NZ291330A - Detection and isolation of the gene brca1 a cancer predisposing gene - Google Patents

Description

New Zealand No. 291330 International No. PCT/US95/10203 TO BE ENTERED AFTER ACCEPTANCE AND PUBLICATION Priority dates: 24.03.1995;29.11.1994;16.09.19Q94;12.08.1 994;02.09.1994:07.060.1995;07.06.1995;07.06.1995; Complete Specification Filed: 11.08.1995 Classification:^) C12N15/12; C12P21/00; C07K14/82; C07K16/32; C12Q1/6B Publication date: 27 May 1998 Journal No.: 1428 NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: 17q-linked breast and ovarian cancer susceptibility gene Name, address and nationality of applicant(s) as in international application form: MYRIAD GENETICS, INC., 390 Wakara Way, Salt Lake City, Utah 84108, United States of America; UNIVERSITY OF UTAH RESEARCH FOUNDATION, Suite 170, 421 Wakara Way, Salt Lake City, Utah 84108, United States of America; UNITED ST' TES OF AMERICA, represented by THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES, Office of Technology Transfer, Suite 325, 6011 Executive Boulevard, Rockville, Maryland 20852, United States of America 2 9 i y W V WO 96/05307 PCTA3S95/10203 tttt.e of the invention 17q-LINKED BREAST AND OVARIAN CANCER SUSCEPTIBILITY GENE 5 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 breast and ovarian cancer predisposing gene (BRCA1), some mutant alleles of which cause susceptibility to 0 cancer, in particular, breast and ovarian cancer. More specifically, the invention relates to germline mutations in the BRCA1 gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The present invention further relates to somatic mutations in the BRCA1 gene in human breast and ovarian cancer and their use in the diagnosis and prognosis of human breast and ovarian cancer. Additionally, the invention relates to somatic mutations in the BRCA1 gene in other 5 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 BRCA1 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 BRCA1 gene for mutations, which are useful for diagnosing the predisposition to breast and ovarian cancer. o 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. b ackground of THE INVENTION The genetics of cancer is complicated, involving multiple dominant, positive regulators of the transformed state (oncogenes) as well as multiple recessive, negative regulators (tumor ; 0 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 tumors, and CDKN2, which was homozygously deleted in 46% of tumor cell lines examined (Kamb et al., 1994). 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 unharmed 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 (RBI); 2) Wilms' tumor (WT1); 3) Li-Fraumeni (TP53); 4) Familial adeno- matous polyposis (APC); 5) Neurofibromatosis type 1 (NF1); 6) Neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau syndrome (VHL); 8) Multiple endocrine neoplasia type 2A (MEN2A); and 9) Melanoma (CDKN2).

Tumor suppressor loci that have been mapped genetically but not yet isolated include genes for: Multiple endocrine neoplasia type 1 (MEN1); Lynch cancer family syndrome 2 (LCFS2); Neuroblastoma (NB); 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 (WT1), ancillary transcription regulators (RBI), GTPase activating proteins or GAPs (NF1), 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 man}' 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, 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 familial breast cancer, but at least 80% of families with both breast and ovarian cancer (Easton et al, 1993).

Intense efforts to isolate the BRCA1 gene have proceeded since it was first mapped in 1990 (Hall et al, 1990; Narod et al, 1991). A second locus, BRCA2, has recently been mapped to chromosome 13q (Wooster et al., 1994) and appears to account for a proportion of early-onset breast cancer roughly equal to BRCA1, 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-Telangectasia 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; Willams 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 I7p (Malkin et al, 1990), a 17q-linked susceptibility locus known as BRCAl (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 I7q21; 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 17q-linked breast cancer predisposing gene (BRCAl) require precise genetic localization studies. The simplest model for the functional role of BRCAl holds that alleles of BRCAl that predispose to cancer are recessive to wild type alleles; that is, cells that contain at least one wild type BRCAl allele are not cancerous. However, cells that contain one wild type BRCAl allele and one predisposing allele may occasionally suffer loss 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 BRCAl and may develop into tumors. According to this model, predisposing alleles of BRCAl are recessive, yet susceptibility to cat.cer 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 BRCAl allele. This model applies to a group 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 also explain the BRCAl function, as has recently been suggested (Smith et al., 1992).

A second possibility is that BRCAl predisposing alleles are truly dominant; that is, a wild type allele of BRCAl 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 BRCAl before giving rise to malignant cells. Instead, mammary cells in predisposed individuals would undergo some other stochastic change(s) leading to cancer.

WO 96/0S307 If BRCAl predisposing alleles are recessive, the BRCAl gene is expected to be expressed in normal mammary tissue but not functionally expressed in mammary tumors. In contrast, if BRCAl predisposing alleles are dominant, the wild type BRCAl gene may or may not be expressed in normal mammary tissue. However, the predisposing allele will likely be expressed in breast tumor cells.

The 17q linkage of BRCAl was independently confirmed in three of five kindreds with both breast cancer and ovarian cancer (Narod et al, 1991). These studies claimed to localize the gene within a very large region, 15 centiMorgans (cM), or approximately 15 million base pairs, to either side of the linked marker pCMM86 (D17S74). However, attempts to define the region further by genetic studies, using markers surrounding pCMMS6, proved unsuccessful. Subsequent studies indicated that the gene was considerably more proximal (Easton et al, 1993) and thai the original analysis was flawed (Margaritte et al., 1992). Hall et al, (1992) recently localized the BRCAl gene to an approximately 8 cM interval (approximately 8 million base pairs) bounded by Mfdl5 (D17S250) on the proximal side and the human GIP gene on the distal side. A slightly narrower interval for the BRCAl locus, based on publicly available data, was agreed upon at the Chromosome 17 workshop in March of 1992 (Fain, 1992). The size of these regions and the uncertainty associated with them has made it exceedingly difficult to design and implement physical mapping and/or cloning strategies for isolating the BRCAl gene.

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 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.

SUMMARY OF TTTF. 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 (BRCAl), some alleles of which cause susceptibility to cancer, in particular breast and ovarian cancer. More specifically, the present invention relates to germline mutations in the BRCAl gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The irivention further relates to somatic mutations in the BRCAl gene in human breast cancer and their use in the diagnosis and prognosis of human breast and ovarian cancer. Additionally, the invention relates to somatic mutations in the BRCAl 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 BRCAl 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 BRCAl gene for mutations, which are useful for diagnosing the predisposition to breast and ovarian cancer.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the order of loci neighboring BRCAl as determined by the chromosome 17 workshop. Figure 1 is reproduced from Fain, 1992.

