INHERITED AND SOMATIC MUTATIONS OF APC GENE IN COLORECTAL CANCER OF HUMANS

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of grants awarded by the National Institutes of Health. TECHNICAL AREA OF THE INVENTION

The invention relates to the area of cancer diagnostics and ther¬ apeutics. More particularly, the invention relates to detection of the germline and somatic alterations of wild- type APC genes. In addition, it relates to therapeutic intervention to restore the function of APC gene product. BACKGROUND OF THE INVENTION

According to the model of Knudson for tumorigenesis (Cancer Research, Vol. 45, p. 1482, 1985), there are tumor suppressor genes in all normal cells which, when they become non-functional due to muta¬ tion, cause neoplastic development. Evidence for this model has been found in the cases of retinoblastoma and colorectal tumors. The impli¬ cated suppressor genes in those tumors, RB, p53, DCC and MCC, were found to be deleted or altered in many cases of the tumors studied. (Hansen and Cavenee, Cancer Research, Vol.. 47, pp. 5518-5527 (1987); Baker et al., Science, Vol.. 244, p. 217 (1989); Fearon et al., Science, Vol. 247, p. 49 (1990); Kinzler et al. Science Vol. 251. p. 1366 (1991).)

In order to fully understand the pathogenesis of tumors, it will be necessary to identify the other suppressor genes that play a role in the tumorigenesis process. Prominent among these is the one(s) pre¬ sumptively located at 5q2l. Cytogenetic (Herrera et al., Am J. Med. Genet., Vol. 25, p. 473 (1986) and linkage (Leppert et al., Science, Vol. 238, p. 1411 (1987); Bodmer et al., Nature, Vol. 328, p. 614 (1987)) stud¬ ies have shown that this chromosome region harbors the gene

responsible for familial adenomatous polyposis (FAP) and Gardner's Syndrome (GS). FAP is an autosomal-dominant, inherited disease in which affected individuals develop hundreds to thousands of adenomatous polyps, some of which progress to malignancy. GS is a variant of FAP in which dέsmoid tumors, osteomas and other soft tissue tumors occur together with multiple adenomas of the colon and rec¬ tum. A less severe form of polyposis has been identified in which only a few (2-40) polyps develop. This condition also is familial and is linked to the same chromosomal markers as FAP and GS (Leppert et al., New England Journal of Medicine, Vol. 322, pp. 904-908, 1990.) Additionally, this chromosomal region is often deleted from the adenomas (Vogelstein et al., N. Engl. J. Med., Vol. 319, p. 525 (1988)) and carcino¬ mas (Vogelstein et al., N. Engl. J. Med., Vol. 319, p. 525 (1988); Solomon et al., Nature, Vol. 328, p. 616 (1987); Sasaki et al., Cancer Research, Vol. 49, p. 4402 (1989); Delattre et al., Lancet, Vol. 2, p. 353 (1989); and Ashton-Rickardt et al., Oncogene, Vol. 4, p. 1169 (1989)) of patients without FAP (sporadic tumors). Thus, a putative suppressor gene on chromosome 5q21 appears to play a role in the early stages of colorectal neoplasia in both sporadic and familial tumors.

Although the MCC gene has been identified on 5q2l as a candi¬ date suppressor gene, it does not appear to be altered in FAP or GS patients. Thus there is a need in the art for investigations of this chro¬ mosomal region to identify genes and to determine if any of such genes are associated with FAP and/or GS and the process of tumorigenesis. SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method for diagnosing and prognosing a neoplastic tissue of a human.

It is another object of the invention to provide a method of detecting genetic predisposition to cancer.

It is another object of the invention to provide a method of sup¬ plying wild-type APC gene function to a cell which has lost said gene function.

It is yet another object of the invention to provide a kit for determination of the nueleotide sequence of APC alleles by the polymerase chain reaction.

It is still another object of the invention to provide nucleic acid probes for detection of mutations in the human APC gene.

It is still another object of the invention to provide a cDNA mol¬ f ecule encoding the APC gene product.

It is yet another object of the invention to provide a preparation of the human APC protein.

It is another object of the invention to provide a method of screening for genetic predisposition to cancer.

It is an object of the invention to provide methods of testing therapeutic agents for the ability to suppress neoplasia.

It is still another object of the invention to provide animals car¬ rying mutant APC alleles.

These and other objects of the invention are provided by one or more of the embodiments which are described below. In one embodi¬ ment of the present invention a method of diagnosing or prognosing a neoplastic tissue of a human is provided comprising: detecting somatic alteration of wild-type APC genes or their expression products in a sporadic colorectal cancer tissue, said alteration indicating neoplasia of the tissue.

In yet another embodiment a method is provided of detecting genetic predisposition to cancer in a human including familial adenomatous polyposis (FAP) and Gardner's Syndrome (GS), comprising: isolating a human sample selected from the group consisting of blood and fetal tissue; detecting alteration of wild-type APC gene coding sequences or their expression products from the sample, said alteration indicating genetic predisposition to cancer.

In another embodiment of the present invention a method is provided for supplying wild-type APC gene function to a cell which has lost said gene function by virtue of a mutation in the APC gene, com¬ prising: introducing a wild-type APC gene into a cell which has lost said gene function such that said wild-type gene is expressed in the

) i cell.

In another embodiment a method of supplying wild-type APC gene function to a cell is provided comprising: introducing a portion of a wild-type APC gene into a cell which has lost said gene function such

that said portion is expressed in the cell, said portion encoding a part of the APC protein which is required for non-neoplastic growth of said cell. APC protein can also be applied to cells or administered to ani¬ mals to remediate for mutant APC genes. Synthetic peptides or drugs can also be used to mimic APC function in cells which have altered APC expression.

In yet another embodiment a pair of single stranded primers is provided for determination of the nucleotide sequence of the APC gene by polymerase chain reaction. The sequence of said pair of single stranded DNA primers is derived from chromosome 5q band 21, said pair of primers allowing synthesis of APC gene coding sequences.

In still another embodiment of the invention a nucleic acid probe is provided which is complementary to human wild-type APC gene cod¬ ing sequences and which can form mismatches with mutant APC genes, thereby allowing their detection by enzymatic or chemical cleavage or by shifts in electrophoretic mobility.

In another embodiment of the invention a method is provided for detecting the presence of a neoplastic tissue in a human. The method comprises isolating a body sample from a human; detecting in said sam¬ ple alteration of a wild-type APC gene sequence or wild-type APC expression product, said alteration indicating the presence of a neoplastic tissue in the human.

In still another embodiment a cDNA molecule is provided which comprises the coding sequence of the APC gene.

In even another embodiment a preparation of the human APC protein is provided which is substantially free of other human proteins. The amino acid sequence of the protein is shown in Figure 3 or 7.

In yet another embodiment of the invention a method is provided for screening for genetic predisposition to cancer, including familial adenomatous polyposis (FAP) and Gardner's Syndrome (GS), in a human. The method comprises: detecting among kindred persons the presence of a DNA polymorphism which is linked to a mutant APC allele in an individual having a genetic predisposition to cancer, said kindred being genetically related to the individual, the presence of said polymorphism suggesting a predisposition to cancer.

In another embodiment of the invention a method of testing therapeutic agents for the ability to suppress a neoplastically trans¬ formed phenotype is provided. The method comprises: applying a test f substance to a cultured epithelial cell which carries a mutation in an APC allele; and determining whether said test substance suppresses the neoplastically transformed phenotype of the cell.

In another embodiment of the invention a method of testing therapeutic agents for the ability to suppress a neoplastically trans¬ formed phenotype is provided. The method comprises: administering a test substance to an animal which carries a mutant APC allele; and determining whether said test substance prevents or suppresses the growth of tumors.

In still other embodiments of the invention transgenic animals are provided. The animals carry a mutant APC allele from a second animal species or have been genetically engineered to contain an inser¬ tion mutation which disrupts an APC allele.

The present invention provides the art with the information that the APC gene, a heretofore unknown gene is, in fact, a target of muta- tional alterations on chromosome 5q21 and that these alterations are associated with the process of tumorigenesis. This information allows highly specific assays to be performed to assess the neoplastic status of a particular tissue or the predisposition to cancer of an individual. This invention has applicability to Familial Adenomatous Polyposis, sporadic colorectal cancers, Gardner's Syndrome, as well as the less severe familial polyposis discusses above. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows an overview of yeast artificial chromosome (YAC) contigs. Genetic distances between selected RFLP markers rom within the contigs are shown in centiMorgans.

Figure IB shows a detailed map of the three central contigs.

] The position of the six identified genes from within the FAP region is shown: the 5' and 3' ends of the transcripts from these genes have in general not yet been isolated, as indicated by the string of dots sur¬ rounding the bars denoting the genes' positions. Selected restriction

endonuclease recognition sites are indicated. B, BssH2; S, Sstπ; M, MM; N, Nrul.

Figure 2 shows the sequence of TBl and TB2 genes. The cDNA sequence of the TBl gene was determined from the analysis of 11 cDNA clones derived from normal colon and liver, as described in the text. A total of 2314 bp were contained within the overlapping cD A clones, defining an ORF of 424 amino acids beginning at nucleotide 1. Only the predicted amino acids from the ORF are shown. The carboxy-terminal end of the ORF has apparently been identified, but the 5* end of the TBl transcript has not yet been precisely determined. The cDNA sequence of the TB2 gene was determined from the YS-39 clone derived as described in the text. This clone consisted of 2300 bp and defined an ORF of 185 amino acids beginning at nucleotide 1. Only the predicted amino acids are shown. The carboxy terminal end of the ORF has apparently been identified, but the 5' end of the TB2 transcript has not been precisely determined.

Figure 3 shows the sequence of the APC gene product. The cDNA sequence was determined through the analysis of 87 cDNA clones derived from normal colon, liver, and brain. A total of 8973 bp were contained within overlapping cDNA clones, defining an ORF of 2842 amino acids. In frame stop codons surrounded this ORF, as described in the text, suggesting that the entire APC gene product was represented in the ORF illustrated. Only the predicted amino acids are shown.

Figure 4 shows the local similarity between human APC and ral2 of yeast. Local similarity among the APC and MCC genes and the m3 muscarinic acetylcholine receptor is shown. The region of the mAChR shown corresponds to that responsible for coupling the receptor to G proteins. The connecting lines indicate identities; dots indicate related amino acids residues.

Figure 5 shows the genomic map of the 1200 kb Notl fragment at the FAP locus. The Notl fragment is shown as a bold line. Relevant parts of the deletion chromosomes from patients 3214 and 3824 are shown as stippled lines. Probes used to characterize the Notl ragment and the deletions, and three YACs from which subclones were obtained, are shown below the restriction map. The chimeric end of YAC

183H12 is indicated by a dotted line. The orientation and approximate position of MCC are indicated above the map.

Figure 6 shows the DNA sequence and predicted amino acid sequence of DPI (TB2). The nucleotide numbering begins at the most 5' nucleotide isolated. A proposed initiation methionine (base 77) is indi¬ cated in bold type. The entire coding sequence is presented.

Figure 7 shows the cDNA and predicted amino acid sequence of DP2.5 (APC). The nucleotide numbering begins at the proposed initia¬ tion methionine. The nucleotides and amino acids of the alternatively spliced exon (exon 9; nucleotide positions 934-1236) are presented in lower case letters. At the 3' end, a poly(A) addition signal occurs at 9530, and one cDNA clone has a poly(A) at 9563. Other cDNA clones extend beyond 9563, however, and their consensus sequence is included here.

Figure 8 shows the arrangement of exons in DP2.5 (APC). (A) Exon 9 corresponds to nucleotides 933-1312; exon 9a corresponds to nucleotides 1236-1312. The stop codon in the cDNA is at nucleotide 8535. (B) Partial intronic sequence surrounding each exon is shown. DETAILED DESCRIPTION

It is a discovery of the present invention that mutational events associated with tumorigenesis occur in a previously unknown gene on chromosome 5q named here the APC (Adenomatous Polyposis Coli) gene. Although it was previously known that deletion of alleles on chromosome 5q were common in certain types of cancers, it was not known that a target gene of these deletions was the APC gene. Fur¬ ther it was not known that other types of mutational events in the APC gene are also associated with cancers. The mutations of the APC gene can involve gross rearrangements, such as insertions and deletions. Point mutations have also been observed.

According to the diagnostic and prognostic method of the present invention, alteration of the wild-type APC gene is detected. "Alteration of a wild-type gene" according to the present invention encompasses all forms of mutations — including deletions. The alter¬ ation may be due to either rearrangements such as insertions, inver¬ sions, and deletions, or to point mutations. Deletions may be of the

entire gene or only a portion of the gene. 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. If only a single allele is somatically mutated, an early neoplastic state is indicated. However, if both alleles are mutated then a late neoplastic state is indicated. The finding of APC mutations thus provides both diagnostic and prognostic information. An APC allele which is not deleted (e.g., that on the sister chromosome to a chromosome carrying an APC 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 APC gene product. However, mutations leading to non- unctional 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 diminu¬ tion of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the APC gene product.

In order to detect the alteration of the wild-type APC gene in a tissue, it is helpful to isolate the tissue free from surrounding normal tissues. Means for enriching a tissue preparation for tumor cells are known in the art. For example, the tissue may be isolated from paraf¬ fin or cryostat sections. Cancer cells may also be separated rom nor¬ mal cells by flow cytometry. These as well as other techniques for separating tumor 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 APC allele (or alleles) and sequencing that allele(s) using techniques well known in the art. Alternatively, the polymerase chain reaction (PCR) can be used to amplify gene sequences directly from a genomic DNA preparation from the tumor tissue. The DNA sequence of the amplified sequences can then be determined. The polymerase chain reaction itself is well known in the art. See, e.g., Saiki et al., Science, Vol. 239, p. 487, 1988; U.S. 4,683,203; and U.S. 4,683,195.

Specific primers which can be used in order to amplify the gene will be discussed in more detail below. The ligase chain reaction, which is known in the art, can also be used to amplify APC sequences. See Wu et al., Genomics. Vol. 4, pp. 560-569 (1989). In addition, a technique known as allele specific PCR can be used. (See Ruano and Kidd, Nucleic Acids Research, Vol. 17, p. 8392, 1989.) According to this technique, primers are used which hybridize at their 3' ends to a par¬ ticular APC mutation. If the particular APC 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., Nucleic Acids Research, Vol. 17, p.7, 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 f agment. Such a method is particularly useful for screening among kindred persons of an affected individual for the presence of the APC mutation found in that individual. Single stranded conformation polymorphism (SSCP) analysis can also be used to detect base change variants of an allele. (Orita et al., Proc. Natl. Acad. Sci. USA Vol. 86, pp. 2766-2770, 1989, and Genomics, Vol. 5, pp. 874-879, 1989.) Other techniques for detecting insertions and deletions as are known in the art can be used.

Alteration of wild-type genes can also be detected on the basis of the alteration of a wild-type expression product of the gene. Such expression products include both the APC mRNA as well as the APC protein product. The sequences of these products are shown in Figures 3 and 7. Point mutations may be detected by amplifying and sequencing the mRNA or via molecular cloning of cDNA made from the mRNA. The sequence of the cloned cDNA can be determined using DNA sequencing techniques which are well known in the art. The cDNA can also be sequenced via the polymerase chain reaction (PCR) which will be discussed in more detail below.

Mismatches, according to the present invention are hybridized nucleic acid duplexes which are not 100% homologous. The lack of

total homology may be due to deletions, insertions, inversions, substitu¬ tions or frameshift mutations. Mismatch detection can be used to detect point mutations in the gene or its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to per¬ form on a large number of tumor samples. An example of a mismatch cleavage technique is the RNase protection method, which is described in detail in Winter et al., Proc. Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985 and Meyers et al., Science, Vol. 230, p. 1242, 1985. In the practice of the present invention the method involves the use of a labeled riboprobe which is complementary to the human wild-type APC gene coding sequence. The riboprobe and either mRNA or DNA isolated from the tumor tissue are annealed (hybridized) together and subse¬ quently 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 APC mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the APC mRNA or gene it will be desirable to use a number of these probes to screen the whole mRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mis¬ matches, through enzymatic or chemical cleavage. See, e.g., Cotton et al., Proc. Natl. Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad. Sci. USA, Vol. 72, p. 989, 1975. Alternatively, mis¬ matches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, Human Genetics, Vol. 42, p. 726, 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 APC gene can also be detected using Southern hybridiza¬ tion, especially if the changes are gross rearrangements, such as dele¬ tions and insertions.

DNA sequences of the APC gene which have been amplified by use of polymerase chain reaction may also be screened using allele-spe¬ cific probes. These probes are nucleic acid oligomers, each of which contains a region of the APC gene sequence harboring a known muta¬ tion. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the APC gene sequence. By use of a bat¬ tery of such allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in the APC gene. Hybridization of allele-specific probes with amplified APC sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization condi¬ tions indicates the presence of the same mutation in the tumor tissue as in the allele-specific probe.

Alteration of APC mRNA expression can be detected by any technique known in the art. These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild- type APC gene.

Alteration of wild-type APC genes can also be detected by screening for alteration of wild-type APC protein. For example, monoclonal antibodies immunoreactive with APC can be used to screen a tissue. Lack of cognate antigen would indicate an APC mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant APC gene product. Such immunological assays can be done in any convenient format known in the art. These include West¬ ern blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered APC protein can be used to detect alteration of wild-type APC genes. Functional assays can be used, such as protein binding determinations. For example, it is believed that APC protein oligomerizes to itself and/or MCC protein or binds to a G protein. Thus, an assay for the ability to bind to wild type APC or MCC protein or that G protein can be employed. In addition, assays can be used which detect APC biochemical function. It is believed that APC is involved in phospholipid metabolism. Thus, assaying the enzymatic products of the involved phospholipid metabolic pathway can be used to

determine APC activity. Finding a mutant APC gene product indicates alteration of a wild-type APC gene.

Mutant APC 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 APC 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 APC 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 APC genes or gene products.

The methods of diagnosis of the present invention are applicable to any tumor in which APC has a role in tumorigenesis. Deletions of chromosome arm 5q have been observed in tumors of lung, breast, colon, rectum, bladder, liver, sarcomas, stomach and prostate, as well as in leukemias and lymphomas. Thus these are likely to be tumors in which APC has a role. The diagnostic method of the present invention is useful for clinicians so that they can decide upon an appropriate course of treatment. For example, a tumor displaying alteration of both APC alleles might suggest a more aggressive therapeutic regimen than a tumor displaying alteration of only one APC allele.

The primer pairs of the present invention are useful or determi¬ nation of the nucleotide sequence of a particular APC allele using the polymerase chain reaction. The pairs of single stranded DNA primers can be annealed to sequences within or surrounding the APC gene on chromosome 5q in order to prime amplifying DNA synthesis of the APC gene itself. A complete set of these primers allows synthesis of all of the nucleotides of the APC 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. Sucn primers anneal only to particular APC mutant alleles, and thus will only ampli y 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 APC sequences or sequences adjacent to APC except 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 prim¬ ers can be made using oligonucleotide synthesizing machines which are commercially available. Given the sequence of the APC open reading frame shown in Figure 7, 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 hybrid¬ ization 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 APC gene or mRNA using other tech¬ niques. Mismatches can be detected using either enzymes (e.g., SI nuclease), chemicals (e.g., hydroxylamine or osmium tetroxide and piperidine), or changes in electrophoretic mobility of mismatched hybrids as compared to totally matched hybrids. These techniques are known in the art. See, Cotton, supra, Shenk, supra. Myers, supra. Win¬ ter, supra, and Novack et al., Proc. Natl. Acad. Sci. USA, Vol. 83, p. 586, 1986. Generally, the probes are complementary to APC gene cod¬ ing sequences, although probes to certain introns are also contem¬ plated. An entire battery of nucleic acid probes is used to compose a kit for detecting alteration of wild-type APC genes. The kit allows for hybridization to the entire APC gene. The probes may overlap with each other or be contiguous.

If a riboprobe is used to detect mismatches with mRNA, it is complementary to the mRNA of the human wild-type APC gene. The riboprobe thus is an anti-sense probe in that it does not code for the APC protein because it is of the opposite polarity to the sense strand. The riboprobe generally will be labeled with a radioactive, colorimetric, or fluorometric material, which can be accomplished by

any means known in the art. If the riboprobe is used to detect mis¬ matches with DNA it can be of either polarity, sense or anti-sense. Similarly, DNA probes also may be used to detect mismatches.

Nucleic acid probes may also be complementary to mutant alleles of the APC gene. These are useful to detect similar mutations in other patients on the basis of hybridization rather than mismatches. These are discussed above and referred to as allele-specific probes. As mentioned above, the APC probes can also be used in Southern hybrid¬ izations to genomic DNA to detect gross chromosomal changes such as deletions and insertions. The probes can also be used to select cDNA clones of APC genes from tumor and normal tissues. In addition, the probes can be used to detect APC mRNA in tissues to determine if expression is diminished as a result of alteration of wild-type APC genes. Provided with the APC coding sequence shown in Figure 7 (SEQ ID NO: 1), design of particular probes is well within the skill of the ordinary artisan.

According to the present invention a method is also provided of supplying wild-type APC function to a cell which carries mutant APC alleles. Supplying such function should suppress neoplastic growth of the recipient cells. The wild-type APC 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 portion is introduced and expressed in a cell carrying a mutant APC allele, the gene portion should encode a part of the APC protein which is required for non-neoplastic growth of the cell. More preferred is the situation where the wild-type APC gene or a part of it is introduced into the mutant cell in such a way that it recombines with the endogenous mutant APC gene present in the cell. Such recombination requires a double recombination event which results in the correction of the APC gene mutation. Vectors for introduction of genes both for recombina¬ tion 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 co-precipitation and viral transduction are known in the art and the choice of method is

within the competence of the routineer. Cells transformed with the wild-type APC gene can be used as model systems to study cancer remission and drug treatments which promote such remission.

Similarly, cells and animals which carry a mutant APC 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 APC mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the APC allele. After a test substance is applied to the cells, the neoplastically transformed pheno¬ type of the cell will be determined. Any trait of neoplastically trans¬ formed 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 APC alleles, usu¬ ally from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous APC gene(s) of the animals may be disrupted by insertion or deletion mutation. 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 therapeu¬ tic agent for the treatment of FAP and/or sporadic cancers.

Polypeptides which have APC activity can be supplied to cells which carry mutant or missing APC alleles. The sequence of the APC protein is disclosed in Figure 3 or 7 (SEQ ID NO: 7 or 1). These two sequences differ slightly and appear to be indicate the existence of two different forms of the APC protein. Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors. Alternatively, APC can be extracted from APC- producing mammalian cells such as brain cells. In addition, the tech¬ niques of synthetic chemistry can be employed to synthesize APC pro¬ tein. Any of such techniques can provide the preparation of the present invention which comprises the APC protein. The preparation

is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.

Active APC molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some such active molecules may be taken up by cells, actively or by diffusion. Extracellular application of APC gene product may be suffi¬ cient to affect tumor growth. Supply of molecules with APC activity should lead to a partial reversal of the neoplastic state. Other mole¬ cules with APC activity may also be used to effect such a reversal, for example peptides, drugs, or organic compounds.

The present invention also provides a preparation of antibodies immunoreactive with a human APC protein. The antibodies may be polyclonal or monoclonal and may be raised against native APC pro¬ tein, APC fusion proteins, or mutant APC proteins. The antibodies should be immunoreactive with APC epitopes, preferably epitopes not present on other human proteins. In a preferred embodiment of the invention the antibodies will immunoprecipitate APC proteins from solution as well as react with APC protein on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, the antibod¬ ies will detect APC proteins in paraffin or frozen tissue sections, using immunocytoehemical techniques. Techniques for raising and purifying antibodies are well known in the art and any such techniques may be chosen to achieve the preparation of the invention.

Predisposition to cancers as in FAP and GS can be ascertained by testing any tissue of a human for mutations of the APC gene. For example, a person who has inherited a germline APC 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 diag¬ nosis can be accomplished by testing fetal cells, placental cells, or amniotic fluid for mutations of the APC gene. Alteration of a wild- type APC allele, whether for example, by point mutation or by dele¬ tion, can be detected by any of the means discussed above.

Molecules of cDNA according to the present invention are intron-f ree, APC gene coding molecules. They can be made by reverse

transcriptase using the APC mRNA as a template. These molecules can be propagated in vectors and cell lines as is known in the art. Such molecules have the sequence shown in SEQ ID NO: 7. The cDNA can also be made using the techniques of synthetic chemistry given the sequence disclosed herein.

A short region of homology has been identifiied between APC and the human m3 muscarinic acetylcholine receptor (mAChR). This homology was largely confined to 29 residues in which 6 out of 7 amino acids (EL(GorA)GLQA) were identical (See Figure 4). Initially, it was not known whether this homology was significant, because many other proteins had higher levels of global homology (though few had six out of seven contiguous amino acids in common). However, a study on the sequence elements controlling G protein activation by mAChR subtypes (Lechleiter et al., EMBO J., p. 4381 (1990)) has shown that a 21 amino acid region from the m3 mAChR completely mediated G protein speci¬ ficity when substituted for the 21 amino acids of m2 mAChR at the analogous protein position. These 21 residues overlap the 19 amino acid homology between APC and m3 mAChR.

This connection between APC and the G protein activating region of mAChR is intriguing in light of previous investigations relat¬ ing G proteins to cancer. For example, the RAS oncogenes, which are often mutated in colorectal cancers (Vogelstein, et al., N. Engl. J. Med., Vol. 319, p. 525 (1988); Bos et al., Nature Vol. 327, p. 293 (1987)), are members of the G protein family (Bourne, et al., Nature, Vol. 348, p. 125 (1990)) as is an in vitro transformation suppressor (Noda et al., Proc. Natl. Acad. Sci. USA, Vol. 86, p. 162 (1989)) and genes mutated in hormone producing tumors (Candis et al., Nature, Vol. 340, p. 692 (1989); Lyons et al., Science, Vol. 249, p. 655 (1990)). Additionally, the gene responsible for neurof ibromatosis (presumably a tumor suppressor gene) has been shown to activate the GTPase activity of RAS (Xu et al., Cell, Vol. 63, p. 835 (1990); Martin et al., Cell, Vol. 63, p. 843 (1990); Ballester et al., Cell, Vol. 63, p. 851 (1990)). Another interesting link between G proteins and colon cancer involves the drug sulindac. This agent has been shown to inhibit the growth of benign colon tumors in patients with FAP, presumably by virtue of its activity as a

cyclooxygenase inhibitor (Waddell et al., J. Surg. Oncology 24(1), 83 (1983); Wadell, et al., Am. J. Surg., 157(1), 175 (1989); Charneau et al., Gastroenterologie Clinique at Biologique 14(2), 153 (1990)). Cyclooxygenase is required to convert arachidonic acid to prostaglandins and other biologically active molecules. G proteins are known to regulate phospholipase A2 activity, which generates arachidonic acid from phospholipids (Role et al., Proc. Natl. Acad. Sci. USA, Vol. 84, p. 3623 (1987); Kurachi et al., Nature, Vol. 337, 12 555 (1989)). Therefore we propose that wild-type APC protein functions by interacting with a G protein and is involved in phospholipid metabolism.

