleic acid into a cell or organism (including, without limitation, in the form of viral vectors or naked DNA). Cells may also be cultured ex vivo in the presence of proteins of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

4.9.3 Effective Dosage

Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from appropriate in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that can be used to more accurately determine useful doses in humans. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture (i.e., the concentration of the test compound which achieves a half-maximal inhibition of the protein's biological activity). Such information can be used to more accurately determine useful doses in humans.

A therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms or a prolongation of survival in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds which exhibit high therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g., Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1. Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the desired effects, or minimal effective concentration (MEC). The MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compounds should be administered using a regimen which maintains plasma levels above the MEC for 10-90% of the time, preferably between 30-90% and most preferably between 50-90%. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

An exemplary dosage regimen for polypeptides or other compositions of the invention will be in the range of about 0.01 μg/kg to 100 mg/kg of body weight daily, with the preferred dose being about 0.1 μg/kg to 25 mg/kg of patient body weight daily, varying in adults and children. Dosing may be once daily, or equivalent doses may be delivered at longer or shorter intervals.

The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's age and weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

4.9.4 Packaging

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

4.10 Antibodies

Another aspect of the invention is an antibody that specifically binds the polypeptide of the invention. Such antibodies include 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 and/or antigen-binding sequences, which specifically recognize a polypeptide of the invention. 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 Fv, are also provided by the invention. The term “specific for” indicates that the variable regions of the antibodies of the invention recognize and bind polypeptides of the invention exclusively (i.e., able to distinguish the polypeptide of the invention from other similar polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but 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. (Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, NY (1988), Chapter 6. Antibodies that recognize and bind fragments of the polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, full length polypeptides of the invention. As with antibodies that are specific for full length polypeptides of the invention, antibodies of the invention that recognize fragments are those which can distinguish polypeptides from the same family of polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.

Non-human antibodies may be humanized by any methods known in the art. 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.

Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of a polypeptide of the invention), diagnostic purposes to detect or quantitate a polypeptide of the invention, as well as purification of a polypeptide of the invention. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific. The invention further provides a hybridoma that produces an antibody according to the invention. Antibodies of the invention are useful for detection and/or purification of the polypeptides of the invention.

Polypeptides of the invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the protein. Such antibodies may be obtained using either the entire protein or fragments thereof as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154 (1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987).

Monoclonal antibodies binding to the protein of the invention may be useful diagnostic agents for the immunodetection of the protein. Neutralizing monoclonal antibodies binding to the protein may also be useful therapeutics for both conditions associated with the protein and also in the treatment of some forms of cancer where abnormal expression of the protein is involved. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against the protein may be useful in detecting and preventing the metastatic spread of the cancerous cells, which may be mediated by the protein. In general, techniques for preparing polyclonal and monoclonal antibodies as well as hybridomas capable of producing the desired antibody are well known in the art (Campbell, A. M., Monoclonal Antibodies Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984); St. Groth et al., J. Immunol. 35:1-21 (1990); Kohler and Milstein, Nature 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 4:72 (1983); Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), pp. 77-96).

Any animal (mouse, rabbit, etc.) which is known to produce antibodies can be immunized with a peptide or polypeptide of the invention. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide. One skilled in the art will recognize that the amount of the protein encoded by the ORF of the present invention used for immunization will vary based on the animal which is immunized, the antigenicity of the peptide and the site of injection. The protein that is used as an immunogen may be modified or administered in an adjuvant in order to increase the protein's antigenicity. Methods of increasing the antigenicity of a protein are well known in the art and include, but are not limited to, coupling the antigen with a heterologous protein (such as globulin or -galactosidase) or through the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag14 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells. Any one of a number of methods well known in the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, Western blot analysis, or radioimmunoassay (Lutz et al., Exp. Cell Research. 175:109-124 (1988)). Hybridomas secreting the desired antibodies are cloned and the class and subclass is determined using procedures known in the art (Campbell, A. M., Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984)). Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to proteins of the present invention.

For polyclonal antibodies, antibody-containing antiserum is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures. The present invention further provides the above-described antibodies in delectably labeled form. Antibodies can be delectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art, for example, see (Sternberger, L. A. et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972); Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify cells or tissues in which a fragment of the polypeptide of interest is expressed. The antibodies may also be used directly in therapies or other diagnostics. The present invention further provides the above-described antibodies immobilized on a solid support. Examples of such solid supports include plastics such as polycarbonate, complex carbohydrates such as agarose and Sepharose®, acrylic resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (Weir, D. M. et al., “Handbook of Experimental Immunology” 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986); Jacoby, W. D. et al., Meth. Enzym. 34 Academic Press, N.Y. (1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as well as for immuno-affinity purification of the proteins of the present invention.

4.11 Computer Readable Sequences

In one application of this embodiment, a nucleotide sequence of the present invention can be recorded on computer readable media. As used herein, “computer readable media” refers to any medium which can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable mediums can be used to create a manufacture comprising computer readable medium having recorded thereon a nucleotide sequence of the present invention. As used herein, “recorded” refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the nucleotide sequence information of the present invention.

A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon a nucleotide sequence of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the nucleotide sequence information of the present invention on computer readable medium. The sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g. text file or database) in order to obtain computer readable medium having recorded thereon the nucleotide sequence information of the present invention.

By providing any of the nucleotide sequences SEQ ID NOs: 1-1104 or a representative fragment thereof; or a nucleotide sequence at least 95% identical to any of the nucleotide sequences of the SEQ ID NOs: 1-1104 in computer readable form, a skilled artisan can routinely access the sequence information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. The examples which follow demonstrate how software which implements the BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et al., Comp. Chem. 17:203-207 (1993)) search algorithms on a Sybase system is used to identify open reading frames (ORFs) within a nucleic acid sequence. Such ORFs may be protein encoding fragments and may be useful in producing commercially important proteins such as enzymes used in fermentation reactions and in the production of commercially useful metabolites.

As used herein, “a computer-based system” refers to the hardware means, software means, and data storage means used to analyze the nucleotide sequence information of the present invention. The minimum hardware means of the computer-based systems of the present invention comprises a central processing unit (CPU), input means, output means, and data storage means. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. As stated above, the computer-based systems of the present invention comprise a data storage means having stored therein a nucleotide sequence of the present invention and the necessary hardware means and software means for supporting and implementing a search means. As used herein, “data storage means” refers to memory which can store nucleotide sequence information of the present invention, or a memory access means which can access manufactures having recorded thereon the nucleotide sequence information of the present invention.

As used herein, “search means” refers to one or more programs which are implemented on the computer-based system to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of a known sequence which match a particular target sequence or target motif. A variety of known algorithms are disclosed publicly and a variety of commercially available software for conducting search means are and can be used in the computer-based systems of the present invention. Examples of such software includes, but is not limited to, Smith-Waterman, MacPattern (EMBL), BLASTN and BLASTA (NPOLYPEPTIDEIA). A skilled artisan can readily recognize that any one of the available algorithms or implementing software packages for conducting homology searches can be adapted for use in the present computer-based systems. As used herein, a “target sequence” can be any nucleic acid or amino acid sequence of six or more nucleotides or two or more amino acids. A skilled artisan can readily recognize that the longer a target sequence is, the less likely a target sequence will be present as a random occurrence in the database. The most preferred sequence length of a target sequence is from about 10 to 300 amino acids, more preferably from about 30 to 100 nucleotide residues. However, it is well recognized that searches for commercially important fragments, such as sequence fragments involved in gene expression and protein processing, may be of shorter length.

As used herein, “a target structural motif,” or “target motif,” refers to any rationally selected sequence or combination of sequences in which the sequence(s) are chosen based on a three-dimensional configuration which is formed upon the folding of the target motif. There are a variety of target motifs known in the art. Protein target motifs include, but are not limited to, enzyme active sites and signal sequences. Nucleic acid target motifs include, but are not limited to, promoter sequences, hairpin structures and inducible expression elements (protein binding sequences).

4.12 Triple Helix Formation

In addition, the fragments of the present invention, as broadly described, can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which methods are based on the binding of a polynucleotide sequence to DNA or RNA. Polynucleotides suitable for use in these methods are preferably 20 to 40 bases in length and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 15241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Olmno, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide.

4.13 Diagnostic Assays and Kits

The present invention further provides methods to identify the presence or expression of one of the ORFs of the present invention, or homolog thereof, in a test sample, using a nucleic acid probe or antibodies of the present invention, optionally conjugated or otherwise associated with a suitable label.

In general, methods for detecting a polynucleotide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polynucleotide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polynucleotide of the invention is detected in the sample. Such methods can also comprise contacting a sample under stringent hybridization conditions with nucleic acid primers that anneal to a polynucleotide of the invention under such conditions, and amplifying annealed polynucleotides, so that if a polynucleotide is amplified, a polynucleotide of the invention is detected in the sample.

In general, methods for detecting a polypeptide of the invention can comprise contacting a sample with a compound that binds to and forms a complex with the polypeptide for a period sufficient to form the complex, and detecting the complex, so that if a complex is detected, a polypeptide of the invention is detected in the sample.

In detail, such methods comprise incubating a test sample with one or more of the antibodies or one or more of the nucleic acid probes of the present invention and assaying for binding of the nucleic acid probes or antibodies to components within the test sample.

Conditions for incubating a nucleic acid probe or antibody with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid probe or antibody used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats can readily be adapted to employ the nucleic acid probes or antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The test samples of the present invention include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the system utilized.

In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention. Specifically, the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising one of the probes or antibodies of the present invention; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound probe or antibody.

In detail, a compartment kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allows one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the antibodies used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or probe. Types of detection reagents include labeled nucleic acid probes, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the disclosed probes and antibodies of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

4.14 Medical Imaging

The novel polypeptides and binding partners of the invention are useful in medical imaging of sites expressing the molecules of the invention (e.g., where the polypeptide of the invention is involved in the immune response, for imaging sites of inflammation or infection). See, e.g., Kunkel et al., U.S. Pat. No. 5,413,778. Such methods involve chemical attachment of a labeling or imaging agent, administration of the labeled polypeptide to a subject in a pharmaceutically acceptable carrier, and imaging the labeled polypeptide in vivo at the target site.

4.15 Screening Assays

Using the isolated proteins and polynucleotides of the invention, the present invention further provides methods of obtaining and identifying agents which bind to a polypeptide encoded by an ORF corresponding to any of the nucleotide sequences set forth in the SEQ ID NOs: 1-1104, or bind to a specific domain of the polypeptide encoded by the nucleic acid. In detail, said method comprises the steps of:

    • (a) contacting an agent with an isolated protein encoded by an ORF of the present invention, or nucleic acid of the invention; and
    • (b) determining whether the agent binds to said protein or said nucleic acid.

In general, therefore, such methods for identifying compounds that bind to a polynucleotide of the invention can comprise contacting a compound with a polynucleotide of the invention for a time sufficient to form a polynucleotide/compound complex, and detecting the complex, so that if a polynucleotide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

Likewise, in general, therefore, such methods for identifying compounds that bind to a polypeptide of the invention can comprise contacting a compound with a polypeptide of the invention for a time sufficient to form a polypeptide/compound complex, and detecting the complex, so that if a polypeptide/compound complex is detected, a compound that binds to a polynucleotide of the invention is identified.

Methods for identifying compounds that bind to a polypeptide of the invention can also comprise contacting a compound with a polypeptide of the invention in a cell for a time sufficient to form a polypeptide/compound complex, wherein the complex drives expression of a receptor gene sequence in the cell, and detecting the complex by detecting reporter gene sequence expression, so that if a polypeptide/compound complex is detected, a compound that binds a polypeptide of the invention is identified.

Compounds identified via such methods can include compounds which modulate the activity of a polypeptide of the invention (that is, increase or decrease its activity, relative to activity observed in the absence of the compound). Alternatively, compounds identified via such methods can include compounds which modulate the expression of a polynucleotide of the invention (that is, increase or decrease expression relative to expression levels observed in the absence of the compound). Compounds, such as compounds identified via the methods of the invention, can be tested using standard assays well known to those of skill in the art for their ability to modulate activity/expression.

The agents screened in the above assay can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques.

For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to the protein encoded by the ORF of the present invention. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the particular protein. For example, one skilled in the art can readily adapt currently available procedures to generate peptides, pharmaceutical agents and the like, capable of binding to a specific peptide sequence, in order to generate rationally designed antipeptide peptides, for example see Hurby et al., Application of Synthetic Peptides: Antisense Peptides, “In Synthetic Peptides, A User's Guide, W.H. Freeman, NY (1992), pp. 289-307, and Kaspczak et al., Biochemistry 28:9230-8 (1989), or pharmaceutical agents, or the like.

In addition to the foregoing, one class of agents of the present invention, as broadly described, can be used to control gene expression through binding to one of the ORFs or EMFs of the present invention. As described above, such agents can be randomly screened or rationally designed/selected. Targeting the ORF or EMF allows a skilled artisan to design sequence specific or element specific agents, modulating the expression of either a single ORF or multiple ORFs which rely on the same EMF for expression control. One class of DNA binding agents are agents which contain base residues which hybridize or form a triple helix formation by binding to DNA or RNA. Such agents can be based on the classic phosphodiester, ribonucleic acid backbone, or can be a variety of sulfhydryl or polymeric derivatives which have base attachment capacity.

Agents suitable for use in these methods preferably contain 20 to 40 bases and are designed to be complementary to a region of the gene involved in transcription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Triple helix-formation optimally results in a shut-off of RNA transcription from DNA, while antisense RNA hybridization blocks translation of an mRNA molecule into polypeptide. Both techniques have been demonstrated to be effective in model systems. Information contained in the sequences of the present invention is necessary for the design of an antisense or triple helix oligonucleotide and other DNA binding agents.

Agents which bind to a protein encoded by one of the ORFs of the present invention can be used as a diagnostic agent. Agents which bind to a protein encoded by one of the ORFs of the present invention can be formulated using known techniques to generate a pharmaceutical composition.

4.16 Use of Nucleic Acids as Probes

Another aspect of the subject invention is to provide for polypeptide-specific nucleic acid hybridization probes capable of hybridizing with naturally occurring nucleotide sequences. The hybridization probes of the subject invention may be derived from any of the nucleotide sequences SEQ ID NOs: 1-1104. Because the corresponding gene is only expressed in a limited number of tissues, a hybridization probe derived from of any of the nucleotide sequences SEQ ID NOs: 1-1104 can be used as an indicator of the presence of RNA of cell type of such a tissue in a sample.

Any suitable hybridization technique can be employed, such as, for example, in situ hybridization. PCR as described in U.S. Pat. Nos. 4,683,195 and 4,965,188 provides additional uses for oligonucleotides based upon the nucleotide sequences. Such probes used in PCR may be of recombinant origin, may be chemically synthesized, or a mixture of both. The probe will comprise a discrete nucleotide sequence for the detection of identical sequences or a degenerate pool of possible sequences for identification of closely related genomic sequences.

Other means for producing specific hybridization probes for nucleic acids include the cloning of nucleic acid sequences into vectors for the production of mRNA probes. Such vectors are known in the art and are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate radioactively labeled nucleotides. The nucleotide sequences may be used to construct hybridization probes for mapping their respective genomic sequences. The nucleotide sequence provided herein may be mapped to a chromosome or specific regions of a chromosome using well known genetic and/or chromosomal mapping techniques. These techniques include in situ hybridization, linkage analysis against known chromosomal markers, hybridization screening with libraries or flow-sorted chromosomal preparations specific to known chromosomes, and the like. The technique of fluorescent in situ hybridization of chromosome spreads has been described, among other places, in Verma et al (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York N.Y.

Fluorescent in situ hybridization of chromosomal preparations and other physical chromosome mapping techniques may be correlated with additional genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981f). Correlation between the location of a nucleic acid on a physical chromosomal map and a specific disease (or predisposition to a specific disease) may help delimit the region of DNA associated with that genetic disease. The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals.

4.17 Preparation of Support Bound Oligonucleotides

Oligonucleotides, i.e., small nucleic acid segments, may be readily prepared by, for example, directly synthesizing the oligonucleotide by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer.

Support bound oligonucleotides may be prepared by any of the methods known to those of skill in the art using any suitable support such as glass, polystyrene or Teflon. One strategy is to precisely spot oligonucleotides synthesized by standard synthesizers. Immobilization can be achieved using passive adsorption (Inouye & Hondo, (1990) J. Clin. Microbiol. 28(6) 1469-72); using UV light (Nagata et al., 1985; Dahlen et al., 1987; Morrissey & Collins, (1989) Mol. Cell Probes 3(2) 189-207) or by covalent binding of base modified DNA Seller et al., 1988; 1989); all references being specifically incorporated herein.

Another strategy that may be employed is the use of the strong biotin-streptavidin interaction as a linker. For example, Broude et al. (1994) Proc. Natl. Acad. Sci. USA 91(8) 3072-6, describe the use of biotinylated probes, although these are duplex probes, that are immobilized on streptavidin-coated magnetic beads. Streptavidin-coated beads may be purchased from Dynal, Oslo. Of course, this same linking chemistry is applicable to coating any surface with streptavidin. Biotinylated probes may be purchased from various sources, such as, e.g., Operon Technologies (Alameda, Calif.).

Nunc Laboratories (Naperville, Ill.) is also selling suitable material that could be used. Nunc Laboratories have developed a method by which DNA can be covalently bound to the microwell surface termed Covalink NH. CovaLink NH is a polystyrene surface grafted with secondary amino groups (>NH) that serve as bridge-heads for further covalent coupling. CovaLink Modules may be purchased from Nunc Laboratories. DNA molecules may be bound to CovaLink exclusively at the 5′-end by a phosphoramidate bond, allowing immobilization of more than 1 pmol of DNA (Rasmussen et al., (1991) Anal. Biochem. 198(1) 138-42).

