US20030045704A1 - Chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes - Google Patents

Chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes Download PDF

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US20030045704A1
US20030045704A1 US09/988,687 US98868701A US2003045704A1 US 20030045704 A1 US20030045704 A1 US 20030045704A1 US 98868701 A US98868701 A US 98868701A US 2003045704 A1 US2003045704 A1 US 2003045704A1
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hpc2
gene
sequence
elac2
elac1
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Sean Tavtigian
David Teng
Jacques Simard
Johanna Rommens
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Myriad Genetics Inc
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • tumor predisposition genes Some of the tumor predisposition genes which have been cloned and characterized influence susceptibility to: 1) Retinoblastoma (RB1); 2) Wilms' tumor (WT1); 3) Li-Fraumeni (TP53); 4) Familial adenomatous polyposis (APC); 5) Neurofibromatosis type 1 (NF1); 6) Neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau syndrome (VHL); 8) Multiple endocrine neoplasia type 2A (MEN2A); 9) Melanoma (CDKN2 and CDK4); 10) Breast and ovarian cancer (BRCA1 and BRCA2); 11) Cowden disease (MMAC1); 12) Multiple endocrine neoplasia (MEN1);
  • ADH Alcohol dehydrogenase
  • acetaldehyde a chemical which is both mutagenic and carcinogenic in lab animals.
  • the enzyme encoded by the ADH3 1 allele oxidizes ethanol relatively quickly, whereas the enzyme encoded by the ADH3 2 allele oxidizes ethanol more slowly.
  • ADH3 1 homozygotes presumably have a high capacity for synthesis of acetaldehyde; those who also drink heavily are at increased risk for oral cavity, esophageal, and (in women) breast cancer relative to ADH3 2 homozygotes who drink equally heavily (Harty et al., 1997; Hori et al., 1997; Shields, 1997).
  • the acetyltransferases encoded by N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) catalyze the acetylation of numerous xenobiotics including the aromatic amine carcinogens derived from smoking tobacco products.
  • the risk of hormone related cancers such as breast and prostate cancer may be modulated by allelic variants in enzymes that play a role in estrogen or androgen metabolism, or variants in proteins that mediate the biological effects of estrogens or androgens.
  • a polymorphic CAG repeat in the human androgen receptor gene encodes a polymorphic polyglutamine tract near the amino-terminus of the protein. The length of the polyglutamine tract is inversely correlated with the transcriptional activation activity of the androgen receptor and thus one aspect of the biological response to androgens.
  • Men whose androgen receptor contains a relatively short polyglutamine tract are at higher risk for prostate cancer, especially high stage/high histologic grade prostate cancer, than men whose androgen receptor contains a relatively long polyglutamine tract (Giovannucci et al., 1997).
  • Prostate cancer is the most common cancer in men in many western countries, and the second leading cause of cancer deaths in men. It accounts for more than 40,000 deaths in the US annually. The number of deaths is likely to continue rising over the next 10 to 15 years. In the US, prostate cancer is estimated to cost $1.5 billion per year in direct medical expenses. In addition to the burden of suffering, it is a major public-health issue. Numerous studies have provided evidence for familial clustering of prostate cancer, indicating that family history is a major risk factor for this disease (Cannon et al., 1982; Steinberg et al., 1990; Carter et al, 1993).
  • Prostate cancer has long been recognized to be, in part, a familial disease with a genetic component (Woolf, 1960a; Cannon et al., 1982; Carter et al., 1992). Numerous investigators have examined the evidence for genetic inheritance and concluded that the data are most consistent with dominant inheritance for a major susceptibility locus or loci. Woolf (1960b), described a relative risk of 3.0 of developing prostate cancer among first-degree relatives of prostate cancer cases in Utah using death certificate data.
  • Inherited prostate cancer susceptibility accounted for a significant proportion of early-onset disease, and overall was responsible for 9% of prostate occurrence by age 85.
  • Recent results demonstrate that at least four loci exist which convey susceptibility to prostate cancer as well as other cancers. These loci are HPC1 on chromosome 1q24, (Smith et al., 1996), HPCX on chromosome Xq27-28 (Xu et al., 1998), PCAP at 1q42 (Berthon et al., 1998) and CAPB at 1p36 (Gibbs et al., 1999a). All four suggestions of linkage for prostate cancer predisposition were the result of hints arising from genome-wide searches.
  • the Utah population provides a unique resource for examining the genetic basis of disease.
  • Extended high risk pedigrees containing many cases can be ascertained as units instead of by expansion from individual probands. While these pedigrees are an extremely powerful resource for linkage studies, they also allow analysis of segregation of moderate risk sequence variants through multiple generations of both cases and their unaffected relatives.
  • Detection of genetic linkage for prostate cancer susceptibility to a defined segment of a chromosome requires that DNA sequence variants within that chromosomal segment confer the cancer susceptibility. This is usually taken to mean that the causal sequence variant(s) will either alter the expression of one or more linked genes or will alter the function of one of the linked genes. However, detection of the genetic linkage does not necessarily provide evidence for what class of gene (i.e. tumor suppressor, oncogene, or risk modifier) is affected by the causal sequence variant(s).
  • class of gene i.e. tumor suppressor, oncogene, or risk modifier
  • HPC2 17p-linked prostate cancer predisposing gene
  • the present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human prostate cancer predisposing gene (HPC2), some alleles of which cause susceptibility to cancer, in particular prostate cancer. More specifically, the present invention relates to germline mutations in the HPC2 gene and their use in the diagnosis of predisposition to prostate cancer. The invention also relates to presymptomatic therapy of individuals who carry deleterious alleles of the HPC2 gene. The invention further relates to somatic mutations in the HPC2 gene in human prostate cancer and their use in the diagnosis and prognosis of human prostate cancer.
  • HPC2 human prostate cancer predisposing gene
  • the invention relates to somatic mutations in the HPC2 gene in other human cancers and their use in the diagnosis and prognosis of human cancers.
  • the invention also relates to the therapy of human cancers which have a mutation in the HPC2 gene, including gene therapy, protein replacement therapy, protein mimetics, and inhibitors.
  • the invention further relates to the screening of drugs for cancer therapy.
  • the invention also relates to the screening of the HPC2 gene for mutations, which are useful for diagnosing the predisposition to prostate cancer.
  • the HPC2 gene is useful as a marker for the HPC2 locus and as a marker for prostate cancer.
  • a paralog of HPC2 as well as orthologs of HPC2 in mouse, chimpanzee and gorilla have been isolated and characterized.
  • FIG. 1 is a multipoint linkage analysis of 4 kindreds that show suggestive evidence for linkage to the HPC2 prostate cancer susceptibility locus relative to chromosome 17p markers.
  • FIGS. 2 A-B are diagrams showing the order of genetic markers and recombinant boundaries neighboring HPC2, a schematic map of BACs spanning the HPC2 region, a schematic map of transcription units within the HPC2 region, and two diagrams of the HPC2 transcription unit showing the locations of the exons of HPC2 relative to the BAC to which it maps and relative to each other. The individual exons are numbered.
  • FIG. 3 shows recombinant, physical and transcript maps centered at the human ELAC2 locus on chromosome 17p.
  • the top portion shows genetic markers and recombinants. Microsatellite markers developed at Myriad Genetics, Inc. are given as 17-MYR####. Nested within the arrows that represent meiotic recombinants are the pedigree in which the recombinant occurred and, in parentheses, the number of cases who carry the haplotype on which the recombinant occurred.
  • the second portion of the figure shows a BAC contig tiling path across this interval. The T7 end of each BAC is denoted with an arrowhead.
  • the third portion of the figure shows transcription units identified in the interval.
  • the bottom portion of the figure is an expanded view of a 40 kb segment at the SP6 end of BAC 31k12 showing the relative positions of two exons of the gene 04CG09 and all of the coding exons of ELAC2.
  • FIG. 4 is an alignment of the sequence of exon 1 of the human HPC2 gene with exon 1 of the mouse HPC2 gene.
  • the figure also shows an alignment of the peptide sequence encoded by exon 1 of the human HPC2 gene with the peptide sequence encoded by exon 1 of the mouse HPC2 gene.
  • the human DNA sequence is SEQ ID NO:210; the human amino acid sequence is SEQ ID NO:211; the mouse DNA sequence is SEQ ID NO:212 and the mouse amino acid sequence is SEQ ID NO:213.
  • FIGS. 5 A-B show kindreds 4102 and 4289.
  • the pedigrees have been genotyped over a 20 cM interval extending from D17S786 to D17S805.
  • Haplotypes are represented by the bars; the dark gray haplotype segregating in each pedigree is the mutation bearing chromosome.
  • the relative position of ELAC2 is denoted by * (white on black or white on gray).
  • FIG. 5A shows kindred 4102.
  • the dark bar denotes the 1641 insG bearing haplotype.
  • Individuals 061 and 107 carry part of the frameshift haplotype, but neither carries the frameshift due to recombination events. There are no data to distinguish which of the founders, individuals 050 and 051, carried the frameshift.
  • the second shared haplotype in kindred 4102 is denoted by a light gray bar. Again, there are no data to distinguish which of the founders, individuals 005 and 006, carried the haplotype.
  • FIG. 5B shows kindred 4289. Individuals 064, 066, 067, 068 and 072 share a recombinant chromosome that carries the His 781 missense change.
  • FIGS. 6 A-B are a multiple protein alignment of ELAC/2 family members.
  • ELAC2 family members were selected from human (HSA), mouse (MMU), C. elegans (CEL), A. thaliana (ATH) and S. cerevisiae (SCE).
  • the A. thaliana genome encodes more than one family member; gi6850339 was selected because it aligned with fewer gaps.
  • ELAC1 family members were selected from human, E. coli (Es_c), the blue-green algae Synechocystis (Syn) and the archaebacterium Methanobacterium thernoautotrophicum (Met). Alignments were based on BLASTp searches and then optimized by inspection.
  • human ELAC2 is SEQ ID NO:2
  • mouse Elac2 is SEQ ID NO:222
  • C. elegans CE16965 is SEQ ID NO:227
  • A. thaliana gi 6850339 is SEQ ID NO:228,
  • S. cerevisiae YKRO79C is SEQ ID NO:229
  • human ELAC1 is SEQ ID NO:220 , E.
  • coli elaC is SEQ ID NO:230
  • Synechocystis gi2500943 is SEQ ID NO:231
  • Methanobacterium thernoautotrophicum gi2622965 is SEQ ID NO:232.
  • FIG. 7 shows recessive genotype frequencies by birth cohort.
  • FIG. 8 shows the results of association tests.
  • FIG. 9 shows a multiple protein alignment demonstrating conservation of sequence elements between ELAC2, PSO2 and CPSF73 families.
  • the alignments shown for segments of PSO2 and CPSF73 family members were taken from more extensive alignments that contain family members from a larger set of species, analogous to the ELAC1/2 alignments of FIGS. 6 A-B.
  • the seven His or Cys residues that are conserved across two or more of the gene families are marked by down arrows.
  • the position of Ala 541 in human ELAC2 is also marked by a down arrow.
  • the sequences shown in FIG. 9 are partial sequences of the following: human CPSF73 is SEQ ID NO:233 , A.
  • thaliana gi6751699 is SEQ ID NO:234
  • S. cerevisiae YSH1 is SEQ ID NO:235
  • Synechocystis gi2496795 is SEQ ID NO:236
  • Methanobacterium thermoautotrophicum gi2622312 is SEQ ID NO:237
  • human ha3611 is SEQ ID NO:238
  • A. thaliana gi2979557 is SEQ ID NO:239
  • S. cerevisiae PSO2 is SEQ ID NO:240
  • human ELAC2 is SEQ ID NO:2
  • FIG. 10 shows a similarity comparison among the ELAC2 family members aligned in FIGS. 6 A-B.
  • FIGS. 11 A-D shows an analysis of ELAC 1 expression in human tissues.
  • FIGS. 11 A-B show Multiple Tissue Northern (MTN) filters (Clontech) probed with the human ELAC1 ORF. Note that a 3 kb ELAC1 transcript is detected in all tissues.
  • MTN Multiple Tissue Northern
  • FIGS. 11 C-D show the same filters probed with human ⁇ -actin as a loading control.
  • FIG. 12 shows a multiple protein alignment demonstrating similarity between an N-terminal segment of the ELAC2 family members and the sequence context of the histidine motif shared by ELAC1 and ELAC2 family members. Species abbreviations are as in FIGS. 6 A-B.
  • the sequences shown in FIG. 12 are partial sequences of the following: human ELAC2 is SEQ ID NO:2, mouse Elac2 is SEQ ID NO:222 , C. elegans CE16965 is SEQ ID NO:227 , A. thaliana gi6850339 is SEQ ID NO:228 , S.
  • cerevisiae YKRO79C is SEQ ID NO:229
  • human ELAC1 is SEQ ID NO:220
  • E. coli elaC is SEQ ID NO:230
  • Synechocystis gi2500943 is SEQ ID NO:231
  • Methanobacterium thermoautotrophicum gi2622965 is SEQ ID NO:232.
  • FIG. 13 shows the relationship between ELAC1/2, PSO2 and CPSF73 gene family members.
  • the tree is a distance-based depiction of pairwise sequence similarities determined from a manual alignment of the 67 amino acids immediately surrounding the histidine motif.
  • ClustaIX Thimpson et al., 1997) was used to calculate the percent divergence of each sequence on a pairwise basis and neighbor joining (Saitou and Nei, 1987) was applied to the resulting distance matrix.
  • the treefile produced from ClustaIX was visualized using TreeView (Page, 1996) and further edited in a graphics program for aesthetics. The scale bar indicates amino acid substitutions per residue.
  • Table 1 is a compilation of 2-point LOD scores for markers in the HPC2 region.
  • Table 2A lists the family resource used to detect linkage of HPC2 to chromosome 17p.
  • Table 2B lists two-point LOD scores using the Utah age-specific model.
  • Table 3 is a summary of resource genotyped for the association tests.
  • Table 4 is a list of the accession numbers of human EST sequences used to assemble a tentative, partial cDNA sequence of the human HPC2 gene.
  • Table 5 is a list of the primers used for obtaining 5′ RACE products that contained the start codon and part of the 5′ UTR of the human HPC2 gene, primers used to prepare a full length human HPC2 expression construct, and primers used to check the sequence of that construct.
  • Table 6 is a list of the accession numbers of mouse EST sequences used to assemble a tentative, partial cDNA sequence of the mouse HPC2 gene.
  • Table 7 is a list of the primers used for obtaining 5′ RACE products that contained the start codon and part of the 5′ UTR of the mouse HPC2 gene, primers used to prepare a full length mouse HPC2 expression construct, and primers used to check the sequence of that construct.
  • Table 8 is a list of the primers used to mutation screen the human HPC2 gene from genomic DNA.
  • Table 9 is a summary of germline sequence variants of the human HPC2 gene.
  • Table 10 is a list of the allele frequencies of HPC2.
  • SEQ ID NO: 1 is the nucleotide sequence for the human-HPC2 cDNA from the start codon through the stop codon.
  • SEQ ID NO:2 is the amino acid sequence for the human HPC2 protein.
  • SEQ ID NO:3 is the nucleotide sequence for the human HPC2 cDNA from 50 base pairs before the start codon through the end of the 3′ UTR.
  • SEQ ID NO:4 to SEQ ID NO:27 are the sequences of exon 1 to exon 24 of the human HPC2 gene.
  • SEQ ID NO:28 is the genomic sequence of the human HPC2 gene.
  • SEQ ID Nos:29-190 are nucleotide sequences of primers used to identify the human and/or mouse HPC2 genes or to screen for mutations.
  • SEQ ID Nos:191-209 are nucleotide sequences of the HPC2 around and including various sequence variants.
  • SEQ ID NO:210 is the nucleotide sequence of human HPC2 exon 1 and SEQ ID NO:211 is the corresponding amino acid sequence as shown in FIG. 4.
  • SEQ ID NO:212 is nucleotide sequence of mouse HPC2 exon 1 and SEQ ID NO:213 is the corresponding amino acid sequence as shown in FIG. 4.
  • SEQ ID NO:214 is a histidine containing motif found in HPC2/ELAC2 and ELAC1.
  • SEQ ID NO:215 is exon 1 of ELAC1.
  • SEQ ID NO:216 is exon 2 of ELAC1 plus surrounding genomic sequence.
  • SEQ ID NO:217 is exon 3 of ELAC1 plus surrounding genomic sequence.
  • SEQ ID NO:218 is exon 4 of ELAC1 plus surrounding genomic sequence.
  • SEQ ID NO:219 is the cDNA for ELAC1 and SEQ ID NO:220 is the amino acid sequence for ELAC1.
  • SEQ ID NO:221 is the cDNA for mouse ELAC2 and SEQ ID NO:222 is the amino acid sequence for mouse ELAC2.
  • SEQ ID NO:223 is the cDNA for chimpanzee ELAC2 and SEQ ID NO:224 is the amino acid sequence for chimpanzee ELAC2.
  • SEQ ID NO:225 is the cDNA for gorilla ELAC2 and SEQ ID NO:226 is the amino acid sequence for gorilla ELAC2.
  • SEQ ID NOs:227-229 are the amino acid sequences for ELAC2 family member proteins from C. elegans, A. thaliana and S. cerevisiae as shown in FIG. 6A.
  • SEQ ID NOs:230-232 are the amino acid sequences for ELAC1 family member proteins from E. coli , Synechocystis and Methanobacterium thermoautotrophicum as shown in FIGS. 6 A-B.
  • SEQ ID NOs:233-240 are amino acid sequences of proteins from CPSF73 and PSO2 families as shown in FIG. 9. These are, respectively, human CPSF73, A. thaliana gi6751699, S. cerevisiae YSH1, Synechocystis gi2496795, Methanobacterium thermoautotrophicum gi2622312, human ha3611, A. thaliana gi2979557 and S. cerevisiae PSO2.
  • the sequences for the ELAC2 family of FIG. 9 are SEQ ID NO:2 for human, SEQ ID NO:228 for A. thaliana (as for FIGS. 6 A-B) and SEQ ID NO:229 for S. cerevisiae (as for FIGS. 6 A-B).
  • the sequence listing shows the complete sequences of these proteins whereas FIG. 9 shows only portions of each sequence.
  • the present invention provides an isolated polynucleotide comprising all, or a portion of the HPC2 locus or of a mutated HPC2 locus, preferably at least eight bases and not more than about 27 kb in length. Such polynucleotides may be antisense polynucleotides.
  • the present invention also provides a recombinant construct comprising such an isolated polynucleotide, for example, a recombinant construct suitable for expression in a transformed host cell.
  • Also provided by the present invention are methods of detecting a polynucleotide comprising a portion of the HPC2 locus or its expression product in an analyte. Such methods may further comprise the step of amplifying the portion of the HPC2 locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the HPC2 locus.
  • the method is useful for either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer.
  • the HPC2 gene is useful as a marker for the HPC2 locus and as a marker for prostate cancer.
  • the present invention also provides isolated antibodies, preferably monoclonal antibodies, which specifically bind to an isolated polypeptide comprised of at least five amino acid residues encoded by the HPC2 locus.
  • kits for detecting in an analyte a polynucleotide comprising a portion of the HPC2 locus comprising kits comprising a polynucleotide complementary to the portion of the HPC2 locus packaged in a suitable container, and instructions for its use.
  • the present invention further provides methods of preparing a polynucleotide comprising polymerizing nucleotides to yield a sequence comprised of at least eight consecutive nucleotides of the HPC2 locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a sequence comprising at least five amino acids encoded within the HPC2 locus.
  • the present invention further provides methods of screening the HPC2 gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the HPC2 locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the HPC2 locus.
  • Such methods may also include a step of providing the complete set of short polynucleotides defined by the sequence of HPC2 or discrete subsets of that sequence, all single-base substitutions of that sequence or discrete subsets of that sequence, all 1-, 2-, 3-, or 4-base deletions of that sequence or discrete subsets of that sequence, and all 1-, 2-, 3-, or 4-base insertions in that sequence or discrete subsets of that sequence.
  • the method is useful for identifying mutations for use in either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer.
  • the present invention further provides methods of screening suspected HPC2 mutant alleles to identify mutations in the HPC2 gene.
  • the present invention provides methods to screen drugs for inhibition or restoration of HPC2 gene product function as an anticancer therapy.
  • the present invention also provides the means necessary for production of gene-based therapies directed at cancer cells.
  • These therapeutic agents may take the form of polynucleotides comprising all or a portion of the HPC2 locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the HPC2 protein is reconstituted.
  • Therapeutic agents may also take the form of polypeptides based on either a portion of, or the entire protein sequence of HPC2. These may functionally replace the activity of HPC2 in vivo.
  • the present invention provides the sequence of a paralog of HPC2, herein called ELAC1, as well as the sequences of HPC2 orthologs from mouse, chimpanzee and gorilla. These orthologs are named ELAC2.
  • HPC2 locus which predisposes individuals to prostate cancer
  • HPC2 is a gene encoding an HPC2 protein, which has been found to be non-identical to publicly available protein or cDNA sequences. This gene is termed HPC2 herein.
  • mutations in the HPC2 locus in the germline are indicative of a predisposition to prostate cancer.
  • germline mutations in the HPC2 locus are also associated with prostate cancer and other types of cancer.
  • the mutational events of the HPC2 locus can involve deletions, insertions and nucleotide substitutions within the coding sequence and the non-coding sequence.
  • alteration of the wild-type HPC2 locus is detected.
  • the method can be performed by detecting the wild-type HPC2 locus and confirming the lack of a predisposition to cancer at the HPC2 locus.
  • “Alteration of a wild-type gene” encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or of only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germline.
  • Germline mutations can be found in any of a body's tissues and are inherited. If only a single allele is somatically mutated, an early neoplastic state is indicated. However, if both alleles are somatically mutated, then a late neoplastic state is indicated. The finding of HPC2 mutations thus provides both diagnostic and prognostic information. An HPC2 allele which is not deleted (e.g., found on the sister chromosome to a chromosome carrying an HPC2 deletion) can be screened for other mutations, such as insertions, small deletions, and point mutations. It is believed that many mutations found in tumor tissues will be those leading to decreased expression of the HPC2 gene product.
  • Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to reduction or loss of expression of the HPC2 gene product, expression of an altered HPC2 gene product, or to a decrease in mRNA stability or translation efficiency.
  • Useful diagnostic techniques include, but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCA), RNase protection assay, allele-specific oligonucleotide (ASO), dot blot analysis, hybridization using nucleic acid modified with gold nanoparticles and PCR-SSCP, as discussed in detail further below. Also useful is the recently developed technique of DNA microchip technology.
  • Predisposition to cancers can be ascertained by testing any tissue of a human for mutations of the HPC2 gene.
  • a person who has inherited a germline HPC2 mutation would be prone to develop cancers. This can be determined by testing DNA from any tissue of the person's body. Most simply, blood can be drawn and DNA extracted from the cells of the blood.
  • prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for mutations of the HPC2 gene. Alteration of a wild-type HPC2 allele, whether, for example, by point mutation or deletion, can be detected by any of the means discussed herein.
  • CDGE clamped denaturing gel electrophoresis
  • HA heteroduplex analysis
  • CMC chemical mismatch cleavage
  • tissue In order to detect the alteration of the wild-type HPC2 gene in a tissue, it is helpful to isolate the tissue free from surrounding normal tissues.
  • Means for enriching tissue preparation for tumor cells are known in the art.
  • the tissue may be isolated from paraffin or cryostat sections. Cancer cells may also be separated from normal cells by flow cytometry. These techniques, as well as other techniques for separating tumor cells from normal cells, are well known in the art. If the tumor tissue is highly contaminated with normal cells, detection of mutations is more difficult.
  • Detection of point mutations may be accomplished by molecular cloning of the HPC2 allele(s) and sequencing the allele(s) using techniques well known in the art.
  • the gene sequences can be amplified directly from a genomic DNA preparation from the tumor tissue, using known techniques. The DNA sequence of the amplified sequences can then be determined.
  • coli mutS protein (Modrich, 1991); and 6) allele-specific PCR (Ruano and Kidd, 1989).
  • primers are used which hybridize at their 3′ ends to a particular HPC2 mutation. If the particular HPC2 mutation is not present, an amplification product is not observed.
  • Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., 1989. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification.
  • RFLP restriction fragment length polymorphism
  • Such a method is particularly useful for screening relatives of an affected individual for the presence of the HPC2 mutation found in that individual.
  • Other techniques for detecting insertions and deletions as known in the art can be used.
  • SSCA detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing.
  • RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments.
  • DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel.
  • an allele-specific oligonucleotide assay an oligonucleotide is designed which detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal.
  • the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.
  • Mismatches are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of tumor samples.
  • An example of a mismatch cleavage technique is the RNase protection method. In the practice of the present invention, the method involves the use of a labeled riboprobe which is complementary to the human wild-type HPC2 gene coding sequence.
  • the riboprobe and either mRNA or DNA isolated from the tumor tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA.
  • the riboprobe need not be the full length of the HPC2 mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the HPC2 mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.
  • DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986.
  • mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, 1988.
  • the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the HPC2 gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.
  • DNA sequences of the HPC2 gene which have been amplified by use of PCR may also be screened using allele-specific probes.
  • These probes are nucleic acid oligomers, each of which contains a region of the HPC2 gene sequence harboring a known mutation.
  • one oligomer may be about 30 nucleotides in length (although shorter and longer oligomers are also usable as well recognized by those of skill in the art), corresponding to a portion of the HPC2 gene sequence.
  • PCR amplification products can be screened to identify the presence of a previously identified mutation in the HPC2 gene.
  • Hybridization of allele-specific probes with amplified HPC2 sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under high stringency hybridization conditions indicates the presence of the same mutation in the tumor tissue as in the allele-specific probe.
  • the newly developed technique of nucleic acid analysis via microchip technology is also applicable to the present invention.
  • this technique literally thousands of distinct oligonucleotide probes are built up in an array on a silicon chip. Nucleic acid to be analyzed is fluorescently labeled and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique one can determine the presence of mutations or even sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest.
  • the method is one of parallel processing of many, even thousands, of probes at once and can tremendously increase the rate of analysis.
  • the most definitive test for mutations in a candidate locus is to directly compare genomic HPC2 sequences from cancer patients with those from a control population.
  • genomic HPC2 sequences from cancer patients with those from a control population.
  • Mutations from cancer patients falling outside the coding region of HPC2 can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the HPC2 gene.
  • An early indication that mutations in noncoding regions are important may come from Northern blot experiments that reveal messenger RNA molecules of abnormal size or abundance in cancer patients as compared to control individuals.
  • Alteration of HPC2 mRNA expression can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type HPC2 gene. Alteration of wild-type HPC2 genes can also be detected by screening for alteration of wild-type HPC2 protein. For example, monoclonal antibodies immunoreactive with HPC2 can be used to screen a tissue. Lack of cognate antigen would indicate an HPC2 mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant HPC2 gene product. Such immunological assays can be done in any convenient formats known in the art.
  • HPC2 protein can be used to detect alteration of wild-type HPC2 genes.
  • Functional assays such as protein binding determinations, can be used.
  • assays can be used which detect HPC2 biochemical function. Finding a mutant HPC2 gene product indicates alteration of a wild-type HPC2 gene.
  • Mutant HPC2 genes or gene products can also be detected in other human body samples, such as serum, stool, urine and sputum.
  • the same techniques discussed above for detection of mutant HPC2 genes or gene products in tissues can be applied to other body samples. Cancer cells are sloughed off from tumors and appear in such body samples.
  • the HPC2 gene product itself may be secreted into the extracellular space and found in these body samples even in the absence of cancer cells. By screening such body samples, a simple early diagnosis can be achieved for many types of cancers. In addition, the progress of chemotherapy or radiotherapy can be monitored more easily by testing such body samples for mutant HPC2 genes or gene products.
  • the methods of diagnosis of the present invention are applicable to any tumor in which HPC2 has a role in tumorigenesis.
  • the diagnostic method of the present invention is useful for clinicians, so they can decide upon an appropriate course of treatment.
  • the primer pairs of the present invention are useful for determination of the nucleotide sequence of a particular HPC2 allele using PCR.
  • the pairs of single-stranded DNA primers can be annealed to sequences within or surrounding the HPC2 gene on chromosome 17 in order to prime amplifying DNA synthesis of the HPC2 gene itself.
  • a complete set of these primers allows synthesis of all of the nucleotides of the HPC2 gene coding sequences, i.e., the exons.
  • the set of primers preferably allows synthesis of both intron and exon sequences. Allele-specific primers can also be used. Such primers anneal only to particular HPC2 mutant alleles, and thus will only amplify a product in the presence of the mutant allele as a template.
  • primers may have restriction enzyme site sequences appended to their 5′ ends.
  • all nucleotides of the primers are derived from HPC2 sequences or sequences adjacent to HPC2, except for the few nucleotides necessary to form a restriction enzyme site.
  • the primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available. Given the sequence of the HPC2 open reading frame shown in SEQ ID NOs: 1 and 3, design of particular primers is well within the skill of the art.
  • the nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above. The probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the HPC2 gene or mRNA using other techniques.
  • mutant alleles are then sequenced to identify the specific mutation of the particular mutant allele.
  • mutant HPC2 alleles can be initially identified by identifying mutant (altered) HPC2 proteins, using conventional techniques.
  • the mutant alleles are then sequenced to identify the specific mutation for each allele.
  • the mutations, especially those which lead to an altered function of the HPC2 protein, are then used for the diagnostic and prognostic methods of the present invention.
  • “Amplification of Polynucleotides” utilizes methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. Also useful are strand displacement amplification (SDA), thermophilic SDA, and nucleic acid sequence based amplification (3SR or NASBA). These methods are well known and widely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu and Wallace, 1989 (for LCR); U.S. Pat. Nos.
  • Alyte polynucleotide and “analyte strand” refer to a single- or double-stranded polynucleotide which is suspected of containing a target sequence, and which may be present in a variety of types of samples, including biological samples.
  • the present invention also provides polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the HPC2 polypeptides or to polypeptides encoded by paralogs or orthologs of HPC2 and fragments thereof or to polynucleotide sequences from the HPC2 region, or to polynucleotide sequences which are paralogs or orthologs of HPC2, particularly from the HPC2 locus or a portion thereof.
  • the term “antibody” is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities.
  • Polypeptides may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity to the HPC2 polypeptide or fragment or to polypeptides or fragments encoded by paralogs or orthologs of HPC2. Monoclonal antibodies may be made by injecting mice with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with HPC2 polypeptide or fragments thereof. See, Harlow and Lane, 1988. These antibodies will be useful in assays as well as pharmaceuticals.
  • a carrier molecule e.g., keyhole limpet hemocyanin
  • polypeptide Once a sufficient quantity of desired polypeptide has been obtained, it may be used for various purposes.
  • a typical use is the production of antibodies specific for binding. These antibodies may be either polyclonal or monoclonal, and may be produced by in vitro or in vivo techniques well known in the art.
  • an appropriate target immune system typically mouse or rabbit
  • Substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and by other parameters well known to immunologists. Typical sites for injection are in footpads, intramuscularly, intraperitoneally, or intradermally. Of course, other species may be substituted for mouse or rabbit.
  • Polyclonal antibodies are then purified using techniques known in the art, adjusted for the desired specificity.
  • An immunological response is usually assayed with an immunoassay.
  • immunoassays involve some purification of a source of antigen, for example, that produced by the same cells and in the same fashion as the antigen.
  • a variety of immunoassay methods are well known in the art. See, e.g., Harlow and Lane, 1988, or Goding, 1986.
  • Monoclonal antibodies with affinities of 10 ⁇ 8 M ⁇ 1 or preferably 10 ⁇ 9 to 10 ⁇ 10 M ⁇ 1 or stronger will typically be made by standard procedures as described, e.g., in Harlow and Lane, 1988 or Goding, 1986. Briefly, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatants of each clone tested for their production of an appropriate antibody specific for the desired region of the antigen.
  • Binding partner refers to a molecule capable of binding a ligand molecule with high specificity, as for example, an antigen and an antigen-specific antibody or an enzyme and its inhibitor.
  • the specific binding partners must bind with sufficient affinity to immobilize the analyte copy/complementary strand duplex (in the case of polynucleotide hybridization) under the isolation conditions.
  • Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, the numerous, known receptor-ligand couples, and complementary polynucleotide strands.
  • the partners are normally at least about 15 bases in length, and may be at least 40 bases in length. It is well recognized by those of skill in the art that lengths shorter than 15 (e.g., 8 bases), between 15 and 40, and greater than 40 bases may also be used.
  • the polynucleotides may be composed of DNA, RNA, or synthetic nucleotide analogs. Further binding partners can be identified using, e.g., the two-hybrid yeast screening assay as described herein.
  • a “biological sample” refers to a sample of tissue or fluid suspected of containing an analyte polynucleotide or polypeptide from an individual including, but not limited to, e.g., plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.
  • the terms “diagnosing” or “prognosing,” as used in the context of neoplasia, are used to indicate 1) the classification of lesions as neoplasia, 2) the determination of the severity of the neoplasia, or 3) the monitoring of the disease progression, prior to, during and after treatment.
  • Encode A polynucleotide is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof.
  • the anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
  • nucleic acid e.g., an RNA, DNA or a mixed polymer
  • An “isolated” or “substantially pure” nucleic acid is one which is substantially separated from other cellular components which naturally accompany a native human sequence or protein, e.g., ribosomes, polymerases, many other human genome sequences and proteins.
  • the term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • HPC2 Allele refers to normal alleles of the HPC2 locus as well as alleles carrying variations that predispose individuals to develop prostate cancer. Such predisposing alleles are also called “HPC2 susceptibility alleles”.
  • HPC2 Locus each refer to polynucleotides, all of which are in the HPC2 region, that are likely to be expressed in normal tissue, certain alleles of which predispose an individual to develop prostate cancers. Mutations at the HPC2 locus may be involved in the initiation and/or progression of other types of tumors. The locus is indicated in part by mutations that predispose individuals to develop cancer. These mutations fall within the HPC2 region described infra.
  • the HPC2 locus is intended to include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation.
  • the HPC2 locus is intended to include all allelic variations of the DNA sequence.
  • HPC2 is used interchangeably throughout this disclosure with the terms ELAC2 and HPC2/ELAC2. This holds true regardless of whether the term refers to a nucleic acid, allele, gene, locus, protein or peptide.
  • nucleic acid when applied to a nucleic acid, refer to a nucleic acid which encodes an HPC2 polypeptide, fragment, homolog or variant, including, e.g., protein fusions or deletions.
  • the nucleic acids of the present invention will possess a sequence which is either derived from, or substantially similar to a natural HPC2-encoding gene or one having substantial homology with a natural HPC2-encoding gene or a portion thereof.
  • the HPC2 gene or nucleic acid includes normal alleles of the HPC2 gene, including silent alleles having no effect on the amino acid sequence of the HPC2 polypeptide as well as alleles leading to amino acid sequence variants of the HPC2 polypeptide that do not substantially affect its function. These terms also include alleles having one or more mutations which adversely affect the function of the HPC2 polypeptide.
  • a mutation may be a change in the HPC2 nucleic acid sequence which produces a deleterious change in the amino acid sequence of the HPC2 polypeptide, resulting in partial or complete loss of HPC2 function, or may be a change in the nucleic acid sequence which results in the loss of effective HPC2 expression or the production of aberrant forms of the HPC2 polypeptide.
  • HPC2 nucleic acid may be that shown in SEQ ID NOs:1, 3 or 28 or it may be an allele as described above or a variant or derivative differing from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to the nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.
  • nucleic acid according to the present invention may include a sequence different from the sequence shown in SEQ ID NOs:1, 3 or 28 yet encode a polypeptide with the same amino acid sequence as shown in SEQ ID NO:1. That is, nucleic acids of the present invention include sequences which are degenerate as a result of the genetic code.
  • the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequence shown in SEQ ID NO:2.
  • Nucleic acid encoding a polypeptide which is an amino acid sequence variant, derivative or allele of the amino acid sequence shown in SEQ ID NO:2 is also provided by the present invention.
  • the HPC2 gene also refers to (a) any DNA sequence that (i) hybridizes to the complement of the DNA sequences that encode the amino acid sequence set forth in SEQ ID NO:2 under highly stringent conditions (Ausubel et al., 1992) and (ii) encodes a gene product functionally equivalent to HPC2, or (b) any DNA sequence that (i) hybridizes to the complement of the DNA sequences that encode the amino acid sequence set forth in SEQ ID NO:2 under less stringent conditions, such as moderately stringent conditions (Ausubel et al., 1992) and (ii) encodes a gene product functionally equivalent to HPC2.
  • the invention also includes nucleic acid molecules that are the complements of the sequences described herein.
  • the polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art.
  • Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.).
  • uncharged linkages e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.
  • charged linkages e.g., phosphorothioates, phosphorodithioates, etc.
  • pendent moieties
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • the present invention provides recombinant nucleic acids comprising all or part of the HPC2 region or the HPC2 paralog called ELAC1 or the mouse, chimpanzee or gorilla orthologs of HPC2, herein called mouse ELAC2, chimpanzee ELAC2 or gorilla ELAC2.
  • the recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosomal DNA of the host cell.
  • Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation, 1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; 2) is linked to a polynucleotide other than that to which it is linked in nature; or 3) does not occur in nature.
  • nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.
  • nucleic acids comprising sequences otherwise not naturally occurring are provided by this invention.
  • wild-type sequence may be employed, it will often be altered, e.g., by deletion, substitution or insertion.
  • cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR.
  • the choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.
  • the DNA sequences used in this invention will usually comprise at least about five codons (15 nucleotides), more usually at least about 7-15 codons, and most preferably, at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with an HPC2-encoding sequence. In this context, oligomers of as low as 8 nucleotides, more generally 8-17 nucleotides, can be used for probes, especially in connection with chip technology.
  • nucleic acid manipulation techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., 1989 or Ausubel et al., 1992.
  • Reagents useful in applying such techniques such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S. Biochemicals, New England Nuclear, and a number of other sources.
  • the recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.
  • HPC2 Region refers to a portion of human chromosome 17 bounded by the markers D17S947 and D17S799. This region contains the HPC2 locus, including the HPC2 gene.
  • HPC2 locus As used herein, the terms “HPC2 locus”, “HPC2 allele” and “HPC2 region” all refer to the double-stranded DNA comprising the locus, allele, or region, as well as either of the single-stranded DNAs comprising the locus, allele or region.
  • a “portion” of the HPC2 locus or region or allele is defined as having a minimal size of at least about eight nucleotides, or preferably about 15 nucleotides, or more preferably at least about 25 nucleotides, and may have a minimal size of at least about 40 nucleotides. This definition includes all sizes in the range of 8-40 nucleotides as well as greater than 40 nucleotides.
  • this definition includes nucleic acids of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or nucleic acids having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc., nucleotides), or nucleic acids having more than 500 nucleotides.
  • the present invention includes all novel nucleic acids having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28, its complement or functionally equivalent nucleic acid sequences.
  • the present invention does not include nucleic acids which exist in the prior art. That is, the present invention includes all nucleic acids having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28 with the proviso that it does not include nucleic acids existing in the prior art.
  • HPC2 protein or “HPC2 polypeptide” refers to a protein or polypeptide encoded by the HPC2 locus, variants or fragments thereof.
  • polypeptide refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like.
  • polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
  • polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring.
  • polypeptides will be at least about 50% homologous to the native HPC2 sequence, preferably in excess of about 90%, and more preferably at least about 95% homologous.
  • proteins encoded by DNA which hybridize under high or low stringency conditions, to HPC2-encoding nucleic acids and closely related polypeptides or proteins retrieved by antisera to the HPC2 protein(s).
  • HPC2 polypeptide may be that derived from any of the exons described herein which may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated.
  • the polypeptide may, if produced by expression in a prokaryotic cell or produced synthetically, lack native post-translational processing, such as glycosylation.
  • the present invention is also directed to polypeptides which are sequence variants, alleles or derivatives of an HPC2 polypeptide.
  • Such polypeptides may have an amino acid sequence which differs from that derived from any of the exons described herein by one or more of addition, substitution, deletion or insertion of one or more amino acids.
  • Preferred such polypeptides have HPC2 function.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties.
  • 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.
  • Preferred substitutions are ones which are conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and tyrosine, phenylalanine.
  • Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or binding sites on proteins interacting with an HPC2 polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982).
  • hydrophilicity in conferring interactive biological function of a protein is generally understood in the art (U.S. Pat. No. 4,554,101).
  • hydrophobic index or hydrophilicity in designing polypeptides is further discussed in U.S. Pat. No. 5,691,198.
  • the length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.
  • peptide mimetic or mimetic is intended to refer to a substance which has the essential biological activity of an HPC2, ELAC1 or ELAC2 polypeptide.
  • a peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson et al., 1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen, enzyme and substrate or scaffolding proteins.
  • a peptide mimetic is designed to permit molecular interactions similar to the natural molecule.
  • a mimetic may not be a peptide at all, but it will retain the essential biological activity of a natural HPC2, ELAC1 or ELAC2 polypeptide.
  • Probes Polynucleotide polymorphisms associated with HPC2 alleles which predispose to certain cancers or are associated with most cancers are detected by hybridization with a polynucleotide probe which forms a stable hybrid with that of the target sequence, under highly stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be perfectly complementary to the target sequence, high stringency conditions will be used. Hybridization stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe will not be completely complementary. Conditions are chosen which rule out nonspecific/adventitious bindings, that is, which minimize noise.
  • Probes for HPC2 alleles may be derived from the sequences of the HPC2 region, its cDNA, functionally equivalent sequences, or the complements thereof
  • the probes may be of any suitable length, which span all or a portion of the HPC2 region, and which allow specific hybridization to the HPC2 region. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of about 8-30 base pairs, since the hybrid will be relatively stable under even highly stringent conditions. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridizes to the target sequence with the requisite specificity.
  • the probes will include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods. For techniques for preparing and labeling probes see, e.g., Sambrook et al., 1989 or Ausubel et al., 1992. Other similar polynucleotides may be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., by silent changes (thereby producing various restriction sites) or to optimize expression for a particular system. Mutations may be introduced to modify the properties of the polypeptide, perhaps to change ligand-binding affinities, interchain affinities, or the polypeptide degradation or turnover rate.
  • Probes comprising synthetic oligonucleotides or other polynucleotides of the present invention may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labeled by nick translation, Klenow fill-in reaction, or other methods known in the art.
  • Portions of the polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 9 kb, usually fewer than about 1.0 kb, from a polynucleotide sequence encoding HPC2 are preferred as probes. This definition therefore includes probes of sizes 8 nucleotides through 9000 nucleotides.
  • this definition includes probes of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400 or 500 nucleotides or probes having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc., nucleotides), or probes having more than 500 nucleotides.
  • the probes may also be used to determine whether mRNA encoding HPC2 is present in a cell or tissue.
  • the present invention includes all novel probes having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28 its complement or functionally equivalent nucleic acid sequences.
  • the present invention does not include probes which exist in the prior art. That is, the present invention includes all probes having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28 with the proviso that they do not include probes existing in the prior art.
  • primers which may be used for the amplification of all or part of the HPC2 gene.
  • a definition for primers includes primers of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or primers having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc. nucleotides), or primers having more than 500 nucleotides, or any number of nucleotides between 500 and 9000.
  • the primers may also be used to determine whether mRNA encoding HPC2 is present in a cell or tissue.
  • the present invention includes all novel primers having at least 8 nucleotides derived from the HPC2 locus for amplifying the HPC2 gene, its complement or functionally equivalent nucleic acid sequences.
  • the present invention does not include primers which exist in the prior art. That is, the present invention includes all primers having at least 8 nucleotides with the proviso that it does not include primers existing in the prior art.
  • Protein modifications or fragments are provided by the present invention for HPC2, ELAC1 and ELAC2 polypeptides or fragments thereof which are substantially homologous to primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art.
  • a variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 32 P, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand.
  • radioactive isotopes such as 32 P
  • ligands which bind to labeled antiligands e.g., antibodies
  • fluorophores e.g., chemiluminescent agents
  • enzymes chemiluminescent agents
  • antiligands which can serve as specific binding pair members for a labeled ligand.
  • the choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation.
  • Methods of labeling polypeptides are well known in the art. See Sambrook et al., 1989 or
  • the present invention provides for biologically active fragments of the polypeptides.
  • Significant biological activities include ligand-binding, immunological activity and other biological activities characteristic of HPC2, ELAC1 or ELAC2 polypeptides.
  • Immunological activities include both immunogenic function in a target immune system, as well as sharing of immunological epitopes for binding, serving as either a competitor or substitute antigen for an epitope of the HPC2, ELAC1 or ELAC2 protein.
  • epitope refers to an antigenic determinant of a polypeptide.
  • An epitope could comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of such amino acids are known in the art.
  • tandem-repeat polypeptide segments may be used as immunogens, thereby producing highly antigenic proteins.
  • polypeptides will serve as highly efficient competitors for specific binding. Production of antibodies specific for HPC2, ELAC1 or ELAC2 polypeptides or fragments thereof is described below.
  • the present invention also provides for fusion polypeptides, comprising HPC2, ELAC1 or ELAC2 polypeptides and fragments.
  • Homologous polypeptides may be fusions between two or more HPC2, ELAC1 or ELAC2 polypeptide sequences or between the sequences of HPC2, ELAC1 or ELAC2 and a related protein.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. For example, ligand-binding or other domains may be “swapped” between different new fusion polypeptides or fragments.
  • Such homologous or heterologous fusion polypeptides may display, for example, altered strength or specificity of binding.
  • Fusion partners include immunoglobulins, bacterial ⁇ -galactosidase, trpE, protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See Godowski et al., 1988.
  • Fusion proteins will typically be made by either recombinant nucleic acid methods, as described below, or may be chemically synthesized. Techniques for the synthesis of polypeptides are described, for example, in Merrifield, 1963.
  • Protein purification refers to various methods for the isolation of the HPC2, ELAC1 or ELAC2 polypeptides from other biological material, such as from cells transformed with recombinant nucleic acids encoding HPC2, ELAC1 or ELAC2 and are well known in the art.
  • polypeptides may be purified by immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention.
  • immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention.
  • Various methods of protein purification are well known in the art, and include those described in Irishr, 1990 and Scopes, 1982.
  • isolated is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide sequence.
  • a substantially pure protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure.
  • Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for purification.
  • HPC2, ELAC1 or ELAC2 protein is substantially free of naturally associated components when it is separated from the native contaminants which accompany it in its natural state.
  • a polypeptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • a polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an “isolated polypeptide,” as used herein, even if expressed in a homologous cell type. Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.
  • “Recombinant nucleic acid” is a nucleic acid which is not naturally occurring, or which is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.
  • regulatory sequences refers to those sequences normally within 100 kb of the coding region of a locus, but they may also be more distant from the coding region, which affect the expression of the gene (including transcription of the gene, and translation, splicing, stability or the like of the messenger RNA).
  • nucleic acid or fragment thereof is “substantially homologous” (“or substantially similar”) to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.
  • Identity means the degree of sequence relatedness between two polypeptide or two polynucleotides sequences as determined by the identity of the match between two strings of such sequences. Identity can be readily calculated. While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux et al. (1984), BLASTP, BLASTN, FASTA (Altschul et al. (1990); Altschul et al. (1997)).
  • nucleic acid or fragment thereof will hybridize to another nucleic acid (or a complementary strand thereof) under selective hybridization conditions, to a strand, or to its complement.
  • Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa, 1984.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and preferably in excess of 45° C.
  • Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson, 1968.
  • Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes.
  • the preparation of such probes and suitable hybridization conditions are well known in the art.
  • polypeptides when referring to polypeptides, indicate that the polypeptide or protein in question exhibits at least about 30% identity with an entire naturally-occurring protein or a portion thereof, usually at least about 70% identity, more usually at least about 80% identity, preferably at least about 90% identity, and more preferably at least about 95% identity.
  • Homology for polypeptides, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705, as well as the software described above with reference to nucleic acid homology. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • substantially similar function refers to the function of a modified nucleic acid or a modified protein, with reference to the wild-type HPC2, ELAC1 or ELAC2 nucleic acid or wild-type HPC2, ELAC1 or ELAC2 polypeptide.
  • the modified polypeptide will be substantially homologous to the wild-type HPC2, ELAC1 or ELAC2 polypeptide and will have substantially the same function.
  • the modified polypeptide may have an altered amino acid sequence and/or may contain modified amino acids. In addition to the similarity of function, the modified polypeptide may have other useful properties, such as a longer half-life.
  • the similarity of function (activity) of the modified polypeptide may be substantially the same as the activity of the wild-type HPC2, ELAC1 or ELAC2 polypeptide. Alternatively, the similarity of function (activity) of the modified polypeptide may be higher than the activity of the wild-type HPC2, ELAC1 or ELAC2 polypeptide.
  • the modified polypeptide is synthesized using conventional techniques, or is encoded by a modified nucleic acid and produced using conventional techniques.
  • the modified nucleic acid is prepared by conventional techniques. A nucleic acid with a function substantially similar to the wild-type HPC2, ELAC1 or ELAC2 gene function produces the modified protein described above.
  • a polypeptide “fragment,” “portion” or “segment” is a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous ammo acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids.
  • polypeptides of the present invention may be coupled to a solid-phase support, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates.
  • a solid-phase support e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates.
  • Such supports may take the form, for example, of beads, wells, dipsticks, or membranes.
  • Target region refers to a region of the nucleic acid which is amplified and/or detected.
  • target sequence refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions.
  • polynucleotides of the present invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs, usually DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the polynucleotide constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to (with and without integration within the genome) cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention is described, e.g., in Sambrook et al., 1989 or Ausubel et al., 1992.
  • the polynucleotides of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers, 1981 or the triester method according to Matteucci and Caruthers, 1981, and may be performed on commercial, automated oligonucleotide synthesizers.
  • a double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
  • Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.
  • Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences.
  • ARS autonomously replicating sequence
  • Secretion signals may also be included where appropriate, whether from a native HPC2 protein or from other receptors or from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, and thus attain its functional topology, or be secreted from the cell.
  • Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al., 1989 or Ausubel et al. 1992.
  • An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may include, when appropriate, those naturally associated with HPC2, ELAC1 or ELAC2 genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or Ausubel et al., 1992; see also, e.g., Metzger et al., 1988. Many useful vectors are known in the art and may be obtained from such vendors as Stratagene, New England BioLabs, Promega Biotech, and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts.
  • Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., EP 73,675A.
  • Non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al., 1978) or promoters derived from murine Moloney leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. Insect promoters may be derived from baculovirus.
  • the construct may be joined to an ampliflable gene (e.g., DHFR) so that multiple copies of the gene may be made.
  • DHFR e.g., DHFR
  • Enhancers and Eukaryotic Gene Expression Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1983). See also, e.g., U.S. Pat. Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.
  • expression vectors may replicate autonomously, they may also replicate by being inserted into the genome of the host cell, by methods well known in the art.
  • Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene ensures growth of only those host cells which express the inserts.
  • Typical selection genes encode proteins that a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc.; b) complement auxotrophic deficiencies, or c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • the choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.
  • the vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection (see, Kubo et al., 1988), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al., 1989 and Ausubel et al., 1992.
  • the introduction of the polynucleotides into the host cell by any method known in the art, including, inter alia, those described above, will be referred to herein as “transformation.”
  • the cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.
  • nucleic acids and polypeptides of the present invention may be prepared by expressing the HPC2, ELAC 1 or ELAC2 nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells.
  • prokaryotic hosts are strains of Escherichia coli , although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
  • Mammalian or other eukaryotic host cells such as those of yeast, filamentous fungi, plant, insect, or amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. See, Jakoby and Pastan, 1979. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features. An example of a commonly used insect cell line is SF9.
  • Clones are selected by using markers depending on the mode of the vector construction.
  • the marker may be on the same or a different DNA molecule, preferably the same DNA molecule.
  • the transformant may be selected, e.g., by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.
  • Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of HPC2, ELAC1 or ELAC2 polypeptides.
  • HPC2, ELAC1 or ELAC2 gene products can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats and non-human primates, e.g., baboons, monkeys and chimpanzees, may be used to generate HPC2, ELAC 1 or ELAC2 transgenic animals.
  • any technique known in the art may be used to introduce the HPC2, ELAC1 or ELAC2 gene transgene into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985); gene targeting in embryonic stem cells (Thompson et al., 1989); electroporation of embryos (Lo, 1983); and sperm-mediated gene transfer (Lavitrano et al., 1989); etc.
  • pronuclear microinjection U.S. Pat. No. 4,873,191
  • retrovirus mediated gene transfer into germ lines Van der Putten et al., 1985
  • gene targeting in embryonic stem cells Thompson et al., 1989
  • electroporation of embryos Lo, 1983
  • sperm-mediated gene transfer Lavitrano et al., 1989
  • the present invention provides for transgenic animals that carry the HPC2, ELAC1 or ELAC2 transgene in all their cells, as well as animals which carry the transgene in some, but not all of their cells, i.e., mosaic animals.
  • the transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (1992). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • ELAC1 or ELAC2 gene transgene be integrated into the chromosomal site of the endogenous HPC2, ELAC1 or ELAC2 gene
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous HPC2, ELAC1 or ELAC2 gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous HPC2, ELAC1 or ELAC2 gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous HPC2, ELAC1 or ELAC2 gene in only that cell type, by following, for example, the teaching of Gu et al. (1994).
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • the expression of the recombinant HPC2, ELAC1 or ELAC2 gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of HPC2, ELAC1 or ELAC2 gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the HPC2, ELAC1 or ELAC2 transgene product.
  • Antisense polynucleotide sequences are useful in preventing or diminishing the expression of the HPC2, ELAC1 or ELAC2 locus, as will be appreciated by those skilled in the art.
  • polynucleotide vectors containing all or a portion of the HPC2 locus or other sequences from the HPC2 region (particularly those flanking the HPC2 locus) may be placed under the control of a promoter in an antisense orientation and introduced into a cell. Expression of such an antisense construct within a cell will interfere with HPC2 transcription and/or translation and/or replication.
  • HPC2 gene sequences disclosed herein are used to identify homologous HPC2 gene sequences and proteins in other species. These HPC2 gene sequences and proteins are used in the diagnostic/prognostic, therapeutic and drug screening methods described herein for the species from which they have been isolated.
  • a biological sample such as blood is prepared and analyzed for the presence or absence of susceptibility alleles of HPC2.
  • a biological sample of the lesion is prepared and analyzed for the presence or absence of mutant alleles of HPC2. Results of these tests and interpretive information are returned to the health care provider for communication to the tested individual.
  • diagnoses may be performed by diagnostic laboratories, or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self-diagnosis.
  • the screening method involves amplification of the relevant HPC2 sequences.
  • the screening method involves a non-PCR based strategy.
  • Such screening methods include two-step label amplification methodologies that are well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.
  • the most popular method used today is target amplification.
  • the target nucleic acid sequence is amplified with polymerases.
  • One particularly preferred method using polymerase-driven amplification is the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the polymerase chain reaction and other polymerase-driven amplification assays can achieve over a million-fold increase in copy number through the use of polymerase-driven amplification cycles.
  • the resulting nucleic acid can be sequenced or used as a substrate for DNA probes.
  • the biological sample to be analyzed such as blood or serum
  • the biological sample to be analyzed may be treated, if desired, to extract the nucleic acids.
  • the sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting.
  • the targeted region of the analyte nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art.
  • Analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte.
  • the region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of human chromosome 17. Therefore, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome.
  • the stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide.
  • Detection, if any, of the resulting hybrid is usually accomplished by the use of labeled probes.
  • the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly.
  • Suitable labels, and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation, random priming or kinasing), biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly triggered dioxetanes), enzymes, antibodies, gold nanoparticles and the like.
  • Variations of this basic scheme are known in the art, and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labeled moiety. A number of these variations are reviewed in, e.g., Matthews and Kricka, 1988; Landegren et al., 1988; Mifflin, 1989; U.S. Pat. No. 4,868,105, and in EPO Publication No. 225,807.
  • non-PCR based screening assays are also contemplated in this invention.
  • This procedure hybridizes a nucleic acid probe (or an analog such as a methyl phosphonate backbone replacing the normal phosphodiester), to the low level DNA target.
  • This probe may have an enzyme covalently linked to the probe, such that the covalent linkage does not interfere with the specificity of the hybridization.
  • This enzyme-probe-conjugate-target nucleic acid complex can then be isolated away from the free probe enzyme conjugate and a substrate is added for enzyme detection. Enzymatic activity is observed as a change in color development or luminescent output resulting in a 10 3 -10 6 increase in sensitivity.
  • the small ligand attached to the nucleic acid probe is specifically recognized by an antibody-enzyme conjugate.
  • digoxigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate which turns over a chemiluminescent substrate.
  • the small ligand is recognized by a second ligand-enzyme conjugate that is capable of specifically complexing to the first ligand.
  • a well known embodiment of this example is the biotin-avidin type of interactions. For methods for labeling nucleic acid probes and their use in biotin-avidin based assays see Rigby et al., 1977 and Nguyen et al., 1992.
  • the nucleic acid probe assays of this invention will employ a cocktail of nucleic acid probes capable of detecting HPC2.
  • a cocktail of nucleic acid probes capable of detecting HPC2 in one example to detect the presence. of HPC2 in a cell sample, more than one probe complementary to HPC2 is employed and in particular the number of different probes is alternatively 2, 3, or 5 different nucleic-acid probe sequences.
  • the cocktail includes probes capable of binding to the allele-specific mutations identified in populations of patients with alterations in HPC2.
  • any number of probes can be used, and will preferably include probes corresponding to the major gene mutations identified as predisposing an individual to prostate cancer.
  • the neoplastic condition of lesions can also be detected on the basis of the alteration of wild-type HPC2 polypeptide. Such alterations can be determined by sequence analysis in accordance with conventional techniques. More preferably, antibodies (polyclonal or monoclonal) are used to detect differences in, or the absence of, HPC2 peptides. The antibodies may be prepared as discussed above under the heading “Antibodies” and as further shown in Examples 16 and 17. Other techniques for raising and purifying antibodies are well known in the art and any such techniques may be chosen to achieve the preparations claimed in this invention. In a preferred embodiment of the invention, antibodies will immunoprecipitate HPC2 proteins from solution as well as react with HPC2 protein on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, antibodies will detect HPC2 proteins in paraffin or frozen tissue sections, using immunocytochemical techniques.
  • Preferred embodiments relating to methods for detecting HPC2 or its mutations include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies.
  • ELISA enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • Exemplary sandwich assays are described by David et al. in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference, and exemplified in Example 19.
  • This invention is particularly useful for screening compounds by using the HPC2, ELAC1 or ELAC2 polypeptide or binding fragment thereof in any of a variety of drug screening techniques.
  • HPC2, ELAC1 or ELAC2 polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface.
  • One method of drug screening utilizes eucaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays.
  • the present invention provides methods of screening for drugs comprising contacting such an agent with an HPC2, ELAC1 or ELAC2 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the HPC2, ELAC1 or ELAC2 polypeptide or fragment, or (ii) for the presence of a complex between the HPC2, ELAC1 or ELAC2 polypeptide or fragment and a ligand, by methods well known in the art. In such competitive binding assays the HPC2, ELAC1 or ELAC2 polypeptide or fragment is typically labeled.
  • Free HPC2, ELAC1 or ELAC2 polypeptide or fragment is separated from that present in a protein:protein complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to HPC2, ELAC1 or ELAC2 or its interference with HPC2:ligand, ELAC1:ligand or ELAC2:ligand binding, respectively.
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the HPC2, ELAC1 or ELAC2 polypeptides and is described in detail in Geysen (published PCT WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HPC2, ELAC1 or ELAC2 polypeptide and washed. Bound HPC2, ELAC1 or ELAC2 polypeptide is then detected by methods well known in the art.
  • Purified HPC2, ELAC1 or ELAC2 can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the HPC2, ELAC1 or ELAC2 polypeptide on the solid phase.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the HPC2, ELAC1 or ELAC2 polypeptide compete with a test compound for binding to the HPC2, ELAC1 or ELAC2 polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the HPC2, ELAC1 or ELAC2 polypeptide.
  • a further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional HPC2, ELAC1 or ELAC2 gene. These host cell lines or cells are defective at the HPC2, ELAC1 or ELAC2 polypeptide level. The host cell lines or cells are grown in the presence of drug compound. The rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of HPC2, ELAC1 or ELAC2 defective cells.
  • a method of screening for a substance which modulates activity of a polypeptide may include contacting one or more test substances with the polypeptide in a suitable reaction medium, testing the activity of the treated polypeptide and comparing that activity with the activity of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances.