The following are provided for exemplification purposes only and are not intended to limit the scope of the invention which has been described in broad terms above. Example 1:

This example demonstrates the isolation of a 5.5 Mb region of human DNA linked to the FAP locus. Six genes are identified in this region, all of which are expressed in normal colon cells and in colorectal, lung, ad bladder tumors.

The cosmid markers YN5.64 and YN5.48 have previously been shown to delimit an 8 cM region containing the locus for FAP (Nakamura et al., Am. J. Hum. Genet. Vol. 43, p. 638 (1988)). Further linkage and pulse-field gel electrophoresis (PFGE) analysis with addi¬ tional markers has shown that the FAP locus is contained within a 4 eM region bordered by cosmids EF5.44 and L5.99. In order to isolate clones representing a significant portion of this locus, a yeast artificial chro¬ mosome (YAC) library was screened with various 5q21 markers. Twenty-one YAC clones, distributed within six contigs and including 5.5 Mb from the region between YN5.64 and YN5.48, were obtained (Figure 1A).

Three contigs encompassing approximately 4Mb were contained within the central portion of this region. The YAC's constituting these contigs, together with the markers used for their isolation and orienta¬ tions, are shown in Figure 1. These YAC contigs were obtained in the following way. To initiate each contig, the sequence of a genomic

marker cloned from chromosome 5q21 was determined and used to design primers for PCR. PCR was then carried out on pools of YAC clones distributed in micro titer trays as previously described (Anand et al., Nucleic Acids Research, Vol. 18, p. 1951 (1980)). Individual YAC clones from the positive pools were identified by further PCR or hybridization based assays, and the YAC sizes were determined by

PFGE.

To extend the areas covered by the original YAC clones, "chro¬ mosomal walking" was performed. For this purpose, YAC termini were isolated by a PCR based method and sequenced (Riley et al., Nucleic Acids Research, Vol. 18, p. 2887 (1990)). PCR primers based on these sequences were then used to rescreen the YAC library. For example, the sequence from an intron of the FER gene (Hao et al., Mol. Cell. Biol., Vol. 9, p. 1587 (1989)) was used to design PCR primers for isola¬ tion of the 28EC1 and 5EH8 YACs. The termini of the 28EC1 YAC were sequenced to derive markers RHE28 and LHE28, respectively. The sequences of these two markers were then used to isolate YAC clones 15CH12 (from RHE28) and 40CF1 and 29EF1 (from LHE28). These five YACs formed a contig encompassing 1200 kb (contig 1, Figure IB).

Similarly, contig 2 was initiated using cosmid N5.66 sequences, and contig 3 was initiated using sequences both from the MCC gene and from cosmid EF5.44. A walk in the telomeric direction from YAC 14FH1 and a walk in the opposite direction from YAC 39GG3 allowed connection of the initial contig 3 clones through YAC 37HG4 (Figure IB).

Multipoint linkage analysis with the various markers used to define the contigs, combined with PFGE analysis, showed that contigs 1 and 2 were centromeric to contig 3. These contigs were used as tools to orient and/or identify genes which might be responsible for FAP. Six genes were found to lie within this cluster of YACs, as follows:

Contig #1: FER - The FER gene was discovered through its homology to the viral oncogene ABL (Hao et al., supra). It has an intrinsic tyrosine kinase activity, and in situ hybridization with an FER probe showed that the gene was located at 5qll-23 (Morris et al.,

Cytogenet. Cell. Genet., Vol. 53, p. 4, (1990)). Because of the potential role of this oncogene-related gene in neoplasia, we decided to evaluate it further with regards to the FAP locus. A human genomic clone from FER was isolated (MF 2.3) and used to define a restriction fragment length polymorphism (RFLP), and the RFLP in turn used to map FER by linkage analysis using a panel of three generation families. This showed that FER was very tightly linked to previously defined polymorphic markers for the FAP locus. The genetic mapping of FER was complemented by physical mapping using the YAC clones derived from FER sequences (Figure IB). Analysis of YAC contig 1 showed that FER was within 600 kb of cosmid marker M5.28, which maps to within 1.5 Mb of cosmid L5.99 by PFGE of human genomic DNA. Thus, the YAC mapping results were consistent with the FER linkage data and PFGE analyses.

Contig 2: TBl - TBl was identified through a cross-hybridization approach. Exons of genes are often evolutionarily conserved while introns and intergenic regions are much less conserved. Thus, if a human probe cross-hybridizes strongly to the DNA from non-primate species, there is a reasonable chance that it contains exon sequences. Subclones of the cosmids shown in Figure 1 were used to screen South¬ ern blots containing rodent DNA samples. A subelone of cosmid N5.66 (p 5.66-4) was shown to strongly hybridize to rodent DNA, and this clone was used to screen cDNA libraries derived from normal adult colon and fetal liver. The ends of the initial cDNA clones obtained in this screen were then used to extend the cDNA sequence. Eventually, 11 cDNA clones were isolated, covering 2314 bp. The gene detected by these clones was named TBl. Sequence analysis of the overlapping clones revealed an open reading frame (ORF) that extended for 1302 bp starting from the most 5' sequence data obtained (Figure 2A). If this entire open reading frame were translated, it would encode 434 amino acids. The product of this gene was not globally homologous to any other sequence in the current database but showed two significant local similarities to a family of ADP, ATP carrier/translocator proteins and mitochondrial brown fat uncoupling proteins which are widely distrib¬ uted from yeast to mammals. These conserved regions of TBl

(underlined in Figure 2A) may define a predictive motif for this sequence family. In addition, TBl appeared to contain a signal peptide (or mitochondrial targeting sequence) as well as at least 7 transmembrane domains.

Contig 3: MCC, TB2, SRP and APC - The MCC gene was also discovered through a cross-hybridization approach, as described previ¬ ously (Kinzler et al., Science Vol. 251, p. 1366 (1991)). The MCC gene was considered a candidate for causing FAP by virtue of its tight genetic linkage to FAP susceptibility and its somatic mutation in spo¬ radic colorectal carcinomas. However, mapping experiments suggested that the coding region of MCC was approximately 50 kb proximal to the centromeric end of a 200 kb deletion found in an FAP patient. MCC cDNA probes detected a 10 kb mRNA transcript on Northern blot analysis of which 4151 bp, including the entire open reading frame, have been cloned. Although the 3' non-translated portion or an alter¬ natively spliced form of MCC might have extended into this deletion, it was possible that the deletion did not affect the MCC gene product. We therefore used MCC sequences to initiate a YAC contig, and subse¬ quently used the YAC clones to identify genes 50 to 250 kb distal to MCC that might be contained within the deletion.

In a first approach, the insert from YAC24ED6 (Figure IB) was radiolabelled and hybridized to a cDNA library from normal colon. One of the cDNA clones (YS39) identified in this manner detected a 3.1 kb mRNA transcript when used as a probe for Northern blot hybridization. Sequence analysis of the YS39 clone revealed that it encompassed 2283 nucleotides and contained an ORF that extended for 555 bp from the most 5' sequence data obtained. If all of this ORF were translated, it would encode 185 amino acids (Figure 2B). The gene detected by YS39 was named TB2. Searches of nucleotide and protein databases revealed that the TB2 gene was not identical to any previously reported sequences nor were there any striking similarities.

Another clone (YS11) identified through the YAC 24ED6 screen appeared to contain portions of two distinct genes. Sequences from one end of YSll were identical to at least 180 bp of the signal recogni¬ tion particle protein SRP19 (Lingelbach et al. Nucleic Acids Research,

Vol. 16, p. 9431 (1988). A second ORF, from the opposite end of clone YSll, proved to be identical to 78 bp of a novel gene which was inde¬ pendently identified through a second YAC-based approach. For the latter, DNA from yeast cells containing YAC 14FH1 (Figure IB) was digested with EcoRI and subeloned into a plasmid vector. Plasmids that contained human DNA fragments were selected by colony hybridization using total human DNA as a probe. These clones were then used to search for cross-hybridizing sequences as described above for TBl, and the cross-hybridizing clones were subsequently used to screen cDNA libraries. One of the cDNA clones discovered in this way (FH38) con¬ tained a long ORF (2496 bp), 78 bp of which were identical to the above-noted sequences in YSll. The ends of the FH38 cDNA clone were then used to initiate cDNA walking to extend the sequence. Eventually, 85 cDNA clones were isolated from normal colon, brain and liver cDNA libraries and found to encompass 8973 nucleotides of con¬ tiguous transcript. The gene corresponding to this transcript was named APC. When used as probes for Northern blot analysis, APC cDNA clones hybridized to a single transcript of approximately 9.5 kb, suggesting that the great majority of the gene product was represented in the cDNA clones obtained. Sequences from the 5' end of the APC gene were found in YAC 37HG4 but not in YAC 14FH1. However, the 3' end of the APC gene was found in 14FH1 as well as 37HG4. The yeast artificial chromosome of the present invention designated YAC 37HG4 has been deposited with the National Collection of Indus¬ trial and Marine Bacteria (NCIMB), P.O. Box 31, 135 Abbey Road, Aberdeen AB9 8DG, Scotland, prior to the filing of this patent applica¬ tion. The NCIMB Accession Number of YAC clone YAC 37HG4 is 40353. Analogously, the 5' end of the MCC coding region was found in YAC clones 19AA9 and 26GC3 but not 24ED6 or 14FH1, while the 3' end displayed the opposite pattern. Thus, MCC and APC transcription units pointed in opposite directions, with the direction of transcription going from centromeric to telomeric in the case of MCC, and telomeric to centromeric in the case of APC. PFGE analysis of YAC DNA digested with various restriction endonucleases showed that TB2 and SRP were between MCC and APC, and that the 3' ends of the coding

regions of MCC and APC were separated by approximately 150 kb (Figure IB).

Sequence analysis of the APC cDNA clones revealed an open reading frame of 8,535 nucleotides. The 5' end of the ORF contained a methionine codon (codon 1) that was preceded by an in-frame stop codon 9 bp upstream, and the 3' end was followed by several in-frame stop codons. The protein produced by initiation at codon 1 would con¬ tain 2,842 amino acids (Figure 3). The results of database searching with the APC gene product were quite complex due to the presence of large segments with locally biased amino acid compositions. In spite of this, APC could be roughly divided into two domains. The N-terminal 25% of the protein had a high content of leucine residues (12%) and showed local sequence similarities to myosins, various intermediate filament proteins (e.g., desmin, vimentin, neurofilaments) and Drosophila armadillo/human plakoglobin. The latter protein is a com¬ ponent of adhesive junctions (desmosomes) joining epithelial cells (Franke et al., Proc. Natl. Acad. Sci. U.S.A., Vol. 86, p. 4027 (1989); Perfer et al., Cell, Vol. 63, p. 1167 (1990)) The C-terminal 75% of APC (residues 731-2832) is 17% serine by composition with serine residues more or less uniformly distributed. This large domain also contains local concentrations of charged (mostly acidic) and proline residues. There was no indication of potential signal peptides, transmembrane regions, or nuclear targeting signals in APC, suggesting a cytoplasmic localization.

To detect short similarities to APC, a database search was per¬ formed using the PAM-40 matrix (Altschul. J. Mol. Bio., Vol. 219, p. 555 (1991). Potentially interesting matches to several proteins were found. The most suggestive of these involved the ral2 gene product of yeast, which is implicated in the regulation of ras activity (Fukul et al., Mol. Cell. Biol., Vol. 9, p. 5617 (1989)). Little is known about how ral2 might interact with ras but it is interesting to note the positively-charged character of this region in the context of the negatively-charged GAP interaction region of ras. A specific electrostatic interaction between ras and GAP-related proteins has been proposed.

Because of the proximity of the MCC and APC genes, and the fact that both are implicated in colorectal tumorigenesis, we searched for similarities between the two predicted proteins. Bourne has previ¬ ously noted that MCC has the potential to form alpha helical coiled coils (Nature, Vol. 351, p. 188 (1991). Lupas and colleagues have recently developed a program for predicting coiled coil potential from primary sequence data (Science, Vol. 252, p. 1162 (1991) and we have used their program to analyze both MCC and APC. Analysis of MCC indicated a discontinuous pattern of coiled-coil domains separated by putative "hinge" or "spacer" regions similar to those seen in laminin and other intermediate filament proteins. Analysis of the APC sequence revealed two regions in the N-terminal domain which had strong coiled coil-forming potential, and these regions corresponded to those that showed local similarities with myosin and IF proteins on database searching. In addition, one other putative coiled coil region was identified in the central region of APC. The potential for both APC and MCC to form coiled coils is interesting in that such structures often mediate homo- and hetero-oligomerization.

Finally, it had previously been noted that MCC shared a short similarity with the region of the m3 muscarinic acetylcholine receptor (mAChR) known to regulate specificity of G-protein coupling. The APC gene also contained a local similarity to the region of the m3 mAChR that overlapped with the MCC similarity (Figure 4B). Although the similarities to ral2 (Figure 4A) and m3 mAChR (Figure 4B) were not statistically significant, they were intriguing in light of previous obser¬ vations relating G-proteins to neoplasia.

Each of the six genes described above was expressed in normal colon mucosa, as indicated by their representation in colon cDNA libraries. To study expression of the genes in neoplastic colorectal epithelium, we employed reverse transcription-polymerase chain reac¬ tion (PCR) assays. Primers based on the sequences of FER, TBl, TB2, MCC, and APC were each used to design primers for PCR performed with cDNA templates. Each of these genes was found to be expressed in normal colon, in each of ten cell lines derived from colorectal can¬ cers, and in tumor cell lines derived from lung and bladder tumors. The

ten colorectal cancer cell lines included eight from patients with spo¬ radic CRC and two from patients with FAP. Example 2

This example demonstrates a genetic analysis of the role of the FER gene in FAP and sporadic colorectal cancers.

We considered FER as a candidate because of its proximity to the FAP locus as judged by physical and genetic criteria (see Example 1), and its homology to known tyrosine kinases with oncogenic potential. Primers were designed to PCR-amplify the complete coding sequence of FER from the RNA of two colorectal cancer cell lines derived from FAP patients. cDNA was generated from RNA and used as a template for PCR. The primers used were 5--AGAAGGATCCCTTGTGCAGTGTGGA-3' and

5--GACAGGATCCTGAAGCTGAGTTTG-3'. The underlined nucleotides were altered from the true FER sequence to create BamHI sites. The cell lines used were JW and Difi, both derived from colorectal cancers of FAP patients. (C. Paraskeva, B.G. Buckle, D. Sheer, C.B. Wigley, Int. J. Cancer 34, 49 (1984); M.E. Gross et al., Cancer Res. 51, 1452 (1991). The resultant 2554 basepair fragments were cloned and sequenced in their entirety. The PCR products were cloned in the BamHI site of Bluescript SK (Stratagene) and pools of at least 50 clones were sequenced en masse using T7 polymerase, as described in Nigro et al., Nature 342, 705 (1989).

Only a single conservative amino acid change (GTG->CTG, cre¬ ating a val to leu substitution at codon 439) was observed. The region surrounding this codon was then amplified from the DNA of individuals without FAP and this substitution was found to be a common polymorphism, not specifically associated with FAP. Based on these results, we considered it unlikely (though still possible) the FER gene was responsible for FAP. To amplify the regions surrounding codon 439, the following primers were used: 5'-TCAGAAAGTGCTGAAGAG-3' and 5--GGAATAATTAGGTCTCCAA-3'. PCR products were digested with Pstl, which yields a 50 bp fragment if codon 439 is leucine, but 26 and 24 bp fragments if it is valine. The primers used for sequencing were chosen from the FER cDNA sequence in Hao et al., supra.

Example 3

This example demonstrates the genetic analysis of MCC, TB2, SRP and APC in FAP and sporadic colorectal tumors. Each of these genes is linked and encompassed by contig 3 (see Figure 1).

Several lines of evidence suggested that this contig was of par¬ ticular interest. First, at least three of the four genes in this contig were within the deleted region identified in two FAP patients. (See Example 5 infra.) Second, allelic deletions of chromosome 5q21 in spo¬ radic cancers appeared to be centered in this region. (Ashton-Rickardt et al., Oncogene, in press; and Miki et al., Japn. J. Cancer Res., in press.) Some tumors exhibited loss of proximal RFLP markers (up to and potentially including the 5' end of MCC), but no loss of markers distal to MCC. Other tumors exhibited loss of markers distal to and perhaps including the 3' end of MCC, but no loss of sequences proximal to MCC. This suggested either that different ends of MCC were affected by loss in all such cases, or alternatively, that two genes (one proximal to and perhaps including MCC, the other distal to MCC) were separate targets of deletion. Third, clones from each of the six FAP region genes were used as probes on Southern blots containing tumor DNA from patients with sporadic CRC. Only two examples of somatic changes were observed in over 200 tumors studied: a rearrangement/deletion whose centromeric end was located within the MCC gene (Kinzler et al., supra) and an 800 bp insertion within the APC gene between nucleotides 4424 and 5584. Fourth, point mutations of MCC were observed in two tumors (Kinzler et al.) supra strongly suggesting that MCC was a target of mutation in at least some sporadic colorectal cancers.

Based on these results, we attempted to search for subtle alter¬ ations of contig 3 genes in patients with FAP. We chose to examine MCC and APC, rather than TB2 or SRP, because of the somatic muta¬ tions in MCC and APC noted above. To facilitate the identification of subtle alterations, the genomic sequences of MCC and APC exons were determined (see Table I). These sequences were used to design primers for PCR analysis of constitutional DNA from FAP patients.

We first amplified eight exons and surrounding introns of the MCC gene in affected individuals from 90 different FAP kindreds. The PCR products were analyzed by a ribonuclease (RNase) protein assay. In brief, the PCR products were hybridized to in vitro transcribed RNA probes representing the normal genomic sequences. The hybrids were digested with RNase A, which can cleave at single base pair mis¬ matches within DNA-RNA hybrids, and the cleavage products were visualized following denaturing gel electrophoresis. Two separate RNase protection analyses were performed for each exon, one with the sense and one with the antisense strand. Under these conditions, approximately 40% of all mismatches are detectable. Although some amino acid variants of MCC were observed in FAP patients, all such variants were found in a small percentage of normal individuals. These variants were thus unlikely to be responsible for the inheritance of FAP.

We next examined three exons of the APC gene. The three exons examined included those containing nt 822-930, 931-1309, and the first 300 nt of the most distal exon (nt 1956-2256). PCR and RNase protection analysis were performed as described in Kinzler et al. supra, using the primers underlined in Table I. The primers for nt 1956-2256 were 5'-GCAAATCCTAAGAGAGAACAA-3* and

5'-GATGGCAAGCTTGAGCCAG-3 ^* .

In 90 kindreds, the RNase protection method was used to screen for mutations and in an additional 13 kindreds, the PCR products were cloned and sequenced to search for mutations not detectable by RNase protection. PCR products were cloned into a Bluescript vector modi¬ fied as described in T.A. Holton and M.W. Graham, Nucleic Acids Res. 19, 1156 (1991). A minimum of 100 clones were pooled and sequenced. Five variants were detected among the 103 kindreds analyzed. Cloning and subsequent DNA sequencing of the PCR product of patient P21 indicated a C to T transition in codon 413 that resulted in a change from arginine to cysteine. This amino acid variant was not observed in any of 200 DNA samples from individuals without FAP. Cloning and sequencing of the PCR product from patients P24 and P34, who demon¬ strated the same abnormal RNase protection pattern indicated that

both had a C to T transition at codon 301 that resulted in a change from arginine (CGA) to a stop codon (TGA). This change was not present in 200 individuals without FAP. As this point mutation resulted in the predicted loss of the recognition site for the enzyme Taq I, appropriate PCR products could be digested with Taq I to detect the mutation. This allowed us to determine that the stop codon co-segregated with disease phenotype in members of the family of P24. The inheritance of this change in affected members of the pedigree provides additional evidence for the importance of the mutation.

Cloning and sequencing of the PCR product from FAP patient P93 indicated a C to G transversion at codon 279, also resulting in a stop codon (change from TCA to TGA). This mutation was not present in 200 individuals without FAP. Finally, one additional mutation result¬ ing in a serine (TCA) to stop codon (TGA) at codon 712 was detected in a single patient with FAP (patient P60).

The five germline mutations identified are summarized in Table ΠA, as well as four others discussed in Example 9. In addition to these germline mutations, we identified several somatic mutations of MCC and APC in sporadic CRCs. Seventeen MCC exons were exam¬ ined in 90 sporadic colorectal cancers by RNase protection analysis. In each case where an abnormal RNase protection pattern was observed, the corresponding PCR products were cloned and sequenced. This led to the identification of six point mutations (two described previously) (Kinzler et al., supra), each of which was not found in the germline of these patients (Table UB). Four of the mutations resulted in amino acid substitutions and two resulted in the alteration of splice site consensus elements. Mutations at analogous splice site positions in other genes have been shown to alter RNA processing in vivo and in vitro.

Three exons of APC were also evaluated in sporadic tumors. Sixty tumors were screened by RNase protection, and an additional 98 tumors were evaluated by sequencing. The exons examined included nt 822-930, 931-1309, and 1406-1545 (Table I). A total of three mutations were identified, each of which proved to be somatic. Tumor T27 con¬ tained a somatic mutation of CGA (arginine) to TGA (stop codon) at codon 33. Tumor T135 contained a GT to GC change at a splice donor

site. Tumor T34 contained a 5 bp insertion (CAGCC between codons 288 and 289) resulting in a stop at codon 291 due to a frameshift.

We serendipitously discovered one additional somatic mutation in a colorectal cancer. During our attempt to define the sequences and splice patterns of the MCC and APC gene products in colorectal epithelial cells, we cloned cDNA from the colorectal cancer cell line SW480. The amino acid sequence of the MCC gene from SW480 was identical to that previously found in clones from human brain. The sequence of APC in SW480 cells, however, differed significantly, in that a transition at codon 1338 resulted in a change from glutamine (CAG) to a stop codon (TAG). To determine if this mutation was somatic, we recovered DNA from archival paraffin blocks of the origi¬ nal surgical specimen (T201) from which the tumor cell line was derived 28 years ago.

DNA was purified from paraffin sections as described in S.E. Goelz, S.R. Hamilton, and B. Vogelstein. Biochem. Biophys. Res. Comm. 130, 118 (1985). PCR was performed as described in reference 24, using the primers 5--GTTCCAGCAGTGTCACAG-3' and 5--GGGAGATTTCGCTCCTGA-3'. A PCR product containing codon 1338 was amplified from the archival DNA and used to show that the stop codon represented a somatic mutation present in the original pri¬ mary tumor and in cell lines derived from the primary and metastatic tumor sites, but not from normal tissue of the patient.

The ten point mutations in the MCC and APC genes so far dis¬ covered in sporadic CRCs are summarized in Table HB. Analysis of the number of mutant and wild-type PCR clones obtained from each of these tumors showed that in eight of the ten cases, the wild-type sequence was present in approximately equal proportions to the mutant. This was confirmed by RFLP analysis using flanking markers from chromosome 5q which demonstrated that only two of the ten tumors (T135 and T201) exhibited an allelic deletion on chromosome 5q. These results are consistent with previous observations showing that 20-40% of sporadic colorectal tumors had allelic deletions of chromo¬ some 5q. Moreover, these data suggest that mutations of 5q2l genes

are not limited to those colorectal tumors which contain allelic dele ^¬ tions of this chromosome. Example 4

This example characterizes small, nested deletions in DNA from two unrelated FAP patients.

DNA from 40 FAP patients was screened with cosmids that had been mapped into a region near the APC locus to identify small dele¬ tions or rearrangements. Two of these cosmids, L5.71 and L5.79, hybridized with a 1200 kb Notl fragment in DNAs from most of the FAP patients screened.

The DNA of one FAP patient, 3214, showed only a 940 kb Notl fragment instead of the expected 1200 kb fragment. DNA was ana¬ lyzed from four other members of the patient's immediate family; the 940 kb fragment was present in her affected mother (4711), but not in the other, unaffected family members. The mother also carried a nor¬ mal 1200 kb Notl fragment that was transmitted to her two unaffected offspring. These observations indicated that the mutant polyposis allele is on the same chromosome as the 940 kb Notl fragment. A sim¬ ple interpretation is that APC patients 3214 and 4711 each carry a 260 kb deletion within the APC locus.

If a deletion were present, then other enzymes might also be expected to produce fragments with altered mobilities. Hybridization of L5.79 to Nrul-digested DNAs from both affected members of the family revealed a novel Nrul fragment of 1300 kb, in addition to the normal 1200 kb Nrul fragment. Furthermore, MM fragments in patients 3214 and 4711 also showed an increase in size consistent with the deletion of an MM site. The two chromosome 5 homologs of patient 3214 were segregated in somatic cell hybrid lines; HHW1155 (deletion hybrid) carried the abnormal homolog and HHW1159 (normal hybrid) carried the normal homolog.

Because patient 3214 showed only a 940 kb Notl fragment, she had not inherited the 1200 kb fragment present in the unaffected father's DNA. This observation suggests that he must be heterozygous for, and have transmitted, either a deletion of the L5.79 probe region or a variant Notl fragment too large to resolve on the gel system. As

expected, the hybrid cell line HHW1159, which carries the paternal homolog, revealed no resolved Not fragment when probed with L5.79. However, probing of HHW1159 DNA with L5.79 following digestion with other enzymes did reveal restriction fragments, demonstrating the presence of DNA homologous to the probe. The father is, therefore, interpreted as heterozygous for a polymorphism at the Notl site, with one chromosome 5 having a 1200 kb Notl fragment and the other hav¬ ing a fragment too large to resolve consistently on the gel. The latter was transmitted to patient 3214.