The use of CovaLink NH strips for covalent binding of DNA molecules at the 5′-end has been described (Rasmussen et al., (1991). In this technology, a phosphoramidate bond is employed (Chu et al., (1983) Nucleic Acids Res. 11(8) 6513-29). This is beneficial as immobilization using only a single covalent bond is preferred. The phosphoramidate bond joins the DNA to the CovaLink NH secondary amino groups that are positioned at the end of spacer arms covalently grafted onto the polystyrene surface through a 2 nm long spacer arm. To link an oligonucleotide to CovaLink NH via an phosphoramidate bond, the oligonucleotide terminus must have a 5′-end phosphate group. It is, perhaps, even possible for biotin to be covalently bound to CovaLink and then streptavidin used to bind the probes.

More specifically, the linkage method includes dissolving DNA in water (7.5 ng/ul) and denaturing for 10 min. at 95° C. and cooling on ice for 10 min. Ice-cold 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm7), is then added to a final concentration of 10 mM 1-MeIm7. A ss DNA solution is then dispensed into CovaLink NH strips (75 ul/well) standing on ice.

Carbodiimide 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), dissolved in 10 mM 1-MeIm7, is made fresh and 25 ul added per well. The strips are incubated for 5 hours at 50° C. After incubation the strips are washed using, e.g., Nunc-Immuno Wash; first the wells are washed 3 times, then they are soaked with washing solution for 5 min., and finally they are washed 3 times (where in the washing solution is 0.4 N NaOH, 0.25% SDS heated to 50° C.).

It is contemplated that a further suitable method for use with the present invention is that described in PCT Patent Application WO 90/03382 (Southern & Maskos), incorporated herein by reference. This method of preparing an oligonucleotide bound to a support involves attaching a nucleoside 3′-reagent through the phosphate group by a covalent phosphodiester link to aliphatic hydroxyl groups carried by the support. The oligonucleotide is then synthesized on the supported nucleoside and protecting groups removed from the synthetic oligonucleotide chain under standard conditions that do not cleave the oligonucleotide from the support. Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphorate.

An on-chip strategy for the preparation of DNA probe for the preparation of DNA probe arrays may be employed. For example, addressable laser-activated photodeprotection may be employed in the chemical synthesis of oligonucleotides directly on a glass surface, as described by Fodor et al. (1991) Science 251(4995) 767-73, incorporated herein by reference. Probes may also be immobilized on nylon supports as described by Van Ness et al. (1991) Nucleic Acids Res. 19(12) 3345-50; or linked to Teflon using the method of Duncan & Cavalier (1988) Anal. Biochem. 169(1) 104-8; all references being specifically incorporated herein.

To link an oligonucleotide to a nylon support, as described by Van Ness et al. (1991), requires activation of the nylon surface via alkylation and selective activation of the 5′-amine of oligonucleotides with cyanuric chloride.

One particular way to prepare support bound oligonucleotides is to utilize the light-generated synthesis described by Pease et al., (1994) PNAS USA 91(11) 5022-6, incorporated herein by reference). These authors used current photolithographic techniques to generate arrays of immobilized oligonucleotide probes (DNA chips). These methods, in which light is used to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays, utilize photolabile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemistry and versatile combinatorial synthesis strategies. A matrix of 256 spatially defined oligonucleotide probes may be generated in this manner.

4.18 Preparation of Nucleic Acid Fragments

The nucleic acids may be obtained from any appropriate source, such as cDNAs, genomic DNA, chromosomal DNA, microdissected chromosome bands, cosmid or YAC inserts, and RNA, including mRNA without any amplification steps. For example, Sambrook et al. (1989) describes three protocols for the isolation of high molecular weight DNA from mammalian cells (p. 9.14-9.23).

DNA fragments may be prepared as clones in M13, plasmid or lambda vectors and/or prepared directly from genomic DNA or cDNA by PCR or other amplification methods. Samples may be prepared or dispensed in multiwell plates. About 100-1000 ng of DNA samples may be prepared in 2-500 ml of final volume.

The nucleic acids would then be fragmented by any of the methods known to those of skill in the art including, for example, using restriction enzymes as described at 9.24-9.28 of Sambrook et al. (1989), shearing by ultrasound and NaOH treatment.

Low pressure shearing is also appropriate, as described by Schriefer et al. (1990) Nucleic Acids Res. 18(24) 7455-6, incorporated herein by reference). In this method, DNA samples are passed through a small French pressure cell at a variety of low to intermediate pressures. A lever device allows controlled application of low to intermediate pressures to the cell. The results of these studies indicate that low-pressure shearing is a useful alternative to sonic and enzymatic DNA fragmentation methods.

One particularly suitable way for fragmenting DNA is contemplated to be that using the two base recognition endonuclease, CviJI, described by Fitzgerald et al. (1992) Nucleic Acids Res. 20(14) 3753-62. These authors described an approach for the rapid fragmentation and fractionation of DNA into particular sizes that they contemplated to be suitable for shotgun cloning and sequencing.

The restriction endonuclease CviJI normally cleaves the recognition sequence PuGCPy between the G and C to leave blunt ends. A typical reaction conditions, which alter the specificity of this enzyme (CviJI**), yield a quasi-random distribution of DNA fragments form the small molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992) quantitatively evaluated the randomness of this fragmentation strategy, using a CviJI** digest of pUC19 that was size fractionated by a rapid gel filtration method and directly ligated, without end repair, to a lac Z minus M13 cloning vector. Sequence analysis of 76 clones showed that CviJI** restricts pyGCPy and PuGCPu, in addition to PuGCPy sites, and that new sequence data is accumulated at a rate consistent with random fragmentation.

As reported in the literature, advantages of this approach compared to sonication and agarose gel fractionation include: smaller amounts of DNA are required (0.2-0.5 ug instead of 2-5 ug); and fewer steps are involved (no preligation, end repair, chemical extraction, or agarose gel electrophoresis and elution are needed.

Irrespective of the manner in which the nucleic acid fragments are obtained or prepared, it is important to denature the DNA to give single stranded pieces available for hybridization. This is achieved by incubating the DNA solution for 2-5 minutes at 80-90° C. The solution is then cooled quickly to 2° C. to prevent renaturation of the DNA fragments before they are contacted with the chip. Phosphate groups must also be removed from genomic DNA by methods known in the art.

4.19 Preparation of DNA Arrays

Arrays may be prepared by spotting DNA samples on a support such as a nylon membrane. Spotting may be performed by using arrays of metal pins (the positions of which correspond to an array of wells in a microtiter plate) to repeated by transfer of about 20 nl of a DNA solution to a nylon membrane. By offset printing, a density of dots higher than the density of the wells is achieved. One to 25 dots may be accommodated in 1 mm2, depending on the type of label used. By avoiding spotting in some preselected number of rows and columns, separate subsets (subarrays) may be formed. Samples in one subarray may be the same genomic segment of DNA (or the same gene) from different individuals, or may be different, overlapped genomic clones. Each of the subarrays may represent replica spotting of the same samples. In one example, a selected gene segment may be amplified from 64 patients. For each patient, the amplified gene segment may be in one 96-well plate (all 96 wells containing the same sample). A plate for each of the 64 patients is prepared. By using a 96-pin device, all samples may be spotted on one 8×12 cm membrane. Subarrays may contain 64 samples, one from each patient. Where the 96 subarrays are identical, the dot span may be 1 mm2 and there may be a 1 mm space between subarrays.

Another approach is to use membranes or plates (available from NUNC, Naperville, Ill.) which may be partitioned by physical spacers e.g. a plastic grid molded over the membrane, the grid being similar to the sort of membrane applied to the bottom of multiwell plates, or hydrophobic strips. A fixed physical spacer is not preferred for imaging by exposure to flat phosphor-storage screens or x-ray films.

The present invention is illustrated in the following examples. Upon consideration of the present disclosure, one of skill in the art will appreciate that many other embodiments and variations may be made in the scope of the present invention. Accordingly, it is intended that the broader aspects of the present invention not be limited to the disclosure of the following examples. The present invention is not to be limited in scope by the exemplified embodiments which are intended as illustrations of single aspects of the invention, and compositions and methods which are functionally equivalent are within the scope of the invention. Indeed, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the present preferred embodiments. Consequently, the only limitations which should be placed upon the scope of the invention are those which appear in the appended claims.

All references cited within the body of the instant specification are hereby incorporated by reference in their entirety.

5.0 EXAMPLES 5.1 Example 1

Novel Nucleic Acid Sequences Obtained from Various Libraries

A plurality of novel nucleic acids were obtained from cDNA libraries prepared from various human tissues and in some cases isolated from a genomic library derived from human chromosome using standard PCR, SBH sequence signature analysis and Sanger sequencing techniques. The inserts of the library were amplified with PCR using primers specific for the vector sequences which flank the inserts. Clones from cDNA libraries were spotted on nylon membrane filters and screened with oligonucleotide probes (e.g., 7-mers) to obtain signature sequences. The clones were clustered into groups of similar or identical sequences, Representative clones were selected for sequencing.

In some cases, the 5′ sequence of the amplified inserts was then deduced using a typical Sanger sequencing protocol. PCR products were purified and subjected to fluorescent dye terminator cycle sequencing. Single pass gel sequencing was done using a 377 Applied Biosystems (ABI) sequencer to obtain the novel nucleic acid sequences. In some cases RACE (Random Amplification of cDNA Ends) was performed to further extend the sequence in the 5′ direction.

5.2 Example 2

Novel Nucleic Acids

The novel nucleic acids of the present invention of the invention were assembled from sequences that were obtained from a cDNA library by methods described in Example 1 above, and in some cases sequences obtained from one or more public databases. The nucleic acids were assembled using an EST sequence as a seed. Then a recursive algorithm was used to extend the seed EST into an extended assemblage, by pulling additional sequences from different databases (i.e., Hyseq's database containing EST sequences, dbEST version 114, gb pri 114, and UniGene version 101) that belong to this assemblage. The algorithm terminated when there was no additional sequences from the above databases that would extend the assemblage. Inclusion of component sequences into the assemblage was based on a BLASTN hit to the extending assemblage with BLAST score greater than 300 and percent identity greater than 95%.

Using PHRAP (Univ. of Washington) or CAP4 (Paracel), a full length gene cDNA sequence and its corresponding protein sequence were generated from the assemblage. Any frame shifts and incorrect stop codons were corrected by hand editing. During editing, the sequence was checked using FASTY and/or BLAST against Genbank (i.e., dbEST version 117, gb pri 117, UniGene version 117, Genepet release 117). Other computer programs which may have been used in the editing process were phredPhrap and Consed (University of Washington) and ed-ready, ed-ext and gc-zip-2 (Hyseq, Inc.). The full-length nucleotide and amino acid sequences, including splice variants resulting from these procedures are shown in the Sequence Listing as SEQ ID NOS:1-1104.

Table 1 shows the various tissue sources of SEQ ID NO: 1-1104.

The nearest neighbor results for SEQ ID NO: 1-1104 were obtained by a BLASTP version 2.0al 19 MP-WashU search against Genpept release 118, using BLAST algorithm. The nearest neighbor result showed the closest homologue for SEQ ID NO: 1-1104 from Genpept (and contains the translated amino acid sequences for which the nucleic acid sequence encodes). The nearest neighbor results for SEQ ID NO: 1-1104 are shown in Table 2 below.