Figure 2 is a schematic map of YACs which define part of MfdlS-Mfdl 88 region.

Figure 3 is a schematic map of STSs, Pis and BACs in the BRCAl region.

Figure 4 is a schematic map of human chromosome 17. The pertinent region containing BRCAl is expanded to indicate the relative positions of two previously identified genes, CA125 and RNU2, BRCAl spans the marker D17S855.

Figure 5 shows alignment of the BRCAl zinc-finger domain with 3 other zinc-finger domains that scored highest in a Smith-Waterman alignment RPT1 encodes a protein that appears to be a negative regulator of the IL-2 receptor in mouse. RIN1 encodes a DNA-binding protein that includes a RING-frnger motif related to the zinc-finger. RFP1 encodes a putative transcription factor that is the N-terminal domain of the RET oncogene product The bottom line contains the C3HC4 consensus zinc-finger sequence showing the positions of cysteines and one histidine that form the zinc ion binding pocket Figure 6 is a diagram of BRCAl mRN A showing the locations of introns and the variants of BRCAl mRNA produced by alternative splicing. Intron locations are shown by dark triangles and the exons are numbered below the line representing the cDNA. The top cDNA is the composite used to generate the peptide sequence of BRCAl. Alternative forms identified as cDNA clones or hybrid selection clones are shown below.

PCTAJS95/10203 Figure 7 shows the tissue expression pattern of BRCAl. The blot was obtained from Clontech and contains RNA from the indicated tissues. Hybridization conditions were as recommended by the manufacturer using a probe consisting of nucleotide positions 3631 to 3930 of BRCAl. Note that both breast and ovaiy are heterogeneous tissues and the percentage of relevant epithelial cells can be variable. Molecular weight standards are in kilobases.

Figure 8 is a diagram of the 5' untranslated region plus the beginning of the translated region of BRCAl showing the locations of introns and the variants of BRCAl mRNA produced by alternative splicing. Intron locations are shown by broken dashed lines. Six alternate splice forms are shown.

Figure 9A shows a nonsense mutation in Kindred 2082. P indicates the person originally screened, b and c are haplotype carriers, a, d, e, f, and g do not cany the BRCAl haplotype. The C to T mutation results in a stop codon and creates a site for the restriction enzyme Avrll. PCR amplification products are cut with this enzyme. The carriers are heterozygous for the site and therefore show three bands. Non-carriers remain uncut Figure 9B shows a mutation and cosegregation analysis in BRCAl kindreds. Carrier individuals are represented as filled circles and squares in the pedigree diagrams. Frameshift mutation in Kindred 1910. The first three lanes are control, noncarrier samples. Lanes labeled 1-3 contain sequences from carrier individuals. Lane 4 contains DNA from a kindred member who does not carry the BRCAl mutation. The diamond is used to prevent identification of the kindred. The frameshift resulting from the additional C is apparent in lanes labeled 1,2, and 3.

Figure 9C shows a mutation and co segregation analysis in BRCAl kindreds. Carrier individuals are represented as filled circles and squares in the pedigree diagrams. Inferred regulatory mutation in Kindred 2035. ASO analysis of carriers and noncarriers of 2 different polymorphisms (PM1 and PM7) which were examined for heterozygosity in the germline and compared to the heterozygosity of lymphocyte mRNA. The top 2 rows of each panel contain PCR products amplified from genomic DNA and the bottom 2 rows contain PCR products amplified from cDNA. "A" and "G" are the two alleles detected by the ASO. The dark spots indicate that a particular allele is present in the sample. The first three lanes of PM7 represent the three genotypes in the general population.

Figures 10A-10H show genomic sequence of BRCAl. The lower case letters denote intron sequence while the upper case letters denote exon sequence. Indefinite intervals within introns are PCT/US95/I0203 designated with wwwwwvw. Known polymorphic sites are shown as underlined and boldface type.

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 (BRCAl), some alleles of which cause susceptibility to cancer, in particular breast and ovarian cancer. More specifically, the present invention relates to germline mutations in the BRCAl gene and their use in the diagnosis of predisposition to breast and ovarian cancer. The invention further relates to somatic mutations in the BRCAl gene in human breast cancer and their use in the diagnosis and prognosis of human breast and ovarian cancer. Additionally, the invention relates to somatic mutations in the BRCAl 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 BRCAl 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 BRCAl gene for mutations, which are useful for diagnosing the predisposition to breast and ovarian cancer.

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

Also pro vided by the present invention are methods of detecting a polynucleotide comprising a portion of the BRCAl locus or its expression product in an analyte. Such methods may further comprise the step of amplifying the portion of the BRCAl locus, and may further include a step of providing a set of polynucleotides which are primers for amplification of said portion of the BRCAl 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 BRCAl locus.

The present invention also provides kits for detecting in an analyte a polynucleotide comprising a portion of the BRCAl locus, the kits comprising a polynucleotide complementary to the portion of the BRCAl 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 BRCAl locus; and methods of preparing a polypeptide comprising polymerizing amino acids to yield a sequence comprising at least five amino acids encoded within the BRCAl locus.

The present invention further provides methods of screening the BRCAl gene to identify mutations. Such methods may further comprise the step of amplifying a portion of the BRCAl locus, and may further include a step of providing a stv of polynucleotides which are primers for amplification of said portion of the BRCAl 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 BRCAl mutant alleles to identify mutations in the BRCAl gene.

In addition, the present invention provides methods of screening drugs for cancer therapy to identify suitable drugs for restoring BRCAl 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 BRCAl locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the BRCAl protein is reconstituted. Therapeutic agents may also take the form of polypeptides based on either a portion of, or the entire protein sequence of BRCAl. These may functionally replace the activity of BRCAl in vivo.

