POLYMERASE delta MUTATIONS IN COLORECTAL TUMORS WITH REPLICATION ERRORS

Abstract: The human pol delta gene, responsible for a replication error phenotype in some colorectal tumors, can be used for diagnostic and therapeutic purposes. It can be used to demonstrate the existence of germline or somatic mutations in replication error (RER<+>) tumor cells.

Description: POLYMERASE δ MUTATIONS IN COLORECTAL TUMORS WITH REPLICATION ERRORS TECHNICAL TELD OF THE INVENTION The invention relates to a gene which causes repllicatioin errors in colorcctal and other cancers. In addition, it also relates to biochemical tests which can be used to identify drugs for treatment of affected individuals. BACKGROUND QF THE I ENTION Widespread, subtle alterations of the cancer cell genome were first detected in- a subset of sporadic colorectal tumors using the arbitrarily-primed pόlymerase chain reaction (Peinado et al., 1992, "Isolation and characterization of allelic losses and gains in colorectal tumors by arbitrarily primed polymerase chain reaction." ϊ°roc. Natl. Acad. Sci. USA 89, 10065-10069.). These alterations were subsequently found to represent deletions of up to 4 nucleotides in genomic polyA tracts (Ionov et al., 1993, "Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis." Nature 363, 558- 561). Other studies showed that a similar, distinctive subgroup of sporadic tumors had insertions or deletions in a variety of simple repeated sequences, particularly microsatellite sequences consisting of dinucleotide or trinucleotide repeats (Ionov et al., 1993, supra; Thibodeau et al., 1993. "Microsatdlite instability in cancer of the proximal colon." Science 260, 816-819; Aaltonen et al., 1993, "Clues to the pathogehesis of familial colorectal cancer." Science 260, 812-816). Interestingly, these sporadic tumors had certain features in common with those developing in Hereditary Non-Polyposis Colorectal Cancer (HNPCC) kindreds, such as a tendency to be located on the right side of the colon and to be near-diploid. These and other data suggested that HNPCC and a subset of sporadic tumors were associated with a heritable defect causing replication errors (RER) of microsatellites (Ionov et al., 1993, supra; Aaltonen et al., 1993, supra). The mechanism underlying the postulated defect could not be determined from the study of tumor DNA, but studies in simpler organisms provided an intriguing possibility (Levinson and Gutman, 1987, "High frequencies of short frameshifts in poly-CA/TG tandem repeats borne by bacteriophage M13 in Escherichia coli K-12." Nucleic Acids Research 15, 5323-5338; Strand et al., 1993, "Destabilization of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair." Nature 365, 274-276). This work showed that bacteria and yeast containing defective mismatch repair genes manifest instability of dinucleotide repeats. The disruption of genes primarily involved in DNA replication or recombination had no apparent effect on the fidelity of microsatellite replication (reviewed in Kunkel, 1993, "Slippery DNA and diseases." Nature 365, 207-208). These pivotal studies suggested that defective mismatch repair might be responsible for the microsatellite alterations in the tumors from HNPCC patients (Strand et al., 1993, supra). The replication error (RER + ) phenotype is characterized by genetic instability, particularly in microsatellite sequences. This phenotype occurs in both sporadic and hereditary forms of colorectal cancer. Ionov, Y.M. et al. Nature 363, 558-561 (1993). Thibodeau, S.N., Bren, G. & Schaid, D. Science 260, 816-819 (1993). Aaltonen, L.A. et at. Science 260, 812-816 (1993). In the hereditary form, mutations of mismatch repair genes are usually responsible for the instability. In many sporadic tumors, however, mutations of the four known mismatch repair (MMR) genes are apparently absent. Thus there is a need in the art to identify other genes and proteins responsible for the replication error phenotype found in both hereditary and sporadic tumors. Identification of the gene and protein would allow more widespread diagnostic screening for hereditary non-polyposis colorectal cancer than is currently possible. Identification of the involved gene and protein would also enable the rational screening of compounds for use in drug therapy of hereditary non-polyposis colorectal cancer, and would enable gene therapy for affected individuals. SUMMARY OF THE INVENTION It is an object of the invention to provide DNA molecules which contain specific mutations which cause a replication error phenotype in tumors. It is another object of the invention to provide methods of treating persons who are predisposed to hereditary non-polyposis colorectal cancer. It is still another object of the invention to provide methods for determining a predisposition to cancer. It is a further object of the invention to provide methods for screening test compounds to identify therapeutic agents for treating persons predisposed to hereditary non-polyposis colorectal cancer. It is still another object of the invention to provide a mutant protein which is involved in faulty human DNA mismatch repair. It is yet another object of the. invention to provide a transgenic animal for studying potential therapies for hereditary non-polyposis colorectal cancer. It is still another object of the invention to provide a method of treating a person having an RER + tumor to prevent accumulation of somatic mutations leading to resistance to an anti-cancer therapeutic