AU773601B2 - Chromosome 13-linked breast cancer susceptibility gene - Google Patents

Description

TITLE OF THE INVENTION CHROMOSOME 13-LINKED BREAST CANCER SUSCEPTIBILITY GENE FIELD OF THE INVENTION The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human cancer predisposing gene (BRCA2), some mutant alleles of which cause susceptibility to cancer, in particular, breast cancer in females and males. More specifically, the invention relates to germline mutations in the BRCA2 gene and their use in the diagnosis of predisposition to breast cancer. The present invention further relates to somatic mutations in the BRCA2 gene in human breast cancer and their use in the diagnosis and prognosis of human breast cancer. Additionally, the invention relates to somatic mutations in the BRCA2 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the BRCA2 gene, including gene therapy, protein replacement 20 therapy and protein mimetics. The invention further relates to the screening of drugs for cancer therapy; Finally, the invention relates to the screening of the BRCA2 gene for mutations, which are useful for diagnosing the predisposition to breast cancer.

The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference, and for convenience, are referenced by author and date in the following text and respectively grouped in the appended List of References.

-2- BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

The genetics of cancer is complicated, involving multiple dominant, positive regulators of the transformed state .(oncogenes). as well as multiple recessive, negative regulators (tumor suppressor genes). Over one hundred oncogenes have been characterized. Fewer than. a dozen tumor suppressor genes have been identified, but the number is expected to increase beyond fifty (Knudson, 1993).

The involvement of so many genes underscores the complexity of the growth control mechanisms that operate in cells to maintain the integrity of normal tissue. This complexity is :manifest in another way. So far, no single gene has been shown to participate in the development *.of all, or even the majority of human cancers. The most common oncogenic mutations are in the H-ras gene, found in 10-15% of all solid tumors (Anderson et al., 1992). The most frequently mutated tumor suppressor genes are the TP53 gene, homozygously deleted in roughly 50.% of all 15 tumors, and CDKN2, which was homozygously deleted in 46% of tumor cell lines examined (Kamb et al., 1994a). Without a target that is common to all transformed cells, the dream of a "magic bullet" that can destroy or revert cancer cells while leaving normal tissue unhaimed is improbable. The hope for a new generation of specifically targeted antitumor drugs may'rest on the ability to-identify tumor suppressor genes or oncogenes that play general roles in control of cell division.

The tumor suppressor genes which have been cloned and characterized influence susceptibility to: 1) Retinoblastoma (RB1); 2) Wilms' tumor (WTI); 3) Li-Fraumeni .(TP53); 4) Familial adenomatous polyposis (APC); 5) Neurofibromatosis type I (NFI); 6) Neurofibromatosis type 2 7) von Hippel-Lindau syndrome (VHL); 8) Multiple endocrine neoplasia type 2A (MEN2A); and 9) Melanoma (CDKN2).

Tumor suppressoi loci that have been mapped genetically but not yet isolated include genes for Multiple endocrine neoplasia type 1 (MENI); Lynch cancer family syndrome 2 (LCFS2); Neuroblastoma Basal cell nevus syndrome (BCNS); Beckwith-Wiedemann syndrome (BWS); Renal cell carcinoma (RCC); Tuberous sclerosis 1. (TSC1); and Tuberous sclerosis 2 (TSC2). The tumor suppressor genes that have been characterized to date encode products with similarities to a variety of protein types, including DNA binding proteins (WTI), ancillary transcription regulators (RBI), GTPase activating proteins or GAPs (NFI), cytoskeletal components (NF2), membrane bound receptor kinases (MEN2A), cell cycle regulators (CDKN2) and others with no obvious similarity to known proteins (APC and VHL).

In many cases, the tumor suppressor gene originally identified through genetic studies has been shown to be lost or mutated in some sporadic tumors. This result suggests that regions of chromosomal aberration may signify the position of important tumor suppressor genes involved both in genetic predisposition to cancer and in sporadic cancer.

One of the hallmarks of several tumor suppressor genes characterized to date is that they are deleted at high frequency in certain tumor types. The deletions often involve loss of a single allele, a so-called loss of heterozygosity (LOH), but may also involve homozygous deletion of both alleles. For LOH, the remaining allele is presumed to be nonfunctional, either because of a preexisting inherited mutation, or because of a secondary sporadic mutation.

Breast cancer is one of the most significant diseases that affects women. At the current rate, American women have a 1 in 8 risk of developing breast cancer by age 95 (American Cancer Society, 1992). Treatment of breast cancer at later stages is often futile and disfiguring, making early detection a high priority in medical management of the disease. Ovarian cancer, although less frequent than breast cancer, is often rapidly fatal and is the fourth most common cause of cancer mortality in American women. Genetic factors contribute to an ill-defined proportion of breast cancer incidence, estimated to be about 5% of all cases but approximately 25% of cases diagnosed before age 40 (Claus et al., 1991). Breast cancer has been subdivided into two types, 20 early-age onset and late-age onset, based on an inflection in the age-specific incidence curve around age 50. Mutation of one gene, BRCA1, is thought to account for approximately 45% of Sfamilial breast cancer, but at least 80% of families with both breast and ovarian cancer (Easton et al., 1993).

The BRCAI gene has been isolated (Futreal et al., 1994; Miki et al., 1994) following an .25 intense effort following its mapping in 1990 (Hall et al., .1990; Narod et al., 1991). A second ,locus, BRCA2, has recently been mapped to chromosome 13 (Wooster et 1994) and appears to account for a proportion of early-onset breast cancer roughly equal to BRCAI, but confers a lower risk of ovarian cancer. The remaining susceptibility to early-onset breast cancer is divided between as-yet unmapped genes for familial cancer, and rarer germline mutations in genes such as TP53 (Malkin et al., 1990). It has also been suggested that heterozygote carriers for defective forms of the Ataxia-Telangiectasia gene are at higher risk for breast cancer (Swift et al., 1976; Swift et al., 1991). Late-age onset breast cancer is also often familial although the risks in relatives are not as high as those for early-onset breast cancer (Cannon-Albright et al., 1994; Mettlin et al., 1990).

However, the percentage of such cases due to genetic susceptibility is unknown.

Breast cancer has long been recognized to be, in part, a familial disease (Anderson, 1972).

Numerous investigators have examined the evidence for genetic inheritance and concluded that the data are most consistent with dominant inheritance for a major susceptibility locus or loci (Bishop and Gardner, 1980; Go et al., 1983; Williams and Anderson, 1984; Bishop et al., 1988; Newman et al., 1988; Claus et al., 1991). Recent results demonstrate that at least three loci exist which convey susceptibility to breast cancer as well as other cancers. These loci are the TP53 locus on chromosome 17p (Malkin et al., 1990), a 17q-linked susceptibility locus known as BRCAI (Hall et al., 1990), and one or more loci responsible for the unmapped residual. Hall et al. (1990) indicated that the inherited breast cancer susceptibility in kindreds with early age onset is linked to chromosome 17q21; although subsequent studies by this group using a more appropriate genetic model partially refuted the limitation to early onset breast cancer (Margaritte et al., 1992).

Most strategies for cloning the chromosome 13-linked breast cancer predisposing gene (BRCA2) require precise genetic localization studies. The simplest model for the functional role of BRCA2 holds that alleles of BRCA2 that predispose to cancer are recessive to wild type alleles; that is, cells that contain at least one wild type BRCA2 allele are not cancerous. However, cells that contain one wild type BRCA2 allele and one predisposing allele may occasionally suffer loss 20 of the wild type allele either by random mutation or by chromosome loss during cell division (nondisjunction). All the progeny of such a mutant cell lack the wild type function of BRCA2 and may develop into tumors. According to this model, predisposing alleles of BRCA2 are recessive, 'yet susceptibility to cancer is inherited in a dominant fashion: women who possess one predisposing allele (and one wild type allele) risk developing cancer, because their mammary epithelial cells may spontaneously lose the wild type BRCA2 allele. This model applies to a group S"of cancer susceptibility loci known as tumor suppressors or antioncogenes, a class of genes that includes the retinoblastoma gene and neurofibromatosis gene. By inference this model may explain the BRCAl function, as has recently been suggested (Smith et al., 1992).

A second possibility is that BRCA2 predisposing alleles are truly dominant; that is, a wild type allele of BRCA2 cannot overcome the tumor forming role of the predisposing allele. Thus, a cell that carries both wild type and mutant alleles would not necessarily lose the wild type copy of BRCA2 before giving rise to malignant cells. Instead, mammary cells in predisposed individuals would undergo some other stochastic change(s) leading to cancer.

If BRCA2 predisposing alleles are recessive, the BRCA2 gene is expected to be expressed in normal mammary tissue but not functionally expressed in mammary tumors. In contrast, if BRCA2 predisposing alleles are dominant, the wild type BRCA2 gene may or may not be expressed in normal mammary tissue. However, the predisposing allele will likely be expressed in breast tumor cells.

The chromosome 13 linkage of BRCA2 was independently confirmed by studying fifteen families that had multiple cases of early-onset breast cancer cases that were not linked to BRCA1 (Wooster et 1994). These studies claimed to localize the gene within a large region, 6 centiMorgans or approximately 6 million base pairs, between the markers D13S289 and D13S267, placing BRCA2 in a physical region defined by 13q12-13. The size of these regions and the uncertainty associated with them has made it difficult to design and implement physical mapping and/or cloning strategies for isolating the BRCA2 gene. Like BRCA1, BRCA2 appears to confer a high risk of early-onset breast cancer in females. However, BRCA2 does not appear to confer a substantially elevated risk of ovarian cancer, although it does appear to confer an elevated risk of male breast cancer (Wooster, et 1994).

Identification of a breast cancer susceptibility locus would permit the early detection of susceptible individuals and greatly increase our ability to understand the initial steps which lead to 20 cancer. As susceptibility loci are often altered during tumor progression, cloning these genes could also be important in the development of better diagnostic and prognostic products, as well as better cancer therapies.

*o o SUMMARY OF THE INVENTION The present invention relates generally to the field of human genetics. Specifically, the *present invention relates to methods and materials used to isolate and detect a human breast cancer predisposing gene (BRCA2), some alleles of which cause susceptibility to cancer, in particular breast cancer in females and males. More specifically, the present invention relates to germline mutations in the BRCA2 gene and their use in the diagnosis of predisposition to breast cancer. The invention further relates to somatic mutations in the BRCA2 gene in human breast cancer and their 6' use in the diagnosis and prognosis of human breast cancer. Additionally, the invention relates to somatic mutations in the BRCA2 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the BRCA2 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to the screening of drugs for cancer therapy. Finally, the invention relates to the screening of the BRCA2 gene for mutations, which are useful for diagnosing the predisposition to breast cancer.

According to a first aspect, the present invention provides an isolated DNA comprising a cDNA coding for a BRCA2 polypeptide defined by the amino acid sequence set forth in SEQ ID NO:2 or a corresponding RNA.

According to a second aspect, the present invention provides an isolated nucleic .acid which comprises the coding sequence set forth in SEQ ID NO: I from nucleotide i position 229 to nucleotide position 10482 or a corresponding RNA.

According to a third aspect, the present invention provides an isolated nucleic acid selected from the group consisting of: a DNA comprising the coding sequence set forth in SEQ ID NO: 1, from •nucleotide position 229 to nucleotide position 10482 or a corresponding RNA; and 20 a DNA which hybridizes to and is at least 95% complementary compared to an entire DNA coding for a BRCA2 polypeptide as in above or a corresponding RNA, wherein said nucleic acid further comprises a mutation associated with a predisposition to breast cancer, said mutation being selected from: AC at nucleotide positions 277 and 278 deleted; or (ii) four nucleotides at positions 982-985 deleted; or (iii) having four nucleotides at positions 4706-4709 deleted; or (iv) C at nucleotide position 8525 deleted; or five nucleotides at positions 9254-9258 deleted; or (vi) GT at nucleotide positions 4075 and 4076 deleted; or (vii) five nucleotides at positions 999-1003 deleted; or (viii) three nucleotides at positions 4132-4134 deleted; or -6a- (ix) A at position 1493 deleted; or a C instead of an A at position 2411.

According to a fourth aspect, the present invention provides an isolated nucleic acid selected from the group consisting of: a DNA comprising the coding sequence set forth in SEQ ID NO: from nucleotide position 10482 or corresponding RNA; and a DNA which hybridizes to and is at least 95% complementary compared to an entire DNA coding for a BRCA2 polypeptide as in above or a corresponding RNA, wherein said nucleic acid further comprises an alteration, said alteration being selected from: i a C instead of a G at position 451; or a C instead of an A at position 1093; or a C instead of a G at position 1291; or a T instead of a C at position 2117; or an A instead of a G at position 4813; or a G instead of a T at position 5868; or a T instead ofa C at position 5972; or a T instead of a C at position 6328; or S(i) a T instead of a G at position 7049; or 20 a C instead ofa G at position 7491; or S(k) a G instead of an A at position 9537; or a T instead of an A at position 10204; or a G instead of a C at position 10298; or a G instead of an A at position 10462; or an A instead of a G at position 203; or an A instead of a C at position 1342; or a C instead of a T at position 2457; or a G instead of an A at position 3199; or a G instead of an A at position 3624; or a G instead of an A at position 3668; or a C instead of a T at position 4035; or a G instead of an A at position 7470; or 6b a G instead of an A at position 1593; or an A instead of a G at position 4296; or a G instead of an A at position 5691; or a G instead of an A at position 6051; or (aa) a C instead of a T at position 6828; or (bb) a C instead ofa T at position 6921.

According to a fifth aspect, the present invention provides an isolated nucleic acid having at least 15 contiguous nucleotides of a nucleic acid according to the third aspect encompassing a said mutation associated with a predisposition to breast cancer.

According to a sixth aspect, the present invention provides a vector selected from the group consisting of: a vector comprising an isolated nucleic acid according to any one of the first, second, third, fourth or fifth aspects; and a vector comprising an isolated nucleic acid according to any one of the first, second, third or fourth aspects wherein said nucleic acid is operably-linked to a promoter sequence capable of directing expression of said nucleic acid in host cells for said vector.

According to a seventh aspect, the present invention provides host cells transformed with a vector according to the sixth aspect.

According to an eighth aspect, the present invention provides a method for producing a polypeptide encoded by an isolated nucleic acid according to any one of the first, second, third or fourth aspects which comprises culturing the host cells according to the seventh aspect containing an expression vector encoding said polypeptide under conditions suitable for the production of said polypeptide and (ii) recovering said polypeptide.

According to a ninth aspect, the present invention provides a preparation of human BRCA2 polypeptide substantially free of other human proteins, selected from the group consisting of: a polypeptide having the amino acid sequence set forth in SEQ ID NO:2; a mutated or variant human BRCA2 polypeptide obtainable by expression of a nucleic acid according to any one of the second, third or fourth aspects; and a fusion protein containing a polypeptide as defined in or Apr. 2004 15:29 No. 0103 P. 4 -6c According to a tenth aspect, the present invention provides use of a polypeptide according to the ninth aspect as an immunogen for antibody production.

According to an eleventh aspect, the present invention provides a method for determining variation of the nucleotide sequence of a suspected mutant BRCA2 allele associated with predisposition to breast cancer from a known non-mutant wild-type BRCA2-encoding nucleotide sequence comprising nucleotides 229-10482 of SEQ ID NO:1, said method comprising preparing a biological sample containing said suspected mutant BRCA2 allele and comparing the nucleotide sequence of the suspected mutant BRCA2 allele with said non-mutant sequence and identifying difference(s) between the sequences.

According to a twelfth aspect, the present invention provides a nucleic acid comprising a BRCA2 gene which encodes a BRCA2 polypeptide having the amino acid sequence set forth in SEQ ID NO:2 for use in breast cancer gene therapy.

According to a thirteenth aspect, the present invention provides use of a nucleic acid according to the twelfth aspect for the manufacture of a vector preparation for use in breast cancer gene therapy.

According to a fourteenth aspect, the present invention provides use of a polypeptide which is a wild-type BRCA2 polypeptide having an amino acid sequence set forth in SEQ ID NO:2 for the manufacture of a product for use in peptide breast cancer 0e 20 therapy.

According to a fifteenth aspect the present invention provides an isolated nucleic acid comprising a nucleotide sequence of a nucleic acid amplified from a human tissue sample by PCR using a first primer having the sequence of SEQ ID NO: 112 and a second primer having the sequence of SEQ ID N0:113.

According to a sixteenth aspect, the present invention provides a method for 0.0 detecting a mutation in a BRCA2 gene in a patient sample, comprising, *0% 0:: amplifying at least a BRCA2 gene exon selected from the group consisting of exons 21eee.

27; and determining whether the nucleotide sequence of the amplified exon encodes a shorter 3 amino acid sequence, 3o amino acid sequence, COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 Apr. 2004 15:29 No. 0103 P. -6dwherein said BRCA2 gene is on human chromosome 13 between the markers tdj3820 and YS-G-B1OT, and encodes a BRCA2 protein having a total of 3418 amino acids with the N-terminus amino acid being methionine and the C-terminus amino acid being isoleucine.

According to a seventeenth aspect, the present invention provides a method for detecting an alteration in a BRCA2 gene in a patient sample, comprising, amplifying at least a BRCA2 gene exon selected from the group consisting of exons 21- 27; and comparing the nucleotide sequence of the amplified exon with SEQ ID NO:1.

According to an eighteenth aspect, the present invention provides a method for detecting a predisposition for breast cancer and ovarian cancer in a tissue sample derived from a human, which comprises determining in said tissue sample whether there is a germline alteration in exons 21-27 of the BRCA2 gene, which is defined as the gene that is on human chromosome 13 between the markers tdj3820 and YS-G-BlOT, encodes a BRCA2 protein having a total of 3418 amino acids with the N-terminus amino acid being methionine and the C-terminus amino acid being isoleuoine, and comprises a nucleotide sequence of a nucleic acid amplified from a human tissue sample by PCR using a first primer having the sequence of SEQ ID N0:112 and a second primer having o the sequence of SEQ ID NO:113, wherein the presence of said alteration indicates said 0: 20 predisposition to cancer.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

o'oo 25 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic map ofSTSs, Pls, BACs and YACs in the BRCA2 region.

Figure 2 shows the sequence-space relationship between the cDNA clones, hybrid "selected clones, eDNA PCR products and genomic sequences used to assemble the BRCA2 transcript sequence. 2-Br-C:RACE is a biotin-capture RACE product obtained COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 Apr. 2004 15:29 No. 0103 P. 6 6e from both human breast and human thymus cDNA, The cDNA clone X sC713.1 was identified by screening a pool of human testis and HepG2 cDNA libraries with hybridselected clone GT 713. The sequence 1-BR:CGO26 5kb was generated from a PCR product beginning at the exon 7/8 junction (within X sC713.1) and terminating within an hybrid selected clone that is part of exon 11. The sequence of exon 11 was corrected by comparison to hybrid selected clones, genomic sequence in the public domain and radioactive DNA sequencing gels. Hybrid selected clones located within that exon (clone names beginning with nH or GT) are placed below it. The cDNA clones X wCBF1B8.1, X wCBFIAS.1, X wCBF1A5.12, X wCBFlB6.2 and X wCBF1B6.3 were identified by screening a pool of human mammary gland, placenta, testis and HepG2 cDNA libraries with the exon trapped clones wXBF1B8, wXPF1A5 and wXBFIB6. The clone X wCBF1B6.3 is chimeric (indicated by the dashed line), but its 5' end contained an important overlap with X wCBF11AS.1. a denotes the translation initiator, denotes the translation terminator.

Figures 3A-3D show the DNA sequence of the BRCA2 gene (which is also set forth in SEQ ID NO:1).

Figure 4 shows the genomic organization of the BNRCA2 gene. The exons (boxes and/or vertical lines) are parsed across the genomic sequences (ftp://genome.wustl.edu/pub/gscl/brca:) (horizontal lines) such that their sizes and ra*o 20 spacing are proportional. The name of each genomic 0 *000 COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 sequence is given at the left side of the figure. The sequences 92M18.00541 and 92M18.01289 actually overlap. Distances between the other genomic sequences are not known. Neither the public database nor our sequence database contained genomic sequences overlapping with exon 21.

Exons 1, 11 and 21 are numbered. denotes two adjacent exons spaced closely enough that they are not resolved at this scale.

Figures 5A-5D show a loss of heterozygosity (LOH) analysis of primary breast tumors.

Alleles of STR markers are indicated below the chromatogram. Shown are one example of a tumor heterozygous at BRCA2 (Figs. 5A and 5B) and an example of a tumor with LOH at BRCA2 (Figs. 5C and 5D). Fluorescence units are on the ordinate; size in basepairs is on the abscissa. N is for normal (Figs. 5A and 5C) and T is for tumor (Figs. 5B and DETAILED DESCRIPTION OF THE INVENTION The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect a human breast cancer predisposing gene (BRCA2), some alleles of which cause susceptibility to cancer, in particular breast cancer in females and males. More specifically, the present invention relates to germline mutations in the BRCA2 gene and their use in the diagnosis of predisposition to breast cancer. The invention further relates to somatic mutations in the BRCA2 gene in human breast cancer and their 20 use in the diagnosis and prognosis of human breast cancer. Additionally, the invention relates to somatic mutations in the BRCA2 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The invention also relates to the therapy of human cancers which have a mutation in the BRCA2 gene, including gene therapy, protein replacement therapy.and protein mimetics. The invention further relates to the screening of drugs for cancer therapy.

Finally, the invention relates to the screening of the BRCA2 gene for mutations, which are useful for diagnosing the predisposition to breast cancer.

The present invention provides an isolated polynucleotide comprising all, or a portion of the BRCA2 locus or of a mutated BRCA2 locus, preferably at least eight bases and not more than about 100 kb in length. Such polynucleotides may be antisense polynucleotides. The present 30 invention also provides a recombinant construct comprising such an isolated polynucleotide, for example, a recombinant construct suitable for expression in a transformed host cell.

Also provided by the present invention are methods of detecting a polynucleotide comprising a portion of the BRCA2 locus or its expression product in an analyte. Such methods may further comprise the step of amplifying the portion of the BRCA2 locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the BRCA2 locus. The method is useful for either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer.

The present invention also provides isolated antibodies, preferably monoclonal antibodies, which specifically bind to an isolated polypeptide comprised of at least five amino acid residues encoded by the BRCA2 locus.

The present invention also provides kits for detecting in an analyte a polynucleotide comprising a portion of the BRCA2 locus, the kits comprising a polynucleotide complementary to the portion of the BRCA2 locus packaged in a suitable container, and instructions for its use.

The present invention further provides methods of preparing a polynucleotide comprising polymerizing nucleotides to yield a sequence comprised of at least eight consecutive nucleotides of the BRCA2 locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a sequence comprising at least five amino acids encoded within the BRCA2 locus.

The present invention further provides methods of screening the BRCA2 gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the BRCA2 locus, and may further include a step of providing a set of polynucleotides which are primers for 20 amplification of said portion of the BRCA2 locus. The method is useful for identifying mutations for use in either diagnosis of the predisposition to cancer or the diagnosis or prognosis of cancer.

The present invention further provides methods of screening suspected BRCA2 mutant alleles to identify mutations in the BRCA2 gene.

In addition, the present invention provides methods of screening drugs for cancer therapy to identify suitable drugs for restoring BRCA2 gene product function.

Finally, the present invention provides the means necessary for production of gene-based therapies directed at cancer cells. These therapeutic agents may take the form of polynucleotides comprising all or a portion of the BRCA2 locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the BRCA2 protein is reconstituted. Therapeutic 30 agents may also take the form of polypeptides based on either a portion of, or the entire protein sequence of BRCA2. These may functionally replace the activity of BRCA2 in vivo.

It is a discovery of the present invention that the BRCA2 locus which predisposes individuals to breast cancer, is a gene encoding a BRCA2 protein. This gene is termed BRCA2 herein. It.is a discovery of the present invention that mutations in the BRCA2 locus in the germline are indicative of a predisposition to breast cancer in both men and women. Finally, it is a discovery of the present invention that somatic mutations in the BRCA2 locus are also associated with breast cancer and other cancers, which represents an indicator of these cancers or of the prognosis. of these cancers. The mutational events of the BRCA2 locus can involve deletions, insertions and point mutations within the coding sequence and the non-coding sequence.

Starting from a region on human chromosome 13 of the human genome, which has a size estimated at about 6 million base pairs, a smaller region of 1 to 1.5 million bases which contains a genetic locus, BRCA2, which causes susceptibility to cancer, including breast cancer, has been identified.

The region containing the BRCA2 locus was identified using a variety of genetic techniques.

Genetic mapping techniques initially defined the BRCA2 region in terms of recombination with genetic markers. Based upon studies of large extended families ("kindreds") with multiple cases of breast cancer, a chromosomal region has been pinpointed that contains the BRCA2 gene. A region which contains the BRCA2 locus is physically bounded by the markers D13S289 and D13S267.

The use of the genetic markers provided by this invention allowed the identification of clones which cover the region from a human yeast artificial chromosome (YAC) or a human 20 bacterial artificial chromosome (BAC) library. It also allowed for the identification. and preparation of more easily manipulated P1 and BAC clones from this region and the construction of a contig from a subset of the clones. These Pls, YACs and BACs provide the basis for cloning the BRCA2 locus and provide the basis for developing reagents effective, for example, in the diagnosis and treatment of breast and/or ovarian cancer. The BRCA2 gene and other potential susceptibility genes have been isolated from this region. The isolation was done using software trapping (a computational method for identifying sequences likely to contain coding exons, from *o contiguous or discontinuous genomic DNA sequences), hybrid selection techniques and direct screening, with whole or partial cDNA inserts from Pls and BACs, in the region to screen cDNA *libraries. These methods were used to obtain sequences of loci expressed in breast and other tissue.

These candidate loci were analyzed to identify sequences which confer cancer susceptibility. We have discovered that there are mutations in the coding sequence of the BRCA2 locus in kindreds which are responsible for the chromosome 13-linked cancer susceptibility known as BRCA2. The .present invention not only facilitates the early detection of certain cancers, so vital to patient survival, but also permits the detection of susceptible individuals before they develop cancer.

Population Resources Large, well-documented Utah kindreds are especially important in providing good resources for human genetic studies. Each large kindred independently provides the power to detect whether a BRCA2 susceptibility allele is segregating in that family. Recombinants informative for localization and isolation of the BRCA2 locus could be obtained only from kindreds large enough to confirm the presence of a susceptibility allele. Large sibships are especially important for studying breast cancer, since penetrance of the BRCA2 susceptibility allele is reduced both by age and sex, making informative sibships difficult to find. Furthermore, large sibships are essential for constructing haplotypes of deceased individuals by inference from the haplotypes of their close relatives.

While other populations may also provide beneficial-information, such studies generally require much greater effort, and the families are usually much smaller and thus less informative.

Utah's age-adjusted breast cancer incidence is 20% lower than the average U.S. rate. The lower incidence in Utah is probably due largely to an early age at first pregnancy, increasing the probability that cases found in Utah kindreds carry a genetic predisposition.

V t Genetic Mapping Given a set of informative families, genetic markers are essential for linking a disease to a region of a chromosome. Such markers include restriction fragment length polymorphisms (RFLPs) (Botstein et al., 1980), markers with a variable number of tandem repeats (VNTRs) (Jeffreys et al., 1985; Nakamura et al., 1987), and an abundant class of DNA polymorphisms based on short tandem repeats (STRs), especially repeats of CpA (Weber and May, 1989; Litt et al., 1989). To generate a genetic map, one selects potential genetic markers and tests them using DNA extracted from members of the kindreds being studied.

Genetic markers useful in searching for a genetic locus associated with a disease can be 30 selected on an ad hoc basis, by densely covering a specific chromosome, or by detailed analysis of a specific region of a chromosome. A preferred method for selecting genetic markers linked with a disease involves evaluating the degree of informativeness of kindreds to determine the ideal distance between genetic markers of a given degree of polymorphism, then selecting markers from known genetic maps which are ideally spaced for maximal efficiency. Informativeness of kindreds is measured by the probability that the markers will be heterozygous in unrelated individuals. It is also most efficient to use STR markers which are detected by amplification of the target nucleic acid sequence using PCR; such markers are highly informative, easy to assay (Weber and May, 1989), and can be assayed simultaneously using multiplexing strategies (Skolnick and Wallace, 1988), greatly reducing the number of experiments required.

Once linkage has been established, one needs to find markers that flank the disease locus, one or more markers proximal. to the disease locus, and one or more markers distal to the disease locus. Where possible, candidate markers can be selected from a known genetic map.

Where none is known, new markers can be identified by the STR technique, as shown in the Examples.

Genetic mapping is usually an iterative process. In the present invention, it began by defining flanking genetic markers around the BRCA2 locus, then replacing these flanking markers with. other markers that were successively closer to the BRCA2 locus. As an initial step, recombination events, defined by large extended kindreds, helped specifically to localize the BRCA2 locus as either distal or proximal to a specific genetic marker (Wooster et al, 1994).

.00 The region surrounding BRCA2, until the disclosure of the present invention, was not well 20 mapped and there were few markers. Therefore, short repetitive sequences were developed from cosmids, Pis, BACs and YACs, which physically mapto the region and were analyzed in order to develop new genetic markers. Novel STRs were found which were both polymorphic and which mapped to the BRCA2 region.

Physical Mapping Three distinct methods were employed to physically map the region. The first was the use of yeast artificial chromosomes (YACs) to clone the BRCA2 region. The second was the creation of a set of PI, BAC and cosmid clones which cover the region containing the BRCA2 locus.

Yeast Artificial Chromosomes (YACs). Once a sufficiently small region containing the S 30 BRCA2 locus was identified, physical isolation of the DNA in the region proceeded by identifying a set of overlapping YACs which covers the region. Useful YACs can be isolated from known libraries, such as the St. Louis and CEPH YAC libraries, which are widely distributed and contain approximately 50,000 YACs each. The YACs isolated were from these publicly accessible libraries and can be obtained from a number of sources including the Michigan Genome Center.

Clearly, others who had access to these YACs, without the disclosure of the present invention, would not have known the value of the specific YACs we selected since they would not have known which YACs were within, and which YACs outside of, the smallest region containing the BRCA2 locus.

P1 and BAC Clones. In the present invention, it is advantageous to proceed by obtaining P1 and BAC clones to cover this region. The smaller size' of these inserts, compared to YAC inserts, makes them more useful as specific hybridization probes. Furthermore, having the cloned DNA in bacterial cells, rather than in yeast cells, greatly increases the ease with which the DNA of interest can be manipulated, and improves the signal-to-noise ratio of hybridization assays.

P1 and BAC clones are obtained by screening libraries constructed from the total human genome with specific sequence tagged sites (STSs) derived from the YACs, Pls and BACs, isolated as described herein.

These P1 and BAC clones can be compared by interspersed repetitive sequence (IRS) PCR and/or restriction enzyme digests followed by gel electrophoresis and comparison of the resulting DNA fragments ("fingerprints") (Maniatis et al., 1982). The clones can also be characterized by the presence of STSs. The fingerprints are used to define an overlapping contiguous set of clones 20 which covers the region but is not excessively redundant, referred to herein as a "minimum tiling path". Such a minimum tiling path forms the basis for subsequent experiments to identify cDNAs which may originate from the BRCA2 locus.

P1 clones (Sternberg, 1990; Sternberg et al., 1990; Pierce et al., 1992; Shizuya et al., 1992) were isolated by Genome Sciences using PCR primers provided by us for screening. BACs were 25 provided by hybridization techniques in Dr. Mel Simon's laboratory and by analysis of PCR pools in our laboratory. The strategy of using P1 and BAC clones also permitted the covering of the genomic region with an independent set of clones not derived from YACs. This guards against the possibility of deletions in. YACs. These new sequences derived from the P1 and BAC clones provide the material for further screening for candidate genes, as described below.

Gene Isolation.

There are many techniques for testing genomic clones for the presence of sequences likely to be candidates for the coding sequence of a locus one is attempting to isolate, including but not limited to: zoo blots, identifying HTF islands, exon trapping, hybridizing cDNA to Pis, BAC or YACs and screening cDNA libraries.

Zoo blots. The first technique is to hybridize cosmids to Southern blots to identify DNA sequences which are evolutionarily conserved, and which therefore give positive hybridization signals with DNA from species of varying degrees of relationship to humans (such as monkey, cow, chicken, pig, mouse and rat). Southern blots containing such DNA from a variety of species are commercially available (Clonetech, Cat. 7753-1).

Identifying HTF islands. The second technique involves finding regions rich in the nucleotides C and G, which often occur near or within coding sequences. Such sequences are called HTF (HpaI tiny fragment) or CpG islands, as restriction enzymes specific for sites which contain CpG dimers cut frequently in these regions (Lindsay et al., 1987).

Exon trapping. The third technique is exon trapping, a method that identifies sequences in genomic DNA which contain splice junctions and therefore are likely to comprise coding sequences of genes. Exon amplification (Buckler et al., 1991) is used to select and amplify exons from DNA clones described above. Exon amplification is based on the selection of RNA sequences which are flanked by functional 5' and/or 3' splice sites. The products of the exon amplification are used to screen the breast cDNA libraries to identify a manageable number of candidate genes for further study. Exon trapping can also be performed on small segments of sequenced DNA using computer programs or by software trapping.

Hybridizing cDNA to Pls. BACs or YACs. The fourth technique is a modification of the selective enrichment technique which utilizes, hybridization of cDNA to cosmids, Pls, BACs or YACs and permits transcribed sequences to be identified in, and recovered from cloned genomic DNA (Kandpal et al., 1990). The selective enrichment technique, as modified for the present purpose, involves binding DNA from the region of BRCA2 present in a YAC to a column matrix and selecting cDNAs from the relevant libraries which hybridize with the bound DNA, followed by amplification and purification of the bound DNA, resulting in a great enrichment for cDNAs in 30 the region represented by the cloned genomic DNA.

3 0 the region represented by the cloned genomic DNA.