  • test substances may be screened for ability to interact with the polypeptide, e.g., in a yeast two-hybrid system (e.g., Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans, 1992; Lee et al., 1995).
  • This system may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide.
  • the screen could be used to screen test substances for binding to an HPC2, ELAC1 or ELAC2 specific binding partner, or to find mimetics of an HPC2, ELAC1 or ELAC2 polypeptide.
  • the goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, 1991.
  • one first determines the three-dimensional structure of a protein of interest (e.g., HPC2 polypeptide) or, for example, of the HPC2-receptor or ligand complex, by x-ray crystallography, by computer modeling or most typically, by a combination of approaches.
  • peptides e.g., HPC2 polypeptide
  • an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined.
  • Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • improved HPC2, ELAC1 or ELAC2 polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of HPC2, ELAC1 or ELAC2 polypeptide activity By virtue of the availability of cloned HPC2, ELAC1 and ELAC2 sequences, sufficient amounts of the HPC2, ELAC1 or ELAC2 polypeptide may be made available to perform such analytical studies as x-ray crystallography.
  • the knowledge of the HPC2, ELAC1 and ELAC2 protein sequences provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.
  • the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e., manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention extends in various aspects not only to a substance identified using a nucleic acid molecule as a modulator of polypeptide activity, in accordance with what is disclosed herein, but also a phanmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g., for treatment of prostate cancer, use of such a substance in the manufacture of a composition for administration, e.g., for treatment of prostate cancer, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.
  • a substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature.
  • Non-peptide “small molecules” are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use.
  • the designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal.
  • Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property.
  • the pharmacophore Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
  • a range of sources e.g., spectroscopic techniques, x-ray diffraction data and NMR.
  • Computational analysis, similarity mapping which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms
  • other techniques can be used in this modeling process.
  • the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • a method is also provided of supplying wild-type HPC2 function to a cell which carries mutant HPC2 alleles. Supplying such a function should suppress neoplastic growth of the recipient cells.
  • the wild-type HPC2 gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. If a gene fragment is introduced and expressed in a cell carrying a mutant HPC2 allele, the gene fragment should encode a part of the HPC2 protein which is required for non-neoplastic growth of the cell.
  • the wild-type HPC2 gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant HPC2 gene present in the cell.
  • Such recombination requires a double recombination event which results in the correction of the HPC2 gene mutation.
  • Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used.
  • Methods for introducing DNA into cells such as electroporation, calcium phosphate coprecipitation and viral transduction are known in the art, and the choice of method is within the competence of the practitioner.
  • Cells transformed with the wild-type HPC2 gene can be used as model systems to study cancer remission and drug treatments which promote such remission.
  • the HPC2 gene or fragment may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cancer cells.
  • Such gene therapy is particularly appropriate for use in both cancerous and pre-cancerous cells, in which the level of HPC2 polypeptide is absent or diminished compared to normal cells. It may also be useful to increase the level of expression of a given HPC2 gene even in those tumor cells in which the mutant gene is expressed at a “normal” level, but the gene product is not fully functional.
  • Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman (1991) or Culver (1996).
  • Cells from a patient's tumor would be first analyzed by the diagnostic methods described above, to ascertain the production of HPC2 polypeptide in the tumor cells.
  • a virus or plasmid vector (see further details below), containing a copy of the HPC2 gene linked to expression control elements and capable of replicating inside the tumor cells, is prepared.
  • the vector may be replication deficient and is replicated in helper cells for use in gene therapy.
  • Suitable vectors are known, such as disclosed in U.S. Pat. No. 5,252,479 and PCT published application WO 93/07282 and U.S. Pat. Nos.
  • the vector is then injected into the patient, either locally at the site of the tumor or systemically (in order to reach any tumor cells that may have metastasized to other sites). If the transfected gene is not permanently incorporated into the genome of each of the targeted tumor cells, the treatment may have to be repeated periodically.
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods.
  • viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson and Akrigg, 1992; Stratford-Perricaudet et al., 1990; Schneider et al., 1998), vaccinia virus (Moss, 1992; Moss, 1996), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990; Russell and Hirata, 1998), herpes viruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink
  • Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical techniques, for example microinjection (Anderson et al, 1980; Gordon et al, 1980; Brinster et al, 1981; Costantini and Lacy, 1981); membrane fusion-mediated transfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et al, 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1991); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al, 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al, 1989; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al., 1990; Curiel
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the tumor cells and not into the surrounding nondividing cells.
  • the retroviral vector producer cell line can be injected into tumors (Culver et al., 1992). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumors.
  • plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector.
  • the trimolecular complex is then used to infect cells.
  • the adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged.
  • adenovirus based vectors see Schneider et al. (1998) and U.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500.
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992).
  • Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein.
  • the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes HPC2, expression will produce HPC2. If the polynucleotide encodes an antisense polynucleotide or a ribozyme, expression will produce the antisense polynucleotide or ribozyme. Thus in this context, expression does not require that a protein product be synthesized.
  • the vector also contains a promoter functional in eukaryotic cells.
  • the cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters include those described above.
  • the expression vector may also include sequences, such as selectable markers and other sequences described herein.
  • Receptor-mediated gene transfer is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine.
  • Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type.
  • One appropriate receptor/ligand pair may include the estrogen receptor and its ligand, estrogen (and estrogen analogues).
  • These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs.
  • coinfection with adenovirus can be included to disrupt endosome function.
  • the therapy involves two steps which can be performed singly or jointly.
  • prepubescent females who carry an HPC2 susceptibility allele are treated with a gene delivery vehicle such that some or all of their mammary ductal epithelial precursor cells receive at least one additional copy of a functional normal HPC2 allele.
  • the treated individuals have reduced risk of prostate cancer to the extent that the effect of the susceptible allele has been countered by the presence of the normal allele.
  • predisposed young females in particular women who have received the proposed gene therapeutic treatment, undergo hormonal therapy to mimic the effects on the prostate of a full term pregnancy.
  • Peptides which have HPC2, ELAC1 or ELAC2 activity can be supplied to cells which carry mutant or missing HPC2, ELAC1 or ELAC2 alleles.
  • Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors.
  • HPC2, ELAC1 or ELAC2 polypeptide can be extracted from HPC2-, ELAC1- or ELAC2-producing mammalian cells.
  • the techniques of synthetic chemistry can be employed to synthesize HPC2, ELAC1 or ELAC2 protein. Any of such techniques can provide the preparation of the present invention which comprises the HPC2, ELAC1 or ELAC2 protein. Preparation is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.
  • Active HPC2, ELAC1 or ELAC2 molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Extracellular application of the HPC2, ELAC1 or ELAC2 gene product may be sufficient to affect tumor growth. Supply of molecules with HPC2 activity should lead to partial reversal of the neoplastic state. Other molecules with HPC2 activity (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having substantially similar function are also used for peptide therapy.
  • cells and animals which carry a mutant HPC2, ELAC1 or ELAC2 allele can be used as model systems to study and test for substances which have potential as therapeutic agents.
  • the cells are typically cultured epithelial cells. These may be isolated from individuals with HPC2, ELAC1 or ELAC2 mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the HPC2, ELAC1 or ELAC2 allele, as described above.
  • the neoplastically transformed phenotype of the cell is determined. Any trait of neoplastically transformed cells can be assessed, including anchorage-independent growth, tumorigenicity in nude mice, invasiveness of cells, and growth factor dependence. Assays for each of these traits are known in the art.
  • Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant HPC2, ELAC1 or ELAC2 alleles, usually from a second animal species, as well as insertion of disrupted homologous genes.
  • the endogenous HPC2, ELAC1 or ELAC2 gene(s) of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques (Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992) to produce knockout or transplacement animals.
  • a transplacement is similar to a knockout because the endogenous gene is replaced, but in the case of a transplacement the replacement is by another version of the same gene.
  • test substances After test substances have been administered to the animals, the growth of tumors must be assessed. If the test substance prevents or suppresses the growth of tumors, then the test substance is a candidate therapeutic agent for the treatment of the cancers identified herein. These animal models provide an extremely important testing vehicle for potential therapeutic products.
  • transgenic animals are produced which contain a functional transgene encoding a functional HPC2, ELAC1 or ELAC2 polypeptide or variants thereof.
  • Transgenic animals expressing HPC2, ELAC1 or ELAC2 transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of HPC2, ELAC1 or ELAC2.
  • Transgenic animals of the present invention also can be used as models for studying indications such as disease.
  • an HPC2, ELAC1 or ELAC2 transgene is introduced into a non-human host to produce a transgenic animal expressing a human or murine HPC2, ELAC1 or ELAC2 gene.
  • the transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene.
  • Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191; which is incorporated herein by reference), Brinster et al.
  • HPC2, ELAC1 or ELAC2 may be replaced by homologous recombination between the transgene and the endogenous gene; or the endogenous gene may be eliminated by deletion as in the preparation of “knock-out” animals.
  • an HPC2, ELAC1 or ELAC2 gene flanked by genomic sequences is transferred by microinjection into a fertilized egg.
  • the microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene.
  • Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish.
  • transgenic mice are generated which overexpress HPC2 or express a mutant form of the polypeptide.
  • the absence of an HPC2, ELAC1 or ELAC2 in “knock-out” mice permits the study of the effects that loss of HPC2, ELAC1 or ELAC2 protein has on a cell in vivo.
  • Knock-out mice also provide a model for the development of HPC2-related cancers.
  • transgenic animals and cell lines derived from such animals may find use in certain testing experiments.
  • transgenic animals and cell lines capable of expressing wild-type or mutant HPC2, ELAC1 or ELAC2 may be exposed to test substances. These test substances can be screened for the ability to reduce overexpression of wild-type HPC2, ELAC1 or ELAC2 or impair the expression or function of mutant HPC2, ELAC1 or ELAC2.
  • HPC2, ELAC1 or ELAC2 polypeptides, antibodies, peptides and nucleic acids of the present invention can be formulated in pharmaceutical compositions, which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, intrathecal, epineural or parenteral.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions.
  • any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets).
  • tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques.
  • the active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698.
  • the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin.
  • the carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like.
  • the compounds When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.
  • the active agent is preferably administered in a therapeutically effective amount.
  • the actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington 's Pharmaceutical Sciences.
  • targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.
  • these agents could be produced in the target cell, e.g. in a viral vector such as described above or in a cell based delivery system such as described in U.S. Pat. No. 5,550,050 and published PCT application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635, designed for implantation in a patient.
  • the vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are more tissue specific to the target cells.
  • the cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the active agent.
  • the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See for example, EP 425,731A and WO 90/07936.
  • HPC2 is a prostate cancer susceptibility gene.
  • the kindred 4289 allele carries all three missense changes, Leu 217, Thr 541 and His 781.
  • Examination of the pedigree suggests that the allele is dominant and sufficiently deleterious to demonstrate visible segregation with prostate cancer in an extended pedigree.
  • the youngest affected carrier of this variant 4289.003, is homozygous for Leu 217 and Thr 541.
  • the second ovarian cancer case in the pedigree is an obligate carrier of a Leu 217+ Thr 541 allele.
  • the observation of two ovarian cancer cases in this pedigree, both of whom carry deleterious alleles of ELAC2 is consistent with the possibility that the phenotype conferred by deleterious variants in this gene is not restricted to prostate cancer susceptibility.
  • genotype information from pedigree members at multiple moderate risk loci may allow refined definition of the liability classes used by multipoint linkage software, thereby increasing the power of the analysis.
  • Stratification of cases by genotype would also facilitate positional cloning projects by providing another criterion by which to distinguish between true recombinant carriers and confounding sporadic cases.
  • HPC2 alleles are screened for mutations either directly or after cloning the alleles.
  • the alleles are tested for the presence of nucleic acid sequence differences from the normal allele using any suitable technique, including but not limited to, one of the following methods: fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCP), linkage analysis, RNase protection assay, allele specific oligonucleotide (ASO), dot blot analysis and PCR-SSCP analysis.
  • FISH fluorescent in situ hybridization
  • PFGE analysis direct DNA sequencing
  • SSCP single stranded conformation analysis
  • ASO allele specific oligonucleotide
  • RNA transcripts of both the cloned alleles and normal HPC2 gene or appropriate fragment are determined and then compared, or (2) the RNA transcripts of the HPC2 gene or gene fragment are hybridized to single stranded whole genomic DNA from an individual to be tested, and the resulting heteroduplex is treated with Ribonuclease A (RNase A) and run on a denaturing gel to detect the location of any mismatches.
  • RNase A Ribonuclease A
  • the alleles of the HPC2 gene in an individual to be tested are cloned using conventional techniques. For example, a blood sample is obtained from the individual. The genomic DNA isolated from the cells in this sample is partially digested to an average fragment size of approximately 20 kb. Fragments in the range from 18-21 kb are isolated. The resulting fragments are ligated into an appropriate vector. The sequences of the clones are then determined and compared to the normal HPC2 gene.
  • PCRs polymerase chain reactions
  • PCRs can also be performed with primer pairs based on any sequence of the normal HPC2 gene.
  • primer pairs for one of the introns can be prepared and utilized.
  • RT-PCR can also be performed on the mRNA.
  • the amplified products are then analyzed by single stranded conformation polymorphisms (SSCP) using conventional techniques to identify any differences and these are then sequenced and compared to the normal gene sequence.
  • SSCP single stranded conformation polymorphisms
  • the second method employs RNase A to assist in the detection of differences between the normal HPC2 gene and defective genes. This comparison is performed in steps using small ( ⁇ 500 bp) restriction fragments of the HPC2 gene as the probe.
  • the HPC2 gene is digested with a restriction enzyme(s) that cuts the gene sequence into fragments of approximately 500 bp. These fragments are separated on an electrophoresis gel, purified from the gel and cloned individually, in both orientations, into an SP6 vector (e.g., pSP64 or pSP65).
  • the SP6-based plasmids containing inserts of the HPC2 gene fragments are transcribed in vitro using the SP6 transcription system, well known in the art, in the presence of [ ⁇ - 32 P]GTP, generating radiolabeled RNA transcripts of both strands of the gene.
  • RNA transcripts are used to form heteroduplexes with the allelic DNA using conventional techniques. Mismatches that occur in the RNA:DNA heteroduplex, owing to sequence differences between the HPC2 fragment and the HPC2 allele subclone from the individual, result in cleavage in the RNA strand when treated with RNase A. Such mismatches can be the result of point mutations or small deletions in the individual's allele. Cleavage of the RNA strand yields two or more small RNA fragments, which run faster on the denaturing gel than the RNA probe itself.
  • any differences which are found, will identify an individual as having a molecular variant of the HPC2. These variants can take a number of forms. The most severe forms would be frame shift mutations or large deletions which would cause the gene to code for an abnormal protein or one which would significantly alter protein expression. Less severe disruptive mutations would include small in-frame deletions and nonconservative base pair substitutions which would have a significant effect on the protein produced, such as changes to or from a cysteine residue, from a basic to an acidic amino acid or vice versa, from a hydrophobic to hydrophilic amino acid or vice versa, or other mutations which would affect secondary or tertiary protein structure. Silent mutations or those resulting in conservative amino acid substitutions would not generally be expected to disrupt protein function.
  • BAC DNA was purified and directly sequenced as previously described (Couch et al., 1996). DNA sequences at the SP6 and T7 ends of isolated BAC clones were used to develop STSs that were used for mapping and contig extension. Greater than 95% sequence coverage of the FIG. 1 BAC tiling path was obtained by sequencing plasmid sublibraries generated from these clones. The sequence data obtained were assembled into contigs using Acembly, version 4.3 (U. Sauvage, D. Thierry-Mieg and J. EMS-Mieg; Centre National de la Recherche Scientifique, France). Subsequently, a complete sequence of this interval was released by the MIT genome center.
  • Kindred 4320 was also ascertained from a sibship of early onset prostate cancer cases. In this kindred 3 affected brothers and an affected nephew share a piece of chromosome 17p from D17S786 down to and including myr0084. Together, the kindred 4325 and kindred 4320 recombinants define a minimal region of about 1 megabase (FIG. 2A); this localization is well supported by a larger set of recombinants in both directions.
  • haplotypes used for both localization and mutation screening, were defined as haplotypes shared by 4 or more cases and giving a LOD score >1.0 in the pedigree where they were identified, or haplotypes shared by 6 or more cases irrespective of LOD score;
  • Oligonucleotide primer pairs for the markers located in the interval were synthesized and used to screen libraries of bacterial artificial chromosomes (BACs) to identify BACs in the region.
  • the initial set of markers used was D17S969, WI-2437, WI-2335, D17S947, and D17S799 (FIG. 2A).
  • BACs identified with these markers were end-sequenced.
  • PCR primers designed from those end sequences were used as markers to arrange the initial BACs into contigs.
  • the outermost marker from each contig was used in successive rounds of BAC library screening, eventually enabling the completion of a BAC clone contig that spanned the genetically defined interval.
  • a set of overlapping but non-redundant BAC clones that spanned this interval was then selected for use in subsequent molecular cloning protocols such as genomic sequencing.
  • a BLASTn Altschul et al., 1990 search of genomic sequences from BAC 31k12 against dbEST identified two independent sets of human ESTs that, when parsed across the BAC 31k12 genomic sequences, revealed the presence of two independent multi-exon candidate genes, 04CG09 and the HPC2 gene (FIG. 2B).
  • a subset of the EST sequences assigned to HPC2 was assembled to produce a tentative partial cDNA sequence for the gene.
  • the individual exons of the human HPC2 gene were identified by parsing that tentative cDNA sequence across the BAC 31k12 genomic sequence (see schematics in FIG. 2B). After we had identified the HPC2 gene, the MIT genome sequencing completely sequenced another BAC, 597 m12, that also contains all of the exons of HPC2 (GenBank accession # AC005277) The sequence of the human HPC2 gene was corrected both by comparison of the sequences of the individual exons from the tentative cDNA assembly to the corresponding genomic sequences of BACs 31k12 and 597 m12, and by mutation screening the gene from a set of human genomic DNAs (see Example 8).
  • the original tentative human HPC2 cDNA sequence contained neither the start codon nor any of the 5′ UTR. These were obtained by biotin capture 5′ RACE (Tavtigian et al., 1996). Briefly, a biotinylated reverse primer, CA4cg07.BR2, was designed from the sequence of the third exon of the human HPC2 gene and used, along with the anchor primer SampA, for a first round of PCR amplification from human fetal liver cDNA that had been prepared such that the 5′ ends of cDNA molecules are anchored with the sequence stag1.
  • the resulting PCR products were captured on streptavidin paramagnetic particles (Dynal), washed, and used as template in a second round PCR amplification.
  • a phosphorylated reverse primer, CA4cg07.PR2 was designed from the sequence of the second exon of the human HPC2 sequence and used, along with the nested phosphorylated anchor primer SampB, for the second round PCR amplification.
  • the resulting 5′ RACE products were gel purified and sequenced with the primer CA4cg07.PR2 using dye-terminator chemistry and ABI 377 sequencers. Analysis of the sequences of these 5′ RACE products yielded both the start codon and part of the 5′ UTR including an in-frame stop codon (FIG. 4). Sequences of the human primers used for 5′ RACE are given in Table 5.
  • a full length human HPC2 cDNA was amplified from human head and neck cDNA using the primers CA4cg7.ATG and CA4cg7.TGA.
  • the cDNA was ligated into the vector pGEM-T Easy (Promega) and transformed into E. coli .
  • the sequence of the cDNA clone was confirmed by dye terminator sequencing on ABI 377 sequencers. Sequences of primers used to amplify the cDNA construct and confirm the sequence of the cDNA clone are also given in Table 5.
  • a BLAST search of the assembled HPC2 cDNA sequence against dbEST identified 5 mouse ESTs that derived from a very similar gene, the mouse ortholog of HPC2, Mm.HPC2; their accession numbers are listed in Table 6.
  • TABLE 6 Mouse ESTs Used to Assemble a Tentative Partial Mm.HPC2 cDNA Sequence EST Accession # Exon Span AA563096 1 ⁇ 5 AA518169 8 ⁇ 14 AI132016 16 ⁇ 17 AA184645 19 ⁇ 24 AA174437 24 ⁇ 24
  • the original partial Mm.HPC2 cDNA sequence contained the start codon but little of the 5′ UTR. More extensive 5′ UTR sequence was obtained b 5′ RACE.
  • a biotinylated reverse primer, m04cg07BR1 was designed from the sequence of the fourth exon of the mouse HPC2 gene and used, along with the anchor primer 5ampA, for a first round of PCR amplification from mouse embryo cDNA that had been prepared such that the 5′ ends of cDNA molecules are anchored with the sequence 5tag1.
  • the resulting PCR products were captured on streptavidin paramagnetic particles (Dynal), washed, and used as template in a second round PCR amplification.
  • a phosphorylated reverse primer, m04cg07PR1 was designed from the sequence of the third exon of the mouse HPC2 sequence and used, along with the nested phosphorylated anchor primer SampB, for the second round PCR amplification.
  • the resulting 5′ RACE products were gel purified and sequenced with the primers m04cg07PR1 and m04cg07 exon2 rev using dye-terminator chemistry and ABI 377 sequencers. Analysis of the sequences of these 5′ RACE products yielded both the start codon and part of the 5′ UTR including an in-frame stop codon (FIG. 4). Sequences of the primers used for 5′ RACE are given in Table 7.
  • BAC 428n12 was obtained from a mouse genomic library by screening the library by PCR with a pair of primers (04CG7. m11f1 and 04CG7. m11r1, Table 7) derived from exon 11 of the mouse HPC2 cDNA sequence.
  • a primer pair derived from the SP6 end sequence of BAC 428n12 (428n12.S6.F1 and 428n12.S6.F1, Table 7) was used to screen the mouse BAC library by PCR; several overlapping BACs, including BAC 199n11, were identified.
  • BACs 428n12 and 199n11 were sequenced with a series of 13 sequencing primers (mcg7f1 to mcg7r7, Table 7) derived from mouse HPC2 cDNA dye-terminator chemistry and ABI 377 sequencers. A subset of these sequences were assembled into a genomic sequence contig extending from 280 bp upstream of the ATG start codon of exon 1 into exon 3.
  • a full length mouse HPC2 cDNA is amplified from mouse embryo, placenta, or fetal brain cDNA using the primers msCA4cg7.f out and msCA4cg7.r out
  • the cDNA is reamplified with the primers msCA4cg7.ATG and msCA4cg7.TGA.
  • the resulting PCR products are gel purified, ligated into the vector pGEM-T Easy (Promega), and transformed into E. coli .
  • the sequence of the cDNA clone are confirmed dye terminator sequencing on ABI 377 sequencers. Sequences of primers in use to amplify the cDNA construct are also given in Table 7.
  • Prehybridization and hybridization were performed at 42° C. in 50% formamide, 5 ⁇ SSPE, 1.0% SDS, 5 ⁇ Denhardt's mixture, 0.2 mg/mL denatured salmon sperm DNA, and 2 ⁇ g/mL poly(A). Dextran sulfate (4% v/v) was included in the hybridization solution only. The membranes were washed twice in 2 ⁇ SSC/0.1% SDS at 20° C. for 30 minutes, followed by a stringency wash in 0.1 ⁇ SSC/0.1% SDS at 50° C. for 30 minutes.
  • each amplicon (1A-1P, i.e., forward A and reverse P of amplicon 1)
  • 10-20 ng of genomic DNA were subjected to a 25 cycle primary amplification, after which the PCR products were diluted 45-fold and reamplified using nested M13-tailed primers (1B-1Q, 1C-1R i.e., nested forward B and nested reverse Q of amplicon 1 or nested forward C and nested reverse R of amplicon 1) for another 23 cycles.
  • nested M13-tailed primers (1B-1Q, 1C-1R i.e., nested forward B and nested reverse Q of amplicon 1 or nested forward C and nested reverse R of amplicon 1
  • samples were amplified with Taq Platinum (Life Technologies) DNA polymerase; cycling parameters included an initial denaturation step at 95° C. for 3 min, followed by cycles of denaturation at 96° C.
  • Kindred 4102 was ascertained as a high risk cluster with eight prostate cancer cases in a three generation pedigree. Genotyping revealed that six of the eight cases shared a chromosome 17p haplotype. The youngest (age at diagnosis of 46) affected carrier of this shared haplotype, 4102.013 (i.e., kindred #4102, individual #013; FIG. 5A), was selected for mutation screening. On mutations screening lymphocyte DNA from 4102.013, we detected a frameshift, 1641 insG, in ELAC2. A test for segregation revealed that the frameshift was not on the father's chromosome, but rather was inherited through the carrier's mother, 4102.002.
  • Her affected uncle 4102.053 was diagnosed with and died of prostate cancer at age 76 in the 1960s. Genotyping of his children demonstrated that he was an obligate frameshift carrier. In all, there are five male frameshift carriers over age 45 in the pedigree. Of these, three have prostate cancer, the fourth has a PSA of 5.7 at age 71, and the fifth has a PSA of 4.2 at age 74 (FIG. 5 A). The frameshift occurs at His 548, within the histidine motif (FIGS. 6 A-B) and is predicted to be quite disruptive to the protein.
  • missense change occurs in a very highly charged stretch of amino acid residues near the C-terminus of the protein. Arg 781 is conserved in mouse (FIGS. 6 A-B), and the charge character of the sequence segment is conserved in C. elegans . While one cannot definitively predict that this missense change will affect protein function, expansion from a single affected mutation carrier to a pedigree with a LOD score of 1.3 provides good evidence that the mutation is in fact deleterious.
  • the Thr 541 carrier frequency is not significantly higher in the cases than the unaffected pedigree members.
  • both comparisons support the hypothesis that the allele bearing both Thr 541 and Leu 217 is more deleterious than the allele bearing just Leu 217.
  • DNA fragments encoding all or portions of HPC2 are ligated to a two-hybrid DNA-binding domain vector such as pGBT.C such that the coding sequence of HPC2 is in-frame with coding sequence for the Ga14p DNA-binding domain.
  • a plasmid that encodes a DNA-binding domain fusion to a fragment of HPC2 is introduced into the yeast reporter strain (such as 3692) along with a library of cDNAs fused to an activation domain. Transformants are spread onto 20-150 nun plates of selective media, such as yeast minimal media lacking leucine, tryptophan, and histidine, and containing 25 mM 3-amino-1,2,4-triazole.
  • yeast colonies are assayed for expression of the lacZ reporter gene by ⁇ -galactosidase filter assay. Colonies that both grow in the absence of histidine and are positive for production of ⁇ -galactosidase are chosen for further characterization.
  • the activation domain plasmid is purified from positive colonies by the smash-and-grab technique. These plasmids are introduced into E. coli (e.g., DH10B (Gibco BRL) by electroporation and purified from E. coli by the alkaline lysis method. To test for the specificity of the interaction, specific activation domain plasmids are cotransformed into strain J692 with plasmids encoding various DNA-binding domain fusion proteins, including fusions to segments of HPC2 and human lamin C. Transformants from these experiments are assayed for expression of the HIS3 and lacZ reporter genes. Positives that express reporter genes with Hs.HPC2 constructs and not with lamin C constructs encode bona fide HPC2-interacting proteins. These proteins are identified and characterized by sequence analysis of the insert of the appropriate activation domain plasmid.
  • E. coli e.g., DH10B (Gibco BRL) by electroporation and purified
  • the evolutionary history of the genes present in the daughter species can be quite complex because the individual genes can evolve through a diverse set of processes including nucleotide substitution, insertion, deletion, gene duplication, gene conversion, lateral transfer, etc. Even so, the evolutionary history of related genes in related organisms can often be sorted out, especially if the pair/set of species share a relatively recent common ancestor or if the genes being analyzed evolved primarily through nucleotide substitutions and/or small insertions and/or small deletions, but not gene duplications or gene conversions. When, upon analysis, it appears that a single gene in one species and a single gene in another species have evolved from a single gene in a common ancestor species, those genes are termed orthologs.
  • the human HPC2 cDNA sequence was assembled from a combination of ESTs, hybrid selected clones, and 5′ RACE (Rapid Amplification of cDNA Ends) products; the orthologous mouse Elac2 cDNA sequence was assembled from ESTs and 5′ RACE products.
  • Conceptual translation of the human cDNA sequence yielded a protein of 826 amino acids; parsing the cDNA sequence across the corresponding genomic sequence revealed 24 coding exons (FIG. 3).
  • Mouse Elac2 encodes a protein of 831 residues in 25 exons.
  • BLAST Altschul et al., 1990 searches of the ELAC2 sequence against GenBank readily revealed a single ortholog in S.
  • RNA blots Hybridization of RNA blots to labeled fragments of human ELAC2 cDNA revealed a single transcript of approximately 3 kb (FIGS. 11 A-D), in agreement with our full-length cDNA assembly of 2,970 bp.
  • the transcript was detected in all tissues surveyed and, like BRCA1 and BRCA2, was most abundant in testis. The apparent size of the transcript agrees well with our full length cDNA assembly, 2970 bp. There was no evidence from RNA blots, EST sequences, or RT-PCR experiments of significant alternative splicing of the transcript.
  • thermoautotrophicum gi2622965 was added into the multiprotein alignment (FIGS. 6 A-B), it became apparent that two distinct groups of proteins were represented; a group of larger proteins (800-900 aa) restricted to the eukaryotes, and a group of smaller proteins (300 to 400 aa) that align with the C-terminal half of the former group and includes sequences from the eukaryotes, eubacteria, and archaebacteria. As the 363 residue human protein falls into this second group and is more similar to E. coli elaC than is ELAC2, we will refer to it as ELAC1.
  • the second family encodes the 73 kDa subunit of the mRNA cleavage and polyadenylation specificity factor (CPSF73) (Chanfreau et al., 1996; Jenny et al., 1994; Jenny et al., 1996).
  • members of this latter gene family are present in both eukaryotes and archaebacteria, as well as a cyanobacterium.
  • ELAC1/2, PSO2 and CPSF73 are equally similar to each other (FIGS. 9 and 13); indeed they were originally placed in a single COG (Tatusov et al., 1997).
  • YKRO79C Given the phylogenetic conservation of the ELAC1 domain and the observation that S. cerevisiae encodes only a single member of this gene family, YKRO79C, we asked whether it is an essential gene. To answer this question, we performed one-step gene disruption of YKRO79C using URA3 as a selectable marker in yeast diploid cells. Two heterozygote knockout strains were sporulated and tetrads were dissected. Each tetrad yielded 1 or 2 viable haploid colonies; these were all URA and YKRO79C wt. Thus we concluded that, like YSH1, YKRO79C is an essential gene.
  • ELAC 1/2, PSO2 and CPSF73 proteins share a series of sequence features, some shared pairwise between the gene families and others by all three. Strikingly, all three families have three or four conserved histidine or cysteine positions, past the histidine motif, that lie within these shared regions and can be aligned across the gene families (FIG. 9).
  • a number of members of the ELAC1/2 family are annotated in GenBank as sulfatases or sulfatase homologs.
  • the atsA protein contains a histidine motif and has been demonstrated to have aryl sulfatase activity in vitro (Barbeyron et al., 1995), though its sequence does not contain any of the typical sulfatase motifs listed by PROSITE. No other experimentally verified aryl sulfatase contains the histidine motif.
  • the E. coli protein most similar to A.
  • AtsA may well be a diverged member of the ELAC1 gene family (BLASTp and alignment not shown). Accordingly, ELAC1 family members should be tested for aryl sulfatase activity; however, it is not apparent whether ELAC1 and ELAC2 family members have the same substrate.
  • orthologs of HPC2 have been identified in chimpanzee and gorilla. These are ptELAC2 (Pan troglodytes ELAC2) and ggELAC2 (Gorilla gorilla ELAC2).
  • shading criteria were identity (white on black) or conservative substitution (white on gray) for all ELAC2 sequences with a residue at that position, with four of the five sequences actually having to have a residue at that position. Shaded positions in the ELAC2 sequences were propagated into the ELAC1 sequences.
  • two shading criteria were used: (1) Identity or conservative substitution across the ELAC2 N-terminal alignment and identity or conservative substitution across either the ELAC1 or ELAC2 His motif.
  • Identity or conservative substitution across both the ELAC1 and ELAC2 His motif with some conservation across the ELAC2 N-terminal alignment.
  • shading criteria were identity or conservative substitution across two out of the three (CPSF73, PSO2, ELAC2) protein families represented.
  • HPC2 gene The structure and function of HPC2 gene are determined according to the following methods.
  • Mammalian expression vectors containing HPC2 cDNA are constructed and transfected into appropriate prostate carcinoma cells with lesions in the gene. Wild-type HPC2 cDNA as well as altered HPC2 cDNA are utilized. The altered HPC2 cDNA can be obtained from altered HPC2 alleles or produced as described below. Phenotypic reversion in cultures (e.g., cell morphology, doubling time, anchorage-independent growth) and in animals (e.g., tumorigenicity) is examined. The studies will employ both wild-type and mutant forms of the gene.
  • HPC2 protein The ability of HPC2 protein to bind to known and unknown DNA sequences is examined. Its ability to transactivate promoters is analyzed by transient reporter expression systems in mammalian cells. Conventional procedures such as particle-capture and yeast two-hybrid system are used to discover and identify any functional partners. The nature and functions of the partners are characterized. These partners in turn are targets for drug discovery.
  • Recombinant proteins are produced in E. coli , yeast, insect and/or mammalian cells and are used in crystallographic and NMR studies. Molecular modeling of the proteins is also employed. These studies facilitate structure-driven drug design.
  • the URA3 gene was PCR amplified with tailed primers resulting in a product flanked by 42 bp of YKRO79C coding sequences (amino acids 3-16 and 818-831).
  • the resulting PCR product was transformed into yeast diploid strain YPH501 (Stratagene); URA+clones were screened for disruption by the presence of a shorter PCR product at the YKR079C locus.
  • the knock-out clones were further confirmed by sequencing the shorter PCR product for the presence of URA3 sequences.
  • Two heterozygote knockout strains were sporulated and tetrads dissected. Each tetrad yielded 1 or 2 viable colonies. These were genotyped at YKRO79C and tested for growth on URA plates.
  • Segments of HPC2 coding sequence are expressed as fusion protein in E. coli .
  • the overexpressed proteins are purified by gel elution and used to immunize rabbits and mice using a procedure similar to the one described by Harlow and Lane, 1988. This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer, et al., 1993).
  • HPC2 coding sequence was cloned as a fusion protein in plasmid PET5A (Novagen, Inc., Madison, Wis.).
  • the HPC2 incorporated sequences might include SEQ ID NOs:1, 3 or 28 or portions thereof.
  • SDS/PAGE After induction with IPTG, the overexpression of a fusion protein with the expected molecular weight is verified by SDS/PAGE. Fusion proteins are purified from the gel by electroelution. The identification of the protein as the HPC2 fusion product is verified by protein sequencing at the N-terminus. Next, the purified protein is used as immunogen in rabbits.
  • Rabbits are immunized with 100 ⁇ g of the protein in complete Freund's adjuvant and boosted twice in 3 week intervals, first with 100 ⁇ g of immunogen in incomplete Freund's adjuvant followed by 100 ⁇ g of immunogen in PBS. Antibody containing serum is collected two weeks thereafter.
  • Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising intact HPC2 or HPC2 peptides (wild type or mutant) conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known.
  • the immunogen is mixed with an adjuvant.
  • Each mouse receives four injections of 10 to 100 ⁇ g of immunogen and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen.
  • Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.
  • Spleens are removed from immune mice and a single cell suspension is prepared (see Harlow and Lane, 1988). Cell fissions are performed essentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, Md.) are fused with immune spleen cells using polyethylene glycol as described by Harlow and Lane, 1988. Cells are plated at a density of 2 ⁇ 10 5 cells/well in 96 well tissue culture plates. Individual wells are examined for growth and the supernatants of wells with growth are tested for the presence of HPC2 specific antibodies by ELISA or RIA using wild type or mutant HPC2 target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.
  • Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibody for characterization and assay development.
  • Peptides that bind to the HPC2 gene product are isolated from both chemical and phage-displayed random peptide libraries as follows.
  • Fragments of the HPC2 gene product are expressed as GST and His-tag fusion proteins in both E. coli and SF9 cells.
  • the fusion protein is isolated using either a glutathione matrix (for GST fusions proteins) or nickel chelation matrix (for His-tag fusion proteins).
  • This target fusion protein preparation is either screened directly as described below, or eluted with glutathione or imidizole.
  • the target protein is immobilized to either a surface such as polystyrene; or a resin such as agarose; or solid supports using either direct absorption, covalent linkage reagents such as glutaraldehyde, or linkage agents such as biotin-avidin.
  • Two types of random peptide libraries of varying lengths are generated: synthetic peptide libraries that may contain derivatized residues, for example by phosphorylation or myristylation, and phage-displayed peptide libraries which may be phosphorylated. These libraries are incubated with immobilized HPC1 gene product in a variety of physiological buffers. Next, unbound peptides are removed by repeated washes, and bound peptides recovered by a variety of elution reagents such as low or high pH, strong denaturants, glutathione, or imidizole. Recovered synthetic peptide mixtures are sent to commercial services for peptide micro-sequencing to identify enriched residues. Recovered phage are amplified, rescreened, plaque purified, and then sequenced to determined the identity of the displayed peptides.
  • Peptides identified from the above screens are synthesized in larger quantities as biotin conjugates by commercial services. These peptides are used in both solid and solution phase competition assays with HPC1 and its interacting partners identified in yeast 2-hybrid screens. Versions of these peptides that are fused to membrane-permeable motifs (Lin et al., 1995; Rojas et al., 1996) will be chemically synthesized, added to cultured cells and the effects on growth, apoptosis, differentiation, cofactor response, and internal changes will be assayed.
  • Monoclonal antibody is attached to a solid surface such as a plate, tube, bead, or particle.
  • the antibody is attached to the well surface of a 96-well ELISA plate.
  • 100 ⁇ l sample e.g., serum, urine, tissue cytosol
  • HPC2 peptide/protein wild-type or mutant
  • 100 ⁇ L sample e.g., serum, urine, tissue cytosol
  • the sample is incubated for 2 hrs at room temperature. Next the sample fluid is decanted, and the solid phase is washed with buffer to remove unbound material.
  • 100 ⁇ L of a second monoclonal antibody to a different determinant on the HPC2 peptide/protein is added to the solid phase.
  • This antibody is labeled with a detector molecule (e.g., 125-I, enzyme, fluorophore, or a chromophore) and the solid phase with the second antibody is incubated for two hrs at room temperature. The second antibody is decanted and the solid phase is washed with buffer to remove unbound material.
  • a detector molecule e.g., 125-I, enzyme, fluorophore, or a chromophore
  • the amount of bound label which is proportional to the amount of HPC2 peptide/protein present in the sample, is quantified. Separate assays are performed using monoclonal antibodies which are specific for the wild-type HPC2 as well as monoclonal antibodies specific for each of the mutations identified in HPC2.

Abstract

The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human prostate cancer predisposing gene (HPC2), some alleles of which cause susceptibility to cancer, in particular prostate cancer. More specifically, the present invention relates to germline mutations in the HPC2 gene and their use in the diagnosis of predisposition to prostate cancer. The invention also relates to presymptomatic therapy of individuals who carry deleterious alleles of the HPC2 gene. The invention further relates to somatic mutations in the HPC2 gene in human prostate cancer and their use in the diagnosis and prognosis of human prostate cancer. Additionally, the invention relates to somatic mutations in the HPC2 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the HPC2 gene, (including gene therapy, protein replacement therapy, protein mimetics, and inhibitors). The invention further relates to the screening of drugs for cancer therapy. The invention also relates to the screening of the HPC2 gene for mutations, which are useful for diagnosing the predisposition to prostate cancer. In addition, the invention relates to a paralog of human HPC2, the paralog being named ELAC1, and to orthologs of human HPC2, these being mouse Elac2, chimpanzee Elac2 and gorilla Elac2.

Description

    CROSS REFERENCE TO RELATED APPLICATION
  • This application is a divisional of Ser. No. 09/564,805, filed May 5, 2000, which is a continuation-in-part of Ser. No. 09/434,382, filed Nov. 5, 1999 and is related to U.S. provisional patent application Ser. No. 60/107,468, filed Nov. 6, 1998, and priority is claimed thereto under 35 U.S.C. § 119(e). Each of these applications is incorporated herein by reference.[0001]
  • [0002] This application was made with Government support under Grant Nos. CA62154 and CA64477 from the National Institutes of Health. The United States Government has certain rights in this invention.
    • BACKGROUND OF THE INVENTION
  • The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text and respectively grouped in the appended List of References. [0003]
  • The genetics of cancer is complicated, involving the function of three loosely defined classes of genes: (1) dominant, positive regulators of the transformed state (oncogenes); (2) recessive, negative regulators of the transformed state (tumor suppressor genes); and (3) genes that modify risk without playing a direct role in the biology of transformed cells (risk modifiers). [0004]
  • Specific germline alleles of certain oncogenes and tumor suppressor genes are causally associated with predisposition to cancer. This set of genes is referred to as tumor predisposition genes. Some of the tumor predisposition genes which have been cloned and characterized influence susceptibility to: 1) Retinoblastoma (RB1); 2) Wilms' tumor (WT1); 3) Li-Fraumeni (TP53); 4) Familial adenomatous polyposis (APC); 5) Neurofibromatosis type 1 (NF1); 6) Neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau syndrome (VHL); 8) Multiple endocrine neoplasia type 2A (MEN2A); 9) Melanoma (CDKN2 and CDK4); 10) Breast and ovarian cancer (BRCA1 and BRCA2); 11) Cowden disease (MMAC1); 12) Multiple endocrine neoplasia (MEN1); 13) Nevoid basal cell carcinoma syndrome (PTC); 14) Tuberous sclerosis 2 (TSC2); 15) Xeroderma pigmentosum (genes involved in nucleotide excision repair); 16) Hereditary nonpolyposis colorectal cancer (genes involved in mismatch repair). [0005]
  • Specific germline alleles of certain risk modifier genes are also associated with predisposition to cancer, but the increased risk is sometimes only clearly expressed when it is combined with certain environmental, dietary, or other factors. Alcohol dehydrogenase (ADH) oxidizes ethanol to acetaldehyde, a chemical which is both mutagenic and carcinogenic in lab animals. The enzyme encoded by the ADH3[0006] 1 allele oxidizes ethanol relatively quickly, whereas the enzyme encoded by the ADH32 allele oxidizes ethanol more slowly. ADH31 homozygotes presumably have a high capacity for synthesis of acetaldehyde; those who also drink heavily are at increased risk for oral cavity, esophageal, and (in women) breast cancer relative to ADH32 homozygotes who drink equally heavily (Harty et al., 1997; Hori et al., 1997; Shields, 1997). The acetyltransferases encoded by N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2) catalyze the acetylation of numerous xenobiotics including the aromatic amine carcinogens derived from smoking tobacco products. Individuals who are homozygous for slow acetylating forms of NAT2 who are also heavy smokers are at greater risk for lung, bladder, and (in females) breast cancer than individuals who smoke equally heavily but are homozygous for fast acetylating forms of NAT2 (Shields, 1997; Bouchardy et al., 1998).
  • The risk of hormone related cancers such as breast and prostate cancer may be modulated by allelic variants in enzymes that play a role in estrogen or androgen metabolism, or variants in proteins that mediate the biological effects of estrogens or androgens. A polymorphic CAG repeat in the human androgen receptor gene encodes a polymorphic polyglutamine tract near the amino-terminus of the protein. The length of the polyglutamine tract is inversely correlated with the transcriptional activation activity of the androgen receptor and thus one aspect of the biological response to androgens. Men whose androgen receptor contains a relatively short polyglutamine tract are at higher risk for prostate cancer, especially high stage/high histologic grade prostate cancer, than men whose androgen receptor contains a relatively long polyglutamine tract (Giovannucci et al., 1997). [0007]
  • Prostate cancer is the most common cancer in men in many western countries, and the second leading cause of cancer deaths in men. It accounts for more than 40,000 deaths in the US annually. The number of deaths is likely to continue rising over the next 10 to 15 years. In the US, prostate cancer is estimated to cost $1.5 billion per year in direct medical expenses. In addition to the burden of suffering, it is a major public-health issue. Numerous studies have provided evidence for familial clustering of prostate cancer, indicating that family history is a major risk factor for this disease (Cannon et al., 1982; Steinberg et al., 1990; Carter et al, 1993). [0008]
  • Prostate cancer has long been recognized to be, in part, a familial disease with a genetic component (Woolf, 1960a; Cannon et al., 1982; Carter et al., 1992). Numerous investigators have examined the evidence for genetic inheritance and concluded that the data are most consistent with dominant inheritance for a major susceptibility locus or loci. Woolf (1960b), described a relative risk of 3.0 of developing prostate cancer among first-degree relatives of prostate cancer cases in Utah using death certificate data. Relative risks ranging from 3 to 11 for first-degree relatives of prostate cancer cases have been reported (Cannon et al., 1982; Woolf, 1960b; Fincham et al., 1990; Meikle et al., 1985; Krain, 1974; Morganti et al., 1956; Goldgar et al., 1994). Carter et al. (1992) performed segregation analysis on families ascertained through a single prostate cancer proband. The analysis suggested Mendelian inheritance in a subset of families through autosomal dominant inheritance of a rare (q=0.003), high-risk allele with estimated cumulative risk of prostate cancer for carriers of 88% by [0009] age 85. Inherited prostate cancer susceptibility accounted for a significant proportion of early-onset disease, and overall was responsible for 9% of prostate occurrence by age 85. Recent results demonstrate that at least four loci exist which convey susceptibility to prostate cancer as well as other cancers. These loci are HPC1 on chromosome 1q24, (Smith et al., 1996), HPCX on chromosome Xq27-28 (Xu et al., 1998), PCAP at 1q42 (Berthon et al., 1998) and CAPB at 1p36 (Gibbs et al., 1999a). All four suggestions of linkage for prostate cancer predisposition were the result of hints arising from genome-wide searches. Although only the HPC1 linkage has so far been confirmed (Cooney et al., 1997; Neuhausen et al., 1999; Xu and ICPCG, 2000), it is becoming clear that a large number of genes contribute to familial prostate cancer. It also seems clear, both from published hereditary prostate cancer linkage studies and from genotyping of our family resource at the above mentioned loci, that no single predisposition locus mapped to date is by itself responsible for a large portion of familial prostate cancer (Neuhausen et al., 1999; Eeles et al., 1998; Gibbs et al., 1999b; Lange et al., 1999; Berry et al., 2000; Suarez et al., 2000; Goode et al., 2000).
  • A comparison to the cloning of, and risk profile attributed to, breast cancer susceptibility genes provides an instructive example. The profusion of proposed prostate loci, coupled with minimal confirmation or refined localization following initial publication of these linkages, contrasts sharply with studies of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2. Linkage to BRCA1 was first published in 1990 (Hall et al., 1990); groups competing to identify this gene moved swiftly from confirmatory studies through efforts to refine the localization to the gene identification in 1994 (Miki et al., 1994). With expanded genomics resources, the time from linkage (Wooster et al., 1994) to complete cloning (Wooster et al., 1995; Tavtigian et al., 1996) of BRCA2 was only slightly more than 1 year. Ongoing mutation screening and careful modeling of age specific and familial risks indicate that these two genes account for virtually all extended breast and ovarian cancer families (Antoniou et al., 2000) and the majority of breast cancer families with more than five cases, especially those that include an early-onset component (Ford et al., 1998). [0010]
  • Even so, a fraction of familial breast cancer risk is manifest in smaller family clusters with average age at diagnosis. While BRCA1 and BRCA2 only account for a portion of this component of breast cancer risk (Peto et al., 1999), there are no published and confirmed linkages based on these types of pedigrees to date. Standard genetic analysis appears to be limited by the problems of low penetrance and genetic complexity. It is possible that analysis of genetic predisposition in families with excess prostate cancer also reflects these issues. As absence of distinction by age at diagnosis/onset would also be consistent with the influence of multiple susceptibility genes harboring only moderate risk sequence variants, one might therefore ask what relative contribution low frequency high risk variants analogous to mutations in BRCA1/2, versus higher frequency, moderate risk sequence variants, make to the population risk of prostate cancer. [0011]
  • Indeed, evidence that moderate risk sequence variants in a number of specific genes contribute to prostate cancer susceptibility is beginning to accumulate. For example, a polymorphic CAG repeat within the androgen receptor open reading frame encoding a variable length polyglutamine tract shows an inverse relationship between repeat length and the transcriptional transactivation activity of the receptor (Chamberlain et al., 1994; Kazemi-Esfarjani et al., 1995). Accordingly, a series of studies show an association between shorter androgen receptor CAG repeat length and prostate cancer risk (Giovannucci et al., 1997; Stanford et al., 1997), although it is not entirely clear whether the association is with diagnosis of prostate cancer or severity of disease (Bratt et al., 1999). Second, a number of missense variants have been observed in the steroid 5α-reductase type II gene (SRD5A2), responsible for conversion of testosterone to the more active androgen dihydrotestosterone in the prostate (Makridakis et al., 1997). One of these variants, Ala 49 Thr, has been reported to increase the catalytic activity of the enzyme, and is associated with increased risk of advanced prostate cancer (Makridakis et al., 1999; Jaffe et al., 2000). Finally, several groups have reported an excess of prostate cancer in large BRCA2 pedigrees (Sigurdsson et al., 1997; Breast Cancer Linkage Consortium, 1999), though the relative risk that these mutations confer for prostate cancer is considerably lower than for breast cancer. Further, these effects may be variant specific as association has not been confirmed among men who carry the Ashkenazi BRCA2 founder mutation 6174delT (Wilkens et al., 1999; Nastiuk et al., 1999; Hubert et al., 1999). If these and similar sequence variants play a role in a significant fraction of prostate cancer, then models of the genetic component of familial prostate cancer may need to incorporate both linkage evidence for major susceptibility loci and association evidence for moderate risk sequence variants. [0012]
  • The Utah population provides a unique resource for examining the genetic basis of disease. Extended high risk pedigrees containing many cases can be ascertained as units instead of by expansion from individual probands. While these pedigrees are an extremely powerful resource for linkage studies, they also allow analysis of segregation of moderate risk sequence variants through multiple generations of both cases and their unaffected relatives. [0013]
  • Detection of genetic linkage for prostate cancer susceptibility to a defined segment of a chromosome requires that DNA sequence variants within that chromosomal segment confer the cancer susceptibility. This is usually taken to mean that the causal sequence variant(s) will either alter the expression of one or more linked genes or will alter the function of one of the linked genes. However, detection of the genetic linkage does not necessarily provide evidence for what class of gene (i.e. tumor suppressor, oncogene, or risk modifier) is affected by the causal sequence variant(s). [0014]
  • Most strategies for proceeding from genetic linkage of prostate cancer susceptibility to chromosome 17p to identification of the 17p-linked prostate cancer predisposing gene (HPC2) require precise genetic localization studies to define a discrete segment of the chromosome within which the causal sequence variant(s) must map. Gene identification projects based on precise genetic localization are called positional cloning projects. The general strategy in positional cloning is to find all of the genes located within the genetically defined interval, identify sequence variants in and around those genes, and then determine which of those sequence variants either alter the expression or the function of one (or more) of the associated genes. Segregation of such sequence variants with the disease in the linked kindreds must also be demonstrated. We have executed a positional cloning project in the HPC2 region of chromosome 17p and found a gene, herein named HPC2, germline mutations which predisposes individuals to prostate cancer. [0015]
    • SUMMARY OF THE INVENTION
  • The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human prostate cancer predisposing gene (HPC2), some alleles of which cause susceptibility to cancer, in particular prostate cancer. More specifically, the present invention relates to germline mutations in the HPC2 gene and their use in the diagnosis of predisposition to prostate cancer. The invention also relates to presymptomatic therapy of individuals who carry deleterious alleles of the HPC2 gene. The invention further relates to somatic mutations in the HPC2 gene in human prostate cancer and their use in the diagnosis and prognosis of human prostate cancer. Additionally, the invention relates to somatic mutations in the HPC2 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the HPC2 gene, including gene therapy, protein replacement therapy, protein mimetics, and inhibitors. The invention further relates to the screening of drugs for cancer therapy. The invention also relates to the screening of the HPC2 gene for mutations, which are useful for diagnosing the predisposition to prostate cancer. The HPC2 gene is useful as a marker for the HPC2 locus and as a marker for prostate cancer. Finally, a paralog of HPC2 as well as orthologs of HPC2 in mouse, chimpanzee and gorilla have been isolated and characterized.[0016]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a multipoint linkage analysis of 4 kindreds that show suggestive evidence for linkage to the HPC2 prostate cancer susceptibility locus relative to chromosome 17p markers. [0017]
  • FIGS. [0018] 2A-B are diagrams showing the order of genetic markers and recombinant boundaries neighboring HPC2, a schematic map of BACs spanning the HPC2 region, a schematic map of transcription units within the HPC2 region, and two diagrams of the HPC2 transcription unit showing the locations of the exons of HPC2 relative to the BAC to which it maps and relative to each other. The individual exons are numbered.
  • FIG. 3 shows recombinant, physical and transcript maps centered at the human ELAC2 locus on chromosome 17p. The top portion shows genetic markers and recombinants. Microsatellite markers developed at Myriad Genetics, Inc. are given as 17-MYR####. Nested within the arrows that represent meiotic recombinants are the pedigree in which the recombinant occurred and, in parentheses, the number of cases who carry the haplotype on which the recombinant occurred. The second portion of the figure shows a BAC contig tiling path across this interval. The T7 end of each BAC is denoted with an arrowhead. The third portion of the figure shows transcription units identified in the interval. The bottom portion of the figure is an expanded view of a 40 kb segment at the SP6 end of BAC 31k12 showing the relative positions of two exons of the gene 04CG09 and all of the coding exons of ELAC2. [0019]
  • FIG. 4 is an alignment of the sequence of [0020] exon 1 of the human HPC2 gene with exon 1 of the mouse HPC2 gene. The figure also shows an alignment of the peptide sequence encoded by exon 1 of the human HPC2 gene with the peptide sequence encoded by exon 1 of the mouse HPC2 gene. The human DNA sequence is SEQ ID NO:210; the human amino acid sequence is SEQ ID NO:211; the mouse DNA sequence is SEQ ID NO:212 and the mouse amino acid sequence is SEQ ID NO:213.
  • FIGS. [0021] 5A-B show kindreds 4102 and 4289. The pedigrees have been genotyped over a 20 cM interval extending from D17S786 to D17S805. Haplotypes are represented by the bars; the dark gray haplotype segregating in each pedigree is the mutation bearing chromosome. The relative position of ELAC2 is denoted by * (white on black or white on gray).
  • FIG. 5A shows kindred 4102. The dark bar denotes the 1641 insG bearing haplotype. [0022] Individuals 061 and 107 carry part of the frameshift haplotype, but neither carries the frameshift due to recombination events. There are no data to distinguish which of the founders, individuals 050 and 051, carried the frameshift. The second shared haplotype in kindred 4102 is denoted by a light gray bar. Again, there are no data to distinguish which of the founders, individuals 005 and 006, carried the haplotype.
  • FIG. 5B shows kindred 4289. [0023] Individuals 064, 066, 067, 068 and 072 share a recombinant chromosome that carries the His 781 missense change.
  • FIGS. [0024] 6A-B are a multiple protein alignment of ELAC/2 family members. ELAC2 family members were selected from human (HSA), mouse (MMU), C. elegans (CEL), A. thaliana (ATH) and S. cerevisiae (SCE). The A. thaliana genome encodes more than one family member; gi6850339 was selected because it aligned with fewer gaps. ELAC1 family members were selected from human, E. coli (Es_c), the blue-green algae Synechocystis (Syn) and the archaebacterium Methanobacterium thernoautotrophicum (Met). Alignments were based on BLASTp searches and then optimized by inspection. The positions of Ser 217, Ala 541 and Arg 781 in human ELAC2 are marked by down arrows. The sequences shown in FIGS. 6A-B are: human ELAC2 is SEQ ID NO:2, mouse Elac2 is SEQ ID NO:222, C. elegans CE16965 is SEQ ID NO:227, A. thaliana gi 6850339 is SEQ ID NO:228, S. cerevisiae YKRO79C is SEQ ID NO:229, human ELAC1 is SEQ ID NO:220, E. coli elaC is SEQ ID NO:230, Synechocystis gi2500943 is SEQ ID NO:231, and Methanobacterium thernoautotrophicum gi2622965 is SEQ ID NO:232.
  • FIG. 7 shows recessive genotype frequencies by birth cohort. [0025]
  • FIG. 8 shows the results of association tests. [0026]
  • FIG. 9 shows a multiple protein alignment demonstrating conservation of sequence elements between ELAC2, PSO2 and CPSF73 families. The alignments shown for segments of PSO2 and CPSF73 family members were taken from more extensive alignments that contain family members from a larger set of species, analogous to the ELAC1/2 alignments of FIGS. [0027] 6A-B. The seven His or Cys residues that are conserved across two or more of the gene families are marked by down arrows. The position of Ala 541 in human ELAC2 is also marked by a down arrow. The sequences shown in FIG. 9 are partial sequences of the following: human CPSF73 is SEQ ID NO:233, A. thaliana gi6751699 is SEQ ID NO:234, S. cerevisiae YSH1 is SEQ ID NO:235, Synechocystis gi2496795 is SEQ ID NO:236, Methanobacterium thermoautotrophicum gi2622312 is SEQ ID NO:237, human ha3611 is SEQ ID NO:238, A. thaliana gi2979557 is SEQ ID NO:239, S. cerevisiae PSO2 is SEQ ID NO:240, human ELAC2 is SEQ ID NO:2, A. thaliana gi6850339 is SEQ ID NO:228, and S. cerevisiae YKRO79C is SEQ ID NO:229.
  • FIG. 10 shows a similarity comparison among the ELAC2 family members aligned in FIGS. [0028] 6A-B.
  • FIGS. [0029] 11A-D shows an analysis of ELAC 1 expression in human tissues.
  • FIGS. [0030] 11A-B show Multiple Tissue Northern (MTN) filters (Clontech) probed with the human ELAC1 ORF. Note that a 3 kb ELAC1 transcript is detected in all tissues.
  • FIGS. [0031] 11C-D show the same filters probed with human β-actin as a loading control.
  • FIG. 12 shows a multiple protein alignment demonstrating similarity between an N-terminal segment of the ELAC2 family members and the sequence context of the histidine motif shared by ELAC1 and ELAC2 family members. Species abbreviations are as in FIGS. [0032] 6A-B. The sequences shown in FIG. 12 are partial sequences of the following: human ELAC2 is SEQ ID NO:2, mouse Elac2 is SEQ ID NO:222, C. elegans CE16965 is SEQ ID NO:227, A. thaliana gi6850339 is SEQ ID NO:228, S. cerevisiae YKRO79C is SEQ ID NO:229, human ELAC1 is SEQ ID NO:220, E. coli elaC is SEQ ID NO:230, Synechocystis gi2500943 is SEQ ID NO:231, and Methanobacterium thermoautotrophicum gi2622965 is SEQ ID NO:232.
  • FIG. 13 shows the relationship between ELAC1/2, PSO2 and CPSF73 gene family members. The tree is a distance-based depiction of pairwise sequence similarities determined from a manual alignment of the 67 amino acids immediately surrounding the histidine motif. ClustaIX (Thompson et al., 1997) was used to calculate the percent divergence of each sequence on a pairwise basis and neighbor joining (Saitou and Nei, 1987) was applied to the resulting distance matrix. The treefile produced from ClustaIX was visualized using TreeView (Page, 1996) and further edited in a graphics program for aesthetics. The scale bar indicates amino acid substitutions per residue.[0033]
    • BRIEF DESCRIPTION OF THE TABLES
  • Table 1 is a compilation of 2-point LOD scores for markers in the HPC2 region. [0034]
  • Table 2A lists the family resource used to detect linkage of HPC2 to chromosome 17p. [0035]
  • Table 2B lists two-point LOD scores using the Utah age-specific model. [0036]
  • Table 3 is a summary of resource genotyped for the association tests. [0037]
  • Table 4 is a list of the accession numbers of human EST sequences used to assemble a tentative, partial cDNA sequence of the human HPC2 gene. [0038]
  • Table 5 is a list of the primers used for obtaining 5′ RACE products that contained the start codon and part of the 5′ UTR of the human HPC2 gene, primers used to prepare a full length human HPC2 expression construct, and primers used to check the sequence of that construct. [0039]
  • Table 6 is a list of the accession numbers of mouse EST sequences used to assemble a tentative, partial cDNA sequence of the mouse HPC2 gene. [0040]
  • Table 7 is a list of the primers used for obtaining 5′ RACE products that contained the start codon and part of the 5′ UTR of the mouse HPC2 gene, primers used to prepare a full length mouse HPC2 expression construct, and primers used to check the sequence of that construct. [0041]
  • Table 8 is a list of the primers used to mutation screen the human HPC2 gene from genomic DNA. [0042]
  • Table 9 is a summary of germline sequence variants of the human HPC2 gene. [0043]
  • Table 10 is a list of the allele frequencies of HPC2. [0044]
    • SUMMARY OF SEQUENCE LISTING
  • SEQ ID NO: 1 is the nucleotide sequence for the human-HPC2 cDNA from the start codon through the stop codon. [0045]
  • SEQ ID NO:2 is the amino acid sequence for the human HPC2 protein. [0046]
  • SEQ ID NO:3 is the nucleotide sequence for the human HPC2 cDNA from 50 base pairs before the start codon through the end of the 3′ UTR. [0047]
  • SEQ ID NO:4 to SEQ ID NO:27 are the sequences of [0048] exon 1 to exon 24 of the human HPC2 gene.