It is a discovery of the present invention that the BRCAl locus which predisposes individuals to breast cancer and ovarian cancer, is a gene encoding a BRCAl protein, which has been found to have no significant homology with known protein or DNA sequences. This gene is termed BRCAl herein. It is a discovery of the present invention that mutations in the BRCAl locus in the germline are indicative of a predisposition to breast cancer and ovarian cancer. Finally, it is a discovery of the present invention that somatic mutations in the BRCAl locus are also associated with breast cancer, ovarian cancer and other cancers, which represents an indicator of these cancers or of the prognosis of these cancers. The mutational events of the BRCAl locus can involve deletions, insertions and point mutations within the coding sequence and the non-coding sequence.

Starting from a region on the long arm of human chromosome 17 of the human genome, 17q, which has a size estimated at about 8 million base pairs, a region which contains a genetic locus, BRCAl, which causes susceptibility to cancer, including breast and ovarian cancer, has been identified.

The region containing the BRCAl locus was identified using a variety of genetic techniques. Genetic mapping techniques initially defined the BRCAl region in teims of recombination with genetic markers. Based upon studies of large extended families ("kindreds") with multiple cases of breast cancer (and ovarian cancer cases in some kindreds), a chromosomal region has been pinpointed that contains the BRCAl gene as well as other putative susceptibility alleles in the BRCAl locus. Two meiotic breakpoints have been discovered on the distal side of the BRCAl locus which are expressed as recombinants between genetic markers and the disease, and one recombinant on the proximal side of the BRCAl locus. Thus, a region which contains the BRCAl locus is physically bounded by these markers.

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 bacterial artificial chromosome (BAC) library. It also allowed for the identification and preparation of more easily manipulated cosmid, PI and BAC clones from this region and the construction of a contig from a subset of the clones. These cosmids, Pis, YACs and BACs provide the basis for cloning the BRCAl locus and provide the basis for developing reagents effective, for example, in the diagnosis and treatment of breast and/or ovarian cancer. The BRCAl 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 contiguous or discontinuous genomic DNA sequences), hybrid selection techniques and direct screening, with whole or partial cDNA inserts from cosmids, Pis 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 BRCA1 locus in kindreds which are responsible for the 17q-linked cancer susceptibility known as BRCAl. This gene was not known to be in this region. 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 BRCAl susceptibility allele is segregating in that family. Recombinants informative for localization and isolation of the BRCAl 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 BRCAl 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.

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 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 infoimativeness 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, i.e., 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 BRCAl locus, then replacing these flanking markers with other markers that were successively closer to the BRCAl locus. As an initial step, recombination events, defined by large extended kindreds, helped specifically to localize the BRCAl locus as either distal or proximal to a specific genetic marker (Goldgar et al, 1994).

The region surrounding BRCAl, until the disclosure of the present invention, was not well mapped and there were few markers. Therefore, short repetitive sequences on cosmids subcloned from YACs, which had been physically mapped, were analyzed in order to develop new genetic markers. Using this approach, one marker of the present invention, 42D6, was discovered which replaced pCMM86 as the distal flanking marker for the BRCAl region. Since 42D6 is approximately 14 cM from pCMM86, the BRCAl region was thus reduced by approximately 14 centiMorgans (Easton et al, 1993). The present invention thus began by finding a much more closely linked distal flanking marker of the BRCAl region. BRCAl was then discovered to be distal to the genetic marker MfdlS. Therefore, BRCAl was shown to be in a region of 6 to 10 million bases bounded by Mfdl5 and 42D6. Marker Mfdl91 was subsequently discovered to be distal to Mfdl5 and proximal to BRCAl. Thus, MfdlS was replaced with Mfdl91 as the closest proximal genetic marker. Similarly, it was discovered that genetic marker Mfdl88 could replace genetic marker 42D6, narrowing the region containing the BRCAl locus to approximately 1.5 million bases. Then the marker Mfdl91 was replaced with tdjl474 as the proximal marker and Mfdl88 was replaced with U5R as the distal marker, further narrowing the BRCAl region to a small enough region to allow isolation and characterization of the BRCAl locus (see Figure 3), using techniques known in the art and described herein.

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 region which is flanked by tdjl474 and U5R. The second was the creation of a set of PI, BAC and cosmid clones which cover the region containing the BRCAl locus.

Yeast Artificial Chromosomes (YACsV Once a sufficiently small region containing the BRCAl 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 BRCAl locus.

Cosmid. PI and BAC Clones. In the present invention, it is advantageous to proceed by obtaining cosmid, PI, 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. For cosmid subclones of YACs, the DNA is partially digested with the restriction enzyme Sau3A and cloned into the BaraHI site of the pWE15 cosmid vector (Stratagene, cat. #1251201). The cosmids containing human sequences are screened by hybridization with human repetitive DNA (e.g., Gibco/BRL, Human C0t-1 DNA, cat. 5279SA), and then fingerprinted by a variety of techniques, as detailed in the Examples.

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, cosmids or Pis and BACs, isolated as described herein.

These PI, BAC and cosmid 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 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 BRCAl locus.

Coverage of the Gap with PI and BAC Clones. To cover any gaps in the BRCAl contig between the identified cosmids with genomic clones, clones in PI and BAC vectors which contain inserts of genomic DNA roughly twice as large as cosmids for Pis and still greater for BACs (Sternberg, 1990; Sternberg et al., 1990; Pierce et al., 1992; Shizuya et al., 1992) were used. PI clones were isolated by Genome Sciences using PCR primers provided by us for screening. BACs were provided by hybridization techniques in Dr. Mel Simon's laboratory. The strategy of using PI 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 other deletions in YACs that have not been detected. These new sequences derived from the PI 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: a. zoo blots b. identifying HTF islands c. exon trapping d. hybridizing cDNA to cosmids or YACs. e. screening cDNA libraries. (a) Zoo blots. The first technique is to hybridize cosmids to Southern blots to identify DNA sequences which are evolutionary 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). (b) 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 (Hpal tiny fragment) or CpG islands, as restriction enzymes specific for sites which contain CpG dimers cut frequently in these regions (Lindsay et al, 1987). (c) 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. (d) Hybridizing cDNA to Cosmids. Pis. BACs or YACs. The fourth technique is a modification of the selective enrichment technique which utilizes hybridization of cDNA to cosmids. Pis, 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 BRCAl 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 the region represented by the cloned genomic DNA. (e) Identification of cDNAs. The fifth technique is to identify cDNAs that correspond to the BRCAl 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, ovarian cDNA libraries, and any other necessary libraries.