-14- Identification of cDNAs. The fifth technique is to identify cDNAs that correspond to the BRCA2 locus. Hybridization probes containing putative coding sequences, selected using any of the above techniques, are used to screen various libraries, including breast tissue cDNA libraries and any other necessary libraries.

Another variation on the theme of direct selection of cDNA can be used to find candidate genes for BRCA2 (Lovett et al., 1991; Futreal, 1993). This method uses cosmid, PI or BAC DNA as the probe. The probe DNA is digested with a blunt cutting restriction enzyme such as HaeIII.

Double stranded adapters are then ligated onto the DNA and serve as binding sites for primers in subsequent PCR amplification reactions using biotinylated primers. Target cDNA is generated from mRNA derived from tissue samples, breast tissue, by synthesis of either random primed or oligo(dT) primed first strand followed by second strand synthesis. The cDNA ends are rendered blunt and ligated onto double-stranded adapters. These adapters serve as amplification sites for PCR. The target and probe sequences are denatured and mixed with human Cot-I DNA to block repetitive sequences. Solution hybridization is carried out to high Cot-1/2 values to ensure hybridization of rare target cDNA molecules. The annealed material is then captured on avidin beads, washed at high stringency and the retained cDNAs are eluted and amplified by PCR. The selected cDNA is subjected to further rounds of enrichment before cloning into a plasmid vector for analysis.

20 Testing the cDNA for Candidacy Proof that the cDNA is the BRCA2 locus is obtained by finding sequences in DNA extracted from affected kindred members which create abnormal BRCA2 gene products or abnormal levels of BRCA2 gene product. Such BRCA2 susceptibility alleles will co-segregate with the disease in large kindreds. They will also be present at a much higher frequency in non-kindred individuals.

with breast cancer then in individuals in the general population. Finally, since tumors often mutate somatically at loci which are in other instances mutated in the germline, we expect to see normal germline BRCA2 alleles mutated into sequences which are identical or similar to BRCA2 susceptibility alleles in DNA extracted from tumor tissue. Whether one is comparing BRCA2 sequences from tumor tissue to BRCA2 alleles from the germline of the same individuals, or one is 30 comparing germline BRCA2 alleles from cancer cases to those from unaffected individuals, the key is to find mutations which are serious enough to cause obvious disruption to the normal Sfunction of the gene product. These mutations can take a number of forms. The most severe forms would be frame shift mutations or large deletions which would cause the gene to code for an abnormal protein or one which would significantly alter protein expression. Less severe disruptive mutations would include small in-frame deletions and nonconservative base pair substitutions which would have a significant effect on the protein produced, such as changes to or from a cysteine residue, from a basic to an acidic amino acid or vice versa, from a hydrophobic to hydrophilic amino acid or vice versa, or other mutations which would affect secondary, tertiary or quaternary protein structure. Silent' mutations or those resulting in conservative amino acid substitutions would not generally be expected to disrupt protein function.

According to the diagnostic and prognostic method of the present invention, alteration of the wild-type BRCA2 locus is detected. In addition, the method can be performed by detecting the wild-type BRCA2 locus and confirming the lack of a predisposition to cancer at the BRCA2 locus.

"Alteration of a wild-type gene" encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or of only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or' amino acid substitutions. Somatic mutations are those which occur only in certain tissues, in the tumor tissue, and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited. If only a single allele is somatically mutated, an early neoplastic state is indicated. However, if both alleles are somatically mutated, then a late neoplastic state is 20 indicated. The finding of BRCA2 mutations thus provides both diagnostic and prognostic information. A BRCA2 allele which is not deleted found on the sister chromosome to a chromosome carrying a BRCA2 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 BRCA2 gene product. However, mutations leading 25 to non-functional gene products would also lead to a cancerous state. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the BRCA2 gene product, or to a decrease in mRNA stability or translation efficiency.

30 Useful diagnostic techniques include, but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern, blot analysis, single stranded -16conformation analysis (SSCA), RNase protection assay, allele-specific oligonucleotide (ASO), dot blot analysis and PCR-SSCP, as discussed in detail further below.

Predisposition to cancers, such as breast cancer, and the other cancers identified herein, can be ascertained by testing any tissue of a human for mutations of the BRCA2 gene. For example, a person who has inherited a germline BRCA2 mutation would be prone to develop cancers. This can be determined by testing DNA from any tissue of the person's body. Most simply, blood can be drawn and DNA extracted from the cells of the blood. In addition, prenatal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic cells for mutations of the BRCA2 gene. Alteration of a wild-type BRCA2 allele, whether, for example, by point mutation or deletion, can be detected by any of the means discussed herein.

There are several methods that can be used to detect DNA sequence variation. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing can detect sequence variation. For a gene as large as BRCA2, manual sequencing is very labor-intensive, but under optimal conditions, mutations in the coding sequence of a gene are rarely missed. Another approach is the single-stranded conformation polymorphism assay (SSCA) (Orita et al., 1989).

This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCA makes it an attractive, viable alternative to direct sequencing for mutation detection on a research basis. The.

20 fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., 1991), heteroduplex analysis (HA) (White et al., 1992) and chemical mismatch cleavage (CMC) (Grompe et al., 1989). None of the methods described above will detect 25 large deletions, duplications or insertions, nor will they detect a regulatory mutation which affects transcription or translation of the protein. Other methods which might detect these classes of mutations such as a protein truncation assay or the asymmetric assay, detect only specific types of mutations and would not detect missense mutations. A review of currently available methods of detecting DNA sequence variation can be found in a recent review by Grompe (1993). Once a 30 mutation is known, an allele specific detection approach such as allele specific oligonucleotide -17- (ASO) hybridization can. be utilized to rapidly screen large numbers of other samples for that same mutation.

In order to detect the alteration of the wild-type BRCA2 gene in a tissue, it is helpful to isolate the tissue free from surrounding normal tissues. Means for enriching tissue preparation for tumor cells are known in the art. For example, the tissue. may be isolated from paraffin or cryostat sections. Cancer cells may also be separated from normal cells by flow cytometry. These techniques, as well as other techniques for separating tumor cells from normal cells, are well known in the art. If the tumor tissue is highly contaminated with normal cells, detection of mutations is more difficult.

A rapid preliminary analysis to detect polymorphisms in DNA sequences can be performed by looking at a series of Southern blots of DNA cut with one or more restriction enzymes, preferably with a large number of restriction enzymes. Each blot contains a series of normal individuals and a series of cancer cases, tumors, or both. Southern blots displaying hybridizing fragments (differing in length from control DNA when probed with sequences near or including the BRCA2 locus) indicate a possible mutation. If restriction enzymes which produce very large restriction fragments are used, then pulsed field gel electrophoresis (PFGE) is employed.

Detection of point mutations may be accomplished by molecular cloning of the BRCA2 allele(s) and sequencing the allele(s) using techniques well known in the art. Alternatively, the gene sequences can be amplified directly from a genomic DNA preparation from the tumor tissue, 20 using known techniques. The DNA sequence of the amplified sequences can then be determined.

There are six well known methods for a more complete, yet still indirect, test for confirming the presence of a susceptibility allele: 1) single stranded conformation analysis (SSCA) (Orita et al., 1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989); 3) RNase protection assays (Finkelstein et al, 1990; Kinszler et al., 1991); 4) allele-specific oligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteins which recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, 1991); and 6) allele-specific PCR (Rano Kidd, 1989). For allele-specific PCR, primers are used which hybridize at their 3' ends to a particular BRCA2 mutation. If the particular BRCA2 mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Application Publication No. 0332435 and in Newton et al., 1989.

Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism (RFLP) probes for the gene or surrounding marker genes can be used to score alteration of an allele or an insertion in a polymorphic fragment.

Such a method is particularly useful for screening relatives of an affected individual for the presence of the BRCA2 mutation found in that individual. Other techniques for detecting insertions and deletions as known in the art can be used.

In the first three methods (SSCA, DGGE and RNase protection assay), a new electrophoretic band appears. SSCA detects a band which migrates differentially because the sequence change causes a difference in single-strand, intramolecular base pairing. RNase protection involves cleavage of the mutant polynucleotide into two or more smaller fragments. DGGE detects differences in migration rates of mutant sequences compared to wild-type sequences, using a denaturing gradient gel. In an allele-specific oligonucleotide assay, an oligonucleotide is designed which detects a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal. In the mutS assay, the protein binds only to sequences that contain a nucleotide mismatch in a heteroduplex between mutant and wild-type sequences.

Mismatches, according to the present invention, are hybridized nucleic acid duplexes in which the two strands are not 100% complementary. Lack of total homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in the gene or in its mRNA product. While these techniques are less sensitive than sequencing, they are simpler to perform on a large number of tumor samples. An example of a 20 mismatch cleavage technique is the RNase protection method. In the practice of the present 4e*4e@ invention, the method involves the use of a labeled riboprobe which is complementary to the human wild-type BRCA2 gene coding sequence. The riboprobe and either mRNA or DNA isolated from the tumor tissue are annealed (hybridized) together and subsequently digested with the enzyme RNase A which is able to detect some mismatches in a duplex RNA structure. If a mismatch is detected by RNase A, it cleaves at the site of the mismatch. Thus, when the annealed RNA preparation is separated on an electrophoretic gel matrix, if a mismatch has been detected and cleaved by RNase A, an RNA product will be seen which is smaller than the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the full length of the BRCA2 mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment 30 of the BRCA2 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 mismatches, through enzymatic or chemical cleavage. See, Cotton et 1988; Shenk et al., 1975; Novack et al., 1986.

Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, Cariello, 1988. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the BRCA2 gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.

DNA sequences, of the BRCA2 gene which have been amplified by use of PCR may also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the BRCA2 gene sequence harboring a known mutation. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the BRCA2 gene sequence. By use of a battery of such allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in the BRCA2 gene.

Hybridization of allele-specific probes with amplified BRCA2 sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same -mutation in the tumor tissue as in the allele-specific .probe.

S The most definitive test for mutations in a candidate locus is to directly compare genomic 20 BRCA2 sequences from cancer patients with those from a control population. Alternatively, one could sequence messenger RNA after amplification, by PCR, thereby eliminating the necessity of determining the exon structure of the candidate gene.

Mutations from cancer patients falling outside the coding region of BRCA2 can be detected by examining the non-coding regions, such as introns and regulatory sequences near or within the 25 BRCA2 gene. An early indication that mutations in noncoding regions are important may come from Northern blot experiments that reveal messenger RNA molecules of abnormal size or abundance in cancer patients as compared to control individuals.

Alteration of BRCA2 mRNA expression can be detected by any techniques known in the art.

These include Northern blot analysis, PCR amplification and RNase protection. Diminished 30 mRNA expression indicates an alteration of the wild-type BRCA2 gene. Alteration of wild-type BRC2 genes can also be detected by screening for alteration of wild-type BRCA2 protein. For BRCA2 genes can also be detected by screening for alteration of wild-type BRCA2 protein. For example, monoclonal antibodies immunoreactive with BRCA2 can be used to screen a tissue. Lack of cognate antigen would indicate a BRCA2 mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant BRCA2 gene product. Such immunological assays can be done in any convenient formats known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means for detecting an altered BRCA2 protein can be used to detect alteration of wild-type BRCA2 genes. Functional assays, such as protein binding determinations, can be used. In addition, assays can be used which detect BRCA2 biochemical function. Finding a mutant BRCA2 gene product indicates alteration of a wild-type BRCA2 gene.

Mutant BRCA2 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 BRCA2 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 BRCA2 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 BRCA2 genes or gene products.

The methods of diagnosis of the present invention are applicable to any tumor in which BRCA2 has a role in tumorigenesis. The diagnostic method of the present invention is useful for 20 clinicians, so they can decide upon an appropriate course of treatment.

The primer pairs of the present invention are useful for determination of the nucleotide sequence of a particular BRCA2 allele using PCR. The pairs of single-stranded DNA primers can be annealed to sequences within or surrounding the BRCA2 gene on chromosome 13 in order to prime amplifying DNA synthesis of the BRCA2 gene itself. A complete set of these primers 25 allows synthesis of all of the nucleotides of the BRCA2 gene coding sequences, the exons.

The set of primers preferably allows synthesis of both intron and exon sequences. Allele-specific primers can also be used. Such primers anneal only to particular BRCA2 mutant alleles, and thus s will only amplify a product in the presence of the mutant allele as a template.

In order to facilitate subsequent cloning of amplified sequences, primers may have restriction 30 enzyme site sequences appended to their 5' ends. Thus, all nucleotides of the primers are derived from BRCA2 sequences or sequences adjacent to BRCA2, except for the few nucleotides necessary to form a restriction enzyme site. Such enzymes and sites are well known in the art. The primers themselves can be synthesized using techniques which are well known in the art.

Generally, the primers can be made using oligonucleotide synthesizing machines which are commercially available. Given the sequence of the BRCA2 open reading frame shown in SEQ ID NO:1 and in Figure 3, design of particular primers, in addition to those disclosed below, 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 Southem hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above. The probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the BRCA2 gene or mRNA using other techniques.

It has been discovered that individuals with the wild-type BRCA2 gene do not have cancer which results from the BRCA2 allele. However, mutations which interfere with the function of the BRCA2 protein are involved in the pathogenesis of cancer. Thus, the presence of an altered (or a mutant) BRCA2 gene which produces a protein having a loss of function, or altered function, directly correlates to an increased risk of cancer. In order to detect a BRCA2 gene mutation, a biological sample is prepared and analyzed for a difference between the sequence of the BRCA2 allele being analyzed and the sequence of the wild-type BRCA2 allele. Mutant BRCA2 alleles can be initially identified by any of the techniques described above. The mutant alleles are then 20 sequenced to identify the specific mutation of the particular mutant allele. Alternatively; mutant BRCA2 alleles can be initially identified by identifying mutant (altered) BRCA2 proteins, using conventional techniques. The mutant alleles are then sequenced to identify the specific mutation for each allele. The mutations, especially those which lead to an altered function of the BRCA2 protein, are then used for the diagnostic and prognostic methods of the present invention.

Definitions The present invention employs the following definitions: "Amplification ofPolynucleotides" utilizes methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on 3 0 the use of Q-beta replicase. These methods are well known and widely practiced in the art. See, U.S. Patents 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al., 1989a -22- (for LCR). Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from the .BRCA2 region are preferably complementary to, and hybridize specifically to sequences in the BRCA2 region or in regions that flank a target region therein.

BRCA2 sequences generated by amplification may be sequenced directly. Alternatively, but less desirably, the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf, 1986.

"Analyte polynucleotide" and "analyte strand" refer to a single- or double-stranded polynucleotide which is suspected of containing a target sequence, and which may be present in a variety of types of samples, including biological samples.

"Antibodies." The present invention also provides polyclonal and/or monoclonal antibodies and fragments thereof, and immunologic binding equivalents thereof, which are capable of specifically binding to the BRCA2 polypeptides and fragments thereof or to polynucleotide sequences from the BRCA2 region, particularly from the BRCA2 locus or a portion thereof. The term "antibody" is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Polypeptides may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule keyhole limpet hemocyanin) and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity to the BRCA2 polypeptide or fragment. Monoclonal antibodies may be made by 20 injecting mice with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with BRCA2 polypeptide or fragments thereof. See, Harlow Lane, 1988. These antibodies will be useful in assays as well as pharmaceuticals.

Once a sufficient quantity of desired polypeptide has been obtained, it may be used for various purposes. A typical use is the production of antibodies specific for binding. These o antibodies may be either polyclonal or monoclonal, and may be produced by in vitro or in vivo techniques well known in the art. For production of polyclonal antibodies, an appropriate target immune system, typically mouse or rabbit, is selected. Substantially purified antigen is presented to the immune system in a fashion determined by methods appropriate for the animal and by other parameters well known to immunologists. Typical sites for injection are in footpads, intramuscularly, intraperitoneally, or intradermally. Of course, other species may be substituted for mouse or rabbit. Polyclonal antibodies are then purified using techniques known in the art, adjusted for the desired specificity.

An immunological response is usually assayed with an immunoassay. Normally, such immunoassays involve some purification of a source of antigen, for example, that produced by the same cells and in the same fashion as the antigen. A variety of immunoassay methods are well known in the art. See, Harlow Lane, 1988, or Goding, 1986.

Monoclonal antibodies with affinities of 10' 8 M' or preferably 10' 9 to 10- 1 0 Mf or stronger will typically be made by standard procedures as described, in Harlow Lane, 1988 or Goding, 1986. Briefly, appropriate animals will be selected and the desired immunization protocol followed. After the appropriate period of time, the spleens of such animals are excised and individual spleen cells fused, typically, to immortalized myeloma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supematants of each clone tested for their production of an appropriate antibody specific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides, or alternatively, to selection of libraries of antibodies in phage or similar vectors. See Huse et al., 1989. The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance whichprovides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and S" 20 patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Patents'3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be produced (see U.S. Patent 4,816,567).

"Binding partner" refers to a molecule capable of binding a ligand molecule with high specificity, as for example, an antigen and an antigen-specific antibody or an enzyme and its inhibitor. In general, the specific binding partners must bind with sufficient affinity to immobilize the analyte copy/complementary strand duplex (in the case of polynucleotide hybridization) under the isolation conditions. Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, the numerous, known receptor-ligand couples, and complementary polynucleotide strands. In the case of complementary polynucleotide binding s partners, the partners are normally at least about 15 bases in length, and may be at least 40 bases in length. The polynucleotides may be composed of DNA, RNA, or synthetic nucleotide analogs.

A "biological sample" refers to a sample of tissue or fluid suspected of containing an analyte polynucleotide or polypeptide from an individual including, but not limited to, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, blood cells, tumors, organs, tissue and samples of in vitro cell culture constituents.

As used herein, the terms "diagnosing" or "prognosing," as used in the context of neoplasia, are used to indicate 1) the classification of lesions as neoplasia, 2) the determination of the severity of the neoplasia, or 3) the monitoring of the disease progression, prior to, during and after treatment.

"Encode". A polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

"Isolated" or "substantially pure". An "isolated" or "substantially pure" nucleic acid an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components which naturally accompany a native human sequence or protein, ribosomes, 20 polymerases, many other human genome sequences and proteins. The term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs *ooo** biologically synthesized by heterologous systems.

"BRCA2 Allele" refers to normal alleles of the BRCA2 locus as well as alleles carrying variations that predispose individuals to develop cancer of many sites including, for example, breast, ovarian and stomach cancer. Such predisposing alleles are also called "BRCA2 susceptibility alleles".

"BRCA2 Locus," "BRCA2 Gene," "BRCA2 Nucleic Acids" or ".BRCA2 Polynucleotide" each refer to polynucleotides, all of which are in the BRCA2 region, that are likely to be expressed in normal tissue, certain alleles of which predispose an individual to develop breast, ovarian and stomach cancers. Mutations at the BRCA2 locus may be involved in the initiation and/or progression of other types of tumors. The locus is indicated in part by mutations that predispose individuals to develop cancer. These mutations fall within the BRCA2 region described infra. The BRCA2 locus is intended to include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation. The BRCA2 locus is intended to include all allelic variations of the DNA sequence.

These terms, when applied to a nucleic acid, refer to a nucleic acid which encodes a BRCA2 polypeptide, fragment, homolog or variant, including, protein fusions or deletions. The nucleic acids of the present invention will possess a sequence which is either derived from, or substantially similar to a natural BRCA2-encoding gene or one having substantial homology with a natural BRCA2-encoding gene or a portion thereof. The coding sequence for a BRCA2 polypeptide is shown in SEQ ID NO:1 and Figure 3, with the amino acid sequence sh'own in SEQ ID NO:2.

The polynucleotide compositions of this invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages phosphorothioates, phosphorodithioates, etc.), pendent moieties polypeptides), intercalators acridine, psoralen, etc.), chelators, alkylators, and modified linkages alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

SThe -present invention provides recombinant nucleic acids comprising all or part of the BRCA2 region. The recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosomal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin which, by virtue of its origin or manipulation, 1) is not -26associated with all or a portion of a polynucleotide with which it is associated in nature; 2) is linked to a polynucleotide other than that to which it is linked in nature; or 3) does not occur in nature.

Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by this invention. Although the wild-type sequence may be employed, it will often be altered, by deletion, substitution or insertion.

cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, by PCR. The choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.

The DNA sequences used in this invention will usually comprise at least about five codons nucleotides), more usually at least about 7-15 codons, and most preferably, at least about codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a BRCA2encoding sequence.

Techniques for nucleic acid manipulation are described generally, for example, in Sambrook et al., 1989 or Ausubel et al., 1992. Reagents useful in applying such techniques, such as 20 restriction enzymes and the like, are widely known in the art and commercially available from such vendors as New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U. S.

Biochemicals, New England Nuclear, and a number of other sources. The recombinant nucleic acid sequences used to produce fusion proteins of the present invention may be derived from natural or synthetic sequences. Many natural gene sequences are obtainable from various cDNA or from genomic libraries using appropriate probes. See, GenBank, National Institutes of Health.

"BRCA2 Region" refers to a portion of human chromosome 13 bounded by the markers tdj3820 and YS-G-B10T. This region contains the BRCA2 locusincluding the BRCA2 gene.

As used herein, the terms "BRCA2 locus," "BRCA2 allele" and "BRCA2 region" all refer to the double-stranded DNA comprising the locus, allele, or region, as well as either of the 30 single-stranded DNAs comprising the locus, allele or region.

As used herein, a "portion" of the BRCA2 locus or region or allele is defined as having a minimal size of at least about eight nucleotides, or preferably about 15 nucleotides, or more preferably at least about 25 nucleotides, and may have a minimal size of at least about nucleotides.

"BRCA2 protein" or "BRCA2 polypeptide" refer to a protein or polypeptide encoded by the BRCA2 locus, variants or fragments thereof. The term "polypeptide" refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term als6 does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages as well as other modifications known in the art, both naturally and non-naturally occurring. Ordinarily, such polypeptides will be at least about homologous to the native BRCA2 sequence, preferably in excess of about 90%, and more preferably at least about 95% homologous. Also included are proteins encoded by DNA which hybridize under high or low stringency conditions, to BRCA2-encoding nucleic acids and closely related polypeptides or proteins retrieved by antisera to the BRCA2 protein(s).

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically 20 at least about 28 residues, and preferably more than about 35 residues.

"Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.

"Probes". Polynucleotide polymorphisms associated with BRCA2 alleles which predispose to certain cancers or are associated with most cancers are detected by hybridization with a polynucleotide probe which forms a stable hybrid with that of the target sequence, under stringent to moderately stringent hybridization and wash conditions. If it is expected that the probes will be perfectly complementary to the target sequence, stringent conditions will be used. Hybridization stringency may be lessened if some mismatching is expected, for example, if variants are expected with the result that the probe will not be completely complementary. Conditions are chosen which rule out nonspecific/adventitious bindings, that is, which minimize noise. Since such indications -28identify neutral DNA polymorphisms as well as mutations, these indications need further analysis to demonstrate detection of a BRCA2 susceptibility allele.

Probes for BRCA2 alleles may be derived from the sequences of the BRCA2 region or its cDNAs. The probes may be of any suitable length, which span all or a portion of the BRCA2 region, and which allow specific hybridization to the BRCA2 region. If the target sequence contains a sequence identical to that of the probe, the probes may be short, in the range of about 8-30 base pairs, since the hybrid will be relatively stable under even stringent conditions.. If some degree of mismatch is expected with the probe, if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridizes to the target sequence with the requisite specificity.

The probes will include an isolated polynucleotide attached to a label or reporter molecule and may be used to isolate other polynucleotide sequences, having sequence similarity by standard methods. For techniques for preparing and labeling probes see, Sambrook et al., 1989 or Ausubel et al., 1992. Other similar polynucleotides may be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by use of the redundancy in the genetic code. Various codon substitutions may be introduced, by silent changes (thereby producing various restriction sites) or to optimize expression for a particular system. Mutations may be introduced to modify the properties of the polypeptide, perhaps to change ligand-binding affinities, interchain affinities, or the polypeptide degradation or turnover rate.

Probes comprising synthetic oligonucleotides or other polynucleotides of the present invention may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labeled by nick translation, Klenow fill-in reaction, or other methods known in the art.

Portions of the polynucleotide sequence having at least about eight nucleotides, usually at least about 15 nucleotides, and fewer than about 6 kb, usually fewer than about 1.0 kb, from a polynucleotide sequence encoding BRCA2 are preferred as probes. The probes may also be used to determine whether mRNA encoding BRCA2 is present in a cell or tissue.

"Protein modifications or fragments" are provided by the present invention for BRCA2 polypeptides or fragments thereof which are substantially homologous to primary structural sequence but which include, in vivo or in vitro chemical and biochemical modifications or which incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art.

A variety.of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as 32 P, ligands which bind to labeled antiligands antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements; and available instrumentation. Methods of labeling polypeptides are well known in the art.. See, Sambrook et al., 1989 or Ausubel et al, 1992.

Besides substantially full-length polypeptides, the present invention provides for biologically active fragments of the polypeptides. Significant biological activities include ligand-binding, immunological activity and other biological activities characteristic of BRCA2 polypeptides.

Immunological activities include both immunogenic function in a target immune system, as well as sharing of immunological epitopes for binding, serving as either a competitor or substitute antigen for an epitope of the BRCA2 protein. As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope could comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods of determining the spatial 20 conformation of such amino acids are known in the art.

For immunological purposes, tandem-repeat polypeptide segments may be used as immunogens, thereby producing highly antigenic proteins. Alternatively; such polypeptides will serve as highly efficient competitors for specific binding. .Production of antibodies specific for BRCA2 polypeptides or fragments thereof is described below.

The present invention also provides for fusion polypeptides, comprising BRCA2 polypeptides and fragments. Homologous polypeptides may be fusions between two or more BRCA2 polypeptide sequences or between the sequences of BRCA2 and a related protein.

Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. For example, ligand-binding or other domains may be "swapped" between different new.fusion polypeptides or fragments. Such homologous or heterologous fusion polypeptides may display, for example, altered strength or specificity of binding. Fusion partners include.immunoglobulins, bacterial p-galactosidase, trpE, protein A, 3lactamase, alpha amylase, alcohol dehydrogenase and yeast alpha mating factor. See, e.g., Godowski et al., 1988.

Fusion proteins will typically be made by either recombinant nucleic acid methods, as described below, or may be chemically synthesized. Techniques for the synthesis of polypeptides are described, for example, in Merrifield, 1963.

"Protein purification" refers to various methods for the isolation of the BRCA2 polypeptides from other biological material, such as from cells transformed with recombinant nucleic acids encoding BRCA2, and are well known in the art. For example, such polypeptides may be purified by immunoaffinity chromatography employing, the antibodies provided by the present invention. Various methods of protein purification are well known in the art, and include those described in Deutscher, 1990 and Scopes, 1982.

The terms "isolated", "substantially pure", and "substantially homogeneous" are used interchangeably to describe a protein or polypeptide which has been separated from components which accompany it in its natural state. A monomeric protein is substantially pure when at least about 60 to 75% of a sample exhibits a.single polypeptide sequence. A substantially pure protein will typically comprise about 60 to 90% w/w of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein 20 sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art which are utilized for purification.

A BRCA2 protein is substantially free of naturally associated components when it is separated from the native contaminants which accompany it in its natural state. Thus, a polypeptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.

O'S A polypeptide produced as an expression product of an isolated and manipulated genetic sequence is an "isolated polypeptide," as used herein, even if expressed in a homologous cell type.

Synthetically made forms or molecules expressed by heterologous cells are inherently isolated molecules.

"Recombinant nucleic acid" is a nucleic acid which is not naturally occurring, or which is made by the artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated.segments of nucleic acids, by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.

"Regulatory sequences" refers to those sequences normally within 100 kb of the coding region of a locus, but they may also be more distant from the coding region, which affect the expression of the gene (including transcription of the gene, and translation, splicing, stability or the like of the messenger RNA).

"Substantial homology or similarity". A nucleic acid or fragment thereof is "substantially :i homologous" ("or substantially similar") to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about more usually at least about 80%, preferably at least about 90%, and more preferably at least .0 about 95-98% of the nucleotide bases.

Alternatively, substantial homology or (similarity) exists when a nucleic acid or fragment oooo thereof will hybridize to another nucleic acid (or a complementary strand thereof)' under selective hybridization conditions, to a strand, or to its complement. Selectivity of hybridization exists when hybridization which is substantially more selective than total lack of specificity occurs. Typically, 25 selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa, 1984. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

-32- Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions will generally include temperatures in excess of 30 0 C, typically in excess of 37 0 C, and preferably in excess of 45 0 C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, Wetmur Davidson, 1968.

Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes. The preparation of such probes and suitable hybridization conditions are well known in the art.

The terms "substantial homology" or "substantial identity", when referring to polypeptides, indicate that the polypeptide or protein in question exhibits at least about identity with an entire. naturally-occurring protein or a portion thereof, usually at least about identity, and preferably at least about 95% identity.

"Substantially similar function" refers to the function of a modified nucleic acid or a modified protein, with reference to the wild-type BRCA2 nucleic acid or wild-type BRCA2 polypeptide. The modified polypeptide will be substantially homologous to the wild-type BRCA2 polypeptide and will have substantially the same function. The modified polypeptide may have an 20 altered amino acid sequence and/or may contain modified amino acids. In addition to the similarity of function, the modified polypeptide may have other useful properties, such as a longer half-life. The similarity of function (activity) of the modified polypeptide may be substantially the same as the activity of the wild-type BRCA2 polypeptide. Alternatively, the similarity of function (activity) of the modified polypeptide may be higher than the activity of the wild-type BRCA2 25 polypeptide. The modified polypeptide is synthesized using conventional techniques, or is encoded by a modified nucleic acid and produced using conventional techniques. The modified nucleic acid is prepared by conventional techniques. A nucleic acid with a function substantially similar to the wild-type BRCA2 gene function produces the modified protein described above.

Homology, for polypeptides, is typically measured using sequence analysis software. See, 3 0 the Sequence Analysis Software Package of the Genetics Computer Group; University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

A polypeptide "fragment," "portion" or "segment" is a stretch of amino acid residues of at least about five to seven contiguous amino acids, often at least about seven to nine contiguous amino acids, .typically at least about nine to 13 contiguous amino acids and, most preferably, at least about 20 to 30 or more contiguous amino acids.

The polypeptides of the present invention, if soluble, may be coupled to a solid-phase support, nitrocellulose, nylon, column packing materials Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates. Such supports may take the form, for example, of beads, wells, dipsticks, or membranes.

"Target region" refers to a region of the nucleic acid which is amplified and/or detected.

The term "target sequence" refers toa sequence with which a probe or primer will form a stable hybrid under desired conditions.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, and immunology. See, Maniatis et al., 1982; Sambrook et al, 1989; Ausubel et al., 1992; Glover, 20 1985; Anand, 1992; Guthrie Fink, 1991. A general discussion of techniques and materials for human gene mapping, including mapping of human chromosome 13, is provided, in White and Lalouel, 1988.

Preparation of recombinant or chemically synthesized 25 nucleic acids: vectors, transformation, host cells Large amounts of the polynucleotides of the present invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments coding for a desired fragment will be incorporated into recombinant polynucleotide constructs, usually DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the polynucleotide constructs will be suitable for replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to (with and without integration within the genome) cultured mammalian or plant or other eukaryotic cell lines. The purification of nucleic acids produced by the methods of the present invention is described, in Sambrook et al., 1989 or Ausubel et al., 1992.

The polynucleotides of the present invention may also be produced by chemical synthesis, by the phosphoramidite method described by Beaucage Carruthers, 1981 or the triester method according to Matteucci and Caruthers, 1981, and may be performed on commercial, automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single-stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strands together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Polynucleotide constructs prepared for introduction into a prokaryotic or eukaryotic host may comprise a replication system recognized by the host, including the intended polynucleotide fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment.

Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Secretion signals may also be included where appropriate, whether from a native BRCA2 protein or from other 20 receptors or from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, and thus attain its functional topology, or be secreted from the cell. Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al., 1989 or Ausubel et al. 1992.

An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host, and may include, when appropriate, those naturally associated with BRCA2 genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or Ausubel et al., 1992; see also, Metzger et al., 1988. Many useful vectors are known in the art and may be obtained from such vendors as Stratagene, New Ergland BioLabs. Promega Biotech, and others. Promoters such, as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., EP 73,675A. Appropriate nonnative mammalian promoters might include the early and late promoters from SV40 (Fiers et al., 1978) or promoters derived from murine Moloney leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. In addition, the construct may be joined to an amplifiable gene DHFR) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences, see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York (1983).

While such expression vectors may replicate autonomously, they may also replicate by being inserted into the genome of the host cell, by methods well known in the art.

Expression and cloning vectors will likely contain a selectable marker, a gene encoding a protein necessary for survival or growth of a host cell transformed with the vector. The presence of this gene ensures growth of only those host cells which express the inserts. Typical selection genes encode proteins that a) confer resistance to antibiotics or other toxic substances, e.g. ampicillin, neomycin, methotrexate, etc.; b) complement auxotrophic deficiencies, or c) supply critical nutrients not available from complex media, the gene encoding D-alanine racemase for Bacilli. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, by injection (see, Kubo et al., 1988), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other 25 substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome); and other methods. See generally, Sambrook et al., 1989 and Ausubel et al., 1992. The introduction of the polynucleotides into the host cell by any method *known in the art, including, inter alia, those described above, will be referred to herein as "transformation." The cells into which have been introduced nucleic acids described above are .meant to also include the progeny of such cells.

Large quantities of the nucleic acids and polypeptides of the present invention may be prepared by expressing the BRCA2 nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells. The most commonly used prokaryotic hosts are strains of Escherichia coli,. although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, or amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. See, Jakoby and Pastan, 1979. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, to provide higher expression, desirable glycosylation patterns, or other features.

Clones are selected by using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule, preferably the same DNA molecule. In prokaryotic hosts, the transformant may be selected, by resistance to ampicillin, tetracycline or other antibiotics. Production of a particular product based on temperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present 20 invention, but also, for example, in studying the characteristics of BRCA2 polypeptides.