When double digests were used to order restriction sites within the 1200 kb Notl fragment, L5.71 and L5.79 were both found to lie on a 550 kb Notl-Nrul fragment and, therefore, on the same side of an Nrul site in the 1200 kb Notl fragment. To obtain genomic representation of sequences present over the entire 1200 kb Notl fragment, we con¬ structed a library of small-fragment inserts enriched for sequences from this fragment. DNA from the somatic cell hybrid HHW141, which contains about 40% of chromosome 5, was digested with Notl and electrophoresed under pulsed-field gel (PFG) conditions; EcoRI frag¬ ments from the 1200 kb region of this gel were cloned into a phage vector. Probe Map30 was isolated from this library. In normal individ¬ uals probe Map30 hybridizes to the 1200 kb Notl fragment and to a 200 kb Nrul fragment. This latter hybridization places Map30 distal, with respect to the locations of L5.71 and L5.79, to the Nrul site of the 550 kb Notl-Nrul fragment.

Because Map30 hybridized to the abnormal, 1300 kb Nrul frag¬ ment of patient 3214, the locus defined by Map30 lies outside the hypothesized deletion. Furthermore, in normal chromosomes Map30 identified a 200 kb Nrul fragment and L5.79 identified a 1200 kb Nrul fragment; the hypothesized deletion must, therefore, be removing an Nrul site, or sites, lying between Map30 and L5.79, and these two probes must flank the hypothesized deletion. A restriction map of the genomic region, showing placement of these probes, is shown in Figure 5.

A Notl digest of DNA from another FAP patient, 3824, was probed with L5.79. In addition to the 1200 kb normal Notl fragment, a

fragment of approximately 1100 kb was observed, consistent with the presence of a 100 kb deletion in one chromosome 5. In this case, how¬ ever, digestion with Nrul and MM did not reveal abnormal bands, indi¬ cating that if a deletion were present, its boundaries must lie distal to the Nrul and MM sites of the fragments identified by L5.79. Consis¬ tent with this expectation, hybridization of Map30 to DNA from patient 3824 identified a 760 kb MM fragment in addition to the expected 860 kb fragment, supporting the interpretation of a 100 kb deletion in this patient. The two chromosome 5 homologs of patient 3824 were segregated in somatic cell hybrid lines; HHW1291 was found to carry only the abnormal homolog and HHW1290 only the normal homolog.

That the 860 kb MM fragment identified by Map30 is distinct from the 830 kb MM fragment identified previously by L5.79 was dem¬ onstrated by hybridization of Map30 and L5.79 to a Notl-MM double digest of DNA from the hybrid cell (HHW1159) containing the nondeleted chromosome 5 homolog of patient 3214. As previously indi¬ cated, this hybrid is interpreted as missing one of the Notl sites that define the 1200 kb fragment. A 620 kb Notl-MM fragment was seen with probe L5.79, and an 860 kb fragment was seen with Map30. Therefore, the 830 kb MM fragment reeognized by probe L5.79 must contain a Notl site in HHW1159 DNA; because the 860 kb MM fragment remains intact, it does not carry this Notl site and must be distinct from the 830 kb MM fragment. Example 5

This example demonstrates the isolation of human sequences which span the region deleted in the two unrelated FAP patients char¬ acterized in Example 4.

A strong prediction of the hypothesis that patients 3214 and 3824 carry deletions is that some sequences present on normal chromo¬ some 5 homologs would be missing from the hypothesized deletion homologs. Therefore, to develop genomic probes that might confirm the deletions, as well as to identify genes from the region, YAC clones from a contig seeded by cosmid L5.79 were localized from a library containing seven haploid human genome equivalents (Albertsen et al.,

Proc. Natl. Acad. Sci. U.S.A., Vol. 87, pp. 4256-4260 (1990)) with respect to the hypothesized deletions. Three clones, YACs 57B8, 310D8, and 183H12, were found to overlap the deleted region.

Importantly, one end of YAC 57B8 (clone AT57) was found to lie within the patient 3214 deletion. Inverse polymerase chain reaction (PCR) defined the end sequences of the insert of YAC 57B8. PCR primers based on one of these end sequences repeatedly failed to amplify DNA from the somatic cell hybrid (HHW1155) carrying the deleted homolog of patient 3214, but did amplify a product of the expected size from the somatic cell hybrid (HHW1159) carrying the normal chromosome 5 homolog. This result supported the interpreta¬ tion that the abnormal restriction fragments found in the DNA of patient 3214 result from a deletion.

Additional support for the hypothesis of deletion in DNA from patient 3214 came from subcloned fragments of YAC 183H12, which spans the region in question. Yll, an EcoRI fragment cloned from YAC 183H12, hybridized to the normal, 1200 kb Notl fragment of patient 4711, but failed to hybridize to the abnormal, 940 kb Notl frag¬ ment of 4711 or to DNA from deletion cell line HHW1155. This result confirmed the deletion in patient 3214.

Two additional EcoRI fragments from YAC 183H12, Y10 and Y14, were localized within the patient 3214 deletion by their failure to hybridizie to DNA from HHW1155. Probe Y10 hybridizes to a 150 kb Nrul fragment in normal chromosome 5 homologs. Because the 3214 deletion creates the 1300 kb Nrul fragment seen with the probes L5.79 and Map30 that flank the deletion, these Nrul sites and the 150 kb Nrul fragment lying between must be deleted in patient 3214. Furthermore, probe Y10 hybridizes to the same 620 kb Notl-MM fragment seen with probe L5.79 in normal DNA, indicating its location as L5.79-proximal to the deleted MM site and placing it between the Mlul site and the L5.79-proximal Nrul site. The Mlul site must, therefore, lie between the Nrul sites that define the 150 kb Nrul fragment (see Figure 5).

Probe Yll also hybridized to the 150 kb Nrul fragment in the normal chromosome 5 homolog, but failed to hybridize to the 620 kb Notl-MM fragment, placing it L5.79-distal to the Mlul site, but

proximal to the second Nrul site. Hybridization to the same (860 kb) MM fragment as Map30 confirmed the localization of probe Yll L5.79-distal to the Mlul site.

Probe Y14 was shown to be L5.79-distal to both deleted Nrul sites by virtue of its hybridization to the same 200 kb Nrul fragment of the normal chromosome 5 seen with Map30. Therefore, the order of these EcoRI fragments derived from YAC 183H12 and deleted in patient 3214, with respect to L5.79 and Map30, is L5.79-Y10-Yll-Y14-Map30.

The 100 kb deletion of patient 3824 was confirmed by the failure of aberrant restriction fragments in this DNA to hybridize with probe Yll, combined with positive hybridizations to probes Y10 and/or Y14. Y10 and Y14 each hybridized to the 1100 kb Notl fragment of patient 3824 as well as to the normal 1200 kb Notl fragment, but Yll hybrid¬ ized to the 1200 kb fragment only. In the MM digest, probe Y14 hybridized to the 860 kb and 760 kb fragments of patient 3824 DNA, but probe Yll hybridized only to the 860 kb fragment. We conclude that the basis for the alteration in fragment size in DNA from patient 3824 is, indeed, a deletion. Furthermore, because probes Y10 and Y14 are missing from the deleted 3214 chromosome, but present on the deleted 3824 chromosome, and they have been shown to flank probe Yll, the deletion in patient 3824 must be nested within the patient 3214 deletion.

Probes Y10, Yll, Y14 and Map30 each hybridized to YAC 310D8, indicating that this YAC spanned the patient 3824 deletion and at a minimum, most of the 3214 deletion. The YAC characterizations, therefore, confirmed the presence of deletions in the patients and pro¬ vided physical representation of the deleted region. Example 6

This example demonstrates that the MCC coding sequence maps outside of the region deleted in the two FAP patients characterized in Example 4.

An intriguing FAP candidate gene, MCC, recently was ascer¬ tained with cosmid L5.71 and was shown to have undergone mutation in colon carcinomas (Kinzler et al., supra). It was therefore of interest to

map this gene with respect to the deletions in APC patients. Hybrid¬ ization of MCC probes with an overlapping series of YAC clones extending in either direction from L5.71 showed that the 3' end of MCC must be oriented toward the region of the two APC deletions.

Therefore, two 3' cDNA clones from MCC were mapped with respect to the deletions: clone ICI (bp 2378-4181) and clone 7 (bp 2890-3560). Clone ICI contains sequences from the C-terminal end of the open reading frame, which stops at nucleotide 2708, as well as 3' untranslated sequence. Clone 7 contains sequence that is entirely 3' to the open reading frame. Importantly, the entire 3' untranslated sequence contained in the cDNA clones consists of a single 2.5 kb exon. These two clones were hybridized to DNAs from the YACs spanning the FAP region. Clone 7 fails to hybridize to YAC 310D8, although it does hybridize to YACs 183H12 and 57B8; the same result was obtained with the cDNA ICI. Furthermore, these probes did show hybridization to DNAs from both hybrid cell lines (HWW1159 and HWW1155) and the lymphoblastoid cell line from patient 3214, confirming their locations outside the deleted region. Additional mapping experiments suggested that the 3' end of the MCC cDNA clone contig is likely to be located more than 45 kb from the deletion of patient 3214 and, therefore, more than 100 kb from the deletion of patient 3824. Example 7

This example identifies three genes within the deleted region of chromosome 5 in the two unrelated FAP patients characterized in Example 4.

Genomic clones were used to screen cDNA libraries in three separate experiments. One screening was done with a phage clone derived from YAC 310D8 known to span the 260 kb deletion of patient 3214. A large-insert phage library was constructed from this YAC; screening with Yll identified λ205, which mapped within both dele¬ tions. When clone λ205 was used to probe a random-, plus oligo(dT)-, primed fetal brain cDNA library (approximately 300,000 phage), six cDNA clones were isolated and each of them mapped entirely within both deletions. Sequence analysis of these six clones formed a single cDNA contig, but did not reveal an extended open reading frame. One

of the six cDNAs was used to isolate more cDNA clones, some of which crossed the L5.71-proximal breakpoint of the 3824 deletion, as indi¬ cated by hybridization to both chromosome of this patient. These clones also contained an open reading frame, indicating a transcrip- tional orientation proximal to distal with respect to L5.71. This gene was named DPI (deleted in polyposis 1). This gene is identical to TB2 described above. cDNA walks yielded a cDNA contig of 3.0-3.5 kb, and included two clones containing terminal poly(A) sequences. This size corre¬ sponds to the 3.5 kb band seen by Northern analysis. Sequencing of the first 3163 bp of the cDNA contig revealed an open reading frame extending from the first base to nucleotide 631, followed by a 2.5 kb 3' untranslated region. The sequence surrounding the methionine codon at base 77 conforms to the Kozak consensus of an initiation methionine (Kozak, 1984). Failed attempts to walk farther, coupled with the simi¬ larity of the lengths of isolated cDNA and mRNA, suggested that the NH2~terminus of the DPI protein had been reached. Hybridization to a combination of genomic and YAC DNAs cut with various enzymes indi¬ cated the genomic coverage of DPI to be approximately 30 kb.

Two additional probes for the locus, YS-11 and YS-39, which had been ascertained by screening of a cDNA library with an independent YAC probe identified with MCC sequences adjacent to L5.71, were mapped into the deletion region. YS-39 was shown to be a cDNA iden¬ tical in sequence to DPI. Partial characterization of YS-11 had shown that 200 bp of DNA sequence at one end was identical to sequence cod¬ ing for the 19 kd protein of the ribosomal signal recognition particle, SRP19 (Lingelbach et al., supra). Hybridization experiments mapped YS-11 within both deletions. The sequence of this clone, however, was found to be complex. Although 454 bp of the 1032 bp sequence of YS-11 were identical to the GenBank entry for the SRP19 gene, another 578 bp appended 5' to the SRP19 sequence was found to consist of previously unreported sequence containing no extended open reading frames. This suggested that YS-11 was either a chimeric clone con¬ taining two independent inserts or a clone of an incompletely processed or aberrant message. If YS-11 were a conventional chimeric clone, the

independent segments would not be expected to map to the same physi¬ cal region. The segments resulting from anomalous processing of a continuous transcript, however, would map to a single chromosomal region.

Inverse PCR with primers specific to the two ends of YS-11, the SRP19 end and the unidentified region, verified that both sequences map within the YAC 310D8; therefore, YS-11 is most likely a clone of an immature or anomalous mRNA species. Subsequently, both ends were shown to lie with the deleted region of patient 3824, and YS-11 was used to screen for additional cDNA clones.

Of the 14 cDNA clones selected from the fetal brain library, one clone, V5, was of particular interest in that it contained an open read¬ ing frame throughout, although it included only a short identity to the first 78 5' bases of the YS-11 sequence. Following the 78 bp of identi¬ cal sequence, the two cDNA sequences diverged at an AG. Further¬ more, divergence from genomic sequence was also seen after these 78 bp, suggesting the presence of a splice junction, and supporting the view that YS-11 represents an irregular message.

Starting with V5, successive 5' and 3' walks were performed; the resulting cDNA contig consisted of more than 100 clones, which defined a new transcript, DP2. Clones walking in the 5' direction crossed the 3824 deletion breakpoint farthest from L5.71; since its 3' end is closer to this cosmid than its 5' end, the transcriptional orienta¬ tion of DP2 is opposite to that of MCC and DPI.

The third screening approach relied on hybridization with a 120 kb MM fragment from YAC 57B8. This fragment hybridizes with probe Yll and completely spans the 100 kb deletion in patient 3824. the fragment was purified on two preparative PFGs, labeled, and used to screen a fetal brain cDNA library. A number of cDNA clones previ¬ ously identified in the development of the DPI and DP2 contigs were reascertained. However, 19 new cDNA clones mapped into the patient 3824 deletion. Analysis indicated that these 19 formed a new contig, DP3, containing a large open reading frame.

A clone from the 5* end of this new cDNA contig hybridized to the same EcoRI fragment as the 3' end of DP2. Subsequently, the DP2

and DP3 contigs were connected by a single 5' walking step from DP3, to form the single contig DP2.5. The complete nucleotide sequence of DP2.5 is shown in Figure 9.

The consensus cDNA sequence of DP2.5 suggests that the entire coding sequence of DP2.5 has been obtained and is 8532 bp long. The most 5' ATG codon occurs two codons from an in-frame stop and con¬ forms to the Kozak initiation consensus (Kozak, Nucl. Acids. Res., Vol. 12, p. 857-872 1984). The 3' open reading frame breaks down over the final 1.8 kb, giving multiple stops in all frames. A poly(A) sequence was found in one clone approximately l kb into the 3' untranslated region, associated with a polyadenylation signal 33 bp upstream (posi¬ tion 9530). The open reading frame is almost identical to that identi¬ fied as APC above.

An alternatively spliced exon at nucleotide 934 of the DP2.5 transcript is of potential interest, it was first discovered by noting that two classes of cDNA had been isolated. The more abundant cDNA class contains a 303 bp exon not included in the other. The presence in vivo of the two transcripts was verified by an exon connection experi¬ ment. Primers flanking the alternatively spliced exon were used to amplify, by PCR, cDNA prepared from various adult tissues. Two PCR products that differed in size by approximately 300 bases were ampli¬ fied from all the tissues tested; the larger product was always more abundant than the smaller. Example 8

This example demonstrates the primers used to identify subtle mutations in DPI, SRP19, and DP25.

To obtain DNA sequence adjacent to the exons of the genes DPI, DP2.5, and SRP19, sequencing substrate was obtained by inverse PCR amplification of DNAs from two YACs, 310D8 and 183H12, that span the deletions. Ligation at low concentration cyclized the restriction enzyme-digested YAC DNAs. Oligonucleotides with sequencing tails, designed in inverse orientation at intervals along the cDNAs, primed PCR amplification from the cyclized templates. Comparison of these DNA sequences with the cDNA sequences placed exon boundaries at the divergence points. SRP19 and DPI were each shown to have five

exons. DP2.5 consisted of 15 exons. The sequences of the oligonucleotides synthesized to provide PCR amplification primers for the exons of each of these genes are listed in Table III. With the excep¬ tion of exons 1, 3, 4, 9, and 15 of DP2.5 (see below), the primer sequences were located in intron sequences flanking the exons. The 5' primer of exon 1 is complementary to the cDNA sequence, but extends just into the 5' Kozak consensus sequence for the initiator methionine, allowing a survey of the translated sequences. The 5' primer of exon 3 is actually in the 5' coding sequences of this exon, as three separate intronic primers simply would not amplify. The 5' primer of exon 4 just overlaps the 5' end of this exon, and we thus fail to survey the 19 most 5' bases of this exon. For exon 9, two overlapping primer sets were used, such that each had one end within the exon. For exon 15, the large 3' exon of DP2.5, overlapping primer pairs were placed along the length of the exon; each pair amplified a product of 250-400 bases. Example 9

This example demonstrates the use of single stranded conforma¬ tion polymorphism (SSCP) analysis as described by Orita et al. Proc. Natl. Acad. Sci. U.S.A., Vol. 86, pp. 2766-70 (1989) and Genomics, Vol. 5, pp. 874-879 (1989) as applied to DPI, SRP19 and DP2.5.

SSCP analysis identifies most single- or multiple-base changes in DNA fragments up to 400 bases in length. Sequence alterations are detected as shifts in electrophoretic mobility of single-stranded DNA on nondenaturing acrylamide gels; the two complementary strands of a DNA segment usually resolve as two SSCP conformers of distinct mobilities. However, if the sample is from an individual heterozygous for a base-pair variant within the amplified segment, often three or more bands are seen. In some cases, even the sample from a homozygous individual will show multiple bands. Base-pair-change variants are identified by differences in pattern among the DNAs of the sample set.

Exons of the candidate genes were amplified by PCR from the DNAs of 61 unrelated FAP patients and a control set of 12 normal indi¬ viduals. The five exons from DPI revealed no unique conformers in the FAP patients, although common conformers were observed with exons

2 and 3 in some individuals of both affected and control sets, indicating the presence of DNA sequence polymorphisms. Likewise, none of the five exons of SRP19 revealed unique conformers in DNA from FAP patients in the test panel.

Testing of exons 1 through 14 and primer sets A through N of exon 15 of the DP2.5 gene, however, revealed variant conformers spe¬ cific to FAP patients in exons 7, 8, 10, 11, and 15. These variants were in the unrelated patients 3746, 3460, 3827, 3712, and 3751, respectively. The PCR-SSCP procedure was repeated for each of these exons in the five affected individuals and in an expanded set of 48 normal controls. The variant bands were reproducible in the FAP patients but were not observed in any of the control DNA samples. Additional variant con¬ formers in exons 11 and 15 of the DP2.5 gene were seen; however, each of these was found in both the affected and control DNA sets. The five sets of conformers unique to the FAP patients were sequenced to determine the nucleotide changes responsible for their altered mobili¬ ties. The normal conformers from the host individuals were sequenced also. Bands were cut from the dried acrylamide gels, and the DNA was eluted. PCR amplification of these DNAs provided template for sequencing.

The sequences of the unique conformers from exons 7, 8, 10, and 11 of DP2.5 revealed dramatic mutations in the DP2.5 gene. The sequence of the new mutation creating the exon 7 conf ormer in patient 3746 was shown to contain a deletion of two adjacent nucleotides, at positions 730 and 731 in the cDNA sequence (Figure 7). The normal sequence at this splice junction is CAGGGTCA (intronic sequence underlined), with the intron-exon boundary between the two repetitions of AG. The mutant allele in this patient has the sequence CAGGTCA. Although this change is at the 5' splice site, comparison with known consensus sequences of splice junctions would suggest that a functional splice junction is maintained. If this new splice junction were func¬ tional, the mutation would introduce a frameshift that creates a stop codon 15 nucleotides downstream. If the new splice junction were not functional, messenger processing would be significantly altered.

To confirm the 2-base deletion, the PCR product from FAP patient 3746 and a control DNA were electrophoresed on an acrylamide-urea denaturing gel, along with the products of a sequenc¬ ing reaction. The sample from patient 3746 showed two bands differing in size by 2 nucleotides, with the larger band identical in mobility to the control sample; this result was independent confirmation that patient 3746 is heterozygous for a 2 bp deletion.

The unique conformer found in exon 8 of patient 3460 was found to carry a C-T transition, at position 904 in the cDNA sequence of DP2.5 (shown in Figure 7), which replaced the normal sequence of CGA with TGA. This point mutation, when read in frame, results in a stop codon replacing the normal arginine codon. This single-base change had occurred within the context of a CG dimer, a potential hot spot for mutation (Barker et al., 1984).

The conformer unique to FAP patient 3827 in exon 10 was found to contain a deletion of one nucleotide (1367, 1368, or 1369) when com¬ pared to the normal sequence found in the other bands on the SSCP gel. This deletion, occurring within a set of three T's, changed the sequence from CTTTCA to CTTCA; this 1 base frameshift creates a downstream stop within 30 bases. The PCR product amplified from this patient's DNA also was electrophoresed on an acrylamide-urea denaturing gel, along with the PCR product from a control DNA and products from a sequencing reaction. The patient's PCR product showed two bands differing by 1 bp in length, with the larger identical in mobility to the PCR product from the normal DNA; this result confirmed the presence of a 1 bp deletion in patient 3827.

Sequence analysis of the variant conformer of exon 11 from patient 3712 revealed the substitution of a T by a G at position 1500, changing the normal tyrosine codon to a stop codon.

The pair of conformers observed in exon 15 of the DP2.5 gene for FAP patient 3751 also was sequenced. These conformers were found to carry a nucleotide substitution of C to G at position 5253, the third base of a valine codon. No amino acid change resulted from this substitution, suggesting that this conformer reflects a genetically silent polymorphism.

The observation of distinct inactivating mutations in the DP2.5 gene in four unrelated patients strongly suggested that DP2.5 is the gene involved in FAP. These mutations are summarized in Table IIA. Example 10

This example demonstrates that the mutations identified in the DP2.5 (APC) gene segregate with the FAP phenotype.

Patient 3746, described above as carrying an APC allele with a frameshift mutation, is an affected offspring of two normal parents. Colonoscopy revealed no polyps in either parent nor among the patient's three siblings.

DNA samples from both parents, from the patient's wife, and from their three children were examined. SSCP analysis of DNA from both of the patient's parents displayed the normal pattern of conform¬ ers for exon 7, as did DNA from the patients's wife and one of his off¬ spring. The two other children, however, displayed the same new con¬ formers as their affected father. Testing of the patient and his parents with highly polymorphic VNTR (variable number of tandem repeat) markers showed a 99.98% likelihood that they are his biological parents.

These observations confirmed that this novel conformer, known to reflect a 2 bp deletion mutation in the DP2.5 gene, appeared sponta¬ neously with FAP in this pedigree and was transmitted to two of the children of the affected individual. Example 11

This example demonstrates polymorphisms in the APC gene which appear to be unrelated to disease (FAP).

Sequencing of variant conformers found among controls as well as individuals with APC has revealed the following polymorphisms in the APC gene: first, in exon 11, at position 1458, a substitution of T to C creating an Rsal restriction site but no amino acid change; and sec¬ ond, in exon 15, at positions 5037 and 5271, substitutions of A to G and G to T, respectively, neither resulting in amino acid substitutions. These nucleotide polymorphisms in the APC gene sequence may be useful for diagnostic purposes.

Example 12

This example shows the structure of the APC gene.

The structure of the APC gene is schematically shown in Figure 8, with flanking intron sequences indicated.

The continuity of the very large (6.5 kb), most 3' exon in DP2.5 was shown in two ways. First, inverse PCR with primers spanning the entire length of this exon revealed no divergence of the cDNA sequence from the genomic sequence. Second, PCR amplification with converging primers placed at intervals along the exon generated prod¬ ucts of the same size whether amplified from the originally isolated cDNA, cDNA from various tissues, or genomic template. Two forms of exon 9 were found in DP2.5: one is the complete exon; and the other, labeled exon 9A, is the result of a splice into the interior of the exon that deletes bases 934 to 1236 in the mRNA and removes 101 amino acids from the predicted protein (see Figure 7). Example 13

This example demonstrates the mapping of the FAP deletions with respect to the APC exons.

Somatic cell hybrids carrying the segregated chromosomes 5 from the 100 kb (HHW1291) and 260 kb (HHW1155) deletion patients were used to determine the distribution of the APC genes exons across the deletions. DNAs from these cell lines were used as template, along with genomic DNA from a normal control, for PCR-based amplification of the APC exons.

PCR analysis of the hybrids from the 260 kb deletion of patient 3214 showed that all but one (exon 1) of the APC exons are removed by this deletion. PCR analysis of the somatic cell hybrid HHW1291, carry¬ ing the chromosome 5 homolog with the 100 kb deletion from patient 3824, revealed that exons 1 through 9 are present but exons 10 through 15 are missing. This result placed the deletion breakpoint either between exons 9 and 10 or within exon 10. Example 14

This example demonstrates the expression of alternately spliced APC messenger in normal tissues and in cancer cell lines.

Tissues that express the APC gene were identified by PCR amplification of cDNA made to mRNA with primers located within adjacent APC exons. In addition, PCR primers that flank the alterna¬ tively spliced exon 9 were chosen so that the expression pattern of both splice forms could be assessed. All tissue types tested (brain, lung, aorta, spleen, heart, kidney, liver, stomach, placenta, and colonic mucosa) and cultured cell lines (lymphoblasts, HL60, and choriocarcinoma) expressed both splice forms of the APC gene. We note, however, that expression by lymphocytes normally residing in some tissues, including colon, prevents unequivocal assessment of expression. The large mRNA, containing the complete exon 9 rather than only exon 9A, appears to be the more abundant message.

Northern analysis of poly(A)-selected RNA from lymphoblasts revealed a single band of approximately 10 kb, consistent with the size of the sequenced cDNA. Example 15

This example discusses structural features of the APC protein predicted from the sequence.