TABLE 1
HYSEQ
TISSUE RNA LIBRARY
ORIGIN SOURCE NAME SEQ ID NOS:
adult brain GIBCO AB3001 4-5 7 29-30 35 42 52 55-56 90
97 117 133-134 147 149 151 162
170 174 177 193 201 222 250 258
263 285-286 290 295 311-312
323-324 330 336-337 339 348
351-353 360 369 377 379 392 398
408 415 459-461 480 489 496
542-544 547 554 584-585 597 599
606 609 611-616 620 623 649 666
675-676 683 688 691-693 695-696
706 727 735 748 753 756 759 767
771 796 802 805-806 820 823-826
829 838 840 846 869 895 919 924
931 933 948 962-963 969 978-980
984 997-998 1002 1010 1013 1020
1046 1050-1051 1058 1063-1065
1069 1081 1090
adult brain GIBCO ABD003 2-4 6-7 18-22 29-30 52-54 66 74
82 88 93 98 100-102 104 107-110
112-113 117 119 123 127-128
133-135 142 145-147 150-152 157
165 168 170 174 177 181-182 190
193-195 200-202 209 211-215
217-218 220 228 230 236 245-246
250-252 262-264 269 272 274 278
283-286 293-297 299-300 302
305-311 313-314 321 323-327 331
333-335 339-340 343-346 348
350-352 358 363 369 383 392-393
398 401 408-412 419 427 429-430
434 437 443 449-450 457 459-462
470 473 480 484-485 487-488
495-496 500 502 505-506 517
519-521 525 530-532 536 543-546
549 554 559 563 568 582 586-587
589-590 593 596 598-601 603
608-609 611-614 616 619-621
623-626 628-629 632 642-645 650
653-656 664 666-667 672-673 677
679-680 684-688 692-693 695 700
705-708 711-712 717-719 722 724
727 738 748 752-755 767 770-771
774-775 778 786 792 796 798 801
805-806 808 810 813 816 819 823
833-834 838 840 846-847 856 859
867 873 877 879 882-883 889 891
904-906 909 915-916 919 921 931
933 937 942 948 953 957 959 969
971 974 976-979 983-984 996-997
1002 1006-1010 1016 1023 1028
1031 1034 1038 1041 1045-1047
1058 1064 1067 1070 1076 1079-
1080 1084 1090 1100
adult brain Clontech ABR001 3-4 17 21 25 35 52 57 66 78 82
88 115-116 128 143 155 164 180
191 262 274 309 319 338 373 398
484 488 518 550 556 560 565 567
593 607 624 687 692-694 715 724
729 731 764 796 801 810 816
825-826 833-836 921 928-929 970
983 1010 1035 1045 1051 1073-
1074 1090
adult brain Clontech ABR006 9-11 14 25 30 32-33 35 42 47 52
57 66 69-70 88 93-94 100-102
104 115-116 127 180 293-294 340
371 469 483 530 598 706 742 798
802 813 837 856 876 896 916 952
955 975 1002 1007-1009 1014-
1017 1034 1059 1071 1090
adult brain Clontech ABR008 1 3-4 6-11 14 17-20 22 25 27
29-30 35 42-46 49 52-53 55 57
60 63-64 66-67 70 72-76 79-85
88-89 91-94 100-102 111-112
114-118 127-129 136 138 141
143-145 150 152 156-158 162-165
171 177 180-187 190-191 194-195
199-201 203-205 207-209 212-215
217 219 222-226 228 237 241-243
248-250 253 257 261 263-264
266-267 271 274 276 278-279
283-287 289 292-294 296-297
299-300 305-307 309 311-312
314-319 321 323-325 329-331
334-340 343-345 348 351-353 355
361 364 369 371 373-382 384 388
392-393 398-401 404-405 409-418
420-423 426-427 430 434-436 440
442-443 446-448 450 452 457-462
464-466 468-469 471-473 478 480
484 487-488 490 496 499-500 503
510-511 519-522 524-525 527-528
541-544 546-547 550 552-557
559-560 566 568-569 572 574-577
579-580 582-583 586 589 593 595
597-599 601 604 606 608-611
613-614 617-619 622-628 630 632
636 640-641 645 648-650 654 656
658 662 664 668-670 673 675-677
679 684 686-689 691-693 696 700
706-711 715 717-719 723-725
735-736 741 746 748 752 757 759
761-764 766 770-772 774-782
784-785 797-801 803-814 816
820-822 824-827 831-834 837-847
849-852 856 858 861 864 866 869
872-877 880-884 888-889 893 895
900 902-905 911-912 915-916
919-921 924-925 931-932 934-953
956 959-961 966-967 969-971
974-976 982 984-985 989 995-999
1002 1004 1006-1009 1012 1014-
1017 1020 1022 1024-1025 1029
1034-1036 1041 1044-1045 1047-
1048 1055-1057 1059-1060 1063-
1064 1066-1069 1071-1074 1076-
1078 1082 1084 1086-1087 1090
1094-1098 1101
adult brain Clontech ABR011 37-38 182 300 392 624 689-690
748 893
adult brain BioChain ABR012 423 451 1061
adult brain BioChain ABR013 37-38 66 171 272 369 374-376
515 530 757 1010 1104
adult brain Invitrogen ABR014 30 37-38 48 128 137 415 544 626
670 762 952 960 1010 1094
adult brain Invitrogen ABR015 93 108-109 115-116 447 473 670
1010
adult brain Invitrogen ABR016 37-38 52 1010 1024-1025
adult brain Invitrogen ABT004 9-11 19-20 22-23 29-30 35 44
52-53 55-56 64 66 69 72 74 82
102 112 133 135 150 156 164 176
181-183 190 201 206 233 238 274
279 284-286 301 330 334 349 351
353-354 369-371 377-378 392 395
398 405 416 423 427 434 437 450
462 464-465 473 488 499 511 515
522 526 542 554 559 579-580 612
624 636 641-643 647-648 650
655-656 675-676 687 692-693
704-707 709 724-725 740 742 775
779 798 802 804 809-810 812-813
825-826 829 833-836 840 856 859
863 877 882-883 894 914 919 921
944 948 952 970 975 999 1002
1024-1025 1031 1033-1034 1046-
1047 1060 1068-1069 1073-1074
1094-1095
adipocytes Strategene ADP001 4 9-11 52-53 64 73 102 104-105
184-186 194 199 202 224 233 237
279 295 297 299 309 315 325 352
363-364 392 415 432 466 477-478
502 519-521 528 530 543-544 564
567 578-580 621 647 669 673
682-683 687 689 692-693 695 713
715-716 720 727 733 760 767 786
788-791 825-826 829 908-909 918
950 961 987-988 1004 1010 1012
1019 1029 1035 1055-1056 1060-
1061 1088 1099
adrenal gland Clontech ADR002 9-11 15 22 24-25 27 45-46 56-58
64 73 84 89-90 98 100-101 105
108-111 113 119 128 135 147 151
157 165 167 171-172 177 190 193
202 210 221 224 227 248-249 257
264 272 277 279 285-287 297
305-306 308 315 323-324 348 352
361 385 393 396 398 403 416 418
428-430 442 457-462 473 501 514
522 530 533 554 560 568 583 589
599 609-610 617-618 629 635 639
652 654 656 663-664 668 677
679-680 688 691 694 737 742 744
748 760-761 765 801-802 804 810
816 823-824 840 847 852 864 870
877 898 907 913-914 916 921 933
960-961 964 970 975 980 983 997
1014-1015 1017 1020 1032-1033
1035 1038 1055-1056 1059 1068-
1069 1077 1088 1090 1096
adult heart GIBCO AHR001 3 6 9-11 18 22 24-25 27 31 34
41 43 53 56-57 60-61 64 66 70
74 80 82-83 85-86 88-90 104-105
107-113 119-120 123 126 128-129
131 133 136 143 146-147 150-154
157-158 161-164 166-167 170-172
177 180 182 184-187 190 193-194
196-197 201 209 211-215 217 221
224 228 231 236-238 250-253
257-258 260 262-263 265 269 272
274 281-286 288-289 292-297
299-300 303 310 315 323-330 333
335-339 341-342 346-347 352-354
356-357 365-366 369 374-376 379
383 391 393 395 398-400 403-404
409-410 412 414-416 419 422-423
427-428 430 435 437 443 445 449
454-455 459-464 469 472-474 480
487 489 495 497 502 506 511 513
515 522 528 530-532 534-538
542-546 548-549 554 556 560
562-564 566 568 572 575-577
579-580 582-583 586 588 591-592
596-597 599-600 602 606 608-612
614-615 620-622 632 645 648-650
654 656-659 662 664-665 667-673
677 679 686-689 692-695 698 704
707-708 710-711 713 716-725
727-728 731 735 738 743-744 746
748 752-753 756-757 759 761
764-765 772 774-776 778 782-783
793 795 797-798 802 805-806 808
810 813 815 817 820-823 833-834
837-838 840 847 856 858-859 864
866 881 885 888-889 891 895-897
900 905 907 911 916-919 922-925
928-931 933-934 937-938 940
943-944 946-948 951 958 960-961
967 970-972 977 982 988-989 996
998 1007-1011 1014-1015 1020
1022 1024-1027 1029 1034-1035
1039-1040 1044-1046 1049-1050
1055-1056 1059 1061 1064-1065
1068-1070 1073-1074 1079 1087
1090 1094 1100
adult kidney GIBCO AKD001 2-4 6 14 17 21-23 25 29 31-32
35 37-38 41 43-45 47 49-50 53-
55 65 68 71 73-74 78 81-83 88-
89 97-98 104-111 117-118 120
123 125 127-129 133 135-136 138
141 143 145-148 150-151 154-155
157 161-162 164-165 167 171-179
184-187 190-194 197 199 201-204
209 212-215 219 222 224 229-230
232 236 238 240 243 247-252 254
258 262-265 268-269 275-276 278
281-283 285-290 292-296 298-303
310-312 315-321 323-330 333-334
339-340 343-345 351-354 358
360-361 370 374-377 380 384
393-394 396 400-401 403-404 411
415 419-420 422 427 429-430 435
437 439 446-447 449 455-457
459-462 464 470 472 477 479-481
485 488 490 493 495-497 499-504
506 511 514 517 519-523 527-530
533-540 542-546 549-550 552 554
556 559 561-568 571-572 574-576
578-581 583-585 587 591-593
595-597 599-601 605 607 609
612-614 616-622 624 627-629 632
635-636 638 640-645 647-648
650-651 656-657 659 663-665
669-673 675-677 679-680 682-684
686-689 691-696 698 700-701
704-705 707-708 710 713-719 723
726-728 730 732 735 738 742-744
746-753 757 759-762 767 774-775
777-778 781 783 786 788-791 796
798-801 805-808 810 812-814
821-824 829 831-832 835-836
838-839 842-843 846 848 852 856
859 867-868 870 872 881 885-889
891-893 895-900 905-908 910-911
914 916-917 919-920 922-924
928-931 933-934 936-937 943
947-948 950-951 957-958 960
962-964 971 973 977-979 983-985
989 996 998 1001-1003 1007-1010
1013 1019-1020 1024-1025 1029
1031 1039-1040 1043-1047 1049
1057 1059 1063 1065 1068-1069
1084 1087 1090 1094 1096 1098-
1100
adult kidney Invitrogen AKT002 12 14 17-18 22 31 43-45 49 55-
56 60 65 74 88 90 95 104 117
119 123 127-128 146 161-162 178
180 187 190 193 197 203 211-215
221 229 261 263 270 275 279 284
293-294 303-304 307 314 321 333
343-345 377 391 405 419 427 445
452 459-461 472 480 489 499 512
522-523 534-535 537-538 554-555
566 575-577 583 588 590-592 601
606 612 614 621 628 636 639 644
647 649 655 657 687 696 717-718
732 740 744 752 756-757 762 774
788-791 796 799-800 802 804 808
815 823 825-826 835-837 842 848
881 885 888 922-923 931 946 954
962-963 971 975 983-984 989
1002 1010 1014-1015 1020 1034
1039 1041 1045-1046 1059 1068-
1069 1094
adult lung GIBCO ALG001 5 18 24 35 44 54-55 67 82-83
105 110 119 128 133 135 141 143
145 150-151 162 178 187 202
212-215 219 222-223 229 240 259
284 293-294 298 300-301 312
323-324 333 343-345 352 377 393
399 403 427-428 447 450 458 473
488 496 499 502 516-517 522-523
530 537-538 543-544 546 551 565
572 586 590 628 641-644 653 658
664 666 668 674 677 679 683 688
692-693 702 705 733 735 743 748
751 753 761 769 775-778 783 796
815 823 825-826 831 839 846 862
879 889 897 909 931 962-963 970
972 985 996 1010 1021-1022 1043
1064 1069 1073-1074 1095 1100
lymph node Clontech ALN001 2 16 55 77 95 104 107 119 125
135-136 149 155-156 177 201 204
212-215 221-222 298 303 327 333
361 403 409-410 422 477 481 488
516 519-521 523 529 542-543 551
564 590 600 615 650 663 692-693
700 726 738 805-806 840 888 893
915 933 955 964 1014-1015 1029
1043 1064 1067
young liver GIBCO ALV001 15 24 29 32 35 45 47 65 67 71
74 81-82 84 88 105 118 129 133
150 152 157 167 184-186 190 194
197 201 211 224 247 250-253 261
268 278-279 284 298-301 305-306
308 310-311 315 326 339 347 349
360 379-380 382 401 403 412 414
418 422 428-429 450 454 457 463
472 485 493 497 499 526 534-536
556 559 564 572 578 583-585
587-589 591-592 596-597 606 609
611 621 640 644 650 652 657 671
673 684 688 691 695 700 717-718
721-722 740 750 755 759 765 775
778 782 788-791 799-801 805-806
808 810 814 825-826 833-834
845-846 848 885 888-889 898 900
908-909 931 934 952 960 965 969
973 985 1007-1010 1022 1029
1034 1049 1052-1053 1069 1073-
1074 1094
adult liver Invitrogen ALV002 6 29 32 47 49 71 74 82 88 102
104 107-109 128 150 168 177-179
191-192 203 209 219 227 237-239
243 254 257 285-286 308 310 315
322 328 333 340 346 354 384 389
392 405-406 434 437 462 466 478
499 505 511 519-522 532 534-535
543 546 559 564 578 583 589 596
605 608-609 613 633 635 659 665
673 683 694 708 715 719 722 741
752 757 759 762 775 778 782 786
802 808 845 848 884 887-888 897
909 916 931 951 962-963 967 975
998 1005 1010 1014-1015 1020
1052-1053 1065 1094 1099-1100
adult liver Clontech ALV003 358 684 752 808
adult ovary Invitrogen AOV001 3 5-6 8-11 14 17 22 25-26 29 31
33 35 43-45 49-50 52-57 60 63-
65 67 70-71 73 76 82-84 86 88-
90 97-98 100-101 104-109 113
117-120 122-123 125 127-129
136-137 142-145 147 150-153
155-158 161 163-168 170-171
173-175 177-179 183-188 190-191
193-195 197 199 201 203-209
216-219 221-222 224 227 229-231
237-240 242-243 245-246 248-249
251-252 255 257 259 261-264
266-267 269 272 274 276 278-282
284-290 292 295-299 301-304
307-308 310-311 314-318 320
322-325 327-330 332-334 336-346
348-357 359-360 363 365-366
374-377 379-393 397-405 407
409-411 413 416 418-419 427-430
434-435 437 442 447 449 451
456-457 459-462 464 467-468 470
472 474-475 478-480 485 488 490
494-496 498-500 503-504 506-509
511 513-514 516-517 522-523
528-535 537-542 544-549 552 554
556-557 559-560 563-568 572-573
575-580 583-595 597-601 603
606-609 611 613-614 616-626 628
630-633 635-638 640 642-645
648-651 653-654 657-666 668-669
672-673 675-677 679 682-684
686-688 691-697 699-701 704
707-708 712-713 717-719 721-723
725 727 729-735 738-739 742-749
751-753 756-759 761-762 764-765
767-768 774-775 778-779 783-785
787-792 795-797 801-806 808-809
812-816 820-824 831-832 835-838
842 845 847 852 854-855 858-860
862-864 866-867 869 872-874
879-880 884 886-889 891 894-903
906-908 910-915 922-925 928-931
933-934 937 939 942-944 946-948
952 955 957-958 960 965 968
970-973 976-979 981 983-984
988-989 996 998 1002 1007-1011
1013 1017 1019-1020 1022-1023
1028-1029 1031 1033-1041 1046
1049 1051 1055-1056 1061-1065
1067-1069 1073-1074 1076 1079
1084 1090 1095
adult placenta Clontech APL001 24 88 150 177 220 250 301 314
403 439 457 545 575-576 611 634
642-643 649 677 679 689 733 742
891 1010 1059
adult placenta Invitrogen APL002 2 7 12 32 52-53 91 97 104 108-
109 122 128 157 164-165 191 197
309 407 409-410 412 422 430 453
503 528 539-540 542-543 603 635
640-641 663 675-676 692-693 701
721 724 753 757 759 775 828
833-834 849 967 975 1007-1010
1033 1035
adult spleen GIBCO ASP001 6 14 22 31 45 55 59-60 67 77 80
88 105 108-110 120 122 125 147
155 164-167 174 177 179 191 193
205 209 211-215 222 229 233
250-252 258 268 272 281 288-289
297-298 300 309-310 320 330 333
338 340 349 353 355-357 366 382
393 405 412 414 416 422 426-428
430 446 449 458 464 472-473 478
495-496 502 507 515 517 530 533
537-538 543 547 551 559 564 567
569 591-593 599 606 608-610
613-614 621 629 636 644-645 650
664-665 670 677 679-681 683-684
686 692-693 695 698 700 719 724
730-732 734 737 748 757 759 765
778-779 783 792 798 802 804 813
829 852 859 863 866 879 885
908-909 915-916 919 926 931 936
948 959 961 964 985 991 1010
1012 1020 1024-1025 1029 1039
1041 1043 1049-1050 1054 1059
1064 1076 1087 1104
adult testis GIBCO ATS001 3 7 22 43-45 65 71 84 89 100-
101 104-105 107 111 120 128 135
143 147 151 155-156 167 173
176-177 187 197 201 222 227-228
239-240 248-249 251-252 257-258
261 276-279 282 289 292-294 297
307 309 315 325 327-328 333 335
339 346 352 356-357 395 403-405
407 412 415 422 428 430 457 464
468 472-473 487 489 494 496 499
511 516 522 528 530 533 544 551
554 562 590 597 600 603 606 608
613 616 624 637 647 650 658 662
664 668 677 679-680 695 704
717-718 722-723 726-727 733
748-749 753 759 761-762 776 778
786 788-791 793 797 802 830 838
850-851 864 897-898 911 916 919
921 925 928-929 933 943 975
977-979 981-983 997 1001 1007-
1010 1020 1022 1028 1035 1059
1063-1064 1069 1079 1089-1090
1100
Genomic DNA Research BAC001 368 1042 1103
from BAC 63I18 Genetics
(CITB BAC
Library)
Genomic DNA Research BAC002 1102-1103
from BAC 393I6 Genetics
(CITB BAC
Library)
Genomic DNA Research BAC003 1042
from BAC 393I6 Genetics
(CITB BAC
Library)
adult bladder Invitrogen BLD001 21 36 55 87 89 98 104 117 157
176 217-218 238 240 284 293-294
303 313 330 349 353 392 411
439-440 446 457 470 472 522 525
551 578 583 594 605 616 653 664
670 704 713 744 762 771 829 856
874 878 888 960 1059
bone marrow Clontech BMD001 3 6 12 16 25 30-31 43-44 55 57-
58 60 62 67 77 82 84 86 90 95
98-101 103-105 110-111 113 115-
116 119 121 128-130 135-136
145-146 149 151-153 155-156 159
162 167 172 174 177-180 187 190
193 198-199 201 205 210-215 217
219-220 224 230-231 241 243
245-253 257 259-262 264 269 271
278 280-281 285-286 290-296
298-299 307 310-311 314-315 320
327-328 333 335-339 347 352 354
356-357 361 365 383 390 393