agent. These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention an isolated and purified DNA molecule is provided. The molecule has a sequence of at least about 20 nucleotides of a pol δ allele found in a tumor wherein said DNA molecule contains a mutation relative to wild-type pol b shown in SEQ ID NO:l. In yet another embodiment of the invention a method of treating a person predisposed to hereditary non-polyposis colorectal cancer is provided. The method prevents accumulation of somatic mutations. The method involves administering a DNA molecule which has a sequence of at least about 20 nucleotides of pol δ, as shown in SEQ ID NO: 1, to a person having a mutation in a pol δ allele which predisposes the person to hereditary non-polyposis colorectal cancer, wherein said DNA molecule is sufficient to remedy the mutation in a. pol δ allele of the person. In another embodiment of the invention a method is provided for determining a predisposition to cancer. The method involves testing a body sample of a human to ascertain the presence of a mutation in pol δ which affects DNA polymerase delta expression or DNA polymerase delta function, the presence of such a mutation indicating a predisposition to cancer. In still another embodiment of the invention a method is provided for screening to identify therapeutic agents which can prevent or ameliorate tumors. The screening method involves contacting a test compound with a DNA polymerase delta protein or a cell; determining the ability of the DNA polymerase delta protein or the cell to perform DNA mismatch repair, a test compound which increases the ability of said DNA polymerase delta protein or said cell to perform DNA mismatch repair being a potential therapeutic agent. In another embodiment of the invention an isolated and purified mutant protein is provided. The protein has the sequence of a DNA polymerase delta found in a tumor. In still another embodiment of the invention a transgenic animal is provided. The transgenic (nonhuman) animal maintains a pol δ allele in its germline. The pol δ allele is one which is found in humans having hereditary non- polyposis colorectal cancer or in RER + tumors. Also provided are animals which have no wild-type pol δ alleles, due to mutations introduced. Thus the present invention provides the art with the sequence of a mutant gene which is involved in the replication error phenotype of tumors and may also be responsible for some cases of hereditary non-polyposis colorectal cancer. This enables the art to practice a variety of techniques to identify persons at risk of developing a variety of cancers and to treat them to prevent such cancers from actually developing or progressing. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the protein sequence alignment of Exo HI and flanking codons of normal human, calf, S. cerevisia and S. pombe DNA polymerase delta. Amino acids that differed in the studied tumors are indicated. A 1.1 kb fragment of human pol δ cDNA corresponding to codons 227 to 587 was amplified using primers 5'-CCT GGA ACA GGG CAT CCG-3' and 5'-CGA TGA CAG TGG CTC CCG-3'. The nucleotide sequence of codons 499-522 was determined using the primer 5'-TGT CAG CAT GGT GGG CCG-3'. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS It is a discovery of the present invention that mutations in the human gene pol δ are found in human tumors of the RER + phenotype, which appear not to have mutations in the known mismatch repair genes. The cDNA sequence of wild- type pol δ is shown in SEQ ED NO:l. This gene encodes DNA polymerase delta, which contains a 3'-5' exonuclease activity. It is a further discovery of the present invention that mutation of the pol δ gene causes cells to accumulate other mutations. Thus DNA polymerase delta mutations lead to a mismatch repair defect. The replication error phenotype (RER + ) found in both sporadic and hereditary non-polyposis colorectal cancer is characterized by variations (insertions and deletions) in microsatdlite DNA. Useful DNA molecules according to the invention are those which will specifically hybridize to pol δ mutant sequences found in RER + tumors. Typically these are at least about 20 nucleotides in length and have a mutation relative to the wild-type nucleotide. sequence as shown in SEQ ID NO:l. The DNA molecules contain a mutation which has been found in RER + tumors (HNPCC patients or sporadic tumors). Such molecules can be used as allele-specific oligonucleotide probes to track a particular mutation through a family. Such molecules can be labeled, according to any technique known in the art, such as with radiolabels, fluorescent labels, enzymatic labels, sequence tags, etc. According to some aspects of the invention, it is desirable that a wild-type cDNA, which encodes all or a part of the DNA polymerase delta protein as shown in SEQ ID NO:2, be used. To obtain expression of the protein the DNA sequence can be operably linked to appropriate control sequences, such as promoter, Kozak consensus, and. terminator sequences. A person who is predisposed to devdop cancers due to inheritance of a mutant pol δ allele can be treated by administration of a DNA molecule which contains all or a part of the normal pol δ gene sequence as shown in SEQ ID NO:l. A portion of the gene sequence will be useful when it spans the location of the mutation which is present in the mutant allele, so that a double recombination event between the mutant allele and the normal portion "corrects" the