Antisense polynucleotide sequences are useful in preventing or diminishing the expression of the BRCA2 locus, as will be appreciated by those skilled in the art. For example, polynucleotide vectors containing all or a portion of the BRCA2 locus or other sequences from the BRCA2 region (particularly those flanking the BRCA2 locus) may be placed under the control of a promoter in an 25 antisense orientation and introduced into a cell. Expression of such an antisense construct within a cell will interfere with BRCA2 transcription and/or translation and/or replication.

The probes and primers based on the BRCA2 gene sequences disclosed herein are used to e. identify homologous BRCA2 gene sequences and proteins in other species. These BRCA2. gene sequences and proteins are used in the diagnostic/prognostic, therapeutic and drug screening methods described herein for the species from which they have been isolated.

Methods of Use: Nucleic Acid Diagnosis and Diagnostic Kits In order to detect the presence of a BRCA2 allele predisposing an individual to cancer, a biological sample such as blood is prepared and analyzed for the presence or absence of susceptibility alleles of BRCA2. In order to detect the presence of neoplasia, the progression toward malignancy of a precursor lesion, or as a prognostic indicator, a biological sample of the lesion is prepared and analyzed for the presence or absence of mutant alleles of BRCA2. Results of these tests and interpretive information are returned to the health care provider for communication to the tested individual. Such diagnoses may be performed by diagnostic laboratories, or, alternatively, diagnostic kits are manufactured and sold to health care providers or to private individuals for self-diagnosis.

Initially, the screening method involves amplification of the relevant BRCA2 sequences. In another preferred embodiment of the invention, the screening method involves a non-PCR based strategy. Such screening methods include two-step label amplification methodologies that are well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.

i The most popular method used today is target amplification. Here, the target nucleic acid sequence is amplified with polymerases. One particularly preferred method using polymerasedriven amplification is the polymerase chain reaction (PCR). The polymerase chain reaction and other polymerase-driven amplification assays can achieve over a million-fold increase in copy 20 number through the use of polymerase-driven amplification cycles. Once amplified, the resulting nucleic acid can be sequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the. target sequences (for example, in screening for cancer susceptibility), the biological sample to be analyzed, such as blood or serum, may be treated, if desired, to extract the nucleic acids. The sample nucleic acid may be prepared in various ways to facilitate detection of the target sequence; e.g. denaturation, restriction digestion, electrophoresis or dot blotting. The targeted region of the analyte nucleic acid usually must be at least partially single-stranded to form hybrids with the targeting sequence of the probe. If the sequence is naturally single-stranded, denaturation will not be required. However, if the sequence is double-stranded, the sequence will probably need to be denatured. Denaturation can be carried out by various techniques known in the art.

Analyte nucleic acid and probe are incubated under conditions which promote stable hybrid formation of the target sequence in the probe with the putative targeted sequence in the analyte.

The region of the probes which is used to bind to the analyte can be made completely complementary to the targeted region of human chromosome 13. Therefore, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide. These factors are outlined in, for example, Maniatis et al., 1982 and Sambrook et al., 1989. Under certain circumstances, the formation of higher order hybrids, such as triplexes, quadraplexes, etc., may be desired to provide the means of detecting target sequences.

Detection, if any, of the resulting hybrid is usually accomplished by the use of labeled probes. Alternatively, the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly. Suitable labels, and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which may be *.incorporated by known methods nick translation, random priming or kinasing), biotin, fluorescent groups, chemiluminescent groups dioxetanes, particularly triggered dioxetanes), enzymes, antibodies and the like. Variations of this basic scheme are known in the art, and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labeled moiety. A number of these variations are reviewed S" in, Matthews Kricka, 1988; Landegren et al., 1988; Mittlin, 1989; U.S. Patent 4,868,105, and in EPO Publication No. 225,807.

As noted above, non-PCR based screening assays are also contemplated in this invention.

An exemplary non-PCR based procedure is provided in Example 6. This procedure hybridizes a nucleic acid probe (or an analog such as a methyl phosphonate backbone replacing the normal phosphodiester), to the low level DNA target. This probe may have an enzyme covalently linked to the probe, such that the covalent linkage does not interfere with the specificity of the hybridization. This enzyme-probe-conjugate-target nucleic acid complex can then be isolated away from the free probe enzyme conjugate and a substrate is added for enzyme detection.

Enzymatic activity is observed as a change in color development or luminescent output resulting in a 10 -106 increase in sensitivity. For an example relating to preparation of oligodeoxynucleotidealkaline phosphatase conjugates and their use as hybridization probes, see Jablonski et al., 1986.

Two-step label amplification methodologies are known in the art. These assays work on the principle that a small ligand (such as digoxigenin, biotin, or the like) is attached to a nucleic acid probe capable of specifically binding BRCA2. Exemplary probes can be developed on the basis of the sequence set forth in SEQ ID NO: I and Figure 3 of this patent application. Allele-specific probes are also contemplated within the scope of this example, and exemplary allele specific probes include probes encompassing the predisposing mutations described below, including those described in Table 2.

In one example; the small ligand attached to the nucleic acid probe is specifically recognized by an antibody-enzyme conjugate. In one embodiment of this example, digoxigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate which turns over a chemiluminescent substrate. For methods for labeling nucleic acid probes according to this embodiment see Martin et al., 1990. In a second example, the small ligand is recognized by a second ligand-enzyme conjugate that is capable of specifically complexing to the first ligand. A well known embodiment of this example is the biotin-avidin type of interactions.

For methods for labeling nucleic acid probes and their use in biotin-avidin based assays see Rigby et al., 1977 and Nguyen et 1992.

It is also contemplated within the scope of this invention that the nucleic acid probe assays of 20 this invention will employ a cocktail of nucleic acid probes capable of detecting BRCA2. Thus, in one example to detect the presence of BRCA2 in a cell sample, more than one probe complementary to BRCA2 is employed and in particular the number of different probes is alternatively 2, 3, or 5 different nucleic acid probe sequences. In another example, to detect the presence of mutations in the BRCA2 gene sequence in a patient, more than one probe S 25 complementary to BRCA2 is employed where the cocktail includes probes capable of binding to the allele-specific mutations identified in populations of patients with alterations in BRCA2. In this embodiment, any number of probes can be used, and will preferably include probes .*.,corresponding to the major gene mutations identified as predisposing an individual to breast cancer. Some candidate probes contemplated within the scope of the invention include probes that include the allele-specific mutations described below and those that have the BRCA2 regions shown in SEQ ID NO: 1 and Figure 3, both 5' and 3' to the mutation site.

Methods of Use: Peptide Diagnosis and Diagnostic Kits The neoplastic condition of lesions can also be detected on the basis of the alteration of wildtype BRCA2 polypeptide. Such alterations can be determined by sequence analysis in accordance with conventional techniques. More preferably, antibodies (polyclonal or monoclonal) are used to detect differences in, or the absence of BRCA2 peptides. The antibodies may be prepared as discussed above under the heading "Antibodies" and as further shown in Examples 9 and Other techniques for raising and purifying antibodies are well known in the art and any such techniques may be chosen to achieve the preparations claimed in this invention. In a preferred embodiment of the invention; antibodies will immunoprecipitate BRCA2 proteins from solution as well as react with BRCA2 protein on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, antibodies will detect BRCA2 proteins in paraffin or frozen tissue sections, using immunocytochemical techniques.

Preferred embodiments relating to methods for detecting BRCA2 or its mutations include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using S' monoclonal and/or polyclonal antibodies. Exemplary sandwich assays are described by David et al. in U.S. Patent Nos. 4,376,110 and 4,486,530, hereby incorporated by reference, and exemplified in Example 9.

20 Methods of Use: Drug Screening This invention is particularly useful for screening compounds by using the BRCA2 polypeptide or binding fragment thereof in any of a variety of drug screening techniques.

The BRCA2 polypeptide or fragment employed in such a test may either be free in solution, affixed to a solid support, or borne on a cell surface. One method of drug screening utilizes eucaryotic or procaryotic host cells which are stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in competitive binding assays. Such cells, either in viable or fixed form, can be used for standard binding assays. One may measure, for example, for the formation of complexes between a BRCA2 polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a BRCA2 polypeptide or fragment and a known ligand is interfered with by the agent being tested.

Thus, the present invention provides methods of screening for drugs comprising contacting such an agent with a BRCA2 polypeptide or fragment thereof and assaying for the presence of a complex between the agent and the BRCA2 polypeptide or fragment, or (ii) for the presence of a complex between the BRCA2 polypeptide or fragment and a ligand, by methods well known in the art. In such competitive binding assays the BRCA2 polypeptide or fragment is typically labeled.

Free BRCA2 polypeptide or fragment is separated from that present in a protein:protein complex, and the amount of free uncomplexed) label is a measure of the binding of the agent being tested to BRCA2 or its interference with BRCA2:ligand binding, respectively.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the BRCA2 polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on September 13, 1984. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with BRCA2 polypeptide and washed. Bound BRCA2 polypeptide is then detected by methods well known in the art. Purified BRCA2 can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to a a capture antibodies to immobilize the BRCA2 polypeptide on the solid phase.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of specifically binding the BRCA2 polypeptide compete with a test S 20 compound for binding to the BRCA2 polypeptide or fragments thereof. In this manner, the antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants of the BRCA2 polypeptide.

A further technique.for drug screening involves the use of host eukaryotic cell lines or cells (such as described above) which have a nonfunctional BRCA2 gene. These host cell lines or cells 25 are defective at the BRCA2 polypeptide level. The host cell lines or cells are grown in the presence of drug compound. The rate of growth of the host cells is measured to determine if the compound is capable of regulating the growth of BRCA2 defective, cells..

a a *Methods of Use Rational Drug Design The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, enhance or interfere with the function of a polypeptide in vivo. See, Hodgson, 1991. In one approach, one first determines the three-dimensional structure of a protein of interest BRCA2 polypeptide) or, for example, of the BRCA2-receptor or ligand complex, by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins: An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., 1990). In addition, peptides BRCA2 polypeptide) are analyzed by an alanine scan (Wells, 1991). In this technique, an amino acid residue is replaced by Ala, and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.

It is also possible to isolate a target-specific antibody, selected by a functional assay, and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody.

As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an **:'analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.

Thus, one may design drugs which have, improved BRCA2 polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of BRCA2 polypeptide activity. By virtue of the availability of cloned BRCA2 sequences, sufficient amounts of the BRCA2 polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the BRCA2 protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.

Methods of Use: Gene Therapy According to the present invention, a method is also provided of supplying wild-type BRCA2 function to a cell which carries mutant BRCA2 alleles. Supplying such a function should suppress neoplastic growth of the recipient cells. The wild-type BRCA2 gene or a part of the gene may be introduced into the cell in a vector Such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from the extrachromosomal location. If a gene fragment is introduced and expressed in a cell carrying a mutant BRCA2 allele, the gene fragment should encode a part of the BRCA2 protein which is required for nbn-neoplastic growth of the cell.

More preferred is the situation where the wild-type BRCA2 gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant BRCA2 gene present in the cell. Such recombination requires a double recombination event which results in the correction of the BRCA2 gene mutation. Vectors for introduction of genes both for recombination and for extrachromosomal maintenance are known in the art, and any suitable vector may be used.

Methods for introducing DNA into cells such as electroporation, calcium phosphate 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 BRCA2 gene can be used as model systems to study cancer remission and drug treatments which promote such remission.

As generally discussed above, the BRCA2 gene or fragment, where applicable, may be employed in gene therapy methods in order to. increase the amount of the expression products of such genes in cancer cells. Such gene therapy is particularly appropriate for use in both cancerous and pre-cancerous cells, in which the level of BRCA2 polypeptide is absent or diminished compared to normal cells. It may also be useful to increase the level of expression of a given BRCA2 gene even in those tumor cells in which the mutant gene is expressed at a "normal" level, but the gene product is not fully functional.

Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman, 1991. Cells from a patient's tumor would be first analyzed by the diagnostic methods described above, to ascertain the production of BRCA2 polypeptide in the tumor cells. A virus or plasmid vector (see further details below), containing a copy of the BRCA2 S 25 gene linked to expression control elements and capable of replicating inside the tumor cells, is prepared. Suitable vectors are known, such as disclosed in U.S. Patent 5,252,479 and PCT published application WO 93/07282. The vector is then injected into the patient, either locally at the site of the tumor or systemically (in order to reach any tumor cells that may have metastasized to other sites). If the transfected gene is not permanently incorporated into the genome of each of 3 0 the targeted tumor cells, the treatment may have to be repeated periodically.

-44- Gene transfer systems known- in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods. A number of viruses have been used as gene transfer vectors, including papovaviruses, SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpesviruses including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990), and retroviruses of avian (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), murine (Miller, 1992; Miller et al., 1985; Sorge et a., 1984; Mann and Baltimore, 1985; Miller et al., 1988), and human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Most human gene therapy protocols have been based on disabled murine retroviruses.

Nonviral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical techniques, for example microinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster et al., 1981; Constantini and Lacy, 1981); membrane fusion-mediated transfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et al, 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1992); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al., 1990; Wu et al., 1991; Zenke et 1990; Wu et al., 1989b; Wolff et al, 1991; Wagner et al., 1990; Wagner et al, 1991; Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., O*l 1991b). Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery, allowing one to direct the viral vectors to the tumor cells and not into the surrounding nondividing cells. Altematively, the retroviral vector producer cell line can be injected into tumors (Culver et al, 1992). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with .inoperable brain tumors.

In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific to the adenovirus hexon protein, and the resulting complex is bound to an adenovirus vector. The trimolecular 30 complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization, and degradation ofthe endosome before the coupled DNA is damaged.

Liposome/DNA complexes have been shown to be capable of mediating direct in vivo gene transfer. While in standard liposome preparations the gene transfer 'process is nonspecific, localized in vivo uptake and expression have been reported in tumor deposits, for example, following direct in situ administration (Nabel, 1992).

Gene transfer techniques which target DNA directly to breast and ovarian tissues, e.g., epithelial cells of the breast or ovaries, is preferred. Receptor-mediated gene transfer, for example, is accomplished by the conjugation of DNA (usually in the form of covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. One appropriate receptor/ligand pair may include the estrogen receptor and its ligand, estrogen (and estrogen analogues). These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and intemalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, coinfection with adenovirus can be included to disrupt endosome function.

The therapy involves two steps which.can be performed singly or jointly. In the first step, prepubescent females who carry a BRCA2 susceptibility allele are treated with a gene delivery vehicle such that some or all of their mammary ductal epithelial precursor cells receive at least one.

additional copy of a functional normal BRCA2 allele. In this step, the treated individuals have reduced risk of breast cancer to the extent that the effect of the susceptible allele has been 20 countered by the presence of the normal allele. In the second step of a preventive therapy, predisposed young females, in particular, women who have received the proposed gene therapeutic treatment, undergo hormonal therapy to mimic the effects on the breast of a full term pregnancy.

Methods of Use: Peptide Therapy 25 Peptides which have BRCA2 activity can be supplied to cells which carry mutant or missing BRCA2 alleles. The sequence of the BRCA2 protein is disclosed in SEQ ID NO:2. Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors. Altematively, BRCA2 polypeptide can be extracted from BRCA2-producing mammnalian cells. In addition, the techniques of synthetic chemistry can be employed to synthesize BRCA2 protein. Any of such techniques can provide the preparation of the present invention which -46comprises the BRCA2 protein. The preparation is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.

Active BRCA2 molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. Extracellular application of the BRCA2 gene product may be sufficient to affect tumor growth. Supply of molecules with BRCA2 activity should lead to partial reversal of the neoplastic state. Other molecules with BRCA2 activity (for example, peptides, drugs or organic compounds) may also be used to effect such a reversal. Modified polypeptides having -substantially similar function are also used for peptide therapy.

Methods of Use: Transformed Hosts Similarly, cells and animals which carry a mutant BRCA2 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 BRCA2 mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the BRCA2 allele, as described above. After a test substance is applied to the cells, the neoplastically transformed phenotype of the cell is determined. Any trait of neoplastically transformed cells can be assessed, including anchorage-independent growth, tumorigenicity in nude mice, S* invasiveness of cells, and growth factor dependence. Assays for each of these traits are known in 20 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 BRCA2 alleles, usually from a second animal species, as well. as insertion of disrupted homologous genes.

Alternatively, the endogenous BRCA2 gene(s) of the animals may be disrupted by insertion or deletion mutation or other genetic alterations using conventional techniques (Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992). After test substances have been administered to the animals, the growth of tumors must be assessed. If the test substance prevents or suppresses the growth of tumors, then the test substance is a candidate therapeutic agent for the treatment of the cancers identified herein. These animal models provide an extremely important testing vehicle for potential therapeutic products.

The present invention is described by reference to the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below were utilized.

EXAMPLE 1 Ascertain and Study Kindreds Likely to Have a Chromosome 13-Linked Breast Cancer Susceptibility Locus Extensive cancer prone kindreds were ascertained from a defined population providing a large set of extended kindreds with multiple cases of breast cancer and many.relatives available to study. The large number of meioses present in these large kindreds provided the power to detect whether the BRCA2 locus was segregating, and increased the opportunity for informative recombinants to occur within the small region being investigated. This vastly improved the chances of establishing linkage to the BRCA2 region, and greatly facilitated the reduction of the BRCA2 region to a manageable size, which permits identification of the BRCA2 locus itself.

Each kindred was extended through all available connecting relatives, and to all informative first degree relatives of each proband or cancer case. For these kindreds, additional breast cancer cases and individuals with cancer at other sites of interest who also appeared in the kindreds were 20 identified through the tumor registry linked files. All breast cancers reported in the kindred which were not confirmed in the Utah Cancer Registry were researched. Medical records or death certificates were obtained for confirmation of all cancers. Each key connecting individual and all informative individuals were invited to participate by providing a blood sample from which DNA was extracted. We also sampled spouses and relatives of deceased cases so that the genotype of the deceased cases could be inferred from the genotypes of their relatives.

Kindreds which had three or more cancer cases with inferable genotypes were selected for linkage studies to chromosomel 3 markers. These included kindreds originally ascertained from the linked databases for a study of proliferative breast disease and breast cancer (Skolnick et a., 1990). The criterion for selection of these kindreds was the presence of two sisters or a mother and her daughter with breast cancer. Additionally, kindreds which have been studied since 1980 as part of our breast cancer linkage studies and kindreds ascertained from the linked databases for the presence of clusters of male and female breast cancer and self-referred kindreds with early onset breast cancer were included. These kindreds were investigated and expanded in our clinic in the manner described above.

For each sample collected in these kindreds, DNA was extracted from blood or paraffinembedded tissue blocks using standard laboratory protocols. Genotyping in this study was restricted to short tandem repeat (STR) markers since, in general, they have high heterozygosity and PCR methods offer rapid turnaround while using very small amounts of DNA. To aid in this effort, STR markers on chromosome 13 were developed by-screening a chromosome specific cosmid library for clones which contained short tandem repeats of 2, 3 or 4, localized to the short arm in the region of the Rb tumor suppressor locus. Oligonucleotide sequences for markers not developed in our laboratory were obtained frm published reports, or as part of the Breast Cancer LinkageConsortium, or from other investigators. All genotyping films were scored blindly with a standard lane marker used to maintain consistent coding of alleles. Key samples underwent duplicate typing for all relevant markers.

LOD scores for each kindred were calculated for two recombination fraction values, 0.001 and 0.1. (For calculation of LOD scores, see Ott 1985). Likelihoods were computed under the model derived by Claus et al, 1991, which assumes an estimated gene frequency of 0.003, a lifetime risk in female gene carriers of about 0.80, and population based age-specific risks for breast cancer in non-gene carriers. Allele frequencies for the markers used for the LOD score calculations were calculated from our own laboratory typings of unrelated individuals in the CEPH 20 panel (White and Lalouel, 1988).

Kindred 107 is the largest chromosome 13-linked breast cancer family reported to date by any group. The evidence of linkage to chromosome 13 for this family is overwhelming. In smaller Skindreds, sporadic cancers greatly confound the analysis of linkage and the correct identification of Skey recombinants.

2 i5 In order to improve the characterization of our recombinants and define closer flanking markers, a dense map of this relatively small region on chromosome 13 was required. Our approach was to analyze existing STR markers provided by other investigators and any newly developed markers from our laboratory in our chromosome linked kindreds. Figure 1 shows the location of ten markers used in the genetic analysis. Table I gives the LOD scores for linkage for each of the 19 kindreds in our study, which reduced the region to approximately 1.5 Mb.

*S 0 .5 g..

TABLE 1 Haplotype and Phlienotype Data for the 18 Families Number of Cancer Cases(1) Posterior tdj Kindred EBR MBR Q_ LD Probability 3820 SIRs Examined DI3S 4247 260 107* 8001 8004 2044* 2043* 2018 937 1018* 2328 2263 8002 8003 2367 2388 2027* 4328 2355 2327 1019 22 3 2 0 3 0 1 2 0 8 1 4 2 1 I.

3 1 0 3 1 0 9 1 0 11 1 0 2 I 0 2 1 0 2 1 0 6 0 1 3 0 1 4 0 0 4 0 0 3 0 0 11 0 0 2 2 0 5.06 n.d.

n.d.

2.13 0.86 n.d.

n.d.

2.47 0.42 n.d.

n.d.

n.d.

0.40 0.92 0.39 0.44 0.36 1.92 1.00 0.90 0.90 1.00 0.98 0.90 0.90 1.00 0.96 0.90 0.90 0.90 0.85 0.95 0.85 0.87 0.84 0.99 mb DI3S 5370- GA9 5 171 2(C 10 8 3 2 10 7 10 5 4 7 8 6 7. 5 9 6 12 7 10 5 3 8 3 6 8 10 10 5' 8 2 10 5 8 5 8 4 10 5 8 5 10 6 3 4 10 .12 3 7 12 4 10 4 10 7 10 5 4 8 3 7 4 6 3 7 9 5 10 5 D13S DI3S AC6 31 267 6 .5 8 5 8 6 6 5 5 5 5 5 5 6 8 3 5 4 12 5 4 4 12 4 8 4 12 5 8 7 7 4 8 7 12 7 12 5 8 4 8 5 4.

5 12 7 12 5 12 5 8 3 4 Families reported in Wooster et al. (1994). n.d. not determined Excludes cases known to be sporadic do not share the BRCA2 haplotype segregating in the family).

FBR female' breast cancer under 60 years. MBR male breast cancer OV ovarian cancer Posterior probability assumes that, a priori, 90% of families with male breast and early onset female breast cancers that are unlinked to BRCAI are due to BRCA2, and 70% of female breast cancer families unlinked to BRCAI are due to BRCA1.

Table 1 also gives the posterior probability of a kindred having a BRCA2 mutation based on LOD scores and prior probabilities. Four of these markers (D13S171, D13S260, D13S310 and D13S267) were previously known. The other six markers were found as part of our search for BRCA2. We were able to reduce the region to.1..5 megabases based on a recombinant in Kindred 107 with marker tdj3820 at the left boundary, and a second recombinant in Kindred 2043 with marker YS-G-BIOT at the right boundary (see Figure 1) which is at approximately the same location as AC6 and D13S310. Furthermore, a homozygous deletion was found in a pancreatic tumor cell line in the BRCA2 region which may have been driven by BRCA2 itself; this deletion is referred to as the Schutte/Kern deletion in Figure 1 (Schutte et al., 1995). The Schutte/Kem contig in Figure 1 refers to these authors' physical map which covers the deletion.

EXAMPLE 2 Development of Genetic and Physical Resources in the Region of Interest To increase the number of highly polymorphic loci in the BRCA2 region, we developed a number of STR markers in our laboratory from Pls, BACs and YACs which physically map to the region. These markers allowed us to.further refine the region (see Table 1 and the discussion above).

20 STSs in the desired region were used to identify YACs which contained them. These YACs were then used to identify subclones in Pls or BACs. These subclones were then screened for the presence of a short tandem repeats. Clones with a strong signal were selected preferentially, since they were more likely to represent repeats which have a largenumber of repeats and/or are of nearperfect fidelity to the pattern. Both of these characteristics are known to increase the probability of 25 polymorphism (Weber et 1990), These clones were sequenced directly from the vector to locate the repeat. We obtained a unique sequence on one side of the short tandem repeat by using one of a set of possible primers complementary to the end of the repeat. Based on this unique sequence, a primer was made to sequence back across the repeat in the other direction, yielding a unique sequence for design of a second primer flanking it. STRs were then screened for polymorphism on a small group of unrelated individuals and tested against the hybrid panel to confirm their physical localization. New markers which satisfied these criteria were then typed in a -51set of unrelated individuals from Utah to obtain allele frequencies appropriate for the study of this population. Many of the other markers reported in this study were also tested in unrelated individuals to obtain similarly appropriate allele frequencies.

Using the procedure described above, novel STRs were found from these YACs which were both polymorphic and localized to the BRCA2 region. Figure 1 shows a schematic map of STSs, Pls, BACs and YACs in the BRCA2 region.

EXAMPLE 3 Identification of Candidate cDNA Clones for the BRCA2 Locus by Genomic Analysis of the Contig Region 1. General Methods Complete screen of the plausible region.. The first method to identify candidate cDNAs, although labor intensive, used known techniques. The method comprised the screening of P1 and 15 BAC clones in the contig to identify putative coding sequences. The clones containing putative coding sequences were then used as probes on filters of cDNA libraries to identify, candidate cDNA clones for future analysis. The clones were screened for putative coding sequences by either of two methods.

The P1 clones to be analyzed were.digested with a restriction enzyme to release the human DNA from the vector DNA. The DNA was separated on a 14 cm, 0.5% agarose gel run overnight at 20 volts for 16 hours. The human DNA bands were cut out of the gel and electroeluted from the *o, gel wedge at 100 volts for at least two hours in 0.5x Tris Acetate buffer (Maniatis et al., 1982).

The eluted Not I digested DNA (-15 kb to 25 kb) was then digested with EcoRI restriction enzyme to give smaller fragments kb to 5.0 kb) which melt apart more easily for the next step of labeling the DNA with radionucleotides. The DNA fragments were labeled by means of the hexamer random prime labeling method (Boehringer-Mannheim, Cat. #1004760). The labeled *DNA was spermine precipitated (add 100 pl TE, 5 p1l 0.1 M spermine, and 5 pl of 10 mg/ml salmon sperm DNA) to remove unincorporated radionucleotides. The labeled DNA was then resuspended in 100 pl TE, 0.5 M NaCI at 65 0 C for 5 minutes and then blocked with Human Cot-1 DNA for 2-4 hrs. as per the manufacturer's instructions (Gibco/BRL, Cat. #5279SA). The Cot-1 blocked probe was incubated on the filters in the blocking solution ovemight at 42 0 C. The filters -52were washed for 30 minutes at room temperature in 2 x SSC, 0.1% SDS, and then in the same buffer for 30 minutes at 55 0 C. The filters were then exposed 1 to 3 days at -70°C to Kodak film with an intensifying screen. Thus, the blots were hybridized with either the pool of Eco-RI fragments from the insert, or each of the fragments individually.

The human DNA from clones in the region was isolated as whole insert or as EcoRI fragments and labeled as described above. The labeled DNA was used to screen filters of various cDNA libraries under the same conditions described above except that the cDNA filters undergo a more stringent wash of 0.1 x SSC, 0.1% SDS at 65 0 C for30 minutes twice.

Most of the cDNA libraries used to date in our studies (libraries from normal breast tissue, breast tissue from a woman in her eighth month of pregnancy and a breast malignancy) were prepared at Clonetech, Inc. The cDNA library generated from breast tissue of an 8 month pregnant woman is available from Clonetech (Cat. #HL1037a) in the Lambda gt-10 vector, and is grown in C600Hfl bacterial host cells. Normal breast tissue and malignant breast tissue samples were isolated from a 37 year old Caucasian female and one-gram of each tissue was sent to Clonetech 15 for mRNA processing and cDNA library construction. The latter two libraries were generated 'using both random and oligo-dT priming, with size selection of the final products which were then cloned into the Lambda Zap II vector, and grown in XLl-blue strain of bacteria as described by the manufacturer. Additional tissue-specific cDNA libraries include human fetal brain (Stratagene, Cat. 936206), human testis (Clonetech Cat. HL3024), human thymus (Clonetech Cat. HL1127n), 20 human brain (Clonetech Cat. HL11810), human placenta (Clonetech Cat 1075b), and human skeletal muscle (Clonetech Cat. HL 1124b).

The cDNA libraries were plated with their host cells on NZCYM plates, and filter lifts are made in duplicate from each plate as per Maniatis e al. (1982). Insert (human) DNA from the candidate genomic clones was purified and radioactively labeled to high specific activity. The radioactive DNA was then hybridized to the cDNA filters to identify those cDNAs which correspond to genes located within the candidate cosmid clone. cDNAs identified by this method were picked, replated, and screened again with the labeled clone insert or its derived EcoRI fragment DNA to verify their positive status. Clones that were positive after this second ronnd of screening were then grown up and their DNA purified for Southern blot analysis and sequencing.

3 0 Clones were either purified as plasmid through in vivo excision of the plasmid from the Lambda vector as described in the protocols from the manufacturers, or isolated from the Lambda vector as a restriction fragment and subcloned into plasmid vector.

The Southern blot analysis was performed in duplicate, one using the original genomic insert DNAas a probe to verify that cDNA insert contains hybridizing sequences. The second blot was hybridized with cDNA insert DNA from the largest cDNA clone to identify which clones represent the same gene. All cDNAs which hybridize with the genomic clone and. are unique were sequenced and the DNA analyzed to determine if the sequences represent known or unique genes.

All cDNA clones which appear to be unique were further analyzed as candidate BRCA2 loci.

Specifically, the clones are hybridized to Northern blots to look for breast specific expression and differential expression in normal versus breast tumor RNAs. They are also analyzed by PCR on clones in the BRCA2 region to verify their location. To map the extent of the locus, full length cDNAs are isolated and their sequences used as PCR probes on the YACs and the clones surrounding and including the original identifying clones. Intron-exon boundaries are then further defined through sequence analysis.

15 We have screened the normal breast, 8 month pregnant breast and fetal brain cDNA libraries with Eco RI fragments from cosmid BAC and P1 clones in the region. Potential BRCA2 cDNA clones were identified among the three libraries. Clones were picked, replated, and screened again with the original probe to verify that they were positive.

Analysis of hybrid-selected cDNA. cDNA fragments obtained from direct selection were 20 checked by Southern blot hybridization against the probe DNA to verify that they originated from the contig. Those that passed this test were sequenced in their entirety. The set of DNA sequences obtained in this way were then checked against each other to find independent clones that overlapped.

The direct selection of cDNA method (Lovett et al., 1991; Futreal, 1993) is utilized with P1 and BAC DNA as the probe. The probe DNA is digested with a blunt cutting restriction enzyme such as HaeIII. Double-stranded adapters are then ligated onto the DNA and serve as binding sites for primers in subsequent PCR amplification reactions using biotinylated primers. Target cDNA is generated from mRNA derived from tissue samples, breast tissue, by synthesis of-either random primed or oligo(dT) primed first strand, followed by second strand synthesis. The cDNA ends are rendered blunt and ligated onto double-stranded adapters. These adapters serve as amplification sites for PCR. The target and probe sequences are denatured and mixed with human Cot-1 DNA to block repetitive sequences. Solution hybridization is carried out to high Cot-1/2 values to ensure hybridization of rare target cDNA molecules. The annealed material is then captured on avidin beads, washed at high stringency and the retained cDNAs are eluted and amplified by PCR. The selected cDNA is subjected to further rounds of enrichment before cloning into a plasmid vector for analysis.

HTF island analysis. A method for identifying cosmids to use as probes on the cDNA libraries was HTF island analysis. HTF islands are segments of DNA which contain a very high frequency of unmethylated CpG dinucleotides (Tonolio et al., 1990) and are revealed by the clustering of restriction sites of enzymes whose recognition sequences include CpG dinucleotides.

Enzymes known to be useful in HTF-island analysis are AscI, NotI, BssHII, EagI, SacII, NaeI, Narl, SmaI, and Mlul (Anand, 1992).

Analysis of candidate clones. One or more of the candidate genes generated from above were sequenced and the information used for identification and classification of each expressed gene. The DNA sequences were compared to known genes by nucleotide sequence comparisons 15 and by translation in all frames followed by a comparison with known amino acid sequences. This was accomplished using Genetic Data Environment (GDE) version 2.2.software and the Basic Local Alignment Search Tool (Blast) series of client/server software packages BLASTN 1.3.13MP), for sequence comparison against both local and remote sequence databases GenBank), running on Sun SPARC workstations. Sequences reconstructed from collections of 20 cDNA clones identified with the cosmids and Pls have been generated. All candidate genes that *represented new sequences were analyzed further to test-their candidacy for the putative BRCA2 locus.

Mutation screening. To screen for mutations in the affected pedigrees, two different approaches were followed. First, genomic DNA isolated from family members known to carry the 25 susceptibility allele of BRCA2 was used as a template- for amplification of candidate gene sequences by PCR. If the PCR primers flank or overlap an intron/exon boundary, the amplified fragment will be larger than predicted from the cDNA sequence or will not be present in the amplified mixture. By a combination of such amplification experiments and sequencing of P1 or BAC clones using the set of designed primers it is possible to establish the intron/exon structure and ultimately obtain the DNA sequences of genomic DNA from the kindreds.

Asecond approach that is much more rapid if the intron/exon structure of the candidate gene is complex involves sequencing fragments amplified from cDNA synthesized from lymphocyte mRNA extracted from pedigree blood which was used as a substrate for PCR amplification using the set of designed primers. If the candidate gene is expressed to a significant extent in lymphocytes, such experiments usually produce amplified fragments that can be sequenced directly without knowledge of intron/exon junctions.

The products of such sequencing reactions were analyzed by gel electrophoresis to determine positions in the sequence that contain either mutations such as deletions or insertions, or base pair substitutions that cause amino acid changes or other detrimental effects.