399-400 402-404 415 419-420
424-426 428-429 436 445 449
456-457 459-462 464 469-470
473-475 480 483 487 495-497
508-509 511 515 517-518 522-523
528-529 533-540 543-544 551-552
554 563-565 568 575-577 581 588
590 593 598-601 609-611 614
617-618 624 626-628 639-640
642-645 650 656 660 663 668 672
674 677 679 682 687 689 692-693
695 701 704 713 716-719 721 723
725 727 730 732 735 738 741-743
748 750 752 755 759-760 768 795
799-802 804-806 813 815 823-824
831 833-834 852 861 867 886
888-889 891 905-906 913 916-918
930 936 944 951 956 960 968 971
975 977 980-981 983 989-991 998
1004 1007-1010 1017 1021 1029
1033 1036 1039 1043 1046 1051
1057 1059 1062-1064 1069-1070
1084 1090 1093-1094 1097 1099
1104
bone marrow Clontech BMD002 3 7-8 21 25-26 30 33 43-46 57
59-60 70 76-78 88-90 95 99-101
103-104 107-110 114 118 125 128
139-140 146 149 152 165 167 190
194 198 200 206 220 223 227 242
245-246 257 259 266-267 271
274-275 278 293-294 298 319 328
330 335 338 353 356-357 361 366
392 402-403 412 418 421-422 426
445 449 458-462 474 496 499 503
515 519-521 539-540 544 551 554
556 563-564 569 574 577 589
609-610 619 677 679 685 688 691
704 724 730 736 739 743-744 747
777-778 795 801-803 844 846 881
889 912 916-917 926-927 950-951
972-973 975 977 985 1004 1007-
1011 1014-1017 1020 1023 1031
1033 1037 1040 1043 1045-1047
1057 1059-1060 1063 1067 1069
1071 1073-1074 1084 1087 1090
1097 1104
bone marrow Clontech BMD004 177 609 724
bone marrow Clontech BMD007 551 1010
adult colon Invitrogen CLN001 6 32 59 61 67 82 94 129 155 159
177 184-186 266-267 292 313
325-326 346 354 366 379 392
409-410 427 464 470 472 511-512
533-535 539-540 543 551 557 583
605 608-610 640 663 670 680
692-693 705 708 713 715 743-744
752 757-758 829 842 859 874 885
888 909 970 1004-1005 1010 1035
1041 1045 1059
Mixture of 16 Various CTL016 74
tissues-mRNAs* Vendors*
Mixture of 16 Various CTL021 466 821-822 1094
tissues-mRNAs* Vendors*
adult cervix BioChain CVX001 2-3 5 8 15 25 31 35 44-45 49 52
54-55 57 61 71 73 84 88 90 93-
94 100-101 104-105 107-110 113
120 122 135-136 138 145-151 153
156-157 165 167 170 177 180
184-187 190-191 201 205 210
217-219 221-222 233 237 248-249
251-253 257-258 261-262 264-265
269-270 277 279 281-282 284-286
289 292-296 300 322 328 332-333
336-337 343-346 352 354 362 365
373-376 380 388 390-391 393-394
396 404-405 411 413 415-416 421
429-431 437-438 444 449 455
458-461 464 474 477 480-481 483
485 488 490 503 511 513 516-517
519-521 528 532-533 536-540
542-545 549-551 554 556 561
563-565 567 572 575-576 582
584-587 590-593 598-600 603
605-606 609 614 619 621-625 631
635 642-643 645 650-651 654
656-657 659 663 665-666 670 672
677 679 687 692-695 704-705 712
715 721 724 726 733 735 741
743-745 755-756 760 762 764 768
771 787 802 813 818 823 828
835-838 852 856 859 862 864 866
870 886 889 891 900 903 905
910-911 914 916 922-924 930-931
933 948 954-955 958 960 969
977-980 998 1000-1001 1006 1010
1013 1017 1035 1039 1043-1044
1050 1062-1064 1073-1074 1076
1084 1087 1096-1097
diaphragm BioChain DIA002 414 464 673 1100
endothelial Strategene EDT001 2 4-6 8 13 15 24-28 33 44 48
cells 53-55 57 60 63 65 70 73 81-82
84 86 88 90 99-101 105 108-110
113 118-119 123 128 137-138
146-149 151-153 160 162-164
171-172 174-178 184-187 190-193
197 199 201-203 207-209 211-215
217-221 223 237-239 242-243
248-252 255 257 261-262 265 269
272 274 276 280 284-289 292-294
297 299 303 308 310-312 316-319
322 325 333 335-339 341-345 352
355 360 362-364 374-379 389
391-392 395-396 403 414 416 418
420 422 427-430 435 443 452 455
457 459-462 467 470-473 477 485
488 490-492 495-496 499 501-503
506-507 509 511 513-514 517
519-523 527-530 533-535 539-545
547 549 554 556 559 561-564 568
570 572 575-576 579-580 583-585
588 593 595-596 599-600 603
606-609 611 613-615 617-622 626
630 635-636 638-641 644 646-648
651 656-657 660 662-665 670-671
675-677 679-680 683-684 687 689
691-701 704-708 710-713 716 719
721-722 726-734 738 744-745
748-749 751-753 756-759 761-763
765 767 771 775-776 778-779
782-786 788-792 796 798-802
805-806 813-815 817-820 823-824
827 829 833-834 837-838 842 846
849 852 860 872-873 887-888 891
894-896 900 905-906 908 910 914
918-919 922-925 928-931 933-934
936 940 943 947-948 951 955-956
958 960-961 964-967 970-973
975-976 978-980 983 985 988 998
1002 1007-1010 1014-1015 1017
1019-1020 1024-1025 1028 1032-
1033 1035-1037 1040 1045-1046
1049 1057 1059 1064 1066 1069
1088 1090 1097 1099
Genomic clones DNA from EPM001 368 987 993-994 1042 1102-1103
from the short Genetic
arm of Research
chromosome 8
esophagus BioChain ESO002 53 177 545 577 687 695 1087
fetal brain Clontech FBR001 9-11 52 64 85 155 221 239 284
361 392 552 700 719 744 918 941
952 1010 1098
fetal brain Clontech FBR004 4 35 47 76 110 288 323-324 338
350 352 373 469 490 530 852 898
905 922-923 928-929 1077 1101
fetal brain Clontech FBR006 3 6-7 9-11 19-20 25 30 43 46 50
52-53 55 57 64-65 70 72 75 80-
82 84-85 91 95 98 100-101 104
110-111 114-117 128 134 138 141
147 150 157 162-163 169 171 182
184-187 190 193-194 199 205
212-215 219 222 225 237 243
248-250 258 266-267 272 274 281
284-286 292 300 305-306 309 312
316-318 334 336-337 339 346 351
356-357 361 371 373-376 378-379
381 383 388 392 399 404 412 416
418 420 426-428 441-444 447
459-462 464 484 491-492 495
502-503 511 524 528 543 546 549
556-557 569 575-576 579-580 583
589 597 602 608-610 622-623 625
632 637 639 642-643 645-646 648
650 654 656-658 677 679 686 688
692-694 696 701 704 710 712
717-718 720 723 730 735-736 740
745 754 756 759 771 778 798
803-804 808 820 832-838 840
842-845 849 852 856 861-862 867
873 875 877 879-889 900 905
911-912 915-916 919 921 926 935
943-945 948 950 952 956 960-963
971 977 998-999 1004 1007-1010
1016 1024-1025 1029 1031 1034
1040 1046 1059-1061 1063 1066
1069 1071-1072 1076 1082 1086-
1088
fetal brain Clontech FBRS03 194 549 757 877
fetal brain Invitrogen FBT002 2 7 12 19-20 23 30-32 54-56 63
81 92-93 104 108-109 112 117-
118 135 138-140 157 164 168 183
190 193 197 202 233 237-238
248-249 266-267 272 274 300 310
325-326 328 334 351-352 354 364
372-373 382-383 392 401 420 430
466 468 472-473 499-500 510 514
525-526 532 539-540 542-543
582-585 589 606 612 622-624 633
635-636 641 647-649 653 656-657
673 683 687-688 692-693 695 700
702 710-716 733 740 744 757 759
761 767 771 774 779 798 804 807
809 817 825-826 833-834 838
845-846 882-884 887 893-894 909
911 924 947 952 961 964-965 970
975-976 984 1002 1004 1007-1010
1020 1032 1034-1035 1039-1040
1045-1046 1048 1054 1059 1069
1073-1074
fetal heart Invitrogen FHR001 549 724 837 919
fetal kidney Clontech FKD001 3 44 60 65 68 76 96-97 105 110
117 129 143 174 193 197 217 239
248-249 261 277 296 300 328
332-333 336-337 339 361 403 415
464 483 506 518 529 539-540 542
549 553 563 596 616 645 647 656
677 679 695 734 776 792 818 828
975 1002 1021 1045 1059 1061
1066 1081 1093
fetal kidney Clontech FKD002 25 57 100-101 114 259 279 749
820 1014-1015
fetal kidney Invitrogen FKD007 8 361 675-676 687 916 920 1010
fetal lung Clontech FLG001 44 71 100-101 119 147 224 236
281 293-294 303 309 327 329 393
400 403 430 470-471 517 527
534-535 549 579-580 764 867 871
889 895 918 999 1001 1035
fetal lung Invitrogen FLG003 6 17-18 25 46 49 52 57 82 100-
101 104 106 141-142 149 157 162
167 190 206 210 220 222 224 240
258 279 300 322 339 343-345 352
355 393 400 409-410 412 445
450-451 458 490 515 537-538 549
560 608 619 624 633 636 650
675-676 702 704-705 712 715 779
786 859 874 889 904-905 913 948
980 999 1010 1032 1037 1045
1059 1071 1104
fetal lung Clontech FLG004 7 139-140 421 528 820
fetal liver- Soares FLS001 2-5 15 17-18 23 25-26 29-31 33
spleen 35 43-47 49 51-57 59-60 63 65-
66 73-74 76 80 82 84-86 89 91
93 96-111 113-116 118-119 122
126-128 130 133 135-144 146-153
155-157 161-162 164-165 167
174-175 177-180 183-187 189-194
197-199 201-204 206 209 211-215
217 222-224 228-229 237-238 240
243-244 247-252 255 257 259
261-274 276-282 284 287-290
292-298 300-301 303 305-312
314-318 322 325-328 333-339
341-342 346 348 351-352 354
356-357 360 363-364 366 372-377
379 384 390-393 396-397 399-403
409-412 414-415 418 420 422
424-425 427-430 432 434-435
443-450 452 456 458-461 463
467-472 477 480-481 483 485-490
493 495-497 499 503-504 506-509
511-517 519-522 528-532 534-552
554-561 563-568 572 574-580
582-583 587 589-596 599-601 603
606 608-615 617-624 626 632-637
640-645 647-648 650-652 654 656
658 661-665 667-670 673 675-677
679-680 682-684 687-689 691-696
700 702 704-707 713-714 716-719
721 723-724 727-732 735 737-738
741 743-745 747-748 750-753
756-759 761-763 765 767 771
774-776 778-779 786 788-792
796-802 804-808 810-812 814
816-817 820-823 825-826 833-839
842 848-849 852 859 864 866-867
877 879-881 884-889 891 895
897-900 903 905 907 910-911 914
916 918 924 927 931 934 936-937
941 944 948 951 956 958 960-963
967-972 974-975 977-980 984
989-990 996-998 1003-1010 1014-
1015 1017 1020 1022-1023 1028
1031 1033 1035-1037 1039-1040
1043-1050 1052-1053 1055-1057
1059-1060 1062-1064 1066 1068-
1071 1073-1074 1076 1079-1081
1090 1092 1094 1096-1101 1104
normalized Soares FLS002 2 4 7 15 21-22 25-26 29 32 41
fetal liver- 44 47 49-52 54-57 59-60 65-66
spleen 70-71 73-74 76 84-87 95 97 100-
101 103-104 107-110 112 114 117
119 122 125-126 128-129 134-137
139-141 143 145-147 149-150
152-153 155-157 161 164-165
167-168 171 174-175 177-179
183-187 189-191 193-194 199
201-206 209 212-217 219 222
227-229 231-232 234 237-238
240-243 247-253 259-263 266-267
269 271-272 274 278 281-283
285-290 293-294 296-301 303-310
312 315-320 322 325 327-330 332
334-338 341-346 348 351-354
356-357 363-364 367 370 372 378
380 382-385 389 391-393 396
400-401 403-405 407-408 412
415-416 418 421-422 426-429 432
436 438 443 447-450 452 454
457-461 464 466 468 470 475
479-483 486-487 490 493 496-497
503 508-509 511-512 517 519-522
524 528 531 533-541 543-546 550
552 554 556 558-559 561 563
566-567 572 574 577 579-583
586-587 589 591-593 597 600-602
606 608-611 613-619 621-622 624
626-628 630 633-634 637 640 645
648 652-654 658 660-662 664-666
668 671-672 677 679-680 682-684
686 688 691-693 695 697-699 701
704 708 711 713 715-719 721-723
727-730 732-733 737 741 744-745
747-748 751 753-754 756-759
761-763 768 771 776 778 782 786
788-792 794-796 798-803 812
814-816 821-823 829 832-834
837-839 845 847 849 852 861-862
866-867 870-873 879-881 884-886
888 893 895 897 900 903 905 908
916 918 920 924-925 927 930-931
933 935 937 946 948 950-951 954
958 960-961 965 968-969 974-976
978-979 989-991 996 998 1002
1004 1007-1009 1013-1015 1017-
1020 1024-1025 1029 1033-1037
1039-1040 1044-1045 1047 1050
1052-1053 1055-1057 1059 1062
1066 1068-1070 1076 1079-1081
1084 1090 1094 1097 1100
fetal liver- Soares FLS003 25 45 52 54 57 65-66 76 96 98
spleen 103 108-109 114 118 127 177 189
219 268 665 753 1014-1015 1017
1023 1047 1059 1068
fetal liver Invitrogen FLV001 2 17 29 44 47 49 52-55 68 82 86
88 93 103 108-109 115-116 139-
140 164 168 178-180 183 197 202
209 227-228 231 238 243 245-246
248-250 284 309 336-337 349 351
354 367-368 384 401 409-410 416
432 443 445 462 468 470 494 502
514 522 542 546 557-558 560 566
574 579-580 594 596 603 641 650
654 661 664 673 675-676 680 682
684 687 692-693 696 702-703 708
730-731 759 775-776 778-779
807-808 811 833-834 837 842 846
848 872 884-885 916 940 947-948
952 957 970 975 997 1002 1004
1007-1010 1034-1035 1039-1040
1045 1052-1053 1094 1096
fetal liver Clontech FLV002 358 685
fetal liver Clontech FLV004 28 47 52 230 257 278 361 522
532 544 812 926 960 1003 1014-
1015 1017 1045 1057
fetal muscle Invitrogen FMS001 32 34 49 52 56 71 86-87 93 104
110 122 126 136-137 147 151 177
193 204 237 245-246 266-267 274
284 300 303 313 325 339 354 398
409-410 414 417 445 459-461 472
511 522 545 559 600 602 608 622
657 667 673 675-677 679 687-688
695 702 715 723-724 733 749 753
759 772 786 788-791 805-806 810
853 862 887 897 917 930 948 956
960 967 988 1007-1010 1014-1015
1019 1026-1027 1059 1073-1074
1089-1090 1094 1100
fetal muscle Invitrogen FMS002 75 100-101 137 222 276 420 554
687 721 772 956
fetal skin Invitrogen FSK001 1 3 6-8 12 14 18 24-25 28-29
31-32 35 41 43 45 49 52-55 57
61 67 78-79 82-83 85-86 93-94
98 100-101 110 115-116 119 122-
123 128 136 139-140 146 150 164
167 178 180 183 189-191 193 195
201 206 212-215 222 224 231
236-237 243 248-249 257 260 265
271 288-289 292 300 308-309 313
325 327 329 336-337 339 343-346
349 351 354 358 360 363 370 383
386-387 391-392 396 400-401
404-405 409-410 412-413 416 432
434 437 443 448 450 454 459-461
464-465 470 472-473 483 494 496
499 501-502 507 509-511 516 527
542 544 546 548-549 566 579-580
593 595-596 603 605 609-610 615
619-620 622 624 633 635-636 640
647-648 653-654 657 662 664 673
677 679-680 686-687 691 695 701
706 713 715 720 723 729-731 743
746-747 749 751-753 758 761 766
779-780 786 788-792 797 802 808
812 817 820 823 829 837-838
841-842 845 852 856 867 871 878
881 884-885 887 890-894 897
900-901 905 908-909 911 914 918
930 933 948 956-957 964 967 970
972-975 989 1000 1007-1012
1014-1015 1019-1020 1032 1034
1036 1039-1041 1044-1047 1055-
1056 1059 1061 1063 1066 1068
1070 1073-1074 1077 1089-1090
1096 1101
fetal skin Invitrogen FSK002 3 8 12 14 27 43 45 58 73 75 82
88 100-101 107 110 124 128 189
219 261 275 451 495 530 626 765
798 844 1007-1010 1016 1023-
1025 1033 1037 1040 1059-1061
1067-1068 1081 1087
fetal spleen BioChain FSP001 26 265 598 614 797
umbilical cord BioChain FUC001 6-8 18 24 29 41 44-46 52-53 59
63 65 67 77 80 82-84 86 88 90
94 98 101 104-110 112 118-120
128 133 135 137 141 147 156-157
160 162 164 169 178 183 199 201
212-215 222 230-231 237 250-253
264-267 269 283 292 297-298 310
315 323-325 328 338-339 343-345
349-350 356-357 366 369 378 380
383 385-387 391 394 397 403-404
409-411 416 433-434 446 449 455
459-461 464 473-475 477 480
487-488 499-502 507 518 528 530
533 543 546 549 560 564 573
579-580 582-585 588 591-592 596
599 612 617-620 623 627 635 640
647 653 664 671 677 679 685
687-688 692-693 695 704 711 713
725 734-735 748 750-752 762
765-766 778 783-786 801-802 804
823 833-837 840 843 845 852 860
886 893-894 897 905 907 913
924-925 933-934 936 969 973
976-977 988 1006 1010 1034 1036
1040 1047 1049 1061 1063 1065
1071 1088 1090 1094 1099
fetal brain GIBCO HFB001 3-4 12 14 19-22 25 27 29-32 35
37-38 44-45 50 52-53 55 57 60
63 73 82 88-90 93 98 105 108-
109 112 115-117 123 125 127-129
131-137 144 146-147 149 151-152
156-157 160-162 164-165 167 170
173-174 176-178 180-181 183-186
190 193-194 197 200-201 204-205
212-215 218-219 221-222 231
242-243 245-246 248-258 261 264
266-267 269 272 276 278 281
283-286 288-289 291-297 299-301
308 310 314 321 323-325 328-330
332-333 338 340 346 349-350
352-354 358 360 363 369 371
374-376 379 381 392-393 397 401
405 411 413-416 418 420 422
427-430 434-435 443 447 450 454
456 459-461 468 473 475 480 484
487-490 495-496 499 504-506 509
511 515 519-522 524-535 539-541
543-545 547 549 554 556 561-562
564 568 571-572 575-576 578-580
583-586 590 593-595 597 599-603
606 609-610 612 614 617-620
622-625 635 639-647 650-651 654
656-659 662 665-666 668 670
673-674 677 679 682 684 686-689
692-697 699-701 704 706-707 711
714 717-719 721-723 726 729 735
738 741-742 746 748 753 757 759
761-762 767-768 770-771 775 778
783 786 792 796-798 802-804
810-811 816 823-824 827 829 832
837-840 842 845-846 848 856 859
864 882-883 889 891 896-898 900
905 908-909 915-916 919 922-924
930-931 933 936 939-941 943-944
948 957-958 961 966 969 972
975-979 983-984 995 997-998
1002 1006 1010 1017 1028-1029
1031-1034 1036 1039 1045-1047
1049 1055-1056 1059-1060 1063-
1065 1069 1076 1079 1084 1088
1100 1104
macrophage Invitrogen HMP001 177 312 360 544 563 620 675-676
1069