  • SEQ ID NO:28 is the genomic sequence of the human HPC2 gene. [0049]
  • SEQ ID NOs:29-190 are nucleotide sequences of primers used to identify the human and/or mouse HPC2 genes or to screen for mutations. [0050]
  • SEQ ID NOs:191-209 are nucleotide sequences of the HPC2 around and including various sequence variants. [0051]
  • SEQ ID NO:210 is the nucleotide sequence of [0052] human HPC2 exon 1 and SEQ ID NO:211 is the corresponding amino acid sequence as shown in FIG. 4.
  • SEQ ID NO:212 is nucleotide sequence of [0053] mouse HPC2 exon 1 and SEQ ID NO:213 is the corresponding amino acid sequence as shown in FIG. 4.
  • SEQ ID NO:214 is a histidine containing motif found in HPC2/ELAC2 and ELAC1. [0054]
  • SEQ ID NO:215 is [0055] exon 1 of ELAC1.
  • SEQ ID NO:216 is [0056] exon 2 of ELAC1 plus surrounding genomic sequence.
  • SEQ ID NO:217 is [0057] exon 3 of ELAC1 plus surrounding genomic sequence.
  • SEQ ID NO:218 is [0058] exon 4 of ELAC1 plus surrounding genomic sequence.
  • SEQ ID NO:219 is the cDNA for ELAC1 and SEQ ID NO:220 is the amino acid sequence for ELAC1. [0059]
  • SEQ ID NO:221 is the cDNA for mouse ELAC2 and SEQ ID NO:222 is the amino acid sequence for mouse ELAC2. [0060]
  • SEQ ID NO:223 is the cDNA for chimpanzee ELAC2 and SEQ ID NO:224 is the amino acid sequence for chimpanzee ELAC2. [0061]
  • SEQ ID NO:225 is the cDNA for gorilla ELAC2 and SEQ ID NO:226 is the amino acid sequence for gorilla ELAC2. [0062]
  • SEQ ID NOs:227-229 are the amino acid sequences for ELAC2 family member proteins from [0063] C. elegans, A. thaliana and S. cerevisiae as shown in FIG. 6A.
  • SEQ ID NOs:230-232 are the amino acid sequences for ELAC1 family member proteins from [0064] E. coli, Synechocystis and Methanobacterium thermoautotrophicum as shown in FIGS. 6A-B.
  • SEQ ID NOs:233-240 are amino acid sequences of proteins from CPSF73 and PSO2 families as shown in FIG. 9. These are, respectively, human CPSF73, [0065] A. thaliana gi6751699, S. cerevisiae YSH1, Synechocystis gi2496795, Methanobacterium thermoautotrophicum gi2622312, human ha3611, A. thaliana gi2979557 and S. cerevisiae PSO2. The sequences for the ELAC2 family of FIG. 9 are SEQ ID NO:2 for human, SEQ ID NO:228 for A. thaliana (as for FIGS. 6A-B) and SEQ ID NO:229 for S. cerevisiae (as for FIGS. 6A-B). The sequence listing shows the complete sequences of these proteins whereas FIG. 9 shows only portions of each sequence.
    • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides an isolated polynucleotide comprising all, or a portion of the HPC2 locus or of a mutated HPC2 locus, preferably at least eight bases and not more than about 27 kb in length. Such polynucleotides may be antisense polynucleotides. The present invention also provides a recombinant construct comprising such an isolated polynucleotide, for example, a recombinant construct suitable for expression in a transformed host cell. [0066]
  • Also provided by the present invention are methods of detecting a polynucleotide comprising a portion of the HPC2 locus or its expression product in an analyte. Such methods may further comprise the step of amplifying the portion of the HPC2 locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the HPC2 locus. The method is useful for either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer. The HPC2 gene is useful as a marker for the HPC2 locus and as a marker for prostate cancer. [0067]
  • The present invention also provides isolated antibodies, preferably monoclonal antibodies, which specifically bind to an isolated polypeptide comprised of at least five amino acid residues encoded by the HPC2 locus. [0068]
  • The present invention also provides kits for detecting in an analyte a polynucleotide comprising a portion of the HPC2 locus, the kits comprising a polynucleotide complementary to the portion of the HPC2 locus packaged in a suitable container, and instructions for its use. [0069]
  • The present invention further provides methods of preparing a polynucleotide comprising polymerizing nucleotides to yield a sequence comprised of at least eight consecutive nucleotides of the HPC2 locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a sequence comprising at least five amino acids encoded within the HPC2 locus. [0070]
  • The present invention further provides methods of screening the HPC2 gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the HPC2 locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the HPC2 locus. Such methods may also include a step of providing the complete set of short polynucleotides defined by the sequence of HPC2 or discrete subsets of that sequence, all single-base substitutions of that sequence or discrete subsets of that sequence, all 1-, 2-, 3-, or 4-base deletions of that sequence or discrete subsets of that sequence, and all 1-, 2-, 3-, or 4-base insertions in that sequence or discrete subsets of that sequence. The method is useful for identifying mutations for use in either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer. [0071]
  • The present invention further provides methods of screening suspected HPC2 mutant alleles to identify mutations in the HPC2 gene. [0072]
  • In addition, the present invention provides methods to screen drugs for inhibition or restoration of HPC2 gene product function as an anticancer therapy. [0073]
  • The present invention also provides the means necessary for production of gene-based therapies directed at cancer cells. These therapeutic agents may take the form of polynucleotides comprising all or a portion of the HPC2 locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the HPC2 protein is reconstituted. Therapeutic agents may also take the form of polypeptides based on either a portion of, or the entire protein sequence of HPC2. These may functionally replace the activity of HPC2 in vivo. [0074]
  • Finally, the present invention provides the sequence of a paralog of HPC2, herein called ELAC1, as well as the sequences of HPC2 orthologs from mouse, chimpanzee and gorilla. These orthologs are named ELAC2. [0075]
  • It is a discovery of the present invention that the HPC2 locus which predisposes individuals to prostate cancer, is a gene encoding an HPC2 protein, which has been found to be non-identical to publicly available protein or cDNA sequences. This gene is termed HPC2 herein. It is a discovery of the present invention that mutations in the HPC2 locus in the germline are indicative of a predisposition to prostate cancer. Finally, it is a discovery of the present invention that germline mutations in the HPC2 locus are also associated with prostate cancer and other types of cancer. The mutational events of the HPC2 locus can involve deletions, insertions and nucleotide substitutions within the coding sequence and the non-coding sequence. [0076]
  • Useful Diagnostic Techniques [0077]
  • According to the diagnostic and prognostic method of the present invention, alteration of the wild-type HPC2 locus is detected. In addition, the method can be performed by detecting the wild-type HPC2 locus and confirming the lack of a predisposition to cancer at the HPC2 locus. “Alteration of a wild-type gene” encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or of only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited. If only a single allele is somatically mutated, an early neoplastic state is indicated. However, if both alleles are somatically mutated, then a late neoplastic state is indicated. The finding of HPC2 mutations thus provides both diagnostic and prognostic information. An HPC2 allele which is not deleted (e.g., found on the sister chromosome to a chromosome carrying an HPC2 deletion) can be screened for other mutations, such as insertions, small deletions, and point mutations. It is believed that many mutations found in tumor tissues will be those leading to decreased expression of the HPC2 gene product. However, mutations leading to non-functional gene products would also lead to a cancerous state. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to reduction or loss of expression of the HPC2 gene product, expression of an altered HPC2 gene product, or to a decrease in mRNA stability or translation efficiency. [0078]
  • Useful diagnostic techniques include, but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCA), RNase protection assay, allele-specific oligonucleotide (ASO), dot blot analysis, hybridization using nucleic acid modified with gold nanoparticles and PCR-SSCP, as discussed in detail further below. Also useful is the recently developed technique of DNA microchip technology. [0079]
  • Predisposition to cancers, such as prostate cancer, and the other cancers identified herein, can be ascertained by testing any tissue of a human for mutations of the HPC2 gene. For example, a person who has inherited a germline HPC2 mutation would be prone to develop cancers. This can be determined by testing DNA from any tissue of the person's body. Most simply, blood can be drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for mutations of the HPC2 gene. Alteration of a wild-type HPC2 allele, whether, for example, by point mutation or deletion, can be detected by any of the means discussed herein. [0080]
  • There are several methods that can be used to detect DNA sequence variation. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect sequence variation. For a gene as large as HPC2, manual sequencing is very labor-intensive, but under optimal conditions, mutations in the coding sequence of a gene are rarely missed. Another approach is the single-stranded conformation polymorphism assay (SSCA) (Orita et al., 1989). This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCA makes it an attractive, viable alternative to direct sequencing for mutation detection on a research basis. The fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., 1991), heteroduplex analysis (HA) (White et al., 1992) and chemical mismatch cleavage (CMC) (Grompe et al., 1989). None of the methods described above will detect large deletions, duplications or insertions, nor will they detect a regulatory mutation which affects transcription or translation of the protein. Other methods which might detect these classes of mutations such as a protein truncation assay or the asymmetric assay, detect only specific types of mutations and would not detect missense mutations. A review of currently available methods of detecting DNA sequence variation can be found in a recent review by Grompe (1993). Once a mutation is known, an allele specific detection approach such as allele specific oligonucleotide (ASO) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation. Such a technique can utilize probes which are labeled with gold nanoparticles to yield a visual color result (Elghanian et al., 1997). [0081]
  • In order to detect the alteration of the wild-type HPC2 gene in a tissue, it is helpful to isolate the tissue free from surrounding normal tissues. Means for enriching tissue preparation for tumor cells are known in the art. For example, the tissue may be isolated from paraffin or cryostat sections. Cancer cells may also be separated from normal cells by flow cytometry. These techniques, as well as other techniques for separating tumor cells from normal cells, are well known in the art. If the tumor tissue is highly contaminated with normal cells, detection of mutations is more difficult. [0082]
  • Detection of point mutations may be accomplished by molecular cloning of the HPC2 allele(s) and sequencing the allele(s) using techniques well known in the art. Alternatively, the gene sequences can be amplified directly from a genomic DNA preparation from the tumor tissue, using known techniques. The DNA sequence of the amplified sequences can then be determined. [0083]
  • There are six well known methods for a more complete, yet still indirect, test for confirming the presence of a susceptibility allele: 1) single-stranded conformation analysis (SSCA) (Orita et al., 1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989); 3) RNase protection assays (Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specific oligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteins which recognize nucleotide mismatches, such as the [0084] E. coli mutS protein (Modrich, 1991); and 6) allele-specific PCR (Ruano and Kidd, 1989). For allele-specific PCR, primers are used which hybridize at their 3′ ends to a particular HPC2 mutation. If the particular HPC2 mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., 1989. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment. Such a method is particularly useful for screening relatives of an affected individual for the presence of the HPC2 mutation found in that individual. Other techniques for detecting insertions and deletions as known in the art can be used.
  • In the first three methods (SSCA, DGGE and RNase protection assay), a new electrophoretic band appears. SSCA detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay, an oligonucleotide is designed which detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences. [0085]
  • Mismatches, according to the present invention, are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of tumor samples. An example of a mismatch cleavage technique is the RNase protection method. In the practice of the present invention, the method involves the use of a labeled riboprobe which is complementary to the human wild-type HPC2 gene coding sequence. The riboprobe and either mRNA or DNA isolated from the tumor tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the full length of the HPC2 mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the HPC2 mRNA or gene, it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches. [0086]
  • In similar fashion, DNA probes can be used to detect mismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986. Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, 1988. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the HPC2 gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions. [0087]
  • DNA sequences of the HPC2 gene which have been amplified by use of PCR may also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the HPC2 gene sequence harboring a known mutation. For example, one oligomer may be about 30 nucleotides in length (although shorter and longer oligomers are also usable as well recognized by those of skill in the art), corresponding to a portion of the HPC2 gene sequence. By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in the HPC2 gene. Hybridization of allele-specific probes with amplified HPC2 sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under high stringency hybridization conditions indicates the presence of the same mutation in the tumor tissue as in the allele-specific probe. [0088]
  • The newly developed technique of nucleic acid analysis via microchip technology is also applicable to the present invention. In this technique, literally thousands of distinct oligonucleotide probes are built up in an array on a silicon chip. Nucleic acid to be analyzed is fluorescently labeled and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique one can determine the presence of mutations or even sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest. The method is one of parallel processing of many, even thousands, of probes at once and can tremendously increase the rate of analysis. Several papers have been published which use this technique. Some of these are Hacia et al., 1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et al., 1996; DeRisi et al., 1996; Lipshutz et al., 1995. This method has already been used to screen people for mutations in the breast cancer gene BRCA1 (Hacia et al., 1996). This new technology has been reviewed in a news article in Chemical and Engineering News (Borman, 1996) and been the subject of an editorial (Nature Genetics, 1996). Also see Fodor (1997). [0089]
  • The most definitive test for mutations in a candidate locus is to directly compare genomic HPC2 sequences from cancer patients with those from a control population. Alternatively, one could sequence messenger RNA after amplification, e.g., by PCR, thereby eliminating the necessity of determining the exon structure of the candidate gene. [0090]
  • Mutations from cancer patients falling outside the coding region of HPC2 can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the HPC2 gene. An early indication that mutations in noncoding regions are important may come from Northern blot experiments that reveal messenger RNA molecules of abnormal size or abundance in cancer patients as compared to control individuals. [0091]
  • Alteration of HPC2 mRNA expression can be detected by any techniques known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type HPC2 gene. Alteration of wild-type HPC2 genes can also be detected by screening for alteration of wild-type HPC2 protein. For example, monoclonal antibodies immunoreactive with HPC2 can be used to screen a tissue. Lack of cognate antigen would indicate an HPC2 mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant HPC2 gene product. Such immunological assays can be done in any convenient formats known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered HPC2 protein can be used to detect alteration of wild-type HPC2 genes. Functional assays, such as protein binding determinations, can be used. In addition, assays can be used which detect HPC2 biochemical function. Finding a mutant HPC2 gene product indicates alteration of a wild-type HPC2 gene. [0092]
  • Mutant HPC2 genes or gene products can also be detected in other human body samples, such as serum, stool, urine and sputum. The same techniques discussed above for detection of mutant HPC2 genes or gene products in tissues can be applied to other body samples. Cancer cells are sloughed off from tumors and appear in such body samples. In addition, the HPC2 gene product itself may be secreted into the extracellular space and found in these body samples even in the absence of cancer cells. By screening such body samples, a simple early diagnosis can be achieved for many types of cancers. In addition, the progress of chemotherapy or radiotherapy can be monitored more easily by testing such body samples for mutant HPC2 genes or gene products. [0093]
  • The methods of diagnosis of the present invention are applicable to any tumor in which HPC2 has a role in tumorigenesis. The diagnostic method of the present invention is useful for clinicians, so they can decide upon an appropriate course of treatment. [0094]
  • The primer pairs of the present invention are useful for determination of the nucleotide sequence of a particular HPC2 allele using PCR. The pairs of single-stranded DNA primers can be annealed to sequences within or surrounding the HPC2 gene on [0095] chromosome 17 in order to prime amplifying DNA synthesis of the HPC2 gene itself. A complete set of these primers allows synthesis of all of the nucleotides of the HPC2 gene coding sequences, i.e., the exons. The set of primers preferably allows synthesis of both intron and exon sequences. Allele-specific primers can also be used. Such primers anneal only to particular HPC2 mutant alleles, and thus will only amplify a product in the presence of the mutant allele as a template.
  • In order to facilitate subsequent cloning of amplified sequences, primers may have restriction enzyme site sequences appended to their 5′ ends. Thus, all nucleotides of the primers are derived from HPC2 sequences or sequences adjacent to HPC2, except for the few nucleotides necessary to form a restriction enzyme site. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques which are well known in the art. Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available. Given the sequence of the HPC2 open reading frame shown in SEQ ID NOs: 1 and 3, design of particular primers is well within the skill of the art. [0096]
  • The nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above. The probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the HPC2 gene or mRNA using other techniques. [0097]
  • It has been discovered that individuals with the wild-type HPC2 gene do not have cancer which results from the HPC2 allele. However, mutations which interfere with the function of the HPC2 protein are involved in the pathogenesis of cancer. Thus, the presence of an altered (or a mutant) HPC2 gene which produces a protein having a loss of function, or altered function, directly correlates to an increased risk of cancer. In order to detect an HPC2 gene mutation, a biological sample is prepared and analyzed for a difference between the sequence of the HPC2 allele being analyzed and the sequence of the wild-type HPC2 allele. Mutant HPC2 alleles can be initially identified by any of the techniques described above. The mutant alleles are then sequenced to identify the specific mutation of the particular mutant allele. Alternatively, mutant HPC2 alleles can be initially identified by identifying mutant (altered) HPC2 proteins, using conventional techniques. The mutant alleles are then sequenced to identify the specific mutation for each allele. The mutations, especially those which lead to an altered function of the HPC2 protein, are then used for the diagnostic and prognostic methods of the present invention. [0098]
  • Definitions [0099]
  • The present invention employs the following definitions: [0100]
  • “Amplification of Polynucleotides” utilizes methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. Also useful are strand displacement amplification (SDA), thermophilic SDA, and nucleic acid sequence based amplification (3SR or NASBA). These methods are well known and widely practiced in the art. See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu and Wallace, 1989 (for LCR); U.S. Pat. Nos. 5,270,184 and 5,455,166 and Walker et al., 1992 (for SDA); Spargo et al., 1996 (for thermophilic SDA) and U.S. Pat. No. 5,409,818, Fahy et al., 1991 and Compton, 1991 for 3SR and NASBA. Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from the HPC2 region or HPC2 paralogs or orthologs are preferably complementary to, and hybridize specifically to sequences in the HPC2 region or paralog or ortholog region or in regions that flank a target region therein. HPC2 sequences or paralog or ortholog sequences generated by amplification may be sequenced directly. Alternatively, but less desirably, the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf, 1986. [0101]
  • “Analyte polynucleotide” and “analyte strand” refer to a single- or double-stranded polynucleotide which is suspected of containing a target sequence, and which may be present in a variety of types of samples, including biological samples. [0102]
  • “Antibodies.” The present invention also provides polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the HPC2 polypeptides or to polypeptides encoded by paralogs or orthologs of HPC2 and fragments thereof or to polynucleotide sequences from the HPC2 region, or to polynucleotide sequences which are paralogs or orthologs of HPC2, particularly from the HPC2 locus or a portion thereof. The term “antibody” is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Polypeptides may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity to the HPC2 polypeptide or fragment or to polypeptides or fragments encoded by paralogs or orthologs of HPC2. Monoclonal antibodies may be made by injecting mice with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with HPC2 polypeptide or fragments thereof. See, Harlow and Lane, 1988. These antibodies will be useful in assays as well as pharmaceuticals. [0103]
  • Once a sufficient quantity of desired polypeptide has been obtained, it may be used for various purposes. A typical use is the production of antibodies specific for binding. These antibodies may be either polyclonal or monoclonal, and may be produced by in vitro or in vivo techniques well known in the art. For production of polyclonal antibodies, an appropriate target immune system, typically mouse or rabbit, is selected. Substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and by other parameters well known to immunologists. Typical sites for injection are in footpads, intramuscularly, intraperitoneally, or intradermally. Of course, other species may be substituted for mouse or rabbit. Polyclonal antibodies are then purified using techniques known in the art, adjusted for the desired specificity. [0104]
  • An immunological response is usually assayed with an immunoassay. Normally, such immunoassays involve some purification of a source of antigen, for example, that produced by the same cells and in the same fashion as the antigen. A variety of immunoassay methods are well known in the art. See, e.g., Harlow and Lane, 1988, or Goding, 1986. [0105]
  • Monoclonal antibodies with affinities of 10[0106] −8 M−1 or preferably 10−9 to 10−10 M−1 or stronger will typically be made by standard procedures as described, e.g., in Harlow and Lane, 1988 or Goding, 1986. Briefly, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatants of each clone tested for their production of an appropriate antibody specific for the desired region of the antigen.
  • Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides, or alternatively, to selection of libraries of antibodies in phage or similar vectors. See Huse et al., 1989. The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be produced (see U.S. Pat. No. 4,816,567). [0107]
  • “Binding partner” refers to a molecule capable of binding a ligand molecule with high specificity, as for example, an antigen and an antigen-specific antibody or an enzyme and its inhibitor. In general, the specific binding partners must bind with sufficient affinity to immobilize the analyte copy/complementary strand duplex (in the case of polynucleotide hybridization) under the isolation conditions. Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, the numerous, known receptor-ligand couples, and complementary polynucleotide strands. In the case of complementary polynucleotide binding partners, the partners are normally at least about 15 bases in length, and may be at least 40 bases in length. It is well recognized by those of skill in the art that lengths shorter than 15 (e.g., 8 bases), between 15 and 40, and greater than 40 bases may also be used. The polynucleotides may be composed of DNA, RNA, or synthetic nucleotide analogs. Further binding partners can be identified using, e.g., the two-hybrid yeast screening assay as described herein. [0108]
  • A “biological sample” refers to a sample of tissue or fluid suspected of containing an analyte polynucleotide or polypeptide from an individual including, but not limited to, e.g., plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents. [0109]
  • As used herein, the terms “diagnosing” or “prognosing,” as used in the context of neoplasia, are used to indicate 1) the classification of lesions as neoplasia, 2) the determination of the severity of the neoplasia, or 3) the monitoring of the disease progression, prior to, during and after treatment. [0110]
  • “Encode”. A polynucleotide is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom. [0111]
  • “Isolated” or “substantially pure”. An “isolated” or “substantially pure” nucleic acid (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components which naturally accompany a native human sequence or protein, e.g., ribosomes, polymerases, many other human genome sequences and proteins. The term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. [0112]
  • “HPC2 Allele” refers to normal alleles of the HPC2 locus as well as alleles carrying variations that predispose individuals to develop prostate cancer. Such predisposing alleles are also called “HPC2 susceptibility alleles”. [0113]
  • “HPC2 Locus”, “HPC2 Gene”, “HPC2 Nucleic Acids” or “HPC2 Polynucleotide” each refer to polynucleotides, all of which are in the HPC2 region, that are likely to be expressed in normal tissue, certain alleles of which predispose an individual to develop prostate cancers. Mutations at the HPC2 locus may be involved in the initiation and/or progression of other types of tumors. The locus is indicated in part by mutations that predispose individuals to develop cancer. These mutations fall within the HPC2 region described infra. The HPC2 locus is intended to include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation. The HPC2 locus is intended to include all allelic variations of the DNA sequence. [0114]
  • The term HPC2 is used interchangeably throughout this disclosure with the terms ELAC2 and HPC2/ELAC2. This holds true regardless of whether the term refers to a nucleic acid, allele, gene, locus, protein or peptide. [0115]
  • These terms, when applied to a nucleic acid, refer to a nucleic acid which encodes an HPC2 polypeptide, fragment, homolog or variant, including, e.g., protein fusions or deletions. The nucleic acids of the present invention will possess a sequence which is either derived from, or substantially similar to a natural HPC2-encoding gene or one having substantial homology with a natural HPC2-encoding gene or a portion thereof. [0116]
  • The HPC2 gene or nucleic acid includes normal alleles of the HPC2 gene, including silent alleles having no effect on the amino acid sequence of the HPC2 polypeptide as well as alleles leading to amino acid sequence variants of the HPC2 polypeptide that do not substantially affect its function. These terms also include alleles having one or more mutations which adversely affect the function of the HPC2 polypeptide. A mutation may be a change in the HPC2 nucleic acid sequence which produces a deleterious change in the amino acid sequence of the HPC2 polypeptide, resulting in partial or complete loss of HPC2 function, or may be a change in the nucleic acid sequence which results in the loss of effective HPC2 expression or the production of aberrant forms of the HPC2 polypeptide. [0117]
  • The HPC2 nucleic acid may be that shown in SEQ ID NOs:1, 3 or 28 or it may be an allele as described above or a variant or derivative differing from that shown by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to the nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code. [0118]
  • Thus, nucleic acid according to the present invention may include a sequence different from the sequence shown in SEQ ID NOs:1, 3 or 28 yet encode a polypeptide with the same amino acid sequence as shown in SEQ ID NO:1. That is, nucleic acids of the present invention include sequences which are degenerate as a result of the genetic code. On the other hand, the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from the amino acid sequence shown in SEQ ID NO:2. Nucleic acid encoding a polypeptide which is an amino acid sequence variant, derivative or allele of the amino acid sequence shown in SEQ ID NO:2 is also provided by the present invention. [0119]
  • The HPC2 gene also refers to (a) any DNA sequence that (i) hybridizes to the complement of the DNA sequences that encode the amino acid sequence set forth in SEQ ID NO:2 under highly stringent conditions (Ausubel et al., 1992) and (ii) encodes a gene product functionally equivalent to HPC2, or (b) any DNA sequence that (i) hybridizes to the complement of the DNA sequences that encode the amino acid sequence set forth in SEQ ID NO:2 under less stringent conditions, such as moderately stringent conditions (Ausubel et al., 1992) and (ii) encodes a gene product functionally equivalent to HPC2. The invention also includes nucleic acid molecules that are the complements of the sequences described herein. [0120]
  • The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. [0121]
  • The present invention provides recombinant nucleic acids comprising all or part of the HPC2 region or the HPC2 paralog called ELAC1 or the mouse, chimpanzee or gorilla orthologs of HPC2, herein called mouse ELAC2, chimpanzee ELAC2 or gorilla ELAC2. The recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosomal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation, 1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; 2) is linked to a polynucleotide other than that to which it is linked in nature; or 3) does not occur in nature. Where nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T. [0122]
  • Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by this invention. Although the wild-type sequence may be employed, it will often be altered, e.g., by deletion, substitution or insertion. [0123]
  • cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR. The choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences. [0124]
  • The DNA sequences used in this invention will usually comprise at least about five codons (15 nucleotides), more usually at least about 7-15 codons, and most preferably, at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with an HPC2-encoding sequence. In this context, oligomers of as low as 8 nucleotides, more generally 8-17 nucleotides, can be used for probes, especially in connection with chip technology. [0125]
  • Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S. Biochemicals, New England Nuclear, and a number of other sources. The recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health. [0126]
  • “HPC2 Region” refers to a portion of [0127] human chromosome 17 bounded by the markers D17S947 and D17S799. This region contains the HPC2 locus, including the HPC2 gene.
  • As used herein, the terms “HPC2 locus”, “HPC2 allele” and “HPC2 region” all refer to the double-stranded DNA comprising the locus, allele, or region, as well as either of the single-stranded DNAs comprising the locus, allele or region. [0128]
  • As used herein, a “portion” of the HPC2 locus or region or allele is defined as having a minimal size of at least about eight nucleotides, or preferably about 15 nucleotides, or more preferably at least about 25 nucleotides, and may have a minimal size of at least about 40 nucleotides. This definition includes all sizes in the range of 8-40 nucleotides as well as greater than 40 nucleotides. Thus, this definition includes nucleic acids of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or nucleic acids having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc., nucleotides), or nucleic acids having more than 500 nucleotides. The present invention includes all novel nucleic acids having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28, its complement or functionally equivalent nucleic acid sequences. The present invention does not include nucleic acids which exist in the prior art. That is, the present invention includes all nucleic acids having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28 with the proviso that it does not include nucleic acids existing in the prior art. [0129]
  • “HPC2 protein” or “HPC2 polypeptide” refers to a protein or polypeptide encoded by the HPC2 locus, variants or fragments thereof. The term “polypeptide” refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring. Ordinarily, such polypeptides will be at least about 50% homologous to the native HPC2 sequence, preferably in excess of about 90%, and more preferably at least about 95% homologous. Also included are proteins encoded by DNA which hybridize under high or low stringency conditions, to HPC2-encoding nucleic acids and closely related polypeptides or proteins retrieved by antisera to the HPC2 protein(s). [0130]
  • An HPC2 polypeptide may be that derived from any of the exons described herein which may be in isolated and/or purified form, free or substantially free of material with which it is naturally associated. The polypeptide may, if produced by expression in a prokaryotic cell or produced synthetically, lack native post-translational processing, such as glycosylation. Alternatively, the present invention is also directed to polypeptides which are sequence variants, alleles or derivatives of an HPC2 polypeptide. Such polypeptides may have an amino acid sequence which differs from that derived from any of the exons described herein by one or more of addition, substitution, deletion or insertion of one or more amino acids. Preferred such polypeptides have HPC2 function. [0131]
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties. 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. Preferred substitutions are ones which are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and tyrosine, phenylalanine. [0132]
  • Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or binding sites on proteins interacting with an HPC2 polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982). Alternatively, the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The importance of hydrophilicity in conferring interactive biological function of a protein is generally understood in the art (U.S. Pat. No. 4,554,101). The use of the hydrophobic index or hydrophilicity in designing polypeptides is further discussed in U.S. Pat. No. 5,691,198. [0133]
  • The length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. [0134]
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. [0135]
  • The term peptide mimetic or mimetic is intended to refer to a substance which has the essential biological activity of an HPC2, ELAC1 or ELAC2 polypeptide. A peptide mimetic may be a peptide-containing molecule that mimics elements of protein secondary structure (Johnson et al., 1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen, enzyme and substrate or scaffolding proteins. A peptide mimetic is designed to permit molecular interactions similar to the natural molecule. A mimetic may not be a peptide at all, but it will retain the essential biological activity of a natural HPC2, ELAC1 or ELAC2 polypeptide. [0136]
  • “Probes”. Polynucleotide polymorphisms associated with HPC2 alleles which predispose to certain cancers or are associated with most cancers are detected by hybridization with a polynucleotide probe which forms a stable hybrid with that of the target sequence, under highly stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be perfectly complementary to the target sequence, high stringency conditions will be used. Hybridization stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe will not be completely complementary. Conditions are chosen which rule out nonspecific/adventitious bindings, that is, which minimize noise. (It should be noted that throughout this disclosure, if it is simply stated that “stringent” conditions are used that is meant to be read as “high stringency” conditions are used.) Since such indications identify neutral DNA polymorphisms as well as mutations, these indications need further analysis to demonstrate detection of an HPC2 susceptibility allele. An example of high stringency conditions is to hybridize to filter bound DNA in 0.5 M NaHPO[0137] 4, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C. and to wash in 0.1× SSC/0.1% SDS at 68° C. (Ausubel et al., 1992). Less stringent conditions, such as moderately stringent conditions, are defined as above but with the wash step being in 0.2× SSC/0.1% SDS at 42° C.
  • Probes for HPC2 alleles may be derived from the sequences of the HPC2 region, its cDNA, functionally equivalent sequences, or the complements thereof The probes may be of any suitable length, which span all or a portion of the HPC2 region, and which allow specific hybridization to the HPC2 region. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of about 8-30 base pairs, since the hybrid will be relatively stable under even highly stringent conditions. If some degree of mismatch is expected with the probe, i.e., if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridizes to the target sequence with the requisite specificity. [0138]
  • The probes will include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods. For techniques for preparing and labeling probes see, e.g., Sambrook et al., 1989 or Ausubel et al., 1992. Other similar polynucleotides may be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by use of the redundancy in the genetic code. Various codon substitutions may be introduced, e.g., by silent changes (thereby producing various restriction sites) or to optimize expression for a particular system. Mutations may be introduced to modify the properties of the polypeptide, perhaps to change ligand-binding affinities, interchain affinities, or the polypeptide degradation or turnover rate. [0139]
  • Probes comprising synthetic oligonucleotides or other polynucleotides of the present invention may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labeled by nick translation, Klenow fill-in reaction, or other methods known in the art. [0140]
  • Portions of the polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 9 kb, usually fewer than about 1.0 kb, from a polynucleotide sequence encoding HPC2 are preferred as probes. This definition therefore includes probes of [0141] sizes 8 nucleotides through 9000 nucleotides. Thus, this definition includes probes of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400 or 500 nucleotides or probes having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc., nucleotides), or probes having more than 500 nucleotides. The probes may also be used to determine whether mRNA encoding HPC2 is present in a cell or tissue. The present invention includes all novel probes having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28 its complement or functionally equivalent nucleic acid sequences. The present invention does not include probes which exist in the prior art. That is, the present invention includes all probes having at least 8 nucleotides derived from any of SEQ ID NOs:1 or 3-28 with the proviso that they do not include probes existing in the prior art.
  • Similar considerations and nucleotide lengths are also applicable to primers which may be used for the amplification of all or part of the HPC2 gene. Thus, a definition for primers includes primers of 8, 12, 15, 20, 25, 40, 60, 80, 100, 200, 300, 400, 500 nucleotides, or primers having any number of nucleotides within these ranges of values (e.g., 9, 10, 11, 16, 23, 30, 38, 50, 72, 121, etc. nucleotides), or primers having more than 500 nucleotides, or any number of nucleotides between 500 and 9000. The primers may also be used to determine whether mRNA encoding HPC2 is present in a cell or tissue. The present invention includes all novel primers having at least 8 nucleotides derived from the HPC2 locus for amplifying the HPC2 gene, its complement or functionally equivalent nucleic acid sequences. The present invention does not include primers which exist in the prior art. That is, the present invention includes all primers having at least 8 nucleotides with the proviso that it does not include primers existing in the prior art. [0142]
  • “Protein modifications or fragments” are provided by the present invention for HPC2, ELAC1 and ELAC2 polypeptides or fragments thereof which are substantially homologous to primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as [0143] 32P, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods of labeling polypeptides are well known in the art. See Sambrook et al., 1989 or Ausubel et al., 1992.
  • Besides substantially full-length polypeptides, the present invention provides for biologically active fragments of the polypeptides. Significant biological activities include ligand-binding, immunological activity and other biological activities characteristic of HPC2, ELAC1 or ELAC2 polypeptides. Immunological activities include both immunogenic function in a target immune system, as well as sharing of immunological epitopes for binding, serving as either a competitor or substitute antigen for an epitope of the HPC2, ELAC1 or ELAC2 protein. As used herein, “epitope” refers to an antigenic determinant of a polypeptide. An epitope could comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of such amino acids are known in the art. [0144]
  • For immunological purposes, tandem-repeat polypeptide segments may be used as immunogens, thereby producing highly antigenic proteins. Alternatively, such polypeptides will serve as highly efficient competitors for specific binding. Production of antibodies specific for HPC2, ELAC1 or ELAC2 polypeptides or fragments thereof is described below. [0145]
  • The present invention also provides for fusion polypeptides, comprising HPC2, ELAC1 or ELAC2 polypeptides and fragments. Homologous polypeptides may be fusions between two or more HPC2, ELAC1 or ELAC2 polypeptide sequences or between the sequences of HPC2, ELAC1 or ELAC2 and a related protein. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. For example, ligand-binding or other domains may be “swapped” between different new fusion polypeptides or fragments. Such homologous or heterologous fusion polypeptides may display, for example, altered strength or specificity of binding. Fusion partners include immunoglobulins, bacterial β-galactosidase, trpE, protein A, β-lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See Godowski et al., 1988. [0146]
  • Fusion proteins will typically be made by either recombinant nucleic acid methods, as described below, or may be chemically synthesized. Techniques for the synthesis of polypeptides are described, for example, in Merrifield, 1963. [0147]
  • “Protein purification” refers to various methods for the isolation of the HPC2, ELAC1 or ELAC2 polypeptides from other biological material, such as from cells transformed with recombinant nucleic acids encoding HPC2, ELAC1 or ELAC2 and are well known in the art. For example, such polypeptides may be purified by immunoaffinity chromatography employing, e.g., the antibodies provided by the present invention. Various methods of protein purification are well known in the art, and include those described in Deutscher, 1990 and Scopes, 1982. [0148]
  • The terms “isolated”, “substantially pure”, and “substantially homogeneous” are used interchangeably to describe a protein or polypeptide which has been separated from components which accompany it in its natural state. A monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a single polypeptide sequence. A substantially pure protein will typically comprise about 60 to 90% W/W of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for purification. [0149]
  • An HPC2, ELAC1 or ELAC2 protein is substantially free of naturally associated components when it is separated from the native contaminants which accompany it in its natural state. Thus, a polypeptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. [0150]
  • A polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an “isolated polypeptide,” as used herein, even if expressed in a homologous cell type. Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules. [0151]
  • “Recombinant nucleic acid” is a nucleic acid which is not naturally occurring, or which is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. [0152]
  • “Regulatory sequences” refers to those sequences normally within 100 kb of the coding region of a locus, but they may also be more distant from the coding region, which affect the expression of the gene (including transcription of the gene, and translation, splicing, stability or the like of the messenger RNA). [0153]
  • “Substantial homology or similarity”. A nucleic acid or fragment thereof is “substantially homologous” (“or substantially similar”) to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases. [0154]
  • Identity means the degree of sequence relatedness between two polypeptide or two polynucleotides sequences as determined by the identity of the match between two strings of such sequences. Identity can be readily calculated. While there exist a number of methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Methods commonly employed to determine identity between two sequences include, but are not limited to those disclosed in [0155] Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D. (1988). Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Such methods are codified in computer programs. Preferred computer program methods to determine identity between two sequences include, but are not limited to, GCG program package (Devereux et al. (1984), BLASTP, BLASTN, FASTA (Altschul et al. (1990); Altschul et al. (1997)).
  • Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof will hybridize to another nucleic acid (or a complementary strand thereof) under selective hybridization conditions, to a strand, or to its complement. Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs. Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa, 1984. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. [0156]
  • Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30° C., typically in excess of 37° C., and preferably in excess of 45° C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson, 1968. [0157]
  • Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes. The preparation of such probes and suitable hybridization conditions are well known in the art. [0158]
  • The terms “substantial homology” or “substantial identity”, when referring to polypeptides, indicate that the polypeptide or protein in question exhibits at least about 30% identity with an entire naturally-occurring protein or a portion thereof, usually at least about 70% identity, more usually at least about 80% identity, preferably at least about 90% identity, and more preferably at least about 95% identity. [0159]
  • Homology, for polypeptides, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705, as well as the software described above with reference to nucleic acid homology. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. [0160]
  • “Substantially similar function” refers to the function of a modified nucleic acid or a modified protein, with reference to the wild-type HPC2, ELAC1 or ELAC2 nucleic acid or wild-type HPC2, ELAC1 or ELAC2 polypeptide. The modified polypeptide will be substantially homologous to the wild-type HPC2, ELAC1 or ELAC2 polypeptide and will have substantially the same function. The modified polypeptide may have an altered amino acid sequence and/or may contain modified amino acids. In addition to the similarity of function, the modified polypeptide may have other useful properties, such as a longer half-life. The similarity of function (activity) of the modified polypeptide may be substantially the same as the activity of the wild-type HPC2, ELAC1 or ELAC2 polypeptide. Alternatively, the similarity of function (activity) of the modified polypeptide may be higher than the activity of the wild-type HPC2, ELAC1 or ELAC2 polypeptide. The modified polypeptide is synthesized using conventional techniques, or is encoded by a modified nucleic acid and produced using conventional techniques. The modified nucleic acid is prepared by conventional techniques. A nucleic acid with a function substantially similar to the wild-type HPC2, ELAC1 or ELAC2 gene function produces the modified protein described above. [0161]
  • A polypeptide “fragment,” “portion” or “segment” is a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous ammo acids, typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids. [0162]
  • The polypeptides of the present invention, if soluble, may be coupled to a solid-phase support, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates. Such supports may take the form, for example, of beads, wells, dipsticks, or membranes. [0163]
  • “Target region” refers to a region of the nucleic acid which is amplified and/or detected. The term “target sequence” refers to a sequence with which a probe or primer will form a stable hybrid under desired conditions. [0164]
  • The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology. See, e.g., Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992; Glover, 1985; Anand, 1992; Guthrie and Fink, 1991. A general discussion of techniques and materials for human gene mapping, including mapping of [0165] human chromosome 1, is provided, e.g., in White and Lalouel, 1988.
  • Preparation of Recombinant or Chemically Synthesized Nucleic Acids; Vectors, Transformation, Host Cells [0166]
  • Large amounts of the polynucleotides of the present invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs, usually DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the polynucleotide constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to (with and without integration within the genome) cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention is described, e.g., in Sambrook et al., 1989 or Ausubel et al., 1992. [0167]
  • The polynucleotides of the present invention may also be produced by chemical synthesis, e.g., by the phosphoramidite method described by Beaucage and Caruthers, 1981 or the triester method according to Matteucci and Caruthers, 1981, and may be performed on commercial, automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence. [0168]
  • Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Secretion signals may also be included where appropriate, whether from a native HPC2 protein or from other receptors or from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, and thus attain its functional topology, or be secreted from the cell. Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al., 1989 or Ausubel et al. 1992. [0169]
  • An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may include, when appropriate, those naturally associated with HPC2, ELAC1 or ELAC2 genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or Ausubel et al., 1992; see also, e.g., Metzger et al., 1988. Many useful vectors are known in the art and may be obtained from such vendors as Stratagene, New England BioLabs, Promega Biotech, and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., EP 73,675A. Appropriate non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al., 1978) or promoters derived from murine Moloney leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. Insect promoters may be derived from baculovirus. In addition, the construct may be joined to an ampliflable gene (e.g., DHFR) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences, see also [0170] Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1983). See also, e.g., U.S. Pat. Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.
  • While such expression vectors may replicate autonomously, they may also replicate by being inserted into the genome of the host cell, by methods well known in the art. [0171]
  • Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene ensures growth of only those host cells which express the inserts. Typical selection genes encode proteins that a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc.; b) complement auxotrophic deficiencies, or c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art. [0172]
  • The vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection (see, Kubo et al., 1988), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al., 1989 and Ausubel et al., 1992. The introduction of the polynucleotides into the host cell by any method known in the art, including, inter alia, those described above, will be referred to herein as “transformation.” The cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells. [0173]
  • Large quantities of the nucleic acids and polypeptides of the present invention may be prepared by expressing the HPC2, [0174] ELAC 1 or ELAC2 nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells. The most commonly used prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.
  • Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, or amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. See, Jakoby and Pastan, 1979. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features. An example of a commonly used insect cell line is SF9. [0175]
  • Clones are selected by using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule, preferably the same DNA molecule. In prokaryotic hosts, the transformant may be selected, e.g., by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker. [0176]
  • Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of HPC2, ELAC1 or ELAC2 polypeptides. [0177]
  • The HPC2, ELAC1 or ELAC2 gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats and non-human primates, e.g., baboons, monkeys and chimpanzees, may be used to generate HPC2, [0178] ELAC 1 or ELAC2 transgenic animals.
  • Any technique known in the art may be used to introduce the HPC2, ELAC1 or ELAC2 gene transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., 1985); gene targeting in embryonic stem cells (Thompson et al., 1989); electroporation of embryos (Lo, 1983); and sperm-mediated gene transfer (Lavitrano et al., 1989); etc. For a review of such techniques, see Gordon (1989), which is incorporated by reference herein in its entirety. [0179]
  • The present invention provides for transgenic animals that carry the HPC2, ELAC1 or ELAC2 transgene in all their cells, as well as animals which carry the transgene in some, but not all of their cells, i.e., mosaic animals. The transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (1992). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the HPC2, ELAC1 or ELAC2 gene transgene be integrated into the chromosomal site of the endogenous HPC2, ELAC1 or ELAC2 gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous HPC2, ELAC1 or ELAC2 gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous HPC2, ELAC1 or ELAC2 gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous HPC2, ELAC1 or ELAC2 gene in only that cell type, by following, for example, the teaching of Gu et al. (1994). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. [0180]
  • Once transgenic animals have been generated, the expression of the recombinant HPC2, ELAC1 or ELAC2 gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of HPC2, ELAC1 or ELAC2 gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the HPC2, ELAC1 or ELAC2 transgene product. [0181]
  • Antisense polynucleotide sequences are useful in preventing or diminishing the expression of the HPC2, ELAC1 or ELAC2 locus, as will be appreciated by those skilled in the art. For example, polynucleotide vectors containing all or a portion of the HPC2 locus or other sequences from the HPC2 region (particularly those flanking the HPC2 locus) may be placed under the control of a promoter in an antisense orientation and introduced into a cell. Expression of such an antisense construct within a cell will interfere with HPC2 transcription and/or translation and/or replication. [0182]
  • The probes and primers based on the HPC2 gene sequences disclosed herein are used to identify homologous HPC2 gene sequences and proteins in other species. These HPC2 gene sequences and proteins are used in the diagnostic/prognostic, therapeutic and drug screening methods described herein for the species from which they have been isolated. [0183]
  • Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits [0184]
  • In order to detect the presence of an HPC2 allele predisposing an individual to cancer, a biological sample such as blood is prepared and analyzed for the presence or absence of susceptibility alleles of HPC2. In order to detect the presence of neoplasia, the progression toward malignancy of a precursor lesion, or as a prognostic indicator, a biological sample of the lesion is prepared and analyzed for the presence or absence of mutant alleles of HPC2. Results of these tests and interpretive information are returned to the health care provider for communication to the tested individual. Such diagnoses may be performed by diagnostic laboratories, or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self-diagnosis. [0185]
  • Initially, the screening method involves amplification of the relevant HPC2 sequences. In another preferred embodiment of the invention, the screening method involves a non-PCR based strategy. Such screening methods include two-step label amplification methodologies that are well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity. [0186]
  • The most popular method used today is target amplification. Here, the target nucleic acid sequence is amplified with polymerases. One particularly preferred method using polymerase-driven amplification is the polymerase chain reaction (PCR). The polymerase chain reaction and other polymerase-driven amplification assays can achieve over a million-fold increase in copy number through the use of polymerase-driven amplification cycles. Once amplified, the resulting nucleic acid can be sequenced or used as a substrate for DNA probes. [0187]
  • When the probes are used to detect the presence of the target sequences (for example, in screening for cancer susceptibility), the biological sample to be analyzed, such as blood or serum, may be treated, if desired, to extract the nucleic acids. The sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting. The targeted region of the analyte nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art. [0188]
  • Analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte. The region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of [0189] human chromosome 17. Therefore, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide. These factors are outlined in, for example, Maniatis et al., 1982 and Sambrook et al., 1989. Under certain circumstances, the formation of higher order hybrids, such as triplexes, quadraplexes, etc., may be desired to provide the means of detecting target sequences.
  • Detection, if any, of the resulting hybrid is usually accomplished by the use of labeled probes. Alternatively, the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly. Suitable labels, and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation, random priming or kinasing), biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly triggered dioxetanes), enzymes, antibodies, gold nanoparticles and the like. Variations of this basic scheme are known in the art, and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labeled moiety. A number of these variations are reviewed in, e.g., Matthews and Kricka, 1988; Landegren et al., 1988; Mifflin, 1989; U.S. Pat. No. 4,868,105, and in EPO Publication No. 225,807. [0190]
  • As noted above, non-PCR based screening assays are also contemplated in this invention. This procedure hybridizes a nucleic acid probe (or an analog such as a methyl phosphonate backbone replacing the normal phosphodiester), to the low level DNA target. This probe may have an enzyme covalently linked to the probe, such that the covalent linkage does not interfere with the specificity of the hybridization. This enzyme-probe-conjugate-target nucleic acid complex can then be isolated away from the free probe enzyme conjugate and a substrate is added for enzyme detection. Enzymatic activity is observed as a change in color development or luminescent output resulting in a 10[0191] 3-106 increase in sensitivity. For an example relating to the preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes see Jablonski et al., 1986.
  • Two-step label amplification methodologies are known in the art. These assays work on the principle that a small ligand (such as digoxigenin, biotin, or the like) is attached to a nucleic acid probe capable of specifically binding HPC2. Allele specific probes are also contemplated within the scope of this example and exemplary allele specific probes include probes encompassing the predisposing or potentially predisposing mutations summarized in Table 9 of this patent application. [0192]
  • In one example, the small ligand attached to the nucleic acid probe is specifically recognized by an antibody-enzyme conjugate. In one embodiment of this example, digoxigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate which turns over a chemiluminescent substrate. For methods for labeling nucleic acid probes according to this embodiment see Martin et al., 1990. In a second example, the small ligand is recognized by a second ligand-enzyme conjugate that is capable of specifically complexing to the first ligand. A well known embodiment of this example is the biotin-avidin type of interactions. For methods for labeling nucleic acid probes and their use in biotin-avidin based assays see Rigby et al., 1977 and Nguyen et al., 1992. [0193]
  • It is also contemplated within the scope of this invention that the nucleic acid probe assays of this invention will employ a cocktail of nucleic acid probes capable of detecting HPC2. Thus, in one example to detect the presence. of HPC2 in a cell sample, more than one probe complementary to HPC2 is employed and in particular the number of different probes is alternatively 2, 3, or 5 different nucleic-acid probe sequences. In another example, to detect the presence of mutations in the HPC2 gene sequence m a patient, more than one probe complementary to HPC2 is employed where the cocktail includes probes capable of binding to the allele-specific mutations identified in populations of patients with alterations in HPC2. In this embodiment, any number of probes can be used, and will preferably include probes corresponding to the major gene mutations identified as predisposing an individual to prostate cancer. [0194]
  • Methods of Use: Peptide Diagnosis and Diagnostic Kits [0195]
  • The neoplastic condition of lesions can also be detected on the basis of the alteration of wild-type HPC2 polypeptide. Such alterations can be determined by sequence analysis in accordance with conventional techniques. More preferably, antibodies (polyclonal or monoclonal) are used to detect differences in, or the absence of, HPC2 peptides. The antibodies may be prepared as discussed above under the heading “Antibodies” and as further shown in Examples 16 and 17. Other techniques for raising and purifying antibodies are well known in the art and any such techniques may be chosen to achieve the preparations claimed in this invention. In a preferred embodiment of the invention, antibodies will immunoprecipitate HPC2 proteins from solution as well as react with HPC2 protein on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, antibodies will detect HPC2 proteins in paraffin or frozen tissue sections, using immunocytochemical techniques. [0196]
  • Preferred embodiments relating to methods for detecting HPC2 or its mutations include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al. in U.S. Pat. Nos. 4,376,110 and 4,486,530, hereby incorporated by reference, and exemplified in Example 19. [0197]
  • Methods of Use: Drug Screening [0198]
  • This invention is particularly useful for screening compounds by using the HPC2, ELAC1 or ELAC2 polypeptide or binding fragment thereof in any of a variety of drug screening techniques. [0199]
  • The HPC2, ELAC1 or ELAC2 polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface. One method of drug screening utilizes eucaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, for the formation of complexes between an HPC2, ELAC1 or ELAC2 polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between an HPC2, ELAC1 or ELAC2 polypeptide or fragment and a known ligand is interfered with by the agent being tested. [0200]
  • Thus, the present invention provides methods of screening for drugs comprising contacting such an agent with an HPC2, ELAC1 or ELAC2 polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the HPC2, ELAC1 or ELAC2 polypeptide or fragment, or (ii) for the presence of a complex between the HPC2, ELAC1 or ELAC2 polypeptide or fragment and a ligand, by methods well known in the art. In such competitive binding assays the HPC2, ELAC1 or ELAC2 polypeptide or fragment is typically labeled. Free HPC2, ELAC1 or ELAC2 polypeptide or fragment is separated from that present in a protein:protein complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to HPC2, ELAC1 or ELAC2 or its interference with HPC2:ligand, ELAC1:ligand or ELAC2:ligand binding, respectively. One may also measure the amount of bound, rather than free, HPC2, ELAC1 or ELAC2. It is also possible to label the ligand rather than the HPC2, ELAC1 or ELAC2 and to measure the amount of ligand binding to HPC2, ELAC1 or ELAC2 in the presence and in the absence of the drug being tested. [0201]
  • Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the HPC2, ELAC1 or ELAC2 polypeptides and is described in detail in Geysen (published PCT WO 84/03564). Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HPC2, ELAC1 or ELAC2 polypeptide and washed. Bound HPC2, ELAC1 or ELAC2 polypeptide is then detected by methods well known in the art. [0202]
  • Purified HPC2, ELAC1 or ELAC2 can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the HPC2, ELAC1 or ELAC2 polypeptide on the solid phase. [0203]
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the HPC2, ELAC1 or ELAC2 polypeptide compete with a test compound for binding to the HPC2, ELAC1 or ELAC2 polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the HPC2, ELAC1 or ELAC2 polypeptide. [0204]
  • A further technique for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional HPC2, ELAC1 or ELAC2 gene. These host cell lines or cells are defective at the HPC2, ELAC1 or ELAC2 polypeptide level. The host cell lines or cells are grown in the presence of drug compound. The rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of HPC2, ELAC1 or ELAC2 defective cells. [0205]
  • Briefly, a method of screening for a substance which modulates activity of a polypeptide may include contacting one or more test substances with the polypeptide in a suitable reaction medium, testing the activity of the treated polypeptide and comparing that activity with the activity of the polypeptide in comparable reaction medium untreated with the test substance or substances. A difference in activity between the treated and untreated polypeptides is indicative of a modulating effect of the relevant test substance or substances. [0206]
  • Prior to or as well as being screened for modulation of activity, test substances may be screened for ability to interact with the polypeptide, e.g., in a yeast two-hybrid system (e.g., Bartel et al., 1993; Fields and Song, 1989; Chevray and Nathans, 1992; Lee et al., 1995). This system may be used as a coarse screen prior to testing a substance for actual ability to modulate activity of the polypeptide. Alternatively, the screen could be used to screen test substances for binding to an HPC2, ELAC1 or ELAC2 specific binding partner, or to find mimetics of an HPC2, ELAC1 or ELAC2 polypeptide. [0207]
  • Methods of Use: Rational Drug Design [0208]
  • The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, 1991. In one approach, one first determines the three-dimensional structure of a protein of interest (e.g., HPC2 polypeptide) or, for example, of the HPC2-receptor or ligand complex, by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., 1990). In addition, peptides (e.g., HPC2 polypeptide) are analyzed by an alanine scan (Wells, 1991). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide. [0209]
  • It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the phannacore. [0210]
  • Thus, one may design drugs which have, e.g., improved HPC2, ELAC1 or ELAC2 polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of HPC2, ELAC1 or ELAC2 polypeptide activity. By virtue of the availability of cloned HPC2, ELAC1 and ELAC2 sequences, sufficient amounts of the HPC2, ELAC1 or ELAC2 polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the HPC2, ELAC1 and ELAC2 protein sequences provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography. [0211]
  • Following identification of a substance which modulates or affects polypeptide activity, the substance may be investigated further. Furthermore, it may be manufactured and/or used in preparation, i.e., manufacture or formulation, or a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals. [0212]
  • Thus, the present invention extends in various aspects not only to a substance identified using a nucleic acid molecule as a modulator of polypeptide activity, in accordance with what is disclosed herein, but also a phanmaceutical composition, medicament, drug or other composition comprising such a substance, a method comprising administration of such a composition comprising such a substance, a method comprising administration of such a composition to a patient, e.g., for treatment of prostate cancer, use of such a substance in the manufacture of a composition for administration, e.g., for treatment of prostate cancer, and a method of making a pharmaceutical composition comprising admixing such a substance with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients. [0213]
  • A substance identified as a modulator of polypeptide function may be peptide or non-peptide in nature. Non-peptide “small molecules” are often preferred for many in vivo pharmaceutical uses. Accordingly, a mimetic or mimic of the substance (particularly if a peptide) may be designed for pharmaceutical use. [0214]
  • The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a “lead” compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., pure peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large numbers of molecules for a target property. [0215]
  • There are several steps commonly taken in the design of a mimetic from a compound having a given target property. First, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g., by substituting each residue in turn. Alanine scans of peptide are commonly used to refine such peptide motifs. These parts or residues constituting the active region of the compound are known as its “pharmacophore”. [0216]
  • Once the pharmacophore has been found, its structure is modeled according to its physical properties, e.g., stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g., spectroscopic techniques, x-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process. [0217]
  • In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic. [0218]
  • A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. Alternatively, where the mimetic is peptide-based, further stability can be achieved by cyclizing the peptide, increasing its rigidity. The mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing. [0219]
  • Methods of Use: Gene Therapy [0220]
  • According to the present invention, a method is also provided of supplying wild-type HPC2 function to a cell which carries mutant HPC2 alleles. Supplying such a function should suppress neoplastic growth of the recipient cells. The wild-type HPC2 gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. If a gene fragment is introduced and expressed in a cell carrying a mutant HPC2 allele, the gene fragment should encode a part of the HPC2 protein which is required for non-neoplastic growth of the cell. More preferred is the situation where the wild-type HPC2 gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant HPC2 gene present in the cell. Such recombination requires a double recombination event which results in the correction of the HPC2 gene mutation. Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used. Methods for introducing DNA into cells such as electroporation, calcium phosphate coprecipitation and viral transduction are known in the art, and the choice of method is within the competence of the practitioner. Cells transformed with the wild-type HPC2 gene can be used as model systems to study cancer remission and drug treatments which promote such remission. [0221]
  • As generally discussed above, the HPC2 gene or fragment, where applicable, may be employed in gene therapy methods in order to increase the amount of the expression products of such genes in cancer cells. Such gene therapy is particularly appropriate for use in both cancerous and pre-cancerous cells, in which the level of HPC2 polypeptide is absent or diminished compared to normal cells. It may also be useful to increase the level of expression of a given HPC2 gene even in those tumor cells in which the mutant gene is expressed at a “normal” level, but the gene product is not fully functional. [0222]
  • Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman (1991) or Culver (1996). Cells from a patient's tumor would be first analyzed by the diagnostic methods described above, to ascertain the production of HPC2 polypeptide in the tumor cells. A virus or plasmid vector (see further details below), containing a copy of the HPC2 gene linked to expression control elements and capable of replicating inside the tumor cells, is prepared. Alternatively, the vector may be replication deficient and is replicated in helper cells for use in gene therapy. Suitable vectors are known, such as disclosed in U.S. Pat. No. 5,252,479 and PCT published application WO 93/07282 and U.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500. The vector is then injected into the patient, either locally at the site of the tumor or systemically (in order to reach any tumor cells that may have metastasized to other sites). If the transfected gene is not permanently incorporated into the genome of each of the targeted tumor cells, the treatment may have to be repeated periodically. [0223]
  • Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson and Akrigg, 1992; Stratford-Perricaudet et al., 1990; Schneider et al., 1998), vaccinia virus (Moss, 1992; Moss, 1996), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990; Russell and Hirata, 1998), herpes viruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakefield and Geller, 1987; Freese et al., 1990; Fink et al., 1996), lentiviruses (Naldini et al., 1996), Sindbis and Semliki Forest virus (Berglund et al., 1993), and retroviruses of avian (Bandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al, 1988), and human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Most human gene therapy protocols have been based on disabled murine retroviruses, although adenovirus and adeno-associated virus are also being used. [0224]
  • Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical techniques, for example microinjection (Anderson et al, 1980; Gordon et al, 1980; Brinster et al, 1981; Costantini and Lacy, 1981); membrane fusion-mediated transfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et al, 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1991); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al, 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al, 1989; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al., 1990; Curiel et al., 1991; Curiel et al., 1992). Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the tumor cells and not into the surrounding nondividing cells. Alternatively, the retroviral vector producer cell line can be injected into tumors (Culver et al., 1992). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumors. [0225]
  • In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization, and degradation of the endosome before the coupled DNA is damaged. For other techniques for the delivery of adenovirus based vectors see Schneider et al. (1998) and U.S. Pat. Nos. 5,691,198; 5,747,469; 5,436,146 and 5,753,500. [0226]
  • Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer process is nonspecific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992). [0227]
  • Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein. In viral expression vectors, the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes HPC2, expression will produce HPC2. If the polynucleotide encodes an antisense polynucleotide or a ribozyme, expression will produce the antisense polynucleotide or ribozyme. Thus in this context, expression does not require that a protein product be synthesized. In addition to the polynucleotide cloned into the expression vector, the vector also contains a promoter functional in eukaryotic cells. The cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters include those described above. The expression vector may also include sequences, such as selectable markers and other sequences described herein. [0228]
  • Gene transfer techniques which target DNA directly to prostate tissues, e.g., epithelial cells of the prostate, are preferred. Receptor-mediated gene transfer, for example, is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. One appropriate receptor/ligand pair may include the estrogen receptor and its ligand, estrogen (and estrogen analogues). These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, coinfection with adenovirus can be included to disrupt endosome function. [0229]
  • The therapy involves two steps which can be performed singly or jointly. In the first step, prepubescent females who carry an HPC2 susceptibility allele are treated with a gene delivery vehicle such that some or all of their mammary ductal epithelial precursor cells receive at least one additional copy of a functional normal HPC2 allele. In this step, the treated individuals have reduced risk of prostate cancer to the extent that the effect of the susceptible allele has been countered by the presence of the normal allele. In the second step of a preventive therapy, predisposed young females, in particular women who have received the proposed gene therapeutic treatment, undergo hormonal therapy to mimic the effects on the prostate of a full term pregnancy. [0230]
  • Methods of Use: Peptide Therapy [0231]
  • Peptides which have HPC2, ELAC1 or ELAC2 activity can be supplied to cells which carry mutant or missing HPC2, ELAC1 or ELAC2 alleles. Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors. Alternatively, HPC2, ELAC1 or ELAC2 polypeptide can be extracted from HPC2-, ELAC1- or ELAC2-producing mammalian cells. In addition, the techniques of synthetic chemistry can be employed to synthesize HPC2, ELAC1 or ELAC2 protein. Any of such techniques can provide the preparation of the present invention which comprises the HPC2, ELAC1 or ELAC2 protein. Preparation is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro. [0232]
  • Active HPC2, ELAC1 or ELAC2 molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Extracellular application of the HPC2, ELAC1 or ELAC2 gene product may be sufficient to affect tumor growth. Supply of molecules with HPC2 activity should lead to partial reversal of the neoplastic state. Other molecules with HPC2 activity (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having substantially similar function are also used for peptide therapy. [0233]
  • Methods of Use: Transformed Hosts [0234]
  • Similarly, cells and animals which carry a mutant HPC2, ELAC1 or ELAC2 allele can be used as model systems to study and test for substances which have potential as therapeutic agents. The cells are typically cultured epithelial cells. These may be isolated from individuals with HPC2, ELAC1 or ELAC2 mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the HPC2, ELAC1 or ELAC2 allele, as described above. After a test substance is applied to the cells, the neoplastically transformed phenotype of the cell is determined. Any trait of neoplastically transformed cells can be assessed, including anchorage-independent growth, tumorigenicity in nude mice, invasiveness of cells, and growth factor dependence. Assays for each of these traits are known in the art. [0235]
  • Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant HPC2, ELAC1 or ELAC2 alleles, usually from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous HPC2, ELAC1 or ELAC2 gene(s) of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques (Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992) to produce knockout or transplacement animals. A transplacement is similar to a knockout because the endogenous gene is replaced, but in the case of a transplacement the replacement is by another version of the same gene. After test substances have been administered to the animals, the growth of tumors must be assessed. If the test substance prevents or suppresses the growth of tumors, then the test substance is a candidate therapeutic agent for the treatment of the cancers identified herein. These animal models provide an extremely important testing vehicle for potential therapeutic products. [0236]
  • In one embodiment of the invention, transgenic animals are produced which contain a functional transgene encoding a functional HPC2, ELAC1 or ELAC2 polypeptide or variants thereof. Transgenic animals expressing HPC2, ELAC1 or ELAC2 transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of HPC2, ELAC1 or ELAC2. Transgenic animals of the present invention also can be used as models for studying indications such as disease. [0237]
  • In one embodiment of the invention, an HPC2, ELAC1 or ELAC2 transgene is introduced into a non-human host to produce a transgenic animal expressing a human or murine HPC2, ELAC1 or ELAC2 gene. The transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene. Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191; which is incorporated herein by reference), Brinster et al. 1985; which is incorporated herein by reference in its entirety) and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition (eds., Hogan, Beddington, Costantini and Long, Cold Spring Harbor Laboratory Press, 1994; which is incorporated herein by reference in its entirety). [0238]
  • It may be desirable to replace the endogenous HPC2, ELAC1 or ELAC2 by homologous recombination between the transgene and the endogenous gene; or the endogenous gene may be eliminated by deletion as in the preparation of “knock-out” animals. Typically, an HPC2, ELAC1 or ELAC2 gene flanked by genomic sequences is transferred by microinjection into a fertilized egg. The microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene. Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish. Within a particularly preferred embodiment, transgenic mice are generated which overexpress HPC2 or express a mutant form of the polypeptide. Alternatively, the absence of an HPC2, ELAC1 or ELAC2 in “knock-out” mice permits the study of the effects that loss of HPC2, ELAC1 or ELAC2 protein has on a cell in vivo. Knock-out mice also provide a model for the development of HPC2-related cancers. [0239]
  • Methods for producing knockout animals are generally described by Shastry (1995, 1998) and Osterrieder and Wolf (1998). The production of conditional knockout animals, in which the gene is active until knocked out at the desired time is generally described by Feil et al. (1996), Gagneten et al. (1997) and Lobe and Nagy (1998). Each of these references is incorporated herein by reference. [0240]
  • As noted above, transgenic animals and cell lines derived from such animals may find use in certain testing experiments. In this regard, transgenic animals and cell lines capable of expressing wild-type or mutant HPC2, ELAC1 or ELAC2 may be exposed to test substances. These test substances can be screened for the ability to reduce overexpression of wild-type HPC2, ELAC1 or ELAC2 or impair the expression or function of mutant HPC2, ELAC1 or ELAC2. [0241]
  • Pharmaceutical Compositions and Routes of Administration [0242]
  • The HPC2, ELAC1 or ELAC2 polypeptides, antibodies, peptides and nucleic acids of the present invention can be formulated in pharmaceutical compositions, which are prepared according to conventional pharmaceutical compounding techniques. See, for example, [0243] Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.). The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., intravenous, oral, intrathecal, epineural or parenteral.