Any sequence within the BRCA2 region that is expressed in breast is considered to be a candidate gene for BRCA2. Compelling evidence that a given candidate gene corresponds to BRCA2 comes from a demonstration that kindred families contain defective alleles of the.

candidate.

2. Specific Methods Hybrid selection. Two distinct methods of hybrid selection were used in this work.

Method 1: cDNA preparation and selection. Randomly primed cDNA was prepared from poly (A) RNA of mammary gland, ovary testis, fetal brain and placenta tissues and from total RNA of the cell line Caco-2 (ATCC HTB 37). cDNAs were homopolymer tailed and then S 20 hybrid selected for two consecutive rounds of hybridization to immobilized PI or BAC DNA as described previously. (Parimoo et al., 1991; Rommens et 1994). Groups of two to fdur overlapping P1 and/or BAC clones were used in individual selection experiments. Hybridizing cDNA was collected, passed over a G50 Fine Sephadex column and.amplified using tailed primers. The products were then digested with EcoRI, size selected on agarose gels, and ligated 25 into pBluescript (Stratagene) that had been digested with EcoRI and treated with calf alkaline phosphatase (Boehringer Mannheim). Ligation products were transformed into competent E. coli cells (Life Technologies, Inc.).

Characterization of Retrieved cDNAs. 200 to 300 individual colonies from each ligation (from each 250 kbases of genomic DNA) were picked and gridded into microtiter plates for ordering and storage. Cultures were replica transferred onto Hybond N membranes (Amersham) supported by LB agar with ampicillin. Colonies were allowed to propagate and were -56subsequently lysed with standard procedures. Initial analysis of the cDNA clones involved a prescreen for ribosomal sequences and subsequent cross screenings for detection of overlap and redundancy.

Approximately 10-25% of the clones were eliminated as they hybridized strongly with radiolabeled cDNA obtained from total RNA. Plasmids from 25 to 50 clones from each selection experiment that did not hybridize in prescreening were isolated for further analysis.

The retrieved cDNA fragments were verified to originate from individual starting genomic clones by hybridization to restriction digests of DNAs of the starting clones, of a hamster hybrid cell line (GM10898A) that contains chromosome 13 as its only human material and to human genomic DNA. The clones were tentatively assigned into groups based on the overlapping or non-overlapping intervals of the genomic clones. Of the clones tested, approximately mapped appropriately to the starting clones.

Method 2 (Lovett et al.. 1991): cDNA Preparation. Poly(A) enriched RNA from.human mammary gland, brain, lymphocyte and stomach were reverse-transcribed using the tailed 15 random primer XN 12 [5'-(NH 2 )-GTAGTGCAAGGCTCGAGAACNNNNNNNNNNNN] (SEQ ID NO:3) and Superscript II reverse transcriptase (Gibco BRL). After second strand synthesis and end polishing, the ds cDNA was purified on Sepharose CL-4B columns (Pharmacia). cDNAs were "anchored" by ligation of a double-stranded oligo RP 20 [5'-(NH,)-TGAGTAGAATTCTAACGGCCGTCATTGTTC (SEQ ID NO:4) annealed to 2 (SEQ ID to their 5' ends relative to mRNA) using T4 DNA ligase. Anchored ds cDNA was then repurified on Sepharose CL-4B columns.

25 Selection. cDNAs from mammary gland, brain, lymphocyte and stomach tissues were first amplified using a nested version of RP (RP.A: 5'-TGAGTAGAATTCTAACGGCCGTCAT) (SEQ ID NO:6),and XPCR [5'-(PO 4 )-GTAGTGCAAGGCTCGAGAAC (SEQ ID NO:7)] and purified by fractionation on Sepharose CL-4B. Selection probes were prepared from purified Pls, BACs or PACs by digestion with Hinfl and Exonuclease III. The single-stranded probe was photolabelled with photobiotin (Gibco BRL) according to the manufacturer's recommendations. Probe, cDNA and Cot-1 DNA were hybridized in 2.4M TEA-CL, NaPO 4 ImM EDTA. Hybridized cDNAs were captured on streptavidin-paramagnetic particles (Dynal), eluted, reamplified with a further nested version of RP [RP.B: 5'-(PO 4 )-TGAGTAGAATTCTAACGGCCGTCATTG (SEQ ID NO:8)] and XPCR, and size-selected on Sepharose CL-6B. The selected, amplified cDNA was hybridized with an additional aliquot of probe and Cot-1 DNA. Captured and eluted products were amplified again with RP.B and XPCR, size-selected by gel electrophoresis and cloned into dephosphorylated HinclI cut pUC18. Ligation products were transformed into XL2-Blue ultracompetent cells (Stratagene).

Analysis. Approximately 192 colonies for each single-probe selection experiment were amplified by colony PCR using vector primers and blotted in duplicate onto Zeta Probe nylon filters (Bio-Rad). The filters were hybridized using standard procedures with either random primed Cot-l DNA or probe DNA (P1, BAC or PAC). Probe-positive, Cot-1 negative clones S- were sequenced in both directions using vector primers on an ABI 377 sequencer.

15 Exon Trapping. Exon amplification was performed using a minimally overlapping set of BACs, Pls and PACs in order to isolate a number of gene sequences from the BRCA2 candidate region. Pools of genomic clones were assembled, containing from 100-300 kb of DNA in the form of 1-3 overlapping genomic clones. Genomic clones were digested with PstI or BamHI BglII and ligated into PstI or BamHI sites of the pSPL3 splicing vector. The exon 20 amplification technique was performed (Church et al., 1993) and the end products were cloned in the pAMPl plasmid from the Uracil DNA Glycosylase cloning system (BRL).

Approximately 6000 clones were picked, propagated in 96 well plates, stamped onto filters, and analyzed for the presence of vector and repeat sequences by hybridization. Each clone insert was PCR amplified and tested for redundancy, localization and human specificity by 25 hybridization to grids of exons and dot blots of the parent genomic DNA. Unique candidate exons were sequenced, searched against the databases, and used for hybridization to cDNA libraries.

RACE. The 5' end of BRCA2 was identified by a modified RACE protocol called biotin capture RACE. Poly(A) enriched RNA from human mammary gland and thymus was reverse-transcribed using the tailed random primer XNI2 -GTAGTGCAAGGCTCGAGAACNNNNNNNNNNN (SEQ ID NO:3)] and Superscript II reverse transcriptase (Gibco BRL). The RNA strand was hydrolyzed in NaOH and first strand cDNA'purified by fractionation on Sepharose CL-4B (Pharmacia). First strand cDNAs were "anchored" by. ligation of a double-stranded oligo with a 7 bp random 5' overhang [ds UCA: 5'-CCTTCACACGCGTATCGATTAGTCACNNNNNNN-(NH 2 (SEQ ID NO:9) annealed to 5'-(PO 4 )-GTGACTAATCGATACGCGTGTGAAGGTGC (SEQ ID NO:10)] to their 3' ends using T4 DNA ligase. After ligation, the anchored cDNA was repurified by fractionation on Sepharose CL-4B. The 5' end of BRCA2 was amplified using a biotinylated reverse primer [5'-(B)-TTGAAGAACAACAGGACTTTCACTA] (SEQ ID NO:11) and a nested version of UCA [UCP.A: 5'-CACCTTCACACGCGTATCG (SEQ ID NO:12)]. PCR products were fractionated. on an agarose gel, gel purified, and captured on streptavidinparamagnetic particles (Dynal). Captured cDNA was reamplified using a nested reverse primer (SEQ ID NO:13) and a further nested version of UCA [UCP.B: 5'-CCTTCACACGCGTATCGATTAG] (SEQ ID NO:14)]. This PCR reaction gave a single sharp band on an agarose gel; the DNA was gel purified and sequenced in S* 15 both directions on an ABI 377 sequencer.

cDNA Clones. Human cDNA libraries were screened with 2P-labeled hybrid selected or exon trapped clones. Phage eluted from tertiary plaques were PCR amplified with vectorspecific primers and then sequenced on an ABI 377 sequencer.

Northern Blots. Multiple Tissue Northern (MTN) filters, which are loaded with 2 pg 20 per lane of poly(A) RNA derived from a number of human tissues, were purchased from Clonetech. 32P-random-primer labeled probes corresponding to retrieved cDNAs GT 713 S(BRCA2 exons wCPFIB8.1 end of exon 11 into exon 20), and glyceraldehyde-3phosphate dehydrogenase (GAPDH) were used to probe the filters. Prehybridizations were at 42 0 C in 50% formamide, 5X SSPE, 1% SDS, 5X Denhardt's mixture, 0.2 mg/ml denatured 25 salmon testis DNA and 2 pg/ml poly(A). Hybridizations were in the same solution with the addition of dextran sulfate to 4% and probe. Stringency washes were in 0.1X SSC/0.1% SDS at 0

C.

RT-PCR Analysis. Ten jg of total RNA extracted from five human breast canc e r cell lines (ZR-75-1, T-47D, MDA-MB-231, MDA-MB468 and BT-20) and three human prostate cancer cell lines (LNCaP, DU145 and PC-3) (RNAs provided by Dr. Claude Labrie, CHUL Research Center) were reverse transcribed using the primer mH20-1D05#RA -59- (SEQ ID and Superscript II reverse transcriptase (Gibco BRL). Thereafter, the single strand cDNAs were amplified using the primers CG026#FB: (SEQ ID NO: 16)] and mH20-1D05#RA (this is the primer pair that was used to island hop from the exon 7/8 junction into exon 11; the PCR product is about 1.55 kb). PCR products were fractionated on a 1.2% agarose gel.

PCR Amplification and Mutation Screening. All 26 coding exons of BRCA2 and their associated splice sites were amplified from genomic DNA as described (Kamb et al., 1994b). The DNA sequences of the primers, some of which lie in flanking intron sequence, used for amplification and sequencing appear in Table 2. Some of the exons (2 through 10, 11-5, 11- 6, 11-7 and 23 through 27) were amplified by a simple one-step method. The PCR conditions for those exons were: single.denaturing step of 95 0 C (1 min.); 40 cycles of 96 0 C (6 sec.), T. 55 0 C (15 sec.), 72 0 C (1 min.). Other exons (11-22) required nested reamplification after the primary PCR reaction. In these cases, the initial amplification was carried out with the primers in the first two columns of Table 2 for 19 cycles as described above. Nested reamplification for these exons was carried out for 28 or 32 cycles at the same conditions with the primers appearing in the third column of Table 2. The buffer conditions were as described (Kamb et al., 1994b). The products were purified from 0.8% agarose gels using Qiaex beads (Qiagen). The 20 purified products were analyzed by cycle sequencing with c-P 32 dATP with Ampli-CycleT Sequencing Kit (Perkin Elmer, Branchburg, NJ). The reaction products.were fractionated on 6% polyacrylamide gels. All reactions were loaded adjacent each other, followed by the (C) reactions, etc. Detection of polymorphisms was carried out visually and confirmed on the other strand.

*0 I I EX-2N 2 3 4 6 7 8 9 1 10-2 10-3 I1-i 1 1-2 I11-3 1 1-4 11I-6 11-7 11-8 11-9 11-10 il-Il 12 13 FORWARD PRIMER TGTTGCCATCCTCACAGTAAG* GjGTTAA.AACTAAG .GTGGGA-"' 9 1TfCCCAGTATAGAGGAGA*( 2 1

ATCTAAAGTAGTATTCCAACA'(

23 GAG ATAAGTCAG GTATG ATT'.

3 GGCAATTCAGTAAACGi-AA* 21

OTGTCATGTAATCAAATAGT*(

29

GACCTAGGTTGATTGCA*(

31 1

CTATGAGAAAGGTT*GTCGAC*(

3 AACAGTTGTAGATACCTCTGAA*(31) CAGCATCTTOAATCTCATACAG* AACTTAGTGAAAAATATi AGTGA( 9

AGAACCAACTTTGTCCTTAA(

4 2

ATGGAAAAGAATCAAGATGTAT*(

45

GTGTAAAGCAGCATATAAAAAT*

48 CCATAATTAACACCTAGCCA

GGCTTTTATTCTGCTCATGGC*(

53 AACGGACTTGCTATACTGA *(55)

CAGCTAGCGGGAAAAAAGTA*(

57 GCCTTAGCTTTTTACACAA*(59)

CCATTAAATTGTCCATATCTA*

62

GAAGATAGTACCAAGCAAGTC

6 1)

GTCTTCACTATTCACGTACG*(

6 1)

ACTCMTCAAACATAGGTCA*(

7 1 1TUATGCTGATTCTGTTGTAT' 74 0: to .0* TABLE 2 Pimiers for Amplifying BRCA2 Exons REVERSE PRIMER GTACTGGO1T1TAGCAAGCA*(' 8 A1TTTGCCCAGCATGACACA* 20 GTAGGAAAATGM1CATTAA *(22)

GGGGGTAAAAAAAGGGGAA*

24 1

AAYI'GCCTGTATGAGGCAGA*(

26

AITGTCAGTT-ACTAACACAC*

2

CAGGTTTAGAGACTTMCTC*

30 GTCAAGAAAGGTAAGGTAA *(32)

CCTAGTCTTGCTAGTTCTT-

34 GACMTTGATACCCTGAAATG*(c3) CATGTATACAGATGATGCCTAAG *(38) ATACATC'ITGATTCT=rCCAT*( 40 TTAGATTTGTGT-T1TGGTGAA 4 3 CCTAATGTTATGITCAGAGAG 46 ,CITGCTGCTGTCTACCTG 4 1 CCAAAAAAGT-rAAATCTGACA* *(S2)

CCTCI*GCAGAAGTTTCCTCAC*

54

AGTACCTTGCTCTTTTTCATC*(

56 T-rCGGAGAGATGATMTTGTC* 58 T=T~GATTATATCTCGTTG 60 GACGTAGGTGAATAGTGAAGA 6 3 TGAGAC~iTGGfl'CCTAATAC* 66 CCCCCAAACTGACTACACAA 69 TTGGAGAGGCAGGTGGAT4 2 ATAAAACGGcJAAGTGTTAACT*(75).

NESTED PRIMER

TTTAG;TGAATGTGATTGATCOGT*(

4

I

:TAGCTCT1TITGGGACAAY-C 44 GCTACCTCCAAAACTGTGA *(47)

AGTGGTCTTAAGATAGTCAT*

50 TFATTCTCGTFGTMCCT[A TCAAATTCCTCTAACACTCC (64) AGtAACGAACATrCAGACCAG *(67) AGCATACCAAGI'CTA CTGAAT*(1 7 0 C1'ATAGAGGGAGAACAGA 7 1) CTGTrGAGTTAMTGG'FCCA*( 76

S

S..

S

S S S S S

*SS

TAL 2 (Co* t Prier for *S liy g B S S S EX-QN FORWARD PRIMER 14 GAATACA-AAACAGTACCAGA 1 7

GTCCGACCAGAACTTGAG

80 16 ATG I1 IT IGTAGTGAAGATTCT 83 17 CAGAGAATAGTTGTAGTTGTY 18. TTTTATTCTCAG7TATTCAGTG 89 19 ATATI7JTTAAGGCAGTTCTAGA 9 GATTTTTTA 95 21 CTTTTAGCAGTTATATAGMrC 98 22 TTTGTTGTATTTGTCCI'GMTA 1 01 23 ATCACTTCTTCCATGCATC* 10 4 24 cTrGGTAGCTCCAACTAATC *(1 06

CTATTTTGATTTGCTTTTATTATT*

t10 8 26 TTGGAAACATAAATATGTGGG* t1 10 27 CTACA1TAATTATGATAGGCTNCG**tI 1) REVERSE PRIMER

CACCACCAAAGGGGGAAA*(

7 8

AGCCATTTGTAGGATACTAG*

81 TAGTCGAGGACATTAA*(1 4 AACCTTAACCCATACTGCC*(87) GAAATTGAkGCATCCTTAGTAAL*(90) 'ITACACACACCAAAAAAGTCA*(1 3 CYI'GT-FGCTATrTCTrrGTCTA 96 GCCAGAGAGTCTAAAACAG *(99) A11TTGjTIAGiTAAOGTCA1T-* 102 CCGTGGCTGGTAAATCTG*(1OS) ACCGGTACAAACCTTrCAY[O *(107) GCTATTITCCTrATACTGGAC*( 109) ACTTACAGGAGCCACAFAAC*(" 11) GTAC'rAATGTGT('GTTTGAAA**(' TCAATG3CAAGTTCTTrCGTCAGC* t 14) .NESTED PRJIMER AAA TGAGGGTCTGCAACAAA *(79)

CTACTAGACGGGCGGAG*(

82 CAGTITrGG1IGT-TATAATI-G (Bs)

TTCAGTATCATCCTATGTGG*)

AATITCTAGAGTCACACTTCC*(

9 1)

T'GAAAACTCTI'ATGATATCTGT*(

94 CCCTAGATACTAAAAAATAAAG*(97) C1TITGGGTG1TIATJCT-rG* ')O GTrCTGA-TGCTIIATCC*( 103 Primers with an were used for sequencing.

Primers without an were replaced by the internal nested primer for both* the second round of PCR and'seque ncing.

For large exons requiring internal sequencing primers, primers with an ""were used to amplif' the exon Number in parathensis referes to the SEQ ID NO: for each primer.

-62- EXAMPLE4 Identification ofBRCA2 Assembly of the full-length BRCA2 sequence. The full-length sequence of BRCA2 was assembled by combination of several smaller sequences obtained from hybrid selection, exon trapping, cDNA library screening, genomic sequencing, and PCR experiments using cDNA as template for amplification "island hopping") (Figure The extreme 5' end of the mRNA including the predicted translational start site was identified by a modified 5'RACE protocol (Stone et al., 1995). The first nucleotide in the sequence (nucleotide 1) is a non-template G, an indication that the mRNA cap is contained in the sequence. One of the exons (exon 11) located on the interior of the BRCA2 cDNA is nearly 5 kb. A portion of exon 11 was identified by analysis of roughly 900 kb of genomic sequence in the public domain (ftp://genome.wustl.edu/ pub/gscl/brca). This genomic sequence was condensed with genomic sequence determined by us into a set of 160 sequence contigs. When the condensed genomic sequence was scanned for 5 open reading frames (ORFs), a contiguous stretch of nearly 5 kb was identified that was spanned by long ORFs. This sequence was linked together by island hopping experiments with two previously identified candidate gene fragments. The current composite BRCA2 cDNA sequence consists of 11,385 bp, but does not include the polyadenylation signal or poly(A) tail. This cDNA sequence is set forth in SEQ ID NO:1 and Figure 3.

Structure of the BRCA2 gene and BRCA2 polvpeptide. Conceptual translation of the ScDNA revealed an ORF that began at nucleotide 229 and encoded a predicted protein of 3418 amino acids. The peptide bears no discernible similarity to other proteins apart from sequence composition. There is no signal sequence at the amino terminus, and no obvious membrane- *spanning regions. Like BRCA1, the BRCA2 protein is highly charged. Roughly one quarter of the residues are acidic or basic.

The BRCA2 gene structure was determined by comparison of cDNA and genomic sequences. BRCA2 is composed of 27 exons distributed over roughly 70 kb of genomic DNA.

A CpG-rich region at the 5' end of BRCA2 extending upstream suggests the presence of regulatory signals often associated with CpG "islands." Based on Southern blot experiments, 30 BRCA2 appears to be unique, with no close homologs in the human genome.

Expression studies of BRCA2. Hybridization of labeled cDNA to human multiple tissue Northern filters revealed an 11-12 kb transcript that was detectable in testis only. The size of the this transcript suggests that little of the BRCA2 mRNA sequence is missing from our composite cDNA. Because the Northern filters did not include mammary gland RNA, RT-PCR experiments using a BRCA2 cDNA amplicon were performed on five breast and three prostate cancer cell line RNAs. All of the lines produced positive signals. In addition, PCR of a BRCA2 amplicon (1-BrCG026 5kb) and 5' RACE were used to compare mammary gland and thymus cDNA as templates for amplification. In both cases, the product amplified more efficiently from breast than from thymus.

Germline mutations in. BRCA2. Individuals from eighteen putative BRCA2 kindreds were screened for BRCA2 germline mutations by DNA sequence analysis (Wooster et al., 1994).. Twelve kindreds have at least one case of male breast cancer, four have two or more cases; and, four include at least one individual affected with ovarian cancer who shares the S* linked BRCA2 haplotype. Each of the 18 kindreds has a posterior probability of harboring a 15 BRCA2 mutation of at. least 69%, and nine kindreds have posterior probabilities greater than 90%. Based on these combined probabilities, 16 of 18 kindreds are expected to segregate BRCA2 mutations. The entire coding sequence and associated splice junctions were screened for mutations in multiple individuals from nine kindreds using either cDNA orgenomic DNA (Table Individuals from the remaining nine kindreds were screened for mutations using only 20 genomic DNA. These latter screening experiments encompassed 99% of the coding sequence (all exons excluding exon 15) and all but two of the splice junctions.

SQ@

sa 0 O

S

5 0 0 0 @0 6 5* 0 @0 05 0 0 0 0 0* 50 5 S C 50 S TABLE 3 Set of Families Screened for BRCA2 Mutations

FBC

Family EBC <0Yrs QY MB.C LOD UT-107' UT-1018' UT-2044' UT-2367' UT-2327 UT-2388' UT-2328' UT-4328' MI-1016' 2 CU-1592 UT-2043 2 IC-2204 2 MS-075 2 UT-10192 UT-2027 2 UT-2263 2 UT-21712 5.06 2.47 2.13 2.09 1.92 0.92 0.21 0.18 0.04 1.09 0.99 0.86 0.51 0.50 nd 0.39 nd nd Prior Probability 1.00 1.00 1.00 0.99 0.99 0.92 0.87 0.69 0.81 1.00 0.94 0.97 0.98 0.93 0.95 0.79 0.9 nd BRCA2 Mutation 277 delAC 982 del4 4706 del4

IR

ND

ND

ND

ND

ND

8525 delC 9254 del 5 4075 delGT 999 del5 6174 delT 4132 del3

ND

ND

ND

Exon Codon 17 252 1493 2766 3009 1283 257 1982 1302 Effect termination codon at 29 termination codon at 275 terminatin codon at 1502 termination codon at 2776 termination codon at 3015 termination codon at 1285 termination codon at 273 termination codon at 2003 deletion of thr13 02 2 Families screened for complete coding sequence and with informative cDNA sample.

Families screened for all BRCA2 exons except 15 and for which there was no informative cDNA sample available.

IR inferred regulatory mutation ND none detected nd not determined FBC Female Breast Cancer Ov Ovarian Cancer MBC Male Breast Cancer Sequence alterations were identified in 9 of 18 kindreds. All except one involved nucleotide deletions that altered the reading frame, leading to truncation of the predicted BRCA2 protein. The single exception contained a deletion of three nucleotides (kindred 1019). All nine mutations differed from one another.

A subset of kindreds was tested for transcript loss. cDNA samples were available for a group of nine kindreds, but three of the nine kindreds in the group contained frameshift mutations. Specific polymorphic sites know to be heterozygous in genomic DNA were examined in cDNA from kindred individuals. The appearance of hemizygosity at these polymorphic sites was interpreted as evidence for a mutation leading to reduction in mRNA levels. -In only one of the six cases with no detectable sequence alteration (kindred 2367) could such a regulatory mutation be inferred. In addition, one of the three kindreds with a frameshift mutation (kindred 2044) displayed signs of transcript loss. This implies that some mutations in the BRCA2 coding sequence may destabilize the transcript in addition to disrupting the protein sequence. Such mutations have been observed in BRCA1 (Friedman et al., 1995). Thus, 56% of the kindreds (10 of 18) contained an altered BRCA2 gene.

Role of BRCA2 in Cancer. Most tumor suppressor genes identified to date give rise to protein products that are absent, nonfunctional, or reduced in function. The majority of TP53 mutations are missense; some of these have been shown to produce abnormal p53 molecules that interfere with the function of the wildtype product (Shaulian et al, 1992; Srivastava et al., 1993).

20 A similar dominant negative mechanism of action has been proposed for some adenomatous polyposis coli (APC) alleles that produce truncated molecules (Su et al., 1993), and for point mutations in the Wilms' tumor gene (WTI) that alter DNA binding of the protein (Little et al.,.

1993). The nature of the mutations observed in the BRCA2 coding sequence is consistent with Sproduction of either dominant negative proteins or nonfunctional proteins.

EXAMPLE

SAnalysis of the BRCA2 Gene The structure and function of BRCA2 gene are determined according to the following S 30 methods.

-66- Biological Studies. Mammalian expression vectors containing BRCA2 cDNA are constructed and transfected into appropriate breast carcinoma cells with lesions in the gene. Wildtype BRCA2 cDNA as well as altered BRCA2 cDNA are utilized. The altered BRCA2 cDNA can be obtained from altered BRCA2 alleles or produced as described below. Phenotypic reversion in cultures cell morphology, doubling time, anchorage-independent growth) and in animals tumorigenicity) is examined. The studies will employ both wild-type and mutant forms (Section B) of the gene.

Molecular Genetics Studies. In vitro mutagenesis is performed to construct deletion mutants and missense mutants (by single base-pair substitutions in individual codons and cluster charged alanine scanning mutagenesis). The mutants are used in biological, biochemical and biophysical studies.

Mechanism Studies. The ability of BRCA2 protein to bind to known and unknown DNA sequences is examined. Its ability to transactivate promoters is analyzed by transient reporter expression systems in mammalian cells. Conventional procedures such as particle-capture and yeast two-hybrid system are used to discover and identify any functional partners. The nature and functions of the partners are characterized. These partners in turn are targets for drug discovery.

Structural Studies. Recombinant proteins are produced in E coli, yeast, insect and/or mammalian cells and are used in crystallographical and NMR studies. Molecular modeling of the proteins is also employed. These studies facilitate structure-driven drug design.

EXAMPLE

Two Step Assay to Detect the Presence of BRCA2 in a Sample Patient sample is processed according to the method disclosed by Antonarakis et al. (1985), separated through a 1% agarose gel and transferred to nylon membrane for Southern blot analysis.

Membranes are UV cross linked at 150 mJ using a GS Gene Linker (Bio-Rad). A BRCA2 probe selected from the sequence shown in Figure 3 is subcloned into pTZ18U. The phagemids are transformed into E. coli MV1190 infected with M13K07 helper phage (Bio-Rad, Richmond, CA).

Single stranded DNA is isolated according to standard procedures (see Sambrook et al., 1989).

Blots are prehybridized for 15-30 min at 65 0 C in 7% sodium dodecyl sulfate (SDS) in 0.5 M NaPO 4 The methods follow those described by Nguyen et al., 1992. The blots are hybridized overnight at 65 0 C in 7% SDS, 0.5 M NaPO 4 with 25-50 ng/ml single stranded probe DNA. Posthybridization washes consist of two 30 min washes in 5% SDS, 40 mM NaPO 4 at 65 0 C, followed by two 30 min washes in 1% SDS, 40 mM NaPO 4 at 65 0

C.

Next the blots are rinsed with phosphate buffered saline (pH 6.8) for 5 min at room temperature and incubated with 0.2% casein in PBS for 30-60 min at room temperature and rinsed in PBS for 5 min. The blots are then preincubated for 5-10 minutes in a shaking water bath at 0 C with. hybridization buffer consisting of 6 M urea, 0.3 M NaC1, and 5X Denhardt's solution (see Sambrook, et al., 1989). The buffer is removed and replaced with 50-75 pl/cm 2 fresh hybridization buffer plus 2.5 nM of the covalently cross-linked oligonucleotide-alkaline phosphatase conjugate with the nucleotide sequence complementary to the universal primer site (UP-AP, Bio-Rad). The blots are hybridized for 20-30 min at 45°C and post hybridization washes are incubated at 45C as two 10 min washes in 6 M urea, lx standard saline citrate (SSC), 0.1% SDS and one 10 min wash in Ix SSC, 0.1% Triton X-100. The blots are rinsed for 10 min at room temperature with Ix SSC.

Blots are incubated for 10 min at room temperature with shaking in the substrate buffer consisting of 0.1 M diethanolamine, 1 mM MgCl 2 0.02% sodium azide, pH 10.0. Individual blots are placed in heat sealable bags with substrate buffer and 0.2 mM AMPPD spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane, disodium salt, Bio-Rad).

After a 20 min incubation at room temperature with shaking, the excess AMPPD solution is removed. The blot is exposed to X-ray film overnight. Positive bands indicate the presence of .BRCA2.

EXAMPLE 7 Generation of Polvclonal Antibody against BRCA2 Segments of BRCA2 coding sequence are expressed as fusion protein in E. coli. The overexpressed protein is purified by gel elution and used to immunize rabbits and mice using a procedure similar to the one described by Harlow and Lane, 1988. This procedure has been shown to generate Abs against various other proteins (for example, see Kraemer et al, 1993).

Briefly, a stretch of BRCA2 coding sequence selected from the sequence shown in Figure 3 is cloned as a fusion protein in plasmid PET5A (Novagen, Inc., Madison, WI). After induction with IPTG, the overexpression of a fusion protein with the expected molecular weight is verified by SDS/PAGE. Fusion protein is purified from the gel by electroelution. The identification of the protein as the BRCA2 fusion product is verified by protein sequencing at the N-terminus. Next, the purified protein is used as immunogen in rabbits. Rabbits are immunized with 100 4ig of the protein in complete Freund's adjuvant and boosted twice in 3 week intervals, first with 100 pg of immunogen in incomplete Freund's adjuvant followed by 100 pg of immunogen in PBS. Antibody containing serum is collected two weeks thereafter.

This procedure is repeated to generate antibodies against the mutant forms of the BRCA2 gene. These antibodies, in conjunction with antibodies to wild type BRCA2; are used to detect the presence and the relative level of the mutant forms in various tissues and biological fluids.

EXAMPLE 8 Generation of Monoclonal Antibodies Specific for BRCA2 Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising intact BRCA2 or BRCA2 peptides (wild type or mutant) conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC as is well known.

The immunogen is mixed with an adjuvant. Each mouse receives four injections of 10 to 100 pg of immunogen and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen. Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.

Spleens are removed from immune mice and a single cell suspension is prepared (see Harlow S and Lane, 1988). Cell fusions are performed essentially as described by Kohler and Milstein, 1975. Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, MD) are fused with immune spleen cells using polyethylene glycol as described by Harlow and Lane, 1988.

Cells are plated at a density of 2x10 5 cells/well in 96 well tissue cultuie plates. Individual wells are examined for growth and the supematants of wells with growth are tested for the preserice of BRCA2 specific antibodies by ELISA or RIA using wild type or mutant BRCA2 target protein.

30 Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.

-69- Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibody for characterization and assay development.

EXAMPLE9 Sandwich Assay for BRCA2 Monoclonal antibody is attached to a solid surface such as a plate, tube, bead, or particle.

Preferably, the antibody is attached to the well surface of a 96-well ELISA plate. 100 gl sample serum, urine, tissue cytosol) containing the BRCA2 peptide/protein (wild-type or mutant) is added to the solid phase antibody. The sample is incubated for 2 hrs at room temperature. Next the sample fluid is decanted, and the solid phase is washed with buffer to remove unbound material. 100 pl of a second monoclonal antibody (to a different determinant on the BRCA2 peptide/protein) is added to the solid phase. This antibody is labeled with a detector molecule enzyme, fluorophore, or a chromophore) and the solid phase with the second antibody is incubated for two hrs at room temperature. The second antibody is decanted and the solid phase is washed with buffer to remove unbound material.

The amount of bound label, which is proportional to the amount of BRCA2 peptide/protein present in the sample, is quantitated. Separate assays are performed using monoclonal antibodies 20 which are specific for the wild-type BRCA2 as well as monoclonal antibodies specific for each of the mutations identified.in BRCA2.

EXAMPLE The 6174delT Mutation is Common in Ashkenazi Jewish Women Affected by Breast Cancer The 6174delT mutation (see Table 3) has been found to be present in many cases of Ashkenazi Jewish women who have had breast cancer (Neuhausen et al., 1996). Two grdups of probands comprised the ascertainment for this study. The first group was ascertained based on 30 both age-of-onset and a positive family history. The first group consisted of probands affected with breast cancer on or before 41 years of age with or without a family history of breast cancer.

Inclusion criteria for the second group were that the proband was affected with breast cancer between the ages of 41 and 51 with one or more first degree relatives affected with breast or ovarian cancer on or before the age of 50; or the proband was affected with breast cancer between the ages of 41 and 51 with two or more second degree relatives affected with breast or ovarian cancer, 1 on or before age 50; or the proband was affected between the ages of 41 and 51 with both primary breast and primary ovarian cancer. Probands were ascertained through medical oncology and genetic counseling clinics, with an effort to offer study participation to all eligible patients.

Family history was obtained by a self-report questionnaire. Histologic confirmation of diagnosis was obtained for probands in all cases. Religious background was confirmed on all probands by self report or interview.

Mutation Detection The BRCA2 6174delT mutation was detected by amplifying genomic DNA from each patient according to standard polymerase chain.reaction (PCR) procedures (Saiki.et al., 1985; Mullis et al., 1986; Weber and May, 1989). The primers.used for the PCR are: BCI 1-RP: GGGAAGCTTCATAAGTCAGTC (SEQ ID NO: 115) (forward primer) and BC 11-LP: TTTGTAATGAAGCATCTGATACC (SEQ ID NO: 116) (reverse primer).

The reactions were performed in a total volume of 10.0 pl containing 20 ng DNA with annealing at 0 C. This produces a PCR product 97 bp long in wild-type samples and 96 bp long when the 20 6174delT mutation is present. The radiolabeled PCR products were electrophoresed on standard 6% polyacrylamide denaturing sequencing gels at 65W for 2 hours. The gels were then dried and S* autoradiographed. All the cases exhibiting the 1 bp deletion were sequenced to confirm the 6174delT mutation. For sequencing, half of the samples were amplified with one set of PCR primers and the coding strand was sequenced and the other half of the samples were amplified with a second set of PCR primers and the noncoding strand was sequenced. For one set the PCR primers were: AATGATGAATGTAGCACGC (SEQ ID NO: 117) (forward primer) and CGORF-RH: GTCTGAATGTTCGTTACT (SEQ ID NO: 118) (reverse primer).