Another variation on the theme of direct selection of cDNA was also used to find candidate genes for BRCAl (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 Haelll. 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, e.g., 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 C0t-1 DNA to block repetitive sequences. Solution hybridization is carried out to high C0t-l/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.

Testing the cDNA for Candidacy Proof that the cDNA is the BRCAl locus is obtained by finding sequences in DNA extracted from affected kindred members which create abnormal BRCAl gene products or abnormal levels of BRCAl gene product Such BRCAl 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 and ovarian 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 BRCAl alleles mutated into sequences which are identical or similar to BRCAl susceptibility alleles in DNA extracted from tumor tissue. Whether one is comparing BRCAl sequences from tumor tissue to BRCAl alleles from the germline of the same individuals, or one is comparing germline BRCAl 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 function 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 BRCAl locus is detected. In addition, the method can be performed by detecting the wild-type BRCAl locus and confirming the lack of a predisposition to cancer at the BRCAl locus. "Alteration of a wild-type gene" encompasses all forms of mutations including deletions, insertions and point mutations in the coding and noncoding regions. Deletions may be of the entire gene or of only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g., in the tumor tissue, and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited. If only a single allele is somatically mutated, an early neoplastic state is it uicated. However, if both alleles are somatically mutated, then a late neoplastic state is indicated. The finding of BRCAl mutations thus provides both diagnostic and prognostic information. A BRCAl allele which is not deleted (e.g., found on the sister chromosome to a chromosome carrying a BRCAl 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 BRCAl gene product However, mutations leading to non-functional gene products would also lead to a cancerous state. Point mutational events may occur in regulatory regions, such as in the promoter of the gene, leading to loss or diminution of expression of the mRNA. Point mutations may also abolish proper RNA processing, leading to loss of expression of the BRCAl gene product, or to a decrease in mRNA stability or translation efficiency.

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

Predisposition to cancers, such as breast and ovarian cancer, and the other cancers identified herein, can be ascertained by testing any tissue of a human for mutations of the BRCAl gene. For PCTA3S95/10203 example, a person who has inherited a germline BRCAl 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 BRCAl gene. Alteration of a wild-type BRCAl 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 BRCAl, manual sequencing is very labor-intensive, but under optimal conditions, mutations in the coding sequence of a gene are rarely missed. Another approach is the single-stranded conformation polymorphism assay (SSCA) (Orita et al., 1989). This method does not detect all sequence changes, especially if the DNA fragment size is greater than 200 bp, but can be optimized to detect most DNA sequence variation. The reduced detection sensitivity is a disadvantage, but the increased throughput possible with SSCA makes it an attractive, viable alternative to direct sequencing for mutation detection on a research basis. The fragments which have shifted mobility on SSCA gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al, 1991), heteroduplex analysis (HA) (White et al., 1992) and chemical mismatch cleavage (CMC) (Grompe et al., 1989). None of the methods described above will detect large deletions, duplications or insertions, nor will they detect a regulatory mutation which affects transcription or translation of the protein. Other methods which might detect these classes of mutations such as a protein truncation assay or the asymmetric assay, detect only specific types of mutations and would not detect missense muiations. A review of currently available methods of detecting DNA sequence variation can be found in a recent review by Grompe (1993). Once a mutation is known, an allele specific detection approach such as allele specific oligonucleotide (ASO) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation.

In order to detect the alteration of the wild-type BRCAl 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 BRCAl 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 BRCAl allele(s) and sequencing the allele(s) using techniques well known in the art Alternatively, the gene sequences can be amplified directly from a genomic DNA preparation from the tumor tissue, using known techniques. The DNA sequence of the amplified sequences can then be determined.

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 31 ends to a particular BRCAl mutation. If the particular BRCAl 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 BRCAl 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 niunber of tumor samples. An example of a mismatch cleavage technique is the RNase protection method. In the practice of the present invention, the method involves the use of a labeled riboprobe which is complementary to die human wild-type BRCAl 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 that the full length duplex RNA for the riboprobe and the mRNA or DNA. The riboprobe need not be the full length of the BRCAl mRNA or gene but can be a segment of either. If the riboprobe comprises only a segment of the BRCAl 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 rrismatches, through enzymatic or chemical cleavage. See, e.g., Cotton et al, 1988; Shenk et al, 1975; Novack et al, 1986. Alternatively, mismatches can be detected by shifts in the electrophoretic mobility of mismatched duplexes relative to matched duplexes. See, e.g., Cariello, 1988. With either riboprobes or DNA probes, the cellular mRNA or DNA which might contain a mutation can be amplified using PCR (see below) before hybridization. Changes in DNA of the BRCAl gene can also be detected using Southern hybridization, especially if the changes are gross rearrangements, such as deletions and insertions.

DNA sequences of the BRCAl 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 BRCAl gene sequence harboring a known mutation. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the BRCAl 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 BRCAl gene. Hybridization of allele-specific probes with amplified BRCAl 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.

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

Mutations from cancer patients falling outside the coding region of BRCAl can be detected bv examining the non-coding regions, such as introns and regulatory sequences near or within the BRCAl 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 BRCAl mRNA expression can be detected by any techniques known in the art These include Northern blot analysis, PCR amplification and RNase protection. Diminished mRNA expression indicates an alteration of the wild-type BRCAl gene. Alteration of wild-type BRCAl genes can also be detected by screening for alteration of wild-type BRCAl protein. For example, monoclonal antibodies immunoreactive with BRCAl can be used to screen a tissue. Lack of cognate antigen would indicate a BRCAl mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant BRCAl 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 BRCAl protein can be used to detect alteration of wild-type BRCAl genes. Functional assays, such as protein binding determinations, can be used. In addition, assays can be used which detect BRCAl biochemical function. Finding a mutant BRCAl gene product indicates alteration of a wild-type BRCAl gene.

Mutant BRCAl 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 BRCAl 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 BRCAl 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 BRCAl genes or gene products.

The methods of diagnosis of the present invention are applicable to any tumor in which BRCAl has a role in tumorigenesis. The diagnostic method of the present invention is useful for clinicians, so they can decide upon an appropriate course of treatment The primer pairs of the present invention are useful for determination of the nucleotide sequence of a particular BRCAl allele using PCR. The pairs of single-stranded DNA primers can be annealed to sequences within or surrounding the BRCAl gene on chromosome 17q21 in order to prime amplifying DNA synthesis of the BRCAl gene itself. A complete set of these primers allows synthesis of all of the nucleotides of the BRCAl gene coding sequences, i.e., the exons. The set of primers preferably allows synthesis of both intron and exon sequences. Allele-specific primers can also be used. Such primers anneal only to particular BRCAl mutant alleles, and thus will only amplify a product in the presence of the mutant allele as a template.