The cDNA consensus sequence of APC predicts that the longer, more abundant form of the message codes for a 2842 or 28444 amino acid peptide with a mass of 311.8 kd. This predicted APC peptide was compared with the current data bases of protein and DNA sequences using both Inteliigenetics and GCG software packages. No genes with a high degree of amino acid sequence similarity were found. Although many short (approximately 20 amino acid) regions of sequence similar¬ ity were uncovered, none was sufficently strong to reveal which, if any, might represent functional homology. Interestingly, multiple simi¬ larities to myosins and keratins did appear. The APC gene also was scanned for sequence motifs of known function; although multiple glycosylation, phosphorylation, and myristoylation sites were seen, their significance is uncertain.

Analysis of the APC peptide sequence did identify features important in considering potential protein structure. Hydropathy plots (Kyte and Doolittle, J. Mol. Biol. Vol. 157, pp. 105-132 (1982)) indicate that the APC protein is notably hydrophilic. No hydrophobic domains

suggesting a signal peptide or a membrane-spanning domain were found. Analysis of the first 1000 residues indicates that α-helical rods may form (Cohen and Parry, Trends Biochem, Sci. Vol. 77, pp. 245-248 (1986); there is a scarcity of proline residues and, there are a number of regions containing heptad repeats (apolar-X-X-apolar-X-X-X). Inter¬ estingly, in exon 9A, the deleted form of exon 9, two heptad repeat regions are reconnected in the proper heptad repeat frame, deleting the intervening peptide region. After the first 1000 residues, the high proline content of the remainder of the peptide suggests a compact rather than a rod-like structure.

The most prominent feature of the second 1000 residues is a 20 amino acid repeat that is iterated seven times with semiregular spacing (Table 4). The intervening sequences between the seven repeat regions contained 114, 116, 151, 205, 107, and 58 amino acids, respectively. Finally, residues 2200-24000 contain a 200 amino acid basic domain.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: ALBERTSEN, HANS ANAND, RAKESH CARLSON, MARY GRODEN, JOANNA HEDGE, PHILIP J. JOSLYN, GEOFF KINZLER, KENNETH MARKHAM, ALEXANDER F. NAKAMURA, YUSUKE THLIVERIS, ANDREW

(ii) TITLE OF INVENTION: INHERITED AND SOMATIC MUTATIONS OF APC GENE IN COLORECTAL CANCER IN HUMANS

(iii) NUMBER OF SEQUENCES: 94

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Banner, Birch, McKie S Beckett

(B) STREET: 1001 G Street, NW

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: USA

(F) ZIP: 20001-4598

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/741,940

(B) FILING DATE: 08-AUG-1991

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kagan, Sarah A.

(B) REGISTRATION NUMBER: 32 , 141

(C) REFERENCE/DOCKET NUMBER: 1107.035574

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202-508-9100

(B) TELEFAX: 202-508-9299

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9606 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

-47-

(vii) IMMEDIATE SOURCE:

(B) CLONE: DP2.5(APC)

(ix) FEATURE ^" :

(A) NAME/KEY: CDS

(B) LOCATION: 34..8562

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GGACTCGGAA ATGAGGTCCA AGGGTAGCCA AGG ATG GCT GCA GCT TCA TAT GAT 54

Met Ala Ala Ala Ser Tyr Asp 1 5

CAG TTG TTA AAG CAA GTT GAG GCA CTG AAG ATG GAG AAC TCA AAT CTT 102 Gin Leu Leu Lys Gin Val Glu Ala Leu Lys Met Glu Asn Ser Asn Leu 10 15 20

CGA CAA GAG CTA GAA GAT AAT TCC AAT CAT CTT ACA AAA CTG GAA ACT 150 Arg Gin Glu Leu Glu Asp Asn Ser Asn His Leu Thr Lys Leu Glu Thr 25 30 35

GAG GCA TCT AAT ATG AAG GAA GTA CTT AAA CAA CTA CAA GGA AGT ATT 198 Glu Ala Ser Asn Met Lys Glu Val Leu Lys Gin Leu Gin Gly Ser lie 40 45 50 55

GAA GAT GAA GCT ATG GCT TCT TCT GGA CAG ATT GAT TTA TTA GAG CGT 246 Glu Asp Glu Ala Met Ala Ser Ser Gly Gin lie Asp Leu Leu Glu Arg 60 65 70

CTT AAA GAG CTT AAC TTA GAT AGC AGT AAT TTC CCT GGA GTA AAA CTG 294 Leu Lys Glu Leu Asn Leu Asp Ser Ser Asn Phe Pro Gly Val Lys Leu 75 80 85

CGG TCA AAA ATG TCC CTC CGT TCT TAT GGA AGC CGG GAA GGA TCT GTA 342 Arg Ser Lys Met Ser Leu Arg Ser Tyr Gly Ser Arg Glu Gly Ser Val 90 95 100

TCA AGC CGT TCT GGA GAG TGC AGT CCT GTT CCT ATG GGT TCA TTT CCA 390 Ser Ser Arg Ser Gly Glu Cys Ser Pro Val Pro Met Gly Ser Phe Pro 105 110 115

AGA AGA GGG TTT GTA AAT GGA AGC AGA GAA AGT ACT GGA TAT TTA GAA 438 Arg Arg Gly Phe Val Asn Gly Ser Arg Glu Ser Thr Gly Tyr Leu Glu 120 125 130 135

GAA CTT GAG AAA GAG AGG TCA TTG CTT CTT GCT GAT CTT GAC AAA GAA 486 Glu Leu Glu Lys Glu Arg Ser Leu Leu Leu Ala Asp Leu Asp Lys Glu 140 145 150

GAA AAG GAA AAA GAC TGG TAT TAC GCT CAA CTT CAG AAT CTC ACT AAA 534 Glu Lys Glu Lys Asp Trp Tyr Tyr Ala Gin Leu Gin Asn Leu Thr Lys 155 160 165

AGA ATA GAT AGT CTT CCT TTA ACT GAA AAT TTT TCC TTA CAA ACA GAT 582 Arg lie Asp Ser Leu Pro Leu Thr Glu Asn Phe Ser Leu Gin Thr Asp 170 175 180

TTG ACC AGA AGG CAA TTG GAA TAT GAA GCA AGG CAA ATC AGA GTT GCG 630 Leu Thr Arg Arg Gin Leu Glu Tyr Glu Ala Arg Gin He Arg Val Ala 185 190 195

ATG GAA GAA CAA CTA GGT ACC TGC CAG GAT ATG GAA AAA CGA GCA CAG 678 Met Glu Glu Gin Leu Gly Thr Cys Gin Asp Met Glu Lys Arg Ala Gin 200 205 210 215

CGA AGA ATA GCC AGA ATT CAG CAA ATC GAA AAG GAC ATA CTT CGT ATA 726 Arg Arg He Ala Arg He Gin Gin He Glu Lys Asp He Leu Arg He 220 225 230

CGA CAG CTT TTA CAG TCC CAA GCA ACA GAA GCA GAG AGG TCA TCT CAG 774 Arg Gin Leu Leu Gin Ser Gin Ala Thr Glu Ala Glu Arg Ser Ser Gin 235 240 245

AAC AAG CAT GAA ACC GGC TCA CAT GAT GCT GAG CGG CAG AAT GAA GGT 822 Asn Lys His Glu Thr Gly Ser His Asp Ala Glu Arg Gin Asn Glu Gly 250 255 260

CAA GGA GTG GGA GAA ATC AAC ATG GCA ACT TCT GGT AAT GGT CAG GGT 870 Gin Gly Val Gly Glu He Asn Met Ala Thr Ser Gly Asn Gly Gin Gly 265 270 275

TCA ACT ACA CGA ATG GAC CAT GAA ACA GCC AGT GTT TTG AGT TCT AGT 918 Ser Thr Thr Arg Met Asp His Glu Thr Ala Ser Val Leu Ser Ser Ser 280 285 290 295

AGC ACA CAC TCT GCA CCT CGA AGG CTG ACA AGT CAT CTG GGA ACC AAG 966 Ser Thr His Ser Ala Pro Arg Arg Leu Thr Ser His Leu Gly Thr Lys 300 305 310

GTG GAA ATG GTG TAT TCA TTG TTG TCA ATG CTT GGT ACT CAT GAT AAG 1014 Val Glu Met Val Tyr Ser Leu Leu Ser Met Leu Gly Thr His Asp Lys 315 320 325

GAT GAT ATG TCG CGA ACT TTG CTA GCT ATG TCT AGC TCC CAA GAC AGC 1062 Asp Asp Met Ser Arg Thr Leu Leu Ala Met Ser Ser Ser Gin Asp Ser 330 335 340

TGT ATA TCC ATG CGA CAG TCT GGA TGT CTT CCT CTC CTC ATC CAG CTT 1110 Cys He Ser Met Arg Gin Ser Gly Cys Leu Pro Leu Leu He Gin Leu 345 350 355

TTA CAT GGC AAT GAC AAA GAC TCT GTA TTG TTG GGA AAT TCC CGG GGC 1158 Leu His Gly Asn Asp Lys Asp Ser Val Leu Leu Gly Asn Ser Arg Gly 360 365 370 375

AGT AAA GAG GCT CGG GCC AGG GCC AGT GCA GCA CTC CAC AAC ATC ATT 1206 Ser Lys Glu Ala Arg Ala Arg Ala Ser Ala Ala Leu His Asn He He 380 385 390

CAC TCA CAG CCT GAT GAC AAG AGA GGC AGG CGT GAA ATC CGA GTC CTT 1254 His Ser Gin Pro Asp Asp Lys Arg Gly Arg Arg Glu He Arg Val Leu 395 400 405

CAT CTT TTG GAA CAG ATA CGC GCT TAC TGT GAA ACC TGT TGG GAG TGG 1302 His Leu Leu Glu Gin He Arg Ala Tyr Cys Glu Thr Cys Trp Glu Trp 410 415 420

CAG GAA GCT CAT GAA CCA GGC ATG GAC CAG GAC AAA AAT CCA ATG CCA 1350 Gin Glu Ala His Glu Pro Gly Met Asp Gin Asp Lys Asn Pro Met Pro 425 430 435

_G CT CCT GTT GAA CAT CAG ATC TGT CCT GCT GTG TGT GTT CTA ATG AAA 1398 Ala Pro Val Glu His Gin He Cys Pro Ala Val Cys Val Leu Met Lys 440 445 450 455

CTT TCA TTT GAT GAA GAG CAT AGA CAT GCA ATG AAT GAA CTA GGG GGA 1446 Leu Ser Phe Aβp Glu Glu His Arg His Ala Met Asn Glu Leu Gly Gly 460 465 470

CTA CAG GCC ATT GCA GAA TTA TTG CAA GTG GAC TGT GAA ATG TAT GGG 1494 Leu Gin Ala He Ala Glu Leu Leu Gin Val Aβp Cys Glu Met Tyr Gly 475 480 485

CTT ACT AAT GAC CAC TAC AGT ATT ACA CTA AGA CGA TAT GCT GGA ATG 1542 Leu Thr Asn Asp His Tyr Ser He Thr Leu Arg Arg Tyr Ala Gly Met 490 495 500

GCT TTG ACA AAC TTG ACT TTT GGA GAT GTA GCC AAC AAG GCT ACG CTA 1590 Ala Leu Thr Asn Leu Thr Phe Gly Asp Val Ala Asn Lys Ala Thr Leu 505 510 515

TGC TCT ATG AAA GGC TGC ATG AGA GCA CTT GTG GCC CAA CTA AAA TCT 1638 Cys Ser Met Lys Gly Cys Met Arg Ala Leu Val Ala Gin Leu Lys Ser 520 525 530 535

GAA AGT GAA GAC TTA CAG CAG GTT ATT GCA AGT GTT TTG AGG AAT TTG 1686 Glu Ser Glu Aβp Leu Gin Gin Val He Ala Ser Val Leu Arg Asn Leu 540 545 550

TCT TGG CGA GCA GAT GTA AAT AGT AAA AAG ACG TTG CGA GAA GTT GGA 1734 Ser Trp Arg Ala Asp Val Asn Ser Lys Lys Thr Leu Arg Glu Val Gly 555 560 565

AGT GTG AAA GCA TTG ATG GAA TGT GCT TTA GAA GTT AAA AAG GAA TCA 1782 Ser Val Lys Ala Leu Met Glu Cys Ala Leu Glu Val Lys Lys Glu Ser 570 575 580

ACC CTC AAA AGC GTA TTG AGT GCC TTA TGG AAT TTG TCA GCA CAT TGC 1830 Thr Leu Lys Ser Val Leu Ser Ala Leu Trp Asn Leu Ser Ala His Cys 585 590 595

ACT GAG AAT AAA GCT GAT ATA TGT GCT GTA GAT GGT GCA CTT GCA TTT 1878 Thr Glu Asn Lys Ala Asp He Cys Ala Val Aβp Gly Ala Leu Ala Phe 600 605 610 615

TTG GTT GGC ACT CTT ACT TAC CGG AGC CAG ACA AAC ACT TTA GCC ATT 1926 Leu Val Gly Thr Leu Thr Tyr Arg Ser Gin Thr Asn Thr Leu Ala He 620 625 630

ATT GAA AGT GGA GGT GGG ATA TTA CGG AAT GTG TCC AGC TTG ATA GCT 1974 He Glu Ser Gly Gly Gly He Leu Arg Asn Val Ser Ser Leu He Ala 635 640 645

ACA AAT GAG GAC CAC AGG CAA ATC CTA AGA GAG AAC AAC TGT CTA CAA 2022 Thr Asn Glu Asp His Arg Gin He Leu Arg Glu Asn Asn Cys Leu Gin 650 655 660

ACT TTA TTA CAA CAC TTA AAA TCT CAT AGT TTG ACA ATA GTC AGT AAT 2070 Thr Leu Leu Gin His Leu Lys Ser His Ser Leu Thr He Val Ser Asn 665 670 675

GCA TGT GGA ACT TTG TGG AAT CTC TCA GCA AGA AAT CCT AAA GAC CAG 2118 Ala Cyβ Gly Thr Leu Trp Aβn Leu Ser Ala Arg Aβn Pro Lye Asp Gin 680 685 690 695

GAA GCA TTA TGG GAC ATG GGG GCA GTT AGC ATG CTC AAG AAC CTC ATT 2166 Glu Ala Leu Trp Asp Met Gly Ala Val Ser Met Leu Lye Aβn Leu He 700 705 710

CAT TCA AAG CAC AAA ATG ATT GCT ATG GGA AGT GCT GCA GCT TTA AGG 2214 His Ser Lys His Lys Met He Ala Met Gly Ser Ala Ala Ala Leu Arg 715 720 725

AAT CTC ATG GCA AAT AGG CCT GCG AAG TAC AAG GAT GCC AAT ATT ATG 2262 Aβn Leu Met Ala Aβn Arg Pro Ala Lys Tyr Lys Asp Ala Asn He Met 730 735 740

TCT CCT GGC TCA AGC TTG CCA TCT CTT CAT GTT AGG AAA CAA AAA GCC 2310 Ser Pro Gly Ser Ser Leu Pro Ser Leu His Val Arg Lys Gin Lys Ala 745 750 755

CTA GAA GCA GAA TTA GAT GCT CAG CAC TTA TCA GAA ACT TTT GAC AAT 2358 Leu Glu Ala Glu Leu Asp Ala Gin His Leu Ser Glu Thr Phe Asp Asn 760 765 770 775

ATA GAC AAT TTA AGT CCC AAG GCA TCT CAT CGT AGT AAG CAG AGA CAC 2406 He Asp Asn Leu Ser Pro Lye Ala Ser His Arg Ser Lys Gin Arg His 780 785 790

AAG CAA AGT CTC TAT GGT GAT TAT GTT TTT GAC ACC AAT CGA CAT GAT 2454 Lys Gin Ser Leu Tyr Gly Aβp Tyr Val Phe Asp Thr Asn Arg His Asp 795 800 805

GAT AAT AGG TCA GAC AAT TTT AAT ACT GGC AAC ATG ACT GTC CTT TCA 2502 Asp Asn Arg Ser Aβp Aβn Phe Aβn Thr Gly Aβn Met Thr Val Leu Ser 810 815 820

CCA TAT TTG AAT ACT ACA GTG TTA CCC AGC TCC TCT TCA TCA AGA GGA 2550 Pro Tyr Leu Asn Thr Thr Val Leu Pro Ser Ser Ser Ser Ser Arg Gly 825 830 835

AGC TTA GAT AGT TCT CGT TCT GAA AAA GAT AGA AGT TTG GAG AGA GAA 2598 Ser Leu Aβp Ser Ser Arg Ser Glu Lys Asp Arg Ser Leu Glu Arg Glu 840 845 850 855

CGC GGA ATT GGT CTA GGC AAC TAC CAT CCA GCA ACA GAA AAT CCA GGA 2646 Arg Gly He Gly Leu Gly Asn Tyr His Pro Ala Thr Glu Asn Pro Gly 860 865 870

ACT TCT TCA AAG CGA GGT TTG CAG ATC TCC ACC ACT GCA GCC CAG ATT 2694 Thr Ser Ser Lys Arg Gly Leu Gin He Ser Thr Thr Ala Ala Gin He 875 880 885

GCC AAA GTC ATG GAA GAA GTG TCA GCC ATT CAT ACC TCT CAG GAA GAC 2742 Ala Lye Val Met Glu Glu Val Ser Ala He His Thr Ser Gin Glu Asp 890 895 900

AGA AGT TCT GGG TCT ACC ACT GAA TTA CAT TGT GTG ACA GAT GAG AGA 2790 Arg Ser Ser Gly Ser Thr Thr Glu Leu His Cys Val Thr Asp Glu Arg 905 910 915

AAT GCA CTT AGA AGA AGC TCT GCT GCC CAT ACA CAT TCA AAC ACT TAC 2838 Aβn Ala Leu Arg Arg Ser Ser Ala Ala Hie Thr Hie Ser Aβn Thr Tyr 920 925 930 935

AAT TTC ACT AAG TCG GAA AAT TCA AAT AGG ACA TGT TCT ATG CCT TAT 2886 Aβn Phe Thr Lys Ser Glu Aβn Ser Aβn Arg Thr Cys Ser Met Pro Tyr 940 945 950

GCC AAA TTA GAA TAC AAG AGA TCT TCA AAT GAT AGT TTA AAT AGT GTC 2934 Ala Lys Leu Glu Tyr Lye Arg Ser Ser Asn Asp Ser Leu Asn Ser Val 955 960 965

AGT AGT AAT GAT GGT TAT GGT AAA AGA GGT CAA ATG AAA CCC TCG ATT 2982 Ser Ser Aβn Aβp Gly Tyr Gly Lys Arg Gly Gin Met Lys Pro Ser He 970 975 980

GAA TCC TAT TCT GAA GAT GAT GAA AGT AAG TTT TGC AGT TAT GGT CAA 3030 Glu Ser Tyr Ser Glu Aβp Asp Glu Ser Lys Phe Cyβ Ser Tyr Gly Gin 985 990 995

TAC CCA GCC GAC CTA GCC CAT AAA ATA CAT AGT GCA AAT CAT ATG GAT 3078 Tyr Pro Ala Aβp Leu Ala Hie Lye He Hie Ser Ala Asn Hie Met Aβp 1000 1005 1010 1015

GAT AAT GAT GGA GAA CTA GAT ACA CCA ATA AAT TAT AGT CTT AAA TAT 3126 Aβp Asn Aβp Gly Glu Leu Aβp Thr Pro He Asn Tyr Ser Leu Lys Tyr 1020 1025 1030

TCA GAT GAG CAG TTG AAC TCT GGA AGG CAA AGT CCT TCA CAG AAT GAA 3174 Ser Asp Glu Gin Leu Asn Ser Gly Arg Gin Ser Pro Ser Gin Asn Glu 1035 1040 1045

AGA TGG GCA AGA CCC AAA CAC ATA ATA GAA GAT GAA ATA AAA CAA AGT 3222 Arg Trp Ala Arg Pro Lys His He He Glu Aβp Glu He Lys Gin Ser 1050 1055 1060

GAG CAA AGA CAA TCA AGG AAT CAA AGT ACA ACT TAT CCT GTT TAT ACT 3270 Glu Gin Arg Gin Ser Arg Asn Gin Ser Thr Thr Tyr Pro Val Tyr Thr 1065 1070 1075

GAG AGC ACT GAT GAT AAA CAC CTC AAG TTC CAA CCA CAT TTT GGA CAG 3318 Glu Ser Thr Asp Asp Lys Hie Leu Lye Phe Gin Pro Hie Phe Gly Gin 1080 1085 1090 1095

CAG GAA TGT GTT TCT CCA TAC AGG TCA CGG GGA GCC AAT GGT TCA GAA 3366 Gin Glu Cys Val Ser Pro Tyr Arg Ser Arg Gly Ala Asn Gly Ser Glu 1100 1105 1110

ACA AAT CGA GTG GGT TCT AAT CAT GGA ATT AAT CAA AAT GTA AGC CAG 3414 Thr Asn Arg Val Gly Ser Asn His Gly He Asn Gin Asn Val Ser Gin 1115 1120 1125

TCT TTG TGT CAA GAA GAT GAC TAT GAA GAT GAT AAG CCT ACC AAT TAT 3462 Ser Leu Cys Gin Glu Asp Asp Tyr Glu Asp Asp Lys Pro Thr Asn Tyr 1130 1135 1140

AGT GAA CGT TAC TCT GAA GAA GAA CAG CAT GAA GAA GAA GAG AGA CCA 3510 Ser Glu Arg Tyr Ser Glu Glu Glu Gin His Glu Glu Glu Glu Arg Pro 1145 1150 1155

ACA AAT TAT AGC ATA AAA TAT AAT GAA CAG AAA CGT CAT CTC GAT CAG 3558 Thr Asn Tyr Ser He Lys Tyr Aβn Glu Glu Lye Arg Hie Val Aβp Gin 1160 1165 1170 1175

CCT ATT GAT TAT AGT TTA AAA TAT GCC ACA GAT ATT CCT TCA TCA CAG 3606 Pro He Asp Tyr Ser Leu Lye Tyr Ala Thr Aβp He Pro Ser Ser Gin 1180 1185 1190

AAA CAG TCA TTT TCA TTC TCA AAG AGT TCA TCT GGA CAA AGC AGT AAA 3654 Lys Gin Ser Phe Ser Phe Ser Lys Ser Ser Ser Gly Gin Ser Ser Lys 1195 1200 1205

ACC GAA CAT ATG TCT TCA AGC AGT GAG AAT ACG TCC ACA CCT TCA TCT 3702 Thr Glu His Met Ser Ser Ser Ser Glu Aβn Thr Ser Thr Pro Ser Ser 1210 1215 1220

AAT GCC AAG AGG CAG AAT CAG CTC CAT CCA AGT TCT GCA CAG AGT AGA 3750 Asn Ala Lys Arg Gin Asn Gin Leu His Pro Ser Ser Ala Gin Ser Arg 1225 1230 1235

AGT GGT CAG CCT CAA AAG GCT GCC ACT TGC AAA GTT TCT TCT ATT AAC 3798 Ser Gly Gin Pro Gin Lys Ala Ala Thr Cys Lye Val Ser Ser He Aβn 1240 1245 1250 1255

CAA GAA ACA ATA CAG ACT TAT TGT GTA GAA GAT ACT CCA ATA TGT TTT 3846 Gin Glu Thr He Gin Thr Tyr Cys Val Glu Asp Thr Pro He Cys Phe 1260 1265 1270

TCA AGA TGT AGT TCA TTA TCA TCT TTG TCA TCA GCT GAA GAT GAA ATA 3894 Ser Arg Cys Ser Ser Leu Ser Ser Leu Ser Ser Ala Glu Asp Glu He 1275 1280 1285

GGA TGT AAT CAG ACG ACA CAG GAA GCA GAT TCT GCT AAT ACC CTG CAA 3942 Gly Cys Aβn Gin Thr Thr Gin Glu Ala Aβp Ser Ala Aβn Thr Leu Gin 1290 1295 1300

ATA GCA GAA ATA AAA GGA AAG ATT GGA ACT AGG TCA GCT GAA GAT CCT 3990 He Ala Glu He Lys Gly Lys He Gly Thr Arg Ser Ala Glu Aβp Pro 1305 1310 1315

GTG AGC GAA GTT CCA GCA GTG TCA CAG CAC CCT AGA ACC AAA TCC AGC 4038 Val Ser Glu Val Pro Ala Val Ser Gin Hie Pro Arg Thr Lys Ser Ser 1320 1325 1330 1335

AGA CTG CAG GGT TCT AGT TTA TCT TCA GAA TCA GCC AGG CAC AAA GCT 4086 Arg Leu Gin Gly Ser Ser Leu Ser Ser Glu Ser Ala Arg His Lys Ala 1340 1345 1350

GTT GAA TTT CCT TCA GGA GCG AAA TCT CCC TCC AAA AGT GGT GCT CAG 4134 Val Glu Phe Pro Ser Gly Ala Lys Ser Pro Ser Lye Ser Gly Ala Gin 1355 1360 1365

ACA CCC AAA AGT CCA CCT GAA CAC TAT GTT CAG GAG ACC CCA CTC ATG 4182 Thr Pro Lys Ser Pro Pro Glu His Tyr Val Gin Glu Thr Pro Leu Met 1370 1375 1380

TTT AGC AGA TGT ACT TCT GTC AGT TCA CTT GAT AGT TTT GAG AGT CGT 4230 Phe Ser Arg Cys Thr Ser Val Ser Ser Leu Asp Ser Phe Glu Ser Arg 1385 1390 1395

TCG ATT GCC AGC TCC GTT CAG AGT GAA CCA TGC AGT GGA ATG GTA AGT 4278 Ser He Ala Ser Ser Val Gin Ser Glu Pro Cye Ser Gly Met Val Ser 1400 1405 1410 1415

GGC ATT ATA AGC CCC AGT GAT CTT CCA GAT AGC CCT GGA CAA ACC ATG 4326 Gly He He Ser Pro Ser Asp Leu Pro Aβp Ser Pro Gly Gin Thr Met 1420 1425 1430

CCA CCA AGC AGA AGT AAA ACA CCT CCA CCA CCT CCT CAA ACA GCT CAA 4374 Pro Pro Ser Arg Ser Lys Thr Pro Pro Pro Pro Pro Gin Thr Ala Gin 1435 1440 1445