defect present in the person. A portion of the gene can also be usefully administered when it encodes enough of the protein to express a functional 3'-5' exonuclease enzyme. Such a portion need not necessarily recombine with the mutant allele, but can be maintained at a separate locus in the genome or on an independently replicating vector. Means for administering DNA to humans are known in the art, and any can be used as is convenient. A variety of vectors are also known for this purpose. According to some techniques vectors are not required. Such techniques are well known to those of skill in the art. Also contemplated as part of the present invention is the use of a combined anti-neoplastic therapy regimen. Such a combined regimen is useful for patients having an RER + tumor, whether sporadic or associated with HNPCC. The regimen combines any standard anti-neoplastic therapy to which a patient can become resistant and pol δ gene therapy, as described above. By remedying the defect present in RER + cells, i.e., a pol δ mutation, the likelihood of the tumor developing a resistance mutation is greatly diminished. By delaying or preventing the onset of resistance, the lives of cancer patients can be prolonged. In addition, such prevention of resistance allows a greater degree of tumor destruction by the therapeutic agent. Examples of anti-neoplastic therapies which can be combined with pol δ gene therapy are hormones, radiation, cytotoxic drugs, cytotoxins, and antibodies. Body samples can be tested to determine whether the pol δ gene is normal or mutant. Mutations are those deviations from the sequence shown in SEQ ID NO: 1 which are associated with disease and which cause a change in pol δ protein function or expression. Such mutations include, but are not limited to, nonconservative amino add substitutions, ddetions, premature terminations and frameshifts. It is believed that such mutations reside in the domains termed Exo I, Exo π, and Exo m. However, mutations in other sites could affect the exonuclease activity of DNA polymerase ddta. Suitable body samples for testing include those comprising DNA, RNA, or protein, obtained from biopsies, blood, prenatal, or embryonic tissues, for example. Provided with the information that a defect causing HNPCC and/or sporadic RER + tumors is in an exonuclease which affects DNA mismatch repair, one can perform assays on test compounds and compositions to determine if the compounds will remedy the defect. Such therapeutic compounds could bind to missense DNA polymerase delta mutant proteins to restore the proteins to the normal, active conformation. Alternatively, such therapeutic compounds could stimulate the expression of alternate pathways for mismatch repair. Screening for such therapeutic compounds could be performed by contacting test compounds with cells, either normal cells or those with a pol δ mutation found in a tumor. The mismatch repair ability of the- cells which were contacted with the test compounds is compared with the ability of the same cells which were not contacted with the test compounds. Such activity can be tested as is known in the art. See, for example, Levinson and Gutman, 1987, and Strand et al., 1993. Observation of changes in microsatellite DNA in cells is one way of assessing mismatch repair activity. Another approach is to assay DNA mismatch repair in vitro in nuclear extracts. See Holmes, 1990; Thomas, 1991; and Fang, 1993. Exonudease activity can also be assayed. Provided with the cDNA sequence of pol δ and the amino acid sequence of the protein, one of ordinary skill in the art can readily produce DNA polymerase delta protein, isolated and purified from other human proteins. For example, recombinant cells or organisms can be used to produce the protein in bacteria, yeast, or other convenient cell system. The isolated and purified protein can be used in screening for new therapeutic agents, for example, in in vitro 1026 - 8 " PCI7US96/00005 assays of DNA mismatch repair. The protein can also be used to raise antibodies against pol δ. Therapeutic administration of the protein is also contemplated. Transgenic animals are also contemplated by the present invention. These animals have pol δ alldes which are associated with HNPCC or sporadic tumors inserted in their germline. Such animals provide modd systems for testing drugs and other therapeutic agents to prevent or retard the devdσpment of tumors. Also contemplated are genetically engineered animals which contain one or more mutations in their own pol δ genes. The mutations will be engineered to correspond to mutations found in pol δ alldes which are found in HNPCC-affected individuals or in other human RER + tumors. Animals with both native pol δ alldes inactivated and containing a human wild-type or mutant pol δ allde are particularly desirable. Examples Mutator phenotypes have been extensivdy studied in unicellular organisms such as E. coli and S. cerevisiae. In both of these organisms, mutations in the 3'-5* exonuclease "proofreading" domain of DNA polymerases can cause a mutator phenotype with similarities to that observed in RER + cancers. Particularly intriguing was the observation that mutations affecting the exonuclease activity associated with E. coli pol III or S. cerevisiae pol δ can cause a mismatch repair defiriency or microsatellite instability. Schaaper, R-M. & Radman, M. EMBO J 8, 3511-3516 (1989); Strand, M., et al. Nature 365, 274-276 (1993). These observations prompted us to study the exonuclease domain of human pol δ Eight colorectal cancer cell lines which displayed microsatellite instability (Shibata, D. et al. Nature Genet. 