In order to facilitate subsequent cloning of amplified sequences, primers may have restriction enzyme site sequences appended to their 5' ends. Thus, all nucleotides of the primers are derived from BRCAl sequences or sequences adjacent to BRCAl, 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 BRCAl open reading frame shown in SEQ ID NO:l, design of particular primers is well within the skill of the art.

The nucleic acid probes provided by the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA and in the RNase protection method for detecting point mutations already discussed above. The probes can be used to detect PCR amplification products. They may also be used to detect mismatches with the BRCAl gene or mRNA using other techniques.

It has been discovered that individuals with the wild-type BRCAl gene do not have cancer which results from the BRCAl allele. However, mutations which interfere with the function of the BRCAl protein are involved in the pathogenesis of cancer. Thus, the presence of an altered (or a mutant) BRCAl 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 BRCAl gene mutation, a biological sample is prepared and analyzed for a difference between the sequence of the BRCAl allele being analyzed and the sequence of the wild-type BRCAl allele. Mutant BRCAl alleles can be initially identified by any of the techniques described above. The mutant alleles are then sequenced to identify the specific mutation of the particular mutant allele. Alternatively, mutant BRCAl alleles can be initially identified by identifying mutant (altered) BRCAl 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 BRCAl protein, are then used for the diagnostic and prognostic methods of the present invention.

Definitions The present invention employs the following definitions: "Amplification of Polynucleotides" utilizes methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. These methods are well known and widely practiced in the art See, e.g., U.S. Patents 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al., 1989a (for LCR). Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from the BRCAl region are preferably complementary to, and hybridize specifically to sequences in the BRCAl region or in regions that flank a target region therein. BRCAl 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 BRCAl polypeptides and fragments thereof or to polynucleotide sequences from the BRCAl region, particularly from the BRCAl locus or a portion thereof. The term "antibody" is used both to refer to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Polypeptides may be prepared synthetically in a peptide synthesizer and coupled to a carrier molecule (e.g., keyhole limpet hemocyanin) and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity to the BRCAl polypeptide or fragment Monoclonal antibodies may be made by injecting mice with the protein polypeptides, fusion proteins or fragments thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with BRCAl 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 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 ana in the same fashion as the antigen. A variety of immunoassay methods are well known in the art. See, e.g., Harlow & Lane, 1988, or Goding, 1986.

Monoclonal antibodies with affinities of 10"8 M"' or preferably 10"9 to 10"10 M"1 or stronger will typically be made by standard procedures as described, e.g., 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 supernatants of each clone tested for their production of an appropriate antibody specific for the desired region of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides, or alternatively, to selection of libraries of antibodies in phage or similar vectors. See Huse et al., 1989. The polypeptides and antibodies of the present invention may be used with or without modification. Frequently, polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles and the like. Patents teaching the use of such labels include U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996345; 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 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, e.g., plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genito urinary 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 (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components which naturally accompany a native human sequence or protein, e.g., ribosomes, polymerases, many other human genome sequences and proteins. The term embraces a nucleic acid sequence or protein which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

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

"BRCAl Locus," "BRCAl Gene," "BRCAl Nucleic Acids" or "BRCAl Polynucleotide" each refer to polynucleotides, all of which are in the BRCAl region, that are likely to be expressed in normal tissue, certain alleles of which predispose an individual to develop breast, ovarian, colorectal and prostate cancers. Mutations al the BRCAl 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 BRCAl region described infra. The BRCAl locus is intended to include coding sequences, intervening sequences and regulatory elements controlling transcription and/or translation. The BRCAl 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 BRCAl polypeptide, fragment, homolog or variant, including, e.g., protein fusions or deletions. The nucleic acids of the present invention will possess a sequence which is either derived from, or substantially similar to a natural BRCAl -encoding gene ot one having substantial homology with a natural BRCAl-encoding gene or a portion thereof. The coding sequence for a BRCAl polypeptide is shown in SEQ ID NO:l, with the amino acid sequence shown 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, intemucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. 0 The present invention provides recombinant nucleic acids comprising all or part of the BRCAl 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 5 associated with all or a portion of a polynucleotide with which it is associated in nature; 2) is linked to a polynucleotide other than that to which it is linked in nature; or 3) does not occur in nature.

Therefore, recombinant nucleic acids comprising sequences otherwise not naturally occurring are provided by this invention. Although the wild-type sequence may be employed, it will often be altered, e.g., by deletion, substitution or insertion. 0 cDNA or genomic libraries of various types may be screened as natural sources of the nucleic acids of the present invention, or such nucleic acids may be provided by amplification of sequences resident in genomic DNA or other natural sources, e.g., by PCR. The choice of cDNA libraries normally corresponds to a tissue source which is abundant in mRNA for the desired proteins. Phage libraries are normally preferred, but other types of libraries may be used. Clones of a library are spread onto plates, transferred to a substrate for screening, denatured and probed for the presence of desired sequences.

The DNA sequences used in this invention will usually comprise at least about five codons (15 nucleotides), more usually at least about 7-L5 codons, and most preferably, at least about 35 codons. One or more introns may also be present. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with a BRCAl -encoding 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 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.

"BRCAl Region" refers to a portion of human chromosome 17q21 bounded by the markers tdj 1474 and U5R. This region contains the BRCAl locus, including the BRCAl gene.

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

As used herein, a "portion" of the BRCAl locus or region or allele is defined as having a minimal size of at least about eight nucleotides, or preferably about 15 nucleotides, or more preferably at least about 25 nucleotides, and may have a minimal size of at least about 40 nucleotides.

"BRCAl protein" or "BRCAl polypeptide" refer to a protein or polypeptide encoded by the BRCAl locus, variants or fragments thereof. The term "polypeptide" refers to a polymer of amino acids and its equivalent and does not refer to a specific length of the product; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not refer to, or exclude modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages as well as other modifications known in the art, both 5 naturally and non-naturally occurring. Ordinarily, such polypeptides will be at least about 50% I homologous to the native BRCAl 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 BRCAl-encoding nucleic acids and closely related polypeptides or proteins retrieved by antisera to the BRCAl protein(s). 10 The length of polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.

"Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is 15 operably linked to a coding sequence if the promoter affects its transcription or expression.

"Probes". Polynucleotide polymorphisms associated with BRCAl 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 2 0 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 identify neutral DNA polymorphisms as well as mutations, these indications need further analysis 2 5 to demonstrate detection of a BRCAl susceptibility allele.

Probes for BRCAl alleles may be derived from the sequences of the BRCAl region or its cDNAs. The probes may be of any suitable length, which span all or a portion of the BRCAl region, and which allow specific hybridization to the BRCAl region. If the target sequence contains a sequence identical to that of the probe, the probes may be short, e.g., in the range of 3 0 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, i.e., if it is suspected that the probe will hybridize to a variant region, a longer probe may be employed which hybridizes to the target sequence with the requisite specificity.

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

Probes comprising synthetic oligonucleotides or other polynucleotides of the present invention may be derived from naturally occurring or recombinant single- or double-stranded polynucleotides, or be chemically synthesized. Probes may also be labeled by nick translation, fClenow 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 BRCAl are preferred as probes. The probes may also be used to determine whether mRNA encoding BRCAl is present in a cell or tissue.

"Protein modifications or fragments" are provided by the present invention for BRCAl polypeptides or fragments thereof which are substantially homologous to primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate unusual amino acids. Such modifications include, for example, acetylation, carboxylation. phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those well skilled in the art A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as P, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability PCT/U S95/10203 requirements, and available instrumentation. Methods of labeling polypeptides are well known in the art. See, e.g., 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 BRCAl 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 BRCAl protein. As used herein, "epitope" refers to an antigenic determinant of a polypeptide. An epitope could comprise three amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least five such amino acids, and more usually consists of at least 8-10 such amino acids. Methods of determining the spatial conformation of such amino acids are known in the art 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 BRCAl polypeptides or fragments thereof is described below.

The present invention also provides for fusion polypeptides, comprising BRCAl polypeptides and fragments. Homologous polypeptides may be fusions between two or more BRCAl polypeptide sequences or between the sequences of BRCAl 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 domain* 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 (J-galactosidase, trpE, protein A, |J-lactamase, 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 BRCAl polypeptides from other biological material, such as from cells transformed with recombinant nucleic acids encoding BRCAl, and are well known in the art. For example, such polypeptides may be purified by immunoaffmity chromatography employing, e.g., the antibodies provided by the present invention. Various methods of protein purification are well known in the art, and include those described in Deutscher, 1990 and Scopes, 1982.

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

A BRCAl 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 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 arc inherently isolated molecules.

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

"Regulator}' sequences" refers to those sequences normally within 100 kb of the ending 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 homologous" ("or substantially similar") to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at ler'n about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.

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

Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art Stringent temperature conditions will generally include temperatures in excess of 30°C, typically in excess of 37°C, and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur & 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 30% identity with an entire naturally-occurring protein or a portion thereof, usually at least about 70% 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 BRCAl nucleic acid or wild-type BRCAl polypeptide. The modified polypeptide will be substantially homologous to the wild-type BRCAl polypeptide and will have substantially the same function. The modified polypeptide may have an altered amino acid sequence and/or may contain modified amino acids. In addition to the similarity of function, the modified polypeptide may have other useful properties, such as a longer half-life. The similarity of function (activity) of the modified polypeptide may be substantially the same as the activity of the wild-type BRCAl polypeptide. Alternatively, the similarity of function (activity) of the modified polypeptide may be higher than the activity of the wild-type BRCAl 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 BRCAl gene function produces the modified protein described above.

Homology, for polypeptides, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, 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, e.g., nitrocellulose, nylon, column packing materials (e.g., Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gels, cells, or other substrates. Such supports may take the fo; m, 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 to a 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, e.g., Maniatis et al., 1982; Sambrook et al, 1989; Ausubel et al, 1992; Glover, 1985; Anand, 1992; Guthrie & Fink, 1991. A general discussion of techniques and materials for human gene mapping, including mapping of human chromosome 17q, is provided, e.g., in White andLalouel, 1988.

Preparation of recombinant or chemically synthesized 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, e.g., in Sambrook et al., 1989 or Ausubel etal, 1992.

The polynucleotides of the present invention may also be produced by chemical synthesis, e.g., 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, polyadenylalion sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Secretion signals may also be included where appropriate, whether from a native BRCAl protein or from other receptors or from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes, and thus attain its functional topology, or be secreted from the cell. Such vectors may be prepared by means of standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al., 1989 or Ausubel et al. 1992.

An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host and may include, when appropriate, those naturally associated with BRCAl genes. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al.. 1989 or Ausubel et al., 1992; see also, e.g., Metzger et al., 1988. Many useful vectors are known in the art and may be obtained from such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others. Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al, EP 73,675A. Appropriate non-native mammalian promoters might include the early and late promoters from SV40 (Fiers et al, 1978) or promoters derived from murine Moloney leukemia virus, mouse tumor virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. In addition, the construct may be joined to an amplifiable gene (e.g., 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, e.g., the gene encoding D-alanine racemase for Bacilli. The choice of the proper selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art The vectors containing the nucleic acids of interest can be transcribed in vitro, and the resulting RNA introduced into the host cell by well-known methods, e.g., by injection (see, Kubo et al, 1988), or the vectors can be introduced directly into host cells by methods well known in the art, which vary depending on the type of cellular host, including elcctroporalion; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent such as a retroviral genome); and other methods. See generally, Sambrook et al, 1989 and Ausubel et al., 1992. The introduction of the polynucleotides into the host cell by any method known in the art, including, inter alia, those described above, will be referred to herein as "transformation." The cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.

Large quantities of the nucleic acids and polypeptides of the present invention may be prepared by expressing the BRCAl 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 pjeudomonas 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 Pas tan, 1979. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although it will be appreciated by the skilled practitioner that other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns, or other features.

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

Prokaiyotic or eukaryotic cell s transformed with the polynucleotides of the present invention will be useful not only for the production of the nucleic acids and polypeptides of the present invention, but also, for example, in studying the characteristics of BRCAl polypeptides.