infant brain Soares IB2002 3-4 6-7 12 19-20 22 25 29-30 35
44-45 50 52-53 56-57 65 70 74
80 82 85 87-88 90 92-93 96 102
104-105 108-109 112 117 123
127-128 135 138-140 143-144
146-147 149-150 152 156-157 161
164 166 168 171 177 181-182
184-187 190 193-194 199-201
205-209 211-215 222 225-226 237
239-240 242-243 248-252 255
257-258 260-263 266-267 269
272-274 284-286 288-290 293-294
297 299-301 304 309 315 319
323-325 328-334 336-337 339-340
343-346 348-357 371 373 377 379
381 385 389 396-398 401 405
412-415 417-420 423 427 429 435
439 443 447 453-454 456 462
464-465 469 472-473 477-478 484
488 493 496 499 510-511 515
519-521 525-528 530 533-535
539-540 545 547 549-550 555 557
560-561 563 571 573 575-576
579-581 583 588-589 597 603-604
606 609-612 614-615 617-619
621-623 625 628 632-633 635-637
640-641 646-649 653-656 664 666
668 673-677 679 684 686-687
691-694 702 704 706-707 712-713
721 724 735 739-740 743-744
746-748 752-753 757-759 762
770-771 774 778 797-802 807-809
812-813 819 821-827 829 838 840
842-843 852-859 861 863-864
872-873 877 879 882-883 885-886
888 893 897-898 900 907-911
915-916 919 921 924 931 937 939
941 946 948 951-952 959 964 969
971 973 975 977-979 984-985 995
997 1003-1004 1006-1010 1019
1022 1031-1032 1035 1039 1046
1052-1053 1055-1056 1058-1060
1064 1071 1073-1074 1078 1090
1094 1097-1098 1104
infant brain Soares IB2003 4 7 12 19-20 22-23 25 30 44 49-
50 55 57 76 82-83 88-89 127-129
146 150 152-153 164 177 181 190
194 205 209 211-215 226 239
248-252 261 263 266-267 269
272-273 278 283 289 293-294 297
299-301 328 330-331 340 346 348
352-353 356-357 360 371 398 401
411-412 414 418 443 459-461
464-465 469 480 491-492 496 509
530-531 549 554 560 567 574
579-580 583 589 597 604 606
608-610 619 622-623 633 637 641
649 664 666 684 686-687 692-693
697 699 705 712 721 725 731 744
746 798 803-804 809 812 816
833-834 838 840-842 849 856 859
863 882-883 885 888 891 895 898
909-910 915 918-919 930-931 941
948 974 978-979 995 1004 1017
1023 1034 1037 1040 1046-1047
1055-1056 1068-1069 1078 1090
1094
infant brain Soares IBM002 47 50 226 239 285-286 331 338
348 499 641 692-693 717-718 819
853 898 948 984 995 1010 1017
1087
infant brain Soares IBS001 7 44 120 226 239 300 330 349
351 353-354 504 636 653 802 819
840 877 882-883 885 919 948 955
961 977 1007-1009 1014-1015
1032
lung Strategene LFB001 6 29 43 101 104 110 120 136 146
fibroblast 167 172 177 190 193 202 210
212-215 217-219 238 241 243 262
276-277 284 292 308 325 343-345
374-376 395 403 429 432 459-461
478-480 495 522 528-532 534
539-541 554 561 588 621 640
642-643 651 662-663 671 687
692-693 695 712-713 721 727 741
753 759 761 771 801-802 823
825-827 829 884 912 918 922-923
936 969 975 985 1001 1010 1024-
1025 1033
lung tumor Invitrogen LGT002 2 5 14-15 17-18 21 25 31-33 36
41 43-44 49 53-54 57 62-63 71
73 77 83 88 97 100-101 104 106-
110 117-118 124-125 128 136 142
146-151 154-157 161-162 164-165
167 170-172 174 177-180 183-186
189-190 193 196-198 201 205 209
216 219 222 229 231 233 240
242-243 248-252 261-262 264-265
272-273 278 284 288-289 292 296
298 300 303 307-308 310-311 322
325 327 329 334-335 339 343-346
349 351 354 365-366 378 382 388
392-393 396 399-400 402-403
406-407 412 418-423 427 434 436
447 449-450 457-462 468-469
471-473 475 481 485 487-488
494-497 499 509 511 513-514 517
523-524 526-530 534-535 537-540
542-546 549 551 559 563-565 567
572 575-576 582-583 587 589
591-592 595 597-598 603 605-607
609 611-612 614 617-619 621-622
624 634 637 640 642-643 648 650
653-654 656 658 664-666 668 671
673-674 677 679-680 682 684
686-688 692-695 697 700-701 706
708 710-712 714 717-718 721 726
729 733 735-738 740 743-744 748
752-753 756-760 762-763 767 774
776 778 784-786 793 795-796 798
801 805-806 808 815 823-824 828
830-831 833-834 837 841-842
845-847 856 859 861 867 884-885
887-889 891 893 897 903 905 908
911 916 924 931 937 942 947-948
955 957-960 966 968 970-971 973
975 978-979 985 990 995 998
1002 1007-1011 1019 1028-1029
1031 1034-1036 1039-1041 1043-
1045 1047 1055-1056 1059 1068-
1069 1073-1075 1084 1087 1090
1092 1094 1099
lymphocytes ATCC LPC001 12 21 25 30 44 55 57 63 73 77-
78 86 88 95 97 100-101 104-105
119 121-122 125 128-129 149 177
190 201-202 205 212-215 224
232-236 239 250 253 265 279 290
292-294 298 300 305-306 308
314-315 325 327 333 336-338
341-342 372 379 401 407 439 448
459-461 464 470 473-474 477 485
487 507 523 534 551 554 561
566-568 577 583 591-592 596
608-610 615 662 677 679 683 688
691-695 721 738 743 767 796 824
829-830 832 837 847 852 866 888
894 905 915 921 926 931 934 940
948 950 955 959-961 980-983 998
1002 1007 1009-1010 1029 1035-
1036 1038 1040-1041 1047 1057
1059 1069 1071 1073-1074 1076-
1077 1079 1084 1087 1096
leukocyte GIBCO LUC001 1-3 6-7 13-15 21-22 25 27-31
43-44 53-58 60-61 73 77 79 82-
84 86 88-90 95-99 104-105 108-
109 111 113 115-116 119-125
127-128 130 133 135-138 142-146
148-153 155 157-159 161-165 170
173-175 177-178 183-187 190-193
197 201-202 204-206 209 211-215
217 222-224 228-230 232-235
242-243 247 250-253 255 258
260-262 265 269 272-274 278-279
281 283-284 288-290 292-298 300
304-306 311 314-315 320 322-328
333-334 338-339 341-346 349 351
356-357 360 364-366 374-378 380
382 388 390 392 399-404 409-410
413-420 422-423 427-430 433 435
437 440 443 449-450 452 454-462
464 468-470 472-474 477 480 483
485 487-488 495-496 499 502 504
511 513-515 517 519-521 523 528
530-545 548-549 551-556 562-564
566-577 579-581 583 587 589-601
603 606-612 614 617-619 622-623
628-629 632 635-636 640 642-643
645 647-649 651 654 657-658 660
662 664 672-677 679-680 682-683
686-689 691-697 699 702 705-707
710-711 713 715 717-719 721-724
726 729 731 735 738 741-743
747-748 750-751 753 757 759 761
767 774-775 778-780 782 786
795-796 799-802 804-806 811-812
814 818 821-822 824-826 829-834
841 847 852 857 859-863 865-867
869 871-872 875 884 887-889 891
894-895 900 905 908-910 912-916
920-926 928-932 937 942 944 946
948 951-952 954-955 959-960
962-965 967 970-974 977-983
989-990 996 998 1002-1003 1007-
1010 1012 1014-1020 1023-1025
1029 1031 1033-1037 1039 1043-
1047 1049-1051 1055-1057 1059-
1060 1063-1064 1067 1069-1071
1076-1077 1084 1090 1094 1100
leukocyte Clontech LUC003 45 78 104-105 129 135 150-151
155 177 212-215 229 251-252 257
265 285-286 298-299 301 308 310
325 343-345 351 361 380 400 448
457 459-461 487 533 536 541-542
564 590-592 602 636 639 677 679
689 802 808 859 872 905 926-927
931 965 1036 1043 1047 1049
1067
melanoma from Clontech MEL004 9-11 18 24 45 53-54 56 60 36
cell line 89-90 104 119 122 128 130 141
ATCC # CRL 155 164 173 177 201 203 221 223
1424 243 251-252 264 278 290 292 325
339 341-342 346 418-419 421 443
462 471 474 485 517 530 541 544
554 563 568 572 590 601 619 621
645 663 672 677 679 686-687 713
719 726 738 744 757 763 777
788-791 796 825-827 838 845 852
861 879 884 889 903-904 906
915-916 925 928-929 934 943 968
970 983 1001 1010 1020 1024-
1025 1035 1045 1057 1076-1077
1084 1094 1097
mammary gland Invitrogen MMG001 1-2 4-5 7 12 14 17-18 21 23 29
32-33 35-36 41 44 49 52-56 61
63 66-68 70-71 79 82 86 88-89
97 104 108-111 115-118 122 125-
129 136-138 148 150 153 156
159-160 162 164 168-169 172 177
180 183-186 190-191 193-194 197
202 204 209 212-216 222 224 229
231 233 238 248-250 263 265-267
274 279 284 295 300 305-306 310
312-318 322 325-326 328-330 334
336-337 339 341-342 346 349
351-354 356-358 361 364 370
377-378 388-389 392 396 400-401
404 406-414 416-418 420 422 427
431-432 434 439 443 445 450 454
464 468 470-473 478 488 491-492
494 499 502-507 512-514 522 527
539-543 548-549 551 554-555 557
563 566 574 578-585 589 591-592
596 603 606 608-610 613 622-624
633 635-636 640-641 644 647-648
650 652-653 657 664-665 671-676
680-684 687-688 692-694 696
701-702 704-705 711 713 715
720-721 724 727 731 733 744
746-747 752 757-764 766-767 774
776 778-780 784-791 796-797
801-808 810-812 820-822 825-826
828-831 833-834 837-838 842 848
856 859 861-862 872-874 879-881
885-888 896-897 901 905-907
911-912 914 921 924 930-931 937
943-944 948-949 951-952 960-970
973 975-976 988 997 1004 1006-
1010 1013-1015 1020 1024-1025
1032-1035 1039-1042 1044-1045
1049 1059-1060 1067 1069 1071
1073-1074 1079 1096
induced neuron Strategene NTD001 3 13 31 44 48 50 53 55 96 98
cells 166 171 207-208 217 221 224 242
262 272 289 323-325 332 418
459-462 464 476 484 506 511 541
543 560 623 640 672 677 679
729-731 744 761 793 882-883 895
912 936 943 964 978-979 981 984
1001 1010 1039 1049 1103
retinoid acid Strategene NTR001 56 105 180 300 359 415 686-687
induced 888
neuronal cells
neuronal cells Strategene NTU001 5 7 9-11 48 64 66 80 88-90 128
139-140 144 162 177 180 184-186
193 212-215 248-250 274 279 284
300 325 340 382 384 399 427 455
476 543 589 635 641 664 687
692-693 713 753 757 811 837 874
908 915 924 936 961 973 985-986
1001 1007-1010 1019-1020 1040
1045 1055-1056 1068 1073-1074
1090 1103
pituitary Clontech PIT004 52 82 93 104 128 744 784-785
gland 962-963 1002 1010 1068
placenta Clontech PLA003 43 528 591-592 970 1007-1009
1059
prostate Clontech PRT001 3 31 46 55-56 73 104 108-110
135-136 143 151 163 171 174
179-180 187 196 201 206-208 222
295 327 333 336-337 343-345 392
413 451 455 473 505 546 556 559
575-576 590 597 625 632 650-651
675-676 689 713 721 733 742 750
784-785 801 814 831 885 887 891
922-923 931 948 962-963 977 985
1003 1023 1031 1034 1039 1050
1052-1053 1057
rectum Invitrogen REC001 14 17 41 46 61 68 82 88 94 104
115-116 120 122 138 142 157 191
212-215 222 243 261 265 279 300
305-306 310 323-325 329 336-337
351 401-403 405 409-410 422 432
434 440 446-447 450 454 458 474
504 510 528 534 536 566 588 594
598 635-636 647 673 683 708 711
721-722 753 756 759 764 775
797-798 802 819 828 842 848 861
867 874 876-878 894 909 914 930
934 961 1007-1010 1013 1024-
1025 1040-1041 1045 1059 1063
1065
salivary gland Clontech SAL001 6 24 27 33 84 111 122 147 161
168 170 175 210 230 245-246
248-249 266-267 301 314 329 443
446 448 452 455 472 485 494 511
533 550 564 591-592 612 620 650
704 708 713 728 734 744 755 792
839 861 870 924 971-972 978-979
988 1005 1010 1023 1026-1027
1036 1049 1063 1069
skin ATCC SFB001 1010 1090
fibroblast
skin ATCC SFB002 420 713 943
fibroblast
skin ATCC SFB003 391 808 1040
fibroblast
small Clontech SIN001 7 13 44 71 82 86 88 90 97 100-
intestine 101 104 108-109 119 126 128 152
177 190 193 196 198 218 222 224
230 235 239 245-246 261 268 281
288 303 305-306 310 328 338 363
392-393 397 402 434 448 464 483
514 527 542 544 551 572 577
579-580 586 589 591-592 599 606
622 684-685 687 695-696 714-716
732 744 778 786 805-806 820 837
858 888 908 924 937 954 969
1006 1010 1016 1031 1033 1039
1041 1064 1077 1088 1090 1094
1098
skeletal Clontech SKM001 2 34 86 88 98 114 126 133 144
muscle 162 177-178 212-215 325 339
355-357 398 457 464 470 481 515
590 609 637 650 669 677 679 708
735 752 804-806 810 823 917-918
958 974 988 1011 1019 1026-1027
1033 1100 1104
skeletal Clonetech SKMS03 1100
muscle
spinal cord Clontech SPC001 45 52 60 65 93 100-101 104 107
122 141 147 161 163 180 183 187
193 199 201 212-215 218 222 224
226 231 235 243 245-246 269 284
293-294 300 302 307 315 327-328
330 336-337 352-353 355 361
363-364 391 394 398 400 403
412-413 418 433-434 437 459-461
465 473 475 494 505 517 519-521
532-534 539-540 543 546 549
556-557 560 569 583 591-593 604
606 611 621 629 631 657 672 677
679-680 685 701 727 741 748 754
761 764 766 771 774 786 792 798
805-806 817 823 840 859 863
881-883 904 914 931 933 973 984
997 1002 1010 1013 1017 1020
1040 1043-1044 1046 1049 1058
1065 1090 1097-1098
adult spleen Clontech SPLc01 21 98 100-101 105 530 551 1010
1020 1023 1034 1045-1047 1061
1077 1087
stomach Clontech STO001 31 35 77 100-101 104 120 136
149 153 210 274 297 308 329
343-345 406 423 464 466 469 531
606 647 651 653-654 663 696 700
720 730 752 760 824 859 897
924-925 949 967 970-971 1012
1039 1049 1059
Mixture of 16 Various SUP002 4 37-38 47-48 94 108-109 472
tissues-mRNAs* Vendors* 544 859 1010 1016 1102
Mixture of 16 Various SUP005 88 99 108-109 115-116 195 547
tissues-mRNAs* Vendors* 1010 1064 1078
Mixture of 16 Various SUP008 19-20 24 82 98 104 115-116 157
tissues-mRNAs* Vendors* 326 705 852 952 1068 1073-1074
Mixture of 16 Various SUP009 12 43 104 472 850-851 1010 1040
tissues-mRNAs* Vendors* 1043 1090
thalamus Clontech THA002 6-7 14 24 29 37-38 52 61 66 69
93-94 115-116 132 151 157 182
200 206 224 243 283 285-286 325
333 348 363 398 409-411 420 458
466 468 478 512 568 575-576
584-585 648 684 686-688 691 694
722 744 786 818 833-834 839-840
846 861-862 866 882-883 897 909
939 970 995 1035 1045 1059
thymus Clonetech THM001 3 6 15 24 31 44 84 104 119 139-
140 142 145 157 161 167 177 180
183 191 201 210 212-215 221 223
229 231 243 260 262 266-267 281
289 298 316-318 323-325 327 335
339 346 352 380 392 400 404 412
419 423 430 443 446 450-451
459-461 464 468-469 481 485 487
494 509 511 513 530 536 543-544
549 551 555-556 563 569 572 577
584-585 598 614 617-618 654 670
673 677 679 686 702 717-718 721
748 755 762 792 796 802 805-806
808 829-831 847 852 861 866 884
895 898 902 905 907-908 910-911
916 961 967 972 1010 1019 1039
1057 1059 1073-1074
thymus Clontech THMc02 7 15 23 25-26 33 43-46 55 57 73
76 84 88-90 98 104-105 110 119
128-130 135 138-141 144 146
148-150 157 162 171 174 178 180
187 190 193-194 199 201 205-206
209 212-215 224 229-230 241-242
245-246 248-249 251-253 262-263
272 280-281 283 289 308-310 312
314 325 328 333 336-339 347 349
351 355-357 360 380 382 385 390
400 404 409-410 415 422-423
427-428 434-437 441 443 447-449
451 456-463 471 473-474 481 483
485 495 508 514 519-521 526 533
539-541 544 546 549 555-556 566
575-576 583 593-594 598 600 615
620 628 636 645 648 650 654 656
662 668 673 675-677 679 686
695-696 702 704 727 729 762-763
768 778 786 798 803-806 811 820
829-830 833-834 852 861 864-866
875 885 887 895 900 905 909 912
916 924-933 951-952 957 961
970-971 974 980 998 1007-1010
1012 1014-1015 1017 1020 1024-
1025 1029 1036 1040-1041 1045
1047 1050 1060 1064 1067-1069
1076 1079-1080 1082 1087 1090
1092-1093 1097-1098
thyroid gland Clontech THR001 5 8 14-15 17 21 23 27 31-32 36
41 44-46 55-56 58 67 71 73-74
85 88-89 95 98 104-105 107-110
117-120 124-126 128-129 132
146-148 150-152 156 158-159
161-162 164-168 170-171 174-175
177-178 180 183-187 190-191 193
199 201-202 204 210-215 222 224
238 240 245-246 251-252 264 270
272 278-279 281 283 287 289
292-294 296-300 305-306 308
312-313 320 325 327-329 333 339
343-346 348 350 352 363 372-376
378 380 382 391 393 405 411-413
416 418 422 424-425 427 429 434
443 445 447 449 454 457 464 469
479-480 483 487 489 508-509 511
513 518 523 526-530 532 534
542-545 552 559-560 563 565-566
572-573 575-577 579-580 583 593
595-596 600-601 609-610 613-614
616 619-621 624 626 628-629 635
640 644 648 650-651 653 665-666
668-669 674 677 679 688 691-693
698 702 708 712 721 726-727 732
735 738 740-741 743 745 747 757
759-760 766-768 777 783-786
794-795 801-802 805-806 813 815
817-818 820 823 825-826 831-832
835-836 838-839 845 850-853 856
859-862 866-870 879 881 886 889
893 897-898 903 906 908 911 920
922-923 925 931 935 937 944
947-948 950 952 961-963 973
976-979 989 996 1004 1010 1020-
1022 1024-1025 1032 1035 1039
1055-1057 1060-1062 1064 1071
1084 1098
trachea Clontech TRC001 4 25 45 50 57 88 98 119-120 128
146 148 165 170 236 255 264 269
274 284 289 303 363 384 403 495
544 551 563 579-580 599 603 609
619 622 734 764 769 788-791 802
897 904 918 922-923 927 971
1002 1075 1077 1096
uterus Clontech UTR001 60 82 94 112 120 122 126 147
165 167 173 177 180 187 193 197
201 205 217-218 236 278 287 310
338 346 404 435 457 464 518 530
542 557 562 599 616 621 624 683
697 699 706 738 764 796 813 859
908 948 969 977 1000 1010 1013
1033-1034 1065