6, 273-281 (1994)) and were negative in screens for mutations of the four known human genes were studied. All cell lines (HCT15, DLD-1, LS180, RKO, 587X, VACO 444, VACO 457, and VACO 481) were examined for gene mutations by reverse transcriptase- polymerase chain reaction (RT-PCR) analysis to detect abnormal transcripts and by the in vitro synthesized protein assay to detect abnormal polypeptides. These assays detect over 90% of the mutations previously identified in the human mismatch repair (MMR) genes. Liu, B. et al. Cancer Research 54, 4590-4594 (1994). Additionally, the sequences of the entire coding regions of the MMR genes in 587X, VACO 444, VACO 457, and VACO 481 were determined. No gene abnormalities were detected by any of these assays in any of the cell lines. RNA from these lines was used to amplify a 1.1 kb fragment of pol δ cDNA (see description of Figure 1). This fragment contains the three highly conserved regions, termed Exo I, Exo π, and Exo HI, thought to be involved in 3'-5' exonuclease activity. Chung, D.W. et al. Proc. Natl. Acad. Sci. USA 88, 11197-11201 (1991); Yang, C.-L. et al. Nucleic Acids Res. 20, 735-745 (1992). The sequence of the RT-PCR products revealed variants in three of the lines, each of which was confirmed in separate RT-PCR and sequencing reactions. The variant in the tumor from 587X resulted in an Asp to Asn substitution at codon 502 due to a G to A transition. Codon 502 is just proximal to the Exo HI domain (Figure 1). This variant was also found in the normal colon of patient 587X. A second variant was found in both cell lines DLD-1 and HCT15, resulting in an Arg to His substitution at codon 506 due to a G to A transition at a CpG site. Codon 506 is within the Exo m domain at a position conserved throughout evolution (Fig. 1). The DLD-1 and HCT15 lines probably originated from the same patient, as they were derived from the same laboratory, have identical mutations in RAS and p53 (Shibata, D. et al. Nature Genet. 6, 273-281 (1994)), have identical alldes detected by four minisatdlite markers (unpublished data), and have the same/rø/ δ sequence at codon 506. Fresh cultures of DLD-1 and HCT15 were obtained from the American Type Culture Collection, and the same pol δ variant was found. Normal tissue from the patient giving rise to these cell lines was not available. Neither of the variants at codons 502 or 506, nor any other variations between codons 499 and 522, were found in 100 control chromosomes. Though the variants detected are not common polymorphisms, they should be interpreted cautiously.- The codon 502 variant was found in the normal colon of patent 587X, who had no family history of neoplasia and did not develop cancer until the age of 70. The codon 506 mutation in DLD-1/HCT15 could have been somatic. However, none of dght RER + tumors from patents with germline mismatch repair gene mutations had a variation in the Exo m region of pol δ, and clonal mutations in nonmicrosatellite sequences are uncommon even in RER + tumors. There was a remarkable similarity between the hypermutability in DLD-1/HCT15 and that in S. cerevisiae strains harboring mutations in the exonuclease domain of pol δ. DLD-1/HCT15 has a relatively low rate of microsatellite instability compared to tumor cell lines with mutations of hMSH2 or hMLHI, but an equivalently elevated rate of mutation at the HPRT locus. Shibata, supra; Bhattacharyya, N.P. el al. Proc. Natl. Acad. Sci. USA 91, 6319-6323 (1994). Analogously, S. cerevisiae pol δ exonuclease mutants have a relatively low level of microsatellite instability compared to yMSH2 and yMLHl mutants (Strand, supra) but a high rate of chromosomal mutability. Simon, M., et al., EMBO J. 10, 2165-2170 (1991). Indeed, exonuclease defiάent mutants of E. coli and S. cerevisiae, including Exo m region mutants, are among the most potent imitators known. Schaaper, supra, Strand, supra, Bhattacharyya, supra, Simon, supra, and Foury, F. & Vanderstraeten, S. EMBO J. 11, 2717-2726 (1992). The HPRT mutational spectrum in DLD-1/HCT15 is also different than that in lines with a MMR gene mutation, analogous to the difference between the mutational spectra of yeast strains with pol δ and MMR gene mutations. Strand, supra. It has also been shown that extracts of DLD-1/HCT15 have a deficiency in mismatch repair in vitro. Umar, A. et al. J. Biol. Chem. 269, 14367-14370 (1994). How could a mutation in the exonuclease domain of a polymerase result in such a deficiency? It has been suggested that a proofreading defect could lead to numerous mispairs, resulting in saturation of the mismatch repair apparatus. Schaaper, supra. Indeed; an exonuclease mutant of E. coli has been shown to result in a profound mismatch repair deficiency during active growth that can be corrected by overexpression of mismatch repair genes. Schaaper, supra. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Vogelβtβin, Bert Kinzler, Kenneth . (ii) TITLE OF INVENTION: POLYMERASE DELTA MUTATIONS IN COLORECTAL TUMORS WITH REPLICATION ERRORS (ill) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Banner & Allegretti, Ltd. (B) STREET: 1001 G Street N.W. (C) CITY: Washington (D) STATE: D.C. (E) COUNTRY: U.S.A. (F) ZIP: 20001 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 2-JAN-96 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Kagan, Sarah A. (B) REGISTRATION NUMBER: 32,141 (C) REFERENCE/DOCKET NUMBER: 01107.53505 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 202-508-9100 (B) TELEFAX: 202-508-9299 (C) TELEX: 197430 BBMB UT (2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3435 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 43..3364 (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l: ACGGCGGCGT AGGCTGTGGC GGGAAACGCT GTTTGAAGCG GG ATG GAT GGC AAG 54 Met Asp Gly Lye 1 CGG CGG CCA GGC CCA GGG CCC GGG GTG CCC CCA AAG CGG GCC CGT GGG 102 Arg Arg Pro Gly Pro Gly Pro Gly Val Pro Pro Lye Arg Ala Arg Gly 5 10 15 20 GGC CTC TGG GAT GAT GAT GAT GCA CCT TOG CCA TCC CAA TTC GAG GAG ISO Gly Leu Trp Asp Asp Asp Asp Ala Pro Trp Pro Ser Gin Phe Glu Glu 25 30 35 GAC CTG GCA CTG ATG GAG GAG ATG GAG GCA GAλ CAC AGG CTG CAG GAG 198 Asp Leu Ala Leu Met Glu Glu Met Glu Ala Glu His Arg Leu Gin Glu 40 45 50 CAG GAG GAG GAG GAG CTG CAG TCA GTC CTG GAG βββ GTT GCA GAC GGG 246 Gin Glu Glu Glu Glu Leu Gin Ser Val Leu Glu Gly Val Ala Asp Gly 55 60 65 CAG GTC CCA CCA TCA GCC ATA GAT CCT CGC TGG CTT CGG CCC ACA CCA 294 Gin Val Pro Pro Ser Ala lie Asp Pro Arg Trp Leu Arg Pro Thr Pro 70 75 80 CCA GCG CTG GAC CCC CAG ACA GAG CCC CTC ATC TTC CAA CAG TTG GAG 342 Pro Ala Leu Asp Pro Gin Thr Glu Pro Leu He Phe Gin Gin Leu Glu 85 90 95 100 ATT GAC CAT TAT GTG GGC CCA GCG CAG CCT GTG CCT GGG GGG CCC CCA 390 He Asp His Tyr Val Gly Pro Ala Gin Pro Val Pro Gly Gly Pro Pro 105 110 115 CCA TCC CGC GGC TCC GTG CCT GTG CTC CGC GCC TTC GGG GTC ACC GAT 438 Pro Ser Arg Gly Ser Val Pro Val Leu Arg Ala Phe Gly Val Thr Asp 120 125 130 GAG GGG TTC TCT GTC TGC TGC CAC ATC CAC GGC TTC GCT CCC TAC TTC 486 Glu Gly Phe Ser Val Cys Cys His He His Gly Phe Ala Pro Tyr Phe 135 140 145 TAC ACC CCA GCG CCC CCT GGT TTC GGG CCC GAG CAC ATG GGT GAC CTG 534 Tyr Thr Pro Ala Pro Pro Gly Phe Gly Pro Glu His Met Gly Asp Leu 150 155 160 CAA CGG GAG CTG AAC TTG GCC ATC AGC CGG GAC AGT CGC GGG GGG AGG 582 Gin Arg Glu Leu Asn Leu Ala He Ser Arg Asp Ser Arg Gly Gly Arg 165 170 175 180 GAG CTG ACT GGG CCG GCC GTG CTG GCT GTG GAλ CTG TGC TCC C6A GAG 630 Glu Leu Thr Gly Pro Ala Val Leu Ala Val Glu Leu Cys Ser Arg Glu 185 190 195 AGC ATG TTT GGG TAC CAC GGG CAC GGC CCC TCC CCG TTC CTG CGC ATC 678 Ser Met Phe Gly Tyr His Gly His Gly Pro Ser Pro Phe Leu Arg He 200 205 210 ACC GTG GCG CTG CCG CGC CTC GTG GCC CCG GCC CGC CGT CTC CTG GAλ 726 Thr Val Ala Leu Pro Arg Leu Val Ala Pro Ala Arg Arg Leu Leu Glu 215 220 225 CAG GGC ATC CGT GTG GCA GGC CTG GGC ACG CCC AGC TTC GCG CCC TAC 774 Gin Gly He Arg Val Ala Gly Leu Gly Thr Pro Ser Phe λla Pro Tyr 230 235 240 GAG GCC AAC GTC GλC TTT GAG ATC CGG TTC ATG GTG GλC ACG GAC ATC 822 Glu λla λsn Val λsp Phe Glu He Arg Phe Met Val Asp Thr Asp He 245 250 255 260 GTC GGC TGC AAC TGG CTG GAG CTC CCA GCT GGG λλλ TλC GCC CTG λGG 870 Val Gly Cys Aβn Trp Leu Glu Leu Pro Ala Gly Lye Tyr Ala Leu Arg 265 270 275 CTG λλG GλG λλG GCT λCG CAG TGC CλG CTG GλG GCG GλC GTG CTG TGG 918 Leu Lys Glu Lye λla Thr Gin Cys Gin Leu Glu λla λsp Val Leu Trp 280 285 290 TCT GλC GTG GTC λGT CAC CCA CCG Gλλ GGG CCA TGG CAG CGC ATT GCG 966 Ser λsp Val Val Ser His Pro Pro Glu Gly Pro Trp Gin λrg He λla 295 300 305 CCC TTG CGC GTG CTC λGC TTC GAT ATC GAG TGC GCC GGC CGC λλλ GGC 1014 Pro Leu λrg Val Leu Ser Phe λβp He Glu Cys λla Gly λrg Lys Gly 310 315 320 λTC TTC CCT GλG CCT GλG CGG GλC CCT GTC λTC CλG λTC TGC TCG CTG 1062 He Phe Pro Glu Pro Glu λrg λsp Pro Val He Gin He Cys Ser Leu 325 330 335 340 GGC CTG CGC TGG GGG GAG CCG GλG CCC TTC CTλ CGC CTG GCG CTC λCC 1110 Gly Leu λrg Trp Gly Glu Pro Glu Pro Phe Leu λrg Leu λla Leu Thr 345 350 355 CTG CGG CCC TGT GCC CCC λTC CTG GGT GCC λλG GTG CAG λGC TλC GλG 1158 Leu λrg Pro Cys λla Pro He Leu Gly λla Lys Val Gin Ser Tyr Glu 360 365 370 λλG GλG GλG GλC CTG CTG CλG GCC TGG TCC λCC TTC λTC CGT λTC ATG 1206 Lys Glu Glu Asp Leu Leu Gin λla Trp Ser Thr Phe He λrg He Met 375 380 385 GλC CCC GλC GTG λTC λCC GGT TλC AAC ATC CAG AAC TTC GAC CTT CCG 1254 Asp Pro Asp Val He Thr Gly Tyr Asn He Gin Asn Phe Asp Leu Pro 390 395 400 TAC CTC λTC TCT CGG GCC CλG λCC CTC λλG GTλ CAA ACA TTC CCT TTC 1302 Tyr Leu He Ser λrg λla Gin Thr Leu Lys Val Gin Thr Phe Pro Phe 405 410 415 420 CTG GGC CGT GTG GCC GGC CTT TGC TCC λλC λTC CGG GλC TCT TCλ TTC 1350 Leu Gly λrg Val λla Gly Leu Cys Ser λsn He λrg λsp Ser Ser Phe 425 430 435 CλG TCC λλG CλG ACG GGC CGG CGG GAC λCC λλG GTT GTC λGC ATG GTG 1398 Gin Ser Lys Gin Thr Gly Arg Arg λsp Thr Lys Val Val Ser Met Val 440 445 450 GGC CGC GTG CλG ATG GAC ATG CTG CλG GTG CTG CTG CGG GλG TλC λλG 1446 Gly λrg Val Gin Met λsp Met Leu Gin Val Leu Leu λrg Glu Tyr Lys 455 460 465 CTC CGC TCC CAC ACG CTC AλT GCC GTG λGC TTC CAC TTC CTG GGC GλG 1494 Leu λrg Ser His Thr Leu λβn λla Val Ser Phe His Phe Leu Gly Glu 470 475 480 CλG λλG GλG GλC GTG CλG CAC λGC λTC λTC λCC GAC CTG CAG AλT GGG 1542 Gin Lys Glu λβp Val Gin His Ser He He Thr λβp Leu Gin λsn Gly 485 490 495 500 AAC GAC CAG λCC CGC CGC CGC CTG GCT GTG TλC TGC CTG λλG GλT GCC 1590 λsn λβp Gin Thr λrg λrg λrg Leu λla Val Tyr Cys Leu Lye λsp λla 505 510 515 TλC CTG CCλ CTG CGG CTG CTG GλG CGG CTC λTG GTG CTG GTG λλC GCC 1638 Tyr Leu Pro Leu λrg Leu Leu Glu λrg Leu Met Val Leu Val λβn λla 520 525 530 GTG GAG ATG GCG AGO GTC ACT GGC GTG CCC CTC λGC TλC CTG CTC λGT 1686 Val Glu Met λla λrg Val Thr Gly Val Pro Leu Ser Tyr Leu Leu Ser 535 540 545 CGT GGC CλG CAG GTC λλλ GTC GTλ TCC CλG CTG TTG CGG CλG GCC λTG 1734 λrg Gly Gin Gin Val Lys Val Val Ser G n Leu Leu λrg Gin λla Met 550 555 560 CλC βλβ GGG CTG