ACC AAG CGA GAA GTA CCT AAA AAT AAA GCA CCT ACT GCT GAA AAG AGA 4422 Thr Lys Arg Glu Val Pro Lye Aβn Lye Ala Pro Thr Ala Glu Lys Arg 1450 1455 1460

GAG AGT GGA CCT AAG CAA GCT GCA GTA AAT GCT GCA GTT CAG AGG GTC 4470 Glu Ser Gly Pro Lye Gin Ala Ala Val Asn Ala Ala Val Gin Arg Val 1465 1470 1475

CAG GTT CTT CCA GAT GCT GAT ACT TTA TTA CAT TTT GCC ACA GAA AGT 4518 Gin Val Leu Pro Aβp Ala Aβp Thr Leu Leu Hie Phe Ala Thr Glu Ser 1480 1485 1490 1495

ACT CCA GAT GGA TTT TCT TGT TCA TCC AGC CTG AGT GCT CTG AGC CTC 4566 Thr Pro Asp Gly Phe Ser Cys Ser Ser Ser Leu Ser Ala Leu Ser Leu 1500 1505 1510

GAT GAG CCA TTT ATA CAG AAA GAT GTG GAA TTA AGA ATA ATG CCT CCA 4614 Asp Glu Pro Phe He Gin Lys Aβp Val Glu Leu Arg He Met Pro Pro 1515 1520 1525

GTT CAG GAA AAT GAC AAT GGG AAT GAA ACA GAA TCA GAG CAG CCT AAA 4662 Val Gin Glu Asn Asp Asn Gly Aβn Glu Thr Glu Ser Glu Gin Pro Lys 1530 1535 1540

GAA TCA AAT GAA AAC CAA GAG AAA GAG GCA GAA AAA ACT ATT GAT TCT 4710 Glu Ser Asn Glu Asn Gin Glu Lys Glu Ala Glu Lys Thr He Asp Ser 1545 1550 1555

GAA AAG GAC CTA TTA GAT GAT TCA GAT GAT GAT GAT ATT GAA ATA CTA 4758 Glu Lys Asp Leu Leu Asp Asp Ser Aβp Aβp Asp Aβp He Glu He Leu 1560 1565 1570 1575

GAA GAA TGT ATT ATT TCT GCC ATG CCA ACA AAG TCA TCA CGT AAA GGC 4806 Glu Glu Cyβ He He Ser Ala Met Pro Thr Lys Ser Ser Arg Lys Gly 1580 1585 1590

AAA AAG CCA GCC CAG ACT GCT TCA AAA TTA CCT CCA CCT GTG GCA AGG 4854 Lys Lys Pro Ala Gin Thr Ala Ser Lys Leu Pro Pro Pro Val Ala Arg 1595 1600 1605

AAA CCA AGT CAG CTG CCT GTG TAC AAA CTT CTA CCA TCA CAA AAC AGG 4902 Lys Pro Ser Gin Leu Pro Val Tyr Lys Leu Leu Pro Ser Gin Asn Arg 1610 1615 1620

TTG CAA CCC CAA AAG CAT GTT AGT TTT ACA CCG GGG GAT GAT ATG CCA 4950 Leu Gin Pro Gin Lys His Val Ser Phe Thr Pro Gly Asp Asp Met Pro 1625 1630 1635

CGG GTG TAT TGT GTT GAA GGG ACA CCT ATA AAC TTT TCC ACA GCT ACA 4998 Arg Val Tyr Cys Val Glu Gly Thr Pro He Aβn Phe Ser Thr Ala Thr 1640 1645 1650 1655

TCT CTA AGT GAT CTA ACA ATC GAA TCC CCT CCA AAT GAG TTA GCT GCT 5046 Ser Leu Ser Aβp Leu Thr He Glu Ser Pro Pro Aβn Glu Leu Ala Ala 1660 1665 1670

GGA GAA GGA GTT AGA GGA GGA GCA CAG TCA GGT GAA TTT GAA AAA CGA 5094 Gly Glu Gly Val Arg Gly Gly Ala Gin Ser Gly Glu Phe Glu Lye Arg 1675 1680 1685

GAT ACC ATT CCT ACA GAA GGC AGA AGT ACA GAT GAG GCT CAA GGA GGA 5142 Asp Thr He Pro Thr Glu Gly Arg Ser Thr Aβp Glu Ala Gin Gly Gly 1690 1695 1700

AAA ACC TCA TCT GTA ACC ATA CCT GAA TTG GAT GAC AAT AAA GCA GAG 5190 Lys Thr Ser Ser Val Thr He Pro Glu Leu Asp Asp Asn Lys Ala Glu 1705 1710 1715

GAA GGT GAT ATT CTT GCA GAA TGC ATT AAT TCT GCT ATG CCC AAA GGG 5238 Glu Gly Asp He Leu Ala Glu Cys He Asn Ser Ala Met Pro Lys Gly 1720 1725 1730 1735

AAA AGT CAC AAG CCT TTC CGT GTG AAA AAG ATA ATG GAC CAG GTC CAG 5286 Lys Ser His Lys Pro Phe Arg Val Lys Lys He Met Asp Gin Val Gin 1740 1745 1750

CAA GCA TCT GCG TCG TCT TCT GCA CCC AAC AAA AAT CAG TTA GAT GGT 5334 Gin Ala Ser Ala Ser Ser Ser Ala Pro Asn Lys Aβn Gin Leu Asp Gly 1755 1760 1765

AAG AAA AAG AAA CCA ACT TCA CCA GTA AAA CCT ATA CCA CAA AAT ACT 5382 Lys Lys Lys Lye Pro Thr Ser Pro Val Lys Pro He Pro Gin Asn Thr 1770 1775 1780

GAA TAT AGG ACA CGT GTA AGA AAA AAT GCA GAC TCA AAA AAT AAT TTA 5430 Glu Tyr Arg Thr Arg Val Arg Lys Asn Ala Asp Ser Lys Asn Asn Leu 1785 1790 1795

AAT GCT GAG AGA GTT TTC TCA GAC AAC AAA GAT TCA AAG AAA CAG AAT 5478 Aβn Ala Glu Arg Val Phe Ser Asp Asn Lys Asp Ser Lys Lye Gin Asn 1800 1805 1810 1815

TTG AAA AAT AAT TCC AAG GAC TTC AAT GAT AAG CTC CCA AAT AAT GAA 5526 Leu Lye Asn Asn Ser Lye Aβp Phe Aβn Aβp Lye Leu Pro Asn Aβn Glu 1820 1825 1830

GAT AGA GTC AGA GGA AGT TTT GCT TTT GAT TCA CCT CAT CAT TAC ACG 5574 Aβp Arg Val Arg Gly Ser Phe Ala Phe Aβp Ser Pro Hie Hie Tyr Thr 1835 1840 1845

CCT ATT GAA GGA ACT CCT TAC TGT TTT TCA CGA AAT GAT TCT TTG AGT 5622 Pro He Glu Gly Thr Pro Tyr Cys Phe Ser Arg Asn Asp Ser Leu Ser 1850 1855 1860

TCT CTA GAT TTT GAT GAT GAT GAT GTT GAC CTT TCC AGG GAA AAG GCT 5670 Ser Leu Asp Phe Asp Asp Asp Asp Val Asp Leu Ser Arg Glu Lys Ala 1865 1870 1875

GAA TTA AGA AAG GCA AAA GAA AAT AAG GAA TCA GAG GCT AAA GTT ACC 5718 Glu Leu Arg Lys Ala Lye Glu Asn Lye Glu Ser Glu Ala Lys Val Thr 1880 1885 1890 1895

AGC CAC ACA GAA CTA ACC TCC AAC CAA CAA TCA GCT AAT AAG ACA CAA 5766 Ser His Thr Glu Leu Thr Ser Asn Gin Gin Ser Ala Aβn Lys Thr Gin 1900 1905 1910

GCT ATT GCA AAG CAG CCA ATA AAT CGA GGT CAG CCT AAA CCC ATA CTT 5814 Ala He Ala Lys Gin Pro He Asn Arg Gly Gin Pro Lye Pro He Leu 1915 1920 1925

CAG AAA CAA TCC ACT TTT CCC CAG TCA TCC AAA GAC ATA CCA GAC AGA 5862 Gin Lys Gin Ser Thr Phe Pro Gin Ser Ser Lys Asp He Pro Aβp Arg 1930 1935 1940

GGG GCA GCA ACT GAT GAA AAG TTA CAG AAT TTT GCT ATT GAA AAT ACT 5910 Gly Ala Ala Thr Asp Glu Lys Leu Gin Asn Phe Ala He Glu Asn Thr 1945 1950 1955

CCA GTT TGC TTT TCT CAT AAT TCC TCT CTG AGT TCT CTC AGT GAC ATT 5958 Pro Val Cys Phe Ser His Aβn Ser Ser Leu Ser Ser Leu Ser Aβp He 1960 1965 1970 1975

GAC CAA GAA AAC AAC AAT AAA GAA AAT GAA CCT ATC AAA GAG ACT GAG 6006 Aβp Gin Glu Aβn Aβn Aβn Lye Glu Asn Glu Pro He Lys Glu Thr Glu 1980 1985 1990

CCC CCT GAC TCA CAG GGA GAA CCA AGT AAA CCT CAA GCA TCA GGC TAT 6054 Pro Pro Asp Ser Gin Gly Glu Pro Ser Lys Pro Gin Ala Ser Gly Tyr 1995 2000 2005

GCT CCT AAA TCA TTT CAT GTT GAA GAT ACC CCA GTT TGT TTC TCA AGA 6102 Ala Pro Lys Ser Phe His Val Glu Aβp Thr Pro Val Cyβ Phe Ser Arg 2010 2015 2020

AAC AGT TCT CTC AGT TCT CTT AGT ATT GAC TCT GAA GAT GAC CTG TTG 6150 Asn Ser Ser Leu Ser Ser Leu Ser He Asp Ser Glu Asp Asp Leu Leu 2025 2030 2035

CAG GAA TGT ATA AGC TCC GCA ATG CCA AAA AAG AAA AAG CCT TCA AGA 6198 Gin Glu Cys He Ser Ser Ala Met Pro Lys Lye Lye Lye Pro Ser Arg 2040 2045 2050 2055

CTC AAG GGT GAT AAT GAA AAA CAT AGT CCC AGA AAT ATG GGT GGC ATA 6246 Leu Lye Gly Aβp Aβn Glu Lye His Ser Pro Arg Asn Met Gly Gly He 2060 2065 2070

TTA GGT GAA GAT CTG ACA CTT GAT TTG AAA GAT ATA CAG AGA CCA GAT 6294 Leu Gly Glu Asp Leu Thr Leu Asp Leu Lys Aβp He Gin Arg Pro Aβp 2075 2080 2085

TCA GAA CAT GGT CTA TCC CCT GAT TCA GAA AAT TTT GAT TGG AAA GCT 6342 Ser Glu His Gly Leu Ser Pro Asp Ser Glu Asn Phe Aβp Trp Lye Ala 2090 2095 2100

ATT CAG GAA GGT GCA AAT TCC ATA GTA AGT AGT TTA CAT CAA GCT GCT 6390 He Gin Glu Gly Ala Asn Ser He Val Ser Ser Leu His Gin Ala Ala 2105 2110 2115

GCT GCT GCA TGT TTA TCT AGA CAA GCT TCG TCT GAT TCA GAT TCC ATC 6438 Ala Ala Ala Cys Leu Ser Arg Gin Ala Ser Ser Asp Ser Asp Ser He 2120 2125 2130 2135

CTT TCC CTG AAA TCA GGA ATC TCT CTG GGA TCA CCA TTT CAT CTT ACA 6486 Leu Ser Leu Lys Ser Gly He Ser Leu Gly Ser Pro Phe Hie Leu Thr 2140 2145 2150

CCT GAT CAA GAA GAA AAA CCC TTT ACA AGT AAT AAA GGC CCA CGA ATT 6534 Pro Aβp Gin Glu Glu Lys Pro Phe Thr Ser Asn Lys Gly Pro Arg He 2155 2160 2165

CTA AAA CCA GGG GAG AAA AGT ACA TTG GAA ACT AAA AAG ATA GAA TCT 6582 Leu Lys Pro Gly Glu Lye Ser Thr Leu Glu Thr Lys Lye He Glu Ser 2170 2175 2180

GAA AGT AAA GGA ATC AAA GGA GGA AAA AAA GTT TAT AAA AGT TTG ATT 6630 Glu Ser Lys Gly He Lys Gly Gly Lys Lys Val Tyr Lys Ser Leu He 2185 2190 2195

ACT GGA AAA GTT CGA TCT AAT TCA GAA ATT TCA GGC CAA ATG AAA CAG 6678 Thr Gly Lys Val Arg Ser Asn Ser Glu He Ser Gly Gin Met Lys Gin 2200 2205 2210 2215

CCC CTT CAA GCA AAC ATG CCT TCA ATC TCT CGA GGC AGG ACA ATG ATT 6726 Pro Leu Gin Ala Asn Met Pro Ser He Ser Arg Gly Arg Thr Met He 2220 2225 2230

CAT ATT CCA GGA GTT CGA AAT AGC TCC TCA AGT ACA AGT CCT GTT TCT 6774 His He Pro Gly Val Arg Asn Ser Ser Ser Ser Thr Ser Pro Val Ser 2235 2240 2245

AAA AAA GGC CCA CCC CTT AAG ACT CCA GCC TCC AAA AGC CCT AGT GAA 6822 Lys Lys Gly Pro Pro Leu Lys Thr Pro Ala Ser Lye Ser Pro Ser Glu 2250 2255 2260

GGT CAA ACA GCC ACC ACT TCT CCT AGA GGA GCC AAG CCA TCT GTG AAA 6870 Gly Gin Thr Ala Thr Thr Ser Pro Arg Gly Ala Lye Pro Ser Val Lye 2265 2270 2275

TCA GAA TTA AGC CCT GTT GCC AGG CAG ACA TCC CAA ATA GGT GGG TCA 6918 Ser Glu Leu Ser Pro Val Ala Arg Gin Thr Ser Gin He Gly Gly Ser 2280 2285 2290 2295

AGT AAA GCA CCT TCT AGA TCA GGA TCT AGA GAT TCG ACC CCT TCA AGA 6966 Ser Lys Ala Pro Ser Arg Ser Gly Ser Arg Asp Ser Thr Pro Ser Arg 2300 2305 2310

CCT GCC CAG CAA CCA TTA AGT AGA CCT ATA CAG TCT CCT GGC CGA AAC 7014 Pro Ala Gin Gin Pro Leu Ser Arg Pro He Gin Ser Pro Gly Arg Asn 2315 2320 2325

TCA ATT TCC CCT GGT AGA AAT GGA ATA AGT CCT CCT AAC AAA TTA TCT 7062 Ser He Ser Pro Gly Arg Asn Gly He Ser Pro Pro Aβn Lye Leu Ser 2330 2335 2340

CAA CTT CCA AGG ACA TCA TCC CCT AGT ACT GCT TCA ACT AAG TCC TCA 7110 Gin Leu Pro Arg Thr Ser Ser Pro Ser Thr Ala Ser Thr Lys Ser Ser 2345 2350 2355

GGT TCT GGA AAA ATG TCA TAT ACA TCT CCA GGT AGA CAG ATG AGC CAA 7158 Gly Ser Gly Lye Met Ser Tyr Thr Ser Pro Gly Arg Gin Met Ser Gin 2360 2365 2370 2375

CAG AAC CTT ACC AAA CAA ACA GGT TTA TCC AAG AAT GCC AGT AGT ATT 7206 Gin Asn Leu Thr Lye Gin Thr Gly Leu Ser Lye Aβn Ala Ser Ser He 2380 2385 2390

CCA AGA AGT GAG TCT GCC TCC AAA GGA CTA AAT CAG ATG AAT AAT GGT 7254 Pro Arg Ser Glu Ser Ala Ser Lye Gly Leu Aβn Gin Met Aβn Aβn Gly 2395 2400 2405

AAT GGA GCC AAT AAA AAG GTA GAA CTT TCT AGA ATG TCT TCA ACT AAA 7302 Aβn Gly Ala Aβn Lye Lys Val Glu Leu Ser Arg Met Ser Ser Thr Lys 2410 2415 2420

TCA AGT GGA AGT GAA TCT GAT AGA TCA GAA AGA CCT GTA TTA GTA CGC 7350 Ser Ser Gly Ser Glu Ser Asp Arg Ser Glu Arg Pro Val Leu Val Arg 2425 2430 2435

CAG TCA ACT TTC ATC AAA GAA GCT CCA AGC CCA ACC TTA AGA AGA AAA 7398 Gin Ser Thr Phe He Lys Glu Ala Pro Ser Pro Thr Leu Arg Arg Lys 2440 2445 2450 2455

TTG GAG GAA TCT GCT TCA TTT GAA TCT CTT TCT CCA TCA TCT AGA CCA 7446 Leu Glu Glu Ser Ala Ser Phe Glu Ser Leu Ser Pro Ser Ser Arg Pro 2460 2465 2470

GCT TCT CCC ACT AGG TCC CAG GCA CAA ACT CCA GTT TTA AGT CCT TCC 7494 Ala Ser Pro Thr Arg Ser Gin Ala Gin Thr Pro Val Leu Ser Pro Ser 2475 2480 2485

CTT CCT GAT ATG TCT CTA TCC ACA CAT TCG TCT GTT CAG GCT GGT GGA 7542 Leu Pro Aβp Met Ser Leu Ser Thr Hie Ser Ser Val Gin Ala Gly Gly 2490 2495 2500

TGG CGA AAA CTC CCA CCT AAT CTC AGT CCC ACT ATA GAG TAT AAT GAT 7590 Trp Arg Lye Leu Pro Pro Aβn Leu Ser Pro Thr He Glu Tyr Aβn Asp 2505 2510 2515

GGA AGA CCA GCA AAG CGC CAT GAT ATT GCA CGG TCT CAT TCT GAA AGT 7638 Gly Arg Pro Ala Lye Arg Hie Aβp He Ala Arg Ser Hie Ser Glu Ser 2520 2525 2530 2535

CCT TCT AGA CTT CCA ATC AAT AGG TCA GGA ACC TGG AAA CGT GAG CAC 7686 Pro Ser Arg Leu Pro He Aβn Arg Ser Gly Thr Trp Lye Arg Glu Hie 2540 2545 2550

AGC AAA CAT TCA TCA TCC CTT CCT CGA GTA AGC ACT TGG AGA AGA ACT 7734 Ser Lye Hie Ser Ser Ser Leu Pro Arg Val Ser Thr Trp Arg Arg Thr 2555 2560 2565

GGA AGT TCA TCT TCA ATT CTT TCT GCT TCA TCA GAA TCC AGT GAA AAA 7782 Gly Ser Ser Ser Ser He Leu Ser Ala Ser Ser Glu Ser Ser Glu Lye 2570 2575 2580

GCA AAA AGT GAG GAT GAA AAA CAT GTG AAC TCT ATT TCA GGA ACC AAA 7830 Ala Lys Ser Glu Asp Glu Lys Hie Val Aβn Ser He Ser Gly Thr Lys 2585 2590 2595

CAA AGT AAA GAA AAC CAA GTA TCC GCA AAA GGA ACA TGG AGA AAA ATA 7878 Gin Ser Lys Glu Aβn Gin Val Ser Ala Lye Gly Thr Trp Arg Lye He 2600 2605 2610 2615

AAA GAA AAT GAA TTT TCT CCC ACA AAT AGT ACT TCT CAG ACC GTT TCC 7926 Lys Glu Aβn Glu Phe Ser Pro Thr Asn Ser Thr Ser Gin Thr Val Ser 2620 2625 2630

TCA GGT GCT ACA AAT GGT GCT GAA TCA AAG ACT CTA ATT TAT CAA ATG 7974 Ser Gly Ala Thr Asn Gly Ala Glu Ser Lye Thr Leu He Tyr Gin Met 2635 2640 2645

GCA CCT GCT GTT TCT AAA ACA GAG GAT GTT TGG GTG AGA ATT GAG GAC 8022 Ala Pro Ala Val Ser Lys Thr Glu Aβp Val Trp Val Arg He Glu Asp 2650 2655 2660

TGT CCC ATT AAC AAT CCT AGA TCT GGA AGA TCT CCC ACA GGT AAT ACT 8070 Cys Pro He Aβn Aβn Pro Arg Ser Gly Arg Ser Pro Thr Gly Aβn Thr 2665 2670 2675

CCC CCG GTG ATT GAC AGT GTT TCA GAA AAG GCA AAT CCA AAC ATT AAA 8118 Pro Pro Val He Asp Ser Val Ser Glu Lys Ala Asn Pro Asn He Lys 2680 2685 2690 2695

GAT TCA AAA GAT AAT CAG GCA AAA CAA AAT GTG GGT AAT GGC AGT GTT 8166 Aβp Ser Lys Asp Asn Gin Ala Lys Gin Asn Val Gly Asn Gly Ser Val 2700 2705 2710

CCC ATG CGT ACC GTG GGT TTG GAA AAT CGC CTG ACC TCC TTT ATT CAG 8214 Pro Met Arg Thr Val Gly Leu Glu Asn Arg Leu Thr Ser Phe He Gin 2715 2720 2725

GTG GAT GCC CCT GAC CAA AAA GGA ACT GAG ATA AAA CCA GGA CAA AAT 8262 Val Asp Ala Pro Asp Gin Lys Gly Thr Glu He Lys Pro Gly Gin Aβn 2730 2735 2740

AAT CCT GTC CCT GTA TCA GAG ACT AAT GAA AGT CCT ATA GTG GAA CGT 8310 Asn Pro Val Pro Val Ser Glu Thr Asn Glu Ser Pro He Val Glu Arg 2745 2750 2755

ACC CCA TTC AGT TCT AGC AGC TCA AGC AAA CAC AGT TCA CCT AGT GGG 8358 Thr Pro Phe Ser Ser Ser Ser Ser Ser Lye Hie Ser Ser Pro Ser Gly 2760 2765 2770 2775

ACT GTT GCT GCC AGA GTG ACT CCT TTT AAT TAC AAC CCA AGC CCT AGG 8406 Thr Val Ala Ala Arg Val Thr Pro Phe Aβn Tyr Aβn Pro Ser Pro Arg 2780 2785 2790

AAA AGC AGC GCA GAT AGC ACT TCA GCT CGG CCA TCT CAG ATC CCA ACT 8454 Lys Ser Ser Ala Aβp Ser Thr Ser Ala Arg Pro Ser Gin He Pro Thr 2795 2800 2805

CCA GTG AAT AAC AAC ACA AAG AAG CGA GAT TCC AAA ACT GAC AGC ACA 8502 Pro Val Asn Asn Asn Thr Lye Lye Arg Aβp Ser Lye Thr Aβp Ser Thr 2810 2815 2820

GAA TCC AGT GGA ACC CAA AGT CCT AAG CGC CAT TCT GGG TCT TAC CTT 8550 Glu Ser Ser Gly Thr Gin Ser Pro Lye Arg Hie Ser Gly Ser Tyr Leu 2825 2830 2835

GTG ACA TCT GTT TAAAAGAGAG GAAGAATGAA ACTAAGAAAA TTCTATGTTA 8602

Val Thr Ser Val

2840

ATTACAACTG CTATATAGAC ATTTTGTTTC AAATGAAACT TTAAAAGACT GAAAAATTTT 8662

GTAAATAGGT TTGATTCTTG TTAGAGGGTT TTTGTTCTGG AAGCCATATT TGATAGTATA 8722

CTTTGTCTTC ACTGGTCTTA TTTTGGGAGG CACTCTTGAT GGTTAGGAAA AAATAGAAAG 8782

CCAAGTATGT TTGTACAGTA TGTTTTACAT GTATTTAAAG TAGCATCCCA TCCCAACTTC 8842

CTTAATTATT GCTTGTCTAA AATAATGAAC ACTACAGATA GGAAATATGA TATATTGCTG 8902

TTATCAATCA TTTCTAGATT ATAAACTGAC TAAACTTACA TCAGGGGAAA ATTGGTATTT 8962

ATGCAAAAAA AAAATGTTTT TGTCCTTGTG AGTCCATCTA ACATCATAAT TAATCATGTG 9022

GCTGTGAAAT TCACAGTAAT ATGGTTCCCG ATGAACAAGT TTACCCAGCC TGCTTTGCTT 9082

ACTGCATGAA TGAAACTGAT GGTTCAATTT CAGAAGTAAT GATTAACAGT TATGTGGTCA 9142

CATGATGTGC ATAGAGATAG CTACAGTGTA ATAATTTACA CTATTTTGTG CTCCAAACAA 9202

AACAAAAATC TGTGTAACTG TAAAACATTG AATGAAACTA TTTTACCTGA ACTAGATTTT 9262

ATCTGAAAGT AGGTAGAATT TTTGCTATGC TGTAATTTGT TGTATATTCT GGTATTTGAG 9322

GTGAGATGGC TGCTCTTTAT TAATGAGACA TGAATTGTGT CTCAACAGAA ACTAAATGAA 9382

CATTTCAGAA TAAATTATTG CTGTATGTAA ACTGTTACTG AAATTGGTAT TTGTTTGAAG 9442

GGTTTGTTTC ACATTTGTAT TAATTAATTG TTTAAAATGC CTCTTTTAAA AGCTTATATA 9502

AATTTTTTCT TCAGCTTCTA TGCATTAAGA GTAAAATTCC TCTTACTGTA ATAAAAACAT 9562

TGAAGAAGAC TGTTGCCACT TAACCATTCC ATGCGTTGGC ACTT 9606

(2 ) INFORMATION FOR SEQ ID NO: 2 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 2843 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

( ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :

Met Ala Ala Ala Ser Tyr Asp Gin Leu Leu Lye Gin Val Glu Ala Leu 1 5 10 15

Lye Met Glu Asn Ser Asn Leu Arg Gin Glu Leu Glu Asp Asn Ser Asn 20 25 30

His Leu Thr Lys Leu Glu Thr Glu Ala Ser Asn Met Lys Glu Val Leu 35 40 45

Lys Gin Leu Gin Gly Ser He Glu Asp Glu Ala Met Ala Ser Ser Gly

50 55 60

_G ln He Asp Leu Leu Glu Arg Leu Lye Glu Leu Aβn Leu Aβp Ser Ser 65 70 75 80

Aβn Phe Pro Gly Val Lye Leu Arg Ser Lye Met Ser Leu Arg Ser Tyr 85 90 95

Gly Ser Arg Glu Gly Ser Val Ser Ser Arg Ser Gly Glu Cyβ Ser Pro 100 105 110

Val Pro Met Gly Ser Phe Pro Arg Arg Gly Phe Val Aβn Gly Ser Arg 115 120 125

Glu Ser Thr Gly Tyr Leu Glu Glu Leu Glu Lye Glu Arg Ser Leu Leu 130 135 140

Leu Ala Asp Leu Asp Lye Glu Glu Lys Glu Lys Aβp Trp Tyr Tyr Ala 145 150 155 160

Gin Leu Gin Asn Leu Thr Lys Arg He Asp Ser Leu Pro Leu Thr Glu 165 170 175

Asn Phe Ser Leu Gin Thr Asp Leu Thr Arg Arg Gin Leu Glu Tyr Glu 180 185 190

Ala Arg Gin He Arg Val Ala Met Glu Glu Gin Leu Gly Thr Cys Gin 195 200 205

Asp Met Glu Lys Arg Ala Gin Arg Arg He Ala Arg He Gin Gin He 210 215 220

Glu Lys Asp He Leu Arg He Arg Gin Leu Leu Gin Ser Gin Ala Thr 225 230 235 240

Glu Ala Glu Arg Ser Ser Gin Asn Lys Hie Glu Thr Gly Ser His Asp 245 250 255

Ala Glu Arg Gin Asn Glu Gly Gin Gly Val Gly Glu He Asn Met Ala 260 265 270

Thr Ser Gly Asn Gly Gin Gly Ser Thr Thr Arg Met Asp His Glu Thr 275 280 285

Ala Ser Val Leu Ser Ser Ser Ser Thr Hie Ser Ala Pro Arg Arg Leu 290 295 300

Thr Ser His Leu Gly Thr Lys Val Glu Met Val Tyr Ser Leu Leu Ser 305 310 315 320

Met Leu Gly Thr His Asp Lys Asp Asp Met Ser Arg Thr Leu Leu Ala 325 330 335

Met Ser Ser Ser Gin Asp Ser Cyβ He Ser Met Arg Gin Ser Gly Cys 340 345 350

Leu Pro Leu Leu He Gin Leu Leu His Gly Asn Asp Lys Asp Ser Val 355 360 365

Leu Leu Gly Asn Ser Arg Gly Ser Lye Glu Ala Arg Ala Arg Ala Ser 370 375 380

Ala Ala Leu His Asn He He His Ser Gin Pro Asp Asp Lys Arg Gly 385 390 395 400

Arg Arg Glu He Arg Val Leu Hie Leu Leu Glu Gin He Arg Ala Tyr 405 410 415

Cys Glu Thr Cyβ Trp Glu Trp Gin Glu Ala Hie Glu Pro Gly Met Aβp 420 425 430

Gin Aβp Lys Asn Pro Met Pro Ala Pro Val Glu Hie Gin He Cys Pro 435 440 445

Ala Val Cyβ Val Leu Met Lye Leu Ser Phe Asp Glu Glu Hie Arg His 450 455 460

Ala Met Aβn Glu Leu Gly Gly Leu Gin Ala He Ala Glu Leu Leu Gin 465 470 475 480

Val Aβp Cys Glu Met Tyr Gly Leu Thr Aβn Aβp His Tyr Ser He Thr 485 490 495

Leu Arg Arg Tyr Ala Gly Met Ala Leu Thr Asn Leu Thr Phe Gly Asp 500 505 510

Val Ala Asn Lys Ala Thr Leu Cys Ser Met Lys Gly Cys Met Arg Ala 515 520 525

Leu Val Ala Gin Leu Lys Ser Glu Ser Glu Aβp Leu Gin Gin Val He 530 535 540

Ala Ser Val Leu Arg Asn Leu Ser Trp Arg Ala Asp Val Asn Ser Lys 545 550 555 560

Lys Thr Leu Arg Glu Val Gly Ser Val Lye Ala Leu Met Glu Cys Ala 565 570 575

Leu Glu Val Lys Lys Glu Ser Thr Leu Lys Ser Val Leu Ser Ala Leu 580 585 590

Trp Asn Leu Ser Ala His Cyβ Thr Glu Aen Lys Ala Asp He Cys Ala 595 600 60S

Val Asp Gly Ala Leu Ala Phe Leu Val Gly Thr Leu Thr Tyr Arg Ser 610 615 620

Gin Thr Asn Thr Leu Ala He He Glu Ser Gly Gly Gly He Leu Arg 625 630 635 640

Asn Val Ser Ser Leu He Ala Thr Aβn Glu Aβp Hie Arg Gin He Leu 645 650 655

Arg Glu Asn Asn Cyβ Leu Gin Thr Leu Leu Gin His Leu Lys Ser Hie 660 665 670

Ser Leu Thr He Val Ser Aβn Ala Cyβ Gly Thr Leu Trp Aβn Leu Ser 675 680 685

Ala Arg Asn Pro Lys Asp Gin Glu Ala Leu Trp Asp Met Gly Ala Val 690 695 700

Ser Met Leu Lys Asn Leu He His Ser Lys His Lys Met He Ala Met 705 710 715 720

Gly Ser Ala Ala Ala Leu Arg Asn Leu Met Ala Asn Arg Pro Ala Lys 725 730 735

Tyr Lye Aβp Ala Aβn He Met Ser Pro Gly Ser Ser Leu Pro Ser Leu 740 745 750

Hie Val Arg Lye Gin Lye Ala Leu Glu Ala Glu Leu λβp Ala Gin Hie 755 760 765

Leu Ser Glu Thr Phe Aβp Aβn He Asp Asn Leu Ser Pro Lys Ala Ser 770 775 780

His Arg Ser Lys Gin Arg His Lys Gin Ser Leu Tyr Gly Asp Tyr Val 785 790 795 800

Phe Asp Thr Asn Arg His Aβp Aβp Aβn Arg Ser Aβp Asn Phe Asn Thr 805 810 815

Gly Asn Met Thr Val Leu Ser Pro Tyr Leu Asn Thr Thr Val Leu Pro 820 825 830

Ser Ser Ser Ser Ser Arg Gly Ser Leu Asp Ser Ser Arg Ser Glu Lys 835 840 845

Asp Arg Ser Leu Glu Arg Glu Arg Gly He Gly Leu Gly Asn Tyr His 850 855 860

Pro Ala Thr Glu Asn Pro Gly Thr Ser Ser Lys Arg Gly Leu Gin He 865 -870 875 880

Ser Thr Thr Ala Ala Gin He Ala Lys Val Met Glu Glu Val Ser Ala 885 890 895

He His Thr Ser Gin Glu Aβp Arg Ser Ser Gly Ser Thr Thr Glu Leu 900 905 910

His Cyβ Val Thr Asp Glu Arg Asn Ala Leu Arg Arg Ser Ser Ala Ala 915 920 925

His Thr His Ser Asn Thr Tyr Asn Phe Thr Lye Ser Glu Asn Ser Aβn 930 935 940

Arg Thr Cyβ Ser Met Pro Tyr Ala Lys Leu Glu Tyr Lys Arg Ser Ser 945 950 955 960

Aβn Aβp Ser Leu Asn Ser Val Ser Ser Asn Asp Gly Tyr Gly Lys Arg 965 970 975

Gly Gin Met Lys Pro Ser He Glu Ser Tyr Ser Glu Asp Asp Glu Ser 980 985 990

Lye Phe Cyβ Ser Tyr Gly Gin Tyr Pro Ala Aβp Leu Ala Hie Lye He 995 1000 1005

His Ser Ala Asn His Met Asp Asp Aβn Aβp Gly Glu Leu Asp Thr Pro 1010 1015 1020

He Asn Tyr Ser Leu Lys Tyr Ser Asp Glu Gin Leu Asn Ser Gly Arg 1025 1030 1035 1040

Gin Ser Pro Ser Gin Asn Glu Arg Trp Ala Arg Pro Lys His He He 1045 1050 1055

Glu Asp Glu He Lys Gin Ser Glu Gin Arg Gin Ser Arg Asn Gin Ser 1060 1065 1070

Thr Thr Tyr Pro Val Tyr Thr Glu Ser Thr Aβp Asp Lys Hie Leu Lye 1075 1080 1085

Phe Gin Pro Hie Phe Gly Gin Gin Glu Cyβ Val Ser Pro Tyr Arg Ser 1090 1095 1100

Arg Gly Ala Asn Gly Ser Glu Thr Asn Arg Val Gly Ser Aβn Hie Gly 1105 1110 1115 1120

He Asn Gin Asn Val Ser Gin Ser Leu Cyβ Gin Glu Aβp Aβp Tyr Glu 1125 1130 1135

Aβp Asp Lys Pro Thr Aβn Tyr Ser Glu Arg Tyr Ser Glu Glu Glu Gin 1140 1145 1150

Hie Glu Glu Glu Glu Arg Pro Thr Aβn Tyr Ser He Lye Tyr Aβn Glu 1155 1160 1165

Glu Lys Arg His Val Asp Gin Pro He Asp Tyr Ser Leu Lys Tyr Ala 1170 1175 1180

Thr Asp He Pro Ser Ser Gin Lys Gin Ser Phe Ser Phe Ser Lys Ser 1185 1190 1195 1200

Ser Ser Gly Gin Ser Ser Lys Thr Glu His Met Ser Ser Ser Ser Glu 1205 ^* 1210 1215

Asn Thr Ser Thr Pro Ser Ser Aen Ala Lye Arg Gin Aβn Gin Leu Hie 1220 1225 1230

Pro Ser Ser Ala Gin Ser Arg Ser Gly Gin Pro Gin Lys Ala Ala Thr 1235 1240 1245

Cys Lys Val Ser Ser He Asn Gin Glu Thr He Gin Thr Tyr Cys Val 1250 1255 1260

Glu Asp Thr Pro He Cys Phe Ser Arg Cyβ Ser Ser Leu Ser Ser Leu 1265 1270 1275 1280

Ser Ser Ala Glu Asp Glu He Gly Cyβ Asn Gin Thr Thr Gin Glu Ala 1285 1290 1295

Asp Ser Ala Asn Thr Leu Gin He Ala Glu He Lys Gly Lys He Gly 1300 1305 1310

Thr Arg Ser Ala Glu Asp Pro Val Ser Glu Val Pro Ala Val Ser Gin 1315 1320 1325

His Pro Arg Thr Lys Ser Ser Arg Leu Gin Gly Ser Ser Leu Ser Ser 1330 1335 1340

Glu Ser Ala Arg His Lys Ala Val Glu Phe Pro Ser Gly Ala Lys Ser 1345 1350 1355 1360

Pro Ser Lys Ser Gly Ala Gin Thr Pro Lys Ser Pro Pro Glu Hie Tyr 1365 1370 1375

Val Gin Glu Thr Pro Leu Met Phe Ser Arg Cyβ Thr Ser Val Ser Ser 1380 1385 1390

Leu Asp Ser Phe Glu Ser Arg Ser He Ala Ser Ser Val Gin Ser Glu 1395 1400 1405

Pro Cys Ser Gly Met Val Ser Gly He He Ser Pro Ser Asp Leu Pro 1410 1415 1420

Asp Ser Pro Gly Gin Thr Met Pro Pro Ser Arg Ser Lye Thr Pro Pro 1425 1430 1435 1440

Pro Pro Pro Gin Thr Ala Gin Thr Lye Arg Glu Val Pro Lye Aβn Lye 1445 1450 1455

Ala Pro Thr Ala Glu Lys Arg Glu Ser Gly Pro Lys Gin Ala Ala Val 1460 1465 1470

Asn Ala Ala Val Gin Arg Val Gin Val Leu Pro Asp Ala Aβp Thr Leu 1475 1480 1485

Leu His Phe Ala Thr Glu Ser Thr Pro Asp Gly Phe Ser Cys Ser Ser 1490 1495 1500

Ser Leu Ser Ala Leu Ser Leu Asp Glu Pro Phe He Gin Lys Aβp Val 1505 1510 1515 1520

Glu Leu Arg He Met Pro Pro Val Gin Glu Aβn Aβp Asn Gly Asn Glu 1525 1530 1535

Thr Glu Ser Glu Gin Pro Lys Glu Ser Asn Glu Asn Gin Glu Lys Glu 1540 1545 1550

Ala Glu Lys Thr He Asp Ser Glu Lys Aβp Leu Leu Aβp Aβp Ser Aβp 1555 1560 1565

Asp Asp Asp He Glu He Leu Glu Glu Cys He He Ser Ala Met Pro 1570 1575 1580

Thr Lys Ser Ser Arg Lys Gly Lye Lye Pro Ala Gin Thr Ala Ser Lye 1585 1590 1595 1600

Leu Pro Pro Pro Val Ala Arg Lye Pro Ser Gin Leu Pro Val Tyr Lye 1605 1610 1615

Leu Leu Pro Ser Gin Asn Arg Leu Gin Pro Gin Lys His Val Ser Phe 1620 1625 1630

Thr Pro Gly Asp Asp Met Pro Arg Val Tyr Cys Val Glu Gly Thr Pro 1635 1640 1645

He Asn Phe Ser Thr Ala Thr Ser Leu Ser Aβp Leu Thr He Glu Ser 1650 1655 1660

Pro Pro Asn Glu Leu Ala Ala Gly Glu Gly Val Arg Gly Gly Ala Gin 1665 1670 1675 1680

Ser Gly Glu Phe Glu Lys Arg Asp Thr He Pro Thr Glu Gly Arg Ser 1685 1690 1695

Thr Asp Glu Ala Gin Gly Gly Lys Thr Ser Ser Val Thr He Pro Glu 1700 1705 1710

Leu Asp Asp Asn Lys Ala Glu Glu Gly Asp He Leu Ala Glu Cys He 1715 1720 1725

Asn Ser Ala Met Pro Lys Gly Lys Ser His Lys Pro Phe Arg Val Lys 1730 1735 1740

Lyβ He Met Aβp Gin Val Gin Gin Ala Ser Ala Ser Ser Ser Ala Pro 1745 1750 1755 1760

Aβn Lys Asn Gin Leu Aβp Gly Lye Lye Lys Lys Pro Thr Ser Pro Val 1765 1770 1775

Lys Pro He Pro Gin Asn Thr Glu Tyr Arg Thr Arg Val Arg Lye Aβn 1780 1785 1790

Ala Aβp Ser Lys Asn Asn Leu Aβn Ala Glu Arg Val Phe Ser Aβp Aβn 1795 1800 1805

Lys Asp Ser Lys Lys Gin Aβn Leu Lys Aβn Aβn Ser Lys Aβp Phe Asn 1810 1815 1820

Asp Lys Leu Pro Aβn Aβn Glu Aβp Arg Val Arg Gly Ser Phe Ala Phe 1825 1830 1835 1840

Aβp Ser Pro Hie Hie Tyr Thr Pro He Glu Gly Thr Pro Tyr Cyβ Phe 1845 1850 1855

Ser Arg Aβn Asp Ser Leu Ser Ser Leu Aβp Phe Aβp Aβp Aβp Aβp Val 1860 1865 1870

Asp Leu Ser Arg Glu Lys Ala Glu Leu Arg Lys Ala Lys Glu Aβn Lys 1875 1880 1885

Glu Ser Glu Ala Lys Val Thr Ser Hie Thr Glu Leu Thr Ser Aβn Gin 1890 1895 1900

Gin Ser Ala Aβn Lys Thr Gin Ala He Ala Lye Gin Pro He Asn Arg 1905 1910 1915 1920

Gly Gin Pro Lye Pro He Leu Gin Lys Gin Ser Thr Phe Pro Gin Ser 1925 1930 1935

Ser Lys Aβp He Pro Asp Arg Gly Ala Ala Thr Asp Glu Lye Leu Gin 1940 1945 1950

Asn Phe Ala He Glu Aβn Thr Pro Val Cyβ Phe Ser Hie Aβn Ser Ser 1955 1960 1965

Leu Ser Ser Leu Ser Aβp He Aβp Gin Glu Aβn Aβn Asn Lys Glu Asn 1970 1975 1980

Glu Pro He Lye Glu Thr Glu Pro Pro Aβp Ser Gin Gly Glu Pro Ser 1985 1990 1995 2000

Lye Pro Gin Ala Ser Gly Tyr Ala Pro Lye Ser Phe Hie Val Glu Aβp 2005 2010 2015

Thr Pro Val Cyβ Phe Ser Arg Aβn Ser Ser Leu Ser Ser Leu Ser He 2020 2025 2030

Asp Ser Glu Asp Asp Leu Leu Gin Glu Cyβ He Ser Ser Ala Met Pro 2035 2040 2045

Lys Lys Lys Lys Pro Ser Arg Leu Lys Gly Asp Asn Glu Lys His Ser 2050 2055 2060

Pro Arg Asn Met Gly Gly He Leu Gly Glu Asp Leu Thr Leu Asp Leu 2065 2070 2075 2080

Lvβ Aβp He Gin Arg Pro Aβp Ser Glu Hie Gly Leu Ser Pro Aβp Ser 2085 2090 2095

Glu Aβn Phe Aβp Trp Lye Ala He Gin Glu Gly Ala Asn Ser He Val 2100 2105 2110

Ser Ser Leu His Gin Ala Ala Ala Ala Ala Cys Leu Ser Arg Gin Ala 2115 2120 2125

Ser Ser Aβp Ser Aβp Ser He Leu Ser Leu Lys Ser Gly He Ser Leu 2130 2135 2140

Gly Ser Pro Phe His Leu Thr Pro Asp Gin Glu Glu Lys Pro Phe Thr 2145 2150 2155 2160

Ser Aβn Lys Gly Pro Arg He Leu Lys Pro Gly Glu Lye Ser Thr Leu 2165 2170 2175

Glu Thr Lye Lye He Glu Ser Glu Ser Lye Gly He Lye Gly Gly Lye 2180 2185 2190

Lye Val Tyr Lye Ser Leu He Thr Gly Lys Val Arg Ser Asn Ser Glu 2195 2200 2205

He Ser Gly Gin Met Lye Gin Pro Leu Gin Ala Aβn Met Pro Ser He 2210 2215 2220

Ser Arg Gly Arg Thr Met He His He Pro Gly Val Arg Asn Ser Ser 2225 2230 2235 2240

Ser Ser Thr Ser Pro Val Ser Lys Lys Gly Pro Pro Leu Lye Thr Pro 2245 2250 2255

Ala Ser Lys Ser Pro Ser Glu Gly Gin Thr Ala Thr Thr Ser Pro Arg 2260 2265 2270

Gly Ala Lys Pro Ser Val Lys Ser Glu Leu Ser Pro Val Ala Arg Gin 2275 2280 2285

Thr Ser Gin He Gly Gly Ser Ser Lys Ala Pro Ser Arg Ser Gly Ser 2290 2295 2300

Arg Asp Ser Thr Pro Ser Arg Pro Ala Gin Gin Pro Leu Ser Arg Pro 2305 2310 2315 2320

He Gin Ser Pro Gly Arg Asn Ser He Ser Pro Gly Arg Asn Gly He 2325 2330 2335

Ser Pro Pro Asn Lys Leu Ser Gin Leu Pro Arg Thr Ser Ser Pro Ser 2340 2345 2350

Thr Ala Ser Thr Lys Ser Ser Gly Ser Gly Lye Met Ser Tyr Thr Ser 2355 2360 2365

Pro Gly Arg Gin Met Ser Gin Gin Asn Leu Thr Lys Gin Thr Gly Leu 2370 2375 2380

Ser Lys Asn Ala Ser Ser He Pro Arg Ser Glu Ser Ala Ser Lys Gly 2385 2390 2395 2400

Leu Asn Gin Met Asn Asn Gly Asn Gly Ala Asn Lys Lys Val Glu Leu 2405 2410 2415

Ser Arg Met Ser Ser Thr Lys Ser Ser Gly Ser Glu Ser λβp λrg Ser 2420 2425 2430

Glu Arg Pro Val Leu Val Arg Gin Ser Thr Phe He Lye Glu λla Pro 2435 2440 2445

Ser Pro Thr Leu Arg Arg Lye Leu Glu Glu Ser Ala Ser Phe Glu Ser 2450 2455 2460

Leu Ser Pro Ser Ser Arg Pro Ala Ser Pro Thr λrg Ser Gin λla Gin 2465 2470 2475 2480

Thr Pro Val Leu Ser Pro Ser Leu Pro λβp Met Ser Leu Ser Thr Hie 2485 2490 2495

Ser Ser Val Gin λla Gly Gly Trp λrg Lye Leu Pro Pro λβn Leu Ser 2500 2505 2510

Pro Thr He Glu Tyr λβn λsp Gly λrg Pro λla Lys λrg Hie λep He 2515 2520 2525 λla λrg Ser Hie Ser Glu Ser Pro Ser λrg Leu Pro He λβn λrg Ser 2530 2535 2540

Gly Thr Trp Lye λrg Glu Hie Ser Lye Hie Ser Ser Ser Leu Pro λrg 2545 2550 2555 2560

Val Ser Thr Trp λrg λrg Thr Gly Ser Ser Ser Ser He Leu Ser λla 2565 2570 2575

Ser Ser Glu Ser Ser Glu Lys Ala Lye Ser Glu Aβp Glu Lys His Val 2580 2585 2590

Aβn Ser He Ser Gly Thr Lye Gin Ser Lys Glu Aβn Gin Val Ser Ala 2595 2600 2605

Lys Gly Thr Trp Arg Lys He Lys Glu Asn Glu Phe Ser Pro Thr λsn 2610 2615 2620

Ser Thr Ser Gin Thr Val Ser Ser Gly λla Thr λβn Gly λla Glu Ser 2625 2630 2635 2640

Lys Thr Leu He Tyr Gin Met λla Pro λla Val Ser Lye Thr Glu λβp 2645 2650 2655

Val Trp Val λrg He Glu λβp Cyβ Pro He λβn λβn Pro λrg Ser Gly 2660 2665 2670 λrg Ser Pro Thr Gly λβn Thr Pro Pro Val He λsp Ser Val Ser Glu 2675 2680 2685

Lys λla λsn Pro λsn He Lys λsp Ser Lys λβp λβn Gin λla Lye Gin 2690 2695 2700 λsn Val Gly λsn Gly Ser Val Pro Met λrg Thr Val Gly Leu Glu λsn 2705 2710 2715 2720 λrg Leu Thr Ser Phe He Gin Val λsp λla Pro λsp Gin Lys Gly Thr 2725 2730 2735

Glu He Lys Pro Gly Gin Asn Asn Pro Val Pro Val Ser Glu Thr Asn 2740 2745 2750

Glu Ser Pro He Val Glu λrg Thr Pro Phe Ser Ser Ser Ser Ser Ser 2755 2760 2765

Lys His Ser Ser Pro Ser Gly Thr Val λla λla λrg Val Thr Pro Phe 2770 2775 2780 λβn Tyr λsn Pro Ser Pro λrg Lys Ser Ser λla λβp Ser Thr Ser λla 2785 2790 2795 2800 λrg Pro Ser Gin He Pro Thr Pro Val λβn λβn λsn Thr Lys Lys λrg 2805 2810 2815 λsp Ser Lys Thr λβp Ser Thr Glu Ser Ser Gly Thr Gin Ser Pro Lys 2820 2825 2830 λrg His Ser Gly Ser Tyr Leu Val Thr Ser Val 2835 2840

(2) INFORMλTION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3172 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: DP1(TB2)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCλTIQN: 1..630

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GCA GTC GCC GCT CCA GTC TAT CCG GCA CTA GGA ACA GCC CCG GGN GGC 48 λla Val Ala Ala Pro Val Tyr Pro Ala Leu Gly Thr λla Pro Gly Gly 1 5 10 15

GAG ACG GTC CCC GCC ATG TCT GCG GCC ATG AGG GAG AGG TTC GAC CGG 96 Glu Thr Val Pro Ala Met Ser Ala Ala Met λrg Glu Arg Phe Asp Arg 20 25 30

TTC CTG CAC GAG AAG AAC TGC ATG ACT GAC CTT CTG GCC AAG CTC GAG 144 Phe Leu Hie Glu Lys Asn Cys Met Thr λβp Leu Leu λla Lye Leu Glu 35 40 45

GCC AλA ACC GGC GTG AλC λGG λGC TTC λTC GCT CTT GGT GTC λTC GGλ 192 λla Lys Thr Gly Val Asn Arg Ser Phe He Ala Leu Gly Val He Gly 50 55 60

CTG GTG GCC TTG TAC CTG GTG TTC GGT TAT GGA GCC TCT CTC CTC TGC 240 Leu Val Ala Leu Tyr Leu Val Phe Gly Tyr Gly Ala Ser Leu Leu Cys 65 70 75 80

λλC CTG λTλ GGλ TTT GGC TλC CCλ GCC TλC λTC TCλ λTT λλλ GCT λTλ 288 λβn Leu He Gly Phe Gly Tyr Pro λla Tyr He Ser He Lye λla He 85 90 95

GλG λGT CCC AλC λλA Gλλ GλT GλT λCC CλG TGG CTG λCC TλC TGG GTλ 336 Glu Ser Pro λβn Lye Glu λβp λβp Thr Gin Trp Leu Thr Tyr Trp Val 100 105 110

GTG TλT GGT GTG TTC λGC λTT GCT Gλλ TTC TTC TCT GλT λTC TTC CTG 384 Val Tyr Gly Val Phe Ser He λla Glu Phe Phe Ser λβp He Phe Leu 115 120 125

TCλ TGG TTC CCC TTC TλC TλC ATG CTG λG TGT GGC TTC CTG TTG TGG 432 Ser Trp Phe Pro Phe Tyr Tyr Met Leu Lye Cye Gly Phe Leu Leu Trp 130 135 140

TGC λTG GCC CCG λGC CCT TCT λλT GGG GCT Gλλ CTG CTC TλC λλG CGC 480 Cyβ Met λla Pro Ser Pro Ser λβn Gly λla Glu Leu Leu Tyr Lye λrg 145 150 155 160