  • For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, melts, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, WO 96/11698. [0244]
  • For parenteral administration, the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid. [0245]
  • The active agent is preferably administered in a therapeutically effective amount. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in [0246] Remington 's Pharmaceutical Sciences.
  • Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells. [0247]
  • Instead of administering these agents directly, they could be produced in the target cell, e.g. in a viral vector such as described above or in a cell based delivery system such as described in U.S. Pat. No. 5,550,050 and published PCT application Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635, designed for implantation in a patient. The vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are more tissue specific to the target cells. The cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the active agent. Alternatively, the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See for example, EP 425,731A and WO 90/07936. [0248]
  • As disclosed in the following Examples, on the basis of segregating mutations of HPC2 in [0249] kindreds 4102 and 4289, plus association between carriage of the common missense changes Leu 217 and Thr 541 with a diagnosis of prostate cancer, we conclude that HPC2 is a prostate cancer susceptibility gene.
  • While a 1641 insG frameshift found in kindred 4102 will clearly disrupt protein function, this is not obviously the case for the His 781 missense change in kindred 4289. Interestingly, this missense change occurred on a chromosome that also carries Leu 217 and [0250] Thr 541. Thus one might entertain an additive hypothesis to explain the relative strength of the three missense bearing alleles that we have observed. Substitution of Leu for Ser 217 may change the character of a normally hydrophilic segment of the protein; the phenotype conferred is sufficiently modest that it is only detected when the variant is homozygous. Ala 541 is immediately adjacent to the histidine motif. At the position corresponding to Ala 541 in the ELAC1/2, CPSF73 and PSO2 gene families, the most common residue is alanine; when not alanine, the residue is hydrophobic, amide, or basic (FIGS. 6A-B, 9 and 12). Although threonine is observed at this position in other histidine motif containing gene families, it is rare or absent in these three closely related gene families. Thus, from sequence conservation considerations, it is quite reasonable that the Leu 217+ Thr 541 allele should be more deleterious than Leu 217 alone, apparently sufficiently deleterious to be detected in a co-dominant to dominant association test. The kindred 4289 allele carries all three missense changes, Leu 217, Thr 541 and His 781. Examination of the pedigree suggests that the allele is dominant and sufficiently deleterious to demonstrate visible segregation with prostate cancer in an extended pedigree. Interestingly, the youngest affected carrier of this variant, 4289.003, is homozygous for Leu 217 and Thr 541. Thus his mother, the second ovarian cancer case in the pedigree, is an obligate carrier of a Leu 217+ Thr 541 allele. The observation of two ovarian cancer cases in this pedigree, both of whom carry deleterious alleles of ELAC2, is consistent with the possibility that the phenotype conferred by deleterious variants in this gene is not restricted to prostate cancer susceptibility.
  • The potential contributions of the androgen receptor CAG repeat and SRDSA2 Ala 49 Thr missense change to prostate cancer risk were first detected in association studies using sporadic cases and unaffected controls. However, straightforward deduction from the considerable literature on sib pair analyses would predict that such sequence variants should be enriched among affected sibs versus isolated cases, and it follows that such sequence variants should contribute to a larger fraction of familial than truly sporadic prostate cancer cases. Thus one might expect genotypes at moderate risk susceptibility genes such as the androgen receptor, SRD5A2, and the common missense changes in HPC2/ELAC2, to confound linkage studies aimed at detecting and localizing lower prevalence, higher risk susceptibility genes. However, inclusion of genotype information from pedigree members at multiple moderate risk loci may allow refined definition of the liability classes used by multipoint linkage software, thereby increasing the power of the analysis. Stratification of cases by genotype would also facilitate positional cloning projects by providing another criterion by which to distinguish between true recombinant carriers and confounding sporadic cases. [0251]
  • The genetic data presented demonstrate that there are deleterious sequence variants in HPC2/ELAC2 that contribute to prostate cancer risk. Elucidating the functional alteration by which a moderate risk sequence variant such as Leu 217 contributes to a late onset pathology could prove difficult because its manifestation could be quite subtle. However, a mutation as dramatic as a frameshift leading to protein truncation within the likely active site of an enzyme should have a more easily detected effect on cell physiology. Conservation of the C-terminal domain of the gene through the eubacteria and archaebacteria, combined with the observation that the [0252] S. cerevisiae ortholog YRK079C is essential, emphasize that the function of the ELAC1/2 gene family is of fundamental biological interest.
  • The identification of the association between the HPC2 gene mutations and prostate cancer permits the early presymptomatic screening of individuals to identify those at risk for developing prostate cancer. To identify such individuals, HPC2 alleles are screened for mutations either directly or after cloning the alleles. The alleles are tested for the presence of nucleic acid sequence differences from the normal allele using any suitable technique, including but not limited to, one of the following methods: fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single stranded conformation analysis (SSCP), linkage analysis, RNase protection assay, allele specific oligonucleotide (ASO), dot blot analysis and PCR-SSCP analysis. Also useful is the recently developed technique of DNA microchip technology. For example, either (1) the nucleotide sequence of both the cloned alleles and normal HPC2 gene or appropriate fragment (coding sequence or genomic sequence) are determined and then compared, or (2) the RNA transcripts of the HPC2 gene or gene fragment are hybridized to single stranded whole genomic DNA from an individual to be tested, and the resulting heteroduplex is treated with Ribonuclease A (RNase A) and run on a denaturing gel to detect the location of any mismatches. Two of these methods can be carried out according to the following procedures. [0253]
  • The alleles of the HPC2 gene in an individual to be tested are cloned using conventional techniques. For example, a blood sample is obtained from the individual. The genomic DNA isolated from the cells in this sample is partially digested to an average fragment size of approximately 20 kb. Fragments in the range from 18-21 kb are isolated. The resulting fragments are ligated into an appropriate vector. The sequences of the clones are then determined and compared to the normal HPC2 gene. [0254]
  • Alternatively, polymerase chain reactions (PCRs) are performed with primer pairs for the 5 region or the exons of the HPC2 gene. PCRs can also be performed with primer pairs based on any sequence of the normal HPC2 gene. For example, primer pairs for one of the introns can be prepared and utilized. Finally, RT-PCR can also be performed on the mRNA. The amplified products are then analyzed by single stranded conformation polymorphisms (SSCP) using conventional techniques to identify any differences and these are then sequenced and compared to the normal gene sequence. [0255]
  • Individuals can be quickly screened for common HPC2 gene variants by amplifying the individual s DNA using suitable primer pairs and analyzing the amplified product, e.g., by dot-blot hybridization using allele-specific oligonucleotide probes. [0256]
  • The second method employs RNase A to assist in the detection of differences between the normal HPC2 gene and defective genes. This comparison is performed in steps using small (˜500 bp) restriction fragments of the HPC2 gene as the probe. First, the HPC2 gene is digested with a restriction enzyme(s) that cuts the gene sequence into fragments of approximately 500 bp. These fragments are separated on an electrophoresis gel, purified from the gel and cloned individually, in both orientations, into an SP6 vector (e.g., pSP64 or pSP65). The SP6-based plasmids containing inserts of the HPC2 gene fragments are transcribed in vitro using the SP6 transcription system, well known in the art, in the presence of [α-[0257] 32P]GTP, generating radiolabeled RNA transcripts of both strands of the gene.
  • Individually, these RNA transcripts are used to form heteroduplexes with the allelic DNA using conventional techniques. Mismatches that occur in the RNA:DNA heteroduplex, owing to sequence differences between the HPC2 fragment and the HPC2 allele subclone from the individual, result in cleavage in the RNA strand when treated with RNase A. Such mismatches can be the result of point mutations or small deletions in the individual's allele. Cleavage of the RNA strand yields two or more small RNA fragments, which run faster on the denaturing gel than the RNA probe itself. [0258]
  • Any differences which are found, will identify an individual as having a molecular variant of the HPC2. These variants can take a number of forms. The most severe forms would be frame shift mutations or large deletions which would cause the gene to code for an abnormal protein or one which would significantly alter protein expression. Less severe disruptive mutations would include small in-frame deletions and nonconservative base pair substitutions which would have a significant effect on the protein produced, such as changes to or from a cysteine residue, from a basic to an acidic amino acid or vice versa, from a hydrophobic to hydrophilic amino acid or vice versa, or other mutations which would affect secondary or tertiary protein structure. Silent mutations or those resulting in conservative amino acid substitutions would not generally be expected to disrupt protein function. [0259]
  • Genetic testing will enable practitioners to identify individuals at risk prostate cancer, at, or even before, birth. Presymptomatic diagnosis of these epilepsies will enable prevention of these disorders. [0260]
    • EXAMPLES
  • The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized. [0261]
    • Example 1 Linkage Analysis
  • All participants signed informed consent documents. This research project has the approval of the University of Utah School of Medicine Institutional Review Board. Ninety-seven percent of cancer cases have been confirmed through medical records (and/or through the Utah Cancer Registry for prostate cancer cases diagnosed in Utah). Two-point linkage analysis was performed with the package LINKAGE (Lathrop et al., 1984) using the FASTLINK implementation (Cottingham et al., 1993; Schaffer et al., 1994). The statistical analysis for the inheritance of susceptibility to prostate cancer used a model that assumes age-specific incidence rates from the Utah Cancer Registry, and a relative risk of 2.5 for first-degree relatives. Susceptibility to prostate cancer was assumed due to a dominant allele with a population frequency of 0.003. The details of the model are more thoroughly defined in Neuhausen et al. (1999). Marker allele frequencies were estimated from unrelated individuals present in the pedigrees. Linkage in the presence of heterogeneity was assessed by the admixture test (A-test) of Ott (1986), using HOMOG, which postulates two family types, linked and unlinked. Three-point linkage analysis was performed using VITESSE (O'Connell and Weeks, 1995). [0262]
    • Example 2 Physical Mapping
  • BAC DNA was purified and directly sequenced as previously described (Couch et al., 1996). DNA sequences at the SP6 and T7 ends of isolated BAC clones were used to develop STSs that were used for mapping and contig extension. Greater than 95% sequence coverage of the FIG. 1 BAC tiling path was obtained by sequencing plasmid sublibraries generated from these clones. The sequence data obtained were assembled into contigs using Acembly, version 4.3 (U. Sauvage, D. Thierry-Mieg and J. Thierry-Mieg; Centre National de la Recherche Scientifique, France). Subsequently, a complete sequence of this interval was released by the MIT genome center. [0263]
    • Example 3 Genetic Localization of HPC2
  • A. Early Studies [0264]
  • A set of high risk prostate cancer kindreds has been collected in Utah since 1990 for the purpose of localization of prostate cancer susceptibility loci. In February 1996, linkage analysis of data from a genome scan performed on a subset of the families noted evidence for linkage with markers on chromosome 17p. Subsequent analysis of more markers in this region of chromosome 17p in a larger set of families has led to strong linkage evidence for a susceptibility gene. [0265]
    TABLE 1
    Chromosome 17p Two-point Linkage Evidence
    Marker 17p map position Heterogeneity Lod Score
    D17S786 20.0 4.21
    Myr 0022 25.5 3.99
    Myr 0088 27.0 3.46
    D17S947 31.6 2.32
    Myr 0084 31.9 3.02
    Myr 0079 32.0 0.99
    D17S805 43.6 2.25
  • The study of specific kindreds with strong evidence of linkage to chromosome 17p allows the definition of a most likely region for the susceptibility locus by identifying the smallest inherited piece of chromosome 17p shared by the prostate cancer cases in the kindred. The minimal genetically defined region is based on a telomeric recombinant in kindred 4325 and a centromeric recombinant in kindred 4320. [0266] Kindred 4325 was ascertained from a sibship of early onset prostate cancer cases. There are 6 affected brothers in this family, one of whom also has an affected son. Five of the 6 affected brothers, and the affected son, all share the same piece of chromosome 17p from somewhere below marker myr0065 down to and including marker D17S805. Kindred 4320 was also ascertained from a sibship of early onset prostate cancer cases. In this kindred 3 affected brothers and an affected nephew share a piece of chromosome 17p from D17S786 down to and including myr0084. Together, the kindred 4325 and kindred 4320 recombinants define a minimal region of about 1 megabase (FIG. 2A); this localization is well supported by a larger set of recombinants in both directions.
  • B. Recent Studies [0267]
  • We originally performed a genome-wide search for prostate cancer predisposition loci using a small set of Utah high risk prostate cancer pedigrees and a set of 300 polymorphic markers. The pedigrees were not selected for early age of cancer onset, but were a subset of families ascertained using the Utah Population Database. The first eight pedigrees analyzed gave suggestive evidence of linkage on chromosome 17p near marker D17S520, although significance was not established. We increased the density of markers in the region and expanded the analysis to 33 pedigrees (Table 2A). Analysis of the additional data, using a dominant model integrated with Utah age-specific incidence, yielded the two-point linkage evidence shown in Table 2B. A maximum two-point LOD score of 4.5 was observed at marker D17S1289, theta=0.07, and a maximum three-point LOD score of 4.3 was observed using the markers D17S1289 and D17S921. Based on these data, we initiated a positional cloning project, focusing on the interval between D17S1289 and D17S921. [0268]
    TABLE 2A
    Family Resource Used to Detect Linkage to 17p
    Number of pedigrees 3.3
    Total number of cases 338
    Total number of typed cases 188
    Mean number of cases/pedigree (range) 10.2  (2-29)
    Mean number of typed cases/pedigree (range) 5.7  (1-16)
    Mean age of typed cases at diagnosis (range) 68.3 (35-88)
    TABLE 2B
    Two-point LOD Scores Using Utah Age-specific Model
    distance Heterogeneity
    Marker (cM)† Max LOD¥ (theta) LOD (alpha, theta)
    D17S796 — 0.11 (.37) 0.10 (1.00, 0.4)
    D17S952 10.2 0.90 (.17) 0.87 (1.00, 0.2)
    D17S786 10.4 0.00 (.50) 0.95 (0.20, 0.0)
    D17S945 12.7 0.38 (.28) 1.41 (0.25, 0.0)
    D17S520 15.0 0.69 (.26) 0.64 (1.00, 0.3)
    D17S974 15.1 1.01 (.20) 1.20 (0.40, 0.01)
    D17S1289 15.2 4.53 (.07) 4.43 (1.00, 0.1)
    D17S1159 15.4 0.50 (.27) 1.38 (0.25, 0.0)
    GATA134G03 15.7 0.48 (.20) 0.78 (0.75, 0.2)
    D17S954 16.2 0.00 (.50) 0.11 (0.40, 0.2)
    D17S969 18.2 0.54 (.21) 0.55 (0.85, 0.2)
    D17S799 22.0 0.30 (.26) 0.44 (0.70, 0.2)
    D17S921 25.2 1.41 (.10) 1.42 (0.95, 0.1)
    D17S953 29.2 1.04 (.25) 0.94 (1.00, 0.3)
    D17S925 31.2 0.02 (.45) 0.00 (1.00, 0.0)
    D17S798 36.2 0.02 (.43) 0.02 (1.00, 0.4)
  • In order to refine the localization of the implied susceptibility gene, we expanded to the set of 127 families (Table 3) which have now been typed at both this locus and the HPC1 locus. Although the overall data set neither provides significant LOD score evidence for linkage on [0269] chromosome 17 nor provides sufficient evidence for de novo identification of the HPC1 locus (Neuhausen et al., 1999), complete haplotyping of the pedigree resource revealed a similar number of prostate cancer-associated haplotypes at each locus.
    TABLE 3
    Summary of Resource Genotyped for the Association Tests
    Number of pedigrees 127
    Total number of cases 2,402
    Total number of typed cases 700
    Total number of typed pedigree unaffecteds 3,295
    Total number of typed divergent controls 243
    Mean number of cases/pedigree (range) 18.3  (3-74)
    Mean number of typed cases/pedigree (range) 5.5  (1-34)
    Mean age of typed cases at diagnosis (range) 66.5 (39-88)
  • Early in our analysis, we observed that at both 17p and HPC1 many of our pedigrees segregate haplotypes that are shared by four or more cases, but also contain enough noncarrying cases with respect to either locus to eliminate any linkage evidence within the pedigree, as estimated by LOD score. For instance, 12 affected individuals from kindred 4333 share an HPC1 haplotype and 9 affecteds in kindred 4344 share a 17p haplotype, but neither pedigree shows LOD score evidence for linkage at either locus. While we recognize that this phenomenon may be due simply to lack of linkage, we hypothesized that the underlying cause is actually genetic complexity that is greater than the linkage models can accommodate. We subsequently used multipoint haplotyping software (Thomas et al., 2000) to define segregating haplotypes, and then classified those haplotypes into three groups, depending on strength of evidence: [0270] group 1 haplotypes, used for both localization and mutation screening, were defined as haplotypes shared by 4 or more cases and giving a LOD score >1.0 in the pedigree where they were identified, or haplotypes shared by 6 or more cases irrespective of LOD score; group 2 haplotypes, used for mutation screening only, were defined as haplotypes shared by 4 cases with 0.5<LOD <1.0 in the pedigree where they were identified, or haplotypes shared by 5 cases with LOD <1.0; and finally, haplotypes that failed to meet either of the above criteria.
  • Considering [0271] group 1 and 2 haplotypes together, evidence at HPC1 and 17p is quite similar: 43 haplotypes at HPC1 versus 42 at 17p and 258 affected haplotype carriers at HPC1 versus 232 at 17p. Focusing on the group 1 haplotpes, evidence at HPC1 is relatively stronger: 26 group 1 haplotypes at HPC1 versus 18 at 17p and an average of 7.2 affected carriers per group 1 haplotype at HPC1 versus 6.6 at 17p. However, there is one other critical difference between the linkage evidence for the two regions. At HPC1, meiotic recombinant mapping using the group 1 haplotypes has thus far failed to define a consistent region. This is also reflected in the ICPCG HPC1 study (Xu, 2000); in this work, most of the evidence for linkage comes from a combination of the Utah and Hopkins data sets, but the locations with the best evidence for linkage in each of the individual sets map approximately 15 cM apart. In contrast, recombinant mapping in affected carriers of 17p group 1 haplotypes defined a consistent region (FIG. 3). As a result, we were able to focus our contig assembly, transcript map development, and mutation screening efforts on an approximately 1 MB interval centered on D17S947 (FIG. 3).
  • One of the genes mapping near D17S947 shares amino acid sequence similarity with members of the NCBI Cluster of Orthologous Groups (Tatusov et al., 1997) COG1234, typified by the uncharacterized [0272] E. coli ORF elaC and the uncharacterized S. cerevisiae ORF YKRO79C. On mutation screening this candidate gene from the genomic DNA of prostate cancer cases carrying 17p group 1 haplotypes, a germline frameshift mutation, 1641 insG, was found in a carrier from kindred 4102. Following detection of this frameshift, the gene, which we shall refer to as ELAC2 because it is the larger of two human genes that we have found that are homologs of E. coli elaC, was subjected to careful sequence and intense genetic analyses.
    • Example 4 Contig Assembly and Genomic Sequencing in the Minimal Genetically Defined HPC2 Region
  • Contig Assembly. [0273]
  • Given a genetically defined interval flanked by meiotic recombinants, one needs to generate a contig of genomic clones that spans that interval. Publicly available resources, such as the Whitehead integrated maps of the human genome (e.g., the [0274] WICGR Chr 17 map) provide aligned chromosome maps of genetic markers, other sequence tagged sites (STSs), radiation hybrid map data, and CEPH yeast artificial chromosome (YAC) clones.
  • Oligonucleotide primer pairs for the markers located in the interval were synthesized and used to screen libraries of bacterial artificial chromosomes (BACs) to identify BACs in the region. The initial set of markers used was D17S969, WI-2437, WI-2335, D17S947, and D17S799 (FIG. 2A). BACs identified with these markers were end-sequenced. PCR primers designed from those end sequences were used as markers to arrange the initial BACs into contigs. The outermost marker from each contig was used in successive rounds of BAC library screening, eventually enabling the completion of a BAC clone contig that spanned the genetically defined interval. A set of overlapping but non-redundant BAC clones that spanned this interval (FIG. 2A) was then selected for use in subsequent molecular cloning protocols such as genomic sequencing. [0275]
  • Genomic Sequencing. [0276]
  • Given a tiling path of BAC clones across a defined interval, one useful gene finding strategy is to generate an almost complete genomic sequence of that interval. Two types of random genomic clone sublibraries were prepared from each BAC on the tiling path; these were Sau 3A partial digest libraries with inserts in the 5 to 8 kb size range, and random shear libraries with inserts in the 1.0 to 1.5 kb size range. Plasmid DNA from individual clones from the Sau 3A sublibraries sufficient in number to generate an, on average, lx redundant sequence of each BAC was prepared using an Autogen robotic plasmid preparation machine (Integrated Separation Systems). Insert DNA from individual clones from the random shear sublibraries sufficient in number to generate an, on average, 5× redundant sequence of each BAC, was prepared by PCR with vector primers directly from aliquots of bacterial cultures of each individual clone. The resulting DNA templates were subjected to DNA sequencing from both ends with M13 forward or reverse fluorescent dye-labeled primers on ABI 377 sequencers. [0277]
  • These sequences were assembled into sequence contigs using the program Acem.bly (Thierry-Mieg et al., 1995; Durbin and Thierry-Mieg, 1991). The genomic sequence contigs were placed in a Genetic Data Environment (GDE) (Smith et al., 1994) local database for subsequent similarity searches. Similarities among genomic DNA sequences and GenBank entries—both DNA and protein—were identified using BLAST (Altschul et al., 1990). The DNA sequences were also characterized with respect to short period repeats, CpG content, and long open reading frames. [0278]
    • Example 5 Sequence Assembly of the Human HPC2 Gene
  • A BLASTn (Altschul et al., 1990) search of genomic sequences from BAC 31k12 against dbEST identified two independent sets of human ESTs that, when parsed across the BAC 31k12 genomic sequences, revealed the presence of two independent multi-exon candidate genes, 04CG09 and the HPC2 gene (FIG. 2B). A subset of the EST sequences assigned to HPC2 (Table 4) was assembled to produce a tentative partial cDNA sequence for the gene. [0279]
    TABLE 4
    Human ESTs Used to Assemble a Tentative Partial Human HPC2
    cDNA Sequence
    EST Accession # Exon Span
    AA679618
    1→6
    Z17886 4→8
    W37591  7→12
    AA310236 12→16
    R55841 15→19
    T34216 18→21
    AA634909 20→24
    AA504412 23→24
    R42795   24→polyA
  • The individual exons of the human HPC2 gene were identified by parsing that tentative cDNA sequence across the BAC 31k12 genomic sequence (see schematics in FIG. 2B). After we had identified the HPC2 gene, the MIT genome sequencing completely sequenced another BAC, 597 m12, that also contains all of the exons of HPC2 (GenBank accession # AC005277) The sequence of the human HPC2 gene was corrected both by comparison of the sequences of the individual exons from the tentative cDNA assembly to the corresponding genomic sequences of BACs 31k12 and 597 m12, and by mutation screening the gene from a set of human genomic DNAs (see Example 8). [0280]
  • The original tentative human HPC2 cDNA sequence contained neither the start codon nor any of the 5′ UTR. These were obtained by [0281] biotin capture 5′ RACE (Tavtigian et al., 1996). Briefly, a biotinylated reverse primer, CA4cg07.BR2, was designed from the sequence of the third exon of the human HPC2 gene and used, along with the anchor primer SampA, for a first round of PCR amplification from human fetal liver cDNA that had been prepared such that the 5′ ends of cDNA molecules are anchored with the sequence stag1. The resulting PCR products were captured on streptavidin paramagnetic particles (Dynal), washed, and used as template in a second round PCR amplification. A phosphorylated reverse primer, CA4cg07.PR2, was designed from the sequence of the second exon of the human HPC2 sequence and used, along with the nested phosphorylated anchor primer SampB, for the second round PCR amplification. The resulting 5′ RACE products were gel purified and sequenced with the primer CA4cg07.PR2 using dye-terminator chemistry and ABI 377 sequencers. Analysis of the sequences of these 5′ RACE products yielded both the start codon and part of the 5′ UTR including an in-frame stop codon (FIG. 4). Sequences of the human primers used for 5′ RACE are given in Table 5.
  • A full length human HPC2 cDNA was amplified from human head and neck cDNA using the primers CA4cg7.ATG and CA4cg7.TGA. The cDNA was ligated into the vector pGEM-T Easy (Promega) and transformed into [0282] E. coli. The sequence of the cDNA clone was confirmed by dye terminator sequencing on ABI 377 sequencers. Sequences of primers used to amplify the cDNA construct and confirm the sequence of the cDNA clone are also given in Table 5.
    TABLE 5
    Primers Used in 5′ RACE, cDNA Cloning and
    Sequence Confirmation of a Full-length Human HPC2 cDNA
    Sequence (SEQ ID NO:)
    5′RACE PRIMERS
    5tag1 CAG GAA TTC AGC ACA TAC TCA TTG TTC Agn n (29)
    5AmpA CAG GAA TTC AGC ACA TAC TCA (30)
    5AmpB (P)TT CAG CAC ATA CTC ATT GTT CA (31)
    CA4cg07.BR2 (B)TG AAC GCC TTC TCC ACA GT (32)
    CA4cg07.PR2 (P)GT ACC CGC TGC CAC CAC (33)
    EXPRESSION CONSTRUCT PRIMERS
    CA4cg7.ATG GCT AGG ATC CGC CAC CAT GTG GGC GCT TTG CTC (34)
    CA4cg7.TGA GCT ACT CGA GTC ACT GGG CTC TGA CCT TC (35)
    SEQUENCING PRIMERS
    M13F20 GTA AAA CGA CGG CCA GT (36)
    M13R20 GGA AAC AGC TAT GAC CAT G (37)
    CA4cg7F1 TGC GCA CGC GAG AGA AG (38)
    CA4cg7R1 CGC TTC TCT CGC GTG CG (39)
    CA4cg7F2 TCT AAT GTT GGG GGC TTA (40)
    CA4cg7R2 TAA GCC CCC AAC ATT AGA (41)
    CA4cg7F3 TGA AAA TGA GCC ACA CCT (42)
    CA4cg7R3 AGG TGT GGC TCA TTT TCA (43)
    CA4cg7F4 CAT TCA ACC CAT CTG TGA (44)
    CA4cg7R4 TCA CAG ATG GGT TGA ATG (45)
    CA4cg7F5 TGA ATG CCT CCT CAA GTA (46)
    CA4cg7R5 TAC TTG AGG AGG CAT TCA (47)
    CA4cg7F6 GCT ACT GGA CTG TGG TGA (48)
    CA4cg7R6 TCA CCA CAG TCC AGT AGC (49)
    CA4cg7F7 TGG AAG AGT TTC AGA CCT G (50)
    CA4cg7R7 CAG GTC TGA AAC TCT TCC A (51)
    CA4cg7F8 CGC AGG GAC GCA CCA TA (52)
    CA4cg7R8 GGT TGA ACT CGG AGA AGA (53)
    CA4cg7F9 CAA CTG GAA AAA TAC CTC G (54)
    CA4cg7F10 GCA GAG TCC AGA AAG GC (55)
    CA4cg7F11 AGA GGA AAC TTC TTG GTG C (56)
    CA4cg7F12 ACC AAG GAA AGG CAG ATG (57)
    CA4cg7F13 GTC AAC ATA AGC CCC GAC (58)
    CA4cg7F14 GGC TGC TGT GTT TGT GTC (59)
    CA4cg7R14 GAA GGC ATT TGG CAG GA (60)
    CA4cg7F15 TAT GAT TCC TGC CAA ATG (61)
    CA4cg7R15 TCC AGC CAG AGG TGT GC (62)
    CA4cg7F16 TGC GAG GCT CTG GTC CG (63)
    CA4cg7R16 GGG CAT TGT TGG AAA GTC (64)
    CA4cg7F17 TGT TTG CTG GCG ACA TC (65)
    • Example 6 Sequence Assembly of the Mouse HPC2 Gene
  • A BLAST search of the assembled HPC2 cDNA sequence against dbEST identified 5 mouse ESTs that derived from a very similar gene, the mouse ortholog of HPC2, Mm.HPC2; their accession numbers are listed in Table 6. [0283]
    TABLE 6
    Mouse ESTs Used to Assemble a Tentative Partial Mm.HPC2
    cDNA Sequence
    EST Accession # Exon Span
    AA563096
    1→5
    AA518169  8→14
    AI132016 16→17
    AA184645 19→24
    AA174437 24→24
  • The original partial Mm.HPC2 cDNA sequence contained the start codon but little of the 5′ UTR. More extensive 5′ UTR sequence was obtained [0284] b 5′ RACE. Brifly, a biotinylated reverse primer, m04cg07BR1, was designed from the sequence of the fourth exon of the mouse HPC2 gene and used, along with the anchor primer 5ampA, for a first round of PCR amplification from mouse embryo cDNA that had been prepared such that the 5′ ends of cDNA molecules are anchored with the sequence 5tag1. The resulting PCR products were captured on streptavidin paramagnetic particles (Dynal), washed, and used as template in a second round PCR amplification. A phosphorylated reverse primer, m04cg07PR1, was designed from the sequence of the third exon of the mouse HPC2 sequence and used, along with the nested phosphorylated anchor primer SampB, for the second round PCR amplification. The resulting 5′ RACE products were gel purified and sequenced with the primers m04cg07PR1 and m04cg07 exon2 rev using dye-terminator chemistry and ABI 377 sequencers. Analysis of the sequences of these 5′ RACE products yielded both the start codon and part of the 5′ UTR including an in-frame stop codon (FIG. 4). Sequences of the primers used for 5′ RACE are given in Table 7.
  • More extensive 5′ UTR sequence, sequence that may be from the promoter, and the sequences of [0285] intron 1 and intron 2 of the mouse HPC2 gene were obtained by genomic sequencing. BAC 428n12 was obtained from a mouse genomic library by screening the library by PCR with a pair of primers (04CG7. m11f1 and 04CG7. m11r1, Table 7) derived from exon 11 of the mouse HPC2 cDNA sequence. A primer pair derived from the SP6 end sequence of BAC 428n12 (428n12.S6.F1 and 428n12.S6.F1, Table 7) was used to screen the mouse BAC library by PCR; several overlapping BACs, including BAC 199n11, were identified. BACs 428n12 and 199n11 were sequenced with a series of 13 sequencing primers (mcg7f1 to mcg7r7, Table 7) derived from mouse HPC2 cDNA dye-terminator chemistry and ABI 377 sequencers. A subset of these sequences were assembled into a genomic sequence contig extending from 280 bp upstream of the ATG start codon of exon 1 into exon 3.
  • A full length mouse HPC2 cDNA is amplified from mouse embryo, placenta, or fetal brain cDNA using the primers msCA4cg7.f out and msCA4cg7.r out The cDNA is reamplified with the primers msCA4cg7.ATG and msCA4cg7.TGA. The resulting PCR products are gel purified, ligated into the vector pGEM-T Easy (Promega), and transformed into [0286] E. coli. The sequence of the cDNA clone are confirmed dye terminator sequencing on ABI 377 sequencers. Sequences of primers in use to amplify the cDNA construct are also given in Table 7.
    TABLE 7
    Primers Used in 5′ RACE and cDNA Cloning of a Full-length Mouse HPC2 cDNA
    Sequence (SEQ ID NO:)
    5′RACE PRIMERS
    5tag1 CAG GAA TTC AGC ACA TAC TCA TTG TTC Agn n (66)
    5AmpA CAG GAA TTC AGC ACA TAC TCA (67)
    5AmpB (P)TT CAG CAC ATA CTC ATT GTT CA (68)
    m04cg07BR1 (B)CA GAA CAC ATT TGG GAA GC (69)
    m04cgO7PR1 (P)GA TGT TGT CCA AGC GAG C (70)
    BAC library screening primers
    04CG7.m11f1 TGA CAC AGA GCA CCT GA (71)
    04GG7.m11r1 GAA GAT GTC AGG GTG GA (72)
    428n12.S6.F1 CAG GCA TAC CAC TAC AGA (73)
    428n12.S6.R1 TAT CAA CTT CTA GGC AAG TG (74)
    Genomic sequencing primers
    mcg7f1 GCA CCA TGT CGC AGG GTT C (75)
    mcg7r1 GAA CCC TGC GAC ATG GTG C (76)
    mcg7f2 TCG CAG GGT TCG GCT CGT C (77)
    mcg7r2 AAC CCT GCG ACA TGG TGC G (78)
    mcg7f3 AAA GAC CCA CTG CGA CAC C (79)
    mcg7r3 GCA GGT GTC GCA GTG GGT C (80)
    mcg7f4 CCG AAC ACC GTG TAC GTG CA (81)
    mcg7r4 GAG GTA GAG GGT GTT CGG G (82)
    mcg7f5 GTC TTG TCG GAA TAG AAC AGG (83)
    mcg7r5 CTG TTG TAT TCC GAG AAG AC (84)
    mcg7f6 AAG GCG TCC AAC GAC TTA TG (85)
    mcg7r6 AGT CGT TGG ACG CCT TCT CC (86)
    mcg7r7 TCC GAG TCA GAA AGA TGT TG (87)
    EXPRESSION CONSTRUCT PRIMERS
    PRIMARY PCR
    msCA4cg7.f out GCC TTG TGA GCC TGG TG (88)
    msCA4cg7.r out AGG AAG TGA GCA GAG CG (89)
    SECONDARY PCR
    msCA4cg7ATG GCT AAA GCT TGC CAC CAT GTG GGC GCT CCG CTC (90)
    msCA4cg7.TGA GCT ACT CGA GTG ACA CTC GCG CTC CTA (91)
    SEQUENCING PRIMERS
    m04cg07 exon2 rev GCC TTC TCC GCA GTT A (92)
    • Example 7 Northern Blots
  • Prehybridization and hybridization were performed at 42° C. in 50% formamide, 5× SSPE, 1.0% SDS, 5× Denhardt's mixture, 0.2 mg/mL denatured salmon sperm DNA, and 2 μg/mL poly(A). Dextran sulfate (4% v/v) was included in the hybridization solution only. The membranes were washed twice in 2× SSC/0.1% SDS at 20° C. for 30 minutes, followed by a stringency wash in 0.1× SSC/0.1% SDS at 50° C. for 30 minutes. [0287]
    • Example 8 Mutation Screening of the Human HPC2 Gene
  • Using genomic DNAs from prostate kindred members, prostate cancer affecteds, and tumor cell lines as templates, nested PCR amplifications were performed to generate PCR products to screen for mutations in the HPC2 gene. The primers listed in Table 8 were used to amplify segments of the HPC2 gene. Using the outer primer pair for each amplicon (1A-1P, i.e., forward A and reverse P of amplicon 1), 10-20 ng of genomic DNA were subjected to a 25 cycle primary amplification, after which the PCR products were diluted 45-fold and reamplified using nested M13-tailed primers (1B-1Q, 1C-1R i.e., nested forward B and nested reverse Q of [0288] amplicon 1 or nested forward C and nested reverse R of amplicon 1) for another 23 cycles. In general, samples were amplified with Taq Platinum (Life Technologies) DNA polymerase; cycling parameters included an initial denaturation step at 95° C. for 3 min, followed by cycles of denaturation at 96° C. (12 s), annealing at 55° C. (15 s) and extension at 72° C. (30-60 s). After the PCR reactions, excess primers and deoxynucleotide triphosphates were digested with exonuclease I (United States Biochemicals) and shrimp alkaline phosphatase (Amersham). PCR products were sequenced with M13 forward or reverse fluorescent (Big Dye, ABI) dye-labeled primers on ABI 377 sequencers. Chromatograms were analyzed for the presence of polymorphisms or sequence aberrations in either the Macintosh program Sequencher (Gene Codes) or the Java program Mutscreen. We obtained more than 95% double strand sequence coverage for the entire open reading frame of all samples screened.
    TABLE 8
    Primers Used to Mutation Screen the HPC2 Gene from Genomic DNA
    Exon/Primer name Sequence (SEQ ID NO:)
    HPC2 exon 1
    ca4cg7.m1Anew CCG CTT GAG ACG CTC TAG TAT (93)
    ca4cg7.m1P GCT CCG AAA GTG CTG ACA G (94)
    ca4cg7.m1Bnew GTT TTC CCA GTC ACG ACG TTT CTA TTG GAT GAG CAG CCT (95)
    ca4cg7.m1Qnew AGG AAA CAG CTA TGA CCA TGC CTG CGA TAT GGT GCG TC (96)
    ca4cg7.m1C GTT TTC CCA GTC ACG ACG CTC AGT TTT GGT GGA GAC G (97)
    ca4cg7.mRnew AGG AAA CAG CTA TGA CCA TGT GCC CCG ATG CTC AGA G (98)
    HPC2 exons 2&3 (primary)
    ca4cg7.m2&23 A2 AAT GGT GTC AGA GAG TTT ACA G (99)
    ca4cg7.m2&23P GCT ATT TGG GAG GCT GAG G (100)
    HPC2 exon 2 (nested)
    ca4cg7.m2B GTT TTC CCA GTC ACG ACG AAT GGT GTC AGA GAG TTT ACA G (101)
    ca4cg1.m2Q AGG AAA CAG CTA TGA CCA TGA ACA AGG ACC ACT TTT GCT AT (102)
    HPC2 exon 3 (nested)
    ca4cg7.m23B GTT TTC CCA GTC ACG ACG TTT ATA GCA AAA GTG GTC CTT G (103)
    ca4cg7.m23Q AGG AAA CAG CTA TGA CCA TGA GAC TTC CCA CCA GCC TC (104)
    HPC2 exon 4
    ca4.cg07.m24A CCT TGC TGC TTC ACC CTA G (105)
    ca4.cg07.m24P TGC TTT ATA TGT GCT GCT ACG (106)
    ca4.cg07.m24B GTT TTC CCA GTC ACG ACG CAT CTT CCC TGG TTG TAC TTC (107)
    ca4.cg07.m24Q AGG AAA CAG CTA TGA CCA TCT GGA GGG CAG AAG ACT GAT (108)
    HPC2 exon 5
    ca4cg7.m3A CTA CAT TTG TTC AAC CAT AAC TG (109)
    ca4cg7.m3P GAT TTT GAG GTT TGA TGT TGA TG (110)
    ca4cg7.m3B GTT TTC CCA GTC ACG ACG CAT TTG TTC AAC CAT AAC TGC (111)
    ca4cg7.m3Q AGG AAA CAG CTA TGA CCA TAT TTG AGA GGT CAG GGC ATA (112)
    HPC2 exon 6
    ca4cg7.m4A TCG TGT CAG ATT CCC ACC ATA (113)
    ca4cg7.m4P AGG CAT AAG TCA GAC ATC CGT (114)
    ca4cg7.m4B GTT TTC CCA GTC ACG ACG GTT ACT CTT CCC ACA CAT CTT C (115)
    ca4cg7.m4Q AGG AAA CAG CTA TGA CCA TCA CAG CAA GTG TTC AGT TTC TA (116)
    HPC2 exon 7
    ca4cg7.m5A CAT TCC CAT GTA TGA ACG TCT (117)
    ca4cg7.m5P ATA GTA AGC CCA GGA AGA AGGA (118)
    ca4cg7.m5B GTT TTC CCA GTC ACG ACG CAT TCC CAT GTA TGA ACG TCT (119)
    ca4cg7.m5Q AGG AAA CAG CTA TGA CCA TCT ACA AGC ATT ACA AGG CAG AG (120)
    HPC2 exon 8
    ca4cg7.m6A AGT GTC TTC AGC CTT TGT ATT G (121)
    ca4cg7.m6P ATC TGC TAT CTC TTC TTG TCT CA (122)
    ca4cg7.m6B GTT TTC CCA GTC ACG ACG ATC GGG TCA TAA TCA GTC TGT G (123)
    ca4cg7.m6Q AGG AAA CAG CTA TGA CCA TAT CTC TTC TTG TCT CAG GTA ACA (124)
    HPC2 exons 9&10 (primary)
    ca4cg7.m7&8A CTT CTG AAA GCA ATA AAC GCA T (125)
    ca4cg7.m7&8P GAT GTC CAA ACT GTT CCA CG (126)
    HPC2 exon 9 (nested)
    ca4cg7.m7B GTT TTC CCA GTC ACG ACG TAA AAC CAA CCT TCT TCA TTA G (127)
    ca4cg7.m7Q AGG AAA CAG CTA TGA CCA TAG CAA TGA TGG GAG CGA TG (128)
    HPC2 exon 10 (nested)
    ca4cg7.m8B GTT TTC CCA GTC ACG ACG GGC TTC TGG GGA CTC ACT G (129)
    ca4cg7.m8Q AGG AAA CAG CTA TGA CCA TCC TTC AAA AGT GGT GTC TGT AG (130)
    HPC2 exon 11
    ca4.cg07.m9A GTA TCC ACA AAG AGA CCA GAA G (131)
    ca4.cg07.m9P CAC CAA CTA CCA ACA GTG ACT TA (132)
    ca4.cg07.m9B GTT TTC CCA GTC ACG ACG GCT CAC TGG ATA GGA TAT GTC AT (133)
    ca4.cg07.m9Q AGG AAA CAG CTA TGA CCA TCC AGA AAC ACA GCT CTT GCC (134)
    HPC2 exon 12
    ca4.cg07.m10A GCT TGC CAG ATA CAG GAA TC (135)
    ca4.cg07.m10P ACA GAA AGT TTA GGC AGG TG (136)
    ca4.cg07.m10B GTT TTC CCA GTC ACG ACG ACG ATA CCC CTC CCT GGC T (137)
    ca4.cg07.m10Q AGG AAA CAG CTA TGA CCA TAC AGA AAG TTT AGG CAG GTG (138)
    HPC2 exons 13&14 (primary)
    ca4.cg07.m11&12A CCT CTC ACT CTT CCC AGC AC (139)
    ca4.cg07.m11&12P GGA GTA GGC TGC TTT TCT AAA T (140)
    HPC2 exon 13 (nested)
    ca4.cg07.m11B GTT TTC CCA GTC ACG ACG GAA CAC CTC ATC CTC ATT ACC A (141)
    ca4.cg07.m11Q AGG AAA CAG CTA TGA CCA TAA GAG ACA AAA CAC ATT CAT GG (142)
    HPC2 exon 14 (nested)
    ca4.cg07.m12B GTT TTC CCA GTC ACG ACG GTT TCC GCT GTA AGG TAG TGT (143)
    ca4.cg07.m12Q AGG AAA CAG CTA TGA CCA TCT GGA ACA TTT ACT ATG TGG CTA (144)
    HPC2 exon 15
    ca4.cg07.m13A TGC TAG TGG GTA GAG GTC AG (145)
    ca4.cg07.m13P ACT GAA AGC CAG GTT AGA ATG (146)
    ca4.cg07.m13B GTT TTC CCA GTC ACG ACG ACC CTG TCC GTC ACC TGA G (147)
    ca4.cg07.m13Q AGG AAA CAG CTA TGA CCA TCC CAC CAG CAC TCC ACT TA (148)
    HPC2 exon 16
    ca4cg07.m14A TGT GAA GAC GGG ATA ACC TGA (149)
    ca4cg07.m14P GAC AGG GCT TGA TAC CGCA (150)
    ca4cg07.m148 GTT TTC CCA GTC ACG ACG ATG CTG GCT CAC TTT TGA CC (151)
    ca4cg07.m140 AGG AAA CAG CTA TGA CCA TGAC TGG TGA GTA CAG CAG GA (152)
    HPC2 exon 17
    ca4.cg07.m15A CCA GCC TTT GTG TAA GTC TAC (153)
    ca4.cgQ7.m15P TCT GGG CAA GTT TGG AAG C (154)
    ca4.cg07.m15B GTT TTC CCA GTC ACG ACG TCC AAA GCA GAC ATC AGC CTC (155)
    ca4.cg07.m15Q AGG AAA CAG CTA TGA CCA TGG AGG AAA AGA CGC AGC CA (156)
    HPC2 exon 18
    ca4.cg07.m16A CGC TTT CTG CCT GTG ACA T (157)
    ca4.cg07.m16P TTC TGT CCT TCA GCC AAT GC (158)
    ca4.cg07.m16B GTT TTC CCA GTC ACG ACG TTA GAG GCT GGT GGG TGA C (159)
    ca4.cg07.m16Q AGG AAA CAG CTA TGA CCA TCA TCT CAA TAA AAA CTG GAG TGC (160)
    HPC2 exon 19
    ca4.cg07.m17A CAC TTG ATG GGC GTT CTG AG (161)
    ca4.cg07.m17P TTC TGT CCT TCA GCC AAT GC (162)
    ca4.cg07.m17B GTT TTC CCA GTC ACG ACG TTC CAG CGG TTT ACA CAT CA (163)
    ca4.cg07.m17Q AGG AAA CAG CTA TGA CCA TTA CCC CAG TGT CCA CCT TG (164)
    HPC2 exons 20&21 (primary)
    CA4CG7.m18&22A GGG TTC TCC AGC CAA AGA CT (165)
    CA4CG7.m18&22P CTG AGT CTC CTG CCT CTG C (166)
    HPC2 exon 20 (nested)
    ca4.cg07.m18B GTT TTC CCA GTC ACG ACG GGG TTC TCC AGC CAA AGA CT (167)
    ca4.cg07.m18Q AGG AAA CAG CTA TGA CCA TGT GGG GCT GGA AGG CTC TG (168)
    HPC2 exon 21 (nested)
    ca4.cg07.m22B GTT TTC CCA GTC ACG ACG AAG AGG TAA GGG GCA CAG C (169)
    ca4.cg07.m22Q AGG AAA CAG CTA TGA CCA TCT GAG TCT CCT GCC TCT GC (170)
    HPC2 exon 22
    ca4.cg07.m19A GCT GAG TGT TGA GAC CAG GA (171)
    ca4.cg07.m19P AGA CAA ACG ACG GCT GCT C (172)
    ca4.cg07.m19B GTT TTC CCA GTC ACG ACG TTG AGA CCA GGA AAC AGC AC (173)
    ca4.cg07.m19Q AGG AAA CAG CTA TGA CCA TGA GAG GAT GTG GGC GAC AA (174)
    HPC2 exon 23
    ca4.cg07.m20A GGG AGA TGG TGC TGG CTA C (175)
    ca4.cg07.m20P CCT GGT TAG TGA TGG GTA GAT (176)
    ca4.cg07.m20B GTT TTC CCA GTC ACG ACG CAG GGT CTG TGC CAC TGT C (177)
    ca4.cg07.m20Q AGG AAA CAG CTA TGA CCA TCT CAG TGT GTA GAG TCC TGT C (178)
    HPC2 exon 24 splice acceptor and open reading frame
    ca4.cg07.m21A TTG ATT TTG AGA GCA TCT GGA C (179)
    ca4.cg07.m21P CTC GGA CAC TTA GAC CCA CTG (180)
    ca4.cg07.m21B1 GTT TTC CCA GTC ACG ACG TGC ATC CCT TCC AGC TCC T (181)
    ca4.cg07.m21Q AGG AAA CAG CTA TGA CCA TGA CAC ACA GCC TTC TGA GTT CA (182)
    ca4.cg07.m21C GTT TTC CCA GTC ACG ACG CCA CAC AGA GGA GCC ACA G (183)
    ca4.cg07.m21R AGG AAA CAG CTA TGA CCA TAC CAG TCC TAA GAG GCA TCT ATA (184)
    HPC2 exon 24 3′untranslated region
    ca4.cg07.m21.3′UTR A CCA CAC AGA GGA GCC ACA G (185)
    ca4.cg07.m21.3′UTR P CCA GAG GTG CTC ACT ACG AC (186)
    ca4.cg07.m21.3′UTR B GTT TTC CCA GTC ACG ACG AGG TCA GAG CCC AGT GAA GAT (187)
    ca4.cg07.m21.3′UTR Q AGG AAA CAG CTA TGA CCA TCA TCT GCT TGC TTC CGT GTG (188)
    ca4.cg07.m21.3′UTR C GTT TTC CCA GTC ACG ACG TCA GGA TAG GTG GTA TGG AGC (189)
    ca4.cg07.m21.3′UTR R AGG AAA CAG CTA TGA CCA TCG GAC ACT TAG ACC CAC TGA T (190)
  • [0289]
    TABLE 9
    Sequence Variants
    Variant name Sequence (SEQ ID NO:) Coding effect*
    C650T AGACTCCGAGTYGAATGAAAATG (191) Ser217Leu
    A1560G GGTGAGGGCACRTTTGGGCAGCT (192) Thr520Thr
    G1621A GCACCCTGGCTRCTGTGTTTGTG (193) Ala541Thr
    1641 insG (normal) GTGTCCCACCTG-CACGCAGATCA (194)
    (with insertion of G) GTGTCCCACCTGGCACGCAGATCA (195) frameshift
    C1722T AAGCCGCTTCAYCCTTTGCTGGT (196) His574His
    A1893G GCTGTTGCGAACRTGTGATTTGGA (197) Thr631Thr
    C2632G GAGGCTTGGGSTCCCACATAAG (198)
    C2687T CCTGGCACAGCYGCGGGCCAGGA (199)
    G2801A AATCCAGCAAARTGATTCCCTGC (200)
    IVS2 T-11C Taaatgttttytcattcttag (201)
    IVS5 T-14C Ttgctgttgtgyggttttcttgt (202)
    IVS10 23insGAT (normal) ggttttcttgat---tcagcagttaca (203)
    (with insertion of GAT) Ggttttcttgatgattcagcagttaca (204)
    IVS13 C15T Gtgtctcagacyggccccttgtc (205)
    IVS14 A17T Tgccatcttgawctaatggaatc (206)
    IVS14 T-8C Cttctctctctycctgcagggat (207)
    IVS16 C41T Catcaagggcaygtttacttttt (208)
    IVS19 C26G Cagccttgcccsctgggctgttg (209)
  • Kindred 4102 was ascertained as a high risk cluster with eight prostate cancer cases in a three generation pedigree. Genotyping revealed that six of the eight cases shared a chromosome 17p haplotype. The youngest (age at diagnosis of 46) affected carrier of this shared haplotype, 4102.013 (i.e., kindred #4102, individual #013; FIG. 5A), was selected for mutation screening. On mutations screening lymphocyte DNA from 4102.013, we detected a frameshift, 1641 insG, in ELAC2. A test for segregation revealed that the frameshift was not on the father's chromosome, but rather was inherited through the carrier's mother, 4102.002. Her affected uncle 4102.053 was diagnosed with and died of prostate cancer at age 76 in the 1960s. Genotyping of his children demonstrated that he was an obligate frameshift carrier. In all, there are five male frameshift carriers over [0290] age 45 in the pedigree. Of these, three have prostate cancer, the fourth has a PSA of 5.7 at age 71, and the fifth has a PSA of 4.2 at age 74 (FIG. 5A). The frameshift occurs at His 548, within the histidine motif (FIGS. 6A-B) and is predicted to be quite disruptive to the protein.
  • As the [0291] frameshift 1641 insG was found in an individual with early onset prostate cancer, we screened an additional 45 prostate cancer cases with early age at diagnosis (Dx≦55 years), irrespective of evidence of linkage to any locus, for mutations in ELAC2. An alteration, Arg 781 His, was identified in individual 4289.003, diagnosed with prostate cancer at age 50. Upon expansion of his pedigree, the mutation was traced back four generations to 4289.006, who had affected descendants from five known wives. Prostate cancer cases who carry the missense change have been found among the descendants from three of these five marriages. Of thirteen prostate cancer cases in the pedigree, six carry the missense change, three are unknown, and four are non-carriers. In addition, a female carrier of this missense change, 4289.183, was diagnosed with ovarian cancer at age 43 (FIG. 5B). Within the generations with phenotype information, there are only two unaffected male mutation carriers over age 45; 4289.068 (PSA of 0.6 at age 60) and 4289.063, who died of a heart attack at age 62. We have no additional information on 4289.063; however, two of 4289.063's sons and a grandson are carriers who have been diagnosed with prostate cancer. The missense change occurs in a very highly charged stretch of amino acid residues near the C-terminus of the protein. Arg 781 is conserved in mouse (FIGS. 6A-B), and the charge character of the sequence segment is conserved in C. elegans. While one cannot definitively predict that this missense change will affect protein function, expansion from a single affected mutation carrier to a pedigree with a LOD score of 1.3 provides good evidence that the mutation is in fact deleterious.
  • The identification of two mutations provides strong evidence that ELAC2 is a prostate cancer susceptibility gene. However, after screening 42 haplotypes with evidence for linkage at 17p, we have found only these two high-risk mutations. Thus it seems that only a small fraction of prostate cancer pedigrees segregate obvious mutations in the ELAC2 coding sequence. We do not yet know what fraction of the pedigrees harbor subtle gene rearrangements or regulatory mutations. [0292]
  • Taken together, the observation that the frameshift HPC2 1641insG segregates with prostate cancer across three generations of kindred 4102, and the inference from shared sequence similarity that the frameshift HPC2 1641insG must be deleterious to the function of the HPC2 protein, establish that deleterious germline mutations in the HPC2 gene confer susceptibility to prostate cancer. [0293]
    • Example 9 Common Missense Changes in HPC2
  • When our original set of linked pedigrees was screened for mutations in ELAC2, we observed several occurrences of the non-conservative missense change Ser 217 Leu. This missense change is embedded in an extremely hydrophilic segment of the protein sequence. Like the common human allele, the mouse and C. elegans residues at this position are also serine. Although the sequence of this segment is not well conserved, its hydrophilic character is (FIGS. [0294] 6A-B); thus substitution of a bulky hydrophobic residue for Ser 217 could result in structural consequences to the protein.