This results in an amplified product of 342 bp in wild-type and 341 bp for samples containing the 6174delT mutation. For this set of samples the amplified DNA was sequenced using the CGORF- RH primer for the sequencing primer. The other half of the samples were amplified using the BC11-RP forward primer and the CGORF-RH reverse primer resulting in a fragment of 183 bp in wild-type samples and 182 bp in samples containing the 6174delT mutation. This was sequenced using BC11-RP as the sequencing primer.

Results Six out of eighty women of Ashkenazi Jewish ancestry with breast cancer before the age of 42 had the 6174deIT mutation. This compares to zero cases of the mutation being present in a control group of non-Jewish women who had breast cancer before the age of 42. These cases were ascertained without regard to family history. Table 4 shows the results of the study. Four of the six cases with the 6174delT mutation had a family history of breast or ovarian cancer in a first or second degree relative. In each of two kindreds where multiple samples were available for analysis, the 6174delT mutation co-segregated with two or more cases of breast or ovarian cancer.

A second cohort of 27 Ashkenazim with breast cancer at age 42-50 and a history of at least one additional relative affected with breast or ovarian cancer provided an additional estimate of the frequency of the 6174delT mutation. In this group of 27 women, two were heterozygous for the BRCA2 6174delT mutation. One of these individuals had first degree relatives with both ovarian and breast cancer. From the data presented, and assuming a penetrance similar to BRCAI mutations (Offit et al., 1996; Langston et al., 1996), the frequency of the 6174delT mutation in Ashkenazim can be estimated to be approximately 3 per thousand. However, if the penetrance of 20 this mutation is lower than BRCA1, then the frequency of this mutation will be higher. A more precise estimate of the carrier frequency of the 6174delT mutation in individuals of Ashkenazi Jewish ancestry will emerge from large-scale population studies.

e* *l -72- TABLE4 Number of subjects Number with Group tested. n= 6174delT. n= Group la Diagnosis before age 42, Non-Jewisha 93 0 (0) Group lb Diagnosis before age 42, Jewisha .80 .6 (8) Before age 37 40 4 age 37-41 40 2 Group 2 Diagnosis ages 42-50 and family history positiveb 27 2 (27) Key: a Ascertained regardless of family history Family history for this group was defined as one first degree or two second degree relatives diagnosed with breast or ovarian cancer, one before age o o*oo o *o

O'.

o -73- EXAMPLE 11 BRCA2 Shows a Low Somatic Mutation Rate in Breast Carcinoma and Other Cancers Including Ovarian and Pancreatic Cancers BRCA2 is a tumor suppressor gene. A homozygous deletion of this gene may lead to breast cancer as well as other cancers. A homozygous deletion in a pancreatic xenograft was instrumental in the effort to isolate BRCA2 by positional cloning. Cancer may also result if there is a loss of one BRCA2 allele and a mutation in the remaining allele (loss of heterozygosity or LOH).

Mutations in both alleles may also lead to development of cancer. For studies here, an analysis of 150 cell lines derived from different cancers revealed no cases in which there was a homozygous loss of the BRCA2 gene. Because homozygous loss is apparently rare, investigations were made to study smaller lesions such as. point mutations in BRCA2. Since compound mutant heterozygotes and mutant homozygotes are rare, tumor suppressor gene inactivation nearly always involves LOH. The remaining allele, if inactive, typically contains disruptive mutations. To identify these it is useful to preselect tumors or cell lines that exhibit LOH at the locus of interest.

Identification of tumors and cell lines that exhibit LOH S: A group of 104 primary breast tumor samples and a set of 269 cell lines was tested for LOH in the BRCA2 region. For primary tumors, amplifications of three short tandem repeat markers 20 (STRs) were compared quantitatively using fluorescence. Approximately 10 ng of genomic DNA was amplified by PCR with the following three sets of fluorescently tagged STRs: mM4247.4A.2FI ACCATCAAACACATCATCC (SEQ ID NO: 119) mM4247.4A.2R2 AGAAAGTAACTTGGAGGGAG (SEQ IDNO: 120) STR257-FC CTCCTGAAACTGTTCCCTTGG (SEQ IDNO: 121) 25 STR257-RD TAATGGTGCTGGGATATTTGG (SEQ ID NO: 122) mMB561A-3.1FA2 GAATGTCGAAGAGCTTGTC (SEQ IDNO: 123) mMB561A-3.1RB AAACATACGCTTAGCCAGAC (SEQ IDNO: 124) The PCR products were resolved using an ABI 377 sequencer and quantified with Genescan software (ABI). For tumors, clear peak height differences between alleles amplified from normal and tumor samples were scored as having LOH. For cell lines, if one STR was heterozygous, the sample was scored as non-LOH. In only one case was a cell line or tumor miscalled based on later analysis of single base polymorphisms. The heterozygosity indices for the markers are: 'STR4247 0.89; STR257 0.72; STR561A 0.88 Neuhausen, personal communication; B. Swedlund, unpublished data). Based on their combined heterozygosity indices, the chance that the markers are all homozygous in a particular individual (assuming linkage equilibrium) is only one in 250.

Due to the presence of normal cells in the primary tumor sample, LOH seldom eliminates the signal entirely from the allele lost in the tumor. Rather, the relative intensities of the two alleles are altered. This can be seen clearly by comparing the allelic peak heights from normal tissue with peak heights from the tumor (Figs. 5A-5D). Based on this analysis, 30 tumors were classified as having LOH at the BRCA2 locus (Table a figure that is similar to previous estimates (Collins et al., 1995; Cleton-Jansen et al., 1995).

LOH was assessed in the set of cell lines in a different fashion. Since homozygosity of all three STRs was improbable, and since normal cells were not present, apparent homozygosity at all STRs was interpreted as LOH in the BRCA2 region. Using this criterion, 85/269 of the cell lines exhibited LOH (see Table The frequencies varied according to the particular tumor cell type under consideration. For example, 4/6 ovarian cell lines and 31/62 lung cancer lines displayed LOH compared with 17/81 melanoma lines and 2/11 breast cancer lines.

Sequence Analysis of LOH Primary Breast Tumors and Cell Lines The 30 primary breast cancers identified above which showed LOH in the BRCA2 region were screened by DNA sequence analysis for sequence variants. Greater than 95% of the coding 20 sequence and splice junctions was examined. DNA sequencing was carried out either on the ABI 377 (Applied Biosystems Division, Perkin-Elmer) or manually. For the radioactive mutation screen, the amplified products were purified by Qiagen beads (Qiagen, Inc.). DNA sequence was generated using the Cyclist sequencing kit (Stratagene) and resolved on 6% polyacrylamide gels.

In parallel, non-radioactive sequencing using fluorescent labeling dyes was performed using the 25 TaqFS sequencing kit followed by electrophoresis on ABI 377 sequencers. Samples were gridded into 96-well trays to facilitate PCR and sequencing. Dropouts of particular PCR and sequencing reactions were repeated until >95% coverage was obtained for every sample. Sequence information was analyzed with the Sequencher software (Gene Codes Corporation). All detected mutations were confirmed by sequencing a newly amplified PCR product to exclude the possibility that the sequence alteration was due to a PCR artifact.

Astrocytoma B ladder Breast Colon Glioma Lung Lymphoma M elanoma Neuroblastoma, Ovarian Pancreatic Prostate Renal Total Primary Breast #LOH/# Screened 6/19 6/17 2/11 2/8 11/36 31/62 0/4 17/81 1/10 4/6 1/3 0/2 4/10 8 5/2 69 TABLE Pecntge LOH 32% 18% 31% 0% 21% 67% 33% 33% (avg.=28%) 30/104 29% LOH analysis of cell lines and primary breast tumors. Percentage LOH was calculated two ways: as total and as a mean of percentages (avg.).

0*0*0 '0 0 000* .00.

Of the 30 samples, two specimens contained frameshift mutations, one a nonsense mutation, and two contained missense changes (although one of these tumors also contained a frameshift).

The nonsense mutation would delete 156 codons at the C-terminus suggesting that the C-terminal end of BRCA2 is important for tumor suppressor activity. All sequence variants were also present in the corresponding normal DNA from these cancer patients. To exclude the unlikely possibility that preselection for LOH introduced a systematic bias against detecting mutations dominant behavior of mutations, compound heterozygotes), 12 samples shown to be heterozygous at BRCA2 were also screened. Three of these revealed missense changes that were also found in the normal samples. Thus, in a set of 42 breast carcinoma samples, 30 of which displayed LOH at the BRCA2 locus, no somatic mutations were identified. The frameshift and nonsense changes are likely to be predisposing mutations that influenced development of breast cancer in these patients. The missense variants are rare; they were each observed only once during analysis of 115 chromosomes. From these data- it is not possible to distinguish between rare neutral polymorphisms and predisposing mutations.

Of the 85 cell lines which displayed LOH (see Table 58 were also screened for sequence changes. Greater than 95% of the coding sequence of each sample was screened. Only a single frameshift mutation was identified by this DNA sequence analysis. This mutation (6174delT) was present in a pancreatic cancer line and it is identical to one found in the BTl11 primary tumor sample and to a previously detected germline frameshift (Tavtigian et al., 1996). This suggests that 20 this particular frameshift may be a relatively common germline BRCA2 mutation. In addition, a number of missense sequence variants were detected (Tables 6A and 6B).

Detection of a probable germline BRCA2 mutation in a pancreatic tumor cell line suggests that BRCA2 mutations may predispose to panEreatic cancer, a possibility that has not been explored thoroughly. This mutation also adds weight to the involvement of BRCA2 in sporadic pancreatic cancer, implied previously by the homozygous deletion observed in a pancreatic xenograft (Schutte et al., 1995). Because only three pancreatic cell lines were examined in our study, further investigation of BRCA2 mutations in pancreatic cancers is warranted.

Sample TIpe 4G1 2F8 BT110 4F8 BT163 1D6 BT333 2A2 214 BT111 4G3 1B7 BT118 BT115 1E4 BT110 Renal Ovarian Lung Primary breast Ovarian Primary breast Bladder Primary breast Glioma Lung Primary breast Pancreatic Astrocytoma Primary breast Primary breast Melanoma Primary breast Breast Primary breast

LQH

yes yes yes yes .yes no no no yes yes yes.

yes yes no yes yes yes yes yes TABLE6A Change G451C A1093C G1291C 1493delA C2117T A2411C G4813A T5868G C5972T C5972T 6174delT 6174delT C6328T G7049T G7491C A9537G A10204T C10298G A10462G Effec Ala Pro Asn His Val Leu Frameshift Thr Ile Asp Ala Gly Arg Asn Lys Thr -Met Thr Met Frameshift Frameshift Arg Cys Gly Val Gin His lie Met Lys Stop Thr Arg Ile Val Germline yes yes yes yes yes yes yes yes *r Germline mutations identified in BRCA2. Listed are the mutation positions based on the Genbank entry of BRCA2 (Schutte et al., 1995).

Position 5'UTR(203) PM(1342) PM(2457).

PM(3199) PM(3624) PM(3668) PM(4035) PM(7470) 1593 4296 5691 6051 6828 6921 TABLE 6B Change Effect

G/A

C/A His Asn T/C silent A/G Asn Asp A/G silent A/G Asn Ser T/C silent A/G silent A G silent G A silent A G silent A -G silent T C silent C silent Frequency 0.32 (0.26) 0.32 (0.37) 0.04 (0.05) 0.04 (0.08) 0.35 0(0.15) 0.24 (0.10) 0.26 (0.15) <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Common polymorphisms and silent substitutions detected in BRCA2 by DNA sequencing. Since some rare silent variants may affect gene function splicing (Richard and Beckmann, 1995)), these are not preceded by The frequencies of polymorphisms shown involve the second of the nucleotide pair. Frequencies reported in a previous study are shown in parentheses (Tavtigian et al., 1996). Numbering is as in Table 6A.

Industrial Utility As previously described above, the present invention provides materials and methods for use in testing BRCA2 alleles of an individual and an interpretation of the normal or predisposing nature of the alleles. Individuals at higher than normal risk might modify their lifestyles appropriately. In the case of BRCA2, the most significant non-genetic risk factor is the protective effect of an early, full term pregnancy. Therefore, women at risk could consider early childbearing or a therapy designed to simulate the hormonal effects of an early full-term pregnancy. Women at high risk would also strive for early detection and would be more highly motivated to learn and practice breast self examination. Such women would also be highly motivated to have regular mammograms, perhaps starting at an earlier age than the general population. Ovarian screening could also be undertaken at greater frequency. Diagnostic methods based on sequence analysis of the BRCA2 locus could also be applied to tumor detection and classification. Sequence analysis could be used to diagnose precursor lesions. With the evolution of the method and the accumulation of information about BRCA2 and other causative loci, it could become possible to separate cancers into benign and malignant.

Women with breast cancers may follow different surgical procedures if they are predisposed, and therefore likely to have additional cancers, than if they are not predisposed. Other therapies may be developed, using either peptides or small molecules (rational drug design). Peptides could be the missing gene product itself or a portion of the missing gene product. Alternatively, the therapeutic agent could be another molecule that mimics the deleterious gene's function, either a peptide or a nonpeptidic molecule that seeks to counteract the deleterious effect of the inherited locus. The therapy could also be gene based, through introduction of a normal BRCA2 allele into individuals to make a protein which will counteract the effect of the deleterious allele. These gene therapies may take many forms and may be directed either toward preventing the tumor from forming, curing a cancer once it has occurred, or stopping a cancer from metastasizing.

It will be appreciated that the methods and compositions of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the I 30 invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

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List of Patents and Patent Applications: U.S. Patent No. 3,817,837 U.S. Patent No. 3,850,752 U.S. Patent No. 3,939,350 U.S. Patent No. 3,996,345 U.S. Patent No. 4,275,149 U.S. Patent No. 4,277,437 U.S. Patent No. 4,366,241 U.S. Patent No. 4,376,110 U.S. Patent No. 4,486,530 U.S. Patent No. 4,683,195 U.S. Patent No. 4,683,202 U.S. Patent No. 4,816,567 30 U.S. Patent No. 4,868,105 U.S. Patent No. 5,252,479 S.oo*** EPO Publication No. 225,807 European Patent Application Publication No. 0332435

C

•Geysen, PCT published application WO 84/03564, published 13 September 1984 Hitzeman et al., EP 73,675A PCT published application WO 93/07282 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Myriad Genetics, Inc.

The Trustees of the University of Pennsylvania Endo Recherche, Inc.

HSC Research Development Limited Partnership (ii) TITLE OF INVENTION: Chromosome 13-Linked Breast Cancer Susceptibility Gene (iii) NUMBER OF SEQUENCES: 124 (iv) CORRESPONDENCE

ADDRESS:

ADDRESSEE: Venable, Baetjer, Howard Civiletti STREET: 1201 New York Avenue Suite 1001 CITY: Washington STATE: DC COUNTRY: USA ZIP: 22204 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: Word for Windows (vi) CURRENT APPLICATION

DATA:

APPLICATION NUMBER: WO FILING DATE: 27-DEC-1996

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Ihnen, Jeffrey L.

REGISTRATION NUMBER: 28,957 REFERENCE/DOCKET NUMBER: 24884-116802-WO (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: 202-962-4810 TELEFAX: 202-962-8300 *45 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 11385 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear

M

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: CDS LOCATION: 229. .10482 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: GGTGGCGCOA OCTTCTGAA.A CTAGGCGGCA GAGG;CGGAGC CGCTGTGGCA CTGCTGCGCC TCTGCTGCGC CTCGGGTGTC TTTTGCGGCG GTGGGTCGCC GCCGGGAGAA GCGTOAGGGG ACAGATTTGT GACCGGCGCG GTTTTTGTCA GCTTACTCCG GCCAAAAAAG AACTOCACCT CTGGAGCGGA CTTATTTACC AAGCATTGGA GGAATATCGT AGGTAAAA ATG CCT ATT Met Pro Ile GGA TCC AAA GAG AGO CCA ACA TI'T TTT GAA ATT TTT AAG ACA' CGC TGC Gly Ser Lys Glu Arg Pro Thr Phe Phe Glu Ile Phe Lys Thr Arg Cys 510 AAC AAA OCA GAT TTA OGA CCA ATA AGT CTT AAT TGG TTT GAA GAA CTT 120 237 Asn Lys Ala Asp Leu Gly Pro Ile Ser Leu Asn Trp Phe Giu Glu Leu 33 0 00 TCT TCA GAA OCT CCA CCC TAT AAT TCT Ser Ser Giu Ala Pro Pro Tyr Asn Ser GAA CCT OCA GAA OAA TCT GAA Giu Pro Ala Olu Giu Ser Glu 45 so 333 381 429 CAT AkA AAC His Lys Asn AAT TAC GAA CCA AAC CTA TTT .AAA ACT CCA CAA AGO Asn Tyr 01u Pro Asn Leu Phe Lys Thr Pro Gin Arg AAA CCA TCT Lys Pro Ser TAT AAT CAG CTG Tyr Asn Gin Leu TCA ACT CCA ATA Ser Thr Pro Ile TTC AAA GAG Phe Lys Giu CAA 000 CTG ACT CTG Gin Gly Leu Thr Leu CCO CTO Pro Leu TAC CAA TCT CCT OTA AAA GAA TTA OAT Tyr Gin Ser Pro Val Lys Oiu Leu Asp TTC AAA TTA GAC Phe Lys Leu Asp OGA AGO AAT OTT Oiy Arg Asn Val AAT AOT AGA CAT Asn Ser Arg His

AAA

Lys 115 AGT CTT COC ACA OTO AAA ACT AAA ATO OAT CAA OCA GAT Ser Leu Arg Thr Val Lys Thr Lys Met Asp Gin Ala Asp 120 125 OAT OTT TCC Asp Val Ser 130 TOT .CCA CTT CTA AAT TCT TOT CTT Cys Pro Leu Leu Asn Ser Cys Leu 133 AGT OAA AGT CCT OTT OTT CTA CAA Ser Olu Ser Pro Val Val Leu Gin TGT ACA CAT Cys Thr His 150 TTG TTT CAT Leu Phe His 165 GTA ACA*CCA CAA AGA GAT AAG TCA GTG GTA TGT GGG AGT Val Thr Pro Gin Arg Asp Lys Ser Val Val Cys Gly Ser ACA CCA AAG Thr Pro Lys GTG AAG GGT CGT Val Lys Gly Arg CAG ACA CCA AAA CAT Gin Thr 175 Pro Lys His

ATT

Ile 180 TCT GAA AGT Ser Giu Ser CTA GGA Leu Gly 185 GCT GAG GTG GAT CCT Ala Giu Val Asp Pro 190 GAT ATO TCT TGG Asp Met Ser Trp AGT TCT TTA GCT Ser Ser Leu Ala

ACA

Thr 200 CCA CCC ACC CTT Pro Pro Thr Leu AGT TCT Ser Ser 205 ACT GTG CTC Thr Val Leu ATA OTC Ile Val 210 AGA AAT CAA Arg Asn Glu AAT GTC AAA Asn Val Lys 230 GCA TCT CAA ACT Ala Ser Glu Thr GTA TTT Val Phe 220 CCT CAT CAT Pro His Asp ACT ACT OCT Thr Thr Ala 225 AAC AAA AAT Lys Lys Asn AGC TAT TTT TCC AAT CAT CAT GAA AGT Ser Tyr Phe Ser Asn His Asp Glu Ser CAT AGA TTT ATC OCT TCT Asp Arg Phe Ile Ala Ser ACA CAC ACT GAA Thr Asp Ser Glu ACA AAT CAA AGA Thr ASn Gin Arg 9 9@9* 9 0 *9*rO.

*99900 9 099@ 9

OAA

Glu 260 OCT OCA ACT CAT Ala Ala Ser His

GGA

Oly 265 TTT GGA AAA ACA Phe Gly Lys Thr GGG AAT TCA TTT Gly Asn Ser Phe OTA AAT AGC TGC 35 Val Asn Ser Cys

AAA

Lys 280 CAC CAC ATT GGA Asp His Ile Oly

AAC

Lys 285 OAA OAT OAA Olu Asp Glu TTT TCA TTA Phe Ser Leu 310 TAT OAA ACA OTT Tyr Glu Thr Val TGT TTT TCT AAA Cys Phe Ser Lys OTA OAT Val Asp 300 AGA ACA Arg Thr ATT TTC lie Phe TCA ATO CCA Ser Met Pro ACC TCT OAA Thr Ser Clu AAA AAT CTA Lys Asn Leu 320 CAT GAA OCA His Clu Ala 335 AAT CTC CTA Asn Val Leu 290 CAA OAT AGT Glu Asp Ser 305 CAA AAA OTA Cmn Lys Val AAC CCT OAT Asn Ala Asp 1005 1053 1101 1149 1197 1245 1293 AGA ACT Arg Thr 325 AGC AAC ACT AGG Ser Lys Thr Arg AAA AAA Lys Lys 330 0 6@ 99 9 c 0 0699 50 OAA Olu 340 TGT GAA AAA Cys Glu Lys TCT AAA AAC CAA GTG AAA GAA Ser Lys Asn 345 Gin Val Lys AAA TAC TCA Lys Tyr Ser TTT OTA Phe Val 355 OTA OCA Val Ala .3 TCT CAA GTG GAA Ser Glu Val Glu CCA AAT OAT ACT CAT CCA Pro Asr Asp Thr Asp Pro .36.0365 TTA OAT TCA AAT Leu Asp Ser Asn 1341 -92- CAT CAG AAG CCC TTT His Gin Lys Pro Phe 375 GAG AGT GGA AGT Giu Ser Gly Ser .380 GAC AMA ATC TCC Asp Lys Ile Ser CAA CTA ACC CTT Gin Leu Thr Leu 400 MAG GMA GTT Lys Giu Vai 385 TCA GGT CTA Ser Giy Leu 1389 1.437 GTA CCG TCT Val Pro Ser 390 TTG GCC TGT GMA TGG TCT Leu Aia Cys Giu Trp Ser 395 MAT GGA Asn Gly 405 GCC CAG ATG GAG Ala Gin Met Giu ATA CCC CTA TTG CAT ATT TCT TCA TGT Ile Pro Leu Leu His Ile Ser Ser Cys 415 1485

GAC

Asp 420 CMA MT ATT TCA Gin Asn Ile Ser AMA GAC CTA Lys Asp Leu TTA GAC Leu Asp 430 ACA GAG MAC AMA Thr Giu Asn Lys

AGA

Arg 435 1.533 1.581.

MAG AMA GAT TTT Lys Lys Asp Phe ACT TCA GAG MAT Thr Ser Giu Asn

TCT

S er 445 TTG CCA CGT ATT Leu Pro Arg Ile TCT AGC Ser Ser 450 CTA CCA AMA Leu Pro Lys AGA GAT GMA Arg Asp Giu 470 GAG MAG CCA TTA Giu Lys Pro Leu

MAT

Asn 460 GAG GMA ACA GTG Giu Giu Thr Val GTA MAT MAG Vai Asn Lys 465 ATT CTT GCA Ile Leu Ala 1.629 1.677 GAG CAG CAT Giu Gin His CTT GMA Leu Giu 475 TCT CAT ACA GAC Ser His Thr Asp 0e S S OS 0e 0

OS..

0 0 0 0 150000

S

0 105505 0 *055 I S 0 0 6*O* 0 0@@S 30 GTA MAG CAG Val Lys Gin 485 GCA ATA TCT GGA ACT TCT CCA GTG GCT Ala Ile Ser Giy Thr Ser Pro Val Ala 490 495 TCT TCA TTT CAG Ser Ser Phe Gin 1.725 1.773

GGT

35 Gly 500 ATC AMA MG TCT ATA TTC AGA ATA Ile Lys Lys Ser Ile Phe Arg Ile 505 AGA GMA Arg Giu 52.0 TCA CCT AMA GAG Ser Pro Lys Giu

ACT

Thr 52.5 TTC MAT GCA AGT Phe Asn Ala Ser TCA GGT CAT ATG Ser Gly His Met

ACT

Thr 525 GAT CCA MAC TTT Asp Pro Asn Phe AMA AMA Lys Lys 530 GMA ACT GMA Giu Thr Giu CAG MAG GAG Gin Lys G2.u .550 TCT GMA AGT GGA Ser Giu Ser Gly GMA ATA CAT ACT Giu Ile His Thr GTT TGC TCA Val Cys Ser 545 GGA AGC TGG Giy Ser Trp 1.821.

1.869 1.917 GAC TCC TTA TGT CCA MAT TTA ATT GAT Asp Ser Leu Cys Pro Asn Leu Ile Asp 555 CCA GCC Pro Ala ACC ACC ACA CAG MAT TCT GTA GCT TTG Thr Thr Thr Gin Asn Ser Val Ala Leu 570 MAT GCA GGT TTA Asn Aia Giy Leu 1.965 ATA TCC ACT TTG MAA MAG AMA ACA MAT M-G TTT ATT TAT GCT ATA CAT Ile Ser Thr Leu Lys Lys Lys Thr Asn Lys Phe Ile Tyr Ala Ile His 580 585 590 9 202.3 GAT GAA ACA Asp Giu Thr GAA CTA ATT Giu Leu Ile

TCT

Ser TAT AAA Tyr Lys 600 GGA AAA AAA Giy Lys Lys

ATA

Ile 605 CCG AAA GAC CAA Pro Lys Asp Gin AAA TCA Lys Ser 610 2061 2109 AAC TGT TCA GCC CAG TTT GAA GCA AAT GCT TTT GAA GCA Asn Cys Ser Ala Gin Phe Giu Ala Asn Ala Phe Giu Aia 615 620 .625 CCA CTT ACA TTT Pro Leu Thr Phe 630 GCA AAT GCT Ala Asn Ala GAT TCA GGT Asp Ser Gly 635 TTA TTG CAT Leu Leu His 640 TCT TCT GTG Ser Ser Vai TTG TCC TTA Leu Ser Leu 2157 2205 AAA AGA Lys Arg 645 AGC TGT TCA CAG Ser Cys Ser Gin GAT TCT GAA GAA Asp Ser Giu Giu CCA ACT Pro Thr 655

ACT

Thr 660 AGC TCT TTT GGG Ser Ser Phe Gly

ACA

Thr 665 ATT CTG AGG AAA Ile Leu Arg Lys TCT AGA AAT Ser Arg Asn GAA ACA Glu Thr 675 GAA GCA Giu Ala 690 2253 2301 TGT TCT AAT AAT Cys Ser Asn Asn GTA ATC TCT CAG Val Ile Ser Gin CTT GAT TAT AA Leu Asp Tyr Lys AAA TGT AAT Lys Cys Asn 30 TCT CTG TCA Ser Leu Ser 710 AAA AAA GTT Lys Lys Val 725 GAA AAA CTA CAG Glu Lys Leu Gin

TTA

Leu 700 TTT ATT ACC CCA Phe Ile Thr Pro GAA GCT GAT Giu Ala Asp 705 CCA AAA AGC Pro Lys Ser TGC CTG CAG GAA Cys Leu Gin Giu CAG TGT GAA AAT Gin Cys Glu Asn 2349 2397 2445 TCA GAT ATA Ser Asp Ile GAA GAG GTC TTG Giu Giu Val Leu

GCT

Al a 735 GCA GCA TGT CAC Ala Ala Cys His GAC TTT CAA TCC Asp Phe Gin Ser

CCA

Pro 40 740 GTA CAA CAT TCA Val Gin His Ser GTG GAA TAC AGT Val Giu Tyr Ser GAT ACT Asp Thr 750 2493 CAG AAA AGT CTT Gin Lys Ser Leu TAT GAT CAT GAA Tyr Asp His Glu GCC AGC ACT CTT Ala Ser Thr Leu ATT TTA Ile Leu 770 ACT CCT ACT Thr Pro Thr GGC AAA GAA Gly Lys Giu 790 AAG GAT GTT CTG Lys Asp Val Leu

TCA

Ser 780 AAC CTA GTC ATG Asn Leu Val Met ATT TCT AGA Ile Ser Arg 785 AAC AAT TAT Asn Asn Tyr 2541 2589 2637 TCA TAC AAA ATG TCA GAC AAG CTC AAA Ser Tyr Lys Met Ser Asp Lys Leu Lys .795 GAA TCT Giu Ser 805 GAT GTT GAA TTA ACC AAA AAT ATT CCC ATG GAA AAG AAT CAA Asp Val Giu Leu Thr Lys Asn Ile Pro Met Giu Lys Asn Gin 810 81-5 2685 GAT GTA Asp Val 820 TGT GCT TTA Cys Ala Leu AAT GAA AAT TAT AAA AAC GTT GAG CTG TTG CCA Asn Glu Asn Tyr Lys Asn Val Giu Leu Leu Pro 825 -830 835 CCT GAA AAA TAC Pro Giu Lys Tyr AGA GTA GCA Arg Val. Ala TCA CCT Ser Pro 845 TCA AGA AAG GTA CAA TTC Ser Arg Lys Val Gin Phe 850 2733 2781 2829 AAC CAA AAC Asn Gin Asn AAT CTA AGA GTA Asn Leu Arg Val CAA AAA AAT CAA Gin Lys Asn Gin GAA GAA ACT Giu Giu Thr 865 GAA CTT TTC Giu Leu Phe ACT TCA ATT TCA AAA ATA ACT GTC Thr Ser Ile 870 Ser Lys Ile Thr AAT CCA GAC Asn Pro Asp TCT GAA Ser Giu 880 TCA GAC Ser Asp 885 AAT GAG AAT Asn Giu Asn AAT TTT Asn Phe 890 AAT ACT Asn-Thr 905 GTC TTC CAA Val. Phe Gin GTA GCT Vai Aia 895 AAT GAA AGG AAT Asn.Giu Arg Asn

AAT

Asn 900 CTT GCT TTA GGA Leu Aia Leu Gly AAG GAA CTT CAT Lys Giu Leu His 910 GAA ACA GAC TTG Giu Thr Asp Leu

ACT

Thr 915 2877 2925 2973 3021 3069 3117 TGT GTA AAC GAA Cys Val Asn Giu ATT TTC AAG AAC Ile Phe Lys Asn ACC ATG OTT TTA Thr Met Val Leu TAT GGA Tyr Gly 930 GAC ACA GOT OAT AAA Asp Thr Oly Asp Lys 935 CAA GCA ACC CAA GTO TCA ATT AAA Gin Ala Thr Gin Val Ser Ile Lys 940 AAA OAT TTG Lys Asp Leu 945 CAG CAT ATA Gin His Ile GTT TAT GTT 35 Val Tyr Val 950 CTT GCA GAG GAO Leu Ala Giu Giu AAA AAT AGT Lys Asn Ser OTA AAO Vai Lys 960 AAA ATO Lys Met 965 ACT CTA GGT CAA Thr Leu Gly Gin TTA AAA TCG OAC Leu Lys Ser Asp

ATC

Ile 975 TCC TTO AAT ATA Ser Leu Asn Ile AAA ATA CCA OAA Lys Ile Pro Glu AAT AAT GAT Asn Asn Asp TAC ATG Tyr Met 990 AAC AAA TGG GCA Asn Lys Trp Ala

GGA

Gly 995 CTC TTA GOT CCA ATT TCA AAT CAC Leu Leu Gly Pro Ile Ser Asn His 1000 AOT TTT OGA Ser Phe Oly 1005 GGT AOC TTC AGA ACA Gly Ser Phe Arg Thr 1010 3165 3213 3261 3309 3357 GCT TCA AAT AAG .GAA ATC AAG CTC TCT GAA CAT AAC Ala Ser Asn Lys Giu Ile Lys Leu Ser Glu His Asn 1015 1020 AAA ATO TTC TTC AAA GAT ATT GAA GAA CAA TAT Lys Met Phe Phe Lys Asp Ile Glu Glu Gin Tyr 1030 .1Q3 5

CCT

Pro ATT AAG AAG AGC Ile Lys Lys Ser 1025 ACT AGT TTA GCT Thr Ser Leu Ala 0.4 0 TOT GTT GAA ATT GTA AAT ACC TTG GCA TTA Cys Val Giu Ile Val. Asn Thr Leu Ala Leu 1045 1050 GAT AAT CAA AAG AAA CTG Asp Asn Gin Lys Lys Leu 1055 3405 3453 AGC AAG CCT Ser Lys Pro 1060 CAG TCA ATT AAT ACT GTA TCT Gln Ser Ile Asn Thr Val Ser 1065 GCA CAT TTA Ala His Leu 1070 CAG AGT AGT Gin Ser Ser 1075 GTA GTT GTT Val Val Val TCT GAT TGT Ser Asp Cys 1080 AAA AAT AGT CAT ATA Lys Asn Ser His Ile 1085 ACC CCT CAG ATG TTA Thr Pro Gin Met Leu 1090 TTT TCC AAG CAG GAT TTT AAT TCA AAC CAT AAT TTA ACA CCT AGC CAA Phe Ser Lys Gin Asp Phe Asn Ser Asn His Asn Leu Thr Pro Ser Gin 1095 1100 1105 AAG GCA GAA ATT ACA GAA CTT TCT ACT ATA TTA GAA GAA TCA GGA AGT Lys Ala Oiu Ile Thr Oiu Leu Ser Thr Ile Leu Giu Oiu Ser Gly Ser 1110 1115 1120 CAG TTT OAA TTT ACT CAG TTT AGA AAA CCA AOC TAC ATA TTG CAG AAG Gin Phe Oiu Phe Thr Gin Phe Arg Lys Pro Ser Tyr Ile Leu Gin Lys 1125 1130 1135 AOT ACA TTT OAA OTO CCT GAA AAC CAG ATG ACT ATC TTA AAO ACC ACT Ser Thr Phe Olu Val Pro Oiu Asn Gin Met Thr Ile Leu Lys Thr Thr 1140 1145 1150 1155 3501 3549 3597 3645 3693 3741 3789

S

TCT GAG AA.