* The 16 tissue-mRNAs and their vendor source, are as follows: 1) Normal adult brain mRNA (Invitrogen), 2) normal adult kidney mRNA (Invitrogen), 3) normal adult liver mRNA (Invitrogen), 4) normal fetal brain mRNA (Invitrogen), 5) normal fetal kidney mRNA (Invitrogen), 6) normal fetal liver mRNA (Invitrogen), 7) normal fetal skin mRNA (Invitrogen),
#8) human adrenal gland mRNA (Clontech), 9) human bone marrow mRNA (Clontech), 10) human leukemia lymphablastic mRNA (Clontech), 11) human thymus mRNA (Clontech), 12) human lymph node mRNA (Clontech), 13) human spinal cord mRNA (Clontech), 14) human thyroid mRNA (Clontech), 15) human esophagus mRNA (BioChain), 16) human conceptional umbilical cord mRNA (BioChain).

TABLE 2
CORRESPONDING
SEQ ID NO. IN SMITH-
SEQ U.S.S.N. ACCESSION WATERMAN %
ID NO 09/552,317 NUMBER DESCRIPTION SCORE IDENTITY
1 152 AL122081 Homo sapiens 1930 100
hypothetical protein
2 167 AF212921 Mus musculus MMTV 484 94
receptor variant 1
3 205 Z75330 Homo sapiens nuclear 6492 99
protein SA-1
4 210 AL008583 Homo sapiens 2133 99
dJ327J16.3 (supported
by GENSCAN, FGENES and
GENEWISE)
5 225 AK000381 Homo sapiens unnamed 1028 98
protein product
6 226 AK000418 Homo sapiens unnamed 1747 100
protein product
7 264 AF156598 Mus musculus p53- 997 65
regulated DDA3
8 268 AK001463 Homo sapiens unnamed 1131 100
protein product
9 293 AB033039 Homo sapiens KIAA1213 2438 100
protein
10 293 AB033039 Homo sapiens KIAA1213 1510 74
protein
11 293 AB033039 Homo sapiens KIAA1213 2415 98
protein
12 302 AK001184 Homo sapiens unnamed 2830 99
protein product
13 311 AB021643 Homo sapiens 2761 99
gonadotropin inducible
transcription
repressor-3
14 352 AL122089 Homo sapiens 593 100
hypothetical protein
15 358 AC007228 Homo sapiens BC37295_1 1178 44
16 368 L29154 Homo sapiens 439 84
immunoglobulin heavy
chain VDJ region
17 393 AB037780 Homo sapiens KIAA1359 1439 74
protein
18 477 AK000404 Homo sapiens unnamed 1177 99
protein product
19 508 L22557 Rattus norvegicus 1949 85
calmodulin-binding
protein
20 508 L22557 Rattus norvegicus 2363 92
calmodulin-binding
protein
21 515 AK002158 Homo sapiens unnamed 1588 99
protein product
22 578 AL080076 Homo sapiens 107 30
hypothetical protein
23 588 AJ251516 Mus musculus cysteine 1460 99
and histidine-rich
protein
24 591 AL117551 Homo sapiens 1773 100
hypothetical protein
25 593 AB033076 Homo sapiens KIAA1250 6286 100
protein
26 594 AK000625 Homo sapiens unnamed 721 100
protein product
27 619 AF161420 Homo sapiens HSPC302 2623 97
28 620 AL117477 Homo sapiens 2551 100
hypothetical protein
29 654 AK001782 Homo sapiens unnamed 1161 100
protein product
30 692 D25217 Homo sapiens KIAA0027 1911 100
protein
31 753 AB041581 Mus musculus unnamed 1758 95
protein product
32 758 X03414 Drosophila 316 45
melanogaster Kr
polypeptide
33 787 AF151079 Homo sapiens HSPC245 643 100
34 833 AK000643 Homo sapiens unnamed 614 53
protein product
35 838 AB029022 Homo sapiens KIAA1099 1095 61
protein
36 870 AF213465 Homo sapiens dual 2016 100
oxidase
37 891 AF181562 Homo sapiens proSAAS 1319 100
38 891 AF181562 Homo sapiens proSAAS 1024 99
39 921 AB020671 Homo sapiens KIAA0864 5438 99
protein
40 924 AB033051 Homo sapiens KIAA1225 4438 100
protein
41 932 AB011105 Homo sapiens KIAA0533 8255 100
protein
42 942 AB032983 Homo sapiens KIAA1157 2231 100
protein
43 958 AF139077 Homo sapiens M5-14 1463 98
44 968 AK001366 Homo sapiens unnamed 2940 97
protein product
45 992 AF198454 Homo sapiens 3927 100
epithelial protein
lost in neoplasm beta
46 1025 AK001753 Homo sapiens unnamed 217 68
protein product
47 1074 AF169017 Homo sapiens 2717 98
formiminotransferase
cyclodeaminase
48 1104 A95106 unidentified RED ALPHA 1202 99
49 1114 AL137479 Homo sapiens 1942 100
hypothetical protein
50 1144 AF072372 Mus musculus lysosomal 3388 97
trafficking regulator 2
51 1262 M14912 Homo sapiens pol 132 86
52 1318 AF090934 Homo sapiens PRO0518 382 100
53 1319 X66363 Homo sapiens 2499 100
serine/threonine
protein kinase
54 1328 AF072758 Mus musculus fatty 2097 87
acid transport protein
3; FATP3
55 1436 AB014514 Homo sapiens KIAA0614 8406 100
protein
56 1464 AE003453 Drosophila 654 51
melanogaster CG10509
gene product
57 1584 AB033076 Homo sapiens KIAA1250 6286 100
protein
58 1617 AB033067 Homo sapiens KIAA1241 4229 99
protein
59 1724 D88585 Chlorocebus aethiops 401 36
hepatitis A virus
receptor
60 1728 AF208845 Homo sapiens BM-003 1375 99
61 1772 AB015427 Homo sapiens zinc 3934 100
finger protein 219
62 1809 X57821 Homo sapiens 797 76
immunoglobulin lambda
light chain
63 1868 AF043695 Caenorhabditis elegans 555 43
Similar to
mitochondrial carrier
protein
64 1898 AB033039 Homo sapiens KIAA1213 2438 100
protein
65 1926 AK000279 Homo sapiens unnamed 3271 99
protein product
66 1965 AF178432 Homo sapiens SH3 3700 100
protein
67 1967 AB033099 Homo sapiens KIAA1273 3082 99
protein
68 1995 AF181721 Homo sapiens RU2S 2254 100
69 2005 AL133093 Homo sapiens 2241 100
hypothetical protein
70 2027 U48238 Mus musculus zinc 749 63
finger protein neuro-
d4
71 2055 AL133105 Homo sapiens 1783 99
hypothetical protein
72 2103 AB032958 Homo sapiens KIAA1132 9116 100
protein
73 2106 AE003528 Drosophila 472 25
melanogaster CG5018
gene product
74 2166 AK001713 Homo sapiens unnamed 5323 99
protein product
75 2175 AB010266 Mus musculus tenascin-X 10246 64
76 2176 AE003746 Drosophila 363 40
melanogaster CG5986
gene product
77 2194 AL163206 Homo sapiens protein 1944 100
with homology to
KIAA0790
78 2236 AB033020 Homo sapiens KIAA1194 2918 99
protein
79 2250 AL122081 Homo sapiens 1930 100
hypothetical protein
80 2300 AL133572 Homo sapiens 3303 100
hypothetical protein
81 2323 AB033107 Homo sapiens KIAA1281 3228 100
protein
82 2340 AB030183 Mus musculus contains 380 42
transmembrane (TM)
region
83 2371 Z18361 Ovis aries 184 82
trichohyalin
84 2399 AJ010045 Mus musculus guanine 1470 58
nucleotide-exchange
factor
85 2411 AF176529 Mus musculus F-box 2072 94
protein FBX13
86 2428 AF210842 Homo sapiens HARP 4880 100
87 2430 AL031658 Homo sapiens 776 98
dJ310013.7 (novel
protein similar to H. roretzi
HRPET-3)
88 2439 X57398 Homo sapiens pm5 6131 99
protein
89 2447 AE003779 Drosophila 1670 62
melanogaster CG2118
gene product
90 2461 AL122097 Homo sapiens 3213 99
hypothetical protein
91 2487 AE003801 Drosophila 247 38
melanogaster CG14490
gene product
92 2492 AB033072 Homo sapiens KIAA1246 4087 99
protein
93 2512 AB033103 Homo sapiens KIAA1277 5252 99
protein
94 2564 AF117946 Homo sapiens Link 2363 100
guanine nucleotide
exchange factor II
95 2678 AL133087 Homo sapiens 4159 99
hypothetical protein
96 2816 AK001529 Homo sapiens unnamed 1420 99
protein product
97 2818 AL137530 Homo sapiens 433 94
hypothetical protein
98 2819 AB028942 Homo sapiens KIAA1019 7437 98
protein
99 2943 AF189722 Homo sapiens PDZ- 1688 99
binding kinase
100 3137 AE003450 Drosophila 681 48
melanogaster CG2221
gene product
101 3137 AE003450 Drosophila 716 38
melanogaster CG2221
gene product
102 3160 AK000708 Homo sapiens unnamed 1103 99
protein product
103 3323 Y07829 Homo sapiens RING 2201 99
finger protein
104 3360 AB007931 Homo sapiens KIAA0462 11741 99
protein
105 3362 U41387 Homo sapiens Gu 4021 99
protein
106 3417 AF023674 Homo sapiens 3783 100
nephrocystin
107 3418 AF146760 Homo sapiens septin 2- 2284 100
like cell division
control protein
108 3442 Z66524 Caenorhabditis elegans 1190 48
Homology with Squid
retinal-binding
protein (PIR Acc. No.
A53057)˜cDNA EST
yk463d10.3 comes from
this gene˜cDNA EST
yk663h12.3 comes from
this gene
109 3442 Z66524 Caenorhabditis elegans 848 42
Homology with Squid
retinal-binding
protein (PIR Acc. No.
A53057)˜cDNA EST
yk463d10.3 comes from
this gene˜cDNA EST
yk663h12.3 comes from
this gene
110 3444 M26576 Homo sapiens alpha-1 9412 99
type IV collagen
111 3855 AF113536 Homo sapiens MO25 1381 81
protein
112 3863 AJ271385 Homo sapiens UDP-N- 733 46
acetyl-alpha-D-
galactosamine:polypeptide
N-
acetylgalactosaminyltransferase 8
113 4090 AF105228 Bos taurus tuftelin 285 33
114 4105 U32614 Mus musculus SOX6 2855 96
115 4142 X14971 Mus musculus alpha- 4897 98
adaptin (A) (AA 1-977)
116 4142 X53773 Rattus norvegicus 3979 82
alpha-c large chain
(AA 1-938)
117 4149 AF034746 Mus musculus LNXp70 2922 88
118 4196 AC006551 Arabidopsis thaliana 214 34
Hypothetical protein
119 4202 AF229032 Mus musculus piL 2077 93
120 4274 AF056035 Rattus norvegicus s- 2662 85
nexilin
121 4304 AK000080 Homo sapiens unnamed 3037 99
protein product
122 4306 D88158 Sus scrofa cytochrome 474 47
b561
123 4311 AF161445 Homo sapiens HSPC327 1606 100
124 4321 AL133112 Homo sapiens 1861 100
hypothetical protein
125 4323 AL137432 Homo sapiens 3002 100
hypothetical protein
126 4332 AF186461 Rattus norvegicus ring 204 22
finger protein Fxy
127 4488 AE003749 Drosophila 422 33
melanogaster CG13644
gene product
128 4588 D87438 Homo sapiens Similar 4069 100
to a C. elegans protein
in cosmid C14H10
129 5569 D87442 Homo sapiens KIAA0253 3682 100
130 5573 Z15005 Homo sapiens CENP-E 13305 99
131 5577 M59216 Homo sapiens gamma- 2477 100
aminobutyric acid
receptor beta-1
subunit
132 5579 D31884 Homo sapiens KIAA0063 518 55
133 5582 AF188706 Homo sapiens g20 188 49
protein
134 3583 AB029030 Homo sapiens KIAA1107 6581 99
protein
135 5584 D87446 Homo sapiens Similar 9196 99
to a C. elegans protein
encoded in cosmid
C27F2 (U40419)
136 5585 AF047663 Caenorhabditis elegans 225 37
W09G12.7 gene product
137 5591 AC002398 Homo sapiens F25965_1 1018 100
138 5593 AB023215 Homo sapiens KIAA0998 6323 99
protein
139 5594 AF223408 Homo sapiens B99 3686 99
140 5594 AF223408 Homo sapiens B99 2878 88
141 5598 D83781 Homo sapiens the 6859 99
KIAA0197 gene is
expressed
ubiquitously.; the
KIAA0197 protein has
histidine acid
phosphatase signature
at amino acid
positions 1047-1061.
142 5602 U53450 Rattus norvegicus Jun 196 49
dimerization protein 1
JDP-1
143 5605 AL117233 Homo sapiens 3564 99
hypothetical protein
144 5608 U38253 Rattus norvegicus 1203 89
initiation factor eIF-
2B gamma subunit
145 5617 AE003538 Drosophila 354 44
melanogaster CG10191
gene product
146 5620 AB020694 Homo sapiens KIAA0887 2328 100
protein
147 5622 AB029025 Homo sapiens KIAA1102 4394 100
protein
148 5623 AL137255 Homo sapiens 2636 100
hypothetical protein
149 5624 AB018289 Homo sapiens KIAA0746 5223 99
protein
150 5625 D38549 Homo sapiens ha1025 is 6533 99
new
151 5627 AF241230 Homo sapiens TAK1- 3656 100
binding protein 2
152 5628 AK000759 Homo sapiens unnamed 3306 100
protein product
153 5630 AL117665 Homo sapiens 6463 100
hypothetical protein
154 5632 AF161544 Homo sapiens HSPCO59 434 77
155 5640 AJ238248 Homo sapiens centaurin 3986 99
beta2
156 5641 AB007929 Homo sapiens KIAA0460 4781 99
protein
157 5643 AF161381 Homo sapiens HSPC263 1404 100
158 5647 AF223468 Homo sapiens AD021 1314 100
protein
159 5649 AF203343 Mus musculus RIBP 115 39
160 5658 X57527 Homo sapiens alpha 4166 99
1(VIII) collagen
161 5659 Y19062 Homo sapiens 39k3 2475 100
protein
162 5667 AK000566 Homo sapiens unnamed 1053 100
protein product
163 5672 AL021918 Homo sapiens b34I8.1 4184 100
(Kruppel related Zinc
Finger protein 184)
164 5674 AB020706 Homo sapiens KIAA0899 4732 100
protein
165 5678 AB040915 Homo sapiens KIAA1482 2828 99
protein
166 5680 AE001448 Helicobacter pylori 698 37
J99 THREONINE SYNTHASE
167 5684 AF226614 Homo sapiens 2929 100
ferroportin1
168 5686 Z93241 Homo sapiens 513 96
dJ222E13.1 (novel
protein with some
similarity to
Drosophila KRAKEN)
169 5694 AF036977 Homo sapiens unknown 1812 100
170 5698 AK001746 Homo sapiens unnamed 141 45
protein product
171 5699 AF108843 Homo sapiens env 320 47
protein
172 5712 AF069781 Drosophila 653 43
melanogaster Bem46-
like protein
173 5719 U95098 Xenopus laevis mitotic 1200 70
phosphoprotein 44
174 5720 X70944 Homo sapiens PTB- 3883 100
associated splicing
factor
175 5727 AE003741 Drosophila 456 44
melanogaster CG13832
gene product
176 5730 AF195833 Mus musculus cell 2693 93
adhesion molecule
nectin-3 alpha
177 5734 AJ249732 Homo sapiens G8 669 100
protein
178 5738 AF208861 Homo sapiens BM-019 1629 100
179 5739 L09708 Homo sapiens 4022 100
complement component
C2
180 5740 AF156961 Homo sapiens gag 106 47
181 5744 X66285 Mus musculus HC1 ORF 115 44
182 5748 D00189 Rattus norvegicus 5227 99
Na+, K+-ATPase alpha-
subunit
183 5749 U10185 Xenopus laevis XPMC2 1020 53
protein
184 5750 AB019038 Homo sapiens beta-1,4 781 77
mannosyltransferase
185 5750 AB019038 Homo sapiens beta-1,4 1347 100
mannosyltransferase
186 5750 AB019038 Homo sapiens beta-1,4 1520 99
mannosyltransferase
187 5761 X84908 Homo sapiens 5729 99
phosphorylase kinase
188 5762 X52851 Homo sapiens 650 76
peptidylprolyl
isomerase
189 5767 AJ245671 Homo sapiens 3064 100
hypothetical protein
190 5773 AC004447 Homo sapiens KIAA0365 4963 99
191 5783 U04706 Bos taurus 50 kDa 1749 78
protein
192 5784 AF092207 Rattus norvegicus 1180 84
unknown
193 5788 AK001934 Homo sapiens unnamed 1368 100
protein product
194 5798 AK000284 Homo sapiens unnamed 3385 97
protein product
195 5802 AF247042 Homo sapiens tandem 2186 99
pore domain potassium
channel TRAAK
196 5807 AF114494 Homo sapiens putative 1284 99
tyrosine phosphatase