CTG λTG CCC GTG GTG λλG TCλ GλG GGC GGC Gλβ GλC 1782 His Glu Gly Leu Leu Met Pro Val Val Lys Ser Glu Gly Gly Glu λsp 565 570 575 580 TλC λCG GGλ GCC ACT GTC λTC Gλβ CCC CTC λλλ GGG TλC TλC GλC GTC 1830 Tyr Thr Gly λla Thr Val He Glu Pro Leu Lys Gly Tyr Tyr λsp Val 585 590 595 CCC λTC GCC ACC CTG GAC TTC TCC TCG CTG TAC CCG TCC λTC λTβ λTG 1878 Pro He λla Thr Leu λβp Phe Ser Ser Leu Tyr Pro Ser He Met Met 600 605 610 GCC CλC AAC CTG TGT TAC ACC ACG CTC CTT CGG CCC GGG ACT GCA CAG 1926 Ala His Asn Leu Cys Tyr Thr Thr Leu Leu λrg Pro Gly Thr λla βln 615 620 625 λλλ CTC GGC CTG λCT GλG GλT CλG TTC λTC λGG λCC CCC λCC GGG GλC 1974 Lys Leu βly Leu Thr βlu λβp Gin Phe He λrg Thr Pro Thr βly λsp 630 635 640 βλβ TTT GTG λλG λCC TCλ GTG CGG λλG GGG CTG CTG CCC CAG ATC CTG 2022 Glu Phe Val Lys Thr Ser Val λrg Lye Gly Leu Leu Pro Gin He Leu 645 650 655 660 GλG AAC CTG CTC ACT GCC CGG λλG λGG GCC λλG GCC GλG CTG GCC λλG 2070 Glu λsn Leu Leu Ser λla λrg Lys λrg λla Lye λla Glu Leu λla Lye 665 670 675 GλG λCλ GλC CCC CTC CGG CGC CλG GTC CTG GλT ββλ CGG CλG CTG GCG 2118 Glu Thr λβp Pro Leu λrg λrg Gin Val Leu λβp Gly λrg Gin Leu λla 680 685 690 CTG λλG GTG λGC GCC AAC TCC GTλ TλC GGC TTC ACT GGC GCC CAG GTG 2166 Leu Lye Val Ser λla λβn Ser Val Tyr Gly Phe Thr Gly λla Gin Val 695 700 705 GGC λλG TTG CCC TGC CTG GAG ATC TCλ CλG λGC CTC ACG GGG TTC Gβλ 2214 βly Lye Leu Pro Cys Leu βlu He Ser βln Ser Val Thr βly Phe βly 710 715 720 CGT CλG λTG λTC βλβ λλλ λCC AAG CAG CTG GTG βλβ TCT λλβ TλC λCλ 2262 λrg βln Met He βlu Lye Thr Lys βln Leu Val βlu Ser Lye Tyr Thr 725 730 735 740 GTG Gλβ AAT ββC TAC λCC λCC AGT GCC λλG GTG GTG TAT GGT GAC ACT 2310 Val Glu λβn Gly Tyr Ser Thr Ser λla Lye Val Val Tyr Gly λβp Thr 745 750 755 βλC TCC GTC λTG TGC Cβλ TTC GβC GTG TCC TCG GTG GCT GλG GCG λTG 2358 λβp Ser Val Met Cys λrg Phe βly Val Ser Ser Val λla βlu λla Met 760 765 770 GCC CTG GGG CGG βλβ GCC GCG βλC TGG GTG TCλ GGT CλC TTC CCG TCG 2406 λla Leu Gly λrg Glu λla λla λsp Trp Val Ser Gly His Phe Pro Ser 775 780 785 CCC λTC CGG CTG CλG TTT Gλβ λλG GTC TλC TTC CCλ TλC CTG CTT λTC 2454 Pro He λrg Leu Glu Phe Glu Lys Val Tyr Phe Pro Tyr Leu Leu He 790 795 800 λGC λλG λλG CGC TλC GCC GGC CTG CTC TTC TCC TCC CGG CCC GλC GCC 2502 Ser Lye Lye λrg Tyr λla Gly Leu Leu Phe Ser Ser λrg Pro λβp λla 805 810 815 820 CλC GλC CGC λTG βλC TGC λλG GGC CTG βλG GCC GTG CGC λββ βλC AAC 2550 Hie λβp λrg Met λβp Cyβ Lye Cly Leu βlu λla Val λrg λrg λβp λβn 825 830 835 TGC CCC CTC GTG GCC λλC CTG GTC λCT GCC TCλ CTG CGC CGC CTG CTC 2598 Cyβ Pro Leu Val λla λβn Leu Val Thr λla Ser Leu λrg λrg Leu Leu 840 845 850 λTC GλC Cβλ βλC CCT Cλβ GGC GCG GTG GCT CλC GCλ CλG βλC GTC λTC 2646 He λβp λrg λβp Pro βlu βly λla Val λla Hie λla Gin λβp Val He 855 860 865 TCG GλC CTG CTG TGC AAC CGC ATC CAT λTC TCC CAG CTG GTC λTC λCC 2694 Ser λsp Leu Leu Cyβ λβn λrg He λβp He Ser Gin Leu Val He Thr 870 875 880 λλG βλG CTG λCC CGC GCG GCC TCC CλC TλT GCC GGC λλG CλG GCC CAC 2742 Lye Glu Leu Thr λrg λla λla Ser λβp Tyr λla Gly Lys Gin λla His 885 890 895 900 GTG GλG CTG GCC Gλβ λGG λTG λGG λλG CGG GλC CCC GGG λGT GCG CCC 2790 Val Glu Leu λla Glu λrg Met λrg Lys λrg λsp Pro Gly Ser λla Pro 905 910 915 λGC CTG GGC GλC CGC CTC CCC TλC GTG λTC λTC λGT GCC GCC λλG GGT 2838 Ser Leu Gly λsp λrg Val Pro Tyr Val He He Ser λla λla Lye Gly 920 925 930 GTG GCC GCC TλC λTG λλG TCG GλG GAC CCG CTG TTC GTG CTG GAG CλC 2886 Val λla λla Tyr Met Lye Ser Glu λβp Pro Leu Phe Val Leu Glu Hie 935 940 945 ACC CTG CCC ATT GAC λCG CλC TλC TλC CTG GλG CAG CAG CTG GCC λλG 2934 Ser Leu Pro He λβp Thr Gin Tyr Tyr Leu Glu Gin Gin Leu λla Lye 950 955 960 CCC CTC CTG CGC λTC TTC GλG CCC λTC CTG GGC GλG GGC CGT GCC GλG 2982 Pro Leu Leu λrg He Phe Glu Pro He Leu Gly Glu Gly λrg λla Glu 965 970 975 980 GCT GTG CTλ CTG CGG GGG GλC CλC ACG CGC TGC AAG ACG GTG CTC ACG 3030 λla Val Leu Leu λrg Gly λβp Hie Thr λrg Cyβ Lye Thr Val Leu Thr 985 990 995 GGC λλG GTG GGC GβC CTC CTG GCC TTC GCC λλλ CGC CGC AAC TGC TGC 3078 Gly Lys Val Gly Gly Leu Leu Ala Phe λla Lys Arg Arg λsn Cys Cys 1000 1005 1010 ATT GGC TGC CGC λCλ GTG CTC λGC CλC CλG GGλ GCC GTG TGT GλG TTC 3126 He Gly Cys λrg Thr Val Leu Ser His Gin Gly λla Val Cyβ Glu Phe 1015 1020 1025 TGC CAG CCC CGG GAG TCT GAG CTG TλT CλG λλG GλG GTA TCC CAT CTG 3174 Cyβ Gin Pro Arg βlu Ser βlu Leu Tyr Gin Lys Glu Val Ser His Leu 1030 1035 1040 AλT GCC CTG Gλβ βλG CGC TTC TCG CGC CTC TGG λCG CλG TGC CλG CGC 3222 λβn λla Leu Glu Glu λrg Phe Ser λrg Leu Trp Thr Gin Cyβ Gin λrg 1045 1050 1055 1060 TGC CAG GGC AGC CTG CAC GAG GAC GTC ATC TGC λCC λGC CGG GλC TGC 3270 Cyβ Gin Gly Ser Leu Hie Glu λβp Val He Cys Thr Ser λrg λβp Cys 1065 1070 1075 CCC λTC TTC TλC λTG CCC λλβ λλβ GTG CGG λλG βλC CTG Gλλ βλC Cλβ 3318 Pro He Phe Tyr Met λrg Lye Lye Val λrg Lye λβp Leu βlu λβp βln 1080 1085 1090 βλβ CλC CTC CTG CGG CGC TTC GGλ CCC CCT Gβλ CCT βλβ GCC TGG T 3364 βlu βln Leu Leu λrg λrg Phe Gly Pro Pro Gly Pro Glu λla Trp 1095 1100 1105 βλCCTTGCλλ GCλTCCCλTβ GGGCGGGGGC βββλCCλβββ λβλλTTλλTλ λλβTTCTGGλ 3424 CTTTTGCTλC λ 3435 (2) INFORMλTION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1107 amino acidβ (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met λβp βly Lye λrg λrg Pro βly Pro βly Pro βly Val Pro Pro Lye 1 5 10 15 λrg λla λrg Gly Gly Leu Trp λβp λβp λβp λβp λla Pro Trp Pro Ser 20 25 30 βln Phe βlu βlu λβp Leu λla Leu Met Glu Glu Met Glu λla Glu Hie 35 40 45 λrg Leu βln βlu βln βlu βlu βlu βlu Leu βln Ser Val Leu βlu Gly 50 55 60 Val λla λβp βly βln Val Pro Pro Ser λla He λβp Pro λrg Trp Leu 