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

The probes and primers based on the BRCAl gene sequences disclosed herein are used to identify homologous BRCAl gene sequences and proteins in other species. These BRCAl 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 BRCAl 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 BRCAl. 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 BRCAl. 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, altematively, 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 BRCAl 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.

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

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 earned 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 17q. Therefore, high stringency conditions are desirable in order to prevent false positives. However, conditions of high stringency are used only if the probes are complementary to regions of the chromosome which are unique in the genome. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, base composition, probe length, and concentration of formamide. These factors are outlined in, for example, Maniatis et al, 1982 and Sambrook et al., 1989. Under certain circumstances, the formation of higher order hybrids, such as triplexes, quadraplexes. etc., may be desired to provide the means of detecting target sequences.

Detection, if any, of the resulting hybrid is usually accomplished by the use of labeled probes. Alternatively, the probe may be unlabeled, but may be detectable by specific binding with a ligand which is labeled, either directly or indirectly. Suitable labels, and methods for labeling probes and ligands are known in the art, and include, for example, radioactive labels which may be incorporated by known methods (e.g., nick translation, random priming or kinasing), biotin, fluorescent groups, chemiluminescent groups (e.g., dioxetanes, particularly triggered dioxetanes), enzymes, antibodies and the like. Variations of this basic scheme are known in the art, and include those variations that facilitate separation of the hybrids to be detected from extraneous materials and/or that amplify the signal from the labeled moiety. A number of these variations are reviewed in, e.g., Matthews & 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 11. 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 103- i 06 increase in sensitivity. For an example relating to the preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes see Jablonski et al., 1986.

Two-step label amplification methodologies are known in the art These assays work on the principle that a small ligand (such as digoxigenin, biotin, or the like) is attached to a nucleic acid probe capable of specifically binding BRCAl. Exemplary probes are provided in Table 9 of this patent application and additionally include the nucleic acid probe corresponding to nucleotide positions 3631 to 3930 of SEQ ID NO:l. Allele specific probes are also contemplated within the scope of this example and exemplary allele specific probes include probes encompassing the predisposing mutations summarized in Tables 11 and 12 of this patent application.

In one example, the small ligand attached to the nucleic acid probe is specifically recognized by an antibody-enzyme conjugate. In one embodiment of this example, digoxigenin is attached to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate which turns over a chemiluminescent substrate. For methods for labeling nucleic acid probes according to this embodiment see Martin et al., 1990. In a second example, the small ligand is recognized by a second ligand-enzyme conjugate that is capable of specifically complexing to the first ligand. A well known embodiment of this example is the biotin-avidin type of interactions. For methods for labeling nucleic acid probes and their use in biotin-avidin based assays see Rigby et al, 1977 and Nguyen et al., 1992.

It is also contemplated within the scope of this invention that the nucleic acid probe assays of this invention will employ a cocktail of nucleic acid probes capable of detecting BRCAl. Thus, in one example to detect the presence of BRCAl in a cell sample, more than one probe complementary to BRCAl 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 BRCAl gene sequence in a patient, more than one probe complementary to BRCAl is employed where the cocktail includes probes capable of binding to the allele-specific mutations identified in populations of patients with alterations in BRCAl. 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 identified in Tables 11 and 12 and those that have the BRCAl regions corresponding to SEQ ID NO:l both 5' and 3' to the mutation site.

Methods of Use: Peptide Diagnosis and Diagnostic Kite The neoplastic condition of lesions can also be detected on the basis of the alteration of wild-type BRCAl 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 BRCAl peptides. The antibodies may be prepared as discussed above under the heading "Antibodies" and as further shown in Examples 12 and 13.

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 BRCAl proteins from solution as well as react with BRCAl protein on Western or immunoblots of polyacrylamide gels. In another preferred embodiment, antibodies will detect BRCAl proteins in paraffin or frozen tissue sections, using immunocytochemical techniques.

Preferred embodiments relating to methods for detecting BRCAl or its mutations include enzyme linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradiometric assays (IRMA) and immunoenzymatic assays (IEMA), including sandwich assays using monoclonal and/or polycbnal 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 14.

Methods of Ike- Dnip Screening This invention is particularly useful for screening compounds by using the BRCAl polypeptide or binding fragment thereof in any of a variety of drug screening techniques.

The BRCAl 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 procaiyotic 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 BRCAl polypeptide or fragment and the agent being tested, or examine the degree to which the formation of a complex between a BRCAl 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 BRCAl polypeptide or fragment thereof and assaying (i) for the presence of a complex between the agent and the BRCAl polypeptide or fragment, or (ii) for the presence of a complex between the BRCAl polypeptide or fragment and a ligand, by methods well known in the art. In such competitive binding 'wsays the BRCAl polypeptide or fragment is typically labeled. Free BRCAl polypeptide or fragment is separated from that present in a protein:protein complex, and the amount of free (i.e., uncomplexed) label is a measure of the binding of the agent being tested to BRCAl or its interference with BRCAl :ligand binding, respectively.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to the BRCAl polypeptides and is described in detail in Geysen, PCT published application WO 84/03564, published on September 13, 1984. Briefly stated, laige 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 BRCAl polypeptide and washed. Bound BRCAl polypeptide is then detected by methods well known in the art.

Purified BRCAl can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to capture antibodies to immobilize the BRCAl 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 BRCAl polypeptide compete with a test compound for binding to the BRCAl 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 BRCAl 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 BRCAl gene. These host cell lines or cells are defective at the BRCAl 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 BRCAl defective cells.

Methods of Use: Rational Drue 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 (e.g., agonists, antagonists, inhibitors) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g., enhance or interfere with the function of a polypeptide in vivo. See, e.g., Hodgson, 1991. In one approach, one first determines the three-dimensional structure of a protein of interest (e.g., BRCAl polypeptide) or, for example, of the BRCAl-receptor or ligand complex, by x-ray crystallography, by computer modeling or most typically, by a combination of PCTAJS95/10203 approaches. Less often, useful information regarding the structure of a polypeptide may be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al., 1990). In addition, peptides (e.g., BRCAl polypeptide) are analyzed by an alanine scan (Wells, 1991). In this technique, an aminn 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, e.g., improved BRCAl polypeptide activity or stability or which act as inhibitors, agonists, antagonists, etc. of BRCAl polypeptide activity. By virtue of the availability of cloned BRCAl sequences, sufficient amounts of the BRCAl polypeptide may be made available to perform such analytical studies as x-ray crystallography. In addition, the knowledge of the BRCAl protein sequence provided herein will guide those employing computer modeling techniques in place of, or in addition to x-ray crystallography.