ATC λTC CGT CCT TTC TTC CTG λλG CλC GλG TCC CλG λTG GλC λGT GTG 528 He He λrg Pro Phe Phe Leu Lye Hie Glu Ser Gin Met λβp Ser Val 165 170 175

GTC λλG GλC CTT λλλ GλC λλG TCC Aλλ GλG λCT GCλ GλT GCC λTC λCT 576 Val Lys λsp Leu Lys λsp Lye Ser Lye Glu Thr λla λsp λla He Thr 180 185 190

AAA GAA GCG AAG AAA GCT ACC GTG λλT TTA CTG GGT Gλλ GAA AAG AAG 624 Lys Glu λla Lys Lys λla Thr Val λsn Leu Leu Gly Glu Glu Lys Lys 195 200 205 λGC λCC TλλλCCλGλC TλλλCCλGλC TGGλTGGλλ CTTCCTGCCC TCTCTGTλCC 680 Ser Thr 210

TTCCTλCTGG λGCTTGATGT TATATTAGGG λCTGTGGTλT λλTTλTTTTλ λTAλTGTTGC 740

CTTGGAAACA TTTTTGλGλT λTTλλλGλTT GGAATGTGTT GTAλGTTTCT TTGCTTλCTT 800

TTλCTGTCTλ TATλTλTλGG GλGCACTTTA λλCTTλ TGC λGTGGGCλGT GTCCλCGTTT 860

TTGGλλAATG TATTTTGCCT CTGGGTλGGλ λλλGλTGTλT GTTGCTλTCC TGCλGGλλAT 920 λTλλλCTTλλ λλTλλλλTTλ TλTλCCCCλC λGGCTGTGTλ CTTTλCTGGG CTCTCCCTGC 980 λCGSλTTTTC TCTGTλGTTλ CλTTTλGGRT λλTCTTTλTG GTTCTλCTTC CTRTλλTGTλ 1040

CλλTTTTλTλ TλλTTCNGRλ λTGTTTTTλA TGTλTTTGTG CλCλTGTλCλ TλTGGλλλTG 1100

TTλCTGTCTG λCTλCANCλT GCλTCλTGCT CλTGGGGλGG GλGCλGGGGλ λGGTTGTλTG 1160

TGTCλTTTλT λλCTTCTGTλ CλGTλλGλCC λCCTGCCλλλ λGCTGGλGGA ACCλTTGTGC 1220

TGGTGTGGTC TλCTλλλTAA TλCTTTλGGλ λλTλCGTGλT TλλTλTGCλ GTGA CAAλG 1280

TGλGAAATGA AATCGAATGG λGλTTGGCCT GGTTGTTTCC GTλGTλTλTG GCλTλTGAAT 1340

ACCλGGλTλG CTTTλTλAAG CλGTTλGTTλ GTTλGTTλCT CλCTCTλGTG λTλλλTCGGG 1400

AAATTTACAC ACACλCλCλC λCλCλCλCAC ACλCACACAC λCACACACAC ACACACACAG 1460

-70-

AGTλCCCTGT λλCTCTCλλT TCCCTGAAλλ λCTλGTλλTλ CTGTCTTλTC TGCTλTλλλC 1520 TTTλCλTλTT TGTCTλTTGT CλλGλTGCTλ CλNTGGλMNC CλTTTCTGGT TTTλTCTTCλ 1580 NλGSGGλGλN λCλTGTTGλT TTλGTCTTCT TTCCCλλTCT TCTTTTTTλλ MCCAGTTTNA 1640 GGMNCTTCTG RλGATTTGYC CACCTCTGAT TACλTGTλTG TTCTYGTTTG TλTCλTKλGC 1700 λλCAACλTGC TAATGRCGAC ACCTλGCTCT 'RAGMGCAλTT CTGGGAGANT GARAGGNWGT 1760 ATARλGTMNC CCλTλλTCTG CTTGGCλλTA GTTAλGTCλA TCTλTCTTCλ GTTTTTCTCT 1820 GGCCTTTAAG GTCAλACACA AGλGGCTTCC CTλGTTTλCλ λGTCAGAGTC λCTTGTλGTC 1880 CλTTTλλλTG CCCTCλTCCG TλTTCTTTGT GTTGλTλAGC TGCACλKGλC TλCλTλGTλλ 1940 GTλCAGANCλ GTAλλGTTλA NNCGGλTGTC TCCλTTGλTC TGCCAANTCG NTλTλGλGλG 2000 CλλTTTGTCT GGACTAGAAλ λTCTGAGTTT TACACCλTAC TGTTAAGAGT CCTTTTGAλT 2060 TAAACTAGAC TλAAλCλλGT GTλTλλCTλA λCTλλCAλGλ TTλλλTλTCC AGCCλGTλCA 2120 GTATTTTTTA λGGCAAATλλ λGATGATTAG CTCλCCTTGA GNTAACAATC AGGTAAGATC 2180 ATNACAATGT CTCλTGATGT NλANAλTλTT λλAGATλTCλ λTλCTAAGTG λCλGTλTCAC 2240 NNCTAATλTλ λTATGGATCλ GλGCλTTTλT TTTGGGGλGG λAAACλGTGG TGATTACCGG 2300 CλTTTTATTA λλCTTλAAAC TTTGTAGλλA GCAAλCAλAA TTGTTCTTGG GAGAAAATCA 2360 ACTTTTλGλT TλλAλλλλTT TTλλGTAWCT AGGλGTλTTT λAATCCTTTT CCCATλλλTA 2420 AAAGTλCλGT TTTCTTGGTG GCAGAATGAλ λATCAGCλAC NTCTAGCATA TAGλCTλTλT 2480 AλTCλGATTG λCλGCλTλTλ GλATATλTTA TCAGλCλAGA TGλGGλGGTλ CAAAAGTTλC 2540 TλTTGCTCλT λATGACTTAC AGGCTλλAAN TλGNTNTλAA ATλCTATATT AAATTCTGAA 2600 TGCAATTTTT TTTTGTTCCC TTGAGACCAA AλTTTλλGTT λACTGTTGCT GGCAGTCTλA 2660 GTGTAλλTGT TAACλGCλGG AGλλGTTλλG λλTTGλGCAG TTCTGTTGCA TGλTTTCCCλ 2720 AATGλAATAC TGCCTTGGCT AGAGTTTGAA AAACTλλTTG AGCCTGTGCC TGGCTAGλλA 2780 ACλλGCGTTT ATTTGλλTGT GλλTλGTGTT TCλλλGGTλT GTλGTTλCλG λλTTCCTλCC 2840 λλλCλGCTTλ λλTTCTTCλλ GλλλGλATTC CTGCλGCλGT TλTTCCCTTλ CCTGλλGGCT 2900 TCλATCATTT GGλTCλλCλλ CTGCTλCTCT CGGGλλGλCT CCTCTACTCA CAGCTGλλGλ 2960 λλλTGλGCλC λCCCTTCλCλ CTGTTλTCλC CTλTCCTGλA GATGTGλTλC λCTGλATGGA 3020 AλTλAATAGλ TGTλAATAAλ ATTGAGWTCT CATTTλλλλλ λAACCATGTG CCCAATGGGA 3080 AAATGλCCTC λTGTTGTGGT TTλAACAGCA ACTGCλCCCλ CTAGCACAGC CCATTGAGCT 3140 ANCCTATATA TACλTCTCTG TCAGTGCCCC TC 3172

(2 ) INFORMATION FOR SEQ ID NO: 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 210 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Val Ala Ala Pro Val Tyr Pro λla Leu Gly Thr λla Pro Gly Gly 1 5 10 15

Glu Thr Val Pro λla Met Ser λla Ala Met Arg Glu Arg Phe Asp Arg 20 25 30

Phe Leu His Glu Lys Asn Cyβ Met Thr λsp Leu Leu λla Lys Leu Glu 35 40 45 λla Lys Thr Gly Val λsn λrg Ser Phe He λla Leu Gly Val He Gly 50 55 60

Leu Val λla Leu Tyr Leu Val Phe Gly Tyr Gly λla Ser Leu Leu Cys 65 70 75 80 λsn Leu He Gly Phe Gly Tyr Pro Ala Tyr He Ser He Lys Ala He 85 90 95

Glu Ser Pro Asn Lys Glu Asp Asp Thr Gin Trp Leu Thr Tyr Trp Val 100 105 110

Val Tyr Gly Val Phe Ser He Ala Glu Phe Phe Ser Asp He Phe Leu 115 120 125

Ser Trp Phe Pro Phe Tyr Tyr Met Leu Lys Cys Gly Phe Leu Leu Trp 130 135 140

Cys Met Ala Pro Ser Pro Ser λsn Gly λla Glu Leu Leu Tyr Lys λrg 145 150 155 160

He He λrg Pro Phe Phe Leu Lys His Glu Ser Gin Met λsp Ser Val 165 170 175

Val Lys λsp Leu Lys λβp Lys Ser Lys Glu Thr λla λsp λla He Thr 180 185 190

Lys Glu λla Lys Lye λla Thr Val λβn Leu Leu Gly Glu Glu Lye Lys 195 200 205

Ser Thr 210

(2) INFORMλTION FOR SEQ ID NO:5:

(i) SEQUENCE CHARλCTERISTICS:

(A) LENGTH: 434 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: TBl

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Val Ala Pro Val Val Val Gly Ser Gly Arg Ala Pro Arg His Pro Ala 1 5 10 15

Pro λla λla Met His Pro λrg λrg Pro λβp Gly Phe λβp Gly Leu Gly 20 25 30

Tyr λrg Gly Gly λla λrg λsp Glu Gin Gly Phe Gly Gly λla Phe Pro 35 40 45

Ala Arg Ser Phe Ser Thr Gly Ser Asp Leu Gly His Trp Val Thr Thr 50 55 60

Pro Pro Asp He Pro Gly Ser Arg Asn Leu His Trp Gly Glu Lys Ser 65 70 75 80

Pro Pro Tyr Gly Val Pro Thr Thr Ser Thr Pro Tyr Glu Gly Pro Thr 85 90 95

Glu Glu Pro Phe Ser Ser Gly Gly Gly Gly Ser Val Gin Gly Gin Ser 100 105 110

Ser Glu Gin Leu Asn Arg Phe Ala Gly Phe Gly He Gly Leu Ala Ser 115 120 125

Leu Phe Thr Glu Asn Val Leu λla His Pro Cys He Val Leu λrg λrg 130 135 140

Gin Cys Gin Val λsn Tyr His λla Gin Hie Tyr Hie Leu Thr Pro Phe 145 150 155 160

Thr Val He λβn He Met Tyr Ser Phe λβn Lye Thr Gin Gly Pro λrg 165 170 175

Ala Leu Trp Lye Gly Met Gly Ser Thr Phe He Val Gin Gly Val Thr 180 185 190

Leu Gly Ala Glu Gly He He Ser Glu Phe Thr Pro Leu Pro λrg Glu 195 200 205

Val Leu His Lys Trp Ser Pro Lys Gin He Gly Glu His Leu Leu Leu 210 215 220

Lys Ser Leu Thr Tyr Val Val λla Met Pro Phe Tyr Ser λla Ser Leu 225 230 235 240

He Glu Thr Val Gin Ser Glu He He λrg λsp λβn Thr Gly He Leu 245 250 255

Glu Cys Val Lys Glu Gly He Gly λrg Val He Gly Met Gly Val Pro 260 265 270

Hiβ Ser Lye λrg Leu Leu Pro Leu Leu Ser Leu He Phe Pro Thr Val 275 280 285

Leu Hie Gly Val Leu His Tyr He He Ser Ser Val He Gin Lye Phe 290 295 300

Val Leu Leu He Leu Lye λrg Lye Thr Tyr λβn Ser Hie Leu λla Glu 305 310 315 320

Ser Thr Ser Pro Val Gin Ser Met Leu λep λla Tyr Phe Pro Glu Leu 325 330 335

He λla λsn Phe λla λla Ser Leu Cys Ser λsp Val He Leu Tyr Pro 340 345 350

Leu Glu Thr Val Leu His λrg Leu Hie He Gin Gly Thr λrg Thr He 355 360 365

He λβp λsn Thr λβp Leu Gly Tyr Glu Val Leu Pro He λβn Thr Gin 370 375 380

Tyr Glu Gly Met λrg λβp Cyβ He λβn Thr He λrg Gin Glu Glu Gly 385 390 395 400

Val Phe Gly Phe Tyr Lye Gly Phe Gly λla Val He He Gin Tyr Thr 405 410 415

Leu Hie λla λla Val Leu Gin He Thr Lye He He Tyr Ser Thr Leu 420 425 430

Leu Gin

INFORMATION FOR SEQ ID NO: 6 :

( i) SEQUENCE CHΛRλCTERISTICS :

(λ) LENGTH: 185 amino acidβ

(B ) TYPE : amino acid

(C) STRANDEDNESS : single

(D ) TOPOLOGY : linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo βapienβ

(vii) IMMEDIATE SOURCE:

(B) CLONE: YS-39(TB2)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Glu Leu λrg λrg Phe λsp λrg Phe Leu Hie Glu Lye λsn Cys Met Thr 1 5 10 15

Asp Leu Leu Ala Lys Leu Glu λla Lys Thr Gly Val λsn λrg Ser Phe 20 25 30

He Ala Leu Gly Val He Gly Leu Val Ala Leu Tyr Leu Val Phe Gly 35 40 45

Tyr Gly Ala Ser Leu Leu Cyβ λβn Leu He Gly Phe Gly Tyr Pro Ala 50 55 60

Tyr He Ser He Lye λla He Glu Ser Pro λβn Lye Glu λβp λβp Thr 65 70 75 80

Gin Trp Leu Thr Tyr Trp Val Val Tyr Gly Val Phe Ser He λla Glu 85 90 95

Phe Phe Ser λβp He Phe Leu Ser Trp Phe Pro Phe Tyr Tyr He Leu 100 105 110

Lye Cys Gly Phe Leu Leu Trp Cys Met λla Pro Ser Pro Ser λβn Gly 115 120 125 λla Glu Leu Leu Tyr Lys λrg He He λrg Pro Phe Phe Leu Lys His 130 135 140

Glu Ser Gin Met λsp Ser Val Val Lys λsp Leu Lys λβp Lys λla Lys 145 150 155 160

Glu Thr λla λsp λla He Thr Lys Glu λla Lys Lye λla Thr Val λβn 165 170 175

Leu Leu Gly Glu Glu Lye Lye Ser Thr 180 185

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2842 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: APC

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met λla λla λla Ser Tyr λsp Gin Leu Leu Lys Gin Val Glu λla Leu 1 5 10 15

Lys Met Glu Asn Ser Asn Leu Arg Gin Glu Leu Glu Aβp Aβn Ser Aβn 20 25 30

His Leu Thr Lys Leu Glu Thr Glu Ala Ser λsn Met Lys Glu Val Leu 35 40 45

Lys Gin Leu Gin Gly Ser He Glu λsp Glu λla Met λla Ser Ser Gly 50 55 60

Gin He λsp Leu Leu Glu λrg Leu Lys Glu Leu λsn Leu λsp Ser Ser 65 70 75 80

λβn Phe Pro Gly Val Lye Leu λrg Ser Lye Met Ser Leu λrg Ser Tyr 85 90 95

Gly Ser λrg Glu Gly Ser Val Ser Ser λrg Ser Gly Glu Cyβ Ser Pro 100 105 110

Val Pro Met Gly Ser Phe Pro λrg λrg Gly Phe Val λβn Gly Ser λrg 115 120 125

Glu Ser Thr Gly Tyr Leu Glu Glu Leu Glu Lye Glu λrg Ser Leu Leu 130 135 140

Leu λla λβp Leu λβp Lye Glu Glu Lye Glu Lye λsp Trp Tyr Tyr λla 145 150 155 160

Gin Leu Gin λβn Leu Thr Lye λrg He λβp Ser Leu Leu Thr Glu λβn 165 170 175

Phe Ser Leu Gin Thr λsp Met Thr λrg λrg Gin Leu Glu Tyr Glu λla 180 185 190 λrg Gin He λrg Val λla Met Glu Glu Gin Leu Gly Thr Cyβ Gin λβp 195 200 205

Met Glu Lye λrg λla Gin λrg λrg He λla λrg He Gin Gin He Glu 210 215 220

Lys λsp He Leu λrg He λrg Gin Leu Leu Gin Ser Gin λla Thr Glu 225 230 235 240 λla Glu λrg Ser Ser Gin λsn Lys Hie Glu Thr Gly Ser Hie λβp λla 245 250 255

Glu λrg Gin λβn Glu Gly Gin Gly Val Gly Glu He λβn Met λla Thr 260 265 270

Ser Gly λsn Gly Gin Gly Ser Thr Thr λrg Met λsp His Glu Thr λla 275 280 285

Ser Val Leu Ser Ser Ser Ser Thr Hie Ser λla Pro λrg λrg Leu Thr 290 295 300

Ser Hie Leu Gly Thr Lye Val Glu Met Val Tyr Ser Leu Leu Ser Met 305 310 315 320

Leu Gly Thr Hie λβp Lye λβp λβp Met Ser λrg Thr Leu Leu Ala Met 325 330 335

Ser Ser Ser Gin λβp Ser Cyβ He Ser Met λrg Gin Ser Gly Cys Leu 340 345 350

Pro Leu Leu He Gin Leu Leu His Gly Asn Aβp Lye λβp Ser Val Leu 355 360 365

Leu Gly λβn Ser λrg Gly Ser Lye Glu λla λrg λla λrg λla Ser λla 370 375 380 λla Leu Hie λβn He He Hie Ser Gin Pro λsp λβp Lye Arg Gly λrg 385 390 395 400

Arg Glu He Arg Val Leu His Leu Leu Glu Gin He Arg Ala Tyr Cys 405 410 415

_-,7 _-

Thr Cy _β Trp Glu Trp Gin Glu λla Hie Glu Pro ^G ly Met ^λβ p ^G in

Glu

420 425

A _β p Lye A _β n Pro Met Pro Ala Pro Val Glu Hie Gin He Cy ^β Pro Ala — AAΓI 445

435 440

Val _C ys _V al Leu Met Lys Leu Ser Phe A ^β p Glu Glu Hie Arg Hie Ala

450 455

Het Asn _G lu Leu _G ly _G ly Leu Gin λla He Ala ^G lu Leu Leu ^G in Val

465 470 _λ βp Cyβ G Glluu MMeect xTyyrr ^■ G**l ^■ y Leu Thr λβn λep Hie Tyr Ser He Thr Leu

485 490 495 Arg Arg Tyr Ala _G ly Met Ala Leu Thr λen Leu Thr Phe Gly ^λβ p Val

Ala Asn Lys _λ la Thr Leu Cys Ser Met Lys Gly Cys Met ^λ rg ^λ la Leu

515 ^5Z0

Val _λ la Gin Leu Lye Ser Glu Ser Glu λ ^β p Leu ^G in ^G in Val He λla

530 ⁵³⁵

Ser Val Leu λrg _λ βn Leu Ser Trp λrg λla λ ^β p Val ^λβ n Ser Lye Lye 545 ⁵⁵ °

Thr Leu _λ rg _G lu Val _G ly Ser Val Lye λla Leu Met ^G lu ^C y ^{β λ} la Leu 565 ^57U

_G lu Val Lys Lys Glu Ser Thr Leu Lys Ser Val Leu ^S er λla Leu Trp

580 ⁵⁸⁵

A _β n Leu Ser Ala Hie _C yβ Thr Glu Aβn Lye Ala A ^β p II. ^C y ^β Ala Val

595 ⁶⁰ °

A _β p Gly Ala Leu Ala Phe Leu Val Gly Thr Leu Thr Tyr Arg ^S er ^G in

610 ⁶¹⁵

Thr A _β n Thr Leu _λ la II. He Glu Ser Gly Gly ^G ly He Leu Arg Asn 625 ⁶³ °

Val Ser _S er Leu II. Ala Thr Aβn Glu A ^β p Hie Arg ^G in He Leu Arg

_G lu A _β n A _β n Cys Leu _G in Thr Leu Leu Gin His Leu Lys Ser Hie ^S er

660 ⁶⁶⁵

Leu Thr He Val Ser Aβn Ala Cyβ Gly Thr Leu Trp A ^β n Leu ^S er Ala

675 680

Arg A _β n Pro Lye Asp _G in Glu Ala Leu Trp Asp Met Gly λla Val Ser

690 ⁶⁹⁵ Met Leu Lys λ _» s-n _τ L_e_.u _{.. T} li _IP e _H Hiiss _S seerr Lι_vys ^β H ⁿ i_-s Ly ^y s Met He λla Met ^G ^ly

705 ⁷¹⁰ ser _λ la Ala Ala Leu Arg Asn Leu Met Ala Asn Arg Pro Ala Lys Tyr

Lys Asp Ala Asn He Met Ser Pro Gly Ser Ser Leu Pro Ser Leu His

Val λrg Lye Gin Lye λla Leu Glu λla Glu Leu λβp λla Gin Hie Leu 755 760 765

Ser Glu Thr Phe λβp λβn He λβp λβn Leu Ser Pro Lye λla Ser Hie 770 775 780 λrg Ser Lye Gin λrg Hie Lye Gin Ser Leu Tyr Gly λβp Tyr Val Phe 785 790 795 800 λsp Thr λsn λrg Hie λβp λβp Aβn Arg Ser λβp λsn Phe λsn Thr Gly 805 810 815 λβn Met Thr Val Leu Ser Pro Tyr Leu λβn Thr Thr Val Leu Pro Ser 820 825 830

Ser Ser Ser Ser λrg Gly Ser Leu λβp Ser Ser λrg Ser Glu Lye λβp 835 840 845 λrg Ser Leu Glu λrg Glu λrg Gly He Gly Leu Gly λβn Tyr Hie Pro 850 855 860 λla Thr Glu Asn Pro Gly Thr Ser Ser Lye Arg Gly Leu Gin He Ser 865 870 875 880

Thr Thr Ala λla Gin He λla Lye Val Met Glu Glu Val Ser λla He 885 890 895

Hie Thr Ser Gin Glu Asp Arg Ser Ser Gly Ser Thr Thr Glu Leu His 900 905 910

Cys Val Thr λsp Glu λrg λsn λla Leu λrg λrg Ser Ser λla λla His 915 920 925

Thr His Ser λsn Thr Tyr λsn Phe Thr Lye Ser Glu Asn Ser Aβn Arg 930 935 940

Thr Cyβ Ser Met Pro Tyr Ala Lys Leu Glu Tyr Lys Arg Ser Ser Aβn 945 950 955 960

Asp Ser Leu Asn Ser Val Ser Ser Ser λsp Gly Tyr Gly Lys λrg Gly 965 970 975

Gin Met Lys Pro Ser He Glu Ser Tyr Ser Glu λsp λsp Glu Ser Lys 980 985 990

Phe Cys Ser Tyr Gly Gin Tyr Pro Ala Aβp Leu Ala Hie Lye He Hie 995 1000 1005

Ser Ala λβn Hie Met λβp λβp λβn λβp Gly Glu Leu λβp Thr Pro He 1010 1015 1020 λβn Tyr Ser Leu Lye Tyr Ser λβp Glu Gin Leu λβn Ser Gly λrg Gin 1025 1030 1035 1040

Ser Pro Ser Gin λβn Glu λrg Trp λla λrg Pro Lye Hie He He Glu 1045 1050 1055

Asp Glu He Lye Gin Ser Glu Gin λrg Gin Ser λrg λsn Gin Ser Thr 1060 1065 1070

Thr Tyr Pro Val Tyr Thr Glu Ser Thr λsp Asp Lys His Leu Lys Phe 1075 1080 1085

_G ln Pro Hie Phe Gly Gin Gin Glu Cyβ Val Ser Pro Tyr Arg Ser Λrg 1090 1095 1100

Gly λla λβn Gly Ser Glu Thr λβn λrg Val Gly Ser λβn Hie Gly He 1105 1110 1115 1120 _λ βn Gin λen Val Ser Gin Ser Leu Cyβ Gin Glu λβp λβp Tyr Glu λβp 1125 1130 1135 λsp Lys Pro Thr λsn Tyr Ser Glu λrg Tyr Ser Glu Glu Glu Gin His 1140 1145 1150

Glu Glu Glu Glu λrg Pro Thr λβn Tyr Ser He Lye Tyr λβn Glu Glu 1155 1160 1165

Lye λrg His Val λβp Gin Pro He λep Tyr Ser Leu Lye Tyr λla Thr 1170 H75 1180 _λ sp He Pro Ser Ser Gin Lys Gin Ser Phe Ser Phe Ser Lye Ser Ser 1185 1190 1195 1200

_S er Gly Gin Ser Ser Lye Thr Glu Hie Met Ser Ser Ser Ser Glu λβn 1205 1210 1215

Thr Ser Thr Pro Ser Ser λsn λla Lye λrg Gin λsn Gin Leu His Pro 1220 1225 1230

Ser Ser λla Gin Ser λrg Ser Gly Gin Pro Gin Lys λla λla Thr Cys 1235 1240 1245

Lys Val Ser Ser He λsn Gin Glu Thr He Gin Thr Tyr Cyβ Val Glu 1250 1255 1260

Aβp Thr Pro He Cys Phe Ser Arg Cys Ser Ser Leu Ser Ser Leu Ser 1265 1270 1275 1280

Ser Ala Glu Asp Glu He Gly Cys Asn Gin Thr Thr Gin Glu Ala Asp 1285 1290 1295

Ser Ala Asn Thr Leu Gin He Ala Glu He Lys Glu Lye He Gly Thr 1300 1305 1310

Arg Ser Ala Glu Asp Pro Val Ser Glu Val Pro λla Val Ser Gin His 1315 1320 1325

Pro λrg Thr Lye Ser Ser λrg Leu Gin Gly Ser Ser Leu Ser Ser Glu 1330 1335 1340

Ser λla λrg Hie Lye λla Val Glu Phe Ser Ser Gly λla Lye Ser Pro 1345 1350 1355 1360

_S er Lys Ser Gly λla Gin Thr Pro Lys Ser Pro Pro Glu Hie Tyr Val 1365 1370 1375

Gin Glu Thr Pro Leu Met Phe Ser λrg Cys Thr Ser Val Ser Ser Leu 1380 1385 1390 _λ sp Ser Phe Glu Ser λrg Ser He λla Ser Ser Val Gin Ser Glu Pro 1395 1400 1405