  • We analyzed this sequence variant in our pedigree cases, unaffected pedigree members, and an unrelated set of males who have no diagnosis of cancer (divergent controls). The total number of individuals typed exceeded 4,000 (Table 3), with an overall allele frequency of 30% for Leu 217. A logistic regression was performed for disease status to delineate effects of genotypes at Ser 217 Leu versus birth year (a demographic datum collected on all participants). We observed a significant interaction between genotype and birth year (p=0.027), indicating that association tests should be performed which appropriately considered birth cohort. FIG. 7 illustrates this birth effect, showing that genotype frequencies differ across birth cohorts for cases, but appear more uniform for the unaffected controls. We subsequently chose to analyze the effect of genotype in individuals born after 1919, since the data suggest that a different risk pattern may exist for individuals born before this date. [0295]
  • Association tests are consistent with the hypothesis that the Leu 217 variant is deleterious or in disequilibrium with another deleterious variant. Prostate cancer patients born between 1920 and 1959 have a significantly higher proportion of Leu 217 homozygotes than either the divergent controls (57/429 vs. 9/148, p-value=0.026) or the unaffected pedigree members (57/429 vs. 220/2371, p-value=0.013) (FIGS. 7 and 8). That Leu 217 is so common could be explained by the allele contributing to a common disease in a recessive manner. [0296]
  • Upon mutation screening ELAC2 in the set of early onset prostate cancer cases, we also observed several occurrences of a second non-conservative missense change, [0297] Ala 541 Thr. This missense change occurs at the border of the histidine motif (FIGS. 6A-B and 9) and thus may well affect the protein's function. This variant has been examined in the same set of cases and controls, where it has an overall allele frequency of 4%. Thr 541 is in strong disequilibrium with Leu 217; in fact, we have yet to observe a chromosome that carries Thr 541 that does not also carry Leu 217. Another logistic regression was performed to investigate effects of genotypes at Ala 541 Thr. Again, a significant interaction between genotype and birth year was found (p=0.003), along with evidence for an effect of genotype at Ala 541 Thr on disease status. Table 10 shows the allele frequencies.
    TABLE 10
    Allele Definitions
    Allele Defining Sequence Variant(s) Note
    0 wt Matches mouse at
    polymorphic positions
    1 Leu 217 Allele frequency = 26.0%
    2 Leu 217 + Thr 541 Allele frequency = 3.9%
  • The carrier frequency of [0298] Thr 541 is significantly higher in prostate cancer cases than divergent controls such that the variant appears to be dominant and deleterious (carrier frequency of 42/429 vs. 5/148, p-value=0.022) (FIG. 8). In contrast, the Thr 541 carrier frequency is not significantly higher in the cases than the unaffected pedigree members. However, in the comparison between cases and pedigree unaffecteds, when Leu 217 homozygotes are subdivided into Thr 541 carriers and non-carriers, the presence of Thr 541 is associated with a higher odds ratio (2.0 vs. 1.4) and the model remains statistically significant (p-value=0.017, trend test p-value 0.004) (FIG. 8). Thus both comparisons support the hypothesis that the allele bearing both Thr 541 and Leu 217 is more deleterious than the allele bearing just Leu 217.
    • Example 10 Identification of HPC2-interacting Proteins by Two-hybrid Analysis
  • DNA fragments encoding all or portions of HPC2 are ligated to a two-hybrid DNA-binding domain vector such as pGBT.C such that the coding sequence of HPC2 is in-frame with coding sequence for the Ga14p DNA-binding domain. A plasmid that encodes a DNA-binding domain fusion to a fragment of HPC2 is introduced into the yeast reporter strain (such as 3692) along with a library of cDNAs fused to an activation domain. Transformants are spread onto 20-150 nun plates of selective media, such as yeast minimal media lacking leucine, tryptophan, and histidine, and containing 25 mM 3-amino-1,2,4-triazole. After one-week incubation at-30° C. yeast colonies are assayed for expression of the lacZ reporter gene by β-galactosidase filter assay. Colonies that both grow in the absence of histidine and are positive for production of β-galactosidase are chosen for further characterization. [0299]
  • The activation domain plasmid is purified from positive colonies by the smash-and-grab technique. These plasmids are introduced into [0300] E. coli (e.g., DH10B (Gibco BRL) by electroporation and purified from E. coli by the alkaline lysis method. To test for the specificity of the interaction, specific activation domain plasmids are cotransformed into strain J692 with plasmids encoding various DNA-binding domain fusion proteins, including fusions to segments of HPC2 and human lamin C. Transformants from these experiments are assayed for expression of the HIS3 and lacZ reporter genes. Positives that express reporter genes with Hs.HPC2 constructs and not with lamin C constructs encode bona fide HPC2-interacting proteins. These proteins are identified and characterized by sequence analysis of the insert of the appropriate activation domain plasmid.
  • This procedure is repeated with mutant forms of the HPC2 gene, to identify proteins that interact with only the mutant protein or to determine whether a mutant form of the HPC2 protein can or cannot interact with a protein known to interact with wild-type HPC2. [0301]
    • Example 11 Identification and Sequencing of Orthologs and a Paralog of the Human HPC2 Gene
  • All species living on the Earth now are thought to have evolved from a single common ancestor that lived in the distant past, perhaps 3.5 to 4 billion years ago. This means that any pair of species must share a common ancestor species that lived at some time in the past. Admittedly, this view is a bit simplistic because, for instance, the nuclear genomes and mitochondrial genomes of eukaryotes are thought to have independent prokaryotic ancestries. During the evolution of an ancestral species into two or more extant daughter species, the genes present in the genome of the ancestral species evolve into the genes present in the genomes of the daughter species. The evolutionary history of the genes present in the daughter species can be quite complex because the individual genes can evolve through a diverse set of processes including nucleotide substitution, insertion, deletion, gene duplication, gene conversion, lateral transfer, etc. Even so, the evolutionary history of related genes in related organisms can often be sorted out, especially if the pair/set of species share a relatively recent common ancestor or if the genes being analyzed evolved primarily through nucleotide substitutions and/or small insertions and/or small deletions, but not gene duplications or gene conversions. When, upon analysis, it appears that a single gene in one species and a single gene in another species have evolved from a single gene in a common ancestor species, those genes are termed orthologs. [0302]
  • Knowledge of the identity of genes orthologous to disease-related human genes can often be quite useful. [0303]
  • The human HPC2 cDNA sequence was assembled from a combination of ESTs, hybrid selected clones, and 5′ RACE (Rapid Amplification of cDNA Ends) products; the orthologous mouse Elac2 cDNA sequence was assembled from ESTs and 5′ RACE products. Conceptual translation of the human cDNA sequence yielded a protein of 826 amino acids; parsing the cDNA sequence across the corresponding genomic sequence revealed 24 coding exons (FIG. 3). Mouse Elac2 encodes a protein of 831 residues in 25 exons. BLAST (Altschul et al., 1990) searches of the ELAC2 sequence against GenBank readily revealed a single ortholog in [0304] S. cerevisiae (YKRO79C) and a single ortholog in C. elegans (CE16965, CELE04A4.4), but two related sequences in S. pombe and A. thaliana. Alignment of representative family members revealed a block of good conservation near the N-termini and a series of blocks of high similarity across the C-terminal half of the proteins (FIGS. 6A-B and 10).
  • Hybridization of RNA blots to labeled fragments of human ELAC2 cDNA revealed a single transcript of approximately 3 kb (FIGS. [0305] 11A-D), in agreement with our full-length cDNA assembly of 2,970 bp. The transcript was detected in all tissues surveyed and, like BRCA1 and BRCA2, was most abundant in testis. The apparent size of the transcript agrees well with our full length cDNA assembly, 2970 bp. There was no evidence from RNA blots, EST sequences, or RT-PCR experiments of significant alternative splicing of the transcript.
  • In the course of surveying ESTs derived from this gene, we identified a small number of human and rabbit ESTs derived from a second, related gene. The human cDNA sequence of this related gene was assembled from a combination of ESTs and 5′ RACE products. Conceptual translation revealed that the transcript encodes a protein of 363 residues. Radiation hybrid mapping placed the gene at approximately 365 cR on [0306] chromosome 18. When this sequence, along with representative sequences from a eubacterium (E. coli elaC), a cyanobacterium (Synechocystis sp. gi2500943/SLR0050) and an archaebacterium (M. thermoautotrophicum gi2622965) was added into the multiprotein alignment (FIGS. 6A-B), it became apparent that two distinct groups of proteins were represented; a group of larger proteins (800-900 aa) restricted to the eukaryotes, and a group of smaller proteins (300 to 400 aa) that align with the C-terminal half of the former group and includes sequences from the eukaryotes, eubacteria, and archaebacteria. As the 363 residue human protein falls into this second group and is more similar to E. coli elaC than is ELAC2, we will refer to it as ELAC1.
  • The alignment revealed a striking histidine containing motif, φφφ[S/T]HxHxDHxxG (SEQ ID NO:214), where φ can be any large hydrophobic residue, near the N-terminus of the ELAC1 group, and in the C-terminal portion of the ELAC2 group. This motif is reminiscent of the histidine motif found in the metallo-β-lactamases (Melino et al., 1998) and suggests, in accord with the annotation for COG1234 (www.ncbi.nlm.nih.gov/COG/index.html), that the proteins are metal-dependent hydrolases. While assembling the multiple alignment, we observed that the sequence within which the histidine motif is embedded also aligns with the ELAC2 N-terminal conserved block (FIG. 12), leading us to predict that some structural feature of the protein is repeated. Even so, the N-terminal copy of the repeated sequence would not necessarily retain metal-dependent hydrolase activity, as the histidine motif itself is not conserved. [0307]
  • Thorough BLAST searches of GenBank using sequences containing this histidine motif, combined with iterative motif searches (Nevill-Manning et al., 1998) using the eMOTIF SCAN website (http://dna.stanford.edu/scan), revealed two other families of proteins that share extended amino acid sequence similarity with members of COG1234. The similarity includes 4 to 6 shared motifs distributed across the ELAC1 domain (FIG. 9). One such family is the PSO2 (or SNM1) family of DNA inter-strand crosslink repair proteins (Haase et al., 1989; Meniel et al., 1995; Niegemann and Brendel, 1994), present only in eukaryotes. The second family encodes the 73 kDa subunit of the mRNA cleavage and polyadenylation specificity factor (CPSF73) (Chanfreau et al., 1996; Jenny et al., 1994; Jenny et al., 1996). Surprisingly, members of this latter gene family are present in both eukaryotes and archaebacteria, as well as a cyanobacterium. These three gene families, ELAC1/2, PSO2 and CPSF73, are equally similar to each other (FIGS. 9 and 13); indeed they were originally placed in a single COG (Tatusov et al., 1997). While PSO2 is required for repair of DNA inter-strand crosslinks following treatment of cells with, for instance, 8-methoxypsoralen plus Lw-irradiation (Menial et al., 1995), the actual substrate for the protein's presumptive metal-dependent hydrolase activity has not been defined. Similarly, although CPSF73 is a component of the [0308] mRNA 3′ end cleavage and polyadenylation specificity factor, it has neither the 3′ end cleavage nor the polyadenylation activity, and the substrate for its presumptive metal-dependent hydrolase activity is unknown. While the S. cerevisiae CPSF73 ortholog YSH1 (BRR5) is an essential gene, PSO2 is not. Given the phylogenetic conservation of the ELAC1 domain and the observation that S. cerevisiae encodes only a single member of this gene family, YKRO79C, we asked whether it is an essential gene. To answer this question, we performed one-step gene disruption of YKRO79C using URA3 as a selectable marker in yeast diploid cells. Two heterozygote knockout strains were sporulated and tetrads were dissected. Each tetrad yielded 1 or 2 viable haploid colonies; these were all URA and YKRO79C wt. Thus we concluded that, like YSH1, YKRO79C is an essential gene.
  • In addition to the histidine motif and the local sequence context in which it is embedded, [0309] ELAC 1/2, PSO2 and CPSF73 proteins share a series of sequence features, some shared pairwise between the gene families and others by all three. Strikingly, all three families have three or four conserved histidine or cysteine positions, past the histidine motif, that lie within these shared regions and can be aligned across the gene families (FIG. 9). The arrangement is reminiscent of the binuclear zinc binding active site of some metallo-β lactamases (Carfi et al., 1998; Fabiane et al., 1998) and the shared similarity between the metallo-β lactamases and glyoxalase II (Melino et al., 1998). This series of sequence similarities leads to three predictions. First, the extended similarity between the ELAC1/2, PSO2 and CPSF73 protein families suggests that they share a domain of approximately 300 residues, and this domain constitutes a metal-dependent hydrolase that coordinates two-divalent cations in its active site. Second, the overall fold of this domain is likely to be similar to that of the metallo-β lactamases. Third, similarity between the region surrounding the ELAC1/2 histidine motif and the N-terminus of the ELAC2 proteins suggests that these proteins are comprised of two structurally similar domains and arose from a direct repeat/duplication of an ancestral ELAC1-type gene.
  • A number of members of the ELAC1/2 family are annotated in GenBank as sulfatases or sulfatase homologs. The annotation appears to be assigned through sequence similarity to the atsA gene of [0310] Alteromonas carrageenovora. The atsA protein contains a histidine motif and has been demonstrated to have aryl sulfatase activity in vitro (Barbeyron et al., 1995), though its sequence does not contain any of the typical sulfatase motifs listed by PROSITE. No other experimentally verified aryl sulfatase contains the histidine motif. As the E. coli protein most similar to A. carrageenovora atsA is elaC, atsA may well be a diverged member of the ELAC1 gene family (BLASTp and alignment not shown). Accordingly, ELAC1 family members should be tested for aryl sulfatase activity; however, it is not apparent whether ELAC1 and ELAC2 family members have the same substrate.
  • In addition to the paralog and the mouse ortholog mmELAC2 (for [0311] Mus musculus ELAC2), orthologs of HPC2 have been identified in chimpanzee and gorilla. These are ptELAC2 (Pan troglodytes ELAC2) and ggELAC2 (Gorilla gorilla ELAC2).
    • Example 12 Multiple Protein Sequence Alignments
  • For the alignment of FIGS. [0312] 6A-B, shading criteria were identity (white on black) or conservative substitution (white on gray) for all ELAC2 sequences with a residue at that position, with four of the five sequences actually having to have a residue at that position. Shaded positions in the ELAC2 sequences were propagated into the ELAC1 sequences. For the alignment of FIG. 12, two shading criteria were used: (1) Identity or conservative substitution across the ELAC2 N-terminal alignment and identity or conservative substitution across either the ELAC1 or ELAC2 His motif. (2) Identity or conservative substitution across both the ELAC1 and ELAC2 His motif, with some conservation across the ELAC2 N-terminal alignment. For the alignment of FIG. 9, shading criteria were identity or conservative substitution across two out of the three (CPSF73, PSO2, ELAC2) protein families represented.
    • Example 13 Analysis of the HPC2 Gene
  • The structure and function of HPC2 gene are determined according to the following methods. [0313]
  • Biological Studies. [0314]
  • Mammalian expression vectors containing HPC2 cDNA are constructed and transfected into appropriate prostate carcinoma cells with lesions in the gene. Wild-type HPC2 cDNA as well as altered HPC2 cDNA are utilized. The altered HPC2 cDNA can be obtained from altered HPC2 alleles or produced as described below. Phenotypic reversion in cultures (e.g., cell morphology, doubling time, anchorage-independent growth) and in animals (e.g., tumorigenicity) is examined. The studies will employ both wild-type and mutant forms of the gene. [0315]
  • Molecular Genetics Studies. [0316]
  • In vitro mutagenesis is performed to construct deletion mutants and missense mutants (by single base-pair substitutions in individual codons and alanine scanning mutagenesis). The mutants are used in biological, biochemical and biophysical studies. [0317]
  • Mechanism Studies. [0318]
  • The ability of HPC2 protein to bind to known and unknown DNA sequences is examined. Its ability to transactivate promoters is analyzed by transient reporter expression systems in mammalian cells. Conventional procedures such as particle-capture and yeast two-hybrid system are used to discover and identify any functional partners. The nature and functions of the partners are characterized. These partners in turn are targets for drug discovery. [0319]
  • Structural Studies. [0320]
  • Recombinant proteins are produced in [0321] E. coli, yeast, insect and/or mammalian cells and are used in crystallographic and NMR studies. Molecular modeling of the proteins is also employed. These studies facilitate structure-driven drug design.
    • Example 14 S. cerevisiae Gene Knockout
  • The URA3 gene was PCR amplified with tailed primers resulting in a product flanked by 42 bp of YKRO79C coding sequences (amino acids 3-16 and 818-831). The resulting PCR product was transformed into yeast diploid strain YPH501 (Stratagene); URA+clones were screened for disruption by the presence of a shorter PCR product at the YKR079C locus. The knock-out clones were further confirmed by sequencing the shorter PCR product for the presence of URA3 sequences. Two heterozygote knockout strains were sporulated and tetrads dissected. Each tetrad yielded 1 or 2 viable colonies. These were genotyped at YKRO79C and tested for growth on URA plates. [0322]
    • Example 15 Association Tests
  • STSs for Ser 217 Leu and [0323] Ala 541 Thr were amplified by allele specific PCR using fluorescently labeled oligos. Allele calls were made with our automated genotyping system. Genotype calls required good allele calls at both markers. Logistic regression analyses were performed using the SPSS statistical software package. The chi-squared statistics for the 2×2 contingency tables were calculated with the Yates correction. The trend statistic for the 3×2 contingency table was calculated with the Cochran-Armitage trend test (Cochran, 1954; Armitage, 1955) using a simple linear trend (0,1,2) for the row scores.
    • Example 16 Generation of Polyclonal Antibody Against HPC2
  • Segments of HPC2 coding sequence are expressed as fusion protein in [0324] E. coli. The overexpressed proteins are purified by gel elution and used to immunize rabbits and mice using a procedure similar to the one described by Harlow and Lane, 1988. This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer, et al., 1993).
  • Briefly, a stretch of HPC2 coding sequence was cloned as a fusion protein in plasmid PET5A (Novagen, Inc., Madison, Wis.). The HPC2 incorporated sequences might include SEQ ID NOs:1, 3 or 28 or portions thereof. After induction with IPTG, the overexpression of a fusion protein with the expected molecular weight is verified by SDS/PAGE. Fusion proteins are purified from the gel by electroelution. The identification of the protein as the HPC2 fusion product is verified by protein sequencing at the N-terminus. Next, the purified protein is used as immunogen in rabbits. Rabbits are immunized with 100 μg of the protein in complete Freund's adjuvant and boosted twice in 3 week intervals, first with 100 μg of immunogen in incomplete Freund's adjuvant followed by 100 μg of immunogen in PBS. Antibody containing serum is collected two weeks thereafter. [0325]
  • This procedure can be repeated to generate antibodies against mutant forms of the HPC2 protein. These antibodies, in conjunction with antibodies to wild type HPC2, are used to detect the presence and the relative level of the mutant forms in various tissues and biological fluids. [0326]
    • Example 17 Generation of Monoclonal Antibodies Specific for HPC2
  • Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising intact HPC2 or HPC2 peptides (wild type or mutant) conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known. [0327]
  • The immunogen is mixed with an adjuvant. Each mouse receives four injections of 10 to 100 μg of immunogen and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen. Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production. [0328]
  • Spleens are removed from immune mice and a single cell suspension is prepared (see Harlow and Lane, 1988). Cell fissions are performed essentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, Md.) are fused with immune spleen cells using polyethylene glycol as described by Harlow and Lane, 1988. Cells are plated at a density of 2×10[0329] 5 cells/well in 96 well tissue culture plates. Individual wells are examined for growth and the supernatants of wells with growth are tested for the presence of HPC2 specific antibodies by ELISA or RIA using wild type or mutant HPC2 target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.
  • Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibody for characterization and assay development. [0330]
    • Example 18 Isolation of HPC2 Binding Peptides
  • Peptides that bind to the HPC2 gene product are isolated from both chemical and phage-displayed random peptide libraries as follows. [0331]
  • Fragments of the HPC2 gene product are expressed as GST and His-tag fusion proteins in both [0332] E. coli and SF9 cells. The fusion protein is isolated using either a glutathione matrix (for GST fusions proteins) or nickel chelation matrix (for His-tag fusion proteins). This target fusion protein preparation is either screened directly as described below, or eluted with glutathione or imidizole. The target protein is immobilized to either a surface such as polystyrene; or a resin such as agarose; or solid supports using either direct absorption, covalent linkage reagents such as glutaraldehyde, or linkage agents such as biotin-avidin.
  • Two types of random peptide libraries of varying lengths are generated: synthetic peptide libraries that may contain derivatized residues, for example by phosphorylation or myristylation, and phage-displayed peptide libraries which may be phosphorylated. These libraries are incubated with immobilized HPC1 gene product in a variety of physiological buffers. Next, unbound peptides are removed by repeated washes, and bound peptides recovered by a variety of elution reagents such as low or high pH, strong denaturants, glutathione, or imidizole. Recovered synthetic peptide mixtures are sent to commercial services for peptide micro-sequencing to identify enriched residues. Recovered phage are amplified, rescreened, plaque purified, and then sequenced to determined the identity of the displayed peptides. [0333]
  • Use of HPC1 Binding Peptides. [0334]
  • Peptides identified from the above screens are synthesized in larger quantities as biotin conjugates by commercial services. These peptides are used in both solid and solution phase competition assays with HPC1 and its interacting partners identified in yeast 2-hybrid screens. Versions of these peptides that are fused to membrane-permeable motifs (Lin et al., 1995; Rojas et al., 1996) will be chemically synthesized, added to cultured cells and the effects on growth, apoptosis, differentiation, cofactor response, and internal changes will be assayed. [0335]
    • Example 19 Sandwich Assay for HPC2
  • Monoclonal antibody is attached to a solid surface such as a plate, tube, bead, or particle. Preferably, the antibody is attached to the well surface of a 96-well ELISA plate. 100 μl sample (e.g., serum, urine, tissue cytosol) containing the HPC2 peptide/protein (wild-type or mutant) is added to the solid phase antibody. The sample is incubated for 2 hrs at room temperature. Next the sample fluid is decanted, and the solid phase is washed with buffer to remove unbound material. 100 μL of a second monoclonal antibody (to a different determinant on the HPC2 peptide/protein) is added to the solid phase. This antibody is labeled with a detector molecule (e.g., 125-I, enzyme, fluorophore, or a chromophore) and the solid phase with the second antibody is incubated for two hrs at room temperature. The second antibody is decanted and the solid phase is washed with buffer to remove unbound material. [0336]
  • The amount of bound label, which is proportional to the amount of HPC2 peptide/protein present in the sample, is quantified. Separate assays are performed using monoclonal antibodies which are specific for the wild-type HPC2 as well as monoclonal antibodies specific for each of the mutations identified in HPC2. [0337]
  • While the invention has been disclosed in this patent application by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims. [0338]
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  • 1 240 1 2481 DNA Homo sapiens CDS (1)..(2478) 1 atg tgg gcg ctt tgc tcg ctg ctg cgg tcc gcg gcc gga cgc acc atg 48 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 tcg cag gga cgc acc ata tcg cag gca ccc gcc cgc cgc gag cgg ccg 96 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 cgc aag gac ccg ctg cgg cac ctg cgc acg cga gag aag cgc gga ccg 144 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 tcg ggg tgc tcc ggc ggc cca aac acc gtg tac ctg cag gtg gtg gca 192 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 gcg ggt agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc gag ttc 240 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 aac cgg tat ctc ttc aac tgt gga gaa ggc gtt cag aga ctc atg cag 288 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Val Gln Arg Leu Met Gln 85 90 95 gag cac aag tta aag gtt gct cgc ctg gac aac ata ttc ctg aca cga 336 Glu His Lys Leu Lys Val Ala Arg Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110 atg cac tgg tct aat gtt ggg ggc tta agt gga atg att ctt act tta 384 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 aag gaa acc ggg ctt cca aag tgt gta ctt tct gga cct cca caa ctg 432 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 gaa aaa tac ctc gaa gca atc aaa ata ttt tct ggt cca ttg aaa gga 480 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 ata gaa ctg gct gtg cgg ccc cac tct gcc cca gaa tac gag gat gaa 528 Ile Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 acc atg aca gtt tac cag atc ccc ata cac agt gaa cag agg agg gga 576 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 aag cac caa cca tgg cag agt cca gaa agg cct ctc agc agg ctc agt 624 Lys His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 cca gag cga tct tca gac tcc gag tcg aat gaa aat gag cca cac ctt 672 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 cca cat ggt gtt agc cag aga aga ggg gtc agg gac tct tcc ctg gtc 720 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230 235 240 gta gct ttc atc tgt aag ctt cac tta aag aga gga aac ttc ttg gtg 768 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 ctc aaa gca aag gag atg ggc ctc cca gtt ggg aca gct gcc atc gct 816 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 ccc atc att gct gct gtc aag gac ggg aaa agc atc act cat gaa gga 864 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 aga gag att ttg gct gaa gag ctg tgt act cct cca gat cct ggt gct 912 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 gct ttt gtg gtg gta gaa tgt cca gat gaa agc ttc att caa ccc atc 960 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 tgt gag aat gcc acc ttt cag agg tac caa gga aag gca gat gcc ccc 1008 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 gtg gcc ttg gtg gtt cac atg gcc cca gca tct gtg ctt gtg gac agc 1056 Val Ala Leu Val Val His Met Ala Pro Ala Ser Val Leu Val Asp Ser 340 345 350 agg tac cag cag tgg atg gag agg ttt ggg cct gac acc cag cac ttg 1104 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 gtc ctg aat gag aac tgt gcc tca gtt cac aac ctt cgc agc cac aag 1152 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 att caa acc cag ctc aac ctc atc cac ccg gac atc ttc ccc ctg ctc 1200 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400 acc agt ttc cgc tgt aag aag gag ggc ccc acc ctc agt gtg ccc atg 1248 Thr Ser Phe Arg Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405 410 415 gtt cag ggt gaa tgc ctc ctc aag tac cag ctc cgt ccc agg agg gag 1296 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425 430 tgg cag agg gat gcc att att act tgc aat cct gag gaa ttc ata gtt 1344 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile Val 435 440 445 gag gcg ctg cag ctt ccc aac ttc cag cag agc gtg cag gag tac agg 1392 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 agg agt gcg cag gac ggc cca gcc cca gca gag aaa aga agt cag tac 1440 Arg Ser Ala Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 cca gaa atc atc ttc ctt gga aca ggg tct gcc atc ccg atg aag att 1488 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 cga aat gtc agt gcc aca ctt gtc aac ata agc ccc gac acg tct ctg 1536 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510 cta ctg gac tgt ggt gag ggc aca ttt ggg cag ctg tgc cgt cat tac 1584 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His Tyr 515 520 525 gga gac cag gtg gac agg gtc ctg ggc acc ctg gct gct gtg ttt gtg 1632 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 tcc cac ctg cac gca gat cac cac acg ggc ttg cca agt atc ttg ctg 1680 Ser His Leu His Ala Asp His His Thr Gly Leu Pro Ser Ile Leu Leu 545 550 555 560 cag aga gaa cgc gcc ttg gca tct ttg gga aag ccg ctt cac cct ttg 1728 Gln Arg Glu Arg Ala Leu Ala Ser Leu Gly Lys Pro Leu His Pro Leu 565 570 575 ctg gtg gtt gcc ccc aac cag ctc aaa gcc tgg ctc cag cag tac cac 1776 Leu Val Val Ala Pro Asn Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590 aac cag tgc cag gag gtc ctg cac cac atc agt atg att cct gcc aaa 1824 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys 595 600 605 tgc ctt cag gaa ggg gct gag atc tcc agt cct gca gtg gaa aga ttg 1872 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 atc agt tcg ctg ttg cga aca tgt gat ttg gaa gag ttt cag acc tgt 1920 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 ctg gtg cgg cac tgc aag cat gcg ttt ggc tgt gcg ctg gtg cac acc 1968 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650 655 tct ggc tgg aaa gtg gtc tat tcc ggg gac acc atg ccc tgc gag gct 2016 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670 ctg gtc cgg atg ggg aaa gat gcc acc ctc ctg ata cat gaa gcc acc 2064 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu Ala Thr 675 680 685 ctg gaa gat ggt ttg gaa gag gaa gca gtg gaa aag aca cac agc aca 2112 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 acg tcc caa gcc atc agc gtg ggg atg cgg atg aac gcg gag ttc att 2160 Thr Ser Gln Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720 atg ctg aac cac ttc agc cag cgc tat gcc aag gtc ccc ctc ttc agc 2208 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 ccc aac ttc agc gag aaa gtg gga gtt gcc ttt gac cac atg aag gtc 2256 Pro Asn Phe Ser Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750 tgc ttt gga gac ttt cca aca atg ccc aag ctg att ccc cca ctg aaa 2304 Cys Phe Gly Asp Phe Pro Thr Met Pro Lys Leu Ile Pro Pro Leu Lys 755 760 765 gcc ctg ttt gct ggc gac atc gag gag atg gag gag cgc agg gag aag 2352 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 cgg gag ctg cgg cag gtg cgg gcg gcc ctc ctg tcc agg gag ctg gca 2400 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu Leu Ser Arg Glu Leu Ala 785 790 795 800 ggc ggc ctg gag gat ggg gag cct cag cag aag cgg gcc cac aca gag 2448 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 gag cca cag gcc aag aag gtc aga gcc cag tga 2481 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825 2 826 PRT Homo sapiens 2 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Val Gln Arg Leu Met Gln 85 90 95 Glu His Lys Leu Lys Val Ala Arg Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 Ile Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 Lys His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230 235 240 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 Val Ala Leu Val Val His Met Ala Pro Ala Ser Val Leu Val Asp Ser 340 345 350 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400 Thr Ser Phe Arg Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405 410 415 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425 430 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile Val 435 440 445 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 Arg Ser Ala Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His Tyr 515 520 525 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 Ser His Leu His Ala Asp His His Thr Gly Leu Pro Ser Ile Leu Leu 545 550 555 560 Gln Arg Glu Arg Ala Leu Ala Ser Leu Gly Lys Pro Leu His Pro Leu 565 570 575 Leu Val Val Ala Pro Asn Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys 595 600 605 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650 655 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu Ala Thr 675 680 685 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 Thr Ser Gln Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 Pro Asn Phe Ser Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750 Cys Phe Gly Asp Phe Pro Thr Met Pro Lys Leu Ile Pro Pro Leu Lys 755 760 765 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu Leu Ser Arg Glu Leu Ala 785 790 795 800 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825 3 2958 DNA Homo sapiens misc_feature (51)..(2531) coding sequence as in SEQ ID NO1 3 cgcgggcgta ggtgaccggc ggctttctca gttttggtgg agacgggcgc atgtgggcgc 60 tttgctcgct gctgcggtcc gcggccggac gcaccatgtc gcagggacgc accatatcgc 120 aggcacccgc ccgccgcgag cggccgcgca aggacccgct gcggcacctg cgcacgcgag 180 agaagcgcgg accgtcgggg tgctccggcg gcccaaacac cgtgtacctg caggtggtgg 240 cagcgggtag ccgggactcg ggcgccgcgc tctacgtctt ctccgagttc aaccggtatc 300 tcttcaactg tggagaaggc gttcagagac tcatgcagga gcacaagtta aaggttgctc 360 gcctggacaa catattcctg acacgaatgc actggtctaa tgttgggggc ttaagtggaa 420 tgattcttac tttaaaggaa accgggcttc caaagtgtgt actttctgga cctccacaac 480 tggaaaaata cctcgaagca atcaaaatat tttctggtcc attgaaagga atagaactgg 540 ctgtgcggcc ccactctgcc ccagaatacg aggatgaaac catgacagtt taccagatcc 600 ccatacacag tgaacagagg aggggaaagc accaaccatg gcagagtcca gaaaggcctc 660 tcagcaggct cagtccagag cgatcttcag actccgagtc gaatgaaaat gagccacacc 720 ttccacatgg tgttagccag agaagagggg tcagggactc ttccctggtc gtagctttca 780 tctgtaagct tcacttaaag agaggaaact tcttggtgct caaagcaaag gagatgggcc 840 tcccagttgg gacagctgcc atcgctccca tcattgctgc tgtcaaggac gggaaaagca 900 tcactcatga aggaagagag attttggctg aagagctgtg tactcctcca gatcctggtg 960 ctgcttttgt ggtggtagaa tgtccagatg aaagcttcat tcaacccatc tgtgagaatg 1020 ccacctttca gaggtaccaa ggaaaggcag atgcccccgt ggccttggtg gttcacatgg 1080 ccccagcatc tgtgcttgtg gacagcaggt accagcagtg gatggagagg tttgggcctg 1140 acacccagca cttggtcctg aatgagaact gtgcctcagt tcacaacctt cgcagccaca 1200 agattcaaac ccagctcaac ctcatccacc cggacatctt ccccctgctc accagtttcc 1260 gctgtaagaa ggagggcccc accctcagtg tgcccatggt tcagggtgaa tgcctcctca 1320 agtaccagct ccgtcccagg agggagtggc agagggatgc cattattact tgcaatcctg 1380 aggaattcat agttgaggcg ctgcagcttc ccaacttcca gcagagcgtg caggagtaca 1440 ggaggagtgc gcaggacggc ccagccccag cagagaaaag aagtcagtac ccagaaatca 1500 tcttccttgg aacagggtct gccatcccga tgaagattcg aaatgtcagt gccacacttg 1560 tcaacataag ccccgacacg tctctgctac tggactgtgg tgagggcaca tttgggcagc 1620 tgtgccgtca ttacggagac caggtggaca gggtcctggg caccctggct gctgtgtttg 1680 tgtcccacct gcacgcagat caccacacgg gcttgccaag tatcttgctg cagagagaac 1740 gcgccttggc atctttggga aagccgcttc accctttgct ggtggttgcc cccaaccagc 1800 tcaaagcctg gctccagcag taccacaacc agtgccagga ggtcctgcac cacatcagta 1860 tgattcctgc caaatgcctt caggaagggg ctgagatctc cagtcctgca gtggaaagat 1920 tgatcagttc gctgttgcga acatgtgatt tggaagagtt tcagacctgt ctggtgcggc 1980 actgcaagca tgcgtttggc tgtgcgctgg tgcacacctc tggctggaaa gtggtctatt 2040 ccggggacac catgccctgc gaggctctgg tccggatggg gaaagatgcc accctcctga 2100 tacatgaagc caccctggaa gatggtttgg aagaggaagc agtggaaaag acacacagca 2160 caacgtccca agccatcagc gtggggatgc ggatgaacgc ggagttcatt atgctgaacc 2220 acttcagcca gcgctatgcc aaggtccccc tcttcagccc caacttcagc gagaaagtgg 2280 gagttgcctt tgaccacatg aaggtctgct ttggagactt tccaacaatg cccaagctga 2340 ttcccccact gaaagccctg tttgctggcg acatcgagga gatggaggag cgcagggaga 2400 agcgggagct gcggcaggtg cgggcggccc tcctgtccag ggagctggca ggcggcctgg 2460 aggatgggga gcctcagcag aagcgggccc acacagagga gccacaggcc aagaaggtca 2520 gagcccagtg aagatctggg agaccctgaa ctcagaaggc tgtgtgtctt ctgccccacg 2580 cacgcacccg tatctgccct ccttgctggt agaagctgaa gagcacggtc ccccaggagg 2640 cagctcagga taggtggtat ggagctgtgc cgaggcttgg gctcccacat aagcactagt 2700 ctatagatgc ctcttaggac tggtgcctgg cacagccgcg ggccaggagg ctgccacacg 2760 gaagcaagca gatgaactaa tttcatttca aggcagtttt taaagaagtc ttggaaacag 2820 acggcggcac ctttcctcta atccagcaaa gtgattccct gcacaccaga gacaagcaga 2880 gtaacaggat cagtgggtct aagtgtccga gacttaacga aaatagtatt tcagctgcaa 2940 taaagattga gtttgcaa 2958 4 295 DNA Homo sapiens misc_feature (51)..(295) exon 1 4 cgcgggcgta ggtgaccggc ggctttctca gttttggtgg agacgggcgc atgtgggcgc 60 tttgctcgct gctgcggtcc gcggccggac gcaccatgtc gcagggacgc accatatcgc 120 aggcacccgc ccgccgcgag cggccgcgca aggacccgct gcggcacctg cgcacgcgag 180 agaagcgcgg accgtcgggg tgctccggcg gcccaaacac cgtgtacctg caggtggtgg 240 cagcgggtag ccgggactcg ggcgccgcgc tctacgtctt ctccgagttc aaccg 295 5 51 DNA Homo sapiens misc_feature (1)..(51) exon 2 5 gtatctcttc aactgtggag aaggcgttca gagactcatg caggagcaca a 51 6 71 DNA Homo sapiens misc_feature (1)..(71) exon 3 6 gttaaaggtt gctcgcctgg acaacatatt cctgacacga atgcactggt ctaatgttgg 60 gggcttaagt g 71 7 65 DNA Homo sapiens misc_feature (1)..(65) exon 4 7 gaatgattct tactttaaag gaaaccgggc ttccaaagtg tgtactttct ggacctccac 60 aactg 65 8 58 DNA Homo sapiens misc_feature (1)..(58) exon 5 8 gaaaaatacc tcgaagcaat caaaatattt tctggtccat tgaaaggaat agaactgg 58 9 69 DNA Homo sapiens misc_feature (1)..(69) exon 6 9 ctgtgcggcc ccactctgcc ccagaatacg aggatgaaac catgacagtt taccagatcc 60 ccatacaca 69 10 120 DNA Homo sapiens misc_feature (1)..(120) exon 7 10 gtgaacagag gaggggaaag caccaaccat ggcagagtcc agaaaggcct ctcagcaggc 60 tcagtccaga gcgatcttca gactccgagt cgaatgaaaa tgagccacac cttccacatg 120 11 59 DNA Homo sapiens misc_feature (1)..(59) exon 8 11 gtgttagcca gagaagaggg gtcagggact cttccctggt cgtagctttc atctgtaag 59 12 59 DNA Homo sapiens misc_feature (1)..(59) exon 9 12 cttcacttaa agagaggaaa cttcttggtg ctcaaagcaa aggagatggg cctcccagt 59 13 73 DNA Homo sapiens misc_feature (1)..(73) exon 10 13 tgggacagct gccatcgctc ccatcattgc tgctgtcaag gacgggaaaa gcatcactca 60 tgaaggaaga gag 73 14 113 DNA Homo sapiens misc_feature (1)..(113) exon 11 14 attttggctg aagagctgtg tactcctcca gatcctggtg ctgcttttgt ggtggtagaa 60 tgtccagatg aaagcttcat tcaacccatc tgtgagaatg ccacctttca gag 113 15 96 DNA Homo sapiens misc_feature (1)..(96) exon 12 15 gtaccaagga aaggcagatg cccccgtggc cttggtggtt cacatggccc cagcatctgt 60 gcttgtggac agcaggtacc agcagtggat ggagag 96 16 139 DNA Homo sapiens misc_feature (1)..(139) exon 13 16 gtttgggcct gacacccagc acttggtcct gaatgagaac tgtgcctcag ttcacaacct 60 tcgcagccac aagattcaaa cccagctcaa cctcatccac ccggacatct tccccctgct 120 caccagtttc cgctgtaag 139 17 86 DNA Homo sapiens misc_feature (1)..(86) exon 14 17 aaggagggcc ccaccctcag tgtgcccatg gttcagggtg aatgcctcct caagtaccag 60 ctccgtccca ggagggagtg gcagag 86 18 119 DNA Homo sapiens misc_feature (1)..(119) exon 15 18 ggatgccatt attacttgca atcctgagga attcatagtt gaggcgctgc agcttcccaa 60 cttccagcag agcgtgcagg agtacaggag gagtgcgcag gacggcccag ccccagcag 119 19 97 DNA Homo sapiens misc_feature (1)..(97) exon 16 19 agaaaagaag tcagtaccca gaaatcatct tccttggaac agggtctgcc atcccgatga 60 agattcgaaa tgtcagtgcc acacttgtca acataag 97 20 139 DNA Homo sapiens misc_feature (1)..(139) exon 17 20 ccccgacacg tctctgctac tggactgtgg tgagggcaca tttgggcagc tgtgccgtca 60 ttacggagac caggtggaca gggtcctggg caccctggct gctgtgtttg tgtcccacct 120 gcacgcagat caccacacg 139 21 39 DNA Homo sapiens misc_feature (1)..(39) exon 18 21 ggcttgccaa gtatcttgct gcagagagaa cgcgccttg 39 22 110 DNA Homo sapiens misc_feature (1)..(110) exon 19 22 gcatctttgg gaaagccgct tcaccctttg ctggtggttg cccccaacca gctcaaagcc 60 tggctccagc agtaccacaa ccagtgccag gaggtcctgc accacatcag 110 23 100 DNA Homo sapiens misc_feature (1)..(100) exon 20 23 tatgattcct gccaaatgcc ttcaggaagg ggctgagatc tccagtcctg cagtggaaag 60 attgatcagt tcgctgttgc gaacatgtga tttggaagag 100 24 121 DNA Homo sapiens misc_feature (1)..(121) exon 21 24 tttcagacct gtctggtgcg gcactgcaag catgcgtttg gctgtgcgct ggtgcacacc 60 tctggctgga aagtggtcta ttccggggac accatgccct gcgaggctct ggtccggatg 120 g 121 25 79 DNA Homo sapiens misc_feature (1)..(79) exon 22 25 ggaaagatgc caccctcctg atacatgaag ccaccctgga agatggtttg gaagaggaag 60 cagtggaaaa gacacacag 79 26 145 DNA Homo sapiens misc_feature (1)..(145) exon 23 26 cacaacgtcc caagccatca gcgtggggat gcggatgaac gcggagttca ttatgctgaa 60 ccacttcagc cagcgctatg ccaaggtccc cctcttcagc cccaacttca gcgagaaagt 120 gggagttgcc tttgaccaca tgaag 145 27 655 DNA Homo sapiens misc_feature (1)..(228) exon 24 27 gtctgctttg gagactttcc aacaatgccc aagctgattc ccccactgaa agccctgttt 60 gctggcgaca tcgaggagat ggaggagcgc agggagaagc gggagctgcg gcaggtgcgg 120 gcggccctcc tgtccaggga gctggcaggc ggcctggagg atggggagcc tcagcagaag 180 cgggcccaca cagaggagcc acaggccaag aaggtcagag cccagtgaag atctgggaga 240 ccctgaactc agaaggctgt gtgtcttctg ccccacgcac gcacccgtat ctgccctcct 300 tgctggtaga agctgaagag cacggtcccc caggaggcag ctcaggatag gtggtatgga 360 gctgtgccga ggcttgggct cccacataag cactagtcta tagatgcctc ttaggactgg 420 tgcctggcac agccgcgggc caggaggctg ccacacggaa gcaagcagat gaactaattt 480 catttcaagg cagtttttaa agaagtcttg gaaacagacg gcggcacctt tcctctaatc 540 cagcaaagtg attccctgca caccagagac aagcagagta acaggatcag tgggtctaag 600 tgtccgagac ttaacgaaaa tagtatttca gctgcaataa agattgagtt tgcaa 655 28 26664 DNA Homo sapiens misc_feature (910)..