197 5818 AE000995 Archaeoglobus fulgidus 153 20
chromosome segregation
protein (smc1)
198 5819 AF062249 Homo sapiens 605 97
immunoglobulin heavy
chain variable region
199 5827 AJ223830 Rattus norvegicus ARE1 2950 98
200 5828 AL133027 Homo sapiens 1224 84
hypothetical protein
201 5842 D87684 Homo sapiens KIAA0242 2566 100
protein
202 5853 AL050318 Homo sapiens 524 79
dJ977B1.3.1 (novel
protein similar to
putative RAB5-
interacting protein
(isoform 1))
203 5861 D49387 Homo sapiens NADP 1616 100
dependent leukotriene
b4 12-
hydroxydehydrogenase
204 5864 AL050022 Homo sapiens 330 34
hypothetical protein
205 5865 AL050267 Homo sapiens 3325 99
hypothetical protein
206 5871 AL137300 Homo sapiens 1056 98
hypothetical protein
207 5873 AK001480 Homo sapiens unnamed 1562 99
protein product
208 5873 AK001480 Homo sapiens unnamed 1082 98
protein product
209 5875 X12966 Homo sapiens 3- 1972 100
oxoacyl-CoA thiolase
propeptide (424 AA)
210 5878 Y09267 Homo sapiens flavin- 2486 100
containing
monooxygenase 2
211 5879 Z11773 Homo sapiens SRE-ZBP 2201 99
212 5880 D84224 Homo sapiens methionyl 4741 99
tRNA synthetase
213 5880 D84224 Homo sapiens methionyl 3887 99
tRNA synthetase
214 5880 D84224 Homo sapiens methionyl 2933 96
tRNA synthetase
215 5880 D84224 Homo sapiens methionyl 4529 99
tRNA synthetase
216 5885 J03244 Bos taurus H+ ATPase 848 77
31 kDa subunit (EC
3.6.1.3)
217 5895 AK001589 Homo sapiens unnamed 2313 100
protein product
218 5898 AL117615 Homo sapiens 3174 99
hypothetical protein
219 5902 AE003735 Drosophila 436 58
melanogaster CG6353
gene product
220 5904 A06669 synthetic construct 2070 99
preTGF-betal
221 5918 AE003487 Drosophila 238 26
melanogaster CG1905
gene product
222 5921 AL110243 Homo sapiens 2275 100
hypothetical protein
223 5927 X60271 Mus musculus c-rel 2264 74
224 5932 AK001475 Homo sapiens unnamed 3025 100
protein product
225 5939 AF131851 Homo sapiens Unknown 262 44
226 5945 AB002320 Homo sapiens KIAA0322 8183 100
227 5946 AE003518 Drosophila 135 22
melanogaster CG6836
gene product
228 5947 AF119855 Homo sapiens PRO1847 265 67
229 5956 M17236 Homo sapiens MHC HLA- 1332 100
DQ alpha precursor
230 5967 AK001345 Homo sapiens unnamed 1453 99
protein product
231 5968 M28515 Mus musculus zinc 225 28
finger protein mfg3
mRNA (put.); putative
232 5975 AB037730 Homo sapiens KIAA1309 515 44
protein
233 5977 AE003464 Drosophila 610 44
melanogaster CG11414
gene product
234 5978 M12140 Homo sapiens pol gene 117 50
protein; Xxx
235 5979 U79267 Homo sapiens unknown 225 56
236 5980 X56681 Homo sapiens junD 373 88
protein
237 5988 AB023151 Homo sapiens KIAA0934 7099 100
protein
238 5989 AL109839 Homo sapiens 877 100
dJ1069P2.3.1 (novel
PABPC1 (poly(A) —
binding protein,
cytoplasmic 1)
(PABPL1) like protein
(putative isoform 1))
239 5991 AE003583 Drosophila 289 42
melanogaster
BcDNA: GH09817 gene
product
240 5997 AF052723 Feline leukemia virus 1547 44
gag-pol precursor
polyprotein gPr80
241 5998 AF161472 Homo sapiens HSPC123 439 45
242 6003 AK000360 Homo sapiens unnamed 796 100
protein product
243 6004 U09848 Homo sapiens zinc 1738 100
finger protein
244 6013 U19177 Homo sapiens Hin-2 55 46
245 6028 AF155113 Homo sapiens NY-REN-55 3603 93
antigen
246 6028 AF155113 Homo sapiens NY-REN-55 3951 99
antigen
247 6029 AL032821 Homo sapiens dJ55C23.1 1821 98
(vanin 1)
248 6031 M69181 Homo sapiens non- 7350 99
muscle myosin B
249 6031 M69181 Homo sapiens non- 7311 98
muscle myosin B
250 6032 X61280 Oryza sativa 143 38
hydroxyproline-rich
glycoprotein
251 6037 AB002330 Homo sapiens KIAA0332 5362 100
252 6037 AB002330 Homo sapiens KIAA0332 4897 97
253 6043 X06745 Homo sapiens DNA 7619 99
polymerase alpha-
subunit (AA 1-1462)
254 6044 AF252292 Homo sapiens PAR6C 1342 100
255 6046 D86984 Homo sapiens similar 2446 100
to yeast adenylate
cyclase (S56776)
256 6048 AF165124 Homo sapiens gamma- 2499 99
aminobutyric acid A
receptor gamma 2
257 6049 AF110267 Rattus norvegicus 2088 89
golgi stacking protein
homolog GRASP55
258 6051 U82319 Homo sapiens novel ORF 342 100
259 6053 Y00816 Homo sapiens CR1 11396 99
precursor protein
260 6060 AJ223948 Homo sapiens RNA 6608 99
helicase
261 6063 Y08612 Homo sapiens 88 kDa 3874 99
nuclear pore complex
protein
262 6066 AB014597 Homo sapiens KIAA0697 5060 100
protein
263 6067 AF129756 Homo sapiens BAT4 1873 98
264 6068 AF131775 Homo sapiens Unknown 1929 99
265 6073 AJ250865 Homo sapiens TESS 2 2348 100
266 6076 Z98885 Homo sapiens dJ522J7.2 5588 100
(bromodomain-
containing 1 (similar
to peregrin, BR140))
267 6076 Z98885 Homo sapiens dJ522J7.2 4167 100
(bromodomain-
containing 1 (similar
to peregrin, BR140))
268 6077 L76571 Homo sapiens nuclear 1355 100
hormone receptor
269 6079 AF091622 Homo sapiens PHD 9054 100
finger protein 3
270 6082 X56807 Homo sapiens 4443 100
desmocollin type 2a
271 6087 AC002464 Homo sapiens organic 1542 99
cation transporter;
50% similarity to
JC4884 (PID: g2143892)
272 6088 AL050272 Homo sapiens 697 99
hypothetical protein
273 6091 AL022329 Homo sapiens 3653 100
bK407F11.2
(adrenergic, beta,
receptor kinase 2)
274 6094 AK000833 Homo sapiens unnamed 2001 98
protein product
275 6101 AJ245600 Homo sapiens 2616 99
hypothetical protein
276 6103 AB041810 Mus musculus unnamed 1468 91
protein product
277 6104 L36531 Homo sapiens integrin 5386 99
alpha 8 subunit
278 6108 AL117646 Homo sapiens 1491 100
hypothetical protein
279 6112 AF218584 Homo sapiens GGA1 3265 100
280 6121 Y13115 Homo sapiens 5071 99
serine/threonine
protein kinase
281 6125 AB018319 Homo sapiens KIAA0776 3960 99
protein
282 6126 AL034452 Homo sapiens 1979 100
dJ682J15.1 (novel
Collagen triple helix
repeat containing
protein)
283 6128 Y14494 Homo sapiens aralar1 3465 99
284 6129 AJ001981 Homo sapiens OXA1L 2603 100
285 6133 A58799 unidentified unnamed 3069 100
protein product
286 6133 A58799 unidentified unnamed 2464 100
protein product
287 6135 AF163572 Homo sapiens Forssman 1865 99
glycolipid synthetase
288 6139 AF161503 Homo sapiens HSPC154 1261 97
289 6141 AB011125 Homo sapiens KIAA0553 5754 100
protein
290 6145 AJ250014 Homo sapiens Familial 3655 99
Cylindromatosis Gene
291 6146 D25217 Homo sapiens KIAA0027 361 94
protein
292 6148 X85786 Homo sapiens binding 3203 100
regulatory factor
293 6149 Y08319 Homo sapiens kinesin-2 3487 99
294 6149 D12644 Mus musculus KIF2 3609 97
protein
295 6153 U28789 Mus musculus PACT 5936 89
296 6159 AL137515 Homo sapiens 1687 100
hypothetical protein
297 6164 AB020705 Homo sapiens KIAA0898 5017 100
protein
298 6167 Y00062 Homo sapiens precursor 3440 99
polypeptide (AA −23 to
1120)
299 6172 AB007941 Homo sapiens KIAA0472 1925 99
protein
300 6173 X98248 Homo sapiens sortilin 4403 99
301 6190 X61100 Homo sapiens 75 kDa 3734 99
subunit NADH
dehydrogenase
precursor
302 6194 S58544 Homo sapiens 75 kda 2125 99
infertility-related
sperm protein
303 6196 AL110265 Homo sapiens 744 100
hypothetical protein
304 6197 X14968 Homo sapiens RII-alpha 2079 100
subunit (AA 1-404)
305 6198 AL050283 Homo sapiens 1983 100
hypothetical protein
306 6198 AL050283 Homo sapiens 1694 100
hypothetical protein
307 6205 AJ011863 Homo sapiens homeobox 3841 99
protein LSX
308 6214 AF098786 Homo sapiens 17 beta- 1754 100
hydroxysteroid
dehydrogenase type VII
309 6215 AL034555 Homo sapiens 4273 100
dJ134O19.3 (zinc
finger protein 151
(pHZ-67))
310 6219 AB011167 Homo sapiens KIAA0595 7678 98
protein
311 6226 U39205 Saccharomyces 277 29
cerevisiae Lpe10p
312 6229 AF041429 Homo sapiens pRGR1 823 99
313 6234 X66357 Homo sapiens 1589 100
serine/threonine
protein kinase
314 6237 Y11284 Homo sapiens AFX1 2571 98
315 6238 AB004884 Homo sapiens PKU-alpha 3718 99
316 6239 AJ002303 Homo sapiens 1020 100
synaptogyrin 1c
317 6239 AJ002304 Homo sapiens 1002 100
synaptogyrin 1b
318 6239 AJ002303 Homo sapiens 933 94
synaptogyrin 1c
319 6240 D87682 Homo sapiens similar 2676 100
to a C. elegans protein
encoded in cosmid
T26A5.
320 6244 M14660 Homo sapiens ISG-K54 2473 99
321 6245 X06661 Homo sapiens calbindin 1358 100
(AA 1-261)
322 6250 AF119900 Homo sapiens PRO2822 185 76
323 6252 AB014527 Homo sapiens KIAA0627 6478 99
protein
324 6252 AB014527 Homo sapiens KIAA0627 6372 98
protein
325 6256 X86691 Homo sapiens Mi-2 10110 99
protein
326 6260 AE003628 Drosophila 985 57
melanogaster CG7475
gene product
327 6261 AF236061 Oryctolagus cuniculus 3830 91
RING-finger binding
protein
328 6264 AB018327 Homo sapiens KIAA0784 5708 100
protein
329 6265 AB018314 Homo sapiens KIAA0771 4949 100
protein
330 6266 AB002318 Homo sapiens KIAA0320 4639 99
331 6270 X14766 Homo sapiens GABA-A 2388 99
receptor alpha 1
subunit
332 6271 AB023177 Homo sapiens KIAA0960 7294 99
protein
333 6272 AB032957 Homo sapiens KIAA1131 8443 100
protein
334 6274 AF007155 Homo sapiens unknown 187 61
335 6276 Z34975 Homo sapiens ldlCp 3733 100
336 6281 AL050306 Homo sapiens dJ475B7.2 3796 100
(novel protein)
337 6281 AL050306 Homo sapiens dJ475B7.2 1942 99
(novel protein)
338 6288 AB014566 Homo sapiens KIAA0666 5541 100
protein
339 6292 AB018353 Homo sapiens KIAA0810 4246 100
protein
340 6294 Z21966 Homo sapiens mPOU 1529 100
homeobox protein
341 6299 AL022395 Homo sapiens 3287 100
dJ273N12.1 (PUTATIVE
protein based on EST
matches)
342 6299 AL022395 Homo sapiens 2403 83
dJ273N12.1 (PUTATIVE
protein based on EST
matches)
343 6312 AL096713 Homo sapiens 7599 99
hypothetical protein
344 6312 AF182316 Homo sapiens myoferlin 6232 99
345 6312 AL096713 Homo sapiens 6120 99
hypothetical protein
346 6322 AK000218 Homo sapiens unnamed 1163 99
protein product
347 6324 D42046 Homo sapiens The 5568 100
ha3631 gene product is
related to
S. cerevisiae protein
encoded in chromosome
VIII.
348 6328 AB023624 Rattus norvegicus SCOP 4792 92
349 6329 X59303 Homo sapiens valyl- 3393 99
tRNA synthetase
350 6331 AC004142 Homo sapiens similar 3676 100
to murine leucine-rich
repeat protein;
possible role in
neural development by
protein-protein
interactions; 93%
similarity to D49802
(PID: g1369906)
351 6333 AC009991 Arabidopsis thaliana 609 51
unknown protein
352 6334 AB018271 Homo sapiens KIAA0728 4316 98
protein
353 6337 AB002318 Homo sapiens KIAA0320 4639 99
354 6339 AB039371 Homo sapiens 2902 99
mitochondrial ABC
transporter 3
355 6346 AK002198 Homo sapiens unnamed 2570 99
protein product
356 6348 AB033087 Homo sapiens KIAA1261 4094 99
protein
357 6348 L14463 Rattus norvegicus 3619 92
‘transducin
358 6350 AC005757 Homo sapiens R32611_1 2779 100
359 6351 S61069 Homo sapiens reverse 252 66
transcriptase
homolog = pol
{retroviral element}
360 6355 AF271388 Homo sapiens CMP-N- 2273 100
acetylneuraminic acid
synthase
361 6362 X79066 Homo sapiens ERF-1 1783 100
362 6368 AF118566 Mus musculus 769 51
hematopoietic zinc
finger protein
363 6369 AB020710 Homo sapiens KIAA0903 4915 99
protein
364 6371 AF143321 Homo sapiens unknown 661 65
365 6376 AF260011 Homo sapiens HSPC087- 8764 99
KIAA0714
366 6379 S83365 Homo sapiens putative 131 49
Rab5-interacting
protein {clone L1-94}
367 6380 AL021878 Homo sapiens 154 68
dJ257I20.4
(transcription factor
20 (AR1) (KIAA0292)
(isoform 2))
368 6381 D90734 Escherichia coli 628 100
ORF_ID: o223#11
369 6392 M58378 Homo sapiens synapsin I 3730 99
370 6395 AF039697 Homo sapiens antigen 508 98
NY-CO-31
371 6397 U09355 Oryctolagus cuniculus 2356 99
protein phosphatase
2A1 B gamma subunit
372 6400 AB002293 Homo sapiens KIAA0295 5054 100
373 6401 AC004774 Homo sapiens Dlx-5 1542 100
374 6411 X90530 Homo sapiens ragB 1926 99
375 6411 X90530 Homo sapiens ragB 1405 99
376 6411 X90530 Homo sapiens ragB 1590 85
377 6416 AL080157 Homo sapiens 2100 94
hypothetical protein
378 6418 AE003628 Drosophila 659 49
melanogaster CG5188
gene product
379 6422 AB007884 Homo sapiens KIAA0424 2757 99
380 6423 AB018323 Homo sapiens KIAA0780 5631 100
protein
381 6426 AF042713 Rattus norvegicus 1337 96
neurexophilin 3
382 6427 AJ131891 Homo sapiens DNA 1451 100
polymerase mu
383 6428 AF221712 Homo sapiens Smad- and 6705 100
Olf-interacting zinc
finger protein
384 6429 X83573 Homo sapiens ARSE 3184 99
385 6430 AJ243274 Homo sapiens AP-2rep 2078 99
protein
386 6432 AL035608 Homo sapiens dJ479J7.1 1440 100
(similar to
CHONDROMODULIN-1)
387 6432 AL035608 Homo sapiens dJ479J7.1 1316 93
(similar to
CHONDROMODULIN-1)
388 6438 AK001444 Homo sapiens unnamed 943 100
protein product
389 6441 AL022237 Homo sapiens 2030 100
bK1191B2.3 (PUTATIVE
novel Acyl Transferase
similar to C. elegans
C50D2.7) (isoform 1))
390 6446 AJ006266 Homo sapiens AND-1 5942 100
protein
391 6454 AL110240 Homo sapiens 704 98
hypothetical protein
392 6459 AL050149 Homo sapiens 2899 100
hypothetical protein
393 6460 AL096772 Homo sapiens 7049 99
dJ365O12.1 (KIAA0758
protein)
394 6461 AB008376 Sus scrofa 17-kDa PKC- 689 91
potentiated inhibitory
protein of PP1
395 6467 M22334 Homo sapiens unknown 796 59
protein
396 6468 AK002144 Homo sapiens unnamed 2719 100
protein product
397 6487 AL117429 Homo sapiens 1077 100
hypothetical protein
398 6491 AB027004 Homo sapiens protein 435 48
phosphatase
399 6506 AL137013 Homo sapiens bA311P8.3 862 100
(probable uracil
phosphoribosyltranferase)
400 6513 AL080141 Homo sapiens 4793 99
hypothetical protein
401 6514 AB035123 Mus musculus GD1 1696 93
alpha/GT1a alpha/GQ1b