_C ys Ser Gly Met Val Ser Gly He He Ser Pro Ser λsp Leu Pro Asp 1410 1415 1420

Ser Pro Gly Gin Thr Met Pro Pro Ser λrg Ser Lye Thr Pro Pro Pro 1425 1430 1435 1440

Pro Pro Gin Thr λla Gin Thr Lye λrg Glu Val Pro Lye λβn Lye λla 1445 1450 1455

Pro Thr λla Glu Lye λrg Glu Ser Gly Pro Lye Gin λla λla Val λβn 1460 1465 1470

Ala Ala Val Gin Arg Val Gin Val Leu Pro Aβp Ala Asp Thr Leu Leu 1475 1480 1485

Hie Phe Ala Thr Glu Ser Thr Pro λβp Gly Phe Ser Cyβ Ser Ser Ser 1490 1495 1500

Leu Ser λla Leu Ser Leu λβp Glu Pro Phe He Gin Lye λβp Val Glu 1505 1510 1515 1520

Leu λrg He Met Pro Pro Val Gin Glu λβn λβp λβn Gly λβn Glu Thr 1525 1530 1535

Glu Ser Glu Gin Pro Lye Glu Ser Asn Glu Aβn Gin Glu Lye Glu Ala 1540 1545 1550

Glu Lye Thr He λβp Ser Glu Lye λβp Leu Leu λβp Asp Ser Aβp λβp 1555 1560 1565

Asp Asp He Glu He Leu Glu Glu Cyβ He He Ser Ala Met Pro Thr 1570 1575 1580

Lye Ser Ser λrg Lye λla Lys Lye Pro λla Gin Thr λla Ser Lye Leu 1585 1590 1595 1600

Pro Pro Pro Val λla λrg Lye Pro Ser Gin Leu Pro Val Tyr Lye Leu 1605 1610 1615

Leu Pro Ser Gin λβn λrg Leu Gin Pro Gin Lys His Val Ser Phe Thr 1620 1625 1630

Pro Gly λsp λsp Met Pro λrg Val Tyr Cyβ Val Glu Gly Thr Pro He 1635 1640 1645

Aβn Phe Ser Thr Ala Thr Ser Leu Ser Aβp Leu Thr He Glu Ser Pro 1650 1655 1660

Pro Aβn Glu Leu Ala Ala Gly Glu Gly Val λrg Gly Gly λla Gin Ser 1665 1670 1675 1680

Gly Glu Phe Glu Lys λrg λsp Thr He Pro Thr Glu Gly λrg Ser Thr 1685 1690 1695 λsp Glu λla Gin Gly Gly Lys Thr Ser Ser Val Thr He Pro Glu Leu 1700 1705 1710

Asp Aβp λβn Lys λla Glu Glu Gly λsp He Leu λla Glu Cys He λβn 1715 1720 1725

Ser λla Met Pro Lys Gly Lys Ser His Lys Pro Phe λrg Val Lys Lys 1730 1735 1740

He Met Asp Gin Val Gin Gin Ala Ser Ala Ser Ser Ser Ala Pro Aβn 1745 1750 1755 1760

Lye Asn Gin Leu λsp Gly Lye Lye Lye Lye Pro Thr Ser Pro Val Lye 1765 1770 1775

Pro He Pro _G in A _β n Thr Glu Tyr Arg Thr Arg Val Arg Lye A ^β n Ala

1780 1785 1790

Asp Ser Lys λsn λsn Leu λβn Ala Glu Arg Val Phe Ser λsp λ ^β n Lye 1795 1800 1805 _λ sp _S er Lys Lys Gin λβn Leu Lye λβn λ ^β n Ser Lye λ ^β p Phe λ ^β n λ ^β p 1810 1815 1820

Lys Leu Pro λ _β n _λβ n Glu λβp λrg Val λrg Gly Ser Phe λla Phe ^λβ p 1825 1830 1835 1840

Ser Pro Hie Hie Tyr Thr Pro He Glu Gly Thr Pro Tyr Cy ^β Phe Ser 1845 1850 1855 _λ rg _λ sn _λ sp Ser Leu Ser Ser Leu λsp Phe λsp λsp λ ^β p λ ^β p Val λ ^β p

1 -8**■6 _" 0■ 1865 1870

Leu Ser Arg Glu Lye Ala Glu Leu λrg Lye λla Lye Glu A ^β n Lys Glu 1875 1880 1885

_S er Glu Ala Lye Val Thr Ser Hie Thr Glu Leu Thr Ser A ^β n Gin Gin 1890 1895 1900

_S er Ala Asn Lys Thr Gin Ala He Ala Lys Gin Pro He Asn λrg Gly 1905 1910 1915 1920

Gin Pro Lys Pro He Leu Gin Lys Gin Ser Thr Phe Pro Gin Ser Ser 1925 1930 1935

Lye _λβ p He Pro _λβ p λrg Gly λla λla Thr λ ^β p Glu Lye Leu Gin Asn 1940 1945 1950

Phe Ala He Glu Asn Thr Pro Val Cys Phe Ser His A ^β n Ser Ser Leu 1955 1960 1965

_S er Ser Leu Ser A _β p He Asp Gin Glu Asn Asn A ^β n Lye Glu Asn Glu

1970 1975 1980

Pro He Lys Glu Thr Glu Pro Pro Asp Ser Gin Gly Glu Pro Ser Lys 1985 1990 1995 2000

Pro Gin Ala Ser Gly Tyr Ala Pro Lys Ser Phe Hie Val Glu Aep Thr

2005 5 2001100 2015

Pro Val Cy _β Phe Ser Arg Asn Ser Ser Leu Ser Ser Leu Ser He λ ^β p

2020 290-15255 2030 ser «. ».P UP — — «- X" •*• "' " ^{r Sβr} I.;."'' ^Pr ° ^Lϊ *

2035 2040

_Ly e Lys Lys Pro S.r Arg e Lys -_y Mr *s„ _& W -is •« "•

2050 ²⁰ "

Arg A _β n Met _G ly _G ly He Leu Gly Glu λ ^β p Le^Thr Leu λ ^β p Leu Ly ^β _£ 2065 ²⁰⁷ °

_Asp lie Gin Arg Pro Asp Ser Glu His Gly^eu Ser Pro Asp ^β jr Clu

_λ βn Phe λβp Trp Lye λla He Gin Glu Gly λla λβn Ser He Val Ser 2100 2105 2110

Ser Leu Hie Gin λla λla λla λla λla Cyβ Leu Ser λrg Gin λla Ser 2115 2120 2125

Ser λβp Ser λβp Ser He Leu Ser Leu Lye Ser Gly He Ser Leu Gly 2130 2135 2140

Ser Pro Phe Hie Leu Thr Pro λβp Gin Glu Glu Lye Pro Phe Thr Ser 2145 2150 2155 2160 λβn Lys Gly Pro λrg He Leu Lys Pro Gly Glu Lys Ser Thr Leu Glu 2165 2170 2175

Thr Lys Lys He Glu Ser Glu Ser Lye Gly He Lye Gly Gly Lye Lye 2180 2185 2190

Val Tyr Lye Ser Leu He Thr Gly Lye Val λrg Ser λβn Ser Glu He 2195 2200 2205

Ser Gly Gin Met Lye Gin Pro Leu Gin Ala λsn Met Pro Ser He Ser 2210 2215 2220 λrg Gly λrg Thr Met He His He Pro Gly Val λrg λβn Ser Ser Ser 2225 2230 2235 2240

Ser Thr Ser Pro Val Ser Lye Lye Gly Pro Pro Leu Lys Thr Pro λla 2245 2250 2255

Ser Lys Ser Pro Ser Glu Gly Gin Thr λla Thr Thr Ser Pro λrg Gly 2260 2265 2270 λla Lys Pro Ser Val Lys Ser Glu Leu Ser Pro Val λla λrg Gin Thr 2275 2280 2285

Ser Gin He Gly Gly Ser Ser Lys λla Pro Ser λrg Ser Gly Ser λrg 2290 2295 2300 λsp Ser Thr Pro Ser λrg Pro λla Gin Gin Pro Leu Ser λrg Pro He 2305 2310 2315 2320

Gin Ser Pro Gly λrg λβn Ser He Ser Pro Gly λrg λsn Gly He Ser 2325 2330 2335

Pro Pro λsn Lye Leu Ser Gin Leu Pro λrg Thr Ser Ser Pro Ser Thr 2340 2345 2350 λla Ser Thr Lye Ser Ser Gly Ser Gly Lye Met Ser Tyr Thr Ser Pro 2355 2360 2365

Gly λrg Gin Met Ser Gin Gin λβn Leu Thr Lye Gin Thr Gly Leu Ser 2370 2375 2380

Lys λsn λla Ser Ser He Pro λrg Ser Glu Ser λla Ser Lys Gly Leu 2385 2390 2395 2400

Asn Gin Met Asn λsn Gly λsn Gly λla λβn Lys Lys Val Glu Leu Ser 2405 2410 2415

Arg her Ser Ser Thr Lys Ser Ser Gly Ser Glu Ser Asp λrg Ser Glu 2420 2425 2430

λrg Pro Val Leu Val λrg Gin Ser Thr Phe He Lye Glu λla Pro Ser 2435 2440 2445

Pro Thr Leu Arg λrg Lys Leu Glu Glu Ser Ala Ser Phe Glu Ser Leu 2450 2455 2460

Ser Pro Ser Ser λrg Pro Ala Ser Pro Thr Arg Ser Gin Ala Gin Thr 2465 2470 2475 2480

Pro Val Leu Ser Pro Ser Leu Pro Aβp Met Ser Leu Ser Thr Hie Ser 2485 2490 2495

Ser Val Gin Ala Gly Gly Trp Arg Lye Leu Pro Pro λβn Leu Ser Pro 2500 2505 2510

Thr He Glu Tyr λβn λβp Gly λrg Pro λla Lye λrg Hie λβp He λla 2515 2520 2525 λrg Ser Hie Ser Glu Ser Pro Ser λrg Leu Pro He λβn λrg Ser Gly 2530 2535 2540

Thr Trp Lye λrg Glu Hie Ser Lye His Ser Ser Ser Leu Pro λrg Val 2545 2550 2555 2560

Ser Thr Trp λrg λrg Thr Gly Ser Ser Ser Ser He Leu Ser λla Ser 2565 2570 2575

Ser Glu Ser Ser Glu Lys λla Lys Ser Glu Asp Glu Lys Hie Val Aβn 2580 2585 2590

Ser He Ser Gly Thr Lye Gin Ser Lye Glu λβn G n Val Ser λla Lye 2595 2600 2605

Gly Thr Trp λrg Lye He Lye Glu λβn Glu Phe Ser Pro Thr λβn Ser 2610 2615 2620

Thr Ser Gin Thr Val Ser Ser Gly Ala Thr Aβn Gly λla Glu Ser Lys 2625 2630 2635 2640

Thr Leu He Tyr Gin Met λla Pro λla Val Ser Lys Thr Glu λsp Val 2645 2650 2655

Trp Val λrg He Glu λsp Cyβ Pro He λβn λβn Pro λrg Ser Gly Arg 2660 2665 2670

Ser Pro Thr Gly Asn Thr Pro Pro Val He λβp Ser Val Ser Glu Lye 2675 2680 2685 λla λβn Pro λβn He Lye λβp Ser Lye λsp Asn Gin λla Lye Gin λβn 2690 2695 2700

Val Gly λβn Gly Ser Val Pro Met λrg Thr Val Gly Leu Glu λβn λrg 2705 2710 2715 2720

Leu λsn Ser Phe He Gin Val λsp λla Pro Asp Gin Lye Gly Thr Glu 2725 2730 2735

He Lys Pro Gly Gin Asn Asn Pro Val Pro Val Ser Glu Thr Asn Glu 2740 2745 2750

_S er Ser He Val Glu λrg Thr Pro Phe Ser Ser Ser Ser Ser Ser Lys 2755 2760 2765

-83-

Hiβ Ser Ser Pro Ser Gly Thr Val λla λla λrg Val Thr Pro Phe λβn 2770 2775 2780

Tyr λβn Pro Ser Pro λrg Lye Ser Ser λla λβp Ser Thr Ser λla λrg 2785 2790 2795 2800

Pro Ser Gin He Pro Thr Pro Val λβn λβn λβn Thr Lye Lye λrg λβp 2805 2810 2815

Ser Lye Thr λβp Ser Thr Glu Ser Ser Gly Thr Gin Ser Pro Lye λrg 2820 2825 2830

Hie Ser Gly Ser Tyr Leu Val Thr Ser Val 2835 2840

(2) INFORMλTION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vii) IMMEDIATE SOURCE:

(B) CLONE: ral2(yeast)

( i) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Leu Thr Gly λla Lye Gly Leu Gin Leu λrg λla Leu λrg λrg He λla 1 5 10 15 λrg He Glu Gin Gly Gly Thr λla He Ser Pro Thr Ser Pro Leu 20 25 30

(2) INFORMλTION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: m3 (mAChR)

( Xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 9 :

Leu Tyr Trp Arg He Tyr Lys Glu Thr Glu Lys Arg Thr Lys Glu Leu 1 5 10 15

λla Gly Leu Gin λla Ser Gly Thr Glu λla Glu Thr Glu 20 25

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(vii) IMMEDIATE SOURCE:

(B) CLONE: MCC

( i) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Leu Tyr Pro Asn Leu Ala Glu Glu Arg Ser Arg Trp Glu Lys Glu Leu 5 10 15

Ala Gly Leu λrg Glu Glu λsn Glu Ser Leu Thr λla Met 20 25

(2) INFORMλTION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GTATCAAGAC TGTGACTTTT λλTTGTλGTT TλTCCλTTTT 40

(2) INFORMλTION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

-85-

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TTTλGλλTTT CλTGTTλλTλ TλTTGTGTTC TTTTTλλCλG 40

(2) INFORMλTION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTλGλTTTTλ λλλλGGTGTT TTλλλλTλAT TTTTTAλGCT 40

(2) INFORMλTION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AAGCAATTGT TGTATλλλλλ CTTGTTTCTA TTTTATTTλG 40

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: GTAACTTTTC TTCATλTλGT λλλCλTTGCC TTGTGTλCTC 40

(2) INFORMλTION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERIS ICS:

(A) LENGTH: 40 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: NNNNNNNNNN NNNGTCCCTT TTTTTAAAAA λλλλλλλTλG 40

(2) INFORMλTION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: GTAAGTAACT TGGCAGTλCλ ACTTATTTGλ λACTTTAλTλ 40

(2) INFORMλTION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

ATACAAGATA TTGATλCTTT TTTλTTλTTT GTGGTTTTλG 40

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

-87-

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GTAλGTTλCT TGTTTCTλλG TGλTλλλλCλ GYGλλGλGCT 40

(2) INFORMλTION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: λλTλλλλACA TλλCTλλTTλ GGTTTCTTGT TTTλTTTTλG 40

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 40 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINAL SOURCE:

(λ) ORGANISM: Homo sapiens

( i) SEQUENCE DESCRIPTION: SEQ ID NO:21:

GTTAGTAAAT TSCCTTTTTT GTTTGTGGGT ATλλλλλTλG 40

(2) INFORMλTION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 40 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ACCATTTTTG CATGTACTGλ TGTTλλCTCC λTCTTλλCλG 40

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GTAAATAAAT TATTTTλTCA TATTTTTTAλ λλTTλTTTλλ 40

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 64 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

( i) SEQUENCE DESCRIPTION: SEQ ID NO:24:

CATGATGTTλ TCTGTATTTA CCTATλGTCT λλATTATλCC λTCTλTλλTG TGCTTλλTTT 60

TTAG 64

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 52 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GTAACAGAAG ATTACAAACC CTGGTCACTA ATGCCλTGλC TACTTTGCTA AG 52

(2 ) INFORMλTION FOR SEQ ID NO: 26:

( i) SEQUENCE CHλRACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic acid

(C) STRλNDEDNESS : single

(D ) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GGATATTAAA GTCGTAATTT TGTTTCTAAA CTCλTTTGGC CCACAG 46

(2) INFORMλTION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GTATGTTCTC TλTλGTGTλC λTCGTλGTGC λTGTTTCλλλ 40

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 56 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

CλTCλTTGCT CTTCλλλTλλ CλλλGCATTλ TGGTTTλTGT TGλTTTTλTT TTTCλG 56

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 43 base paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

( ii) MOLECULE TYPE: CDNA

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

GTAAGACAAA λλTGTTTTTT λλTGλCλTλG ACAATTACTG GTG 43

(2) INFORMλTION FOR SEQ ID NO:30:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 40 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: eingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: TTλGATGATT GTCTTTTTCC TCTTGCCCTT TTTAλλTTλG 40

(2) INFORMλTION FOR SEQ ID NO:31:

(i) SEQUENCE CHARλCTERISTICS:

(A) LENGTH: 44 baβe paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GTATGTTTTT ATλλCλTGTλ TTTCTTAAGλ TλGCTCλGGT λTGλ 44

(2) INFORMλTION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

_. ₉₁ _ 00376

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GCTTGGCTTC λλGTTGNCTT TTTλλTGλTC CTCTλTTCTG TλTTTλλTTT λCλG 54

(2) INFORMλTION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 65 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: GTACTλTTTλ GλλTTTCλCC TGTTTTTCTT TTTTCTCTTT TTCTTTGλGG CλGGGTCTCλ 60 CTCTG 65

(2) INFORMλTION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 52 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: GCAλCTλGTλ TGλTTTTλTG TλTλλλTTλλ TCTλλλλTTG λTTλλTTTCC λG 52

(2) INFORMλTION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 baβe paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

( i) ORIGINAL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: GTACCTTTGA AλλCλTTTλG TλCTλTλλTA TGAλTTTCλT GT 42

(2 ) INFORMλTION FOR SEQ ID NO: 36:

( i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 40 baβe paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

( i) SEQUENCE DESCRIPTION: SEQ ID NO:36: CCAACTCNλλ TTλGλTGλCC CλTλTTCλGλ λACTTACTAG 40

(2) INFORMλTION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

GTATATATAG λGTTTTλTλT TλCTTTTλλλ GTλCλGλλTT CATACTCTCλ λλλλ 54

(2) INFORMλTION FOR SEQ ID NO:38:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 41 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: ATTGTGACCT TAλTTTTGTG λTCTCTTGAT TTTTATTTCλ G 41

(2) INFORMλTION FOR SEQ ID NO:39:

(i) SEQUENCE CHλRλCTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

TCCCCGCCTG CCGCTCTC 18

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 18 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

GCAGCGGCGG CTCCCGTG 8

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 20 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

GTGAACGGCT CTCλTGCTGC 20

(2) INFORMλTION FOR SEQ ID NO:42:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo sapiens

- _ _M _ PCT/-JS92.00376

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

ACGTGCGGGG λGGAATGGA 19

(2) INFORMλTION FOR SEQ ID NO:43:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 24 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: ATGλTλTCTT λCCλλλTGλT λTλC 24

(2) INFORMλTION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

TTATTCCTAC TTCTTCTλTλ CλG 23

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 21 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: TACCCATGCT GGCTCTTTTT C 21

_ ₉₅ .

(2) INFORMλTION FOR SEQ ID NO:46:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 20 baβe paire

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

TGGGGCCATC TTGTTCCTGλ 20

(2) INFORMλTION FOR SEQ ID NO:47:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 22 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: ACATTAGGCA CλAAGCTTGC λλ 22

(2) INFORMλTION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

ATCAAGCTCC AGTλλGλλGG Tλ 22

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: TGCGGCTCCT GGGTTGTTG 19

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARλCTERISTICS:

(A) LENGTH: 20 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: GCCCCTTCCT TTCTGAGGλC 20

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: eingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

TTTTCTCCTG CCTCTTACTG C 21

(2) INFORMλTION FOR SEQ ID NO:52:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 20 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

-97-

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: λTGACACCCC CCλTTCCCTC 20

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

CCACTTAλλG CλCλTλTλTT TλGT 24

(2) INFORMλTION FOR SEQ ID NO:54:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 22 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

GTATGGAAAA TAGTGAAGAA CC 22

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 24 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: TTCTTAAGTC CTGTTTTTCT TTTG 24

-98- PCI7US92/00376

(2 ) INFORMλTION FOR SEQ ID NO: 56:

( i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 23 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: TTTAGAACCT TTTTTGTGTT GTG 23 (2) INFORMλTION FOR SEQ ID NO:57:

(i) SEQUENCE CHλRλCTERISTICS:

(A) LENGTH: 24 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(λ) ORGλNISM: Homo sapiens

( i) SEQUENCE DESCRIPTION: SEQ ID NO:57:

CTCAGATTAT λCACTAλGCC TAAC 24

(2) INFORMλTION FOR SEQ ID NO:58:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 22 base pairβ

(B) TYPE: nucleic acid

(C) STRλNDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(A) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:

CATGTCTCTT ACλGTλGTλC CA 22

(2) INFORMλTION FOR SEQ ID NO:59:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(A) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:

AGGTCCAAGG GTAGCCAAGG 20

(2) INFORMλTION FOR SEQ ID NO:60:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 27 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(A) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: TAAAAATGGA TAAACTACAA TTAAAAG 27

(2) INFORMλTION FOR SEQ ID NO:61:

(i) SEQUENCE CHλRλCTERISTICS:

(A) LENGTH: 24 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:

AAATACAGAA TCATGTCTTG AAGT 24

(2) INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 23 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo sapiens

-100-

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: ACACCTλλAG ATGλCAATTT GAG 23 (2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: TAACTTλGλT λGCAGTλλTT TCCC 24 (2) INFORMATION FOR SEQ ID NO:64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

ACAATAAACT GGAGTACACλ AGG 23

(2) INFORMATION FOR SEQ ID NO:65:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 23 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65 : ATAGGTCATT GCTTCTTGCT GAT 23

- .1„01,- _

(2) INFORMATION FOR SEQ ID NO:66:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINλL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:

TGAATTTTAA TGGATTλCCT λGGT 24

(2) INFORMATION FOR SEQ ID NO:67:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 25 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CTTTTTTTGC TTTTλCTGλT TλλCG 25

(2) INFORMATION FOR SEQ ID NO:68:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: TGTλATTCAT TTTλTTCCTλ λTλGCTC 27

(2) INFORMλTION FOR SEQ ID NO:69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

-102-

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:

GGTλGCCATA GTATGλTTλT TTCT 24

(2) INFORMATION FOR SEQ ID NO:70:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: CTACCTATTT TTATACCCAC λλλC 24

(2) INFORMATION FOR SEQ ID NO:71:

(i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 23 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:

AλGλλλGCCT λCACCλTTTT TGC 23

(2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 23 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

_ - .

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:

GATCλTTCTT λGλλCCλTCT TGC 23

(2) INFORMλTION FOR SEQ ID NO:73:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 24 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:

ACCTλTλGTC TλλλTTλTλC CλTC 24

(2) INFORMATION FOR SEQ ID NO:74:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 20 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:

GTCATGGCAT TAGTGλCCλG 20

(2) INFORMATION FOR SEQ ID NO:75:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: AGTCGTAATT TTGTTTCTAA λCTC 24

(2) INFORMλTION FOR SEQ ID NO:76:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:

TGλλGGλCTC GGATTTCACG C 21

(2) INFORMλTION FOR SEQ ID NO:77:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 23 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: TCλTTCλCTC λCλGCCTGλT GλC 23 (2) INFORMλTION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: GCTTTGAAAC ATGCλCTλCG AT 22 (2) INFORMATION FOR SEQ ID NO:79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: AAACλTCλTT GCTCTTCλλλ TλλC 24

(2) INFORMλTION FOR SEQ ID NO:80:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:

TACCATGATT TAAAAATCCA CCAG 24

(2) INFORMλTION FOR SEQ ID NO:81:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 23 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:

GATGATTGTC TTTTTCCTCT TGC 23

(2) INFORMλTION FOR SEQ ID NO:82:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 24 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapiens

- ^* ,„. PCT/US92/00376

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:

CTGAGCTλTC TTλλGλλλTλ CλTG 24

(2) INFORMATION FOR SEQ ID NO:83:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 25 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: TTTTAAATGA TCCTCTλTTC TGTλT 25

(2) INFORMλTION FOR SEQ ID NO:84:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) HOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:

ACAGAGTCAG λCCCTGCCTC AAAG 24

(2) INFORMATION FOR SEQ ID NO:85:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 23 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: TTTCTATTCT TACTGCTλGC ATT 23

(2) INFORMATION FOR SEQ ID NO:86:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 22 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: λTλCλCλGGT AAGAAATTAG GA 22

(2) INFORMλTION FOR SEQ ID NO:87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:

TλGλTGλCCC λTλTTCTGTT TC 22

(2) INFORMλTION FOR SEQ ID NO:88:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 22 base pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: CλλTTλGGTC TTTTTGλGλG TA 22

(2) INFORMATION FOR SEQ ID NO:89:

(i) SEQUENCE CHλRλCTERISTICS:

(A) LENGTH: 22 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

PCI7US92/00376

-108-

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:

GTTACTGCAT ACACATTGTG AC 22

(2) INFORMATION FOR SEQ ID NO:90:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 23 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNλ

(vi) ORIGINλL SOURCE:

(λ) ORGANISM: Homo sapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:

GCTTTTTGTT TCCTAACATG AλG 23

(2) INFORMATION FOR SEQ ID NO:91:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:

TCTCCCACAG GTλATACTCC C 21

(2) INFORMλTION FOR SEQ ID NO:92:

(i) SEQUENCE CHλRλCTERISTICS: (λ) LENGTH: 21 baβe pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:

GCTAGλλCTG λλTGGGGTλC G 21

(2) INFORMλTION FOR SEQ ID NO:93:

(i) SEQUENCE CHARλCTERISTICS: (λ) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo βapienβ

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: CλGGλCλλλλ TλλTCCTGTC CC 22

(2) INFORMλTION FOR SEQ ID NO:94:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 baβe pairβ

(B) TYPE: nucleic acid

(C) STRANDEDNESS: βingle

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(vi) ORIGINλL SOURCE:

(λ) ORGλNISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: λTTTTCTTλG TTTCλTTCTT CCTC 24

92/13103

ANNEX M3

-110-

In rnatlonal Application No: PCT/