(13104) exon 1 910-1154; exon 2 1736-1786; exon 3 1925-1995; exon 4 3025-3089; exon 5 4361-4418; exon 6 5582-5650; exon 7 7075-7194; exon 8 8186-8244; exon 9 12878-12936; exon 10 13032-13104; 28 tatcaggtga ctgaattcta tattctgaag taggagatac tgttattgct gttattacat 60 tttacacata agaaagctga ggctctgaga ggtcaagatc acgcagctaa caaatgagcc 120 aagactcttg ctttagagct tgtcctctat tcttgctttt ctttccaaaa aacactacaa 180 tttttgtttt gttttgtttt gttttgagac agggtctcga ggtgtcaccc aggctggagt 240 gcagtggcgc gatttcgact caccgcaacc tccgcctccg cgcttaagcg attctcctgc 300 ctcagcctcc caagtagctg ggactacaag ctcgggacac cacgtaaaaa tgatcaagtt 360 ctaacatgta tgcatacgaa ttacaatgga aataaaatta gcaaagcgct tatgctaatg 420 ctcaatacaa ttgatttcct cacatttaat cctcacaacc actacaacca cctctaactc 480 aagctctgag ggactgacgt gcccggagga cacagctctt atctggtgag aacaggagcg 540 ttttagcgaa actccaaact cctaggtccc gccttcccca ggaaggcttt tcctggcact 600 gtgcttccgg aagtcccgcc ccaggagaaa aacagcttcc ggaaaaaatt gcggccggca 660 aaccggaaca gaactagggg cggggccgct tgagacgctc tagtattcct ctactctatg 720 gccactgtca attgacaagt cccgagcggt aaagctcctt tctattggat gagcagcctc 780 gcgtaggcgg gaagctcggt gcacggcgcg ctgattggct ggatcsgcca tgcggagcgg 840 ctaggtggtg cacgggaaac gcgggcgtag gtgaccggcg gctttctcag ttttggtgga 900 gacgggcgca tgtgggcgct ttgctcgctg ctgcggtccg cggccggacg caccatgtcg 960 cagggacgca ccatatcgca ggcacccgcc cgccgcgagc ggccgcgcaa ggacccgctg 1020 cggcacctgc gcacgcgaga gaagcgcgga ccgtcggggt gctccggcgg cccaaacacc 1080 gtgtacctgc aggtggtggc agcgggtagc cgggactcgg gcgccgcgct ctacgtcttc 1140 tccgagttca accggtcagt caacgagcca cgccccgtcc cgctgggccc tcagtgcggc 1200 gcagcctctg agcatcgggg cacctcccag ggcttcggct tccctgcttc acacatgtgg 1260 ttcactgttg cgggggttcg tggagttatg gtgggtggga aatccgagat tctttgcatc 1320 catgtgattt ctgcggatct gtgaagaact tcaggcctgg gtctgagcgt ccttttccca 1380 acccttgggc cccggcctgg ctgtcagcac tttcggagct ccaccctctt ccgtgcaccc 1440 caaggccagt gtgtcgttgt tagcgtgtgg ggtggacaga tctggtgtgt agccggtggt 1500 ggagaaagga ctcattttgt cctagcaccc acacacacag gcccccactc ctctccacct 1560 ctgctaagga gggctcaaaa cccaccagca taaatgtggc tcggtagtcc aacgtggact 1620 tttaattttt ttttcttttt tttttttcca gagtctacaa taaaacatct aattggtgtc 1680 agagagttta cagaataaaa ccttctgaat gtcttgtgta atgtttgtct tgtaggtatc 1740 tcttcaactg tggagaaggc gttcagagac tcatgcagga gcacaagtga gtcagtctct 1800 tgctttcgga gggggagttg attacggggc ttgaaagccg aaatgagagg ccagttgttt 1860 tttatagcaa aagtggtcct tgttctgttc atgttatcct gtttaaatgt tttytcattc 1920 ttaggttaaa ggttgctcgc ctggacaaca tattcctgac acgaatgcac tggtctaatg 1980 ttgggggctt aagtggtgag tatattcttt gcagtgtcag aggctggtgg gaagtctctg 2040 ggattttaac cggctttacc atttttccaa gtctggggtg ggcagctact tttttttttt 2100 tttttttttt tgtcagtggc gtgatcttgg ctcactgcaa cctttgcctt ctgggctcag 2160 gtgatcccct cacctcagcc tcccaaatag ctgggaccac acgtgtgccc catcacacct 2220 ggctaatttt ttttgtatgt tttgtagcga cggggttttg ctatgttgcc caggctggtc 2280 tcaaacttct gcgatcctcc tgtctcggcc tcccagagtg ctgggattac aggcatgagc 2340 caccgcacct ggcctggaat tctttttata ccagcccagt cagcagcagc acagagcatt 2400 aaaagctgtg actcaggaga acagatttta atatggatac cacctcttaa gtgttaccat 2460 ccacttagtt tcttgcgttg cggggacaga gatttgtggc agtaaactgg agagtctagc 2520 agtggtgatt acagttaata tgtttaccgc agacgccatt ggcacattgg cagccacaca 2580 catacccact gtccagatta ccctgtcatt tatgtctatc aaccggaagg tcaggattgt 2640 gttgcagcca aattgtgtgg gcttggtggc atggaccgga aggagtgaag tgttagacca 2700 gtctcccttc tcagggctga gactagggtg aggcacttag ggtgccagcc cttcacttgc 2760 atgattcctt acattttgca cactgggtgc cttgctgctt caccctagtg acagctcagc 2820 ccattctaga ggcatttaaa gaatatttgg tgtctgttac acctctagct ggcatcactt 2880 ctgctctgta catcttccct ggttgtactt ccaaagctgg aaggtggaga tgtagataaa 2940 tagttggatt agtacggggt gctcctcctg ttagtgacga caggtcaaat tgatgagaga 3000 tctgatttta tgcatccttt ttaggaatga ttcttacttt aaaggaaacc gggcttccaa 3060 agtgtgtact ttctggacct ccacaactgg tgagtctttc ctgacacatc tttcaaaagc 3120 aatctttcct tttgtaatat cagtaacaag aattttcctt tttgcaaatc agtcttctgc 3180 cctccagaga tacctggtcg ttgaaacgct tcccctttca agttaaaaag acttgagttc 3240 tgattaacta tgtgaccttg atcaagttac tttacctttc tgagctttag tttattcatc 3300 tataagatga ctatcacgtt tcatagagtt gttaaagatt aaatgacgta gcagcacata 3360 taaagcacta aatcacttta ttagatatat gtttggcacc aagtaggcac acaagaaagg 3420 gcagcttttg tttttattca ataaatttct gacatcttct tacctttcag tccagcttat 3480 tacactcttg agaaggcgtg tgtgtgttgt tgaatataac agttcatttt ccagtcctta 3540 agaagaaagt caccaagacc tgttaagtct ttccccaaaa taacgtttga aatccatcca 3600 tttgtctctt attgaggcct tccttatttc tgttttctat gcctgtaaac tacaatagcc 3660 tcccatattc attctcgcct tcctgtaatc catctgccac acagcagcca gagaggtcac 3720 ttcaagacag aaaagtagtg tgtcacttgc caccctaaag cccttcatgg gctccccatt 3780 gcaatacaat caaaacacct tgatatggcc tacaagtcct gtaggccccg gccgctaccc 3840 acacttccat ctgtacccat cgctgaactg cagctgcatg ggctgactct tatgtccctc 3900 taactccctg gccacttcag gactttcgcc cttccgcggg ttccctctgc ctcttctaat 3960 tgctgcctat attgttactg aaccttcagg gctcagctag agggtcattt actccagaac 4020 tgcctcttct tctctagaca agttggatcc cagccttctg tatttttcat tttccttgca 4080 gagcacttag cataatgcca ctaagctgtt tctgttatcg tgtttccttt tgtctcctcc 4140 actggcctga ttagagcaag gcctccatct ctttttcctg ctatatcctt ggcatctgat 4200 ataatggata ctcagtaaat atttgtaata aatgatgttc aaaatattta ctaagctttg 4260 ttttatgttg atacctattg gtaacctttt aaatacttga atagttgctg tgttctacat 4320 ttgttcaacc ataactgctc atttctttgt ttttcattag gaaaaatacc tcgaagcaat 4380 caaaatattt tctggtccat tgaaaggaat agaactgggt acgtctttgt ctgtgactca 4440 tcctctgcta tttctaactt atatatgccc tgacctctca aattagaatc cattaaaaac 4500 atcaacatca aacctcaaaa tcaaatgctt catcaccacg agattttttt tttttttttt 4560 ttttttggat agagtcttgc tttcttacca ggctggagtg cagtggcatg atctcggctc 4620 actgcaacct ccacctcctg ggttcaagcc attctcccac ctcagcctcc tgagtagcta 4680 ggactacagg cgcatgccat cacgctcagc taattttttg tatttttagt agagacgggg 4740 tttcaccatg ttggccagga tagtctcgat ctcttgacct tgtgatctgc ccgcctcagc 4800 ctcccaaaat gagctaccat gtggctggag atgggatttc taaatagtga cattttctgt 4860 gttcccacct catgctgtaa aaataggggc caggtcggca ggagtgattg aacagctgat 4920 gcctgcctgt gtacatgctg tgtggcattc tccatccaga cggcagggct cctgcctcag 4980 ttccagaggt gcttctcgtc gttgagttgc tttgagttgg gggcgggggt gacaagggtt 5040 ccctagaggt tttgtggcca actttgtaca ttgaaacgca gctccagctg cgcagggggg 5100 cttacagcct cttgatggga agaggcctca ctgaggatgc tagtagggct cttgtcctgg 5160 cactggtgtg tatctgtggc ttgttaatac tcctctttta tagaaacact aatactttgt 5220 ttcaaaatat acatcagctc ttctggtttg cgatgatagg ttccctggct tcactattct 5280 gtttgttaac ttgggtctct gaaagttgag tactagtttc ttgtttttca atttttaacg 5340 gatagtcacc aaagattata atgtcttttc atctggctgt agtaaatata aatggctgac 5400 caaaatacac ttttatttat ttcctaaaaa tggtaatctc cttagaaagt ctggttttcg 5460 tgtcagattc ccaccataat tctgaggcaa ttcagttgct cgtggttggt gatcctgaag 5520 ttactcttcc cacacatctt cactaatgca atcactttgc tgttgtgygg ttttcttgta 5580 gctgtgcggc cccactctgc cccagaatac gaggatgaaa ccatgacagt ttaccagatc 5640 cccatacaca gtgagtatga aagccaggtt tcccaggagg agggtgtacg tcctgagtaa 5700 agaaaacatg gatgaaaata gaaactgaac acttgctgtg ggcaccctgt tttgtgttct 5760 gagcatgatt agaaaattta gttgaggaat gaagatatgg ctcctgccct ggcttataaa 5820 cttacggatg tctgacttat gcctaatgat agtgattatg ctttggaata ttagataatc 5880 aagcactgtt ggtaaataga ttgcattcaa gtttgcacat tcattgcttg gaggtttttt 5940 cccacaggcg taataccctc ttttgatcag acgatcatga agaggtttgc acagatagat 6000 ttttttaaat aaataatgat tacagcaacc taaaagaagt gttgttgggg gttagaagct 6060 cctgcaaatt ccgaagtatc agggccagat gatgtggtct tagcttagga aaagagttag 6120 tcttgtcctt gaacttggct aaagacattc atgtctggtt ttacttacat gtgaagagag 6180 taccaagcag taggggtatt tccttgttag tactaactaa tgtgatgctt actaagtagt 6240 gctgatgggt gacagaccag agcacccagc aaaggccaga gaagtccaga acctggcgag 6300 gagatgaggc ttacactgac tgaaggcaga aggcagcagg gaggagagga atgtgccgga 6360 gcaatggcac aagtgctcct aggccagtgc tgtgatgagc tgatcagcac tcccattgcc 6420 tggcttgctc ctcctgctca gatgccttct ctcacctgac ccctgctgta gccaccccca 6480 gcctgagttg catccacctg tttgttgtcc atttccagca ccctgttctt cgctccatgg 6540 catgtgacag ttaactttca tatgtgattt gcgtgatcga tgttaacatg ctcagttttg 6600 ccgatcactg ttttttcagt gtccagcggc cctcagtgag tgaacttacg ttcattctcg 6660 ttgcagctgt gctttagctt cttagagcag cgaatttttt tcccttgatc ttgagcctta 6720 actaaatgta aaatgaggct ccttcttgag ataggtaccc tttgggtcta tgtgttttag 6780 cgggagtgat gataataaat aagcatgtct acaacccaca tgctgtttag ataacacgtt 6840 gttgagttgg tactgtggcc gaggctgtga gctaagcaga aacataaaca ttaataggac 6900 ataggtgcag cccagaaacc aggtaggaag ttaactaact agttatttcc tactgtatag 6960 taaaaggtgt gctgatttaa ttggcgttct ggcattccca tgtatgaacg tctgggcctt 7020 ggctgtcagc tcaccttgtg cagtgtgtaa tttggtggta tctgtactga ccaggtgaac 7080 agaggagggg aaagcaccaa ccatggcaga gtccagaaag gcctctcagc aggctcagtc 7140 cagagcgatc ttcagactcc gagtygaatg aaaatgagcc acaccttcca catggtaata 7200 gtataaacaa aacagagcag cagaaaggct tgcgttttct taattctctg ccttgtaatg 7260 cttgtagaga gtcattattg taagaaagcc aggtgtgtaa acagatcctt cttcctgggc 7320 ttactataac ttggcccgtt gggggaatga gaagggttgt tgtaaaggtg gcagcctgca 7380 actttaataa tgaccagtcc acagttttgg ccacccaggg tctgggtagg cccaaaactg 7440 tgttctgttt tcccagagga gaacagggcc tgacaaacgg attcattttg tatttttcat 7500 taatgtaaca tttatgcaaa ttttccatta atgtggaaac tataactgct aagccaatga 7560 gacagtcaaa tcagtgagag gctctgcacg tcttccagaa tgacagccca ctgggaaacg 7620 gagttaaaag tccaagatga gatgtagctc aggagtcagg ccgcttcggg agtttgttgt 7680 ccttaacaga aggtcagcgt tggcaaagct cggcagctcc tctttctgtc ctgaggtctt 7740 gtctagtgac tgagaacagg ctgaccccta tgtgctgtcc ttgtttggat ggcaccgggt 7800 aaagactgac accagcattt tctctgcagg cctttgaact tttgtgttat ttcatatatt 7860 atatgtgtta taaagcacat tacaatatat ttttctctgt cttctccagt cctaggtgaa 7920 atgtgtcatt taaaaaaaat ttcacttgcc attctaaagt ttttctggtg agagttttgt 7980 gtttttcatt tacgcaaaca catctccaca taagtaggga aaaaaagtct tcttgagtat 8040 attagtgtct tcagcctttg tattgggaca gtagcgtcca ttaattttta tgtgaagtga 8100 aattaggtat cgggtcataa tcagtctgtg atgtcttcac agctttcaca tttaccttgt 8160 gataatcaag tgtgtttttc ctcaggtgtt agccagagaa gaggggtcag ggactcttcc 8220 ctggtcgtag ctttcatctg taaggtaagg aagactttcc ggagggctgt acatgactgg 8280 ggtcttggtc agcgacctct ggtttgcact ttttcattaa tttgagggta ggcactcctg 8340 ttacctgaga caagaagaga tagcagatct tcagaaaagc tgatggaagg ccgggtgcag 8400 tggctcacgc ctgtaatccc agcactttgg gagtccaagg caggtggatc acgaggtcag 8460 gagtttgaga acagcctgac caacgtggtg aaaccctgtc tgtactaaaa atacaaaaat 8520 tagctgggtg tggtggcgca tgcctgtaat cccagctact tgagaggcca aggcaagaga 8580 atcgcttgaa cacaggaggc ggaggttgca gtgagttgag attgcaccat tgcactccag 8640 cctgggtgac agagcaagac tctctcaaag aaaaaaaaaa ttcgatagaa atgacactgg 8700 caatgagcct gcaacaagta ttactactga cctttcataa ttgtcatcac ttgtaggttt 8760 cagagtttag atgctctgtt tctcaaaata accccatact tttatttcct tttaaatttt 8820 tttccagtgc cctgtcagcc tccgtacatt tttttttttt ttttttgaga ccatgtctgt 8880 ctccatcgcc taggctggag tgtgcagtgg cacaatctcg gctcactgca gcctccacct 8940 cccaggttca agtgattctc ctgcctcagc ctcccaagta gctaggatta taggtgcgcg 9000 ccaccacacc cagttaattt ttgtattttt agtagagatg gggtttcacc atgttggcca 9060 ggctggtttc actcctgacc tcaggtgatc cacccacctt ggcctcccaa aatgctggga 9120 ttacaggcgt gaagcactgt gcctggtcca tattctttta tatttgccaa tgattggtcc 9180 ttttagaatt cagaaattat tgaaggcagc tgtgtttgtt ttccttcaac tccatcaggc 9240 ctttattcaa agtcttttaa ctctgtttta ctttatttca ttcccctgca atagctaagg 9300 tctaacacca gattaattgg aatattagct agcattcaca aaggcctaga tctgtaactc 9360 tgaaattggt caaattccat taaaaatttt tgttacaata agctgtttgt aagatctgac 9420 tagtggctta tttttaatag aattttgcat taaaatttta tcaatacaat ttgcaacaaa 9480 tttgtctaaa tatgtgaaaa gatttcattg cctttttgtg ggcttagatt attttttaat 9540 gttgattttg aaatatattt ggaattgtta tctaaattct aaaagctaca agtgaaaata 9600 ataatgaaag taagtagtta atattagtgg caagatcatt gccagtatca tttctatcga 9660 tttatttgaa taatgtgatt ttcataaaag ttaagtacta ctgttaacag gcttattact 9720 tgtatgtttc tgagttttag atagcaaaat cattttttaa agttttaaaa atattttatt 9780 tttgataatc tatatttata ttgtctgatt tttaaactgt tttctatggt aatctttaaa 9840 tcgtattcct gctttccgga ataggtaaca gtgagcatga tgaaaagtga caagctcact 9900 tttacacact cgggcagttg ccctattatc aggcagccgt tcctgggggc tgccagctgc 9960 ctgccctggc ttttccatct ccttccttgc tgtcttctgc ggctccttct gagggctgct 10020 gtcactggat tagcctataa cgcctttccc ctcttctaat taatttgctg ctctcaggtg 10080 aggttttgga aagcaataaa gctgagctag gtcaagttcc aggagtctct tggcatgagg 10140 acctgaaaaa ctcatctgtt ggaagacctc ggctttgggc agctggtgca ctgttggggc 10200 gttattggct gcgttctggc tctcatcagt cttccagata ctctgcattc ctcagagagg 10260 aacatatctc catgggttga gttcagctcc cagggagatg ggtttccctg ccttaagtcg 10320 gcaagtacct ttttttttct ttttttgaga cagagtctcg ctctgtcacc aggctggagt 10380 gcagtggtgc gatcttggct cactgcaacc tctgcctccc agggtcaagc agttctcctg 10440 cctcagcctc ccgagtagct gggactacag gagcgcacca ccatgcccag ctaatttttg 10500 tattttttta gtagagacgg ggtttcacca tgttggccag gatggtctgg atctcttgat 10560 ttcctgatcc gcctgccttg gcctcccaaa gtgctgggat tacaggcgtg agccatcatg 10620 accagccttt atgtttcttt gtttgttttg tttttctgag atggagtctc gctctgttgc 10680 ccaggctgga gtgcagtgtt gccatctcga cttactgcaa cctctgcctt ccaggttcaa 10740 gtgattcctt gcctcagcct cccgtgtagc tgggatcaca ggtgcctgcc accatgcccg 10800 gctaattttt gtattgttag tagacacagg gtttcgccat gttggccagg ctagtctcga 10860 actcctgacc tcaagtgatc tgccttcctc agcctcctaa agtgctgggg ttacaggagt 10920 gaaccaccat gcccagcctt caattacctt ttatttattt tatttattta tttatttttg 10980 agacggagtc tttctgtgtt gcccaggctg gagtgcggtg gcgcaatctt agctcactgc 11040 aacctcctcc tcccaggctc aagtgattct catgcatcag cttcccgagt agctgggact 11100 tcaggtgccc gccaccacac ttggctaatt tttgtgtttt tagtagagac ggggtttcac 11160 catgttggcc aggctggtct tgaatttctg acctcaaatg atcctcctgc ttcagcctcc 11220 caaagtgctg ggattacagg cgtgagccac tgcccccaac agcaagtacc ttttaaacat 11280 tagagacatt tagttgccat cctcaaaccc gtttgggtgt gtggagagaa tgttgggtcg 11340 tgacatggtt gttagttatc taaagatgtc agccatcaat catcactgtg tgatgtgcac 11400 actgaagctg taatccttca tctaggatga tattttttaa gatggaaaat tctacaaccc 11460 tgagaataag gatttcagat ccaaatttga gactcagccc tacgagtaac tctttaactt 11520 cagagagtta aaagaagatg cacagttgat gaagatttaa aggagaaaat ggaaatcaaa 11580 tgtcatttag cactcaaagg cctacatgtc atttctgaca tttttctgtt tgtgtgaaat 11640 tttttttttc ctataaaatg attgtgaagt tttctggtag aattattgtt tgcctttcta 11700 atgtaatagc atattagggt tttttttttt ttctttttct ttttttgaga cagagtctca 11760 ctctgtcgcc caggctggag tgcagtggca cgatctcggg tcactgcaat cttccgcctc 11820 ctgggttcct gcctcagcct cccgagtagc tgggactaca ggcgcacgtc accacacccg 11880 gctaattttt tgtattttta gtagtgacag ggattcaccg tcttagccag ggtggtcttg 11940 atctcctgac ctcatgatct acccgcctcg gcttcccaaa gtgctgggat tacaggcatg 12000 agccgctgtg cctggctatt agagattttt tattataatt tatctccaag ataaaagcag 12060 tgacattata ttgccacata attgaaaaat acaagagaaa taaaaatcat ccatgctttt 12120 gttagcctat cactgtcatt gaaatattat gttacatggc agtttgcttg ctggttgctc 12180 tgttaggcaa cgctctggtg acattccttt agctattaat tgaggaatgt agaatgacag 12240 aacagtgttt ctcctcaatg atacttgaag gatatttatt aactttcata ttgaattaca 12300 ttttattaaa tttataatga gttaatgctg ggaaataaaa cactgattta agtcattttg 12360 gcttttagta ctaaagcatt tgacaataaa tgacttcttc agaatatggt ataccttctg 12420 aaagcaataa acgcatttta atgaattgta aggaaacaac atcattttat tttttatttt 12480 tttttttgag acagactttc gcttttgttg cctaggctgg agtgcaatgg cgcgatctcg 12540 gctcactgca acatccgcct ctgggttcaa gcgattctcc tgcctcagct tcctgagttg 12600 ctgggattac aggcacgtgc caccacgcct ggccaatttt gtatttttag tagagacggg 12660 gtttctccac gttggtcagg ctggtctcaa actcctgacc tcaggtgatc tgcccgcctc 12720 agcctccgaa agtactggaa ttacaggcgt gagccaccgt gcctggccaa cattattatt 12780 tttttttaat ctagaaaaat acacttctaa gaaaattgat taaaaccaac cttcttcatt 12840 agcccctaag atcacatcta tgttctcttt gttgcagctt cacttaaaga gaggaaactt 12900 cttggtgctc aaagcaaagg agatgggcct cccagtgtga gtgtgggggg taaggcttct 12960 ggggactcac tgggtacacc tgtccactta aggaaatcac atttcacaga ggccttgcct 13020 cttcatttca gtgggacagc tgccatcgct cccatcattg ctgctgtcaa ggacgggaaa 13080 agcatcactc atgaaggaag agaggtgaga tgcctggttt tcttgatnca gcagttacag 13140 gtagggtctg aaatgctggg cagagtctgt cttcttcagg ccctacagac accacttttg 13200 aaggacgtgg aacagtttgg acatcactca gctaagtgat aaaatggcct cttttatctg 13260 tgtttgtccc gcatgtcaac acggctgcat tcgagcattt ttgtagattg tccatttagg 13320 atctagtcac cgtcctcctt aaagggtgca tgctttcctt ggtacttgag ctcaggacag 13380 tgtctaacaa cagaccccat atggatgggc ctggggttta tggtccagag gaatgccaca 13440 gtattctatg tcaagatatt tcctctgact tctgaggaca ttaggaccag tggccacaga 13500 ctgaagaaaa ccttaatgcc aagcctcctt tcctggccag tgtaggcctg aagtgcctca 13560 acctgacagt tacctgttta ggtatccaca aagagaccag aagggtgttg atggtgatgt 13620 gtaaagttgg ttttgtgctt tgtttacctc tcagctcact ggataggata tgtcatgtta 13680 gcagttgcct tgaaggcagt tcagtttggt ggctgagctg tgacccccag tgggcgggct 13740 tatttggttt tgcagatttt ggctgaagag ctgtgtactc ctccagatcc tggtgctgct 13800 tttgtggtgg tagaatgtcc agatgaaagc ttcattcaac ccatctgtga gaatgccacc 13860 tttcagaggt aatgaggggt ctctagggtg ggagaagtga gagctgaaac ccagcccagc 13920 atcgacatgg gcatcttgtg gcaagagctg tgtttctggg aagaccacta tctgggttta 13980 cagttcagag gccggcactc ctgccttaag tcactgttgg tagttggtgg gctccggtgt 14040 acacagcctc aaagtgaaat tagaaaagat tgaaaactag aaacaactga ggactagaaa 14100 ttcaactaga actcttacag ctcttatacc agaagaaatt ctagaacttt tttgaattct 14160 aactaatgcc ccagattatc atttggatta ttttgaactg aattaatttt cttccattac 14220 ctgcattgaa acaaatgagg tgggtcagag tgtgtgagac tgtcgtggtc aagagtccgt 14280 gttatgggat ggactcacag ctggggaatg tcttttgggc taactgccac tctgttgttg 14340 tcctctatcg aagttaacca gttttgcggt tcagctttca ttccagatgg aatcatcttt 14400 gacccaccta tctgagtttg aatcttttcc cccactctta atggtttacc tgtatttttc 14460 ctgttcctag tttgtatcta tctgtatttt ttcacttgtt tttttctact taccacaaca 14520 aatccttttg ggctgctgta ccccttccga gtcagagcgt taggagttgt ttcatggtct 14580 gctttattct ctgtgggtga atttggatgc gctggtagcc ccggctttgt attttaatcc 14640 agttttgggc agcaaaacct cttcaatgaa tcaggtgtca tttgagagcc atgtgtggat 14700 gtgtgatgat gctgggatag ataaaaatag ctactgtgta tatttctttt taaagggaac 14760 tggagggaaa cacatcagca tgttagtaag tggtctgttg tccaggtggt gaaatttcag 14820 atgattttca tttctcgtgc ctgtgtctca ggtcctctgg aaggcagaca ccagggtggc 14880 attggaggtg caggaggttt attcgaggaa atttgactgt gagagaggaa ggagagaggg 14940 agcaggagga ggcagggaga gcctgggtct ggctttgcag gttggacccg tatgagtgga 15000 gagggtagga aggaagtgca gtgctgagaa aggatcagcc aggcctactg gaaagcccag 15060 agcagagctt gccagataca ggaatcccac gtccattgga aatggcccag caccggggtc 15120 tgccgtgagc agcctgctgt gagagcatgg cctgggcgtg gaggctgtca gctcactgca 15180 gtgctgcaga gggccgcacg atacccctcc ctggctgcgt ggtccctgtc ttggtgtgtc 15240 ctgagtctgc atcactttgt aaagccccac tcttctgccc aggtaccaag gaaaggcaga 15300 tgcccccgtg gccttggtgg ttcacatggc cccagcatct gtgcttgtgg acagcaggta 15360 ccagcagtgg atggagaggt atggagccca gcccagcggc acttggggta actcttctgg 15420 gcagtggtgg attccccttt cctcccctcg tgctctttcc agcgctacct acccttctgc 15480 acctgcctaa actttctgtg ggattcctgc cttcccagaa ttctaggctt cccagatctg 15540 tgctacactc gtgaagaaaa tgcaccgcta ggtggcgcag tgtccacacg attccattta 15600 ttttacaccc tccacactct tcagggtgtc tgaacaaata ctgccgtttg gttgaggatt 15660 ccataagtga attccaaaga agagattgca gctataaaat gatagcttcc atttactgaa 15720 tgcccacttt gtgggaggca gtgtgtgaaa tacccttcat ttcacttcat ttcctctagg 15780 gtcgtcgcca gcagccctgg gaggtagatg tttagtcact ggaaggcatc tttttcctcg 15840 gggcatcgct ggccagggcc aggtggagga gtatgagttg agctcgggtg cggggtgacc 15900 ttgggctgct ttttggcccc tgcccgtatc tccccacatg gcccgtttac ctgcccctca 15960 ctccatggcc tgctctcctg ctgtctcttt cattcctcag ggtttgggtc ccctatttgt 16020 atgccctgga catcttcttt ttcttgtttt tcctctcact cttcccagca cacctgaaag 16080 gcagctgagc tagggaacac cgggctttga gacagcagga gtgggaccat gtttggccat 16140 gtagtaacac tgcttggggc aagtcactga actgtttgaa cacctcatcc tcattaccac 16200 tcctgagctc agcaccactc ctcaggggga gctgcctcct aacagacgct gcaaatgccg 16260 ggtctgtttc ttcacaggtt tgggcctgac acccagcact tggtcctgaa tgagaactgt 16320 gcctcagttc acaaccttcg cagccacaag attcaaaccc agctcaacct catccacccg 16380 gacatcttcc ccctgctcac cagtttccgc tgtaaggtag tgtctcagac yggccccttg 16440 tcggcccagc tctcgtcccc tctctttctc tccatgaatg tgttttgtct ctttcagaag 16500 gagggcccca ccctcagtgt gcccatggtt cagggtgaat gcctcctcaa gtaccagctc 16560 cgtcccagga gggagtggca gaggtctgtg ccatcttgaa ctaatggaat cgtctcagtc 16620 gagttgggaa acatttctgt aaatagccac atagtaaatg ttccaggagg ctctccagac 16680 catatggtct ctgttgtaac tattcaactc cgctttgagc acaaaagaaa cacggacaat 16740 aagctaatga atgggcttgg ctgtgtgcca gcgtgaattt atttagaaaa gcagcctact 16800 ccaggctggg ttgaggtggg cggattgggg ccagtagttc tccttttcca aaattgcctt 16860 gcatgggaat agcagtgata gagctcgtgt gtttcacagt atagaaaata ggaaatgtgt 16920 gatgaacaaa gtcacccata atcctgttgc ccagagataa tgattgataa cattttgtgt 16980 ttcttgattt gtgtatgtgg gtttatattg tcagtctttt cctgtatcac taaacagtct 17040 taagtaacaa gatttttatt ggtattccaa atagggatgt ttactcattt gggatgtttc 17100 caattttttg ttgtttttaa tgaatgaaac aataaatgtc ttatatataa atctttgatg 17160 ggaactctgt tcccttcaag tcattcctaa atgtgggatt actggcccag agtgtgagac 17220 ttgttaaggt acttgataaa tgtaagatgc catcttgaaa gcctcttcca gtacaatcca 17280 accaggaaag tgaacagcct tactgcccca catctttatt ttaattaatt aatttattta 17340 ttttatttat ttatttattt ttttgagacg gagtttcact cttgttgccc aggctggagt 17400 gcaatggcgt gatctcagct cactgcaacc tccgcctccc gggttcaagc atttctcctg 17460 ccccagcctc ccgaatagct ggaattacag gcgcctgcta ccacgcccgg ctaatttttt 17520 gtaattttag cagagacggg tttcaccatg ttggcaggct ggtctcgaac tcctgacctc 17580 aggtgatcca cccacctcag cctcccaaag tgttgggatt acaggcgtga gccgtgcccg 17640 gcctgtttta atttttaagg atctgaacct tgattttaag tttcctgccc actccacagt 17700 atttgtatta gaatagagca tgtgctggat tatgactgga tgctgtgtgc tgttgaggtt 17760 gggtagttgg ggccctttaa gagactatac tagcaagact cgggcccaca ggcaacatca 17820 cggggttgaa gaacctggtg tccctttgtt ggcatctgcg caggctctta acacacagca 17880 gcgatacaca gccctagccg acattcagat ttaccttgtg cttgtgaaaa atattgcaca 17940 gggcctgccc tagacctagt gaattagaat cttgagagtt aggcttggga ctcacaagct 18000 cccagatgat tttaatgctc agcgaggttg aagagccgcc tgtccaagga gttgccactc 18060 cgtgtgatct ggggcttgct aggaaagtgg gatctcaggc ctcactgcag agctgccgaa 18120 ctggcttctg cgttttgcca aggttcctgg gtgtgaacat gagtttcaga gtcactcctc 18180 tagggcccct gcttctcagc tcggaccatt gacccctcag aggacatttg gcaacatctg 18240 gaaacgttct tggttgtcac agcctaggag gtgggtagtg gtgctgctag tgggtagagg 18300 tcaggggtac tgcaccagga cagcagcact ggccacagaa aaaaactgtc ttgccctgag 18360 catcagtagt tccccgttga ctggccctga ggcagagcga tgcagcatcc aaaaggcggt 18420 ggagcagacc tgccccagat cctagtcact taaccttcag tgttgatctg aaggaacttc 18480 ctgcagattg tccccctgaa tttattctgg acatccccaa tggggtctgc tgaggccata 18540 taccctgtcc gtcacctgag atgcttctct ctctycctgc agggatgcca ttattacttg 18600 caatcctgag gaattcatag ttgaggcgct gcagcttccc aacttccagc agagcgtgca 18660 ggagtacagg aggagtgcgc aggacggccc agccccagca ggtgagtggg agcccacaga 18720 gcagcctttc tttcctgggc tctgcccctg ctgctgtttt cctagcatta agtggagtgc 18780 tggtggggcg cattctaacc tggcttttca gtctaatcca gggcttctct actcagctct 18840 acattagaat tatagtcatt ggaggagggg gctttgggga gtttaagaat cccaattcct 18900 ggctgggcgc ggtggctcac acctgtaatc ccagcacttc gggaggccga ggcaggtgga 18960 tcgcgaggtc gggagatcga gaccatcctg gctaacatga tgaaaccccg tctctactaa 19020 aaatacaaaa attagctggg cgtggcggcg ggcgcctgta gtcccagcta ctcgggagac 19080 tgaggcagga gaatggcgag aacccgggag gcggagcttg cagtgagcca aggtcgtgcc 19140 actgcgctcc agcctggacg acagagtgag actccgtctc aaaaaaaaaa atcccaattc 19200 ctgtgcccca tcccacccaa tcagagcatt tggcgatggc acccaggcat tcttggcaag 19260 gcacgcactg agtgaaacgt tttagtgaac acctgtggaa agagctctga gcagggactt 19320 ggctggcaga gatctagtcc tggctttgcg gatgcaaatc catggaggat cttggccacg 19380 tcactcaact gaggctgagg gccgggcaca ggctttggaa ccatcgggtc tccctggatt 19440 tgaatcctga ccctgcctct taccatcttc actggagacc tgggcgtctg agcctgtttc 19500 ccccttggga agcagagcat ttcctacctg gtagggctgg gaggatgcga ccgaagtgca 19560 tggtcttgca gtgagagctg gatgcaaggc acacactgtt ctcttgaaat aaatgacagt 19620 tcccagcata aagaaatgtc attttttaaa tgtaaaagaa ttacagcaat tcttttgaag 19680 aaaggactgg agaatttatt tgttcttctt agccttttgg tgacagatag cctgtgggtc 19740 ccacactggt gcgaagtcct ttgtttcaga gcggttgcca ggggcctgcc agtccccctc 19800 ctgggaagct ggatagaact atgttgctta cccatctgtc ttagtctgtg ttttgttatt 19860 ataaaagaat atgtgagact gggtaattta tcaagaaaag aggtgtattt agttcacggt 19920 tctgcgggct gagaattgaa ggtcacggcc ctagcttcca gtgaaggctt ccatgctgca 19980 tcataacgtg gcagaggagc gcaagtagga agtggacgct tgtgaagacg ggataacctg 20040 agctgcactc tggctttata acaacccccc tctcctggga acaaatccat tcccttgaga 20100 agtaatgcag tctcctgaga gccagtactt actactgcag ctccaagcca ctcaggaggg 20160 tccgtccctg tagcccaaac gccttccact aggccccgcc tcccaaaacc gccatactag 20220 ggagcacgtt tccacatgag gtctggggac aaaccaatga cactcaaacc attgcacctt 20280 ctcatggctg catgctggct cacttttgac ccaaaggaat ggattgtttc acatggattt 20340 tttcacagag aaaagaagtc agtacccaga aatcatcttc cttggaacag ggtctgccat 20400 cccgatgaag attcgaaatg tcagtgccac acttgtcaac ataaggtatg ctgctttccc 20460 aggaagcatc cttccatcaa gggcaygttt actttttaaa caaaagtcct gctgtactca 20520 ccagtcgatt tgaaatgcgg tatcaagccc tgtcacttgt catgtcgact ggagtgtcca 20580 ggagaggagc gtggccttac tgcattttat agcctcagta gcaaacttta ccctgggaat 20640 caccaaaatt catcccatga tgtcttttaa taaacagctg attttactgt gggcagtaca 20700 cctagctaag aaattagctc ctttaatttt tacattaatc ctatgaagtg gtgaataact 20760 acccattttg ttgatgagtg acctgatatt cagagaggtg acttgctatg gttcctacag 20820 ctggtaagtg gggcatctga agtttgagcg gggacttggg gtcttgattg ctacatggta 20880 ttgtccccca gccatttgtt ggtagtatgt taaaaagctt tagggttttg cacatttgtg 20940 ttcagaacct ttattggatt ccccttgaca tgttttttag ttgattctct tgggtttgcc 21000 tggggtcatc agcagagaga ttagtcaaat gcgttgtgac atgtacacgt tatctctaca 21060 gatagtatgt gaagaaaata agattgtgaa ttaccaggtt tgttttaaat tttgctctgc 21120 catcttacat gctagtggtg gatgataaac aaccaaatag tgcattaaat atatacagca 21180 gtgacgagat gtgccctgac atcagaaata tacaatctgg ggtgtgtttc tctgtggatg 21240 aggacatgca ataaagcagc ttggagtgag ccggcctctc ccgggggctg agatcctggg 21300 ggaagaaggg ctttttgagt ttgacctgac accctgcgag cagcttttga accagctgaa 21360 gctaatggga aggtgctatt gccaccttgc ctccgcctcc cgactccttt ttcccccaga 21420 aggtaatgtc ttagcaccgg ggcttctctc tgcaaaatgg gtgcagccct ctcagtgttc 21480 gtggctcctc ccagagaatg aaggaggcca gagcgggtca gcactctctc tgccttggag 21540 cagagcttct gaaatggact gcacagcaga atagcccaag aagtttgtca gaatccagac 21600 ttccagagcc ctgcctaaaa ccaagtcaga aaccccgagt gacacctggg agtctgcgtt 21660 aactggctcc ctgaatgaag cacctgcagc ccgccctgca ccaggtgtct ttgaggacat 21720 gagctgagga aaccccgacc acttgcaaag ggggaaaagt ccgatggcag ctggacctag 21780 aaagagtctc atatggccca gtgcctgtcc tggtattttc aacagaggct gtggccacag 21840 tcaatctgca tggtcagatt cattgttagg actaaatgct ttaagcctcc tataaacttt 21900 tttttttttt ttttgatgcc cagcctttgt gtaagtctac ttgaaagggt ttcagggttc 21960 catggatact tctttgctat aaagaggatg acacatgtaa aatcaccttt atggttaaat 22020 taattggctt ttatattagc tcctcaaagc aaagcaggag agacagaaat ttctgcagtt 22080 gcttcttggt cctgtccaaa gcagacatca gcctctgaac catcagcagt cttcctagtg 22140 gcagtgactc tcttcctctt ctcttctgca gccccgacac gtctctgcta ctggactgtg 22200 gtgagggcac rtttgggcag ctgtgccgtc attacggaga ccaggtggac agggtcctgg 22260 gcaccctggc tgctgtgttt gtgtcccacc tgcacgcaga tcaccacacg gtgagtgttg 22320 ggctggacca caaagctgga gcctggagga ggcactgcca cgttgagttg gccctttggc 22380 tgcgtctttt cctccgcttc caaacttgcc cagagctttt gttactcatc tctggctagg 22440 aaatggtttt ttgcaaaact caacatagtc cttctgcgcc acaagaatgt cttctcttcc 22500 tgttcagttc ctttcctgca gcaggacagg tttgagttta cccagccttc cttgagtctt 22560 gaatctcaca cggcctgctc agcggaagct ttgaccggat gcaggaggtg tggctatgag 22620 accctcacct tggtctcctg gggtgccggg ccctgggccg ttgccctctt cccagcacgg 22680 gtcgtgtcgc tttctgcctg tgacatttca gggccatggc gcagggggct cggcctgtgc 22740 cacccccact gcggctgtgt tagaggctgg tgggtgacgt cgggctggca actcctgcaa 22800 gagagagggc tgcagaccct aacccggagg ggatggccct ggggcctggc tgacgcatgt 22860 ctcctgtttc cttgccaggg cttgccaagt atcttgctgc agagagaacg cgccttggta 22920 agtgtggcac ttgatgggcg ttctgagttt cagcggttta cacatcatcc gccatgcctc 22980 ttggcactcc agtttttatt gagatgttct gtcgtcgagt cggcacttgc attttttgtt 23040 ccaggcatct ttgggaaagc cgcttcaccc tttgctggtg gttgccccca accagctcaa 23100 agcctggctc cagcagtacc acaaccagtg ccaggaggtc ctgcaccaca tcaggtgagc 23160 atccagggca gcctggcccg stgggctgtt gcttgctgcc gtctccttca gaagctcaag 23220 gtggacactg gggtagttac caatatcccc cagcagcctt gcccttgaca tggtcccaga 23280 tggcagaagc aggggagaag tgcattggct gaaggacaga aaccattaga tagttcccat 23340 gtaatgctta ttttcttaga agcatttctt cccagtcctc atttgagttc tgagctgctt 23400 tctaaacttc gagcagcttt tcttgatgag acagttccag agccaagcac ccaaatagtg 23460 gctagcacag agaatgtcca tagcaggtgt gtggctagct ggcaggtggc accatcctca 23520 ccccaagggg aaggagtccc ctctgctgga gccatccgtg gcccgtgctg cctgagccgg 23580 aggcagcatt cacctgctgg gtttctccca gtggcctaga ggctttggtt tggctcttta 23640 tatttgactg ctgtttcctc atcatagtga ctatgattta actcatgttt tctcctaaga 23700 atgattttgg ggttctccag ccaaagactt aaactttggt tccagatgtc caagaaacgt 23760 ttattatcat tttaaatgtt ttgtcttttt acagtatgat tcctgccaaa tgccttcagg 23820 aaggggctga gatctccagt cctgcagtgg aaagattgat cagttcgctg ttgcgaacrt 23880 gtgatttgga agaggtaagg ggcacagccg caggcatcat gggggcgagg tggggagcag 23940 agctgcagag ccctccagcc ccaccctttc agtttcagac ctgtctggtg cggcactgca 24000 agcatgcgtt tggctgtgcg ctggtgcaca cctctggctg gaaagtggtc tattccgggg 24060 acaccatgcc ctgcgaggct ctggtccgga tgggtgagta gaggaagaag caagccaccc 24120 tgaggttgct ctggggtttg tgtagctgga ggtgaatgca ggtgggcttg cagggaaacg 24180 tcagcagagg caggagactc aggtccccac cctcagagtc tctggttgtc atcctagtag 24240 gcagacccag ggccagggga gctgagtgtt gagaccagga aacagcacgt gactgaggcc 24300 tgtgtgccgc tctcgcagag aactctgccc tgatccttgt gctgcttctc cagggaaaga 24360 tgccaccctc ctgatacatg aagccaccct ggaagatggt ttggaagagg aagcagtgga 24420 aaagacacac aggtagcaaa ggccggtcag tccttgtcgc ccacatcctc tccctccccc 24480 actacgtgac actgagcagc cgtcgtttgt ctccactgat gtggggctgc cctgcttcct 24540 atcaagggct atgggggctt ccttgacctg tggcagtgct cacaggctct tggcctttat 24600 ttttgcagaa ttttctaagc aagattctag agtgaggcac agttttttga aagcatctag 24660 aaatcggctg aataaactat aagccatgtc agggaattgc caggggaagg cgggggctgg 24720 gggactgaat ttttggctgc taatttcaac gaaagagtgc attaccccag gtgggccctg 24780 tggtttctct tgggtgccct catggacaga tttggcagcc agcacagagg gtgggcttca 24840 tccaggggtg tgtgcgaagg ctctggccct caggggagat tgtgctggct acggaggtgc 24900 ccgttaagaa aacccaccag cttccccggg tgccctggca gttgatggcc agggtctgtg 24960 ccactgtctg ctttgcagtc ttgcagttga gttcagcttc agtctgctct gtccttcacc 25020 tgcagcacaa cgtcccaagc catcagcgtg gggatgcgga tgaacgcgga gttcattatg 25080 ctgaaccact tcagccagcg ctatgccaag gtccccctct tcagccccaa cttcagcgag 25140 aaagtgggag ttgcctttga ccacatgaag gtctgtatgt cacacggaca gcacagggcg 25200 gggacggggc agggagacag gactctacac actgagtagg acggtcagct ggagtttgct 25260 ttcttatttg gggccaccgt gggaaaaggt tatctaccca tcactaacca ggtcgaacca 25320 ccctgggttt gctggtgaga cccacctcct gcaggggcca actagtcttc agtctcagtt 25380 cactggaaat ttctgagaat ccttttaggc ctggactgct cacacagtca tggcatttga 25440 gcctcagcac agacctgtga gacaggtggt tgcctcttgt gagtgggaaa gccaggcctg 25500 acccttggcc ttccggaatg aaggggcaga gccggagcca ggcctcgttt ttcaggagct 25560 tgattttgag agcatctgga ctgctctccc ttccctctcc ggaggccctt agccaggcct 25620 ggggagcctc tgccccttta gagggttccc tccatgccat tcttttttcc atttcagctg 25680 tggcctgttg gcttgtgcca aggaaggggc gttggcgctg ctgtgtgagc acatgactgc 25740 atcccttcca gctcctgtcc cccacccctg cccctctgag acatgtcctt gtcttctatt 25800 gtgtcttcta ggtctgcttt ggagactttc caacaatgcc caagctgatt cccccactga 25860 aagccctgtt tgctggcgac atcgaggaga tggaggagcg cagggagaag cgggagctgc 25920 ggcaggtgcg ggcggccctc ctgtccaggg agctggcagg cggcctggag gatggggagc 25980 ctcagcagaa gcgggcccac acagaggagc cacaggccaa gaaggtcaga gcccagtgaa 26040 gatctgggag accctgaact cagaaggctg tgtgtcttct gccccacgca cgcacccgta 26100 tctgccctcc ttgctggtag aagctgaaga gcacggtccc ccaggaggca gctcaggata 26160 ggtggtatgg agctgtgccg aggcttgggc tcccacataa gcactagtct atagatgcct 26220 cttaggactg gtgcctggca cagccgcggg ccaggaggct gccacacgga agcaagcaga 26280 tgaactaatt tcatttcaag gcagttttta aagaagtctt ggaaacagac ggcggcacct 26340 ttcctctaat ccagcaaagt gattccctgc acaccagaga caagcagagt aacaggatca 26400 gtgggtctaa gtgtccgaga cttaacgaaa atagtatttc agctgcaata aagattgagt 26460 ttgcaattgt gagttctttt gcttcctcct gctgctgcta cagagcaggg tctgctgtgc 26520 accaccttgg agaaggctct ctgtgctgta gtgtggcagc tgcctggtac ccgggtggct 26580 tggaagaagt cagctcccgt cgtagtgagc acctctggaa cctgtcctca gagagccacc 26640 cttattcgcc aagtcttttt gaca 26664 29 31 DNA Homo sapiens 29 caggaattca gcacatactc attgttcagn n 31 30 21 DNA Homo sapiens 30 caggaattca gcacatactc a 21 31 22 DNA Homo sapiens 31 ttcagcacat actcattgtt ca 22 32 19 DNA Homo sapiens 32 tgaacgcctt ctccacagt 19 33 17 DNA Homo sapiens 33 gtacccgctg ccaccac 17 34 33 DNA Homo sapiens 34 gctaggatcc gccaccatgt gggcgctttg ctc 33 35 29 DNA Homo sapiens 35 gctactcgag tcactgggct ctgaccttc 29 36 17 DNA Homo sapiens 36 gtaaaacgac ggccagt 17 37 19 DNA Homo sapiens 37 ggaaacagct atgaccatg 19 38 17 DNA Homo sapiens 38 tgcgcacgcg agagaag 17 39 17 DNA Homo sapiens 39 cgcttctctc gcgtgcg 17 40 18 DNA Homo sapiens 40 tctaatgttg ggggctta 18 41 18 DNA Homo sapiens 41 taagccccca acattaga 18 42 18 DNA Homo sapiens 42 tgaaaatgag ccacacct 18 43 18 DNA Homo sapiens 43 aggtgtggct cattttca 18 44 18 DNA Homo sapiens 44 cattcaaccc atctgtga 18 45 18 DNA Homo sapiens 45 tcacagatgg gttgaatg 18 46 18 DNA Homo sapiens 46 tgaatgcctc ctcaagta 18 47 18 DNA Homo sapiens 47 tacttgagga ggcattca 18 48 18 DNA Homo sapiens 48 gctactggac tgtggtga 18 49 18 DNA Homo sapiens 49 tcaccacagt ccagtagc 18 50 19 DNA Homo sapiens 50 tggaagagtt tcagacctg 19 51 19 DNA Homo sapiens 51 caggtctgaa actcttcca 19 52 17 DNA Homo sapiens 52 cgcagggacg caccata 17 53 18 DNA Homo sapiens 53 ggttgaactc ggagaaga 18 54 19 DNA Homo sapiens 54 caactggaaa aatacctcg 19 55 17 DNA Homo sapiens 55 gcagagtcca gaaaggc 17 56 19 DNA Homo sapiens 56 agaggaaact tcttggtgc 19 57 18 DNA Homo sapiens 57 accaaggaaa ggcagatg 18 58 18 DNA Homo sapiens 58 gtcaacataa gccccgac 18 59 18 DNA Homo sapiens 59 ggctgctgtg tttgtgtc 18 60 17 DNA Homo sapiens 60 gaaggcattt ggcagga 17 61 18 DNA Homo sapiens 61 tatgattcct gccaaatg 18 62 17 DNA Homo sapiens 62 tccagccaga ggtgtgc 17 63 17 DNA Homo sapiens 63 tgcgaggctc tggtccg 17 64 18 DNA Homo sapiens 64 gggcattgtt ggaaagtc 18 65 17 DNA Homo sapiens 65 tgtttgctgg cgacatc 17 66 31 DNA Homo sapiens 66 caggaattca gcacatactc attgttcagn n 31 67 21 DNA Homo sapiens 67 caggaattca gcacatactc a 21 68 22 DNA Homo sapiens 68 ttcagcacat actcattgtt ca 22 69 19 DNA Homo sapiens 69 cagaacacat ttgggaagc 19 70 18 DNA Homo sapiens 70 gatgttgtcc aagcgagc 18 71 17 DNA Homo sapiens 71 tgacacacag cacctga 17 72 17 DNA Homo sapiens 72 gaagatgtca gggtgga 17 73 18 DNA Homo sapiens 73 caggcatacc actacaga 18 74 20 DNA Homo sapiens 74 tatcaacttc taggcaagtg 20 75 19 DNA Homo sapiens 75 gcaccatgtc gcagggttc 19 76 19 DNA Homo sapiens 76 gaaccctgcg acatggtgc 19 77 19 DNA Homo sapiens 77 tcgcagggtt cggctcgtc 19 78 19 DNA Homo sapiens 78 aaccctgcga catggtgcg 19 79 19 DNA Homo sapiens 79 aaagacccac tgcgacacc 19 80 19 DNA Homo sapiens 80 gcaggtgtcg cagtgggtc 19 81 20 DNA Homo sapiens 81 ccgaacaccg tgtacctgca 20 82 19 DNA Homo sapiens 82 caggtacacg gtgttcggg 19 83 21 DNA Homo sapiens 83 gtcttctcgg aatacaacag g 21 84 20 DNA Homo sapiens 84 ctgttgtatt ccgagaagac 20 85 20 DNA Homo sapiens 85 aaggcgtcca acgacttatg 20 86 20 DNA Homo sapiens 86 agtcgttgga cgccttctcc 20 87 20 DNA Homo sapiens 87 tccgagtcag aaagatgttg 20 88 17 DNA Homo sapiens 88 gccttgtcag cctggtg 17 89 17 DNA Homo sapiens 89 aggaagtgag cagagcg 17 90 33 DNA Homo sapiens 90 gctaaagctt gccaccatgt gggcgctccg ctc 33 91 27 DNA Homo sapiens 91 gctactcgag tcacactcgc gctccta 27 92 16 DNA Homo sapiens 92 gccttctccg cagtta 16 93 21 DNA Homo sapiens 93 ccgcctgaga cgctctagta t 21 94 19 DNA Homo sapiens 94 gctccgaaag tgctgacag 19 95 39 DNA Homo sapiens 95 gttttcccag tcacgacgtt tctattggat gagcagcct 39 96 38 DNA Homo sapiens 96 aggaaacagc tatgaccatg cctgcgatat ggtgcgtc 38 97 37 DNA Homo sapiens 97 gttttcccag tcacgacgct cagttttggt ggagacg 37 98 37 DNA Homo sapiens 98 aggaaacagc tatgaccatg tgccccgatg ctcagag 37 99 22 DNA Homo sapiens 99 aatggtgtca gagagtttac ag 22 100 19 DNA Homo sapiens 100 gctatttggg aggctgagg 19 101 40 DNA Homo sapiens 101 gttttcccag tcacgacgaa tggtgtcaga gagtttacag 40 102 41 DNA Homo sapiens 102 aggaaacagc tatgaccatg aacaaggacc acttttgcta t 41 103 40 DNA Homo sapiens 103 gttttcccag tcacgacgtt tatagcaaaa gtggtccttg 40 104 38 DNA Homo sapiens 104 aggaaacagc tatgaccatg agacttccca ccagcctc 38 105 19 DNA Homo sapiens 105 ccttgctgct tcaccctag 19 106 21 DNA Homo sapiens 106 tgctttatat gtgctgctac g 21 107 39 DNA Homo sapiens 107 gttttcccag tcacgacgca tcttccctgg ttgtacttc 39 108 39 DNA Homo sapiens 108 aggaaacagc tatgaccatc tggagggcag aagactgat 39 109 23 DNA Homo sapiens 109 ctacatttgt tcaaccataa ctg 23 110 23 DNA Homo sapiens 110 gattttgagg tttgatgttg atg 23 111 39 DNA Homo sapiens 111 gttttcccag tcacgacgca tttgttcaac cataactgc 39 112 39 DNA Homo sapiens 112 aggaaacagc tatgaccata tttgagaggt cagggcata 39 113 21 DNA Homo sapiens 113 tcgtgtcaga ttcccaccat a 21 114 21 DNA Homo sapiens 114 aggcataagt cagacatccg t 21 115 40 DNA Homo sapiens 115 gttttcccag tcacgacggt tactcttccc acacatcttc 40 116 41 DNA Homo sapiens 116 aggaaacagc tatgaccatc acagcaagtg ttcagtttct a 41 117 21 DNA Homo sapiens 117 cattcccatg tatgaacgtc t 21 118 22 DNA Homo sapiens 118 atagtaagcc caggaagaag ga 22 119 39 DNA Homo sapiens 119 gttttcccag tcacgacgca ttcccatgta tgaacgtct 39 120 41 DNA Homo sapiens 120 aggaaacagc tatgaccatc tacaagcatt acaaggcaga g 41 121 22 DNA Homo sapiens 121 agtgtcttca gcctttgtat tg 22 122 23 DNA Homo sapiens 122 atctgctatc tcttcttgtc tca 23 123 40 DNA Homo sapiens 123 gttttcccag tcacgacgat cgggtcataa tcagtctgtg 40 124 42 DNA Homo sapiens 124 aggaaacagc tatgaccata tctcttcttg tctcaggtaa ca 42 125 22 DNA Homo sapiens 125 cttctgaaag caataaacgc at 22 126 20 DNA Homo sapiens 126 gatgtccaaa ctgttccacg 20 127 40 DNA Homo sapiens 127 gttttcccag tcacgacgta aaaccaacct tcttcattag 40 128 38 DNA Homo sapiens 128 aggaaacagc tatgaccata gcaatgatgg gagcgatg 38 129 37 DNA Homo sapiens 129 gttttcccag tcacgacggg cttctgggga ctcactg 37 130 41 DNA Homo sapiens 130 aggaaacagc tatgaccatc cttcaaaagt ggtgtctgta g 41 131 22 DNA Homo sapiens 131 gtatccacaa agagaccaga ag 22 132 23 DNA Homo sapiens 132 caccaactac caacagtgac tta 23 133 41 DNA Homo sapiens 133 gttttcccag tcacgacggc tcactggata ggatatgtca t 41 134 39 DNA Homo sapiens 134 aggaaacagc tatgaccatc cagaaacaca gctcttgcc 39 135 20 DNA Homo sapiens 135 gcttgccaga tacaggaatc 20 136 20 DNA Homo sapiens 136 acagaaagtt taggcaggtg 20 137 37 DNA Homo sapiens 137 gttttcccag tcacgacgac gatacccctc cctggct 37 138 39 DNA Homo sapiens 138 aggaaacagc tatgaccata cagaaagttt aggcaggtg 39 139 20 DNA Homo sapiens 139 cctctcactc ttcccagcac 20 140 22 DNA Homo sapiens 140 ggagtaggct gcttttctaa at 22 141 40 DNA Homo sapiens 141 gttttcccag tcacgacgga acacctcatc ctcattacca 40 142 41 DNA Homo sapiens 142 aggaaacagc tatgaccata agagacaaaa cacattcatg g 41 143 39 DNA Homo sapiens 143 gttttcccag tcacgacggt ttccgctgta aggtagtgt 39 144 42 DNA Homo sapiens 144 aggaaacagc tatgaccatc tggaacattt actatgtggc ta 42 145 20 DNA Homo sapiens 145 tgctagtggg tagaggtcag 20 146 21 DNA Homo sapiens 146 actgaaagcc aggttagaat g 21 147 37 DNA Homo sapiens 147 gttttcccag tcacgacgac cctgtccgtc acctgag 37 148 38 DNA Homo sapiens 148 aggaaacagc tatgaccatc ccaccagcac tccactta 38 149 21 DNA Homo sapiens 149 tgtgaagacg ggataacctg a 21 150 19 DNA Homo sapiens 150 gacagggctt gataccgca 19 151 38 DNA Homo sapiens 151 gttttcccag tcacgacgat gctggctcac ttttgacc 38 152 39 DNA Homo sapiens 152 aggaaacagc tatgaccatg actggtgagt acagcagga 39 153 21 DNA Homo sapiens 153 ccagcctttg tgtaagtcta c 21 154 19 DNA Homo sapiens 154 tctgggcaag tttggaagc 19 155 39 DNA Homo sapiens 155 gttttcccag tcacgacgtc caaagcagac atcagcctc 39 156 38 DNA Homo sapiens 156 aggaaacagc tatgaccatg gaggaaaaga cgcagcca 38 157 19 DNA Homo sapiens 157 cgctttctgc ctgtgacat 19 158 20 DNA Homo sapiens 158 ttctgtcctt cagccaatgc 20 159 37 DNA Homo sapiens 159 gttttcccag tcacgacgtt agaggctggt gggtgac 37 160 42 DNA Homo sapiens 160 aggaaacagc tatgaccatc atctcaataa aaactggagt gc 42 161 20 DNA Homo sapiens 161 cacttgatgg gcgttctgag 20 162 20 DNA Homo sapiens 162 ttctgtcctt cagccaatgc 20 163 38 DNA Homo sapiens 163 gttttcccag tcacgacgtt ccagcggttt acacatca 38 164 38 DNA Homo sapiens 164 aggaaacagc tatgaccatt accccagtgt ccaccttg 38 165 20 DNA Homo sapiens 165 gggttctcca gccaaagact 20 166 19 DNA Homo sapiens 166 ctgagtctcc tgcctctgc 19 167 38 DNA Homo sapiens 167 gttttcccag tcacgacggg gttctccagc caaagact 38 168 38 DNA Homo sapiens 168 aggaaacagc tatgaccatg tggggctgga aggctctg 38 169 37 DNA Homo sapiens 169 gttttcccag tcacgacgaa gaggtaaggg gcacagc 37 170 38 DNA Homo sapiens 170 aggaaacagc tatgaccatc tgagtctcct gcctctgc 38 171 20 DNA Homo sapiens 171 gctgagtgtt gagaccagga 20 172 19 DNA Homo sapiens 172 agacaaacga cggctgctc 19 173 38 DNA Homo sapiens 173 gttttcccag tcacgacgtt gagaccagga aacagcac 38 174 38 DNA Homo sapiens 174 aggaaacagc tatgaccatg agaggatgtg ggcgacaa 38 175 19 DNA Homo sapiens 175 gggagatggt gctggctac 19 176 21 DNA Homo sapiens 176 cctggttagt gatgggtaga t 21 177 37 DNA Homo sapiens 177 gttttcccag tcacgacgca gggtctgtgc cactgtc 37 178 40 DNA Homo sapiens 178 aggaaacagc tatgaccatc tcagtgtgta gagtcctctc 40 179 22 DNA Homo sapiens 179 ttgattttga gagcatctgg ac 22 180 21 DNA Homo sapiens 180 ctcggacact tagacccact g 21 181 37 DNA Homo sapiens 181 gttttcccag tcacgacgtg catcccttcc agctcct 37 182 41 DNA Homo sapiens 182 aggaaacagc tatgaccatg acacacagcc ttctgagttc a 41 183 37 DNA Homo sapiens 183 gttttcccag tcacgacgcc acacagagga gccacag 37 184 42 DNA Homo sapiens 184 aggaaacagc tatgaccata ccagtcctaa gaggcatcta ta 42 185 19 DNA Homo sapiens 185 ccacacagag gagccacag 19 186 20 DNA Homo sapiens 186 ccagaggtgc tcactacgac 20 187 39 DNA Homo sapiens 187 gttttcccag tcacgacgag gtcagagccc agtgaagat 39 188 39 DNA Homo sapiens 188 aggaaacagc tatgaccatc atctgcttgc ttccgtgtg 39 189 39 DNA Homo sapiens 189 gttttcccag tcacgacgtc aggataggtg gtatggagc 39 190 40 DNA Homo sapiens 190 aggaaacagc tatgaccatc ggacacttag acccactgat 40 191 23 DNA Homo sapiens 191 agactccgag tygaatgaaa atg 23 192 23 DNA Homo sapiens 192 ggtgagggca crtttgggca gct 23 193 23 DNA Homo sapiens 193 gcaccctggc trctgtgttt gtg 23 194 23 DNA Homo sapiens 194 gtgtcccacc tgcacgcaga tca 23 195 24 DNA Homo sapiens 195 gtgtcccacc tggcacgcag atca 24 196 23 DNA Homo sapiens 196 aagccgcttc aycctttgct ggt 23 197 24 DNA Homo sapiens 197 gctgttgcga acrtgtgatt tgga 24 198 22 DNA Homo sapiens 198 gaggcttggg stcccacata ag 22 199 23 DNA Homo sapiens 199 cctggcacag cygcgggcca gga 23 200 23 DNA Homo sapiens 200 aatccagcaa artgattccc tgc 23 201 21 DNA Homo sapiens 201 taaatgtttt ytcattctta g 21 202 23 DNA Homo sapiens 202 ttgctgttgt gyggttttct tgt 23 203 24 DNA Homo sapiens 203 ggttttcttg attcagcagt taca 24 204 27 DNA Homo sapiens 204 ggttttcttg atgattcagc agttaca 27 205 23 DNA Homo sapiens 205 gtgtctcaga cyggcccctt gtc 23 206 23 DNA Homo sapiens 206 tgccatcttg awctaatgga atc 23 207 23 DNA Homo sapiens 207 cttctctctc tycctgcagg gat 23 208 23 DNA Homo sapiens 208 catcaagggc aygtttactt ttt 23 209 23 DNA Homo sapiens 209 cagccttgcc csctgggctg ttg 23 210 350 DNA Homo sapiens CDS (51)..(293) 210 cgcgggcgta ggtgaccggc ggctttctca gttttggtgg agacgggcgc atg tgg 56 Met Trp 1 gcg ctt tgc tcg ctg ctg cgg tcc gcg gcc gga cgc acc atg tcg cag 104 Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met Ser Gln 5 10 15 gga cgc acc ata tcg cag gca ccc gcc cgc cgc gag cgg ccg cgc aag 152 Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro Arg Lys 20 25 30 gac ccg ctg cgg cac ctg cgc acg cga gag aag cgc gga ccg tcg ggg 200 Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro Ser Gly 35 40 45 50 tgc tcc ggc ggc cca aac acc gtg tac ctg cag gtg gtg gca gcg ggt 248 Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala Ala Gly 55 60 65 agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc gag ttc aac 293 Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe Asn 70 75 80 cggtcagtca acgagccacg ccccgtcccg ctgggccctc agtgcggcgc agcctct 350 211 81 PRT Homo sapiens 211 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 Asn 212 326 DNA Mus musculus CDS (51)..(269) 212 tggcggcgtg aggggtctgg ctgccttgtc agcctggtgt ggtcgggtgc atg tgg 56 Met Trp 1 gcg ctc cgc tca ctg ttg cgt ccc ctt ggc ctg cgc acc atg tcg cag 104 Ala Leu Arg Ser Leu Leu Arg Pro Leu Gly Leu Arg Thr Met Ser Gln 5 10 15 ggt tcg gct cgt cgg ccg cgg cca ccc aag gac cca ctg cga cac ctg 152 Gly Ser Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg His Leu 20 25 30 cgt acg cgg gag aag cgc ggc ccg ggt ccc ggg ggc ccg aac acc gtg 200 Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro Asn Thr Val 35 40 45 50 tac ctg cag gtg gtg gcg gcg ggc ggc cgg gac gcg ggg gct gct ctc 248 Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp Ala Gly Ala Ala Leu 55 60 65 tat gtc ttc tcg gaa tac aac aggtcagagt gggccgacag ccctggggga 299 Tyr Val Phe Ser Glu Tyr Asn 70 ttggccccag cgccacgtgc tcgggag 326 213 73 PRT Mus musculus 213 Met Trp Ala Leu Arg Ser Leu Leu Arg Pro Leu Gly Leu Arg Thr Met 1 5 10 15 Ser Gln Gly Ser Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg 20 25 30 His Leu Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro Asn 35 40 45 Thr Val Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp Ala Gly Ala 50 55 60 Ala Leu Tyr Val Phe Ser Glu Tyr Asn 65 70 214 13 PRT Artificial Sequence Description of Artificial SequenceHistidine containing motif. 214 Xaa Xaa Xaa Xaa His Xaa His Xaa Asp His Xaa Xaa Gly 1 5 10 215 127 DNA Homo sapiens misc_feature (1)..(127) Exon 1. 215 tttaatacga ctcactatag ggaatttggc cctcgagnng aattcggcac gagggtagcc 60 ccgcgacagc tgggccgagg gtgcgggcct gcgctccctc ggctcctggc gcgggctcgg 120 ggagagg 127 216 983 DNA Homo sapiens intron (1)..(300) Upstream intron of exon 2. 