Ser Oi Ou TCG ATT GOT 35 Ser Ile Oly TGC AGA GAT Cys Arg Asp 1160 CAG OTA GAC Gin Vai Asp 1175 OCT OAT CTT CAT GTC ATA ATO Ala Asp Leu His Val Ile Met 1165 AAT 0CC CCA Asn Ala Pro 1170 AOC AOC AAO CAA TTT Ser Ser Lys Gin Phe 1180 OAA GOT ACA OTT OAA Oiu Oly Thr Val Olu 1185 ATT AAA COO AAG TTT OCT GOC CTO TTO AAA AAT GAC TOT AAC AAA AOT Ile Lys Arg Lys Phe Ala Oly Leu Leu Lys Asn Asp Cys Asn Lys Ser 1190 1195 1200 OCT TCT GOT TAT TTA ACA OAT OAA AAT OAA OTO 000 TTT AOO GOC TTT Ala Ser Gly Tyr Leu Thr Asp Oiu Asn 0Th Val Oly Phe Arg Giy Phe 1205 1210 1215 TAT TCT OCT CAT GOC ACA AAA CTO AAT OTT TCT ACT OAA OCT CTG CAA Tyr Ser Ala His Oly Thr Lys .Leu Asn Val Ser Thr Olu Ala Leu Gin 1220 1225 1230 1235 50 AAA OCT OTO AAA CTO TTT AGT OAT ATT GAO AAT ATT AOT GAO OAA ACT Lys Ala Vai Lys Leu Phe Ser Asp Ile Oiu Asn Ile Ser Giu Olu Thr 1240 1245 1250 TCT OCA GAO OTA CAT CCA ATA AOT TTA TCT TCA AGT AAA TOT CAT OAT Ser Ala Giu Val His Pro Ile Ser Leu Ser Ser Ser Lys Cys His Asp 1255 1260 -12 611 3837 3885 3933 3981 4029 TCT GTT GTT TCA ATG TTT AAG ATA GAA AAT CAT AAT GAT AAA ACT GTA Ser Val Val Ser Met Phe Lys Ile Glu Asn His Asn Asp Lys Thr Val 1270 1275 1280 AGT GAA AAA AAT AAT AAA TGC CAA Ser Glu Lys Asn Asn Lys Cys Gin 1285 1290 CTG ATA TTA CAA AAT AAT ATT GAA Leu Ile Leu Gin Asn Asn Ile Glu 1295 4077 4125 4173 ATG ACT ACT Met Thr Thr 1300 AAT ACT GAA Asn Thr Glu GGC ACT TTT GTT GAA Gly Thr Phe Val Glu 1305 AAT GAA GAT AAC AAA Asn Glu Asp Asn Lys 1320 GAA ATT ACT GAA AAT TAC Glu Ile Thr Glu Asn Tyr 1310 AAG AGA Lys Arg 1315 TAT ACT GCT GCC AGT Tyr Thr Ala Ala Ser 1325 AGA AAT TCT Arg Asn Ser 1330 4221 CAT AAC TTA GAA TTT GAT GGC AGT GAT TCA AGT AAA AAT GAT ACT GTT His Asn Leu Glu Phe Asp Gly Ser Asp Ser Ser Lys Asn Asp Thr Val 1335 1340 1345 TGT ATT CAT AAA GAT GAA ACG GAC TTG CTA TTT ACT GAT CAG CAC AAC Cys Ile His Lys Asp Glu Thr Asp Leu Leu Phe Thr Asp Gin His Asn 1350 1355 .1360 ATA TGT CTT AAA TTA TCT GGC CAG TTT ATG AAG GAG GGA AAC ACT CAG Ile Cys Leu Lys Leu Ser Gly Gin Phe Met Lys Glu Gly Asn Thr Gin 1365 1370 1375 r 30 ATT AAA Ile Lys 1380 GAA GAT TTG TCA GAT TTA ACT TTT TTG GAA GTT GCG AAA GCT Glu Asp Leu Ser Asp Leu Thr Phe Leu Glu Val Ala Lys Ala 1385 1390 1395 CAA GAA GCA TGT CAT GGT AAT ACT TCA AAT AAA GAA CAG TTA ACT GCT Gin Glu Ala Cys His Gly Asn Thr Ser Asn Lys Glu Gin Leu Thr Ala 1400 1405 1410 ACT AAA ACG GAG CAA AAT ATA AAA GAT TTT GAG ACT TCT GAT ACA TTT Thr Lys Thr Glu Gin Asn Ile Lys Asp Phe Glu Thr Ser Asp Thr Phe 1415 1420 1425 TTT CAG ACT GCA AGT GGG AAA AAT ATT AGT GTC GCC AAA GAG TCA TTT Phe Gin Thr Ala Ser Gly Lys Asn Ile Ser Val Ala Lys Glu Ser Phe 1430 1435 1440 AAT AAA ATT GTA AAT TTC TTT GAT CAG AAA CCA GAA GAA TTG CAT AAC.

Asn Lys Ile Val Asn Phe Phe Asp Gin Lys Pro Glu Glu Leu His Asn 1445 1450 1455 4269 4317 4365 4413 4461 4509 4557 4605 4653 TTT TCC TTA AAT Phe Ser Leu Asn 1460 GAC ATT CTA AGT Asp Ile Leu Ser TCT GAA TTA CAT TCT GAC Ser Glu Leu His Ser Asp 1465 ATA AGA AAG AAC Ile Arg Lys Asn 1470 AAA ATG Lys Met 1475 TAT GAG GAA ACA GAC ATA GTT AAA CAC AAA ATA CTG Tyr Glu Glu Thr Asp Ile Val Lys His Lys Ile Leu 1480 1485 1490 4701 AAA GAA AGT GTC CCA GTT GGT ACT GGA AAT CAA CTA GTG ACC TTC CAG Lys Giu Ser Val Pro Val Giy Thr Gly Asn Gin Leu Val Thr Phe Gin 1495 1500 1505 GGA CAA CCC GAA CGT GAT GAA AAG ATC AA.A GAA CCT ACT CTG TTG GGT Giy Gin Pro Giu Arg Asp Giu Lys Ile Lys Glu Pro Thr Leu Leu Gly 1510 1515 1520 4749 4797 TTT CAT ACA Phe His Thr 1525 GAC AAA GTG Asp Lys Val 1540 GCT AGC GGG AAA AAA Ala"Ser Gly Lys Lys 1530 AAA AAC CTT TTT GAT Lys Asn Leu Phe Asp 1545 GTT AAA ATT GCA AAG GAA TCT TTG Vai Lys Ile Ala Lys Giu Ser Leu 1535 GAA AA.A GAG CAA GGT ACT AGT GAA Giu Lys Giu Gin Gly Thr Ser Giu 1550 1555 4845 4893 ATC ACC AGT TTT AGC CAT CAA TGG GCA AAG ACC Ile Thr Ser Phe Ser His Gin Trp, Ala Lys Thr 1560 1S65 GCC TGT AAA Ala Cys Lys GAC CTT GAA TTA GCA Asp Leu GiuLeu Aia 1575 TGT GAG ACC Cys Glu Thr 1580 CTA AAG TAC AGA GAG Leu Lys Tyr Arg Giu 1570 ATT GAG ATC ACA GCT Ile Glu Ile Thr Aia 1585 AAT AAT GAT AAA AAC Asn Asn Asp Lys Asn 1600 4941 GCC CCA AAG Ala Pro Lys 159c CTT GTT TCT Leu Vai'Ser 1605 TTA TGT AGA 35 Leu Cys Arg 1620 AAA GAA ATG CAG AAT TCT CTC Lys Glu Met Gin Asn Ser Leu 1595 4989 5037 5085 5133 ATT GAG ACT GTG GTG Ile Giu Thr Val Val 1610 CAA ACT GAA AAT CTC Gin Thr Giu Asn Leu 1625 CCA CCT AAG CTC TTA AGT GAT AAT Pro Pro Lys Leu Leu Ser Asp Asn 1615 AAA ACA TCA AAA AGT ATC TTT Lys Thr Ser Lys Ser Ile Phe 1630

TTG

Leu 1635 AAA GTT AAA GTA CAT. GAA AAT GTA GAA AA.A GAA ACA GCA AAA AGT CCT Lys Val Lys Val His Giu Asn Val Glu Lys Glu Thr Ala Lys Ser Pro 40 1640 1645 1650 GCA ACT TGT TAC ACA AAT CAG TCC CCT TAT TCA GTC ATT GAA AAT TCA Ala Thr Cys Tyr Thr Asn Gin Ser Pro Tyr Ser Vai Ile Glu Asn Ser 1655 1660 1665 GCC TTA GCT TTT TAC ACA AGT TGT AGT AGA AAA ACT TCT GTG AGT CAG Ala Leu Ala Phe Tyr Thr Ser Cys Ser Arg Lys Thr Ser Vai Ser Gin 1670 1675 1680 s0 ACT TCA TTA CTT GAA GCA AAA AAA TGG CTT AGA GAA GGA ATA. TTT GAT Thr Ser Leu Leu Giu Ala Lys Lys Trp Leu Arg Giu Gly Ile Phe Asp 1685 1690 1695 GGT CAA CCA GAA AGA ATA AAT ACT GCA GAT TAT GTA GGA AAT TAT TTG Gly Gin Pro Giu Arg Ile Asn Thr Ala Asp Tyr Vai Giy Asn Tyr Leu 1700 1705 1710 1.71.5 5181 5229 5277 5325 5373 TAT GAA AAT AAT TCA AAC AGT ACT ATA GCT GAA AAT GAC Tyr Glu Asn Asn Ser Asn Ser Thr Ile Ala Glu Asn Asp 1720 1725 AAA AAT CAT Lys Asn His 1730 ATG TCT AAC Met Ser Asn 1745 5421 5469 CTC TCC GAA AAA CAA GAT ACT TAT TTA AGT AAC AGT AGC Leu Ser Glu Lys Gin Asp Thr Tyr Leu Ser Asn Ser Ser 1735 1740 AGC TAT TCC TAC CAT TCT GAT Ser Tyr Ser Tyr His Ser Asp 1750 GAG GTA TAT Glu Val Tyr 1755 AAT GAT TCA GGA TAT CTC Asn Asp Ser Gly Tyr Leu 1760 CCA GTA TTG AAG AAT GTT Pro Val Leu Lys Asn Val 1775 5517 5565 TCA AAA AAT AAA Ser Lys Asn Lys 1765 CTT GAT TCT GGT ATT GAG Leu Asp Ser Gly Ile Glu 1770 GAA GAT CAA AAA AAC ACT AGT TTT TCC AAA GTA ATA TCC AAT GTA AAA Glu Asp Gin Lys Asn Thr Ser Phe Ser Lys Val Ile Ser Asn Val Lys 1780 1785 1790 1795 5613 GAT GCA AAT GCA Asp Ala Asn Ala TAC CCA CAA ACT GTA AAT GAA GAT ATT TGC GTT GAG Tyr Pro Gin Thr Val Asn Glu Asp Ile Cys Val Glu 1800 1805 1810 r r GAA CTT GTG ACT AGC TCT TCA CCC TGC AAA AAT AAA AAT GCA GCC ATT Glu Leu Val Thr Ser Ser Ser Pro Cys Lys Asn Lys Asn Ala Ala Ile 1815 1820 1825 AAA TTG TCC ATA TCT AAT AGT AAT AAT TTT GAG GTA GGG CCA CCT GCA Lys Leu Ser Ile Ser Asn Ser Asn Asn Phe Glu Val Gly Pro Pro Ala 1830 1835 1840 TTT AGG ATA GCC AGT GGT AAA ATC GTT TGT GTT TCA CAT GAA ACA ATT Phe Arg Ile Ala Ser Gly Lys Ile Val Cys Val Ser His Glu Thr Ile 1845 1850 1855 5661 5709 5757 5805 AAA AAA GTG AAA GAC ATA TTT ACA GAC Lys Lys Val Lys Asp Ile Phe Thr Asp 40 1860 1865 GAA AAC AAC GAG AAT AAA TCA AAA ATT Glu Asn Asn Glu Asn Lys Ser Lys Ile 1880 AGT TTC AGT AAA GTA ATT AAG Ser Phe Ser Lys Val Ile Lys 1870 1875 TGC CAA ACG AAA ATT ATG GCA Cys Gin Thr Lys Ile Met Ala 1885 1890 5853 5901 r r GGT TGT TAC GAG GCA TTG GAT GAT TCA GAG GAT ATT CTT CAT AAC TCT Gly Cys Tyr Glu Ala Leu Asp Asp Ser Glu Asp Ile Leu His Asn Ser 1895 1900 1905 CTA GAT AAT GAT GAA TGT AGC ACG CAT TCA CAT AAG GTT TTT GCT GAC Leu Asp Asn Asp Glu Cys Ser Thr His Ser His Lys Val Phe Ala Asp 1910 1915 1920 ATT CAG AGT GAA GAA ATT TTA CAA CAT AAC CAA AAT ATG TCT GGA TTG Ile Gin Ser Glu Glu Ile Leu Gin His Asn Gin Asn Met Ser Gly Leu .19.25 .19.30 .1935 5949 5997 6045 GAG AAA Giu Lys 1940 GTT TCT AAA ATA TCA Val. Ser Lys Ile Ser 1945 CCT TGT GAT GTT AGT TTG GAA Pro Cys Asp Val Ser Leu Glu 1950 GGG AAG CTT CAT AAG TCA GTC Gly Lys Leu His Lys Ser Val 1965 ACT TCA Thr Ser 1955 TCA TCT Ser Ser 1970 6093 6141 GAT ATA TGT AAA TGT AGT ATA Asp Ile Cys Lys Cys Ser Ile 1960 GCA AAT ACT TGT GGG Ala Asn Thr Cys Gly 1978 ATT TTT AGC ACA GCA AGT Ile Phe Ser Thr Ala Ser 1980 GGA AAA TCT GTC CAG Gly Lys Ser Val Gin 1985 6189 GTA TCA GAT GCT Val Ser Asp Ala 1990 GAA GAT AGT ACC Giu Asp Ser Thr 2005 GAA CAT TCA GAC Glu His Ser Asp 2020 TCA TTA CAA AAC GCA Ser Leu Gin Asn Ala 1995 AAG CAA GTC TTT TCC Lys Gin Val Phe Ser 2010 CAG CTC ACA AGA GAA Gin Leu Thr Arg Giu 2025 AGA CAA GTG TTT TCT GAA ATA Arg Gin Val Phe Ser Giu Ile 2000 AAA GTA TTG TTT AAA AGT AAC Lys Val Leu Phe Lys Ser Asn 2015 6237 6285 6333 GMA AAT ACT GCT ATA CGT Giu Asn Thr Ala Ile Arg 2030

ACT

Thr 2035 .CCA GAA CAT Pro Giu His TTA ATA TCC CMA Leu Ile Ser Gin 2040 TCA TCT GCT TTC TCT GGA TTT Ser Ser Ala Phe Ser Gly Phe 2055 ATT TTA GMA AGT TCC TTA CAC Ile Leu Giu Ser Ser Leu His 2070 AMA GGC TTT TCA Lys Giy Phe Ser 2045 AGT ACA GCA AGT Ser Thr Ala Ser 2060 AAA GTT MAG GGA Lys Val Lys Gly 2075 TAT MAT GTG GTA MAT Tyr Asn Vai Val Asn 2050 GGA MAG CMA GTT TCC Gly Lys Gin Vai Ser 2065 GTG TTA GAG GMA TTT Val Leu Giu Giu Phe 2080 TC A CCT ACG TCT AGA Ser Pro Thr Ser Arg 2095 6381 6429.

6477 40 GAT TTA ATC Asp Leu Ile 2085 AGA ACT GAG CAT AGT Arg Thr Giu His Ser 2090 CTT CAC TAT Leu His Tyr 6525 4 CMA MT GTA TCA AMA ATA CTT CCT Gin Asn Val Ser Lys Ile Leu Pro 2100 2105 CAC TGT GTA MAC TCA GMA ATG GMA His Cys Val Asn Ser Giu Met Giu 2120 GTT GAT MAG AGA MAC CCA Val Asp Lys Arg Asn Pro 2110

GAG

Giu 2115 AMA ACC TGC AGT AMA GMA Lys Thr Cys Ser Lys Giu 2125 TTT AMA Phe Lys 2130 6573 6621 6669 6717 TTA TCA MAT MAC TTA MAT GTT GMA GGT GGT TCT TCA GMA MT MAT CAC Leu Ser Asn Asn Leu Asn Val Giu Gly Gly Ser Ser Glu Asn Asn His 2135 *2140 2145 TCT ATT AMA GTT TCT CCA TAT CTC TCT CMA TTT CMA CMA GAC AMA CMA Ser Ile Lys Val Ser Pro Tyr Leu Ser Gin Phe Gin Gin Asp Lys Gin 2.1.1;0 .2.95 .2.6.0 -100- CAG TTG GTA TTA GGA ACC AAA GTC Gin Leu Val Leu Gly Thr Lys Val 2165 2170 TTG GGA AAA GAA Leu Giy Lys Giu 2180 AAA ACT GAA ACT Lys Thr Giu Thr CAG GCT TCA CCT Gin Ala Ser Pro 2185 TTT TCT GAT GTT Phe Ser Asp Val 2200 TCA CTT GTT GAG AAC Ser Leu Val Giu Asn 2175 AAA AAC GTA AAA ATO Lys Asn Val Lys Met 2190 CCT GTG AAA ACA AAT Pro Val Lys Thr Asn 2205 ATT CAT GTT Ile His Val GAA ATT GGT Giu Ile Gly 2195 ATA GAA GTT Ile Giu Vai 2210 6765 6813 6861 TGT TCT ACT TAC TCC AAA Cys Ser Thr Tyr Ser Lys 2215 GAT TCA GAA AAC TAC Asp Ser Giu Asn Tyr 2220 GTA GAA ATT GCT Val. Gu Ile Aia 2230 AAA CTG CCA AGT Lys Leu Pro Ser 2245 AAA GCT TTT ATG GAA GAT Lys Ala Phe Met Giu Asp 2235 CAT GCC ACA CAT TCT CTT His Ala Thr His Ser Leu 2250 TTT GAA ACA GAA GCA Phe Giu Thr Giu Ala 12225 GAA CTG ACA GAT TCT Giu Leu Thr Asp Ser 2240 ACA TGT CCC GAA AAT Thr Cys Pro Giu Asn 2255 GAG GAA Giu Giu 2260 ATG GTT TTG Met Val Leu TCA AAT TCA AGA ATT Ser Asn Ser Arg Ile 2265

GGA

Gly 227( AAA AGA AGA Lys Arg Arg AGA AAC TTA Arg Asn Leu GGA GAG Gly Glu 2275 TTA AAT Leu Asn 2290 6909 6957 7005 7053 7101 7149 7197 CCC CTT ATC TTA GTG GGA GAA CCC TCA ATC AAA Pro Leu Ile Leu Val Gly Giu Pro Ser Ile Lys 2280 2285 GAA TTT GAC AGG ATA 35 Giu Phe Asp Arg Ile 2295 AAA AGC ACT CCA GAT Lys Ser Thr Pro Asp 40 2310 ATA GAA AAT CAA GAA Ile Giu Asn Gin Giu 2300 GGC ACA ATA AAA GAT Gly Thr Ile Lys Asp 2315 AAA TCC TTA AAG GCT TCA Lys Ser Leu Lys Ala Ser 2305 CGA AGA TTG TTT ATG CAT Arg Arg Leu Phe Met His 2320 CAT GT T TCT TTA His Val Ser Leu 2325 GAA CGT CAA GAG Giu Arg Gin Giu 2340 GAG CCG ATT ACC TGT Giu Pro Ile Thr Cys 2330 GTA CCC TTT CGC Val Pro Phe Arg 2335 ACA ACT AAG Thr Thr Lys GGT CAA GAA Gly Gin Glu 2355 7245 7293 ATA CAG AAT Ile Gin Asn 2345 CCA AAT TTT ACC GCA CCT Pro Asn Phe Thr Ala Pro 2350 TTT CTG TCT Phe Leu Ser TCA AGC AAT Ser Ser Asn AAA TCT CAT TTG Lys Ser His Leu 2360 TTA GCA GTT TCA Leu Ala Val Ser 2375 TAT GAA CAT CTG ACT TTG GAA AAA TCT Tyr Giu His Leu Thr Leu Giu Lys Ser 2365 2370 GGA CAT CCA TTT TAT CAA GTT TCT GCT Gly His Pro Phe Tyr Gin Val Ser Ala 2-380 .23 8- 7341 7389 1- ACA AGA AAT GAA AAA ATG AGA CAC TTO ATT Th-r Arg Asn Glu Lys Met Arg His Leu Ile 2390 2395 ACT ACA 0CC AGA CCA ACC Thr Thr Gly Arg Pro Thr 2400 TCA CAT TTT CAC AGA OTT Ser His Phe His Arg Val 2415 AAA GTC TTT Lys Val Phe 2405 GTT CCA CCT TTT AAA Val Pro Pro Phe Lys 2410 GTT AGG AAT ATT AAC Val Arg Asn Ile Asn 2425 ACT AAA Thr Lys 7437 7485 7533 OAA CAO TOT Glu Gln Cys 2420 AAC ATT OAT Asn Ile Asp TTO GAG GAA AAC AGA CAA Leu Oiu Oiu Asn Arg Gin 2430 AAO CAA Lys Gin 2435 GOA CAT 0C OJly His Gly 2440 TCT OAT OAT AGT AAA AAT Ser Asp Asp Ser Lys Asn 2445 AAO ATT AAT OAC Lys Ile Asn Asp 2450 AAT GAO ATT CAT CAO Asn Glu Ile His Gin 2455 OTA ACT TTC ACA AAO Val Thr Phe Thr Lys 2470 CTT CAG AAT GCC AGA Leu Gin Asn Ala Arg 2485 TTT AAC AAA AAC AAC Phe Asn Lys Asn Asn 2460 TOT OA-A GAA GAA CCT Cys Oiu Oiu Giu Pro 2475 OAT ATA CAG OAT ATG Asp Ile Gin Asp Met 2490 TCC AAT CAA OCA OCA OCT Ser Asn Gin Ala Ala Ala 2465 TTA OAT TTA ATT ACA AOT Leu Asp Leu Ile Thr Ser 2480 COA ATT AAO AAO AAA CAA Arg Ile Lys Lys Lys Gln 2495 7581 7629 7677 7725 7773 7821 AOO CAA COC OTC Arg GTn Arg Val 2500 TCC ACT CTG CCT 35 Ser Thr Leu Pro TTT CCA CAG Phe Pro Gln 2505 CCA CCC AOT CTG TAT Pro Gly Ser Leu Tyr 2510 CGA ATC Arg Ile 2520 TCT CTG AAA Ser Leu Lys OCA OCA GTA Ala Ala Val 2525 CTT OCA AAA ACA Leu Ala Lys Thr 2515 OGA GOC CAA OTT Oly Gly Gin Val 2530 GOC OTT TCT AAA Oly Val Ser Lys 2545 CCC TCT OCO TOT TCT CAT AAA CAG Pro.Ser Ala Cys Ser His Lys Gin 2535 CTG TAT ACO TAT Leu Tyr Thr Tyr 2540 7869 9* CAT TGC ATA AAA His Cys Ile Lys 2550 ATT AAC AGC AAA AAT Ile Asri Ser Lys Asn 2555 GCA CAG TCT TTT CAG TTT CAC Ala Glu Ser Phe Gin Phe His 2560 ACT GMA OAT TAT TTT OCT Thr Glu Asp Tyr Phe Cly 2565 AAO OAA ACT Lys Olu Ser 2570 TTA TOO ACT OGA Leu Trp Thr Oly 2575 CCC TCC MAT OAT Pro Ser Asn Asp 2590 AAA OGA ATA Lys Oly Ile OGA AAC OCT Gly Lys Ala 2595 7917 7965 8013 50 CAG TTO OCT OAT Gin Leu Ala Asp 2580 GOT OCA TOO CTC ATA Oly Gly Trp Leu Ile 2585 OGA MAA GMA GAA TTT TAT AGO OCT CTG TOT CAC ACT CCA GOT OTO OAT Oly Lys Olu Olu Phe Tyr Arg Ala Leu Cys Asp Thr Pro Gly Val Asp 2600 2605 2610 8061 -102- *CCA AAG CTT ATT TCT Pro Lys Leu Ile Ser .2615 AGA ATT TGG OTT TAT Arg Ile Trp Val Tyr 2620 ATA TOO AAA CTG Ile Try, Lys Leu 2630 AAT AGA TGC CTA Asn Arg Cys Leu 2645 GCA OCT ATG GAA TGT Ala Ala Met Glu Cys 2635 AGC CCA GAA AGO GTG Ser Pro Oiu Arg Val .2650 AAT CAC TAT AGA TGO ATC Asn His Tyr Arg Trp Ile 2625 TTT CCT AAG OAA TTT OCT Phe Pro Lys Giu Phe Ala 2640 CTT CAA CTA AAA TAC AGA Leu Gin Leu Lys Tyr Axg 2655 8109 8157 8205 TAT OAT ACO Tyr Asp Thr 2660 OAA ATT OAT AGA AOC Oiu Ile Asp Arg Ser 2665 AGA AGA TCO OCT ATA Arg Arg Ser Ala Ile 2670 AAA AAG ATA Lys Lys Ile 2675 8253 ATO OAA AGO OAT GAC ACA OCT OCA AAA ACA CTT OTT CTC TOT' OTT TCT Met Oiu Arg Asp Asp Thr Ala Ala Lys Thr Leu Val Leu Cys Val Ser 2680 2685 2690 GAC ATA ATT TCA TTG AOC OCA A-AT ATA TCT GAA ACT TCT AOC AAT AAA Asp Ile Ile Ser Leu Ser Ala Asn Ile Ser Oiu Thr Ser Ser Asn Lys 2695 2700 2705 ACT AGT AOT OCA OAT ACC CAA AAA OTO Thr Ser Ser Ala Asp Thr Gin Lys Val 2710 2715 GGG TOO TAT OCT OTT AAO 0CC CAG TTA Gly Trp Tyr Ala Val Lys Ala Gin Leu 2725 2730 TTA AAG AAT GOC AGA CTG ACA OTT OGT Leu Lys Asn Oly Arg Leu Thr Val Oly 2740 2745 0CC ATT ATT OAA CTT ACA OAT Ala Ile Ile Oiu Leu Thr Asp 2720 OAT CCT CCC CTC TTA OCT OTC Asp Pro Pro Leu Leu Ala Val 2735 8301 8349 8397 8445 8493 CAG AAO ATT ATT Gin Lys Ile Ile 2750 CTT CAT OGA Leu His Oly 2755 4 4* *4

C

OCA OAA CTO OTG OOC TCT Ala Oiu Leu Val Oly Ser 2760 CCT OAT 0CC Pro Asp Ala TOT ACA CCT Cys Thr Pro 2765 CTT OAA 0CC CCA Leu Oiu Aia Pro 2770 8541 OAA TCT CTT ATO tTA AAO Giu Ser Leu Met Leu Lys 2775 45 TOO TAT ACC AAA CTT OGA Trp Tyr Thr Lys Leu Oiy 2790 .50 CCC TTA TCA TCO CTT TTC Pro Leu Ser Ser Leu Phe 2805 ATT TCT OCT AAC AOT Ile Ser Ala Asn Ser 2780 ACT COO CCT OCT COC Thr Arg Pro Ala Arg 2785 TTC TTT CCT Phe Phe Pro 2795 GAC CCT AGA Asp Pro Arg CCT TTT CCT CTO Pro Phe Pro Leu 2800 8589 8637 8685 AOT OAT OGA GOA Ser Asp Oly Gly 2810 AAT OTT GOT TOT OTT OAT Asn Val Oly Cys Val Asp 2815 OTA ATT ATT CAA AGA Val Ile Ile Gin Arg 2820 OCA TAC CCT ATA CAG TOO ATO GAG AAO ACA TCA Ala Tyr Pro Ile Gin Trp Met Oiu Lys Thr Ser 2825 2830 2835 8733 -103- TCT GGA TTA TAC ATA TTT CGC AAT GAA AGA GAG GAA GAA AAG GAA GCA Ser Giy Leu Tyr Ile Phe Arg Asn Giu Arg Giu Giu Giu Lys Giu Ala 2840 2845 2850 8781 8829 GCA AAA TAT GTG GAG Ala Lys Tyr Val Glu 2855 GCC CAA CAA Ala Gin Gin AAG AGA Lys Arg 2860 CTA GAA GCC Leu Giu Ala TTA TTC ACT Leu Phe Thr 2865 AAA ATT CAG GAG Lys Ile Gin Glu 2870 TAT TTA CCA TCA Tyr Leu Pro Ser 2885 GAT GGT GCA GAG Asp Gly Ala Glu 2900 GAA TTT GAA GAA CAT Glu Phe Giu Glu His 2875 GAA GAA AAC ACA ACA AAA CCA Glu Giu Asn Thr Thr Lys Pro 2880 8877 CGT GCA CTA ACA Arg Ala Leu Thr 2890 CTT TAT GAA GCA Leu Tyr Giu Ala 2905 AGA CAG CAA Arg Gin Gin GTT CGT GCT TTG CAA Val Arg Ala Leu Gin 2895 8925 GTG AAG AAT GCA GCA GAC CCA Val Lys Asn Ala Ala Asp Pro 2910

GCT

Ala 2915 TAC CTT GAG GGT TAT TTC Tyr Leu Giu Gly Tyr Phe 2920 CAC AGG CAA ATG TTG AAT His Arg Gin Met Leu Asn 2935 AGT GAA GAG CAG TTA Ser Giu Giu Gin Leu 2925 GAT AAG AAA CAA GCT Asp Lys Lys Gin Ala 2940 TCT GCT GAA CAA AAG Ser Ala Giu Gin Lys 2955 AGA GCC TTG AAT AAT Arg Ala Leu Asn Asn 2930 CAG ATC CAG TTG GAA Gin Ile Gin Leu Glu 2945 GAA CAA GGT TTA TCA Glu Gin Gly Leu Ser 2960 8973 9021 9069 9117 9165 9213 30 ATT AGG AAG GCC ATG GAA Ile Arg Lys Ala Met Glu 2950 AGO GAT GTC Arg Asp Val 2965 AAA GAA AAA Lys Glu Lys 2980 ACA ACC GTG TGG Thr Thr Vai Trp 2970 GAT TCA GTT ATA Asp Ser Val Ile 2985 TTG CGT ATT Leu Arg Ile GTA AGC Val Ser 2975 TAT TCA AAA Tyr Ser Lys CTG AGT ATT TGG CGT Leu Ser Ile Trp Arg 2990 GGA AAG AGA TAC AGA Oly Lys Arg Tyr Arg 3005 TTA TAT TCT CTG Leu Tyr Ser Leu TTA ACA GAA Leu Thr Glu 3000 CCA TCA TCA GAT Pro Ser Ser Asp 2995 ATT TAT CAT CTT Ile Tyr His Leu 3010 AAC ATA CAG TTA Asn Ile Gin Leu 3025 OCA ACT TCA Ala Thr Ser AAA TCT AAA Lys Ser Lys 3015 AGT AAA TCT GAA Ser Lys Ser Glu 3020 AGA GCT Arg Aia 9261 9309 9357 GCA GCG ACA AAA AAA ACT Ala Ala Thr Lys Lys Thr 3030 ATT TTA TTT CAG ATT TAC Ile Leu Phe Gin Ile Tyr 3045 CAG TAT CAA CAA CTA Gin Tyr Gin Gin Leu 3035 CAG CCA CGG GAG CCC Gin Pro Arg Glu Pro 3050 CCG GTT TCA GAT GAA Pro Val Ser Asp Glu 3040 CTT CAC TTC AGC AAA Leu His Phe Ser Lys 3055 9405 -104- TTT TTA OAT CCA GAC TTT CAG Phe Leu Asp Pro Asp Phe Gin 3060 3065 GGA TTT GTC OTT TCT OTT GTO Gly Phe Val Vai Ser Vai Val 3080 TAT TTG TCA GAC GAA TOT TAC Tyr Leu Ser Asp Giu Cys Tyr 3095 CCA TCT TOT TCT GAG GTG GAC CTA ATA Pro Ser Cys Ser Glu Val. Asp Leu Ile 3070 3075 AAA AAA ACA OGA CTT 0CC CCT TTC GTC Lys Lys Thr Oly Leu Aia Pro Phe Val 3085 3090 AAT TTA CTG OCA ATA AAG TTT TOG ATA Asn Leu Leu Ala Ile Lys PhiB Trp Ile 3100 3105 9453 9501 9549 9597 GAC CTT AAT GAO GAC ATT ATT AAO, CCT CAT ATG TTA ATT OCT OCA AOC Asp Leu Asn Oiu Asp Ile Ile Lys Pro His Met Leu Ile Aia Aia Ser 3110 3115 3120 AAC CTC CAG TOG CGA CCA OAA TCC AAA TCA GOC CTT CTT ACT TTA TTT Asn Leu Gin Trp Arg Pro Giu Ser Lys Ser Gly Leu Leu Thr Leu Phe 3125 3130 3135 OCT OGA OAT TTT TCT OTO TTT TCT OCT AOT CCA AAA GAO GOC CAC TTT Ala Giy Asp Phe Ser Val Phe Ser Aia Ser Pro Lys Giu Oly His Phe 3140 3145 3150 3155 9645 9693

S

CAA GAG ACA TTC AAC AAA ATO Gin Giu Thr Phe Asn Lys Met 3160 CTT TGC AAT GAA OCA GAA AAC Leu Cys Asn Giu Ala Giu Asn 3175 OAT CCC AAO TOG TCC ACC CCA 35 Asp Pro Lys Trp Ser Thr Pro 3190 AAA AAT ACT GTT GAO Lys Asn Thr Val Oiu 3165 AAT ATT GAC ATA Asn Ile Asp Ile 3170 AAO CTT ATO CAT ATA CTG CAT OCA AAT Lys Leu Met His Ile Leu His Ala Asn 3180 3185 ACT AAA GAC TOT ACT TCA 000 CCG TAC Thr Lys Asp Cys Thr Ser Gly Pro Tyr 3195 3200 ACT OCT CAA ATC ATT CCT GOT ACA OGA A.AC AAG CTT CTG Thr Ala Gin Ile Ile Pro Oly Thr Gly Asn Lys Leu Leu 3205 3210 3215 CCT AAT TOT GAO Pro Asn Cys Giu 3220 AA.A AGO AAO TCT Lys Arg Lys Ser ATA TAT TAT CAA Ile Tyr Tyr Gin 3225 OTT TCC ACA CCT Val Ser Thr Pro 3240 AGT CCT TTA TCA CTT Ser Pro Leu Ser Leu 3230 GTC TCA GCC CAG ATG Vai Ser Ala Gin Met 3245 OAT GAC CAA AAG AAC Asp Asp Gin Lys Asn 3260 ATO TCT TCT Met Ser Ser TOT ATO 0CC Cys Met Ala 3235 ACT TCA AAG Thr Ser Lys 3250 TGC.AAA AAO Cys Lys Lys 3265 9741 9789 9837 9885 9933 9981 10029 555

S

S. S 5555 :.50 TCT TOT AAA Ser Cys Lys 000 GAG 0 Giu 3255 AAA GAG ATT Lys Giu Ile AGA AGA 0CC TTO OAT TTC TTG AGT AGA CTG CCT TTA CCT CCA CCT OTT Arg Arg Ala Leu Asp Phe Leu Ser Arg Leu Pro Leu Pro Pro Pro Val 3270 3275 3280 10077 -105- AGT CCC ATT Ser Pro Ile 3285 TGT ACA TTT GTT TCT Cys Thr Phe Vai Ser 3290 CCG GCT GCA CAG AAG Pro Ala Ala Gin Lys 3295 GCA TTT CAG Ala Phe Gin CCA CCA AGG AGT TGT GGC ACC AAA TAC GAA ACA CCC ATA AAG AAA AAA Pro Pro Arg 3300 Ser Cys Giy Thr Lys Tyr Glu Thr Pro 3305 3310 Ile Lys Lys Lys 3315 GAA CTG AAT TCT CCT CAG ATG ACT CCA TTT AAA Giu Leu Asn Ser Pro 3320 TCT CTT TTG GAA AGT Ser Leu Leu Giu Ser 3335 AAT ACC CAA GCT CTT Asn Thr Gin Ala Leu 3350 TCT GTC AGT GAA TCC Ser Val Ser Giu Ser 3365 CTC AGA CTG AAA CGA Gin Met Thr Pro Phe Lys 3325 AAT TCA ATA GCT GAC GAA Asn Ser Ile Ala Asp Glu 3340 TTG TCT GGT TCA ACA GGA Leu Ser Gly Ser Thr Gly 3355 AAA TTC AAT GAA ATT Lys Phe Asn Giu Ile 3330 GAA CTT GCA TTG ATA Giu Leu Ala Leu Ile 3345 GAA AAA CAA TTT ATA Glu Lys Gin Phe Ile 3360 10125 10173 10221 10269 10317 10365 10413 10461 10512

ACT

Thr

CGT

AGG ACT GCT CCC ACC AGT TCA GAA GAT TAT Arg Thr Ala Pro Thr Ser Ser Giu Asp Tyr 3370 TGT ACT 3375 ATC AAA GAA CAG GAG ACA TCT CTG Leu Arg Leu Lys Arg Arg Cys Thr Thr Ser Leu Ile Lys Giu Gin Giu 4. 4 4 4.