65 70 75 80 λrg Pro Thr Pro Pro λla Leu λβp Pro Gin Thr Glu Pro Leu He Phe 85 90 95 βln Gin Leu βlu He λβp Hie Tyr Val βly Pro λla Gin Pro Val Pro 100 105 110 Gly Gly Pro Pro Pro Ser λrg Gly Ser Val Pro Val Leu λrg λla Phe 115 120 125 Gly Val Thr λβp Glu Gly Phe Ser Val Cyβ Cyβ Hie He Hie Gly Phe 130 135 140 λla Pro Tyr Phe Tyr Thr Pro λla Pro Pro Gly Phe Gly Pro Glu Hie 145 150 155 160 Met Gly λβp Leu βln λrg βlu Leu λsn Leu λla He Ser Arg Asp Ser 165 170 175 λrg βly βly λrg βlu Leu Thr βly Pro λla Val Leu λla Val βlu Leu 180 185 190 Cyβ Ser λrg Clu Ser Met Phe βly Tyr Hie Gly Hie Gly Pro Ser Pro 195 200 205 Phe Leu λrg He Thr Val λla Leu Pro λrg Leu Val λla Pro λla λrg 210 215 220 λrg Leu Leu Glu βln βly He λrg Val λla Gly Leu Gly Thr Pro Ser 225 230 235 240 Phe λla Pro Tyr Glu λla λβn Val λβp Phe Glu He λrg Phe Met Val 245 250 255 λβp Thr λβp He Val βly Cyβ λβn Trp Leu Clu Leu Pro λla βly Lye 260 265 270 Tyr λla Leu λrg Leu Lye βlu Lye λla Thr Gin Cys Gin Leu Glu λla 275 280 285 λβp Val Leu Trp Ser λβp Val Val Ser Hie Pro Pro Glu Gly Pro Trp 290 295 300 Gin λrg He λla Pro Leu λrg Val Leu Ser Phe λβp He Glu Cyβ λla 305 310 315 320 Gly λrg Lye Gly He Phe Pro Glu Pro βlu λrg λβp Pro Val He Gin 325 330 335 He Cys Ser Leu Gly Leu λrg Trp βly βlu Pro Glu Pro Phe Leu λrg 340 345 350 Leu λla Leu Thr Leu λrg Pro Cys λla Pro He Leu Gly λla Lye Val 355 360 365 Gin Ser Tyr Glu Lye Glu Glu λβp Leu Leu Gin Ala Trp Ser Thr Phe 370 375 380 He Arg He Met Asp Pro λβp Val He Thr Gly Tyr λsn He Gin Asn 385 390 395 400 Phe Aβp Leu Pro Tyr Leu He Ser λrg λla Gin Thr Leu Lye Val Gin 405 410 415 Thr Phe Pro Phe Leu Gly λrg Val λla Gly Leu Cyβ Ser λβn He λrg 420 425 430 λβp Ser Ser Phe Gin Ser Lys Gin Thr Gly λrg Arg Asp Thr Lys Val 435 440 445 Val Ser Met Val Gly λrg Val Gin Met λβp Met Leu Gin Val Leu Leu 450 455 460 λrg Glu Tyr Lye Leu λrg Ser Hie Thr Leu λβn λla Val Ser Phe Hie 465 470 475 480 Phe Leu Gly Glu Gin Lye Glu λβp Val Gin His Ser He He Thr λβp 485 490 495 Leu Gin λβn Gly λβn Asp Gin Thr Arg Arg Arg Leu Ala Val Tyr Cyβ 500 505 510 Leu Lye Aβp Ala Tyr Leu Pro Leu λrg Leu Leu Glu λrg Leu Met Val 515 520 525 Leu Val λβn λla Val Glu Met λla Arg Val Thr Gly Val Pro Leu Ser 530 535 540 Tyr Leu Leu Ser Arg Gly Gin Gin Val Lye Val Val Ser Gin Leu Leu 545 550 555 560 λrg Gin λla Met Hie Glu Gly Leu Leu Met Pro Val Val Lys Ser Glu 565 570 575 Gly Gly Glu λsp Tyr Thr Gly λla Thr Val He Glu Pro Leu Lys Gly 580 585 590 Tyr Tyr λβp Val Pro He λla Thr Leu λβp Phe Ser Ser Leu Tyr Pro 595 600 605 Ser He Met Met λla Hie λβn Leu Cyβ Tyr Thr Thr Leu Leu λrg Pro 610 615 620 βly Thr λla βln Lye Leu βly Leu Thr βlu λβp βln Phe He λrg Thr 625 630 635 640 Pro Thr βly λβp βlu Phe Val Lye Thr Ser Val λrg Lye βly Leu Leu 645 650 655 Pro βln He Leu βlu λβn Leu Leu Ser λla λrg Lye λrg λla Lye λla 660 665 670 βlu Leu λla Lye βlu Thr λβp Pro Leu λrg λrg βln Val Leu λβp βly 675 680 685 λrg βln Leu λla Leu Lye Val Ser λla λβn Ser Val Tyr βly Phe Thr 690 695 700 βly λla βln Val βly Lye Leu Pro Cyβ Leu Clu He Ser βln Ser Val 705 710 715 720 Thr Gly Phe Gly λrg Gin Met He Glu Lye Thr Lye Gin Leu Val βlu 725 730 735 Ser Lye Tyr Thr Val βlu λβn βly Tyr Ser Thr Ser λla Lye Val Val 740 745 750 Tyr Gly Asp Thr λβp Ser Val Met Cyβ λrg Phe Gly Val Ser Ser Val 755 760 765 λla Glu λla Met λla Leu βly λrg βlu λla λla λβp Trp Val Ser Gly 770 775 780 Hie Phe Pro Ser Pro He λrg Leu βlu Phe Glu Lye Val Tyr Phe Pro 785 790 795 800 Tyr Leu Leu He Ser Lye Lye λrg Tyr λla Gly Leu Leu Phe Ser Ser 805 810 815 λrg Pro λβp λla Hie λβp λrg Met λβp Cyβ Lye Gly Leu Glu λla Val 820 825 830 λrg λrg λβp λβn Cyβ Pro Leu Val λla λβn Leu Val Thr λla Ser Leu 835 840 845 λrg λrg Leu Leu He λβp λrg Aβp Pro Clu βly λla Val λla Hie λla 850 855 860 βln λep Val He Ser λβp Leu Leu Cyβ λβn λrg He λβp He Ser βln 865 870 875 880 Leu Val He Thr Lye Glu Leu Thr λrg λla λla Ser λβp Tyr λla Gly 885 890 895 Lye Gin λla Hie Val Glu Leu λla Glu λrg Met λrg Lye λrg λβp Pro 900 905 910 Gly Ser λla Pro Ser Leu Gly λβp λrg Val Pro Tyr Val He He Ser 915 920 925 λla λla Lys Gly Val λla λla Tyr Met Lys Ser Glu λβp Pro Leu Phe 930 935 940 Val Leu Glu Hie Ser Leu Pro He λβp Thr Gin Tyr Tyr Leu Glu Gin 945 950 955 960 Gin Leu λla Lye Pro Leu Leu λrg He Phe Glu Pro He Leu Gly Glu 965 970 975 Gly λrg λla βlu λla Val Leu Leu λrg Gly λβp Hie Thr λrg Cyβ Lye 980 985 990 Thr Val Leu Thr Gly Lye Val Gly Gly Leu Leu λla Phe λla Lye λrg 995 1000 1005 λrg λβn Cyβ Cyβ He Gly Cyβ λrg Thr Val Leu Ser Hie Gin Gly λla 1010 1015 1020 Val Cyβ Glu Phe Cyβ Gin Pro λrg Glu Ser Glu Leu Tyr Gin Lye Glu 1025 1030 1035 1040 Val Ser Hie Leu λβn λla Leu Glu Glu λrg Phe Ser Arg Leu Trp Thr 1045 1050 1055 Gin Cyβ Gin Arg Cyβ Gin Gly Ser Leu Hie Glu λβp Val He Cyβ Thr 1060 1065 1070 Ser λrg λβp Cyβ Pro He Phe Tyr Met λrg Lys Lye Val λrg Lye Aβp 1075 1080 1085 Leu Glu Aβp Gin Glu Gin Leu Leu Arg λrg Phe Gly Pro Pro Gly Pro 1090 1095 1100 Glu Ala Trp 1105 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: λβn Gly λsn Aβn Gin Thr Arg λrg λrg Leu λla Val Tyr Cyβ Leu Lye 1 5 10 15 λβp λla Tyr Leu Pro Leu λrg Leu 20 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: λβn Gly λβn λβp Gin Thr λrg Hie λrg Leu λla Val Tyr Cyβ Leu Lye 1 5 10 15 λβp λla Tyr Leu Pro Leu λrg Leu 20 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 baβe pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CCTGβλλCλG GGCλTCCC 18 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 baβe pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICλL: NO (iv) λNTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CGATGACAGT GGCTCCCG 18 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 baβe pairβ (B) TYPE: nucleic acid (C) STRANDEDNESS: βingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICλL: NO (iv) λNTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGTCAGCλTG GTGGGCCG 18 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Aβn Cly λen Asp βln Thr Arg λrg λrg Leu Ala Val Tyr Cyβ Leu Lye 1 5 10 15 Aβp Ala Phe Leu Pro Leu λrg Leu 20 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) λNTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (λ) ORGANISM: Saccharomyces cereviβiae (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Asn Gly λβp Ser Glu Thr Arg Arg Arg Leu Ala Val Tyr Cyβ Leu Lye 1 5 10 15 Aβp Ala Tyr Leu Pro Leu Arg Leu 20 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (λ) LENGTH: 24 amino acidβ (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICλL: NO (iv) λNTI-SENSE: NO (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGλNISM: Saccharαmyceβ pombe (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: λβn Cly Thr λla λβp Ser λrg λrg λrg Leu λla He Tyr Cyβ Leu Lye 1 5 10 15 λβp λla Tyr Leu Pro βln λrg Leu 20

Claims: CLAIMS 1. An isolated and purified DNA molecule having a sequence of at least about 20 nucleotides of a pol δ allde found in a tumor wherein said DNA molecule contains a mutation relative to wild-type pol δ as shown in SEQ ID NO:l. 2. The DNA molecule of claim 1 wherein the mutation is within codons 227 to 587. 3. The DNA molecule of claim 1 wherein the mutation is within codons 499 and 522. 4. The DNA molecule of claim 1 wherein the mutation is within the Exo in domain. 5. A method of treating a person predisposed to hereditary non- polyposis colorectal cancer to prevent accumulation of somatic mutations, comprising: administering a DNA molecule which has a sequence of at least about 20 nucleotides of pol δ, as shown in SEQ ID NO:l, to a person having a mutation in a pol δ allele which predisposes the person to hereditary non-polyposis colorectal cancer, wherein said DNA molecule is sufficient to remedy the mutation in a pol δ allele of the person. 6. The method of claim 5 wherein said DNA molecule encodes a pol δ protein as shown in SEQ ID NO:2. 7. The method of claim 5 wherein said DNA molecule spans the mutation site in said allele and remedies the mutation by recombining with said mutant allele. 8. The method of claim 5 wherein said DNA molecule encodes a portion of pol δ protein which is sufficient to provide 3'-5' exonuclease activity. 9. A method of determining a predisposition to cancer comprising: testing a body sample of a human to ascertain the presence of a mutation in pol δ which affects DNA polymerase delta expression or DNA polymerase ddta function, the presence of such a mutation indicating a predisposition to cancer. 10. The method of claim 9 wherein the sample is DNA. 11. The method of claim 9 wherein the sample is RNA. 12. The method of claim 9 wherein the sample is protein. 13. The method of claim 9 wherein the sample is isolated from prenatal or embryonic cells. 14. A method of screening to identify therapeutic agents which can prevent or ameliorate tumors, comprising: contacting a test compound with a cell; determining the ability of the cell to perform DNA mismatch repair, a test compound which increases the ability of said cell to perform DNA mismatch repair being a potential therapeutic agent. 15. The method of claim 14 wherein the cell contains a pol δ mutation found in a tumor. 16. A method of screening to identify therapeutic agents which can prevent or ameliorate tumors, comprising: contacting a test compound with a DNA polymerase delta protein; determining the ability of the DNA polymerase ddta to perform DNA mismatch repair, a test compound which increases the ability of said protein to perform DNA mismatch repair being a potential therapeutic agent. 17. The method of claim 16 wherein the DNA polymerase delta contains a mutation found in a RER + tumor. 18. An isolated and purified DNA polymerase delta protein which has a mutant sequence found in a RER + tumor. 19. A transgenic animal wherein the transgene is a human pol δ allele. 20. The transgenic animal of claim 19 wherein the allde is a mutant allele of pol δ found in a patient having hereditary nonpolyposis colorectal cancer or an RER + tumor. 21. A non-human animal which contains no endogenous wild-type pol δ allele. 22. The transgenic animal of claim 19 wherein said animal contains no endogenous wild-type pol δ allde. 23. A method of treating a person having an RER + tumor to prevent accumulation of somatic mutations leading to resistance to an anti-cancer therapeutic agent, comprising: administering to a person having a tumor with an RER + phenotype: (a) a DNA molecule which has a sequence of at least about 20 nudeotides of pol δ, as shown in SEQ ID NO: 1, wherein said DNA molecule is sufficient to remedy a mutation in a pol δ allele of the person; and (b) an anti-neoplastic therapeutic agent. 24. The method of claim 23 wherein the anti-neoplastic therapeutic agent is selected from the group consisting of: a hormone, radiation, a cytotoxic drug, a cytotoxin, and an antibody.

Inventors: KINZLER KENNETH W, VOGELSTEIN BERT, KINZLER, KENNETH, W., VOGELSTEIN, BERT
Applicants: UNIV JOHNS HOPKINS, THE JOHNS HOPKINS UNIVERSITY
Application year: 1996
Main classification: C12N15
IPC codes: A01K67, A61K38, A61K45, A61K48, A61P35, C12N9, C12N15, C12Q1