216 gtctccatag ttttgccttt ttgagaacat catatagtta gaattcagct atagttttta 60 attgcctggg tttggttatt tttgtttgtt tgggtgtgtg aacaattata caagatttgt 120 taacttgtag ttttagccaa gttattaaaa ccttactgtg gatatgtgtg gaatactatg 180 agagaccaag aatccagact gttctaaata accaaaaagt aataatagag ataaatatta 240 caggaatatg tttttggtcc agtgatatga aataatcccc agatgatctt tctgttgcag 300 ggtggaagat gtctatggat gtgacattcc tggggacggg tgcagcatac ccatctccaa 360 cccggggtgc ctctgctgtg gtccttcggt gtgaaggcga gtgctggctc tttgactgtg 420 gggagggaac acagacacag cttatgaaaa gccaacttaa agcaggttag tgtgccttca 480 gctatctcat taagaatttt ttgttgttct gcttcatttt cttggctctc cttggacatt 540 ttgtttagaa acagccctga tggttgcatc ccacttcagt gctacaccct ggtgagactt 600 ggaaggcctg caggcatctg gccacgtcca ctgaacttca tttacttatt tacttgcttt 660 tcatttatcc tgtagatgct gaaagcaagg attcatgtag gcttggggtt tgggaaatgt 720 cgtgggatac accaggcata ttagatgaac actgccttag caaggaagca gtgtacatac 780 ttacctccac caggagatag ttttcatgag aggatgcaaa gggtaggaaa tgtttggagg 840 aggagatgtt gttttcctct tggggttatc aggtaaactt ctcagagaag ttgacctgtg 900 gattgtcaaa gagagagatt tcaggctgag agaagaaggc atttcatcag gggatggagt 960 gagcagagcc acacctggga gat 983 217 1287 DNA Homo sapiens intron (1)..(300) Intron upstream of exon 3. 217 gtgagctatg atcacaccac tgcactccag cctggatgac agagcaagac ctgtctcttt 60 aaaaaaaaaa aaactattaa aaacaaacaa acaaaaaacc acctggtgaa ataaagcctg 120 tcttcttgtt tttggaatca tgtagcaaaa tgtaaatgaa taagtttatg atgataagta 180 gaacttttaa attcaattta ctatttttaa tgtaaattgt taggcttgtt tcaaatagct 240 ttgtatgggt ttttagttaa tgaaaaattt ccaaacgtat ttctctatct caatcaaaag 300 ggagaattac caagatcttc atcacacacc ttcatggaga ccatttcttt ggccttcctg 360 ggctcctctg cacaatcagc ctgcagagtg gctccatggt gtccaaacag cctattgaaa 420 tctatggccc tgtagggctt cgggacttta tctggcgaac catggaactc tctcacacgg 480 agctggtctt ccattatgtg gttcatgaac tggttcctac agcagatcaa tgtcctgcag 540 aagaactaaa agaatttgcg catgtgaata gagcagacag tcctcccaaa gaggaacaag 600 gaagaactat cctgttagac tcagaagaaa actcatacct tctgtttgat gatgaacaat 660 ttgttgtaaa agcatttcgc ctctttcaca gaattccctc atttgggttt tcagtcgtgg 720 aaaagaaacg cccaggtaaa ctcaatgcac agaaacttaa agaccttggt aagtgttttt 780 ttgttttttg ttttttcccg ccttctcatc aatagggctc ctgttgactg aagctataag 840 aaatgtcata gtaaggccag gagttgtggc tcacgcctgt aatcctagca ctttgggagg 900 ccgaggtggg aggatcactt gagttcggga gttcaagacc agcctgggca acatggcgaa 960 accccatctc tactaaaaat acaaaaagta actgggtgtg gtgtcatgtg cctgtagtcc 1020 cagctacttg gggggctgag gcaggaggat cacttgaacc tgggaggtca aggctgcagt 1080 aagccaagat agtgttacta tactccagct tgggtgacaa agcgaaactc tgtctcaaaa 1140 aaaaaaaagt gtcatagtaa gcttccactc ctctatccca ggcctgaaac tgacaatttc 1200 tcacttagtc ctttgtccaa agttgcttat taagaaatcc atggggccaa aaaaatgcta 1260 tttagagcaa acccagtata catttga 1287 218 1378 DNA Homo sapiens intron (1)..(300) Intron upstream of exon 4. 218 tgtgcacagc agagaatcaa gaatgttaca gtgactacaa taaggtccta gtgatactta 60 ggagactaaa acttgtctga catgtatgca tgggaaatgt ttcaagtact aaggcattgc 120 taatatcaat caacactgaa attttaaaaa tgtataaatc cagttttcca caagtagtaa 180 aacatttata acaattatgg atgccttttc cattagctat ttgcaatgct gttaaaatag 240 actcttgaaa agtcataaat tccattccta tgatgtaatg ttatctgcct tcatcattag 300 gtgttccacc aggtcctgcc tatgggaagc tgaaaaatgg aatttctgtt gttctggaaa 360 atggggttac aatttctccc caagatgtct taaaaaagcc tattgttgga agaaaaatct 420 gcatattggg tgactgctct ggggttgtgg gtgatggagg agtaaaactg tgctttgaag 480 cagacctgtt gatccacgaa gcaaccctgg atgatgccca gatggacaaa gcaaaggagc 540 atggccacag cacaccacag atggcagcaa catttgcaaa gttgtgccgt gcaaagaggc 600 tggttctgac tcacttcagt cagaggtaca aaccagttgc cttggccaga gaaggagaaa 660 cagatggcat tgcagaacta aaaaagcaag ctgaatcagt gttagatctc caagaagtga 720 ctctagcaga agattttatg gtgataagca ttccaatcaa gaaatgaaac cagtgttcct 780 gagtgcacac tgacatgtct gtgaatatgt tactgaacct atagtccagt ttttttattt 840 cttgttttag tctgaaatta tttgggccct aataatccta aaaagaatgg agctgcattg 900 atgaattggc tcagtattta aagggagcaa actttttgat aataaatctt tttaagagaa 960 aaaaaaccca gcatcctttt tgaagtccag atttgtcaaa atgatagact attcagttat 1020 acatcttatt ttgtgctact accacagata gccaatattc catgcagtcc tgggcttagc 1080 ttctgcccag ctttattgct gctattggca aagagcacag gactcagccc tcgtggctaa 1140 aaatggtatt ttggcagttt gtattgaatc tgtttgtgtt attaacagaa gagggagaaa 1200 tgtcatgaga cgttggacag gcaggattga tgatagcatg accatagctt tgctggaata 1260 ctgaatgcag ggtttggcta ggtgtttatt ttaacatttt attaaacttt ctatttgggt 1320 cttaacccat ggttctcaac tggggtgaca ctgctcctct agaacaggtt gaaatatg 1378 219 1462 DNA Homo sapiens CDS (136)..(1224) 219 tttaatacga ctcactatag ggaatttggc cctcgagnng aattcggcac gagggtagcc 60 ccgcgacagc tgggccgagg gtgcgggcct gcgctccctc ggctcctggc gcgggctcgg 120 ggagaggggt ggaag atg tct atg gat gtg aca ttc ctg ggg acg ggt gca 171 Met Ser Met Asp Val Thr Phe Leu Gly Thr Gly Ala 1 5 10 gca tac cca tct cca acc cgg ggt gcc tct gct gtg gtc ctt cgg tgt 219 Ala Tyr Pro Ser Pro Thr Arg Gly Ala Ser Ala Val Val Leu Arg Cys 15 20 25 gaa ggc gag tgc tgg ctc ttt gac tgt ggg gag gga aca cag aca cag 267 Glu Gly Glu Cys Trp Leu Phe Asp Cys Gly Glu Gly Thr Gln Thr Gln 30 35 40 ctt atg aaa agc caa ctt aaa gca ggg aga att acc aag atc ttc atc 315 Leu Met Lys Ser Gln Leu Lys Ala Gly Arg Ile Thr Lys Ile Phe Ile 45 50 55 60 aca cac ctt cat gga gac cat ttc ttt ggc ctt cct ggg ctc ctc tgc 363 Thr His Leu His Gly Asp His Phe Phe Gly Leu Pro Gly Leu Leu Cys 65 70 75 aca atc agc ctg cag agt ggc tcc atg gtg tcc aaa cag cct att gaa 411 Thr Ile Ser Leu Gln Ser Gly Ser Met Val Ser Lys Gln Pro Ile Glu 80 85 90 atc tat ggc cct gta ggg ctt cgg gac ttt atc tgg cga acc atg gaa 459 Ile Tyr Gly Pro Val Gly Leu Arg Asp Phe Ile Trp Arg Thr Met Glu 95 100 105 ctc tct cac acg gag ctg gtc ttc cat tat gtg gtt cat gaa ctg gtt 507 Leu Ser His Thr Glu Leu Val Phe His Tyr Val Val His Glu Leu Val 110 115 120 cct aca gca gat caa tgt cct gca gaa gaa cta aaa gaa ttt gcg cat 555 Pro Thr Ala Asp Gln Cys Pro Ala Glu Glu Leu Lys Glu Phe Ala His 125 130 135 140 gtg aat aga gca gac agt cct ccc aaa gag gaa caa gga aga act atc 603 Val Asn Arg Ala Asp Ser Pro Pro Lys Glu Glu Gln Gly Arg Thr Ile 145 150 155 ctg tta gac tca gaa gaa aac tca tac ctt ctg ttt gat gat gaa caa 651 Leu Leu Asp Ser Glu Glu Asn Ser Tyr Leu Leu Phe Asp Asp Glu Gln 160 165 170 ttt gtt gta aaa gca ttt cgc ctc ttt cac aga att ccc tca ttt ggg 699 Phe Val Val Lys Ala Phe Arg Leu Phe His Arg Ile Pro Ser Phe Gly 175 180 185 ttt tca gtc gtg gaa aag aaa cgc cca ggt aaa ctc aat gca cag aaa 747 Phe Ser Val Val Glu Lys Lys Arg Pro Gly Lys Leu Asn Ala Gln Lys 190 195 200 ctt aaa gac ctt ggt gtt cca cca ggt cct gcc tat ggg aag ctg aaa 795 Leu Lys Asp Leu Gly Val Pro Pro Gly Pro Ala Tyr Gly Lys Leu Lys 205 210 215 220 aat gga att tct gtt gtt ctg gaa aat ggg gtt aca att tct ccc caa 843 Asn Gly Ile Ser Val Val Leu Glu Asn Gly Val Thr Ile Ser Pro Gln 225 230 235 gat gtc tta aaa aag cct att gtt gga aga aaa atc tgc ata ttg ggt 891 Asp Val Leu Lys Lys Pro Ile Val Gly Arg Lys Ile Cys Ile Leu Gly 240 245 250 gac tgc tct ggg gtt gtg ggt gat gga gga gta aaa ctg tgc ttt gaa 939 Asp Cys Ser Gly Val Val Gly Asp Gly Gly Val Lys Leu Cys Phe Glu 255 260 265 gca gac ctg ttg atc cac gaa gca acc ctg gat gat gcc cag atg gac 987 Ala Asp Leu Leu Ile His Glu Ala Thr Leu Asp Asp Ala Gln Met Asp 270 275 280 aaa gca aag gag cat ggc cac agc aca cca cag atg gca gca aca ttt 1035 Lys Ala Lys Glu His Gly His Ser Thr Pro Gln Met Ala Ala Thr Phe 285 290 295 300 gca aag ttg tgc cgt gca aag agg ctg gtt ctg act cac ttc agt cag 1083 Ala Lys Leu Cys Arg Ala Lys Arg Leu Val Leu Thr His Phe Ser Gln 305 310 315 agg tac aaa cca gtt gcc ttg gcc aga gaa gga gaa aca gat ggc att 1131 Arg Tyr Lys Pro Val Ala Leu Ala Arg Glu Gly Glu Thr Asp Gly Ile 320 325 330 gca gaa cta aaa aag caa gct gaa tca gtg tta gat ctc caa gaa gtg 1179 Ala Glu Leu Lys Lys Gln Ala Glu Ser Val Leu Asp Leu Gln Glu Val 335 340 345 act cta gca gaa gat ttt atg gtg ata agc att cca atc aag aaa 1224 Thr Leu Ala Glu Asp Phe Met Val Ile Ser Ile Pro Ile Lys Lys 350 355 360 tgaaaccagt gttcctgagt gcacactgac atgtctgtga atatgttact gaacctatag 1284 tccagttttt ttatttcttg ttttagtctg aaattatttg ggccctaata atcctaaaaa 1344 gaatggagct gcattgatga attggctcag tatttaaagg gagcaaactt tttgataata 1404 aatcttttta agagaaaaaa aaaaaaaaga aaaaagatct ataattaagc aggggcat 1462 220 363 PRT Homo sapiens 220 Met Ser Met Asp Val Thr Phe Leu Gly Thr Gly Ala Ala Tyr Pro Ser 1 5 10 15 Pro Thr Arg Gly Ala Ser Ala Val Val Leu Arg Cys Glu Gly Glu Cys 20 25 30 Trp Leu Phe Asp Cys Gly Glu Gly Thr Gln Thr Gln Leu Met Lys Ser 35 40 45 Gln Leu Lys Ala Gly Arg Ile Thr Lys Ile Phe Ile Thr His Leu His 50 55 60 Gly Asp His Phe Phe Gly Leu Pro Gly Leu Leu Cys Thr Ile Ser Leu 65 70 75 80 Gln Ser Gly Ser Met Val Ser Lys Gln Pro Ile Glu Ile Tyr Gly Pro 85 90 95 Val Gly Leu Arg Asp Phe Ile Trp Arg Thr Met Glu Leu Ser His Thr 100 105 110 Glu Leu Val Phe His Tyr Val Val His Glu Leu Val Pro Thr Ala Asp 115 120 125 Gln Cys Pro Ala Glu Glu Leu Lys Glu Phe Ala His Val Asn Arg Ala 130 135 140 Asp Ser Pro Pro Lys Glu Glu Gln Gly Arg Thr Ile Leu Leu Asp Ser 145 150 155 160 Glu Glu Asn Ser Tyr Leu Leu Phe Asp Asp Glu Gln Phe Val Val Lys 165 170 175 Ala Phe Arg Leu Phe His Arg Ile Pro Ser Phe Gly Phe Ser Val Val 180 185 190 Glu Lys Lys Arg Pro Gly Lys Leu Asn Ala Gln Lys Leu Lys Asp Leu 195 200 205 Gly Val Pro Pro Gly Pro Ala Tyr Gly Lys Leu Lys Asn Gly Ile Ser 210 215 220 Val Val Leu Glu Asn Gly Val Thr Ile Ser Pro Gln Asp Val Leu Lys 225 230 235 240 Lys Pro Ile Val Gly Arg Lys Ile Cys Ile Leu Gly Asp Cys Ser Gly 245 250 255 Val Val Gly Asp Gly Gly Val Lys Leu Cys Phe Glu Ala Asp Leu Leu 260 265 270 Ile His Glu Ala Thr Leu Asp Asp Ala Gln Met Asp Lys Ala Lys Glu 275 280 285 His Gly His Ser Thr Pro Gln Met Ala Ala Thr Phe Ala Lys Leu Cys 290 295 300 Arg Ala Lys Arg Leu Val Leu Thr His Phe Ser Gln Arg Tyr Lys Pro 305 310 315 320 Val Ala Leu Ala Arg Glu Gly Glu Thr Asp Gly Ile Ala Glu Leu Lys 325 330 335 Lys Gln Ala Glu Ser Val Leu Asp Leu Gln Glu Val Thr Leu Ala Glu 340 345 350 Asp Phe Met Val Ile Ser Ile Pro Ile Lys Lys 355 360 221 2470 DNA Mus musculus CDS (1)..(2466) 221 atg tgg gcg ctc cgc tca ctg ttg cgt ccc ctt ggc ctg cgc acc atg 48 Met Trp Ala Leu Arg Ser Leu Leu Arg Pro Leu Gly Leu Arg Thr Met 1 5 10 15 tcg cag ggt tcg gct cgt cgg ccg cgg cca ccc aaa gac cca ctg cga 96 Ser Gln Gly Ser Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg 20 25 30 cac ctg cgt acg cgg gag aag cgc ggc ccg ggt ccc ggg ggc ccg aac 144 His Leu Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro Asn 35 40 45 acc gtg tac ctg cag gtg gtg gcg gcg ggc ggc cgg gac gcg ggg gct 192 Thr Val Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp Ala Gly Ala 50 55 60 gct ctc tat gtc ttc tcg gaa tac aac agg tac ctt ttt aac tgc gga 240 Ala Leu Tyr Val Phe Ser Glu Tyr Asn Arg Tyr Leu Phe Asn Cys Gly 65 70 75 80 gaa ggc gtc caa cga ctt atg cag gaa cac aag act gaa agt cgc tcg 288 Glu Gly Val Gln Arg Leu Met Gln Glu His Lys Thr Glu Ser Arg Ser 85 90 95 ctt gac aac atc ttt ctg act cgg atg cat tgg tca aat gtt ggg ggg 336 Leu Asp Asn Ile Phe Leu Thr Arg Met His Trp Ser Asn Val Gly Gly 100 105 110 ttg tgt gga atg att tta act tta aag gaa acc ggg ctt ccc aaa tgt 384 Leu Cys Gly Met Ile Leu Thr Leu Lys Glu Thr Gly Leu Pro Lys Cys 115 120 125 gtt ctg tct gga cca cca cag ctg gag aaa tat cta gaa gca atc aaa 432 Val Leu Ser Gly Pro Pro Gln Leu Glu Lys Tyr Leu Glu Ala Ile Lys 130 135 140 ata ttt tct ggt cca ttg aaa gga ata gaa ctg gcc gtg cgg cct cac 480 Ile Phe Ser Gly Pro Leu Lys Gly Ile Glu Leu Ala Val Arg Pro His 145 150 155 160 tct gca cca gaa tac aag gat gag acc atg act gtt tac cag gtc cct 528 Ser Ala Pro Glu Tyr Lys Asp Glu Thr Met Thr Val Tyr Gln Val Pro 165 170 175 atc cac agt gaa cgg agg tgt gga aag caa cag cca tcc cag agc ccc 576 Ile His Ser Glu Arg Arg Cys Gly Lys Gln Gln Pro Ser Gln Ser Pro 180 185 190 aga aca tct ccc aac agg ctc agt ccc aaa cag tca tcg gac tct gga 624 Arg Thr Ser Pro Asn Arg Leu Ser Pro Lys Gln Ser Ser Asp Ser Gly 195 200 205 tca gct gaa aat ggg cag tgc caa cag gaa agc atg ggg cag gga ccc 672 Ser Ala Glu Asn Gly Gln Cys Gln Gln Glu Ser Met Gly Gln Gly Pro 210 215 220 tcc tta gtg gta gct ttt gtc tgc aag ctt cac ttg agg aaa gga aac 720 Ser Leu Val Val Ala Phe Val Cys Lys Leu His Leu Arg Lys Gly Asn 225 230 235 240 ttc ttg gtg ctt aaa gca aag gag ctg ggc ctt cct gtt ggg acg gcc 768 Phe Leu Val Leu Lys Ala Lys Glu Leu Gly Leu Pro Val Gly Thr Ala 245 250 255 gcc att gca ccc atc att gct gct gtc aag gac ggg aag agt atc act 816 Ala Ile Ala Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr 260 265 270 tac gaa gga aga gag att gct gct gaa gag ctt tgt aca ccc cca gat 864 Tyr Glu Gly Arg Glu Ile Ala Ala Glu Glu Leu Cys Thr Pro Pro Asp 275 280 285 cct ggt ctt gta ttc atc gtg gta gag tgt cct gat gaa gga ttc atc 912 Pro Gly Leu Val Phe Ile Val Val Glu Cys Pro Asp Glu Gly Phe Ile 290 295 300 ctg ccc atc tgt gag aac gac acc ttt aaa agg tac cag gca gag gct 960 Leu Pro Ile Cys Glu Asn Asp Thr Phe Lys Arg Tyr Gln Ala Glu Ala 305 310 315 320 gat gca cct gtg gcg ctg gtg gtc cac ata gcc cca gaa tct gta ctc 1008 Asp Ala Pro Val Ala Leu Val Val His Ile Ala Pro Glu Ser Val Leu 325 330 335 atc gac agc aga tac cag cag tgg atg gag agg ttc ggg cct gac aca 1056 Ile Asp Ser Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr 340 345 350 cag cac ctg att ctg aat gag aat tgc ccc tcg gtc cac aac ctg cgc 1104 Gln His Leu Ile Leu Asn Glu Asn Cys Pro Ser Val His Asn Leu Arg 355 360 365 agc cac aag att cag acc cag ctc agc ctc atc cac cct gac atc ttc 1152 Ser His Lys Ile Gln Thr Gln Leu Ser Leu Ile His Pro Asp Ile Phe 370 375 380 ccc cag ctt acc agc ttc tat agt aag gag gaa ggg tcc acc ctc agc 1200 Pro Gln Leu Thr Ser Phe Tyr Ser Lys Glu Glu Gly Ser Thr Leu Ser 385 390 395 400 gtg cca aca gtt cgg ggt gaa tgc ctc ctc aag tat tca gtc cgc ccc 1248 Val Pro Thr Val Arg Gly Glu Cys Leu Leu Lys Tyr Ser Val Arg Pro 405 410 415 aag aga gag tgg cag agg gat acc aca ctc gac tgc aat act gat gaa 1296 Lys Arg Glu Trp Gln Arg Asp Thr Thr Leu Asp Cys Asn Thr Asp Glu 420 425 430 ttc ata gct gag gcc ttg gag ctc ccc agt ttc cag gag agt gtg gag 1344 Phe Ile Ala Glu Ala Leu Glu Leu Pro Ser Phe Gln Glu Ser Val Glu 435 440 445 gag tat cgg aag aac gtg cag gaa aac cca gcc cca gca gag aaa aga 1392 Glu Tyr Arg Lys Asn Val Gln Glu Asn Pro Ala Pro Ala Glu Lys Arg 450 455 460 agc cag tat cct gaa att gtc ttc ctg ggt acg ggg tct gcc atc cca 1440 Ser Gln Tyr Pro Glu Ile Val Phe Leu Gly Thr Gly Ser Ala Ile Pro 465 470 475 480 atg gag atc cga aat gtc agt tcc aca ctc gtc aac cta agc cct gac 1488 Met Glu Ile Arg Asn Val Ser Ser Thr Leu Val Asn Leu Ser Pro Asp 485 490 495 aag tca gtg ctc ctg gat tgt gga gaa ggc act ttt ggg cag ttg tgc 1536 Lys Ser Val Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys 500 505 510 cgt cat tac gga cag caa ata gac cga gtc tta tgc agc ctc acg gct 1584 Arg His Tyr Gly Gln Gln Ile Asp Arg Val Leu Cys Ser Leu Thr Ala 515 520 525 gtg ttt gtg tcc cac ctg cac gcc gac cac cac acg ggc ttg ctg aat 1632 Val Phe Val Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn 530 535 540 atc ttg ctg cag aga gag cat gcg ttg gca tct ctg ggg aaa ccc ttc 1680 Ile Leu Leu Gln Arg Glu His Ala Leu Ala Ser Leu Gly Lys Pro Phe 545 550 555 560 cag ccc ttg ctt gtg gtg gct cct acc cag ctc agg gcc tgg ctg cag 1728 Gln Pro Leu Leu Val Val Ala Pro Thr Gln Leu Arg Ala Trp Leu Gln 565 570 575 cag tat cac aac cac tgc cag gag att ctg cac cac gtc agt atg att 1776 Gln Tyr His Asn His Cys Gln Glu Ile Leu His His Val Ser Met Ile 580 585 590 cct gcc aaa tgc ctt cag aaa ggg gca gag gtc tcc aat act aca ttg 1824 Pro Ala Lys Cys Leu Gln Lys Gly Ala Glu Val Ser Asn Thr Thr Leu 595 600 605 gaa agg ctg ata agc ttg ctg ttg gaa aca tgt gac tta gaa gaa ttt 1872 Glu Arg Leu Ile Ser Leu Leu Leu Glu Thr Cys Asp Leu Glu Glu Phe 610 615 620 cag acc tgc ctg gta cgg cac tgc aag cat gct ttt ggc tgt gca ctg 1920 Gln Thr Cys Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu 625 630 635 640 gta cat tca tct ggc tgg aaa gtc gtc tac tcg ggg gat acc atg ccc 1968 Val His Ser Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro 645 650 655 tgt gag gct ctg gtc cag atg ggg aaa gat gcc acc ctc ctg ata cat 2016 Cys Glu Ala Leu Val Gln Met Gly Lys Asp Ala Thr Leu Leu Ile His 660 665 670 gaa gcc act ctg gag gat cnc ttg gaa gag gaa gca gta gag agg aca 2064 Glu Ala Thr Leu Glu Asp Xaa Leu Glu Glu Glu Ala Val Glu Arg Thr 675 680 685 cac agc acc acc tcc cag gct att aat gtg ggg atg cgg atg aat gcg 2112 His Ser Thr Thr Ser Gln Ala Ile Asn Val Gly Met Arg Met Asn Ala 690 695 700 gag ttc atc atg ctg aac cac ttc agt cag cgg tac gcn aag atc ccc 2160 Glu Phe Ile Met Leu Asn His Phe Ser Gln Arg Tyr Xaa Lys Ile Pro 705 710 715 720 ctt ttc agc cct gac ttc aac gag aaa gtt ggc atc gcc ttt gac cac 2208 Leu Phe Ser Pro Asp Phe Asn Glu Lys Val Gly Ile Ala Phe Asp His 725 730 735 atg aag gtc tgn ttt gga gac ttc ccg aca gtg ccc aag ctg att ccc 2256 Met Lys Val Xaa Phe Gly Asp Phe Pro Thr Val Pro Lys Leu Ile Pro 740 745 750 cca ctg aag gcc ctg ttt gca ggt gac att gaa gag atg gtg gaa cgc 2304 Pro Leu Lys Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Val Glu Arg 755 760 765 agg gag aag agg gag cta cgg ctg gtg cga gca gcc ctc ctg acc cag 2352 Arg Glu Lys Arg Glu Leu Arg Leu Val Arg Ala Ala Leu Leu Thr Gln 770 775 780 cag gca gac agc cca gag gac aga gaa ccc caa cag aag cgg gcc cac 2400 Gln Ala Asp Ser Pro Glu Asp Arg Glu Pro Gln Gln Lys Arg Ala His 785 790 795 800 aca gat gaa cca cac agc cca cag agc aag aag gag agc gtg gca aac 2448 Thr Asp Glu Pro His Ser Pro Gln Ser Lys Lys Glu Ser Val Ala Asn 805 810 815 act tta gga gcg cga gtg tgag 2470 Thr Leu Gly Ala Arg Val 820 222 822 PRT Mus musculus 222 Met Trp Ala Leu Arg Ser Leu Leu Arg Pro Leu Gly Leu Arg Thr Met 1 5 10 15 Ser Gln Gly Ser Ala Arg Arg Pro Arg Pro Pro Lys Asp Pro Leu Arg 20 25 30 His Leu Arg Thr Arg Glu Lys Arg Gly Pro Gly Pro Gly Gly Pro Asn 35 40 45 Thr Val Tyr Leu Gln Val Val Ala Ala Gly Gly Arg Asp Ala Gly Ala 50 55 60 Ala Leu Tyr Val Phe Ser Glu Tyr Asn Arg Tyr Leu Phe Asn Cys Gly 65 70 75 80 Glu Gly Val Gln Arg Leu Met Gln Glu His Lys Thr Glu Ser Arg Ser 85 90 95 Leu Asp Asn Ile Phe Leu Thr Arg Met His Trp Ser Asn Val Gly Gly 100 105 110 Leu Cys Gly Met Ile Leu Thr Leu Lys Glu Thr Gly Leu Pro Lys Cys 115 120 125 Val Leu Ser Gly Pro Pro Gln Leu Glu Lys Tyr Leu Glu Ala Ile Lys 130 135 140 Ile Phe Ser Gly Pro Leu Lys Gly Ile Glu Leu Ala Val Arg Pro His 145 150 155 160 Ser Ala Pro Glu Tyr Lys Asp Glu Thr Met Thr Val Tyr Gln Val Pro 165 170 175 Ile His Ser Glu Arg Arg Cys Gly Lys Gln Gln Pro Ser Gln Ser Pro 180 185 190 Arg Thr Ser Pro Asn Arg Leu Ser Pro Lys Gln Ser Ser Asp Ser Gly 195 200 205 Ser Ala Glu Asn Gly Gln Cys Gln Gln Glu Ser Met Gly Gln Gly Pro 210 215 220 Ser Leu Val Val Ala Phe Val Cys Lys Leu His Leu Arg Lys Gly Asn 225 230 235 240 Phe Leu Val Leu Lys Ala Lys Glu Leu Gly Leu Pro Val Gly Thr Ala 245 250 255 Ala Ile Ala Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr 260 265 270 Tyr Glu Gly Arg Glu Ile Ala Ala Glu Glu Leu Cys Thr Pro Pro Asp 275 280 285 Pro Gly Leu Val Phe Ile Val Val Glu Cys Pro Asp Glu Gly Phe Ile 290 295 300 Leu Pro Ile Cys Glu Asn Asp Thr Phe Lys Arg Tyr Gln Ala Glu Ala 305 310 315 320 Asp Ala Pro Val Ala Leu Val Val His Ile Ala Pro Glu Ser Val Leu 325 330 335 Ile Asp Ser Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr 340 345 350 Gln His Leu Ile Leu Asn Glu Asn Cys Pro Ser Val His Asn Leu Arg 355 360 365 Ser His Lys Ile Gln Thr Gln Leu Ser Leu Ile His Pro Asp Ile Phe 370 375 380 Pro Gln Leu Thr Ser Phe Tyr Ser Lys Glu Glu Gly Ser Thr Leu Ser 385 390 395 400 Val Pro Thr Val Arg Gly Glu Cys Leu Leu Lys Tyr Ser Val Arg Pro 405 410 415 Lys Arg Glu Trp Gln Arg Asp Thr Thr Leu Asp Cys Asn Thr Asp Glu 420 425 430 Phe Ile Ala Glu Ala Leu Glu Leu Pro Ser Phe Gln Glu Ser Val Glu 435 440 445 Glu Tyr Arg Lys Asn Val Gln Glu Asn Pro Ala Pro Ala Glu Lys Arg 450 455 460 Ser Gln Tyr Pro Glu Ile Val Phe Leu Gly Thr Gly Ser Ala Ile Pro 465 470 475 480 Met Glu Ile Arg Asn Val Ser Ser Thr Leu Val Asn Leu Ser Pro Asp 485 490 495 Lys Ser Val Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys 500 505 510 Arg His Tyr Gly Gln Gln Ile Asp Arg Val Leu Cys Ser Leu Thr Ala 515 520 525 Val Phe Val Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn 530 535 540 Ile Leu Leu Gln Arg Glu His Ala Leu Ala Ser Leu Gly Lys Pro Phe 545 550 555 560 Gln Pro Leu Leu Val Val Ala Pro Thr Gln Leu Arg Ala Trp Leu Gln 565 570 575 Gln Tyr His Asn His Cys Gln Glu Ile Leu His His Val Ser Met Ile 580 585 590 Pro Ala Lys Cys Leu Gln Lys Gly Ala Glu Val Ser Asn Thr Thr Leu 595 600 605 Glu Arg Leu Ile Ser Leu Leu Leu Glu Thr Cys Asp Leu Glu Glu Phe 610 615 620 Gln Thr Cys Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu 625 630 635 640 Val His Ser Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro 645 650 655 Cys Glu Ala Leu Val Gln Met Gly Lys Asp Ala Thr Leu Leu Ile His 660 665 670 Glu Ala Thr Leu Glu Asp Xaa Leu Glu Glu Glu Ala Val Glu Arg Thr 675 680 685 His Ser Thr Thr Ser Gln Ala Ile Asn Val Gly Met Arg Met Asn Ala 690 695 700 Glu Phe Ile Met Leu Asn His Phe Ser Gln Arg Tyr Xaa Lys Ile Pro 705 710 715 720 Leu Phe Ser Pro Asp Phe Asn Glu Lys Val Gly Ile Ala Phe Asp His 725 730 735 Met Lys Val Xaa Phe Gly Asp Phe Pro Thr Val Pro Lys Leu Ile Pro 740 745 750 Pro Leu Lys Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Val Glu Arg 755 760 765 Arg Glu Lys Arg Glu Leu Arg Leu Val Arg Ala Ala Leu Leu Thr Gln 770 775 780 Gln Ala Asp Ser Pro Glu Asp Arg Glu Pro Gln Gln Lys Arg Ala His 785 790 795 800 Thr Asp Glu Pro His Ser Pro Gln Ser Lys Lys Glu Ser Val Ala Asn 805 810 815 Thr Leu Gly Ala Arg Val 820 223 2908 DNA Pan troglodytes CDS (1)..(2478) 223 atg tgg gcg ctt tgc tcg ctg ctg cgg tcc gcg gcc gga cgc acc atg 48 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 tcg cag gga cgc acc ata tcg cag gca ccc gcc cgc cgc gag cgg ccg 96 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 cgc aag gac ccg ctg cgg cac ctg cgc acg cga gag aag cgc gga ccg 144 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 tcg ggg tgc tcc ggc ggc cca aac acc gtg tac ctg cag gtg gtg gca 192 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 gcg ggt agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc gag ttc 240 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 aac cgg tat ctc ttc aac tgt gga gaa ggc att cag aga ctc atg cag 288 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Ile Gln Arg Leu Met Gln 85 90 95 gag cac aag tta aag gtt gct cgc ctg gac aac ata ttc ctg aca cga 336 Glu His Lys Leu Lys Val Ala Arg Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110 atg cac tgg tct aat gtt ggg ggc tta agt gga atg att ctt act tta 384 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 aag gaa acc ggg ctt cca aag tgt gta ctt tct gga cct cca caa ctg 432 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 gaa aaa tac ctc gaa gca atc aaa ata ttt tct ggt cca ttg aaa gga 480 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 ata gaa ctg gct gtg cgg ccc cac tct gcc cca gaa tac gag gat gaa 528 Ile Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 acc atg aca gtt tac cag atc ccc ata cac agt gaa cag agg agg gga 576 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 aag cac caa cca tgg cag agt cca gaa agg cct ctc agc agg ctc agt 624 Lys His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 cca gag cga tct tca gac tcc gag tca aat gaa aat gag cca cac ctt 672 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 cca cat ggt gtt agc cag aga aga ggg gtc agg gac tct tcc ctg gtc 720 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230 235 240 gta gct ttc atc tgt aag ctt cac tta aag aga gga aac ttc ttg gtg 768 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 ctc aaa gca aag gag atg ggc ctc cca gtt ggg aca gct gcc atc gct 816 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 ccc atc att gct gct gtc aag gac ggg aaa agc atc act cat gaa gga 864 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 aga gag att ttg gct gaa gag ctg tgt act cct cca gat cct ggt gct 912 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 gct ttt gtg gtg gta gaa tgt cca gat gaa agc ttc att caa ccc atc 960 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 tgt gag aat gcc acc ttt cag agg tac caa gga aag gca gat gcc ccc 1008 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 gtg gcc ttg gtg gtt cac atg gcc cca gaa tct gtg ctt gtg gac agc 1056 Val Ala Leu Val Val His Met Ala Pro Glu Ser Val Leu Val Asp Ser 340 345 350 agg tac cag cag tgg atg gag agg ttt ggg cct gac acc cag cac ttg 1104 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 gtc ctg aat gag aac tgt gcc tca gtt cac aac ctt cgc agc cac aag 1152 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 att caa acc cag ctc aac ctc atc cac ccg gac atc ttc ccc ctg ctc 1200 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400 acc agt ttc ccc tgt aag aag gag ggc ccc acc ctc agt gtg ccc atg 1248 Thr Ser Phe Pro Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405 410 415 gtt cag ggt gaa tgc ctc ctc aag tac cag ctc cgt ccc agg agg gag 1296 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425 430 tgg cag agg gat gcc att att act tgc aat cct gag gaa ttc ata att 1344 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile Ile 435 440 445 gag gcg ctg cag ctt ccc aac ttc cag cag agt gtg cag gag tac agg 1392 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 agg agt gcg cag gac ggc cca gcc cca gca gag aaa aga agt cag tac 1440 Arg Ser Ala Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 cca gaa atc atc ttc ctt gga aca ggg tct gcc atc ccg atg aag att 1488 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 cga aat gtc agt gcc aca ctt gtc aac ata agc ccc gac acg tct ctg 1536 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510 cta ctg gac tgt ggt gag ggc acg ttt ggg cag ctg tgc cgt cat tac 1584 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His Tyr 515 520 525 gga gac cag gtg gac agg gtc ctg ggc acc ctg gct gct gtg ttt gtg 1632 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 tcc cac ctg cac gca gat cac cac acg ggc ttg cta aat atc ttg ctg 1680 Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn Ile Leu Leu 545 550 555 560 cag aga gaa cga gcc ttg gca tct ttg gga aag ccc ttt cac cct ttg 1728 Gln Arg Glu Arg Ala Leu Ala Ser Leu Gly Lys Pro Phe His Pro Leu 565 570 575 ctg gtg gtt gcc ccc aac cag ctc aaa gcc tgg ctc cag cag tac cac 1776 Leu Val Val Ala Pro Asn Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590 aac cag tgc cag gag gtc ctg cac cac atc agt atg att cct gcc aaa 1824 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys 595 600 605 tgc ctt cag gaa ggg gct gag atc tcc agt cct gca gtg gaa aga ttg 1872 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 atc agt tcg ctg ttg cga aca tgt gat ttg gaa gag ttt cag acc tgt 1920 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 ctg gtg cgg cac tgc aag cat gcg ttt ggc tgt gcg ctg gtg cac acc 1968 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650 655 tct ggc tgg aaa gtg gtc tat tcc ggg gac acc atg ccc tgc gag gct 2016 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670 ctg gtc cgg atg ggg aaa gat gcc acc ctc ctg ata cat gaa gcc acc 2064 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu Ala Thr 675 680 685 ctg gaa gac ggt ttg gaa gag gaa gca gtg gaa aag aca cac agc aca 2112 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 acg tcc caa gcc atc agc gtg ggg atg cgg atg aac gcg gag ttc att 2160 Thr Ser Gln Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720 atg ctg aac cac ttc agc cag cgc tat gcc aag gtc ccc ctc ttc agc 2208 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 ccc aac ttc aac gag aaa gtg gga gtt gcc ttt gac cac atg aag gtc 2256 Pro Asn Phe Asn Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750 tgc ttt gga gac ttt gca aca atg ccc aag ctg att ccc cca ctg aaa 2304 Cys Phe Gly Asp Phe Ala Thr Met Pro Lys Leu Ile Pro Pro Leu Lys 755 760 765 gcc ctg ttt gct ggc gac atc gag gag atg gag gag cgc agg gag aag 2352 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 cgg gag ctg cgg cag gtg cgg gcg gcc ctc ctg tcc agg gag ctg gca 2400 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu Leu Ser Arg Glu Leu Ala 785 790 795 800 ggc ggc ctg gag gat ggg gag cct cag cag aaa cgg gcc cac aca gag 2448 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 gag cca cag gcc aag aag gtc aga gcc cag tgaagatctg ggagaccctg 2498 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825 aattcagaag gctgtgtgtc ttctgcccca cgcacgcacc cgtatctgcc ctccttgctg 2558 gtagaagctg aagagcacgg tcccccagga ggcagctcag gataggtggt atggagctgt 2618 gccaaggctt gggctcccac ataagcacta gtctatagat gcctcttagg actggtgcct 2678 ggcacagccg cgggacagga ggctgccaca cggaagcaag cagatgaact aatttcattt 2738 caaggcagtt tttaaagaag gcttggaaac agacggcagc acctttcctc taatccagca 2798 aagtgattcc ctgcacacca gagacaagca gagtaacagg atcagtgggt ctaagtgtcc 2858 gagacttaac gaaaatagta tttcagctgc aataaagatt gagtttgcaa 2908 224 826 PRT Pan troglodytes 224 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Ile Gln Arg Leu Met Gln 85 90 95 Glu His Lys Leu Lys Val Ala Arg Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 Ile Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 Lys His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230 235 240 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 Val Ala Leu Val Val His Met Ala Pro Glu Ser Val Leu Val Asp Ser 340 345 350 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400 Thr Ser Phe Pro Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405 410 415 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425 430 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile Ile 435 440 445 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 Arg Ser Ala Gln Asp Gly Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His Tyr 515 520 525 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn Ile Leu Leu 545 550 555 560 Gln Arg Glu Arg Ala Leu Ala Ser Leu Gly Lys Pro Phe His Pro Leu 565 570 575 Leu Val Val Ala Pro Asn Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys 595 600 605 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650 655 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu Ala Thr 675 680 685 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 Thr Ser Gln Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 Pro Asn Phe Asn Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750 Cys Phe Gly Asp Phe Ala Thr Met Pro Lys Leu Ile Pro Pro Leu Lys 755 760 765 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu Leu Ser Arg Glu Leu Ala 785 790 795 800 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825 225 2892 DNA Gorilla gorilla CDS (1)..(2478) 225 atg tgg gcg ctt tgc tcg ctg ctg cgg tcc gcg gcc gga cgc acc atg 48 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 tcg cag gga cgc acc ata tcg cag gca ccc gcc cgc cgc gag cgg ccg 96 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 cgc aag gac ccg ctg cgg cac ctg cgc acg cga gag aag cgc gga ccg 144 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 tcg ggg tgc tcc ggg ggc cca aac acc gtg tac ctg cag gtg gtg gca 192 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 gcg ggt agc cgg gac tcg ggc gcc gcg ctc tac gtc ttc tcc gag ttc 240 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 aac cgg tat ctc ttc aac tgt gga gaa ggc gtt cag aga ctc atg cag 288 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Val Gln Arg Leu Met Gln 85 90 95 gag cac aag tta aag gtt gtt cgc ctg gac aac ata ttc ctg aca cga 336 Glu His Lys Leu Lys Val Val Arg Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110 atg cac tgg tct aat gtt ggg ggc tta agt gga atg att ctt act tta 384 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 aag gaa acc ggg ctt cca aag tgt gta ctt tct gga cct cca cag ctg 432 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 gaa aaa tac ctc gaa gca atc aaa ata ttt tct ggt cca ttg aaa gga 480 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 ata gaa ctg gct gtg cgg ccc cac tct gcc cca gaa tac gag gat gaa 528 Ile Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 acc atg aca gtt tac cag atc ccc ata cac agt gaa cag agg agg gga 576 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 agg cac caa cca tgg cag agt cca gaa agg cct ctc agc agg ctc agt 624 Arg His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 cca gag cga tct tca gac tcc gag tcg aat gaa aat gag cca cac ctt 672 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 cca cat ggt gtt agc cag aga aga ggg gtc agg gac tct tcc ctg gtc 720 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230 235 240 gta gct ttc atc tgt aag ctt cac tta aag aga gga aac ttc ttg gtg 768 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 ctc aaa gca aag gag atg ggc ctc cca gtt ggg aca gct gcc atc gct 816 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 ccc atc att gct gct gtc aag gac ggg aaa agc atc act cat gaa gga 864 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 aga gag att ttg gct gaa gag ctg tgt act cct cca gat cct ggt gct 912 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 gct ttt gtg gtg gta gaa tgt cca gat gaa agc ttc att caa ccc atc 960 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 tgt gag aat gcc acc ttt cag agg tac caa gga aag gca gat gcc ccc 1008 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 gtg gcc ttg gtg gtt cac atg gcc cca gaa tct gtg ctt gtg gac agc 1056 Val Ala Leu Val Val His Met Ala Pro Glu Ser Val Leu Val Asp Ser 340 345 350 agg tac cag cag tgg atg gag agg ttt ggg cct gac acc cag cac ttg 1104 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 gtc ctg aat gag aac tgt gcc tca gtt cac aac ctt cgc agc cac aag 1152 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 att caa acc cag ctc aac ctc atc cac ccg gac atc ttc ccc ctg ctc 1200 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400 acc agt ttc ccc tgt aag aag gag ggc ccc acc ctc agt gtg ccc atg 1248 Thr Ser Phe Pro Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405 410 415 gtt cag ggt gaa tgc ctc ctc aag tac cag ctc cgt ccc agg agg gaa 1296 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425 430 tgg cag agg gat gcc att atc act tgc aat cct gag gaa ttc ata gtt 1344 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile Val 435 440 445 gag gcg ctg cag ctt ccc aac ttc cag cag agt gtg cag gag tac agg 1392 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 agg agt gtg cag gac gtc cca gcc cca gca gag aaa aga agt cag tac 1440 Arg Ser Val Gln Asp Val Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 cca gaa atc atc ttc ctt gga aca ggg tct gcc atc ccc atg aag att 1488 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 cga aat gtc agt gcc aca ctt gtc aac ata agc ccc gac acg tct ctg 1536 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510 cta ctg gac tgt ggt gag ggc acg ttt ggg cag ctg tgc cgt cat tac 1584 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His Tyr 515 520 525 gga gac cag gtg gac agg gtc ctg ggc acc ctg gct gct gtg ttt gtg 1632 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 tcc cac ctg cac gca gat cac cac acg ggc ttg cta aat atc ttg ctg 1680 Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn Ile Leu Leu 545 550 555 560 cag aga gaa caa gcc ttg gca tct ttg gga aag ccc ctt cac cct ttg 1728 Gln Arg Glu Gln Ala Leu Ala Ser Leu Gly Lys Pro Leu His Pro Leu 565 570 575 ctg gtg gtt gcc ccc agc cag ctc aaa gcc tgg ctc cag cag tac cac 1776 Leu Val Val Ala Pro Ser Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590 aac cag tgc cag gag gtc ctg cac cac atc agt atg att cct gcc aaa 1824 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys 595 600 605 tgc ctt cag gaa ggg gct gag atc tcc agt cct gca gtg gaa aga ttg 1872 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 atc agt tcg ctg ttg cga aca tgt gat ttg gaa gag ttt cag acc tgt 1920 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 ctg gtg cgg cac tgc aag cat gcg ttt ggc tgt gcg ctg gtg cac acc 1968 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650 655 tct ggc tgg aaa gtg gtc tat tcc ggg gac acc atg ccc tgc gag gct 2016 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670 ctg gtc cgc atg ggg aaa gat gcc acc ctc ctg ata cat gaa gcc acc 2064 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu Ala Thr 675 680 685 ctg gaa gat ggt ttg gaa gag gaa gca gtg gaa aag aca cac agc aca 2112 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 acg tcc caa gcc atc agc gtg ggg atg cgg atg aac gcg gag ttc att 2160 Thr Ser Gln Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720 atg ctg aac cac ttc agc cag cgc tat gcc aag gtc ccc ctc ttc agc 2208 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 ccc aac ttc aac gag aaa gtg gga gtt gcc ttt gac cac atg aag gtc 2256 Pro Asn Phe Asn Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750 tgc ttt gga gac ttt cca aca atg ccc aag ctg att ccc cca ctg aaa 2304 Cys Phe Gly Asp Phe Pro Thr Met Pro Lys Leu Ile Pro Pro Leu Lys 755 760 765 gcc ctg ttt gcc ggc gac atc gag gag atg gag gag cgc agg gag aag 2352 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 cgg gag ctg cgg cag gtg cgg gcg gcc ctc ctg tcc ggg gag ctg gca 2400 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu Leu Ser Gly Glu Leu Ala 785 790 795 800 ggc ggc ctg gag gat ggg gag cct cag cag aaa cgg gcc cac aca gag 2448 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 gag cca cag gcc aag aag gtc aga gcc cag tgaagatctg ggagaccctg 2498 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825 aattcagaag gctgtgtgtc ttctgcccca cgcacgcacc cgtatctgcc ctccttgctg 2558 gtagaagctg aagagcacgg tcccccagga ggcagctcag gataggtggt atggagctgt 2618 gccgaggctt aggctcccac ataagcacta gtctataggt gcctggcaca gccgcgggac 2678 aggaggctgc cacacggaag caagcagatg aactaatttc atttcaaggc agtttttaaa 2738 gaagtcttgg aaacagacgg cagcaccttt cctctaatcc agcaaagtga ttccctgcac 2798 accagagaca agcagagtaa caggatcact gggtctaagt gtccgagact taacgaaaat 2858 agtatttcag ctgcaataaa gattgagttt gcaa 2892 226 826 PRT Gorilla gorilla 226 Met Trp Ala Leu Cys Ser Leu Leu Arg Ser Ala Ala Gly Arg Thr Met 1 5 10 15 Ser Gln Gly Arg Thr Ile Ser Gln Ala Pro Ala Arg Arg Glu Arg Pro 20 25 30 Arg Lys Asp Pro Leu Arg His Leu Arg Thr Arg Glu Lys Arg Gly Pro 35 40 45 Ser Gly Cys Ser Gly Gly Pro Asn Thr Val Tyr Leu Gln Val Val Ala 50 55 60 Ala Gly Ser Arg Asp Ser Gly Ala Ala Leu Tyr Val Phe Ser Glu Phe 65 70 75 80 Asn Arg Tyr Leu Phe Asn Cys Gly Glu Gly Val Gln Arg Leu Met Gln 85 90 95 Glu His Lys Leu Lys Val Val Arg Leu Asp Asn Ile Phe Leu Thr Arg 100 105 110 Met His Trp Ser Asn Val Gly Gly Leu Ser Gly Met Ile Leu Thr Leu 115 120 125 Lys Glu Thr Gly Leu Pro Lys Cys Val Leu Ser Gly Pro Pro Gln Leu 130 135 140 Glu Lys Tyr Leu Glu Ala Ile Lys Ile Phe Ser Gly Pro Leu Lys Gly 145 150 155 160 Ile Glu Leu Ala Val Arg Pro His Ser Ala Pro Glu Tyr Glu Asp Glu 165 170 175 Thr Met Thr Val Tyr Gln Ile Pro Ile His Ser Glu Gln Arg Arg Gly 180 185 190 Arg His Gln Pro Trp Gln Ser Pro Glu Arg Pro Leu Ser Arg Leu Ser 195 200 205 Pro Glu Arg Ser Ser Asp Ser Glu Ser Asn Glu Asn Glu Pro His Leu 210 215 220 Pro His Gly Val Ser Gln Arg Arg Gly Val Arg Asp Ser Ser Leu Val 225 230 235 240 Val Ala Phe Ile Cys Lys Leu His Leu Lys Arg Gly Asn Phe Leu Val 245 250 255 Leu Lys Ala Lys Glu Met Gly Leu Pro Val Gly Thr Ala Ala Ile Ala 260 265 270 Pro Ile Ile Ala Ala Val Lys Asp Gly Lys Ser Ile Thr His Glu Gly 275 280 285 Arg Glu Ile Leu Ala Glu Glu Leu Cys Thr Pro Pro Asp Pro Gly Ala 290 295 300 Ala Phe Val Val Val Glu Cys Pro Asp Glu Ser Phe Ile Gln Pro Ile 305 310 315 320 Cys Glu Asn Ala Thr Phe Gln Arg Tyr Gln Gly Lys Ala Asp Ala Pro 325 330 335 Val Ala Leu Val Val His Met Ala Pro Glu Ser Val Leu Val Asp Ser 340 345 350 Arg Tyr Gln Gln Trp Met Glu Arg Phe Gly Pro Asp Thr Gln His Leu 355 360 365 Val Leu Asn Glu Asn Cys Ala Ser Val His Asn Leu Arg Ser His Lys 370 375 380 Ile Gln Thr Gln Leu Asn Leu Ile His Pro Asp Ile Phe Pro Leu Leu 385 390 395 400 Thr Ser Phe Pro Cys Lys Lys Glu Gly Pro Thr Leu Ser Val Pro Met 405 410 415 Val Gln Gly Glu Cys Leu Leu Lys Tyr Gln Leu Arg Pro Arg Arg Glu 420 425 430 Trp Gln Arg Asp Ala Ile Ile Thr Cys Asn Pro Glu Glu Phe Ile Val 435 440 445 Glu Ala Leu Gln Leu Pro Asn Phe Gln Gln Ser Val Gln Glu Tyr Arg 450 455 460 Arg Ser Val Gln Asp Val Pro Ala Pro Ala Glu Lys Arg Ser Gln Tyr 465 470 475 480 Pro Glu Ile Ile Phe Leu Gly Thr Gly Ser Ala Ile Pro Met Lys Ile 485 490 495 Arg Asn Val Ser Ala Thr Leu Val Asn Ile Ser Pro Asp Thr Ser Leu 500 505 510 Leu Leu Asp Cys Gly Glu Gly Thr Phe Gly Gln Leu Cys Arg His Tyr 515 520 525 Gly Asp Gln Val Asp Arg Val Leu Gly Thr Leu Ala Ala Val Phe Val 530 535 540 Ser His Leu His Ala Asp His His Thr Gly Leu Leu Asn Ile Leu Leu 545 550 555 560 Gln Arg Glu Gln Ala Leu Ala Ser Leu Gly Lys Pro Leu His Pro Leu 565 570 575 Leu Val Val Ala Pro Ser Gln Leu Lys Ala Trp Leu Gln Gln Tyr His 580 585 590 Asn Gln Cys Gln Glu Val Leu His His Ile Ser Met Ile Pro Ala Lys 595 600 605 Cys Leu Gln Glu Gly Ala Glu Ile Ser Ser Pro Ala Val Glu Arg Leu 610 615 620 Ile Ser Ser Leu Leu Arg Thr Cys Asp Leu Glu Glu Phe Gln Thr Cys 625 630 635 640 Leu Val Arg His Cys Lys His Ala Phe Gly Cys Ala Leu Val His Thr 645 650 655 Ser Gly Trp Lys Val Val Tyr Ser Gly Asp Thr Met Pro Cys Glu Ala 660 665 670 Leu Val Arg Met Gly Lys Asp Ala Thr Leu Leu Ile His Glu Ala Thr 675 680 685 Leu Glu Asp Gly Leu Glu Glu Glu Ala Val Glu Lys Thr His Ser Thr 690 695 700 Thr Ser Gln Ala Ile Ser Val Gly Met Arg Met Asn Ala Glu Phe Ile 705 710 715 720 Met Leu Asn His Phe Ser Gln Arg Tyr Ala Lys Val Pro Leu Phe Ser 725 730 735 Pro Asn Phe Asn Glu Lys Val Gly Val Ala Phe Asp His Met Lys Val 740 745 750 Cys Phe Gly Asp Phe Pro Thr Met Pro Lys Leu Ile Pro Pro Leu Lys 755 760 765 Ala Leu Phe Ala Gly Asp Ile Glu Glu Met Glu Glu Arg Arg Glu Lys 770 775 780 Arg Glu Leu Arg Gln Val Arg Ala Ala Leu Leu Ser Gly Glu Leu Ala 785 790 795 800 Gly Gly Leu Glu Asp Gly Glu Pro Gln Gln Lys Arg Ala His Thr Glu 805 810 815 Glu Pro Gln Ala Lys Lys Val Arg Ala Gln 820 825 227 844 PRT Caenorhabditis elegans 227 Met Lys Met Leu Phe Phe Gly Ile Lys Val Ser Arg His Leu Ile Ser 1 5 10 15 Ser Thr Ser Cys Leu Phe Lys Asp Asn Asn Glu Glu Leu Leu Glu Ser 20 25 30 Ile Lys Glu Arg Ile Ala Arg Asn Arg Arg Ile Leu Gln Lys His Ser 35 40 45 Ser Ser His Leu Lys Ala Arg Glu Val Asn Ala Ser Ile Ser Asn Leu 50 55 60 Arg Gln Ser Met Ala Ala Val Gln Lys Lys Gln Lys Ala Ala His Glu 65 70 75 80 Pro Pro Ala Asn Ser Ile Val Asn Ile Pro Ser Gln Val Ser Ile Glu 85 90 95 Val Leu Gly Asn Gly Thr Gly Leu Leu Arg Ala Cys Phe Ile Leu Arg 100 105 110 Thr Pro Leu Lys Thr Tyr Met Phe Asn Cys Pro Glu Asn Ala Cys Arg 115 120 125 Phe Leu Trp Gln Leu Arg Ile Arg Ser Ser Ser Val Val Asp Leu Phe 130 135 140 Ile Thr Ser Ala Asn Trp Asp Asn Ile Ala Gly Ile Ser Ser Ile Leu 145 150 155 160 Leu Ser Lys Glu Ser Asn Ala Leu Ser Thr Arg Leu His Gly Ala Met 165 170 175 Asn Ile Lys His Phe Leu Glu Cys Ile Arg Pro Phe Gln Asp Ser Asp 180 185 190 Tyr Gly Ser Cys Lys Tyr Pro Ser Gln Val Glu Glu Arg Pro Tyr Thr 195 200 205 Met Glu Asn Tyr Glu Asp Ala Gly Leu Lys Val Thr Tyr Ile Pro Leu 210 215 220 Ser Pro Pro Leu Asn Ile Gly Ser Asn Asn Glu Lys Ser Lys Asn Val 225 230 235 240 Lys Val Asn Asn Val Asp Ile Ala Phe Leu Ile Glu Met Lys Glu Ala 245 250 255 Ala Arg Arg Ile Asp Thr Met Lys Leu Met Glu Leu Lys Val Pro Lys 260 265 270 Gly Pro Leu Ile Gly Lys Leu Lys Ser Gly Glu Ala Val Thr Leu Pro 275 280 285 Asp Gly Arg Thr Ile Gln Pro Asp Gln Val Phe Ser Ser Asp Lys Val 290 295 300 Glu Gly Asp Lys Pro Leu Leu Leu Val Thr Glu Cys Thr Thr Glu Asp 305 310 315 320 His Val Lys Ala Leu Ile Asp Ser Ser Ser Leu Gln Pro Phe Leu Asn 325 330 335 Gly Glu Lys Gln Leu Asp Tyr Met Val His Ile Ser Asp Asp Ala Val 340 345 350 Ile Asn Thr Pro Thr Tyr Arg His Leu Met Glu Lys Leu Asn Asn Pro 355 360 365 Ser Ile Thr His Leu Leu Ile Asn Gly Gly Asn Pro Val Ile Pro Ala 370 375 380 Val Glu Ser Val Tyr Lys His Thr Arg Leu Leu Arg Ser Ile Ala Pro 385 390 395 400 Ser Leu Phe Pro Ala Leu His Pro Ile Asp Trp Ser Gly Ile Ile Thr 405 410 415 Gln Asn Glu Glu Leu Ser Gln Arg Gln Asp Gln Phe Ile Arg Val Ala 420 425 430 Pro Met Gln Arg Tyr Trp Met Arg Arg Gly Ala Ser Phe Asn Glu Glu 435 440 445 Pro Ile Val Asn Asn Leu Leu Ala Ala Glu Pro Glu Leu Ser Asp Lys 450 455 460 Ala Lys Glu Leu Ile Lys Glu Tyr Gln Lys Leu Glu Lys Glu Asn Lys 465 470 475 480 Met Asp Cys Glu Phe Pro Lys Leu Thr Phe Phe Gly Thr Ser Ser Ala 485 490 495 Val Pro Ser Lys Tyr Arg Asn Val Thr Gly Tyr Leu Val Glu Ala Ser 500 505 510 Glu Asn Ser Ala Ile Leu Ile Asp Val Gly Glu Gly Thr Tyr Gly Gln 515 520 525 Met Arg Ala Val Phe Gly Glu Asp Gly Cys Lys Gln Leu Leu