4.

4*4* *444*4 4 4 4*44 4. 4 4 4444 9*4* 4 .4.

.4 44 4 4 *4*4 3380 30 AGT TCC CAG Ser Ser Gin 3385 3390 3395 GCC AGT ACG GAA GAA TGT GAG AAA AAT AAG CAG GAC ACA Ala Ser Thr Glu Glu Cys Giu Lys Asn Lys Gin Asp Thr 3400 3405 3410 AAA AAA TAT ATC TAAGCATTTG CAAAGGCGAC AATAAATTAT ATT ACA ACT 35 Ile Thr Thr Lys Lys Tyr Ile 3415 TGACGCTTAA CCTTTCCAGT TTATGTTGCA CAATGAGAAA GCAAAAATCG TTTTGCCCGA AACTAAATGT AATTTATTAA 45 TGCCTGTAAT CCCAACACTT CAAGACCAGC CTGGGCAACA 50 AAAAGAAAAT CTTTTAAATC TAAACATACC ATTTTCTTTT TATTTTGATG CAGATAATTC

TTATAAGACT

AGAAATTAGT

TTCCGTATTG

CTAATCAAGA

TGAGAAGCTG

TAGGGAGACC

TTTGGATTTG

AGATTGTGTC

CTTTTAGTTT

GGAATATAAT

TTCAAATTTA

GTATACTTTT

AAAACATCTT

AGGTGGGAGG

CCCATCTTTA

ATCACTACAA

ATTAAATGGA

AGCTACTATT

TTCAAACCAC

CCTCAGCGTT

GCTTCAGTTG

TGGCTGAGCT

AGTGCTTGAG

CGAAGAAAAA

GTATTATTTT

ATGAGGTCTC

TTAGGGGATT

ACATTAGTAC

TGTGTATCGG

CATATCTTAA

CGGTGGCTCA

GCCAGGAGTT

AAAAAAGGGG

ACAAGTGAAA

TTAGTACAGT

TTTTTTAGAG

10572 10632 10692 10752 10812 10872 10932 10992 11052 211112' GTAACTCACT ATGAAATAGT TCTCCTTAAT GCAAATATGT TCGT.TCTGCT ATAGTTCCAT -106- CCTGTTCAAA AGTCAGGATG AATATGAAGA GTGGTGTTTC CTTTTGAGCA ATTCTTCATC CTTAAGTCAG CATGATTATA AGAAAAATAG AACCCTCAGT GTAACTCTAA TTCCTTTTTA CTATTCCAGT GTGATCTCTG AAATTAAATT ACTTCAACTA AAAATTCAAA TACTTTAAAT CAGAAGATTT CATAGTTAAT TTATTTTTTT TTTCAACAAA ATGGTCATCC AAACTCAAAC TTGAGAAAAT ATCTTGCTTT CAAATTGACA CTA INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 3418 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 11172 11232 11292 11352 11385 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Pro Ile Gly Ser Lys Glu Arg Pro Thr Phe Phe Glu Ile Phe Lys 1 veto 9.00 a 0

B

9**BS* v e B. 4 4 *5*5 Thr 30 Glu Glu Pro 65 Phe Glu 45 Arg Asp Arg Glu Ser 50 Gin Lys Leu His Val 130 Cys Leu Glu Arg Glu Asp Lys 115 Ser Asn Ser His Lys Gin Lvs 100 Ser Cys 5 Lys Ala Asp Ser Giu Ala Lys Asn Asn 55 Pro Ser Tyr 70 Gly Leu Thr 85 Phe Lys Leu Leu Arg Thr Pro Leu Leu 135 Leu Pro 40 Asn Asn Leu Asp Val 120 Gly Pro Ile Ser Leu Asn Trp Phe 25 Pro Tyr Gin Pro Leu 105 Lys Tyr Asr Ser Glu Pro Asn Leu Ala Ser 75 Leu Tyr Gin 90 Gly Arg Asn Thr Lys Met Cys Leu Ser 140 Gin Arg Asp 155 Glu Leu Thr Ser Val Asp 125 Glu Lys Pro Phe Pro Pro Pro 110 Gin Ser Ser Ala Lys Ile Val Asn Ala Pro Val Glu Thr Ile Lys Ser Asp Va1 Val 160 Asn Ser Val Leu Gin Cys Thr His Val Thr Pro 145 150 His Thr Pro Lys Cys Gly Ser Leu Phe Val 170 Lys Gly Arg Gin Thr 175 Pro Lys Ser Trp Leu Ile 210 Ile Ser 180 Ser Ser Arg Asn Glu Ser Leu Ala Glu Glu 215 Gly 185 Pro Ser -1 Ala Pro Glu Thr Thr Ala Asn Val Lys Ser Tyr Phe Ser 225 Lys is Asn Ala Ser Val 260 Ser Phe Lys Val Asn Ser

S

ecg.

0

S

S

C

0*0S

S

*0S*

S

0 Asn Glu -305 Gin Asr Ser Asn Lys.

385 Ser 45 Ser AsnI Ile S 275 Val Leu 290 Asp Ser Lys Vai Ala Asp Phe Val 355 Val Ala 370 Glu Val Gly Leu Ser Cys .ys Arg 435 er Ser Glu Phe Arg Glu 340 Ser His Val Asn Asp 420 Asp Ser Thr 325 Cys Glu Gin Pro Gly 405 Gin Glu Leu 310 Ser Glu Val Lys Ser 390 Ala Asn Cys Val 295 Cys Lys Lys Glu Pro 375 Leu Gin Ile Phe :.His Lys 280 Tyr Phe Thr Ser Pro 360 Phe Ala Met Ser Leu 440 Gl 261 Asr Git Ser Arg Lys 345 Asn Glu Cys Glu lu 425 rhr 250 r Phe His Thr Lys Lys I 330 Asn G Asp I Ser G Glu T 3 Lys I 410 Lys A Ser G Pro L [07- Glu Val Asp Thr Leu Ser 205 Thr Val Phe 220 Asn His Asp 235 Thr Asp Ser Gly Lys Thr Ile Gly Lys 285 lal Vai Asp 300 :ys Arg Thr 15 .ys Ile Phe I 1n Val Lys hr Asp Pro I 365 'ly Ser Asp I 380 rp Ser Gin L 95 le Pro Leu L sp Leu Leu A 4 lu Asn Ser L 445 eu Asn Giu G Pr 19 Se Pr Gli Gl~ Sei 27( Sez Thr Lys His 3lu 350 .eu ~ys .eu eu sp eu lu o Asp Met 0 r Thr Val His Asp Ser Leu 240 Asn Thr 255 Gly Asn *Met Pro Ser Glu Asn Leu 320 Glu Ala 335 Lys Tyr Asp Ser Ile Ser Thr Leu 400 His Ile 415 Thr Glu Pro Arg Thr Val Lys Lys Asp I 450 Leu Pro Lys Ser Giu Lys 455 Val 465 Asn Lys Arg Asp Glu 470 Glu Gin His Leu 460 Ser His Thr Asp Cys 480 Glu 475 -108- Ile Ser Lys Phe Val 545 Gly Ala Ala Gin Phe 30 625 Ser Leu Asn Lys Glu 705 Pro Ala Leu Phe Glu Lys 530 Cys Ser Gly Ile Lys 610 Glu Ser Ser Glu Glu 690 Ala Lys Cys Ala Gin Thr 515 Lys Ser Trp Leu His 595 Ser Ala Val Leu Thr 675 Ala Asp Ser His Val Gly 500 Phe Glu Gin Pro Ile 580 Asp Glu Pro Lys Tir 660 Cys Lys Ser Lys Pro 740 Lys 485 Ile Asn Thr Lys Ala 565 Ser Glu Leu Leu Arg 645 Ser Ser Cys Leu Lys 725 Val Gin Lys Ala Glu Glu 550 Thr Thr Thr Ile Thr 630 Ser Ser Asn Asn Ser 710 Val GIn Ala Lys Ser Ala 535 Asp Thr Leu Phe Asn 615 Phe Cys Phe Asn Lys 695 Cys Ser His Ile Ser Ser Ile 505 Phe Ser 520 Ser Glu Ser Leu Thr Gin Lys Lys 585 Tyr Lys 600 Cys Ser Ala Asn Ser Gin Gly Thr 665 Thr Val 680 Glu Lys Leu Gin Asp Ile Ser Lys 745 Gly Thr 490 Phe Arg Gly His Ser Gly Cys Pro 555 Asn Ser 570 Lys Thr Gly Lys Ala Gin Ala Asp 635 Asn Asp 650 Ile Leu Ile Ser Leu Gin Glu Gly 715 Lys Glu 730 Val Glu Ser Pro Ile Arg Met Thr 525 Leu Glu 540 Asn Leu Val Ala Asn Lys Lys Ile 605 Phe Giu 620 Ser Gly Ser Glu Arg Lys Gin Asp 685 Leu Phe 700 Gin Cys Glu Val Tyr Ser Val Glu 510 Asp Ile Ile Leu Phe 590 Pro Ala Leu Glu Cys 670 Leu Ile Glu Leu Asp Ala 495 Ser Pro His Asp Lys 575 Ile Lys Asn Leu Pro 655 Ser Asp Thr Asn Ala 735 Thr Ser Pro Asn Thr Asn 560 Asn Tyr Asp Ala His 640 Thr Arg Tyr Pro Asp 720 Ala Asp 750 Ala Ser Thr Phe Gln Ser 755 Gin Lys Ser Leu Tyr Asp His Glu -109- Leu Ile Leu Thr Pro Thr Ser Lys Asp Val Leu Ser Asn Leu Val Met 770 775 780 Ile Ser Arg 785 Asn Asn Tyr Lys Asn Gin Leu Leu Pro 835 Val Gin Phe 850 Glu Glu Thr 865 Glu Leu Phe Glu Arg Asn Asp Leu Thr ****915 Leu Tyr Gly 930 Lys Asp Leu S* 35 945 Gln His Ile Leu Asn Ile Trp Ala Gly 995 Phe Arg Thr 1010 Lys Lys Ser 1025 Ser Leu Ala Gly Glu Asp 820 Pro Asn Thr Ser Asn 900 Cys Asp Val Lys Asp 980.

Leu Ala Lys Cys Lys Ser 805 Val Glu Gin Ser Asp 885 Leu Val Thr Tyr Met 965 Lys Leu Ser Met Val 1045 Glu Ser Tyr Lys 790 Asp Val Glu Leu Cys Ala Leu Asn 825 Lys Tyr Met Arg 840 Asn Thr Asn Leu 855 Ile Ser Lys Ile 870 Asn Glu Asn Asn Ala Leu Gly Asn 905 Asn Glu Pro Ile 920 Gly Asp Lys Gin 935 Val Leu Ala Glu 950 Thr Leu Gly Gin Ile Pro Glu Lys 985 Gly Pro Ile Ser 1000 Asn Lys Glu Ile 1015 Phe Phe Lys Asp 1030 Glu Ile Val Asn Met Thr 810 Glu Val Arg Thr Phe 890 Thr Phe Ala Glu Asp 970 Asn Asn Lys Ile Thr Ser Asp Lys Leu 795 Lys Asn Ile Pro Asn Tyr Lys Asn 830 Ala Ser Pro Ser 845 Val Ile Gin Lys 860 Val Asn Pro Asp 875 Val Phe Gin Val Lys Glu Leu His 910 Lys Asn Ser Thr 925 Thr Gin Val Ser 940 Asn Lys Asn Ser 955 Leu Lys Ser Asp Asn Asp Tyr Met 990 His Ser Phe Gly 1005 Leu Ser Glu His 1020 Glu Glu Gin Tyr 1035 Leu Ala Leu Asp Lys Gly 800 Met Glu 815 Val Glu Arg Lys Asn Gin Ser Glu 880 Ala Asn 895 Glu Thr Met Val Ile Lys Val Lys 960 Ile Ser 975 Asn Lys Gly Ser Asn Ile Pro'Thr 1040 Asn Gin 1050 1055 Lys Lys Leu Ser Lys Pro Gln Ser Ile Asn Thr Val Ser Ala His Leu 1060 1065 1070 -110- Gin Ser Ser Val Val Val Ser Asp Cys Lys Asn Ser His Ile Thr Pro 1075 1080 1085 Gin Met Leu Phe Ser Lys Gin Asp Phe Asn Ser Asn His Asn Leu Thr 1090 1095 1100 Pro Ser Gin Lys Ala Glu Ile Thr Glu Leu Ser Thr Ile Leu Glu Glu 1105 1110 1115 1120 Ser Gly Ser Gin Phe Glu Phe Thr Gin Phe Arg Lys Pro Ser Tyr Ile 1125 1130 1135 Leu Gin Lys Ser Thr Phe Glu Val Pro Glu Asn Gin Met Thr Ile Leu 1140 1145 1150 Lys Thr Thr Ser Glu Glu Cys Arg Asp Ala Asp Leu His Val Ile Met 1155 1160 1165 Asn Ala Pro Ser Ile Gly Gin Val Asp Ser Ser Lys Gin Phe Glu Gly 1170 1175 1180 Thr Val Glu Ile Lys Arg Lys Phe Ala Gly Leu Leu Lys Asn Asp Cys 1185 1190 1195 1200 Asn Lys Ser Ala Ser Gly Tyr Leu Thr Asp Glu Asn Glu Val Gly Phe 1205 1210 1215 Arg Gly Phe Tyr Ser Ala His Gly Thr Lys Leu Asn Val Ser Thr Glu S30 1220 1225 1230 Ala Leu Gin Lys Ala Val Lys Leu Phe Ser Asp Ile Glu Asn Ile Ser 1235 1240 1245 Glu Glu Thr Ser Ala Glu Val His Pro Ile Ser Leu Ser Ser Ser Lys 1250 1255 1260 Cys His Asp Ser Val Val Ser Met Phe Lys Ile Glu Asn His Asn Asp 1265 1270 1275 1280 Lys Thr Val Ser Glu Lys Asn Asn Lys Cys Gin Leu Ile Leu Gin Asn 1285 1290 1295 Asn Ile Glu Met Thr Thr Gly Thr Phe Val Glu Glu Ile Thr Glu Asn 1300 1305 1310 Tyr Lys Arg Asn Thr Glu Asn Glu Asp Asn Lys Tyr Thr Ala Ala Ser 1315 1320 1325 Arg Asn Ser His Asn Leu Glu Phe Asp Gly Ser Asp Ser Ser Lys Asn 1330 1335 1340 Asp Thr Val Cys Ile His Lys Asp Glu Thr Asp Leu Leu Phe Thr Asp 1345 1350 1355 1: Gin His Asn Ile Cys Leu Lys Leu Ser Giy Gin Phe Met Lys Giu Gly 1365 1370 1375 Asn Thr Gin Ile Lys Giu Asp Leu Ser Asp Leu Thr Phe Leu Giu Val 51380 1385 1390 Ala Lys Ala Gin Giu Ala Cys His Gly Asn Thr Ser Asn Lys Giu Gin 1395, 1400 1405 Leu Thr Ala Thr Lys Thr Giu Gin Asn Ile Lys Asp Phe Giu Thr Ser 1410 1415 1420 Asp Thr Phe Phe Gin Thr Ala Ser Gly Lys Asn Ile Ser Val Ala Lys 1425 1430 -1435 1440 Giu Ser Phe Asn Lys Ile Val Asn Phe Phe Asp Gin Lys Pro Giu Giu 1445 1450 1455 Leu His Asn Phe Ser Leu Asn Ser Giu Leu His Ser Asp Ile Arg Lys 1460 1465 1470 Asn Lys Met Asp Ile Leu Ser Tyr Giu Giu Thr Asp Ile Val Lys His 1475 1480 1485 Lys Ile Leu Lys Giu Ser Val Pro Vai Gly Thr Gly Asn Gin Leu Val 1490 1495 1500 Thr Phe Gin Gly Gin Pro Giu Arg Asp Giu Lys Ile Lys Giu Pro Thr 1505 1510 I515 1520 Leu Leu Gly Phe His Thr Ala Ser Giy Lys Lys Vai Lys Ile Ala Lys 1525 1530 1535 Glu Ser Leu Asp Lys Val Lys Asn Leu Phe Asp Giu Lys Giu Gin Gly 1540 1545 1550 Thr Ser Giu Ile Thr Ser Phe Ser His Gin Trp Ala Lys Thr Leu Lys .1555 1560 1565 Tyr Arg Giu Ala Cys Lys Asp Leu Giu Leu Ala Cys Giu Thr Ile Giu 1570 1575 1580 Sle Thr Ala Ala Pro Lys Cys Lys Giu Met Gin Asn Ser Leu Asn Asn *1585 1590 1595 1600 Asp Lys Asn Leu Vai Ser Ile Giu Thr Vai Val Pro Pro Lys Leu Leu 1605 1610 1615 *Ser Asp Asn Leu Cys Arg Gin Thr Giu Asn Leu Lys Thr Ser Lys Ser 501620 1625 1630 Ile Phe Leu Lys Val Lys Val His Giu Asn Val Giu Lys Giu Thr Ala 1635 1640 1645 Lys Ser Pro Ala Thr Cys Tyr Thr Asri Gin Ser Pro Tyr Ser Val Ile 1650 1655 .1660 -112- Glu Asn Ser Ala Leu Ala Phe Tyr Thr Ser Cys Ser Arg Lys Thr Ser 1665 1670 1675 1680 Val Ser Gin Thr Ser Leu Leu Glu Ala Lys Lys Trp Leu Arg Glu Gly 1685 1690 1695 Ile Phe Asp Gly Gin Pro Glu Arg Ile Asn Thr Ala Asp Tyr Val Gly 1700 1705 1710 Asn Tyr Leu Tyr Glu Asn Asn Ser Asn Ser Thr Ile Ala Glu Asn Asp 1715 1720 1725 Lys Asn His Leu Ser Glu Lys Gin Asp Thr Tyr Leu Ser Asn Ser Ser 1730 1735 1740 Met Ser Asn Ser Tyr Ser Tyr His Ser Asp Glu Val Tyr Asn Asp Ser 1745 1750 1755 1760 Gly Tyr Leu Ser Lys Asn Lys Leu Asp Ser Gly Ile Glu Pro Val Leu 1765 1770 1775 Lys Asn Val Glu Asp Gin Lys Asn Thr Ser Phe Ser Lys Val Ile Ser 1780 1785 1790 Asn Val Lys Asp Ala Asn Ala Tyr Pro Gin Thr Val Asn Glu Asp Ile 1795 1800 1805 Cys Val Glu Glu Leu Val Thr Ser Ser Ser Pro Cys Lys Asn Lys Asn 1810 1815 1820 Ala Ala Ile Lys Leu Ser Ile Ser Asn Ser Asn Asn Phe Glu Val Gly 1825 1830 1835 1840 Pro Pro Ala Phe Arg Ile Ala Ser Gly Lys Ile Val Cys Val Ser His 1845 1850 1855 Glu Thr Ile Lys Lys Val Lys Asp Ile Phe Thr Asp Ser Phe Ser Lys 1860 1865 1870 Val Ile Lys Glu Asn Asn Glu Asn Lys Ser Lys Ile Cys Gin Thr Lys 1875 1880 1885 Ile Met Ala Gly Cys Tyr Glu Ala Leu Asp Asp Ser Glu Asp Ile Leu 1890 1895 1900 His Asn Ser Leu Asp Asn Asp Glu Cys Ser Thr His Ser His Lys Val 1905 1910 1915 1920 Phe Ala Asp Ile Gin Ser Glu Glu Ile Leu Gin His Asn Gin Asn Met 1925 1930 1935 Ser Gly Leu Glu Lys Val Ser Lys Ile Ser Pro Cys Asp Val Ser Leu 1940 1945 1950 -113- Glu Thr Ser Asp Ile Cys Lys Cys Ser Ile Gly Lys Leu His Lys Ser 1955 1960 1965 Val Ser Ser Ala Asn Thr Cys Gly Ile Phe Ser Thr Ala Ser Gly Lys 1970 1975 1980 Ser Val Gin Val Ser Asp Ala Ser Leu Gin Asn Ala Arg Gin Val Phe 1985 1990 1995 2000 Ser Glu Ile Glu Asp Ser Thr Lys Gin Val Phe Ser Lys Val Leu Phe 2005 2010 2015 Lys Ser Asn Glu His Ser Asp Gin Leu Thr Arg Glu Glu Asn Thr Ala 2020 2025 2030 Ile Arg Thr Pro Glu His Leu Ile Ser Gin Lys Gly Phe Ser Tyr Asn 2035 2040 2045 Val Val Asn Ser Ser Ala Phe Ser Gly Phe Ser Thr Ala Ser Gly Lys 2050 2055 2060 Gin Val Ser Ile Leu Glu Ser Ser Leu His Lys Val Lys Gly Val Leu 2065 2070 2075 2080 Glu Glu Phe Asp Leu Ile Arg Thr Glu His Ser Leu His Tyr Ser Pro 2085 2090 2095 Thr Ser Arg Gln Asn Val Ser Lys Ile Leu Pro Arg Val Asp Lys Arg 30 2100 2105 2110 Asn Pro Glu His Cys Val Asn Ser Glu Met Glu Lys Thr Cys Ser Lys 2115 2120 2125 Glu Phe Lys Leu Ser Asn Asn Leu Asn Val Glu Gly Gly Ser Ser Glu 2130 2135 2140 Asn Asn His Ser Ile Lys Val Ser Pro Tyr Leu Ser Gin Phe Gin Gin 2145 2150 2155 2160 Asp Lys Gin Gin Leu Val Leu Gly Thr Lys Val Ser Leu Val Glu Asn 2165 2170 2175 S' Ile His Val Leu Gly Lys Glu Gin Ala Ser Pro Lys Asn Val Lys Met 2180 2185 2190 Glu Ile Gly Lys Thr Glu Thr Phe Ser Asp Val Pro Val Lys Thr Asn 2195 2200 2205 Ile Glu Val Cys Ser Thr Tyr Ser Lys Asp Ser Glu Asn Tyr Phe Glu 2210 2215 2220 Thr Glu Ala Val Glu Ile Ala Lys Ala Phe Met Glu Asp Asp Glu Leu 2225 2230 2235 2240 Thr Asp Ser Lys Leu Pro Ser His Ala Thr His Ser Leu Phe Thr Cys 2245 2250 2255 -114- Pro Giu Asn Giu Giu Met Val Leu Ser Asn Ser Arg Ile Gly Lys Arg 2260 2265 2270 Arg Gly Giu Pro Leu Ile Leu Val Gly Glu Pro Ser Ile Lys Arg Asn 2275 2280 2285 Leu Leu Asn Giu Phe Asp Arg le Ile Glu Asn Gin Giu Lys Ser Leu 2290 2295 2300 Lys Ala Ser Lys Ser Thr Pro Asp Gly Thr Ilie Lys Asp Arg Arg Leu 2305 2310 2315 2320 Phe Met His His Val Ser Leu Giu Pro Ile Thr Cys Vai Pro Phe Arg 2325 2330 2335 Thr Thr Lys Gilu Arg Gin'Giu Ile Gin Asn Pro Asn Phe Thr Aia Pro 2340 2345 2350 Giy Gin Giu Phe Leu Ser Lys Ser His Leu Tyr Giu His Leu Thr Leu 2.355 2360 2365 Giu Lys Ser Ser Ser Asn Leu Aia Val Ser Gly His Pro Phe Tyr Gin 2370 2375 2380 Val Ser Ala Thr Arg Asn Giu Lys Met Arg His Leu Ile Thr Thr Gly *2385 2390 2395 2400 Arg Pro Thr Lys Val Phe Vai Pro Pro The Lys Thr Lys Ser His Phe 2405 2410 2415 His Arg Val Giu Gin Cys Vai Arg Asn Ilie Asn Leu Giu Giu Asn Arg 2420 2425 2430 Gin Lys Gin Asn Ile Asp Gly His Gly Ser Asp Asp Ser Lys Asn Lys 2435 2440 2445 Ile Asn Asp Asn Giu Ile His Gin Pi-e Asn Lys Asn Asn Ser Asn Gin ***2450 2455 2460 Ala Ala Ala Val Thr Phe Thr Lys Cys Glu Glu Glu Pro Leu Asp Leu 2465 2470 2475 2480 sees 000. le Thr Ser Leu Gin Asn Ala Arg Asp Ile Gin Asp Met Arg Ile Lys 2485 2490 2495 *Lys Lys Gin Arg Gin Arg Val Phe Pro Gin Pro Gly Ser Leu Tyr Leu **2500 2505 2510 s0 Ala Lys Thr Ser Thr Leu Pro Arg Ile Ser Leu Lys Ala Ala Val Gly 2515 2520 2525 Gly Gin Vai Pro Ser Ala Cys Ser His Lys Gin Leu Tyr. Thr Tyr Gly 2530 2535 2540 -115- Val Ser Lys His Cys Ile Lys Ile Asn Ser Lys Asn Ala Glu Ser Phe 2545 2550 2555 2560 Gin Phe His Thr Glu Asp Tyr Phe Gly Lys Glu Ser Leu Trp Thr Gly 2565 2570 2575 Lys Gly Ile Gin Leu Ala Asp Gly Gly Trp Leu Ile Pro Ser Asn Asp 2580 2585 2590 Gly Lys Ala Gly Lys Glu Glu Phe Tyr Arg Ala Leu Cys Asp Thr Pro 2595 2600 2605 Gly Val Asp Pro Lys Leu Ile Ser Arg Ile Trp Val Tyr Asn His Tyr 2610 2615 2620 Arg Trp Ile Ile Trp Lys Leu Ala Ala Met Glu Cys Ala Phe Pro Lys 2625 2630 2635 2640 Glu Phe Ala Asn Arg Cys Leu Ser Pro Glu Arg Val Leu Leu Gin Leu 2645 2650 2655 Lys Tyr Arg Tyr Asp Thr Glu Ile Asp Arg Ser Arg Arg Ser Ala Ile 2660 2665 2670 Lys Lys Ile Met Glu Arg Asp Asp Thr Ala Ala Lys Thr Leu Val Leu 2675 2680 2685 Cys Val Ser Asp Ile Ile Ser Leu Ser Ala Asn Ile Ser Glu Thr Ser 2690 2695 2700 S* Ser Asn Lys Thr Ser Ser Ala Asp Thr Gln Lys Val Ala Ile Ile Glu 2705 2710 2715 2720 Leu Thr Asp Gly Trp Tyr Ala Val Lys Ala Gin Leu Asp Pro Pro Leu 2725 2730 2735 Leu Ala Val Leu Lys Asn Gly Arg Leu Thr Val Gly Gin Lys Ile Ile 2740 2745 2750 Leu His Gly Ala Glu Leu Val Gly Ser Pro Asp Ala Cys Thr Pro Leu 2755 2760 2765

G

l u Ala Pro Glu Ser Leu Met Leu Lys Ile Ser Ala Asn Ser Thr Arg 2770 2775 2780 Pro Ala Arg Trp Tyr Thr Lys Leu Gly Phe Phe Pro Asp Pro Arg Pro 2785 2790 2795 2800 Phe Pro Leu Pro Leu Ser Ser Leu Phe Ser Asp Gly Gly Asn Val Gly 2805 2810 2815 Cys Val Asp Val Ile Ile Gin Arg Ala Tyr Pro Ile Gln Trp Met Glu 2820 2825 2830 Lys Thr Ser Ser Gly Leu Tyr Ile Phe Arg Asn Glu Arg Glu Glu Glu 2835 2840 2845 Lys Glu Ala Ala Lys Tyr Val Glu Ala Gin Gin Lys Arg Leu Glu Ala 2850 2855 2860 Leu Phe Thr Lys Ile Gin Glu Glu Phe Glu Glu His Glu Glu Asn Thr 2865 2870 2875 2880 Thr Lys Pro Tyr Leu Pro Ser Arg Ala Leu Thr Arg Gin Gin Val Arg 2885 2890 2895 Ala Leu Gin Asp Gly Ala Glu Leu Tyr Glu Ala Val Lys Asn Ala Ala 2900 2905 2910 Asp Pro Ala Tyr Leu Glu Gly Tyr Phe Ser Glu Glu Gin Leu Arg Ala 2915 2920 2925 Leu Asn Asn His Arg Gin Met Leu Asn Asp Lys Lys Gin Ala Gin Ile 2930 2935 2940 Gin Leu Glu Ile Arg Lys Ala Met Glu Ser Ala Glu Gin Lys Glu Gin 2945 2950 2955 2960 Gly Leu Ser Arg Asp Val Thr Thr Val Trp Lys Leu Arg Ile Val Ser 2965 2970 2975 Tyr Ser Lys Lys Glu Lys Asp Ser Val Ile Leu Ser Ile Trp Arg Pro 2980 2985 2990 Ser Ser Asp Leu Tyr Ser Leu Leu Thr Glu Gly Lys Arg Tyr Arg Ile 30 2995 3000 3005 Tyr His Leu Ala Thr Ser Lys Ser Lys Ser Lys Ser Glu Arg Ala Asn 3010 3015 3020 35 Ile Gln Leu Ala Ala Thr Lys Lys Thr Gin Tyr Gin Gin Leu Pro Val 3025 3030 3035 3040 Ser Asp Glu Ile Leu Phe Gin Ile Tyr Gin Pro Arg Glu Pro Leu His oo 40 3045 3050 3055 Phe Ser Lys Phe Leu Asp Pro Asp Phe Gin Pro Ser Cys Ser Glu Val 3060 3065 3070 Asp Leu Ile Gly Phe Val Val Ser Val Val Lys Lys Thr Gly Leu Ala S* 45 3075 3080 3085 Pro Phe Val Tyr Leu Ser Asp Glu Cys Tyr Asn Leu Leu Ala Ile Lys 3090 3095 3100 Phe Trp Ile Asp Leu Asn Glu Asp Ile Ile Lys Pro His Met Leu Ile 3105 3110 3115 3120 Ala Ala Ser Asn Leu Gin Trp Arg Pro Glu Ser Lys Ser Gly Leu Leu 3125 3130 3135 -117- Thr Leu Phe Ala Gly Asp Phe Ser Vai Phe Ser Ala Ser Pro Lys Glu 3140 3145 3150 Gly His Phe Gin Giu Thr Phe Asn Lys Met Lys Asn Thr Val Glu Asn 53155 3160 3165 Ile Asp Ile Leu Cys Asn Giu Ala Giu Asn Lys Leu Met His Ile Leu 3170 3175 3180 His Ala Asn.Asp Pro Lys Trp Ser Thr Pro Thr Lys Asp Cys Thr Ser 3185 3190 3195 3200 Gly Pro Tyr Thr Ala Gln Ile Ile Pro Gly Thr Gly Asn Lys Leu Leu 3205 3210 3215 Met Ser Ser Pro Asn Cys Giu Ile Tyr Tyr Gin Ser Pro Leu Ser Leu 3220 3225 3230 Cys Met Ala Lys Arg Lys Ser Vai Ser Thr Pro Val Ser Ala Gin Met 3235 3240 3245 Thr Ser Lys Ser Cys Lys Gly Giu Lys Giu Ile Asp Asp Gin Lys Asn 3250 3255 3260 Cys Lys Lys Arg Arg Ala Leu Asp Phe Leu Ser Arg Leu Pro Leu Pro* 3265 3270 3275 3280 Pro Pro Val Ser Pro Ile Cys Thr Phe Val Ser Pro Ala Ala Gin Lys 303285 3290 3295 Ala Phe Gin Pro Pro Arg Ser Cys Gly Thr Lys Tyr 0Th Thr Pro Ile 3300 3305 3310 *Lys Lys Lys Glu Leu Asn Ser Pro Gin Met Thr Pro Phe Lys Lys Phe 331S 3320 3325 Asn Giu Ile Ser Leu Leu Giu Ser Asn Ser Ile Ala Asp Giu Giu Leu.