alpha synthase
402 6519 K02882 Homo sapiens 2048 100
immunoglobulin delta-
chain
403 6521 X07979 Homo sapiens integrin 4347 99
beta 1 subunit
precursor
404 6532 AJ224819 Homo sapiens tumor 2149 99
suppressor
405 6536 Y07595 Homo sapiens 2373 100
transcription factor
TFIIH
406 6543 D14479 Rattus norvegicus 1428 88
calpain
407 6544 AF161341 Homo sapiens HSPC078 1097 98
408 6548 AF187318 Homo sapiens F-box 1607 100
protein Fbx2
409 6551 AL050369 Homo sapiens 2495 99
hypothetical protein
410 6551 AL050369 Homo sapiens 2135 99
hypothetical protein
411 6552 AE003785 Drosophila 1211 56
melanogaster CG12792
gene product
412 6554 AF091083 Homo sapiens unknown 1514 100
413 6556 AK001708 Homo sapiens unnamed 2334 99
protein product
414 6560 AE003602 Drosophila 462 38
melanogaster CG2109
gene product
415 6563 AB011139 Homo sapiens KIAA0567 4966 99
protein
416 6564 AK001177 Homo sapiens unnamed 1933 100
protein product
417 6567 D63484 Homo sapiens The 4951 99
KIAA0150 gene product
is novel.
418 6573 AB029012 Homo sapiens KIAA1089 5128 100
protein
419 6575 AL035461 Homo sapiens 1562 98
dJ967N21.6 (novel CDP-
alcohol
phosphatidyltransferase
family member
protein)
420 6577 AK001236 Homo sapiens unnamed 1676 99
protein product
421 6593 AF079098 Homo sapiens arginine- 2733 99
tRNA-protein
transferase 1-1p;
ATE1-1p
422 6595 AJ131712 Homo sapiens nucleolar 2793 100
RNA-helicase
423 6599 AJ133115 Homo sapiens TSC-22- 2054 99
like protein
424 6625 X98258 Homo sapiens M-phase 953 100
phosphoprotein 9
425 6625 X98258 Homo sapiens M-phase 564 75
phosphoprotein 9
426 6626 U97191 Caenorhabditis elegans 960 85
strong similarity to
the YPT1 sub-family of
RAS proteins
427 6630 X76057 Homo sapiens 2191 100
phosphomannose
isomerase
428 6631 AE003559 Drosophila 650 31
melanogaster CG8605
gene product
429 6632 X97064 Homo sapiens Sec23 4034 99
protein
430 6633 AF161401 Homo sapiens HSPC283 779 100
431 6634 AJ005642 Rattus rattus serine 717 48
protease
432 6638 M19529 Sus scrofa follistatin A 1906 98
433 6641 AJ249457 Trichomonas vaginalis 183 28
centrin, putative
434 6644 AC004410 Homo sapiens 2094 100
fos39554_1
435 6646 AK000096 Homo sapiens unnamed 2157 99
protein product
436 6648 AF252284 Homo sapiens 4005 100
transcription
specificity factor Sp1
437 6652 Z92825 Caenorhabditis elegans 541 43
predicted using
Genefinder˜cDNA EST
yk315e12.3 comes from
this gene˜cDNA EST
yk315e12.5 comes from
this gene˜cDNA EST
yk605b12.3 comes from
this gene
438 6654 D79205 Homo sapiens ribosomal 160 77
protein L39
439 6657 AL031027 Unknown/ 584 58
prediction = (method: “”
genefinder“”,
version: ““084””,
score: ““67.72””)˜/prediction = (method
440 6658 S49657 Mus sp. mitochondrial 91 35
capsule selenoprotein;
MCS
441 6663 M26312 Oryctolagus cuniculus 82 30
unknown protein
442 6664 L32162 Homo sapiens 574 80
transcription factor
443 6668 AL050060 Homo sapiens 526 99
hypothetical protein
444 6669 AF205936 Mus musculus ADP- 296 39
ribosylation factor-
like membrane-
associated protein
445 6673 AK000387 Homo sapiens unnamed 1136 100
protein product
446 6685 U38934 Gallus gallus histone 625 97
H2A
447 6687 U76374 Mus musculus skm-BOP2 602 31
448 6689 X13403 Homo sapiens Oct-1 3626 100
protein (AA 1-743)
449 6693 AB023139 Homo sapiens KIAA0922 4258 100
protein
450 6698 AE003467 Drosophila 274 27
melanogaster CG7047
gene product
451 6699 AL049176 Homo sapiens dA141H5.1 1401 99
(C-terminal part of a
Chordin LIKE protein
with von Willebrand
factor type C domains)
452 6705 X92475 Homo sapiens ITBA1 1429 100
453 6711 Y16752 Homo sapiens 1422 99
secretagogin
454 6713 X51416 Homo sapiens hormone 2641 97
receptor hERR1 (AA 1-521)
455 6716 AJ006591 Homo sapiens cysteine- 1793 100
rich protein
456 6725 A08695 Homo sapiens rap2 935 100
457 6726 Z12173 Homo sapiens N- 2970 100
acetylglucosamine-6-
sulphatase
458 6727 AL355092 Homo sapiens 924 98
hypothetical protein
459 6730 AB007930 Homo sapiens KIAA0461 7164 100
perotein
460 6730 AB007930 Homo sapiens KIAA0461 6960 99
perotein
461 6730 AB007930 Homo sapiens KIAA0461 6018 89
perotein
462 6732 D38491 Homo sapiens KIAA0117 1119 99
463 6733 AJ012590 Homo sapiens glucose 4155 99
1-dehydrogenase
464 6737 AL080133 Homo sapiens 5677 100
hypothetical protein
465 6745 Z75532 Caenorhabditis elegans 220 35
similar to
mitochrondrial carrier
protein˜cDNA EST
yk264h5.5 comes from
this gene
466 6751 AF207829 Homo sapiens SCAN- 900 100
related protein RAZ1
467 6754 AF061262 Mus musculus semaF 1316 83
cytoplasmic domain
associated protein 2
468 6758 AF220189 Homo sapiens 605 89
uncharacterized
hypothalamus protein
HBEX2
469 6761 AL079292 Homo sapiens 4135 100
hypothetical protein,
similar to (AC007017)
putative RNA helicase A
470 6765 Z22819 Mus musculus Rab24 1042 98
protein
471 6768 Z97029 Homo sapiens 1548 99
ribonuclease HI large
subunit
472 6773 AB025384 Homo sapiens SRp25 962 94
nuclear protein
473 6776 AF024631 Homo sapiens ANG2 2644 100
474 6796 AJ006710 Rattus norvegicus 4508 97
phosphatidylinositol
3-kinase
475 6798 AL137275 Homo sapiens 4310 100
hypothetical protein
476 6823 V00638 bacteriophage lambda 600 100
reading frame ea10
477 6825 AF049103 Homo sapiens 819 100
Huntingtin interacting
protein
478 6826 U50312 Caenorhabditis elegans 92 40
strong similarity to
the a portion of the
triple-helical region
of collagen alpha
chain
479 6839 Z26317 Homo sapiens 4810 99
desmoglein 2
480 6844 AF227899 Homo sapiens breast 4443 99
carcinoma-associated
antigen isoform I
481 6847 AF106037 Homo sapiens 4905 99
adipocyte-derived
leucine aminopeptidase
482 6849 U15155 Gallus gallus 372 37
trypsinogen
483 6854 D86974 Homo sapiens KIAA0220 2870 99
484 6857 AF112201 Homo sapiens neuronal 1053 100
protein NP25
485 6861 AF234765 Rattus norvegicus 958 64
serine-arginine-rich
splicing regulatory
protein SRRP86
486 6873 AF117383 Homo sapiens placental 502 68
protein 13; PP13
487 6875 AK002059 Homo sapiens unnamed 1665 100
protein product
488 6877 AE003438 Drosophila 338 43
melanogaster CG3184
gene product
489 6880 AK000101 Homo sapiens unnamed 814 100
protein product
490 6885 AK000609 Homo sapiens unnamed 1160 100
protein product
491 6890 AB023201 Homo sapiens KIAA0984 3743 98
protein
492 6890 AB023201 Homo sapiens KIAA0984 2361 97
protein
493 6894 AB013885 Homo sapiens beta- 1494 100
ureidopropionase
494 6901 AL096725 Homo sapiens 1901 100
hypothetical protein
495 6904 AK001901 Homo sapiens unnamed 2212 99
protein product
496 6907 AF226077 Homo sapiens CHRAC17 724 99
497 6914 AE003762 Drosophila 646 75
melanogaster CG5590
gene product
498 6917 Z73497 Homo sapiens cU240C2.2 324 100
(Core histone
H2A/H2B/H3/H4)
499 6923 Z83246 Caenorhabditis elegans 891 60
predicted using
Genefinder˜cDNA EST
EMBL: M79771 comes from
this gene
500 6929 X16282 Homo sapiens zinc 1109 99
finger protein (217
AA) (1 is 2nd base in
codon)
501 6931 Z92539 Mycobacterium 300 36
tuberculosis pth
502 6935 M62324 Homo sapiens modulator 2902 96
recognition factor I
503 6940 AC024762 Caenorhabditis elegans 434 43
Hypothetical protein
Y38F2AL.f
504 6945 AL117555 Homo sapiens 321 94
hypothetical protein
505 6946 AC005328 Homo sapiens R26660_2, 865 97
partial CDS
506 6947 AF151075 Homo sapiens HSPC241 686 98
507 6949 L34807 Musca domestica 174 21
transposase
508 6959 AJ271091 Homo sapiens B-ind1 494 42
protein
509 6960 AK001348 Homo sapiens unnamed 1853 99
protein product
510 6962 AJ006692 Homo sapiens ultra 693 74
high sulfer keratin
511 6963 U23037 Oryctolagus cuniculus 3406 90
eIF-2Bepsilon
512 6967 AL136571 Homo sapiens 413 58
hypothetical protein
513 6983 AF151800 Homo sapiens CGI-41 84 35
protein
514 6988 AF198100 Fowlpox virus ORF 567 54
FPV114 HAL3 domain
515 6996 AL137764 Homo sapiens 2162 100
hypothetical protein
516 7003 AB011792 Homo sapiens 274 35
extracellular matrix
protein
517 7016 AB011542 Homo sapiens MEGF9 2097 100
518 7017 AL096744 Homo sapiens 231 68
hypothetical protein
519 7025 AF119664 Homo sapiens 1574 100
transcriptional
regulator protein
HCNGP
520 7025 AF119664 Homo sapiens 1144 89
transcriptional
regulator protein
HCNGP
521 7025 AF119664 Homo sapiens 1448 94
transcriptional
regulator protein
HCNGP
522 7050 X12517 Homo sapiens C protein 918 100
(AA 1-159)
523 7051 AL079277 Homo sapiens 1294 100
hypothetical protein,
similar to (U32865)
linotte protein
524 7055 AF067730 Homo sapiens TLS- 631 57
associated protein
TASR-2
525 7060 U27831 Homo sapiens striatum- 2840 98
enriched phosphatase
526 7064 L26288 Rattus norvegicus CaM- 1416 82
like protein kinase
527 7067 AL032684 Schizosaccharomyces 300 37
pombe hypothetical
protein
528 7071 AL050028 Homo sapiens 671 100
hypothetical protein
529 7072 X78444 Rattus norvegicus 450 73
ribosomal protein L22
530 7073 U27838 Mus musculus glycosyl- 3305 96
phosphatidyl-inositol-
anchored protein
homolog
531 7076 AB037807 Homo sapiens KIAA1386 4001 99
protein
532 7088 AJ276504 Mus musculus 1705 85
phosphorylated adaptor
for RNA export
533 7089 AB033079 Homo sapiens KIAA1253 2398 100
protein
534 7091 U41315 Homo sapiens ZNF127-Xp 2458 93
535 7091 AF192784 Homo sapiens makorin 1 2062 97
536 7104 AE003704 Drosophila 510 44
melanogaster CG3307
gene product
537 7105 Z22968 Homo sapiens M130 6205 100
antigen
538 7105 Z22971 Homo sapiens M130 6380 100
antigen extracellular
variant
539 7109 AL050225 Homo sapiens 1431 99
hypothetical protein
540 7109 AL050225 Homo sapiens 932 99
hypothetical protein
541 7119 Z46522 Drosophila subobscura 237 55
bcn92
542 7120 AE003771 Drosophila 2185 68
melanogaster CG1972
gene product
543 7121 AL021546 Homo sapiens 593 100
Cytochrome C Oxidase
Polypeptide VIa-liver
precursor (EC 1.9.3.1)
544 7126 L02956 Xenopus laevis 1664 87
ribonucleoprotein
545 7127 AF201947 Homo sapiens MEK 616 100
binding partner 1
546 7130 L31783 Mus musculus uridine 1266 92
kinase
547 7131 AK001534 Homo sapiens unnamed 652 97
protein product
548 7144 AE003834 Drosophila 485 57
melanogaster CG8026
gene product
549 7159 AF154108 Homo sapiens tumor 3559 99
necrosis factor type 1
receptor associated
protein
550 7163 AE003066 Drosophila 251 34
melanogaster CG13865
gene product
551 7175 X57807 Homo sapiens 699 91
immunoglobulin lambda
light chain
552 7188 AL031673 Homo sapiens 4066 99
dJ694B14.1 (PUTATIVE
novel KRAB box protein
with 18 C2H2 type Zinc
finger domains)
553 7189 Y11652 Homo sapiens phosphate 238 100
cyclase
554 7190 AF192968 Homo sapiens high- 3041 99
glucose-regulated
protein 8
555 7191 AB020648 Homo sapiens KIAA0841 3237 99
protein
556 7203 AL031427 Homo sapiens 1608 100
dJ167A19.1 (novel
protein)
557 7204 AF151534 Homo sapiens core 1866 100
histone macroH2A2.2
558 7208 AL021331 Homo sapiens 1129 100
dJ366N23.1 (putative
C. elegans UNC-93
(protein 1, C46F11.1)
LIKE protein)
559 7209 X14608 Homo sapiens 3579 100
propionyl-CoA
carboxylase
560 7210 AL110249 Homo sapiens 4488 99
hypothetical protein
561 7216 AC004982 Homo sapiens similar 2038 100
to yeast hypothetical
protein ybk4; similar
to P38164
(PID: g586461)
562 7221 AE003628 Drosophila 148 30
melanogaster CG5676
gene product
563 7230 AE003519 Drosophila 711 75
melanogaster CG4108
gene product
564 7237 X79417 Sus scrofa 40S 687 100
ribosomal protein S12
565 7240 AB023203 Homo sapiens KIAA0986 7551 100
protein
566 7245 AE003684 Drosophila 1106 51
melanogaster CG8412
gene product
567 7250 AL117662 Homo sapiens 1078 99
hypothetical protein
568 7251 AB041261 Homo sapiens calcium- 2903 100
independent
phospholipase A2
569 7255 AK000812 Homo sapiens unnamed 1350 100
protein product
570 7260 Y10936 Homo sapiens 1104 99
hypothetical protein
571 7265 AK000444 Homo sapiens unnamed 2900 99
protein product
572 7268 AK001798 Homo sapiens unnamed 1460 99
protein product
573 7275 AL117635 Homo sapiens 929 99
hypothetical protein
574 7279 M55531 Homo sapiens GLUT5 924 45
protein
575 7283 AL117573 Homo sapiens 2907 99
hypothetical protein
576 7283 AL117573 Homo sapiens 2457 97
hypothetical protein
577 7287 AF237631 Homo sapiens 1798 100
ubiquitous
tropomodulin U-Tmod
578 7301 AF090929 Homo sapiens PRO0477p 653 99
579 7308 AL031228 Homo sapiens 3196 100
dJ1033B10.2 (WD40
protein BING4 (similar
to S. cerevisiae
YER082C, M. sexta
MNG10 and C. elegans
F28D1.1)
580 7308 AL031228 Homo sapiens 2825 96
dJ1033B10.2 (WD40
protein BING4 (similar
to S. derevisiae
YER082C, M. sexta
MNG10 and C. elegans
F28D1.1)
581 7309 AF171102 Homo sapiens retinal 1302 95
degeneration B beta
582 7319 AK001598 Homo sapiens unnamed 2775 100
protein product
583 7320 AJ237946 Homo sapiens DEAD Box 2443 100
Protein 5
584 7326 Z97184 Homo sapiens HKE2 624 100
585 7326 Z97184 Homo sapiens HKE2 409 98
586 7334 AJ245587 Homo sapiens Kruppel- 1942 100
type zinc finger
587 7337 Z22820 Canis familiaris 995 98
Rab22a protein
588 7339 X64701 Haloferax mediterranei 103 28
gvpI
589 7344 L04733 Homo sapiens kinesin 1936 72
light chain
590 7355 AB020681 Homo sapiens KIAA0874 3090 100
protein
591 7363 M55542 Homo sapiens guanylate 2993 98
binding protein
isoform I
592 7363 M55542 Homo sapiens guanylate 2901 96
binding protein
isoform I
593 7365 U41857 Xenopus laevis WD-40 937 53
motifs; up-regulated
by thyroid hormone in
tadpoles
594 7368 M26285 Xenopus laevis myc</