Val Asn 530 535 540 Leu Asn Cys Val Leu Ile Thr His Ala His Gln Asp His Met Asn Gly 545 550 555 560 Leu Tyr Thr Ile Ile Ala Arg Arg Lys Glu Ala Phe Glu Ser Leu Gly 565 570 575 Ala Pro Tyr Arg Pro Leu Val Leu Val Cys Asn Arg Asn Val Leu Lys 580 585 590 Pro Met Lys Thr Tyr Ser Ile Cys Phe Glu Asn Ile Glu His Leu Leu 595 600 605 Glu Ile Val Asp Ile Ser Arg Tyr Pro Leu Thr Pro Pro Gly Ser Pro 610 615 620 Gly Gly Pro Pro Gly Lys Arg Pro Arg Leu Pro Ser Pro His Leu Pro 625 630 635 640 Pro Ser Arg Asp Val Leu Gln Asp Met Ser Ser Ser Phe Asp Lys Lys 645 650 655 Ala Trp Lys Leu Asp Glu Leu Lys Ala Val Gln Val His His Thr Arg 660 665 670 Met Ala Asn Gly Phe Val Met Arg Val Ala Gly Lys Arg Ile Val Phe 675 680 685 Ser Gly Asp Thr Lys Pro Cys Asp Leu Leu Val Glu Glu Gly Lys Asp 690 695 700 Ala Asp Val Leu Val His Glu Ser Thr Phe Glu Asp Gly His Glu Val 705 710 715 720 Asp Met Thr Pro Lys Pro Pro Lys Lys Leu Ala Lys Ile Ser Ser Leu 725 730 735 Ala Asp Ala Met Arg Lys Arg His Ser Thr Met Gly Gln Ala Val Asp 740 745 750 Val Gly Lys Arg Met Asn Ala Lys His Ile Ile Leu Thr His Phe Ser 755 760 765 Ala Arg Tyr Pro Lys Val Pro Val Leu Pro Glu Tyr Leu Asp Lys Glu 770 775 780 Asn Ile Gly Val Ala Met Asp Met Leu Arg Val Arg Phe Asp His Leu 785 790 795 800 Pro Leu Val Ser Lys Leu Leu Pro Ile Phe Arg Glu Val Phe Val Ala 805 810 815 Glu Leu Phe Glu Leu Thr Ile Lys Lys Glu Gln Arg Val Leu Lys Asp 820 825 830 Lys Glu Leu Ser Glu Lys Arg Gly Gln Leu Lys Ala 835 840 228 837 PRT Arabidopsis thaliana 228 Met Glu Asn Asn Glu Ala Thr Asn Gly Ser Lys Ser Ser Ser Asn Ser 1 5 10 15 Phe Val Phe Asn Lys Arg Arg Ala Glu Gly Phe Asp Ile Thr Asp Lys 20 25 30 Lys Lys Arg Asn Leu Glu Arg Lys Ser Gln Lys Leu Asn Pro Thr Asn 35 40 45 Thr Ile Ala Tyr Ala Gln Ile Leu Gly Thr Gly Met Asp Thr Gln Asp 50 55 60 Thr Ser Ser Ser Val Leu Leu Phe Phe Asp Lys Gln Arg Phe Ile Phe 65 70 75 80 Asn Ala Gly Glu Gly Leu Gln Arg Phe Cys Thr Glu His Lys Ile Lys 85 90 95 Leu Ser Lys Ile Asp His Val Phe Leu Ser Arg Val Cys Ser Glu Thr 100 105 110 Ala Gly Gly Leu Pro Gly Leu Leu Leu Thr Leu Ala Gly Ile Gly Glu 115 120 125 Glu Gly Leu Ser Val Asn Val Trp Gly Pro Ser Asp Leu Asn Tyr Leu 130 135 140 Val Asp Ala Met Lys Ser Phe Ile Pro Arg Ala Ala Met Val His Thr 145 150 155 160 Arg Ser Phe Gly Pro Ser Ser Thr Pro Asp Pro Ile Val Leu Val Asn 165 170 175 Asp Glu Val Val Lys Ile Ser Ala Ile Ile Leu Lys Pro Cys His Ser 180 185 190 Glu Glu Asp Ser Gly Asn Lys Ser Gly Asp Leu Ser Val Val Tyr Val 195 200 205 Cys Glu Leu Pro Glu Ile Leu Gly Lys Phe Asp Leu Glu Lys Ala Lys 210 215 220 Lys Val Phe Gly Val Lys Pro Gly Pro Lys Tyr Ser Arg Leu Gln Ser 225 230 235 240 Gly Glu Ser Val Lys Ser Asp Glu Arg Asp Ile Thr Val His Pro Ser 245 250 255 Asp Val Met Gly Pro Ser Leu Pro Gly Pro Ile Val Leu Leu Val Asp 260 265 270 Cys Pro Thr Glu Ser His Ala Ala Glu Leu Phe Ser Leu Lys Ser Leu 275 280 285 Glu Ser Tyr Tyr Ser Ser Pro Asp Glu Gln Thr Ile Gly Ala Lys Phe 290 295 300 Val Asn Cys Ile Ile His Leu Ser Pro Ser Ser Val Thr Ser Ser Pro 305 310 315 320 Thr Tyr Gln Ser Trp Met Lys Lys Phe His Leu Thr Gln His Ile Leu 325 330 335 Ala Gly His Gln Arg Phe Leu Pro Leu Leu Ile Ile Val Ser His Gln 340 345 350 Lys Thr Val Arg Lys Asn Met Ala Phe Pro Ile Leu Lys Ala Ser Ser 355 360 365 Arg Ile Ala Ala Arg Leu Asn Tyr Leu Cys Pro Gln Phe Phe Pro Ala 370 375 380 Pro Gly Phe Trp Pro Ser Gln Leu Thr Asp Asn Ser Ile Ile Asp Pro 385 390 395 400 Thr Pro Ser Asn Lys Phe Asn Leu Arg Pro Val Ala Ile Arg Gly Ile 405 410 415 Asp Arg Ser Cys Ile Pro Ala Pro Leu Thr Ser Ser Glu Val Val Asp 420 425 430 Glu Leu Leu Ser Glu Ile Pro Glu Ile Lys Asp Lys Ser Glu Glu Ile 435 440 445 Lys Gln Phe Trp Asn Lys Gln His Asn Lys Thr Ile Ile Glu Lys Leu 450 455 460 Trp Leu Ser Glu Cys Asn Thr Val Leu Pro Asn Cys Leu Glu Lys Ile 465 470 475 480 Arg Arg Asp Asp Met Glu Ile Val Ile Leu Gly Thr Gly Ser Ser Gln 485 490 495 Pro Ser Lys Tyr Arg Asn Val Ser Ala Ile Phe Ile Asp Leu Phe Ser 500 505 510 Arg Gly Ser Leu Leu Leu Asp Cys Gly Glu Gly Thr Leu Gly Gln Leu 515 520 525 Lys Arg Arg Tyr Gly Leu Asp Gly Ala Asp Glu Ala Val Arg Lys Leu 530 535 540 Arg Cys Ile Trp Ile Ser His Ile His Ala Asp His His Thr Gly Leu 545 550 555 560 Ala Arg Ile Leu Ala Leu Arg Ser Lys Leu Leu Lys Gly Val Thr His 565 570 575 Glu Pro Val Ile Val Val Gly Pro Arg Pro Leu Lys Arg Phe Leu Asp 580 585 590 Ala Tyr Gln Arg Leu Glu Asp Leu Asp Met Glu Phe Leu Asp Cys Arg 595 600 605 Ser Thr Thr Ala Thr Ser Trp Ala Ser Leu Glu Ser Gly Gly Glu Ala 610 615 620 Glu Gly Ser Leu Phe Thr Gln Gly Ser Pro Met Gln Ser Val Phe Lys 625 630 635 640 Arg Ser Asp Ile Ser Met Asp Asn Ser Ser Val Leu Leu Cys Leu Lys 645 650 655 Asn Leu Lys Lys Val Leu Ser Glu Ile Gly Leu Asn Asp Leu Ile Ser 660 665 670 Phe Pro Val Val His Cys Pro Gln Ala Tyr Gly Val Val Ile Lys Ala 675 680 685 Ala Glu Arg Val Asn Ser Val Gly Glu Gln Ile Leu Gly Trp Lys Met 690 695 700 Val Tyr Ser Gly Asp Ser Arg Pro Cys Pro Glu Thr Val Glu Ala Ser 705 710 715 720 Arg Asp Ala Thr Ile Leu Ile His Glu Ala Thr Phe Glu Asp Ala Leu 725 730 735 Ile Glu Glu Ala Leu Ala Lys Asn His Ser Thr Thr Lys Glu Ala Ile 740 745 750 Asp Val Gly Ser Ala Ala Asn Val Tyr Arg Ile Val Leu Thr His Phe 755 760 765 Ser Gln Arg Tyr Pro Lys Ile Pro Val Ile Asp Glu Ser His Met His 770 775 780 Asn Thr Cys Ile Ala Phe Asp Leu Met Ser Ile Asn Met Ala Asp Leu 785 790 795 800 His Val Leu Pro Lys Val Leu Pro Tyr Phe Lys Thr Leu Phe Arg Asp 805 810 815 Glu Met Val Glu Asp Glu Asp Ala Asp Asp Val Ala Met Asp Asp Leu 820 825 830 Lys Glu Glu Ala Leu 835 229 838 PRT Saccharomyces cerevisiae 229 Met Phe Thr Phe Ile Pro Ile Thr His Pro Thr Ser Asp Thr Lys His 1 5 10 15 Pro Leu Leu Leu Val Gln Ser Ala His Gly Glu Lys Tyr Phe Phe Gly 20 25 30 Lys Ile Gly Glu Gly Ser Gln Arg Ser Leu Thr Glu Asn Lys Ile Arg 35 40 45 Ile Ser Lys Leu Lys Asp Ile Phe Leu Thr Gly Glu Leu Asn Trp Ser 50 55 60 Asp Ile Gly Gly Leu Pro Gly Met Ile Leu Thr Ile Ala Asp Gln Gly 65 70 75 80 Lys Ser Asn Leu Val Leu His Tyr Gly Asn Asp Ile Leu Asn Tyr Ile 85 90 95 Val Ser Thr Trp Arg Tyr Phe Val Phe Arg Phe Gly Ile Asp Leu Asn 100 105 110 Asp His Ile Met Lys Asp Lys Glu Val Tyr Lys Asp Lys Ile Ile Ala 115 120 125 Val Lys Ser Phe Asn Val Leu Lys Asn Gly Gly Glu Asp Arg Leu Gly 130 135 140 Val Phe Asp Ser Phe Gln Lys Gly Val Leu Arg Ser Ile Val Ala Lys 145 150 155 160 Met Phe Pro Lys His Ala Pro Thr Asp Arg Tyr Asp Pro Ser Ser Asp 165 170 175 Pro His Leu Asn Val Glu Leu Pro Asp Leu Asp Ala Lys Val Glu Val 180 185 190 Ser Thr Asn Tyr Glu Ile Ser Phe Ser Pro Val Arg Gly Lys Phe Lys 195 200 205 Val Glu Glu Ala Ile Lys Leu Gly Val Pro Lys Gly Pro Leu Phe Ala 210 215 220 Lys Leu Thr Lys Gly Gln Thr Ile Thr Leu Asp Asn Gly Ile Val Val 225 230 235 240 Thr Pro Glu Gln Val Leu Glu Asn Glu Arg His Phe Ala Lys Val Leu 245 250 255 Ile Leu Asp Ile Pro Asp Asp Leu Tyr Leu Asn Ala Phe Val Glu Lys 260 265 270 Phe Lys Asp Tyr Asp Cys Ala Glu Leu Gly Met Val Tyr Tyr Phe Leu 275 280 285 Gly Asp Glu Val Thr Ile Asn Asp Asn Leu Phe Ala Phe Ile Asp Ile 290 295 300 Phe Glu Lys Asn Asn Tyr Gly Lys Val Asn His Met Ile Ser His Asn 305 310 315 320 Lys Ile Ser Pro Asn Thr Ile Ser Phe Phe Gly Ser Ala Leu Thr Thr 325 330 335 Leu Lys Leu Lys Ala Leu Gln Val Asn Asn Tyr Asn Leu Pro Lys Thr 340 345 350 Asp Arg Val Phe Ser Lys Asp Phe Tyr Asp Arg Phe Asp Thr Pro Leu 355 360 365 Ser Arg Gly Thr Ser Met Cys Lys Ser Gln Glu Glu Pro Leu Asn Thr 370 375 380 Ile Ile Glu Lys Asp Asn Ile His Ile Phe Ser Gln Asn Lys Thr Val 385 390 395 400 Thr Phe Glu Pro Phe Arg Met Asn Glu Glu Pro Met Lys Cys Asn Ile 405 410 415 Asn Gly Glu Val Ala Asp Phe Ser Trp Gln Glu Ile Phe Glu Glu His 420 425 430 Val Lys Pro Leu Glu Phe Pro Leu Ala Asp Val Asp Thr Val Ile Asn 435 440 445 Asn Gln Leu His Val Asp Asn Phe Asn Asn Ser Ala Glu Lys Lys Lys 450 455 460 His Val Glu Ile Ile Thr Leu Gly Thr Gly Ser Ala Leu Pro Ser Lys 465 470 475 480 Tyr Arg Asn Val Val Ser Thr Leu Val Lys Val Pro Phe Thr Asp Ala 485 490 495 Asp Gly Asn Thr Ile Asn Arg Asn Ile Met Leu Asp Ala Gly Glu Asn 500 505 510 Thr Leu Gly Thr Ile His Arg Met Phe Ser Gln Leu Ala Val Lys Ser 515 520 525 Ile Phe Gln Asp Leu Lys Met Ile Tyr Leu Ser His Leu His Ala Asp 530 535 540 His His Leu Gly Ile Ile Ser Val Leu Asn Glu Trp Tyr Lys Tyr Asn 545 550 555 560 Lys Asp Asp Glu Thr Ser Tyr Ile Tyr Val Val Thr Pro Trp Gln Tyr 565 570 575 His Lys Phe Val Asn Glu Trp Leu Val Leu Glu Asn Lys Glu Ile Leu 580 585 590 Lys Arg Ile Lys Tyr Ile Ser Cys Glu His Phe Ile Asn Asp Ser Phe 595 600 605 Val Arg Met Gln Thr Gln Ser Val Pro Leu Ala Glu Phe Asn Glu Ile 610 615 620 Leu Lys Glu Asn Ser Asn Gln Glu Ser Asn Arg Lys Leu Glu Leu Asp 625 630 635 640 Arg Asp Ser Ser Tyr Arg Asp Val Asp Leu Ile Arg Gln Met Tyr Glu 645 650 655 Asp Leu Ser Ile Glu Tyr Phe Gln Thr Cys Arg Ala Ile His Cys Asp 660 665 670 Trp Ala Tyr Ser Asn Ser Ile Thr Phe Arg Met Asp Glu Asn Asn Glu 675 680 685 His Asn Thr Phe Lys Val Ser Tyr Ser Gly Asp Thr Arg Pro Asn Ile 690 695 700 Glu Lys Phe Ser Leu Glu Ile Gly Tyr Asn Ser Asp Leu Leu Ile His 705 710 715 720 Glu Ala Thr Leu Glu Asn Gln Leu Leu Glu Asp Ala Val Lys Lys Lys 725 730 735 His Cys Thr Ile Asn Glu Ala Ile Gly Val Ser Asn Lys Met Asn Ala 740 745 750 Arg Lys Leu Ile Leu Thr His Phe Ser Gln Arg Tyr Pro Lys Leu Pro 755 760 765 Gln Leu Asp Asn Asn Ile Asp Val Met Ala Arg Glu Phe Cys Phe Ala 770 775 780 Phe Asp Ser Met Ile Val Asp Tyr Glu Lys Ile Gly Glu Gln Gln Arg 785 790 795 800 Ile Phe Pro Leu Leu Asn Lys Ala Phe Val Glu Glu Lys Glu Glu Glu 805 810 815 Glu Asp Val Asp Asp Val Glu Ser Val Gln Asp Leu Glu Val Lys Leu 820 825 830 Lys Lys His Lys Lys Asn 835 230 311 PRT Escherichia coli 230 Met Lys Arg Asp Glu Leu Met Glu Leu Ile Phe Leu Gly Thr Ser Ala 1 5 10 15 Gly Val Pro Thr Arg Thr Arg Asn Val Thr Ala Ile Leu Leu Asn Leu 20 25 30 Gln His Pro Thr Gln Ser Gly Leu Trp Leu Phe Asp Cys Gly Glu Gly 35 40 45 Thr Gln His Gln Leu Leu His Thr Ala Phe Asn Pro Gly Lys Leu Asp 50 55 60 Lys Ile Phe Ile Ser His Leu His Gly Asp His Leu Phe Gly Leu Pro 65 70 75 80 Gly Leu Leu Cys Ser Arg Ser Met Ser Gly Ile Ile Gln Pro Leu Thr 85 90 95 Ile Tyr Gly Pro Gln Gly Ile Arg Glu Phe Val Glu Thr Ala Leu Arg 100 105 110 Ile Ser Gly Ser Trp Thr Asp Tyr Pro Leu Glu Ile Val Glu Ile Gly 115 120 125 Ala Gly Glu Ile Leu Asp Asp Gly Leu Arg Lys Val Thr Ala Tyr Pro 130 135 140 Leu Glu His Pro Leu Glu Cys Tyr Gly Tyr Arg Ile Glu Glu His Asp 145 150 155 160 Lys Pro Gly Ala Leu Asn Ala Gln Ala Leu Lys Ala Ala Gly Val Pro 165 170 175 Pro Gly Pro Leu Phe Gln Glu Leu Lys Ala Gly Lys Thr Ile Thr Leu 180 185 190 Glu Asp Gly Arg Gln Ile Asn Gly Ala Asp Tyr Leu Ala Ala Pro Val 195 200 205 Pro Gly Lys Ala Leu Ala Ile Phe Gly Asp Thr Gly Pro Cys Asp Ala 210 215 220 Ala Leu Asp Leu Ala Lys Gly Val Asp Val Met Val His Glu Ala Thr 225 230 235 240 Leu Asp Ile Thr Met Glu Ala Lys Ala Asn Ser Arg Gly His Ser Ser 245 250 255 Thr Arg Gln Ala Ala Thr Leu Ala Arg Glu Ala Gly Val Gly Lys Leu 260 265 270 Ile Ile Thr His Val Ser Ser Arg Tyr Asp Asp Lys Gly Cys Gln His 275 280 285 Leu Leu Arg Glu Cys Arg Ser Ile Phe Pro Ala Thr Glu Leu Ala Asn 290 295 300 Asp Phe Thr Val Phe Asn Val 305 310 231 326 PRT Synechocystis sp. 231 Met Glu Ile Thr Phe Leu Gly Thr Ser Ser Gly Val Pro Thr Arg Asn 1 5 10 15 Arg Asn Val Ser Ser Ile Ala Leu Arg Leu Pro Gln Arg Ala Glu Leu 20 25 30 Trp Leu Phe Asp Cys Gly Glu Gly Thr Gln His Gln Phe Leu Arg Ser 35 40 45 Glu Val Lys Ile Ser Gln Leu Thr Arg Ile Phe Ile Thr His Leu His 50 55 60 Gly Asp His Ile Phe Gly Leu Met Gly Leu Leu Ala Ser Ser Gly Leu 65 70 75 80 Ala Gly Ser Gly Gln Gly Ile Glu Ile Tyr Gly Pro Glu Gly Leu Gly 85 90 95 Asp Tyr Leu Glu Ala Cys Cys Arg Phe Ser Ser Thr His Leu Gly Lys 100 105 110 Arg Leu Lys Val His Thr Val Arg Glu Asn Gly Leu Ile Tyr Glu Asp 115 120 125 Lys Asp Phe Gln Val His Cys Gly Leu Leu Lys His Arg Ile Pro Ala 130 135 140 Tyr Gly Tyr Arg Val Glu Glu Lys Gln Arg Pro Gly Arg Phe Asn Val 145 150 155 160 Glu Gln Ala Glu Ala Leu Gly Ile Pro Phe Gly Pro Ile Tyr Gly Gln 165 170 175 Leu Lys Gln Gly Lys Thr Val Thr Leu Glu Asp Gly Arg Arg Ile Arg 180 185 190 Gly Gln Asp Leu Cys Glu Pro Pro Glu Pro Gly Arg Lys Phe Val Tyr 195 200 205 Cys Thr Asp Thr Val Phe Cys Glu Glu Ala Ile Ala Leu Ala Gln Glu 210 215 220 Ala Asp Leu Leu Val His Glu Ala Thr Phe Ala His Gln Asp Ala Gln 225 230 235 240 Leu Ala Phe Asp Arg Leu His Ser Thr Ser Thr Met Ala Ala Gln Val 245 250 255 Ala Leu Leu Ala Asn Val Lys Gln Leu Ile Met Thr His Phe Ser Pro 260 265 270 Arg Tyr Ala Pro Gly Asn Pro Leu Gln Leu Glu Asn Leu Leu Ala Glu 275 280 285 Ala Gln Ala Ile Phe Pro Asn Thr Arg Leu Ala Arg Asp Phe Leu Thr 290 295 300 Val Glu Ile Pro Arg Arg Thr Ala Asp Pro Ala Ile Ala Met Ser Thr 305 310 315 320 Pro Gln Ala Ser Pro Ala 325 232 307 PRT Methanobacterium thermoautotrophicum 232 Met Met Glu Val Thr Phe Leu Gly Thr Ser Ser Ala Val Pro Ser Lys 1 5 10 15 Asn Arg Asn His Thr Ser Ile Ala Leu Arg Ile Pro Gly Glu Ile Phe 20 25 30 Leu Phe Asp Cys Gly Glu Gly Thr Gln Arg Gln Met Ala Leu Ala Gly 35 40 45 Ile Ser Pro Met Lys Val Thr Arg Ile Phe Ile Thr His Leu His Gly 50 55 60 Asp His Ile Leu Gly Ile Pro Gly Met Ile Gln Ser Met Gly Phe Arg 65 70 75 80 Gly Arg Glu Glu Pro Leu Asp Ile Tyr Gly Pro Pro Gly Ile His Glu 85 90 95 Leu His Glu Cys Ile Met Lys Met Gly Tyr Phe Thr Leu Asp Phe Asp 100 105 110 Ile Asn Val His Glu Val Arg Gly Gly Thr Val Val Glu Glu Asp Asp 115 120 125 Tyr Arg Val Thr Ser Ala Pro Ala Ser His Ser Val Phe Asn Leu Ala 130 135 140 Tyr Cys Phe Glu Glu Lys Lys Arg Pro Arg Phe Leu Arg Glu Lys Ala 145 150 155 160 Ile Ala Leu Gly Leu Lys Pro Gly Pro Ala Phe Gly Lys Leu His Arg 165 170 175 Gly Ile Pro Val Arg Val Gly Asp Arg Ile Ile Met Pro Glu Glu Val 180 185 190 Leu Gly Ser Pro Arg Lys Gly Val Lys Val Cys Tyr Ser Gly Asp Thr 195 200 205 Arg Pro Cys Glu Ser Val Ile Lys Leu Ala Glu Gly Ala Glu Leu Leu 210 215 220 Ile His Glu Ser Thr Leu Glu Ala Gly Ser Glu Asp Lys Ala Ala Glu 225 230 235 240 Ser Gly His Ser Thr Ala Arg Glu Ala Ala Glu Val Ala Arg Ser Ala 245 250 255 Gly Val Lys Arg Leu Ile Leu Thr His Leu Ser Thr Arg Tyr Lys Arg 260 265 270 Thr Glu Val Ile Leu Glu Ala Ala Arg Gln Val Phe Pro Val Thr Asp 275 280 285 Val Ala Asp Asp Leu Met Thr Val Glu Val Lys Ala Tyr Asp Ser Ser 290 295 300 Pro Asp Ser 305 233 684 PRT Homo sapiens 233 Met Ser Ala Ile Pro Ala Glu Glu Ser Asp Gln Leu Leu Ile Arg Pro 1 5 10 15 Leu Gly Ala Gly Gln Glu Val Gly Arg Ser Cys Ile Ile Leu Glu Phe 20 25 30 Lys Gly Arg Lys Ile Met Leu Asp Cys Gly Ile His Pro Gly Leu Glu 35 40 45 Gly Met Asp Ala Leu Pro Tyr Ile Asp Leu Ile Asp Pro Ala Glu Ile 50 55 60 Asp Leu Leu Leu Ile Ser His Phe His Leu Asp His Cys Gly Ala Leu 65 70 75 80 Pro Trp Phe Leu Gln Lys Thr Ser Phe Lys Gly Arg Thr Phe Met Thr 85 90 95 His Ala Thr Lys Ala Ile Tyr Arg Trp Leu Leu Ser Asp Tyr Val Lys 100 105 110 Val Ser Asn Ile Ser Ala Asp Asp Met Leu Tyr Thr Glu Thr Asp Leu 115 120 125 Glu Glu Ser Met Asp Lys Ile Glu Thr Ile Asn Phe His Glu Val Lys 130 135 140 Glu Val Ala Gly Ile Lys Phe Trp Cys Tyr His Ala Gly His Val Leu 145 150 155 160 Gly Ala Ala Met Phe Met Ile Glu Ile Ala Gly Val Lys Leu Leu Tyr 165 170 175 Thr Gly Asp Phe Ser Arg Gln Glu Asp Arg His Leu Met Ala Ala Glu 180 185 190 Ile Pro Asn Ile Lys Pro Asp Ile Leu Ile Ile Glu Ser Thr Tyr Gly 195 200 205 Thr His Ile His Glu Lys Arg Glu Glu Arg Glu Ala Arg Phe Cys Asn 210 215 220 Thr Val His Asp Ile Val Asn Arg Gly Gly Arg Gly Leu Ile Pro Val 225 230 235 240 Phe Ala Leu Gly Arg Ala Gln Glu Leu Leu Leu Ile Leu Asp Glu Tyr 245 250 255 Trp Gln Asn His Pro Glu Leu His Asp Ile Pro Ile Tyr Tyr Ala Ser 260 265 270 Ser Leu Ala Lys Lys Cys Met Ala Val Tyr Gln Thr Tyr Val Asn Ala 275 280 285 Met Asn Asp Lys Ile Arg Lys Gln Ile Asn Ile Asn Asn Pro Phe Val 290 295 300 Phe Lys His Ile Ser Asn Leu Lys Ser Met Asp His Phe Asp Asp Ile 305 310 315 320 Gly Pro Ser Val Val Met Ala Ser Pro Gly Met Met Gln Ser Gly Leu 325 330 335 Ser Arg Glu Leu Phe Glu Ser Trp Cys Thr Asp Lys Arg Asn Gly Val 340 345 350 Ile Ile Ala Gly Tyr Cys Val Glu Gly Thr Leu Ala Lys His Ile Met 355 360 365 Ser Glu Pro Glu Glu Ile Thr Thr Met Ser Gly Gln Lys Leu Pro Leu 370 375 380 Lys Met Ser Val Asp Tyr Ile Ser Phe Ser Ala His Thr Asp Tyr Gln 385 390 395 400 Gln Thr Ser Glu Phe Ile Arg Ala Leu Lys Pro Pro His Val Ile Leu 405 410 415 Val His Gly Glu Gln Asn Glu Met Ala Arg Leu Lys Ala Ala Leu Ile 420 425 430 Arg Glu Tyr Glu Asp Asn Asp Glu Val His Ile Glu Val His Asn Pro 435 440 445 Arg Asn Thr Glu Ala Val Thr Leu Asn Phe Arg Gly Glu Lys Leu Ala 450 455 460 Lys Val Met Gly Phe Leu Ala Asp Lys Lys Pro Glu Gln Gly Gln Arg 465 470 475 480 Val Ser Gly Ile Leu Val Lys Arg Asn Phe Asn Tyr His Ile Leu Ser 485 490 495 Pro Cys Asp Leu Ser Asn Tyr Thr Asp Leu Ala Met Ser Thr Val Lys 500 505 510 Gln Thr Gln Ala Ile Pro Tyr Thr Gly Pro Phe Asn Leu Leu Cys Tyr 515 520 525 Gln Leu Gln Lys Leu Thr Gly Asp Val Glu Glu Leu Glu Ile Gln Glu 530 535 540 Lys Pro Ala Leu Lys Val Phe Lys Asn Ile Thr Val Ile Gln Glu Pro 545 550 555 560 Gly Met Val Val Leu Glu Trp Leu Ala Asn Pro Ser Asn Asp Met Tyr 565 570 575 Ala Asp Thr Val Thr Thr Val Ile Leu Glu Val Gln Ser Asn Pro Lys 580 585 590 Ile Arg Lys Gly Ala Val Gln Lys Val Ser Lys Lys Leu Glu Met His 595 600 605 Val Tyr Ser Lys Arg Leu Glu Ile Met Leu Gln Asp Ile Phe Gly Glu 610 615 620 Asp Cys Val Ser Val Lys Asp Asp Ser Ile Leu Ser Val Thr Val Asp 625 630 635 640 Gly Lys Thr Ala Asn Leu Asn Leu Glu Thr Arg Thr Val Glu Cys Glu 645 650 655 Glu Gly Ser Glu Asp Asp Glu Ser Leu Arg Glu Met Val Glu Leu Ala 660 665 670 Ala Gln Arg Leu Tyr Glu Ala Leu Thr Pro Val His 675 680 234 693 PRT Arabidopsis thaliana 234 Met Ala Ser Ser Ser Thr Ser Leu Lys Arg Arg Glu Gln Pro Ile Ser 1 5 10 15 Arg Asp Gly Asp Gln Leu Ile Val Thr Pro Leu Gly Ala Gly Ser Glu 20 25 30 Val Gly Arg Ser Cys Val Tyr Met Ser Phe Arg Gly Lys Asn Ile Leu 35 40 45 Phe Asp Cys Gly Ile His Pro Ala Tyr Ser Gly Met Ala Ala Leu Pro 50 55 60 Tyr Phe Asp Glu Ile Asp Pro Ser Ser Ile Asp Val Leu Leu Ile Thr 65 70 75 80 His Phe His Ile Asp His Ala Ala Ser Leu Pro Tyr Phe Leu Glu Lys 85 90 95 Thr Thr Phe Asn Gly Arg Val Phe Met Thr His Ala Thr Lys Ala Ile 100 105 110 Tyr Lys Leu Leu Leu Thr Asp Tyr Val Lys Val Ser Lys Val Ser Val 115 120 125 Glu Asp Met Leu Phe Asp Glu Gln Asp Ile Asn Lys Ser Met Asp Lys 130 135 140 Ile Glu Val Ile Asp Phe His Gln Thr Val Glu Val Asn Gly Ile Lys 145 150 155 160 Phe Trp Cys Tyr Thr Ala Gly His Val Leu Gly Ala Ala Met Phe Met 165 170 175 Val Asp Ile Ala Gly Val Arg Ile Leu Tyr Thr Gly Asp Tyr Ser Arg 180 185 190 Glu Glu Asp Arg His Leu Arg Ala Ala Glu Leu Pro Gln Phe Ser Pro 195 200 205 Asp Ile Cys Ile Ile Glu Ser Thr Ser Gly Val Gln Leu His Gln Ser 210 215 220 Arg His Ile Arg Glu Lys Arg Phe Thr Asp Val Ile His Ser Thr Val 225 230 235 240 Ala Gln Gly Gly Arg Val Leu Ile Pro Ala Phe Ala Leu Gly Arg Ala 245 250 255 Gln Glu Leu Leu Leu Ile Leu Asp Glu Tyr Trp Ala Asn His Pro Asp 260 265 270 Leu His Asn Ile Pro Ile Tyr Tyr Ala Ser Pro Leu Ala Lys Lys Cys 275 280 285 Met Ala Val Tyr Gln Thr Tyr Ile Leu Ser Met Asn Asp Arg Ile Arg 290 295 300 Asn Gln Phe Ala Asn Ser Asn Pro Phe Val Phe Lys His Ile Ser Pro 305 310 315 320 Leu Asn Ser Ile Asp Asp Phe Asn Asp Val Gly Pro Ser Val Val Met 325 330 335 Ala Thr Pro Gly Gly Leu Gln Ser Gly Leu Ser Arg Gln Leu Phe Asp 340 345 350 Ser Trp Cys Ser Asp Lys Lys Asn Ala Cys Ile Ile Pro Gly Tyr Met 355 360 365 Val Glu Gly Thr Leu Ala Lys Thr Ile Ile Asn Glu Pro Lys Glu Val 370 375 380 Thr Leu Met Asn Gly Leu Thr Ala Pro Leu Asn Met Gln Val His Tyr 385 390 395 400 Ile Ser Phe Ser Ala His Ala Asp Tyr Ala Gln Thr Ser Thr Phe Leu 405 410 415 Lys Glu Leu Met Pro Pro Asn Ile Ile Leu Val His Gly Glu Ala Asn 420 425 430 Glu Met Met Arg Leu Lys Gln Lys Leu Leu Thr Glu Phe Pro Asp Gly 435 440 445 Asn Thr Lys Ile Met Thr Pro Lys Asn Cys Glu Ser Val Glu Met Tyr 450 455 460 Phe Asn Ser Glu Lys Leu Ala Lys Thr Ile Gly Arg Leu Ala Glu Lys 465 470 475 480 Thr Pro Asp Val Gly Asp Thr Val Ser Gly Ile Leu Val Lys Lys Gly 485 490 495 Phe Thr Tyr Gln Ile Met Ala Pro Asp Glu Leu His Val Phe Ser Gln 500 505 510 Leu Ser Thr Ala Thr Val Thr Gln Arg Ile Thr Ile Pro Phe Val Gly 515 520 525 Ala Phe Gly Val Ile Lys His Arg Leu Glu Lys Ile Phe Glu Ser Val 530 535 540 Glu Phe Ser Thr Asp Glu Glu Ser Gly Leu Pro Ala Leu Lys Val His 545 550 555 560 Glu Arg Val Thr Val Lys Gln Glu Ser Glu Lys His Ile Ser Leu Gln 565 570 575 Trp Ser Ser Asp Pro Ile Ser Asp Met Val Ser Asp Ser Ile Val Ala 580 585 590 Leu Ile Leu Asn Ile Ser Arg Glu Val Pro Lys Ile Val Met Glu Glu 595 600 605 Glu Asp Ala Val Lys Ser Glu Glu Glu Asn Gly Lys Lys Val Glu Lys 610 615 620 Val Ile Tyr Ala Leu Leu Val Ser Leu Phe Gly Asp Val Lys Leu Gly 625 630 635 640 Glu Asn Gly Lys Leu Val Ile Arg Val Asp Gly Asn Val Ala Gln Leu 645 650 655 Asp Lys Glu Ser Gly Glu Val Glu Ser Glu His Ser Gly Leu Lys Glu 660 665 670 Arg Val Arg Val Ala Phe Glu Arg Ile Gln Ser Ala Val Lys Pro Ile 675 680 685 Pro Leu Ser Ala Ser 690 235 779 PRT Saccharomyces cerevisiae 235 Met Glu Arg Thr Asn Thr Thr Thr Phe Lys Phe Phe Ser Leu Gly Gly 1 5 10 15 Ser Asn Glu Val Gly Arg Ser Cys His Ile Leu Gln Tyr Lys Gly Lys 20 25 30 Thr Val Met Leu Asp Ala Gly Ile His Pro Ala Tyr Gln Gly Leu Ala 35 40 45 Ser Leu Pro Phe Tyr Asp Glu Phe Asp Leu Ser Lys Val Asp Ile Leu 50 55 60 Leu Ile Ser His Phe His Leu Asp His Ala Ala Ser Leu Pro Tyr Val 65 70 75 80 Met Gln Arg Thr Asn Phe Gln Gly Arg Val Phe Met Thr His Pro Thr 85 90 95 Lys Ala Ile Tyr Arg Trp Leu Leu Arg Asp Phe Val Arg Val Thr Ser 100 105 110 Ile Gly Ser Ser Ser Ser Ser Met Gly Thr Lys Asp Glu Gly Leu Phe 115 120 125 Ser Asp Glu Asp Leu Val Asp Ser Phe Asp Lys Ile Glu Thr Val Asp 130 135 140 Tyr His Ser Thr Val Asp Val Asn Gly Ile Lys Phe Thr Ala Phe His 145 150 155 160 Ala Gly His Val Leu Gly Ala Ala Met Phe Gln Ile Glu Ile Ala Gly 165 170 175 Leu Arg Val Leu Phe Thr Gly Asp Tyr Ser Arg Glu Val Asp Arg His 180 185 190 Leu Asn Ser Ala Glu Val Pro Pro Leu Ser Ser Asn Val Leu Ile Val 195 200 205 Glu Ser Thr Phe Gly Thr Ala Thr His Glu Pro Arg Leu Asn Arg Glu 210 215 220 Arg Lys Leu Thr Gln Leu Ile His Ser Thr Val Met Arg Gly Gly Arg 225 230 235 240 Val Leu Leu Pro Val Phe Ala Leu Gly Arg Ala Gln Glu Ile Met Leu 245 250 255 Ile Leu Asp Glu Tyr Trp Ser Gln His Ala Asp Glu Leu Gly Gly Gly 260 265 270 Gln Val Pro Ile Phe Tyr Ala Ser Asn Leu Ala Lys Lys Cys Met Ser 275 280 285 Val Phe Gln Thr Tyr Val Asn Met Met Asn Asp Asp Ile Arg Lys Lys 290 295 300 Phe Arg Asp Ser Gln Thr Asn Pro Phe Ile Phe Lys Asn Ile Ser Tyr 305 310 315 320 Leu Arg Asn Leu Glu Asp Phe Gln Asp Phe Gly Pro Ser Val Met Leu 325 330 335 Ala Ser Pro Gly Met Leu Gln Ser Gly Leu Ser Arg Asp Leu Leu Glu 340 345 350 Arg Trp Cys Pro Glu Asp Lys Asn Leu Val Leu Ile Thr Gly Tyr Ser 355 360 365 Ile Glu Gly Thr Met Ala Lys Phe Ile Met Leu Glu Pro Asp Thr Ile 370 375 380 Pro Ser Ile Asn Asn Pro Glu Ile Thr Ile Pro Arg Arg Cys Gln Val 385 390 395 400 Glu Glu Ile Ser Phe Ala Ala His Val Asp Phe Gln Glu Asn Leu Glu 405 410 415 Phe Ile Glu Lys Ile Ser Ala Pro Asn Ile Ile Leu Val His Gly Glu 420 425 430 Ala Asn Pro Met Gly Arg Leu Lys Ser Ala Leu Leu Ser Asn Phe Ala 435 440 445 Ser Leu Lys Gly Thr Asp Asn Glu Val His Val Phe Asn Pro Arg Asn 450 455 460 Cys Val Glu Val Asp Leu Glu Phe Gln Gly Val Lys Val Ala Lys Ala 465 470 475 480 Val Gly Asn Ile Val Asn Glu Ile Tyr Lys Glu Glu Asn Val Glu Ile 485 490 495 Lys Glu Glu Ile Ala Ala Lys Ile Glu Pro Ile Lys Glu Glu Asn Glu 500 505 510 Asp Asn Leu Asp Ser Gln Ala Glu Lys Gly Leu Val Asp Glu Glu Glu 515 520 525 His Lys Asp Ile Val Val Ser Gly Ile Leu Val Ser Asp Asp Lys Asn 530 535 540 Phe Glu Leu Asp Phe Leu Ser Leu Ser Asp Leu Arg Glu His His Pro 545 550 555 560 Asp Leu Ser Thr Thr Ile Leu Arg Glu Arg Gln Ser Val Arg Val Asn 565 570 575 Cys Lys Lys Glu Leu Ile Tyr Trp His Ile Leu Gln Met Phe Gly Glu 580 585 590 Ala Glu Val Leu Gln Asp Asp Asp Arg Val Thr Asn Gln Glu Pro Lys 595 600 605 Val Lys Glu Glu Ser Lys Asp Asn Leu Thr Asn Thr Gly Lys Leu Ile 610 615 620 Leu Gln Ile Met Gly Asp Ile Lys Leu Thr Ile Val Asn Thr Leu Ala 625 630 635 640 Val Val Glu Trp Thr Gln Asp Leu Met Asn Asp Thr Val Ala Asp Ser 645 650 655 Ile Ile Ala Ile Leu Met Asn Val Asp Ser Ala Pro Ala Ser Val Lys 660 665 670 Leu Ser Ser His Ser Cys Asp Asp His Asp His Asn Asn Val Gln Ser 675 680 685 Asn Ala Gln Gly Lys Ile Asp Glu Val Glu Arg Val Lys Gln Ile Ser 690 695 700 Arg Leu Phe Lys Glu Gln Phe Gly Asp Cys Phe Thr Leu Phe Leu Asn 705 710 715 720 Lys Asp Glu Tyr Ala Ser Asn Lys Glu Glu Thr Ile Thr Gly Val Val 725 730 735 Thr Ile Gly Lys Ser Thr Ala Lys Ile Asp Phe Asn Asn Met Lys Ile 740 745 750 Leu Glu Cys Asn Ser Asn Pro Leu Lys Gly Arg Val Glu Ser Leu Leu 755 760 765 Asn Ile Gly Gly Asn Leu Val Thr Pro Leu Cys 770 775 236 554 PRT Synechocystis sp. 236 Met Thr Phe Ser Val Pro Thr Gln Gly Lys Ala Phe Ala Asn Ile Ser 1 5 10 15 Phe Leu Pro Tyr Gly Val Gly Pro Arg Asp Gly Gly Ile Cys Leu Glu 20 25 30 Leu His Leu Gly Pro Tyr Arg Ile Leu Leu Asp Cys Gly Leu Glu Asp 35 40 45 Leu Thr Pro Leu Leu Ala Ala Asp Pro Gly Thr Val Asp Leu Val Phe 50 55 60 Cys Ser His Ala His Arg Asp His Gly Leu Gly Leu Trp Gln Phe His 65 70 75 80 Gln Gln Phe Pro His Ile Pro Ile Leu Ala Ser Glu Val Thr Gln Arg 85 90 95 Leu Leu Pro Leu Asn Trp Pro Asp Glu Phe Val Pro Pro Phe Cys Arg 100 105 110 Val Leu Pro Trp Arg Ser Pro Gln Glu Val Leu Pro Gly Leu Thr Val 115 120 125 Glu Leu Leu Pro Ala Gly His Leu Pro Gly Ala Ala Leu Ile Leu Leu 130 135 140 Glu Tyr His Asn Gly Asp Arg Leu Tyr Arg Val Ile Tyr Thr Gly Asp 145 150 155 160 Tyr Cys Leu Ser His Leu Gln Leu Val Asp Gly Leu Ala Leu Thr Pro 165 170 175 Leu Arg Gly Leu Lys Pro Asp Val Leu Ile Leu Glu Gly His Tyr Gly 180 185 190 Asn Arg Arg Leu Pro His Arg Arg Gln Gln Glu Lys Gln Phe Ile Gln 195 200 205 Ala Ile Glu Thr Val Leu Ala Lys Gly Arg Asn Ile Leu Leu Pro Val 210 215 220 Pro Pro Leu Gly Leu Ala Gln Glu Ile Leu Lys Leu Leu Arg Thr His 225 230 235 240 His Gln Phe Thr Gly Arg Gln Val Asn Leu Trp Ala Gly Glu Ser Val 245 250 255 Ala Arg Gly Cys Asp Ala Tyr Gln Gly Ile Ile Asp His Leu Pro Asp 260 265 270 Asn Val Arg Asn Phe Ala Gln His Gln Pro Leu Phe Trp Asp Asp Lys 275 280 285 Val Tyr Pro His Leu Arg Pro Leu Thr Asp Asp Gln Gly Glu Leu Ser 290 295 300 Leu Ser Ala Pro Ser Ile Val Ile Thr Thr Thr Trp Pro Ala Phe Trp 305 310 315 320 Pro Ser Pro Ala Ala Leu Pro Gly Leu Trp Thr Val Phe Met Pro Gln 325 330 335 Leu Leu Thr Leu Pro Ser Cys Leu Val Asn Phe Ala Trp Gln Asp Leu 340 345 350 Glu Glu Phe Pro Lys Tyr Glu Leu Glu Asp Tyr Leu Leu Ala Asp His 355 360 365 Ser Asp Gly Arg Asn Thr Thr Gln Leu Ile His Asn Leu Arg Pro Gln 370 375 380 His Leu Val Phe Val His Gly Gln Pro Ser Asp Ile Glu Asp Leu Thr 385 390 395 400 Ser Leu Glu Glu Leu Gln Ser Arg Tyr Gln Leu His Ser Pro Ala Ala 405 410 415 Gly Asn Ala Val Ala Leu Pro Ile Gly Asp Arg Phe Val Gln Pro Thr 420 425 430 Pro Pro Pro Pro Gln Ile Tyr Glu Gly Glu Ile His Glu Leu Glu Pro 435 440 445 Asn Lys Gln Ile His His Leu Gly Glu Val Val Ile His Leu Asp Gly 450 455 460 Gln Ile Leu Glu Asn Ser Arg Trp Gly Lys Phe Gly Glu Thr Gly Ile 465 470 475 480 Val Gln Ala Arg Trp Gln Gly Glu Glu Leu Val Leu Arg Gly Ile Ser 485 490 495 Gln Arg Glu Leu Leu Lys Gln Asn Gln Ser Ser Lys Arg Pro Val Asp 500 505 510 Phe Asp Cys Cys Ala Asn Cys Arg His Phe Gln His Tyr His Cys Arg 515 520 525 Asn Pro Val Ser Pro Leu Met Gly Leu Glu Val Arg Ala Asp Gly His 530 535 540 Cys Pro Val Phe Glu Ser Val Ala Ser Ser 545 550 237 636 PRT Methanobacterium thermoautotrophicum 237 Met Val Ser Glu Met Leu Glu Glu Ile Lys Arg Thr Ile Met Gln Arg 1 5 10 15 Leu Pro Glu Arg Val Gln Val Ala Lys Val Glu Phe Glu Gly Pro Glu 20 25 30 Val Val Ile Tyr Thr Lys Asn Pro Glu Ile Ile Thr Glu Asn Gly Asn 35 40 45 Leu Ile Arg Asp Ile Ala Lys Asp Ile Arg Lys Arg Ile Ile Ile Arg 50 55 60 Ser Asp Arg Ser Val Leu Met Asp Pro Glu Lys Ala Ile Arg Lys Ile 65 70 75 80 His Glu Ile Val Pro Glu Glu Ala Lys Ile Thr Asn Ile Ser Phe Asp 85 90 95 Asp Val Thr Cys Glu Val Ile Ile Glu Ala Arg Lys Pro Gly Leu Val 100 105 110 Ile Gly Lys Tyr Gly Ser Thr Ser Arg Glu Ile Val Lys Asn Thr Gly 115 120 125 Trp Ala Pro Lys Ile Leu Arg Thr Pro Pro Ile Ser Ser Glu Ile Ile 130 135 140 Glu Arg Ile Arg Arg Thr Leu Arg Lys Asn Ser Lys Glu Arg Lys Lys 145 150 155 160 Ile Leu Gln Gln Leu Gly Asn Arg Ile His Gln Lys Pro Lys Tyr Asp 165 170 175 Asn Asp Trp Ala Arg Leu Thr Ala Met Gly Gly Phe Arg Glu Val Gly 180 185 190 Arg Ser Cys Leu Tyr Leu Gln Thr Pro Asn Ser Arg Val Leu Leu Asp 195 200 205 Cys Gly Val Asn Val Ala Gly Gly Asp Asp Lys Asn Ser Tyr Pro Tyr 210 215 220 Leu Asn Val Pro Glu Phe Thr Leu Asp Ser Leu Asp Ala Val Ile Ile 225 230 235 240 Thr His Ala His Leu Asp His Ser Gly Phe Leu Pro Tyr Leu Tyr His 245 250 255 Tyr Gly Tyr Asp Gly Pro Val Tyr Cys Thr Ala Pro Thr Arg Asp Leu 260 265 270 Met Thr Leu Leu Gln Leu Asp His Ile Asp Ile Ala His Arg Glu Asp 275 280 285 Glu Pro Leu Pro Phe Asn Val Lys His Val Lys Lys Ser Val Lys His 290 295 300 Thr Ile Thr Leu Asp Tyr Gly Glu Val Thr Asp Ile Ala Pro Asp Ile 305 310 315 320 Arg Leu Thr Leu His Asn Ala Gly His Ile Leu Gly Ser Ala Met Ala 325 330 335 His Leu His Ile Gly Asp Gly Gln His Asn Met Val Tyr Thr Gly Asp 340 345 350 Phe Lys Tyr Glu Gln Ser Arg Leu Leu Glu Ala Ala Ala Asn Arg Phe 355 360 365 Pro Arg Ile Glu Thr Leu Val Met Glu Ser Thr Tyr Gly Gly His Glu 370 375 380 Asp Val Gln Pro Ser Arg Asn Arg Ala Glu Lys Glu Leu Val Lys Thr 385 390 395 400 Ile Tyr Ser Thr Leu Arg Arg Gly Gly Lys Ile Leu Ile Pro Val Phe 405 410 415 Ala Val Gly Arg Ala Gln Glu Leu Met Ile Val Leu Glu Glu Tyr Ile 420 425 430 Arg Thr Gly Ile Ile Asp Glu Val Pro Val Tyr Ile Asp Gly Met Ile 435 440 445 Trp Glu Ala Asn Ala Ile His Thr Ala Arg Pro Glu Tyr Leu Ser Lys 450 455 460 Asp Leu Arg Asp Gln Ile Phe His Met Gly His Asn Pro Phe Ile Ser 465 470 475 480 Asp Ile Phe His Lys Val Asn Gly Met Asp Glu Arg Arg Glu Ile Val 485 490 495 Glu Gly Glu Pro Ser Ile Ile Leu Ser Thr Ser Gly Met Leu Thr Gly 500 505 510 Gly Asn Ser Leu Glu Tyr Phe Lys Trp Leu Cys Glu Asp Pro Asp Asn 515 520 525 Ser Leu Val Phe Val Gly Tyr Gln Ala Glu Gly Ser Leu Gly Arg Arg 530 535 540 Ile Gln Lys Gly Trp Lys Glu Ile Pro Leu Lys Asp Glu Asp Asp Lys 545 550 555 560 Met Arg Val Tyr Asn Val Arg Met Asn Ile Lys Thr Ile Glu Gly Phe 565 570 575 Ser Gly His Ser Asp Arg Arg Gln Leu Met Glu Tyr Val Lys Arg Ile 580 585 590 Ser Pro Lys Pro Glu Lys Ile Leu Leu Cys His Gly Asp Asn Tyr Lys 595 600 605 Thr Leu Asp Leu Ala Ser Ser Ile Tyr Arg Thr Tyr Arg Ile Glu Thr 610 615 620 Lys Thr Pro Leu Asn Leu Glu Thr Val Arg Ile Gln 625 630 635 238 1040 PRT Homo sapiens 238 Met Leu Glu Asp Ile Ser Glu Glu Asp Ile Trp Glu Tyr Lys Ser Lys 1 5 10 15 Arg Lys Pro Lys Arg Val Asp Pro Asn Asn Gly Ser Lys Asn Ile Leu 20 25 30 Lys Ser Val Glu Lys Ala Thr Asp Gly Lys Tyr Gln Ser Lys Arg Ser 35 40 45 Arg Asn Arg Lys Arg Ala Ala Glu Ala Lys Glu Val Lys Asp His Glu 50 55 60 Val Pro Leu Gly Asn Ala Gly Cys Gln Thr Ser Val Ala Ser Ser Gln 65 70 75 80 Asn Ser Ser Cys Gly Asp Gly Ile Gln Gln Thr Gln Asp Lys Glu Thr 85 90 95 Thr Pro Gly Lys Leu Cys Arg Thr Gln Lys Ser Gln His Val Ser Pro 100 105 110 Lys Ile Arg Pro Val Tyr Asp Gly Tyr Cys Pro Asn Cys Gln Met Pro 115 120 125 Phe Ser Ser Leu Ile Gly Gln Thr Pro Arg Trp His Val Phe Glu Cys 130 135 140 Leu Asp Ser Pro Pro Arg Ser Glu Thr Glu Cys Pro Asp Gly Leu Leu 145 150 155 160 Cys Thr Ser Thr Ile Pro Phe His Tyr Lys Arg Tyr Thr His Phe Leu 165 170 175 Leu Ala Gln Ser Arg Ala Gly Asp His Pro Phe Ser Ser Pro Ser Pro 180 185 190 Ala Ser Gly Gly Ser Phe Ser Glu Thr Lys Ser Gly Val Leu Cys Ser 195 200 205 Leu Glu Glu Arg Trp Ser Ser Tyr Gln Asn Gln Thr Asp Asn Ser Val 210 215 220 Ser Asn Asp Pro Leu Leu Met Thr Gln Tyr Phe Lys Lys Ser Pro Ser 225 230 235 240 Leu Thr Glu Ala Ser Glu Lys Ile Ser Thr His Ile Gln Thr Ser Gln 245 250 255 Gln Ala Leu Gln Phe Thr Asp Phe Val Glu Asn Asp Lys Leu Val Gly 260 265 270 Val Ala Leu Arg Leu Ala Asn Asn Ser Glu His Ile Asn Leu Pro Leu 275 280 285 Pro Glu Asn Asp Phe Ser Asp Cys Glu Ile Ser Tyr Ser Pro Leu Gln 290 295 300 Ser Asp Glu Asp Thr His Asp Ile Asp Glu Lys Pro Asp Asp Ser Gln 305 310 315 320 Glu Gln Leu Phe Phe Thr Glu Ser Ser Lys Asp Gly Ser Leu Glu Glu 325 330 335 Asp Asp Asp Ser Cys Gly Phe Phe Lys Lys Arg His Gly Pro Leu Leu 340 345 350 Lys Asp Gln Asp Glu Ser Cys Pro Lys Val Asn Ser Phe Leu Thr Arg 355 360 365 Asp Lys Tyr Asp Glu Gly Leu Tyr Arg Phe Asn Ser Leu Asn Asp Leu 370 375 380 Ser Gln Pro Ile Ser Gln Asn Asn Glu Ser Thr Leu Pro Tyr Asp Leu 385 390 395 400 Ala Cys Thr Gly Gly Asp Phe Val Leu Phe Pro Pro Ala Leu Ala Gly 405 410 415 Lys Leu Ala Ala Ser Val His Gln Ala Thr Lys Ala Lys Pro Asp Glu 420 425 430 Pro Glu Phe His Ser Ala Gln Ser Asn Lys Gln Lys Gln Val Ile Glu 435 440 445 Glu Ser Ser Val Tyr Asn Gln Val Ser Leu Pro Leu Val Lys Ser Leu 450 455 460 Met Leu Lys Pro Phe Glu Ser Gln Val Glu Gly Tyr Leu Ser Ser Gln 465 470 475 480 Pro Thr Gln Asn Thr Ile Arg Lys Leu Ser Ser Glu Asn Leu Asn Ala 485 490 495 Lys Asn Asn Thr Asn Ser Ala Cys Phe Cys Arg Lys Ala Leu Glu Gly 500 505 510 Val Pro Val Gly Lys Ala Thr Ile Leu Asn Thr Glu Asn Leu Ser Ser 515 520 525 Thr Pro Ala Pro Lys Tyr Leu Lys Ile Leu Pro Ser Gly Leu Lys Tyr 530 535 540 Asn Ala Arg His Pro Ser Thr Lys Val Met Lys Gln Met Asp Ile Gly 545 550 555 560 Val Tyr Phe Gly Leu Pro Pro Lys Arg Lys Glu Glu Lys Leu Leu Gly 565 570 575 Glu Ser Ala Leu Glu Gly Ile Asn Leu Asn Pro Val Pro Ser Pro Asn 580 585 590 Gln Lys Arg Ser Ser Gln Cys Lys Arg Lys Ala Glu Lys Ser Leu Ser 595 600 605 Asp Leu Glu Phe Asp Ala Ser Thr Leu His Glu Ser Gln Leu Ser Val 610 615 620 Glu Leu Ser Ser Glu Arg Ser Gln Arg Gln Lys Lys Arg Cys Arg Lys 625 630 635 640 Ser Asn Ser Leu Gln Glu Gly Ala Cys Gln Lys Arg Ser Asp His Leu 645 650 655 Ile Asn Thr Glu Ser Glu Ala Val Asn Leu Ser Lys Val Lys Val Phe 660 665 670 Thr Lys Ser Ala His Gly Gly Leu Gln Arg Gly Asn Lys Lys Ile Pro 675 680 685 Glu Ser Ser Asn Val Gly Gly Ser Arg Lys Lys Thr Cys Pro Phe Tyr 690 695 700 Lys Lys Ile Pro Gly Thr Gly Phe Thr Val Asp Ala Phe Gln Tyr Gly 705 710 715 720 Val Val Glu Gly Cys Thr Ala Tyr Phe Leu Thr His Phe His Ser Asp 725 730 735 His Tyr Ala Gly Leu Ser Lys His Phe Thr Phe Pro Val Tyr Cys Ser 740 745 750 Glu Ile Thr Gly Asn Leu Leu Lys Asn Lys Leu His Val Gln Glu Gln 755 760 765 Tyr Ile His Pro Leu Pro Leu Asp Thr Glu Cys Ile Val Asn Gly Val 770 775 780 Lys Val Val Leu Leu Asp Ala Asn His Cys Pro Gly Ala Val Met Ile 785 790 795 800 Leu Phe Tyr Leu Pro Asn Gly Thr Val Ile Leu His Thr Gly Asp Phe 805 810 815 Arg Ala Asp Pro Ser Met Glu Arg Ser Leu Leu Ala Asp Gln Lys Val 820 825 830 His Met Leu Tyr Leu Asp Thr Thr Tyr Cys Ser Pro Glu Tyr Thr Phe 835 840 845 Pro Ser Gln Gln Glu Val Ile Arg Phe Ala Ile Asn Thr Ala Phe Glu 850 855 860 Ala Val Thr Leu Asn Pro His Ala Leu Val Val Cys Gly Thr Tyr Ser 865 870 875 880 Ile Gly Lys Glu Lys Val Phe Leu Ala Ile Ala Asp Val Leu Gly Ser 885 890 895 Lys Val Gly Met Ser Gln Glu Lys Tyr Lys Thr Leu Gln Cys Leu Asn 900 905 910 Ile Pro Glu Ile Asn Ser Leu Ile Thr Thr Asp Met Cys Ser Ser Leu 915 920 925 Val His Leu Leu Pro Met Met Gln Ile Asn Phe Lys Gly Leu Gln Ser 930 935 940 His Leu Lys Lys Cys Gly Gly Lys Tyr Asn Gln Ile Leu Ala Phe Arg 945 950 955 960 Pro Thr Gly Trp Thr His Ser Asn Lys Phe Thr Arg Ile Ala Asp Val 965 970 975 Ile Pro Gln Thr Lys Gly Asn Ile Ser Ile Tyr Gly Ile Pro Tyr Ser 980 985 990 Glu His Ser Ser Tyr Leu Glu Met Lys Arg Phe Val Gln Trp Leu Lys 995 1000 1005 Pro Gln Lys Ile Ile Pro Thr Val Asn Val Gly Thr Trp Lys Ser Arg 1010 1015 1020 Ser Thr Met Glu Lys Tyr Phe Arg Glu Trp Lys Leu Glu Ala Gly Tyr 1025 1030 1035 1040 239 723 PRT Arabidopsis thaliana 239 Met Ser Asn Thr Val Glu Asp Asp Asp Asp Asp Phe Gln Ile Pro Pro 1 5 10 15 Ser Ser Gln Leu Ser Ile Arg Lys Pro Leu His Pro Thr Asn Ala Asn 20 25 30 Asn Ile Ser His Arg Pro Pro Asn Lys Lys Pro Arg Leu Cys Arg Tyr 35 40 45 Pro Gly Lys Glu Asn Val Thr Pro Pro Pro Ser Pro Asp Pro Asp Leu 50 55 60 Phe Cys Ser Ser Ser Thr Pro His Cys Ile Leu Asp Cys Ile Pro Ser 65 70 75 80 Ser Val Asp Cys Ser Leu Gly Asp Phe Asn Gly Pro Ile Ser Ser Leu 85 90 95 Gly Glu Glu Asp Lys Glu Asp Lys Asp Asp Cys Ile Lys Val Asn Arg 100 105 110 Glu Gly Tyr Leu Cys Asn Ser Met Glu Ala Arg Leu Leu Lys Ser Arg 115 120 125 Ile Cys Leu Gly Phe Asp Ser Gly Ile His Glu Asp Asp Glu Gly Phe 130 135 140 Val Glu Ser Asn Ser Glu Leu Asp Val Leu Ile Asn Leu Cys Ser Glu 145 150 155 160 Ser Glu Gly Arg Ser Gly Glu Phe Ser Leu Gly Lys Asp Asp Ser Ile 165 170 175 Gln Cys Pro Leu Cys Ser Met Asp Ile Ser Ser Leu Ser Glu Glu Gln 180 185 190 Arg Gln Val His Ser Asn Thr Cys Leu Asp Lys Ser Tyr Asn Gln Pro 195 200 205 Ser Glu Gln Asp Ser Leu Arg Lys Cys Glu Asn Leu Ser Ser Leu Ile 210 215 220 Lys Glu Ser Ile Asp Asp Pro Val Gln Leu Pro Gln Leu Val Thr Asp 225 230 235 240 Leu Ser Pro Val Leu Lys Trp Leu Arg Ser Leu Gly Leu Ala Lys Tyr 245 250 255 Glu Asp Val Phe Ile Arg Glu Glu Ile Asp Trp Asp Thr Leu Gln Ser 260 265 270 Leu Thr Glu Glu Asp Leu Leu Ser Ile Gly Ile Thr Ser Leu Gly Pro 275 280 285 Arg Lys Lys Ile Val Asn Ala Leu Ser Gly Val Arg Asp Pro Phe Ala 290 295 300 Ser Ser Ala Glu Val Gln Ala Gln Ser His Cys Thr Ser Gly His Val 305 310 315 320 Thr Glu Arg Gln Arg Asp Lys Ser Thr Thr Arg Lys Ala Ser Glu Pro 325 330 335 Lys Lys Pro Thr Ala Asn Lys Leu Ile Thr Glu Phe Phe Pro Gly Gln 340 345 350 Ala Thr Glu Gly Thr Lys Ile Arg Thr Ala Pro Lys Pro Val Ala Glu 355 360 365 Lys Ser Pro Ser Asp Ser Ser Ser Arg Arg Ala Val Arg Arg Asn Gly 370 375 380 Asn Asn Gly Lys Ser Lys Val Ile Pro His Trp Asn Cys Ile Pro Gly 385 390 395 400 Thr Pro Phe Arg Val Asp Ala Phe Lys Tyr Leu Thr Arg Asp Cys Cys 405 410 415 His Trp Phe Leu Thr His Phe His Leu Asp His Tyr Gln Gly Leu Thr 420 425 430 Lys Ser Phe Ser His Gly Lys Ile Tyr Cys Ser Leu Val Thr Ala Lys 435 440 445 Leu Val Asn Met Lys Ile Gly Ile Pro Trp Glu Arg Leu Gln Val Leu 450 455 460 Asp Leu Gly Gln Lys Val Asn Ile Ser Gly Ile Asp Val Thr Cys Phe 465 470 475 480 Asp Ala Asn His Cys Pro Gly Ser Ile Met Ile Leu Phe Glu Pro Ala 485 490 495 Asn Gly Lys Ala Val Leu His Thr Gly Asp Phe Arg Tyr Ser Glu Glu 500 505 510 Met Ser Asn Trp Leu Ile Gly Ser His Ile Ser Ser Leu Ile Leu Asp 515 520 525 Thr Thr Tyr Cys Asn Pro Gln Tyr Asp Phe Pro Lys Gln Glu Ala Val 530 535 540 Ile Gln Phe Val Val Glu Ala Ile Gln Ala Glu Ala Phe Asn Pro Lys 545 550 555 560 Thr Leu Phe Leu Ile Gly Ser Tyr Thr Ile Gly Lys Glu Arg Leu Phe 565 570 575 Leu Glu Val Ala Arg Val Leu Arg Glu Lys Ile Tyr Ile Asn Pro Ala 580 585 590 Lys Leu Lys Leu Leu Glu Cys Leu Gly Phe Ser Lys Asp Asp Ile Gln 595 600 605 Trp Phe Thr Val Lys Glu Glu Glu Ser His Ile His Val Val Pro Leu 610 615 620 Trp Thr Leu Ala Ser Phe Lys Arg Leu Lys His Val Ala Asn Arg Tyr 625 630 635 640 Thr Asn Arg Tyr Ser Leu Ile Val Ala Phe Ser Pro Thr Gly Trp Thr 645 650 655 Ser Gly Lys Thr Lys Lys Lys Ser Pro Gly Arg Arg Leu Gln Gln Gly 660 665 670 Thr Ile Ile Arg Tyr Glu Val Pro Tyr Ser Glu His Ser Ser Phe Thr 675 680 685 Glu Leu Lys Glu Phe Val Gln Lys Val Ser Pro Glu Val Ile Ile Pro 690 695 700 Ser Val Asn Asn Asp Gly Pro Asp Ser Ala Ala Ala Met Val Ser Leu 705 710 715 720 Leu Val Thr 240 661 PRT Saccharomyces cerevisiae 240 Met Ser Arg Lys Ser Ile Val Gln Ile Arg Arg Ser Glu Val Lys Arg 1 5 10 15 Lys Arg Ser Ser Thr Ala Ser Ser Thr Ser Glu Gly Lys Thr Leu His 20 25 30 Lys Asn Thr His Thr Ser Ser Lys Arg Gln Arg Thr Leu Thr Glu Phe 35 40 45 Asn Ile Pro Thr Ser Ser Asn Leu Pro Val Arg Ser Ser Ser Tyr Ser 50 55 60 Phe Ser Arg Phe Ser Cys Ser Thr Ser Asn Lys Asn Thr Glu Pro Val 65 70 75 80 Ile Ile Asn Asp Asp Asp His Asn Ser Ile Cys Leu Glu Asp Thr Ala 85 90 95 Lys Val Glu Ile Thr Ile Asp Thr Asp Glu Glu Glu Leu Val Ser Leu 100 105 110 His Asp Asn Glu Val Ser Ala Ile Glu Asn Arg Thr Glu Asp Arg Ile 115 120 125 Val Thr Glu Leu Glu Glu Gln Val Asn Val Lys Val Ser Thr Glu Val 130 135 140 Ile Gln Cys Pro Ile Cys Leu Glu Asn Leu Ser His Leu Glu Leu Tyr 145 150 155 160 Glu Arg Glu Thr His Cys Asp Thr Cys Ile Gly Ser Asp Pro Ser Asn 165 170 175 Met Gly Thr Pro Lys Lys Asn Ile Arg Ser Phe Ile Ser Asn Pro Ser 180 185 190 Ser Pro Ala Lys Thr Lys Arg Asp Ile Ala Thr Ser Lys Lys Pro Thr 195 200 205 Arg Val Lys Leu Val Leu Pro Ser Phe Lys Ile Ile Lys Phe Asn Asn 210 215 220 Gly His Glu Ile Val Val Asp Gly Phe Asn Tyr Lys Ala Ser Glu Thr 225 230 235 240 Ile Ser Gln Tyr Phe Leu Ser His Phe His Ser Asp His Tyr Ile Gly 245 250 255 Leu Lys Lys Ser Trp Asn Asn Pro Asp Glu Asn Pro Ile Lys Lys Thr 260 265 270 Leu Tyr Cys Ser Lys Ile Thr Ala Ile Leu Val Asn Leu Lys Phe Lys 275 280 285 Ile Pro Met Asp Glu Ile Gln Ile Leu Pro Met Asn Lys Arg Phe Trp 290 295 300 Ile Thr Asp Thr Ile Ser Val Val Thr Leu Asp Ala Asn His Cys Pro 305 310 315 320 Gly Ala Ile Ile Met Leu Phe Gln Glu Phe Leu Ala Asn Ser Tyr Asp 325 330 335 Lys Pro Ile Arg Gln Ile Leu His Thr Gly Asp Phe Arg Ser Asn Ala 340 345 350 Lys Met Ile Glu Thr Ile Gln Lys Trp Leu Ala Glu Thr Ala Asn Glu 355 360 365 Thr Ile Asp Gln Val Tyr Leu Asp Thr Thr Tyr Met Thr Met Gly Tyr 370 375 380 Asn Phe Pro Ser Gln His Ser Val Cys Glu Thr Val Ala Asp Phe Thr 385 390 395 400 Leu Arg Leu Ile Lys His Gly Lys Asn Lys Thr Phe Gly Asp Ser Gln 405 410 415 Arg Asn Leu Phe His Phe Gln Arg Lys Lys Thr Leu Thr Thr His Arg 420 425 430 Tyr Arg Val Leu Phe Leu Val Gly Thr Tyr Thr Ile Gly Lys Glu Lys 435 440 445 Leu Ala Ile Lys Ile Cys Glu Phe Leu Lys Thr Lys Leu Phe Val Met 450 455 460 Pro Asn Ser Val Lys Phe Ser Met Met Leu Thr Val Leu Gln Asn Asn 465 470 475 480 Glu Asn Gln Asn Asp Met Trp Asp Glu Ser Leu Leu Thr Ser Asn Leu 485 490 495 His Glu Ser Ser Val His Leu Val Pro Ile Arg Val Leu Lys Ser Gln 500 505 510 Glu Thr Ile Glu Ala Tyr Leu Lys Ser Leu Lys Glu Leu Glu Thr Asp 515 520 525 Tyr Val Lys Asp Ile Glu Asp Val Val Gly Phe Ile Pro Thr Gly Trp 530 535 540 Ser His Asn Phe Gly Leu Lys Tyr Gln Lys Lys Asn Asp Asp Asp Glu 545 550 555 560 Asn Glu Met Ser Gly Asn Thr Glu Tyr Cys Leu Glu Leu Met Lys Asn 565 570 575 Asp Arg Asp Asn Asp Asp Glu Asn Gly Phe Glu Ile Ser Ser Ile Leu 580 585 590 Arg Gln Tyr Lys Lys Tyr Asn Lys Phe Gln Val Phe Asn Val Pro Tyr 595 600 605 Ser Glu His Ser Ser Phe Asn Asp Leu Val Lys Phe Gly Cys Lys Leu 610 615 620 Lys Cys Ser Glu Val Ile Pro Thr Val Asn Leu Asn Asn Leu Trp Lys 625 630 635 640 Val Arg Tyr Met Thr Asn Trp Phe Gln Cys Trp Glu Asn Val Arg Lys 645 650 655 Thr Arg Ala Ala Lys 660

Claims (3)

What is claimed is:
1. A kit comprising primers for sequencing the region of HPC2 encoding i) amino acid residue 217 or ii) amino acid residue 541.
2. A kit comprising i) primers for sequencing the region of HPC2 encoding amino acid residue 217 and ii) primers for sequencing the region of HPC2 encoding amino acid residue 541.
3. A kit comprising primers for performing an allele specific polymerase chain reaction wherein one primer is complementary to a region of HPC2 comprising the codon encoding i) amino acid residue 217 or ii) amino acid residue 541.
US09/988,687 1998-11-06 2001-11-20 Chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes Abandoned US20030045704A1 (en)

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US09/434,382 US6844189B1 (en) 1998-11-06 1999-11-05 Chromosome 17P-linked prostate cancer susceptibility gene
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US09/988,626 Abandoned US20030044959A1 (en) 1998-11-06 2001-11-20 Chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes
US09/988,686 Abandoned US20030120052A1 (en) 1998-11-06 2001-11-20 Chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes
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US09/988,626 Abandoned US20030044959A1 (en) 1998-11-06 2001-11-20 Chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes
US09/988,686 Abandoned US20030120052A1 (en) 1998-11-06 2001-11-20 Chromosome 17p-linked prostrate cancer susceptibility gene and a paralog and orthologous genes

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US20050075543A1 (en) * 2003-10-03 2005-04-07 Calabrese Charles A. Method of anonymous medical testing and providing the patient with the test results
NZ596070A (en) * 2009-05-08 2013-10-25 Decode Genetics Ehf Genetic variants contributing to risk of prostate cancer

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5075216A (en) * 1988-09-23 1991-12-24 Cetus Corporation Methods for dna sequencing with thermus aquaticus dna polymerase
US5780263A (en) * 1995-06-05 1998-07-14 Human Genome Sciences, Inc. Human CCN-like growth factor

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5075216A (en) * 1988-09-23 1991-12-24 Cetus Corporation Methods for dna sequencing with thermus aquaticus dna polymerase
US5780263A (en) * 1995-06-05 1998-07-14 Human Genome Sciences, Inc. Human CCN-like growth factor

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