3330 3335 3340 Ala Leu Ile Asn Thr Gin Ala Leu Leu Ser Giy Ser Thr Gly Giu Lys 3345 3350 3355 3360 Gin Phe Ile Ser Val Ser Giu Ser Thr Arg Thr Ala Pro Thr Ser Ser 453365 3370 3375 Giu Asp Tyr Leu Arg Leu Lys Arg Arg Cys Thr Thr Ser Leu Ile Lys 3380 3385 3390 *Giu Gin Giu Ser Ser Gin Ala Ser Thr Giu Giu Cys Glu Lys Asn Lys s o 3395 3400 3405 Gin Asp Thr Ile Thr Thr Lys Lys Tyr Ile 3410 3415 INFORMATION FOR SEQ ID NO:3: -118- SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic.acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..2 OTHER INFORMATION: /note= "(NH2) at nucleotide 1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GTAGTGCAAG GCTCGAGAAC NNNNNNNNNN NN 32 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs 30 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 35 DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL

SOURCE:

ORGANISM: Homo sapiens (ix) FEATURE: 45 NAME/KEY: misc_feature LOCATION: 1..2 OTHER INFORMATION: /note= "(NH2) at nucleotide 1" 50 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TGAGTAGAAT TCTAACGGCC GTCATTGTTC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: -119- LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: misc feature LOCATION: 29..30 OTHER INFORMATION: /note= "(NH2) at nucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID GAACAATGAC GGCCGTTAGA ATTCTACTCA INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: S, LENGTH: 25 base pairs 30 TYPE: nucleic acid S(C) STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 35 DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens S* 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TCAGTAGAAT TCTAACGGCC GTCAT INFORMATION FOR SEQ ID NO:7: S* SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -120- (ii) MOLECULE TYPE:.other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..2 OTHER INFORMATION: /note= "(P04) at nucleotide 1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GTAGTGCAAG GCTCGAGAAC INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: other nucleic acid 30 DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: 40 NAME/KEY: miscfeature S(B) LOCATION: 1..2 OTHER INFORMATION: /note= "(P04) at nucleotide 1" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TGAGTAGAAT TCTAACGGCC GTCATTG 27 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 33 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -121- (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: NAME/KEY: misc feature LOCATION: 32..33 OTHER INFORMATION: /note= (NH2) at nucleotide 33" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CCTTCACACG CGTATCGATT AGTCACNNNN NNN 33 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 30 DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (ix) FEATURE: 40 NAME/KEY: misc feature LOCATION: 1..2 OTHER INFORMATION: /note (P04) at nucleotide 1" (xi) SEQUENCE DESCRIPTION: SEQ ID GTGACTAATC GATACGCGTG TGAAGGTGC 29 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 25 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -122- (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc'= "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homos sapiens (ix) FEATURE: NAME/KEY: misc_feature LOCATION: 1..2 OTHER INFORMATION: /note= "Biotinylated at nucleotide l" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TTGAAGAACA ACAGGACTTT CACTA INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH:. 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 30 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO 35 (iv) ANTI-SENSE: NO o (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CACCTTCACA CGCGTATCG 19 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs o TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO -123- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GTTCGTAATT GTTGTTTTTA TGTTCAG 27 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: I CCTTCACACG CGTATCGATT AG 22 35 INFORMATION FOR SEQ ID S* SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid 40 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" 45 (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO 50 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID TTTGGATCAT TTTCACACTG TC -124- INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GTGCTCATAG TCAGAAATGA AG 22 INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid 30 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 35 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: S* 40 ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: S* 45 TCTTCCCATC CTCACAGTAA G 21 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: 50 LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: lineat (ii) MOLECULE TYPE: DNA (genomic) -125- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GTACTGGGTT TTTAGCAAGC A 21 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: o:oo* GGTTAAAACT AAGGTGGGA 19 35 INFORMATION FOR SEQ ID o SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid 40 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 45 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID ATTTGCCCAG

CATGACACA

-126- INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21: TTTCCCAGTA TAGAGGAGA 19 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 35 (iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GTAGGAAAAT GTTTCATTTA A 21 45 INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

-127- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: ATCTAAAGTA GTATTCCAAC A 21 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens 0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GGGGGTAAAA AAAGGGGAA 19 INFORMATION FOR SEQ ID SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single 40 TOPOLOGY: linear 40 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

S* 45 (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID GAGATAAGTC AGGTATGATT INFO20TION FOR SEQ ID NO:26: INFORMATION FOR SEQ ID NO:26: -128- SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: AATTGCCTGT ATGAGGCAGA INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 35 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GGCAATTCAG TAAACGTTAA INFORMATION FOR-SEQ ID NO:28: 45 SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE*TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO -129- (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: ATTGTCAGTT ACTAACACAC INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: SGTGTCATGTA ATCAAATAGT 30 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 0(A) LENGTH: 19 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 40(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 45 (vi) ORIGINAL SOURCE: ORGANISM:. Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID CAGGTTTAGA GACTTTCTC 19 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs -130- TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GGACCTAGGT TGATTGCA 18 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single (D).TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: GTCAAGAAAG GTAAGGTAA 0505 4019 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs 45 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) 50 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens -131- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CTATGAGAAA GGTTGTGAG 19 INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CCTAGTCTTG CTAGTTCTT 19 INFORMATION FOR SEQ ID 0 SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs ooo, TYPE: nucleic acid STRANDEDNESS: single 35 TOPOLOGY: linear oo (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: 45 ORGANISM: homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID AACAGTTGTA GATACCTCTG AA 22 50 0* INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single -132- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: GACTTTTTGA TACCCTGAAA TG 22 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL

SOURCE:

ORGANISM: Homo sapiens 35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: CAGCATCTTG AATCTCATAC AG 22 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single 45 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO S(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens -133- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: CATGTATACA GATGATGCCT AAG 23 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: AACTTAGTGA AAAATATTTA GTGA 24 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: 30 LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 35 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens 45 (xi) SEQUENCE DESCRIPTION: SEQ ID ATACATCTTG ATTCTTTTCC AT 22 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -134- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: TTTAGTGAAT GTGATTGATG GT 22 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: 30 ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: AGAACCAACT TTGTCCTTAA INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: 40 LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: -135- TTAGATTTGT GTTTTGGTTG AA 22 INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: TAGCTCTTTT GGGACAATTC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid 30 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 35 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID ATGGAAAAGA ATCAAGATGT AT 22 INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -136- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CCTAATGTTA TGTTCAGAGA G 21 INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GCTACCTCCA AAACTGTGA 19 INFORMATION FOR SEQ ID NO:48: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GTGTAAAGCA GCATATAAAA AT o -137- INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: CTTGCTGCTG TCTACCTG 18 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 30 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

35 (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens SEQUENCE DESCRIPTION: SEQ ID AGTGGTCTTA AGATAGTCAT INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

-138- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CCATAATTTA ACACCTAGCC A 21 INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52: CCAAAAAAGT TAAATCTGAC A 21 INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO 45 (iv) ANTI-SENSE: NO ORIGINAL SOURCE: oo ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53: GGCTTTTATT CTGCTCATGG C 21 INFORMATION FOR SEQ ID NO:54: -139- SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: CCTCTGCAGA AGTTTCCTCA C 21 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO *i 35 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID 40 SAACGGACTTG CTATTTACTG A 21 INFORMATION FOR SEQ ID NO:56: 45 SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

-140- (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: AGTACCTTGC TCTTTTTCAT C 21 INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CAGCTAGCGG GAAAAAAGTT A 21 INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 45 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: TTCGGAGAGA TGATTTTTGT C 21 INFORMATION FOR SEQ ID NO:59: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs -141- TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: GCCTTAGCTT TTTACACAA 19 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTTGATTA TATCTCGTTG INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs 45 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens -142- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: TTATTCTCGT TGTTTTCCTT A 21 INFORMATION FOR SEQ ID NO:62: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM:. Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: CCATTAAATT GTCCATATCT A 21 INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single 35 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: S* ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: GACGTAGGTG AATAGTGAAG A 21 50 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single -143- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: TCAAATTCCT CTAACACTCC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID GAAGATAGTA CCAAGCAAGT C 21 INFORMATION FOR SEQ ID NO:66: S* S SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single 45 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens -144- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: TGAGACTTTG GTTCCTAATA C 21 INFORMATION FOR SEQ ID NO:67: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: AGTAACGAAC ATTCAGACCA G 21 INFORMATION FOR SEQ ID NO:68: SEQUENCE CHARACTERISTICS: *oo. 30 LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 35 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: GTCTTCACTA TTCACCTACG INFORMATION FOR SEQ ID NO:69: SEQUENCE

CHARACTERISTICS:

LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -145- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CCCCCAAACT GACTACACAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: 30 ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID AGCATACCAA GTCTACTGAA T 21 INFORMATION FOR SEQ ID NO:71: SEQUENCE CHARACTERISTICS: 40 LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 45 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: -146- ACTCTTTCAA ACATTAGGTC A 21 INFORMATION FOR SEQ ID NO:72: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: TTGGAGAGGC AGGTGGAT 18 INFORMATION FOR SEQ ID NO:73: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid 30 STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) 35 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: 40 ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: CTATAGAGGG AGAACAGAT 19 o(2) INFORMATION FOR SEQ ID NO:74: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -147- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: TTTATGCTGA TTTCTGTTGT AT 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid .STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID ATAAAACGGG AAGTGTTAAC T 21 35 INFORMATION FOR SEQ ID NO:76: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: CTGTGAGTTA TTTGGTGCAT -148e INFORMATION FOR SEQ ID NO:77: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: GAATACAAAA CAGTTACCAG A INFORMATION FOR SEQ ID NO:78: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: CACCACCAAA GGGGGAAA INFORMATION FOR SEQ ID NO:79: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

-149- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: AAATGAGGGT CTGCAACAAA INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID GTCCGACCAG AACTTGAG 18 INFORMATION FOR SEQ ID NO:81: 35 SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv).ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: AGCCATTTGT AGGATACTAG INFORMATION FOR SEQ ID NO:82: -150- SEQUENCE CHARACTERISTICS: LENGTH: 17 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: CTACTAGACG GGCGGAG 17 INFORMATION FOR SEQ ID NO:83: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens o S'"0 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: ATGTTTTTGT AGTGAAGATT CT 22 S(2) INFORMATION FOR SEQ ID'NO:84: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs .o TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO -151- (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: TAGTTCGAGA GACAGTTAAG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID CAGTTTTGGT TTGTTATAAT TG 22 INFORMATION FOR SEQ ID NO:86: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO i (iv) ANTI-SENSE: NO 45 (vi) ORIGINAL

SOURCE:

ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: CAGAGAATAG TTGTAGTTGT T 21 INFORMATION FOR SEQ ID NO:87: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs -152- TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: AACCTTAACC CATACTGCC 19 INFORMATION FOR SEQ ID NO:88: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO ORIGINAL SOURCE: ORGANISM: Homo sapiens a (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: TTCAGTATCA TCCTATGTGG INFORMATION FOR SEQ ID NO:89: S* SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL

SOURCE:

ORGANISM: Homo sapiens -153- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: TTTTATTCTC AGTTATTCAG TG 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID GAAATTGAGC ATCCTTAGTA A 21 INFORMATION FOR SEQ ID NO:91: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single 35 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL

SOURCE:

ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: AATTCTAGAG

TCACACTTCC

INFORMATION FOR SEQ ID NO:92: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single -154- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92: ATATTTTTAA GGCAGTTCTA GA 22 INFORMATION FOR SEQ ID NO:93: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: S(A) ORGANISM: Homo sapiens 35 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93: TTACACACAC CAAAAAAGTC A 21 INFORMATION FOR SEQ ID NO:94: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs •o TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens -155- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94: TGAAAACTCT TATGATATCT GT 22 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID TGAATGTTAT ATATGTGACT TTT 23 INFORMATION FOR SEQ ID.NO:96: SEQUENCE CHARACTERISTICS: 30 LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 35 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96: CTTGTTGCTA TTCTTTGTCT A 21 INFORMATION FOR SEQ ID NO:97: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear -156- (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97: CCCTAGATAC TAAAAAATAA AG 22 INFORMATION FOR SEQ ID NO:98: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO S(vi) ORIGINAL SOURCE: s* 30 ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98: i" 35 CTTTTAGCAG TTATATAGTT TC 22 INFORMATION FOR SEQ ID NO:99: SEQUENCE CHARACTERISTICS: 40 LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single o(D) TOPOLOGY: linear 45 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99: -157- GCCAGAGAGT CTAAAACAG 19 INFORMATION FOR SEQ ID NO:100: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100: CTTTGGGTGT TTTATGCTTG INFORMATION FOR SEQ ID NO:101: SEQUENCE CHARACTERISTICS: S LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) 35 (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101: TTTGTTGTAT TTGTCCTGTT TA 22 INFORMATION FOR SEQ ID NO:102: SEQUENCE

CHARACTERISTICS:

LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) -158- (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102: ATTTTGTTAG TAAGGTCATT TTT 23 INFORMATION FOR SEQ ID NO:103: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103; GTTCTGATTG CTTTTTATTC C 21 35 INFORMATION FOR SEQ ID NO:104: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid 40 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: 50 ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104: ATCACTTCTT CCATTGCATC n -159a a a.

S. a

S

INFORMATION FOR SEQ ID NO:105: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (.iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105: CCGTGGCTGG TAAATCTG INFORMATION FOR SEQ ID NO:106: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE:

NO

(vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: CTGGTAGCTC CAACTAATC 45 INFORMATION FOR SEQ ID NO:107: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

-160- (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107: ACCGGTACAA ACCTTTCATT G 21 INFORMATION FOR SEQ ID NO:108: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens Se (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: CTATTTTGAT TTGCTTTTAT TATT 24 INFORMATION FOR SEQ ID NO:109: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~(iii) HYPOTHETICAL: NO 9 (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: 1(A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109: GCTATTTCCT TGATACTGGA C 21 INFORMATION FOR SEQ ID NO:110: -161- SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID N0:110: TTGGAAACAT AAATATGTGG G 21 INFORMATION FOR SEQ ID NO:111: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO 35 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111: ACTTACAGGA GCCACATAAC INFORMATION FOR SEQ ID NO:112: 6 45 SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid *e STRANDEDNESS: single 000* TOPOLOGY: linear 0 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE:

NO

-162- (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: CTACATTAAT TATGATAGGC TCG 23 INFORMATION FOR SEQ ID NO:113: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113: SGTACTAATGT GTGGTTTGAA A 21 30 INFORMATION FOR SEQ ID NO:114: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs S 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 45 (vi) ORIGINAL SOURCE: ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114: TCAATGCAAG TTCTTCGTCA GC 22 INFORMATION FOR SEQ ID NO:115: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs -163- TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115: GGGAAGCTTC ATAAGTCAGT C 21 INFORMATION FOR SEQ ID NO:116: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO 30 (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116: 35 TTTGTAATGA AGCATCTGAT ACC 23 INFORMATION FOR SEQ ID NO:117: SEQUENCE CHARACTERISTICS: 40 LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 45 (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO 50 (iv) ANTI-SENSE:

NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117: AATGATGAAT GTAGCACGC -164- INFORMATION FOR SEQ ID NO:118: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118: GTCTGAATGT TCGTTACT 18 INFORMATION FOR SEQ ID NO:119: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid 30 DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119: ACCATCAAAC ACATCATCC 40 INFORMATION FOR SEQ ID NO:120: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 00 (ii) MOLECULE TYPE: other nucleic acid 50 DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES -165- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:120: AGAAAGTAAC TTGGAGGGAG INFORMATION FOR SEQ ID NO:121: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:121: CTCCTGAAAC TGTTCCCTTG G 21 INFORMATION FOR SEQ ID NO:122: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid 30 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:122: TAATGGTGCT GGGATATTTG G 21 45 INFORMATION FOR SEQ ID NO:123: SEQUENCE CHARACTERISTICS: LENGTH: 19 base pairs TYPE: nucleic acid 50 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO -166- (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:123: GAATGTCGAA GAGCTTGTC INFORMATION FOR SEQ ID NO:124: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid DESCRIPTION: /desc "Primer" (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO:124: AAACATACGC TTAGCCAGAC

Claims (2)

167- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:- 1. An isolated DNA comprising a cDNA coding for a BRCA2 polypeptide defined by the amino acid sequence set forth in SEQ ID NO:2 or a corresponding RNA. 2. An isolated nucleic acid which comprises the coding sequence set forth in SEQ ID NO: 1 from nucleotide position 229 to nucleotide position 10482 or a corresponding RNA. 3. An isolated nucleic acid as claimed in claim 2 which further comprises a mutation associated with a predisposition to breast cancer, said mutation being selected from: AC at nucleotide positions 277 and 278 deleted; or four nucleotides at positions 982-985 deleted; or having four nucleotides at positions 4706-4709 deleted; or C at nucleotide position 8525 deleted; or five nucleotides at positions 9254-9258 deleted; or GT at nucleotide positions 4075 and 4076 deleted; or five nucleotides at positions 999-1003 deleted; or T at nucleotide position 6174 deleted; or three nucleotides at positions 4132-4134 deleted; or A at position 1493 deleted; or a C instead of an A at position 2411. 20 4. An isolated nucleic acid selected from the group consisting of: a DNA comprising the coding sequence set forth in SEQ ID NO: 1, from nucleotide position 229 to nucleotide position 10482 or a corresponding RNA; and a DNA which hybridizes to and is at least 95% complementary compared to an entire DNA coding for a BRCA2 polypeptide as in above or a corresponding RNA, wherein said nucleic acid further comprises a mutation associated with a predisposition to breast cancer, said mutation being selected from: AC at nucleotide positions 277 and 278 deleted; or (ii) four nucleotides at positions 982-985 deleted; or -168- (iii) having four nucleotides at positions 4706-4709 deleted; or (iv) C at nucleotide position 8525 deleted; or five nucleotides at positions 9254-9258 deleted; or (vi) GT at nucleotide positions 4075 and 4076 deleted; or (vii) five nucleotides at positions 999-1003 deleted; or (viii) three nucleotides at positions 4132-4134 deleted; or (ix) A at position 1493 deleted; or a C instead of an A at position 2411. An isolated nucleic acid selected from the group consisting of: 10 a DNA comprising the coding sequence set forth in SEQ ID NO: 1 from S •nucleotide position 10482 or corresponding RNA; and i a DNA which hybridizes to and is at least 95% complementary compared to i an entire DNA coding for a BRCA2 polypeptide as in above or a corresponding RNA, wherein said nucleic acid further comprises an alteration, said alteration being selected from: a C instead of a G at position 451; or a C instead of an A at position 1093; or a C instead ofa G at position 1291; or a T instead of a C at position 2117; or o 20 an A instead ofa G at position 4813; or a G instead ofa T at position 5868; or a T instead of a C at position 5972; or a T instead of a C at position 6328; or a T instead of a G at position 7049; or a C instead of a G at position 7491; or a G instead of an A at position 9537; or a T instead of an A at position 10204; or a G instead of a C at position 10298; or a G instead of an A at position 10462; or an A instead of a G at position 203; or an A instead of a C at position 1342; or a C instead of a T at position 2457; or -169- a G instead of an A at position 3199; or a G instead of an A at position 3624; or a G instead of an A at position 3668; or a C instead of a T at position 4035; or a G instead of an A at position 7470; or a G instead of an A at position 1593; or an A instead of a G at position 4296; or a G instead of an A at position 5691; or a G instead of an A at position 6051; or (aa) a C instead of a T at position 6828; or (bb) a C instead of a T at position 6921. 6. An isolated nucleic acid having at least 15 contiguous nucleotides of a nucleic acid as claimed in claim 4(a) encompassing a said mutation associated with a predisposition to breast cancer. 7. A vector selected from the group consisting of: a vector comprising an isolated nucleic acid as claimed in any one of claims 1 to 6; and C a vector comprising an isolated nucleic acid as claimed in any one of claims 1 to 5 wherein said nucleic acid is operably-linked to a promoter sequence 20 capable of directing expression of said nucleic acid in host cells for said vector. 8. Host cells transformed with a vector as claimed in claim 7. 9. A method for producing a polypeptide encoded by an isolated nucleic acid as claimed in any one of claims 1 to 5 which comprises culturing the host cells of claim 8 containing an expression vector encoding said polypeptide under conditions suitable for the production of said polypeptide and (ii) recovering said polypeptide. A method as claimed in claim 9 which further comprises labelling the recovered polypeptide. 11. A preparation of human BRCA2 polypeptide substantially free of other human proteins, selected from the group consisting of: Apr. 2004 15:29 No, 0103 P, 7 -170- a polypeptide having the amino acid sequence set forth in SEQ ID NO:2; a mutated or variant human BRCA2 polypeptide obtainable by expression of a nucleic acid according to any one of claims 3,4 or 5; and a fusion protein containing a polypeptide as defined in or 12. A preparation as claimed in claim 11 wherein said polypeptide is labelled. 13. Use of a polypeptide as defined in claim 11 as an immunogen for antibody production. 14. A use as claimed in claim 13, wherein one or more antibodies are subsequently labelled or bound to a solid support. 15. A method for determining variation of the nucleotide sequence of a suspected mutant BRCA2 allele associated with predisposition to breast cancer from a known non- mutant wild-type BRCA2-encoding nucleotide sequence comprising nucleotides 229- 10482 of SEQ ID NO: 1, said method comprising preparing a biological sample containing said suspected mutant BRCA2 allele and comparing the nucleotide sequence of the suspected mutant BRCA2 allele with said non-mutant sequence and identifying difference(s) between the sequences. 16. A nucleic acid comprising a BRCA2 gone which encodes a BRCA2 polypeptide *O .O •having the amino acid sequence set forth in SEQ ID NO:2 for use in breast cancer gene therapy. 20 17. Use of a nucleic acid as defined in claim 16 for the manufacture of a vector S" preparation for use in breast cancer gene therapy. 18. Use of a polypeptide which is a wild-type BRCA2 polypeptide having an amino acid sequence set forth in SEQ ID NO:2 for the manufacture of a product for use in *ooo peptide breast cancer therapy. S. 25 19. An isolated nucleic acid comprising a nucleotide sequence of a nucleic acid oo.•amplified from a human tissue sample by PCR using a first primer having the sequence *of SEQ I NO: 112 and a second primer having the sequence of SEQ I NO: 13. of SEQ ID NO: 112 and a second primer having the sequence of SEQ ID N0:1 13. COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 Apr. 2004 15:30 No. 0103 P. 8 -171- The isolated nucleic acid of Claim 19 which hybridizes to a nucleic acid having a sequence of SEQ ID.NO:51 and/or SEQ ID NO:52 at 45 0 C with hybridization buffer consisting of 6M urea, 0.3M NaCi, and 5X Denhardt's solution. 21. The isolated nucleic acid of Claim 19 which is a cDNA or mRNA derived from the BRCA2 gene on human chromosome 13 between the markers tdj3820 and YS-G-B 1OT, and encoding a BRCA2 protein having a total of 3418 amino acids with the N-terminus amino acid being methionine and the C-terminus amino acid being isoleucine. 22. The isolated nucleic acid of any one of Claims 19 or 20, which hybridizes to a probe consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs:98-114, at 45 0 C with hybridization buffer consisting of 6M urea, 0.3M NaCi, and Denbaxdt's solution. 23. A method for detecting a mutation in a BRCA2 gene in a patient sample, comprising, amplifying at least a BRCA2 gene exon selected from the group consisting of exons 21- 27; and determining whether the nucleotide sequence of the amplified exon encodes a shorter amino acid sequence, 0 wherein said BRCA2 gone is on human chromosome 13 between the markers tdj3820 and YS-G-B 1OT, and encodes a BRCA2 protein having a total of 3418 amino acids with 20 the N-terminus amino acid being methionine and the C-terminus amino acid being isoleucine. 24. The method of Claim 23, wherein the mutation is a frameshift mutation or a premature stop codon. 25. A method for detecting an alteration in a BRCA2 gene in a patient sample, 25 comprising, amplifying at least a BRCA2 gene exon selected from the group consisting of exons 21- 27; and comparing the nucleotide sequence of the amplified exon with SEQ ID NO: 1. COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 Apr. 2004 15:30 No. 0103 P. 9 -172- 26. A method for detecting a predisposition for breast cancer and ovarian cancer in a tissue sample derived from a human, which comprises determining in said tissue sample whether there is a germine alteration in exons 21-27 of the BRCA2 gene, which is defined as the gone that is on human chromosome 13 between the markers tdj3820 and YS-G-B1 OT, encodes a BRCA2 protein having a total of 3418 amino acids with the N- teminus amino acid being methionine and the C-terminus amino acid being isoleucine, and comprises a nucleotide sequence of a nucleic acid amplified from a human tissue sample by PCR using a first primer having the sequence of SEQ ID NO: 112 and a second primer having the sequence of SEQ ID NO: 113, wherein the presence of said alteration indicates said predisposition to cancer. 27. The method of Claim 26, wherein said BRCA2 gene hybridizes to a probe comprising the sequence of SEQ ID NO:51 and/or SEQ ID NO:52. 28. The method of Claim 26 or 27, wherein said alteration causes protein truncation in the encoded BRCA2 protein. 29. The method of Claim 26,27 or 28, wherein the alteration is selected from the group consisting of frameshift mutations and premature stop codons. An isolated DNA comprising a cDNA coding for a BRCA2 polypeptide defined by the amino acid sequence set forth in SEQ ID NO:2 or a corresponding RNA, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 31. An isolated nucleic acid which comprises the coding sequence set forth in SEQ ID NO:1 from nucleotide position 229 to nucleotide position 10482 or a corresponding RNA, substantially as herein described with reference to any one of the examples but excluding comparative eamples. 32. An isolated nucleic acid selected from the group consisting of: 9999 COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 Apr. 2004 15 :30 No. 0103 P. -3 a DNA comprising the coding sequence set forth in SEQ ID NO: 1, from nucleotide position 229 to nucleotide position 10482 or a corresponding RNA; and a DNA which hybridizes to and is at least 95% complementary compared to an entire DNA coding for a BRCA2 polypeptide as in above or a corresponding RNA, wherein said nucleic acid ftrther comprises a mutation associated with a predisposition to breast cancer, said mutation being selected from: AC at nucleotide positions 277 and 278 deleted; or (ii) Ibur nucleotides at positions 982-98 5 deleted; or (iii) having four nucleotides at positions 4706-4709 deleted; or (iv) C at nucleotide position 8525 deleted; or five micleotides at positions 9254-9258 deleted;, or (vi) GT at nucleotide positions 4075 and 4076 deleted; or (vii) five nucleotides at positions 999-1003 deleted; or (viii) three nucleotides at positions 4132-4134 deleted; or (ix) A at position 1493 deleted; or a C instead of an A at position 2411 a C instead of an A at position 2411, substantially as herein described with reference to any one or more of the examples :20 but excluding comparative examples. *see 33. An isolated nucleic acid selected from the group consisting of: a DNA comprising the coding sequence set forth in SEQ U) NO: 1 from 0 nucleotide position 10482 or corresponding RNA, and a DNA which hybridizes to and is at least 95% complementary compared to an entire DNA coding for a BRCA2 polypeptide as in above or a corresponding RNA, wherein said nucleic acid fluter comprises an alteration, said alteration being selected from: a Cinstead of a Gatposition 45l; or a C instead of an A at position 1093; or .30 a C instead of a O at position 1291; or a T instead of a C at position 2117; or an A instead of aG at position 4813; or COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 Ap r. 2 00 4 15:3 0 No. 0103 P. 11

174- a G instead of a T at position 5868; or a T instead of a C at position 5972; or a T instead of a C at position 6328; or a T instead of a G at position 70-49; or a C instead of a G at position 7491;1 or a Gi instead of an A at position 9537; or a T instead of an A at position 10204; or (in) a Gi instead of a C at position 10298; or a Gi instead of an A at position 10462; or an A instead of a G at position 203; or an A instead of a Cat position 1342; or a C instead of a T at position 2457; or a G instead of an A at position 3199; or a Gi instea of an A at position 3624; or a Gi instead of an A at position 3668; or a C instead of a T at position 4035; or a Gi instead of an A at position 7470; or a Ginsteadof an Aat position 1593; or :0 an A instead of a Gi at position 4296; or a Ginstead of an Aatposition 569l; or a Ginstead of an Aatposition 6O51;or 0a) aCisedo Ta oiin62;o (bb) a C instead of a T at position 6828;,o substantially as herein described with reference to any one or more of the examples 40625 but excluding comparative examples. 0 0 go*. 34. An isolated nucleic adid having at least 15 contiguous nucleotides of a nucleic acid 0:0 claimed in claim 4(a) encompassing a said mutation associated with a predisposition 0 0 0000to breast cancer, substantially as herein described with reference to arny one or more of the examples but excluding comparative examples& 000 COMS ID No: SMBI-006g6479 Received by IP Australia: Time 15:33 Date 2004-04-05 Apr. 2004 15:31 No. 0103 P. 12 175 A vector comprising an isolated nucleic acid according to any one of claims 1 to 6, substantially as herein described with reference to any one of the examples but excluding comparative examples. 36. Host cells transformed with a vector comprising isolated nucleic acid according to any one of claims 1 to 6, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 37. A method fir producing a polypeptide encoded by an isolated nucleic acid according to any one of claims 1 to 5, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 38. A preparation of human BRCA2 polypeptide substantially free of other human proteins selected as a preparation of human BRCA2 polypeptide substantially free of other human proteins, selected from the group consisting of: a polypeptide having the amino acid sequence set forth in SEQ ID NO:2; a mutated or variant human BRCA2 polypqtide obtainable by expression of a nucleic acid according to any one of claims 3, 4 or 5; and a fusion protein containing a polypeptide as defined in or substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 39. Use of a polypeptide according to claim 11, substanfially as herein described with 2o reference to any one or more of the examples but excluding comparative examples. A method for determining variation of the nucleotide sequence of a suspected mutant BRCA2 allele associated with predisposition to breast cancer from a known non- mutant wild-type BRCA2 encoding nucleotide sequence comprising nucleotides 229- 10482 of SEQ ID substantially as herein described with reference to any one or 25 more of the examples but excluding comparative examples. 41. A nucleic acid comprising a BRCA2 gene which encodes a BRCA2 polypeptide having the amino acid sequence set forth in SEQ ID NO:2 for use in breast cancer gone therapy, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05 2004 15:31 No. 0103 P. 13 -176- 42. Use of a nucleic acid as defined in claim 16, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 43. An isolated nucleic acid comprising an isolated nucleic acid comprising a nucleotide sequence of a nucleic acid amplified from a human tissue sample by PCR using a first primer having the sequence of SEQ ID NO: 112 and a second primer having the sequence of SEQ I]D NO:113, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 44. A method for detecting a mutation in a BRCA2 gene in a patient sample, comprising, amplifying at least a BRCA2 gene exon selected from the group consisting of exons 21- 27; and determining whether the nucleotide sequence of the amplified exon encodes a shorter amino acid sequence, wherein said BRCA2 gene is on human chromosome 13 between the markers tdj3820 and YS-G-B 1OT, and encodes a BRCA2 protein having a total of 3418 amino acids with the N-terminus amino acid being methionine and the C-terminus amino acid being isoleucine, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 45. A method for detecting an alteration in a BRCA2 gene in a patient sample, comprising, amplifying at least a BRCA2 gene exon selected from the group consisting of exons 21- 27; and comparing the nucleotide sequence of the amplified exon with SEQ ID NO:1, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. 46. A method for detecting a predisposition for breast cancer and ovarian cancer in a tissue sample derived from a human, which comprises determining in said tissue sample COMS ID No: SMBI-0696479 Received by IP Australia: Time 15:33 Date 2004-04-05 Apr. 2004 15:31 No. 0103 P. 14 -177- whether there is a germline alteration in exons 21-27 of the BRCA2 gene, which is defined as the gene that is on human chromosome 13 between the markers tdj3820 and YS-G-B 1 0T, encodes a BRCA2 protein having a total of 3418 amino acids with the N- terminus amino acid being methionine and the C-terminus amino acid being isoleucine, and comprises a nucleotide sequence of a nucleic acid amplified from a human tissue sample by PCR using a first primer having the sequence of SEQ ID NO: 112 and a second primer having the sequence of SEQ ID NO:113, wherein the presence of said alteration indicates said predisposition to cancer, substantially as herein described with reference to any one or more of the examples but excluding comparative examples. DATED this 1 st day of April 2004 BALDWIN SHELSTON WATERS Attorneys for: MYRIAD GENETICS, INC. AND HSC RESEARCH DEVELOPMENT LIMITED PARTNERSHIP AND ENDO RECHERCHE INC. AND THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA o COMS ID No: SMBI-00696479 Received by IP Australia: Time 15:33 Date 2004-04-05