Methods of I Jse: Gene Therapy According to the present invention, a method is also provided of supplying wild-type BRCAl function to a cell which carries mutant BRCAl alleles. Supplying such a function should suppress neoplastic growth of the recipient cells. The wild-type BRCAl gene or a part of the gene may be introduced into the cell in a vector such that the gene remains extrachiomosomal. 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 BRCAl allele, the gene fragment should encode a part of the BRCAl protein which is required for non-neoplastic growth of the cell. More preferred is the situation where the wild-type BRCAl gene or a part thereof is introduced into the mutant cell in such a way that it recombines with the endogenous mutant BRCAl gene present in the cell. Such recombination requires a double recombination event which results in the correction of the BRCAl 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 ait, and the choice of method is within the competence of the routineer. Cells transformed with the wild-type BRCAl gene can be used as model systems to study cancer remission and drug treatments which promote such remission.

As generally discussed above, the BRCAl 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 BRCAl polypeptide is absent or diminished compared to normal cells. It may also be useful to increase the level of expression of a given BRCAl 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 BRCAl polypeptide in the tumor cells. A virus or plasmid vector (see further details below), containing a copy of the BRCAl 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 the targeted tumor cells, the treatment may have to be repealed periodically.

Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and nonviral transfer methods. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia and Kapikian, 1992; Quantin et al, 1992; Rosenfeld et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Mu2yczka, 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 al., 1984; Mann and Baltimore, 1985; Miller et al, 1988), and human origin (Shimada et al., 1991; Helseth et al, 1990; Page et al, 1990; Buchschacher and Panganiban, 1992). Most human gene therapy protocols have been based on disabled murine retroviruses.

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 (Feigner 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 al., 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., 1991b). Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome deliveiy, allowing one to direct the viral vectors to the tumor cells and not into the surrounding nondividing cells. Alternatively, the retroviral vector producer cell line can be injected into tumors (Culver et al., 1992). Injection of producer cells would then provide a continuous source of vector particles. This technique has been approved for use in humans with inoperable brain tumors.

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

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 internalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, coinfection with adenovirus can be included to disrupt endosome function.

The therapy involves two steps which can be performed singly or jointly. In the first step, prepubescent females who carry a BRCAl 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 BRCAl 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 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 Peptides which have BRCAl activity can be supplied to cells which carry mutant or missing BRCAl alleles. The sequence of the BRCAl protein is disclosed (SEQ ID NO:2). Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors. Alternatively, BRCAl polypeptide can be extracted from BRCAl-producing mammalian cells. In addition, the techniques of synthetic chemistry can be employed to synthesize BRCAl protein. Any of such techniques can provide the preparation of the present invention which comprises the BRCAl protein. The preparation is substantially free of other human proteins. This is most readily accomplished by synthesis in a microorganism or in vitro.

Active BRCAl 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 BRCAl gene product may be sufficient to affect tumor growth. Supply of molecules with BRCAl activity should lead to partial reversal of the neoplastic state. Other molecules with BRCAl 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 T Jse: Transformed Hosts Similarly, cells and animals which cany a mutant BRCAl allele can be used as model systems to study and test for substances which have potential as therapeutic agents. The cells are typically cul' ured epithelial cells. These may be isolated from individuals with BRCAl mutations, either somatic or germline. Alternatively, Jie cell line can be engineered to cany the mutation in the BRCAl allele, as described above. After a test substance is applied to the cells, the neoplas-tically transformed phenotype of the cell is determined. Any trait of neoplastically transformed cells can be assessed, including anchorage-independent growth, tumorigenicity in nude mice, invasiveness of cells, and growth factor dependence. Assays for each of these traits are known in the art.

Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant BRCAl alleles, usually from a second animal species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous BRCAl 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.

PCT/Ui»95/J0203 RXAMPLF. 1 Ascertain and Study Kindreds Likely to Have a 17q-Linked Breast Cancer Susceptibility I^ncus 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 BRCAl 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 BRCAl region, and greatly facilitated the reduction of the BRCAl region to a manageable size, which permits identification of the BRCAl 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 (e.g. ovarian) who also appeared in the kindreds were 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.

Ten kindreds which had three or more cancer cases with inferable genotypes were selected for linkage studies to 17q markers from a set of 29 kindreds originally ascertained from the linked databases for a study of proliferative breast disease and breast cancer (Skolnick et al., 1990). The criterion for selection of these kindreds was the presence of two sisters or a mother and her daughter with breast cancer. Additionally, two kindreds which have been studied since 1980 as p"j"t of our breast cancer linkage studies (K1001, K9018), six kindreds ascertained from the linked databases for the presence of clusters of breast and/or ovarian cancer (K2019, K2073, K2079, K2080, K2039, K2082) and a self-referred kindred with early onset breast cancer (K2035) were included. These kindreds were investigated and expanded in our clinic in the manner described above. Table 1 displays the characteristics of these 19 kindreds which are the subject of subsequent examples. In Table 1, for each kindred the total number of individuals in our database, the number of typed individuals, and the minimum, median, and maximum age at diagnosis of breast/ovarian cancer are reported. Kindreds are sorted in ascending order of median age al diagnosis of breast cancer. Four women diagnosed with both ovarian and breast cancer are counted in both categories. table i rteflriripfcjon of fchp 19 Kindreds Breast Ovarian No. of tnHi vi dual a Age at Dx Age at Dx kindred Sample a Aff.

Min.

Med.

Max.

# Aff.

HilLa.

Med. 1910 4 27 34 49 - - - - 1001 133 98 13 28 37 64 — •• * 2035 42 8 28 37 45 1 60 2027 21 11 4 34 38 41 - — • 9018 54 17 9 40 72 2 46 48 50 1925 50 27 4 39 42 53 - - - - 1927 49 29 32 42 51 — • 1911 28 21 7 28 42 76 — — 1929 16 11 4 34 43 73 - — • 1901 19 31 44 76 • " 2082 180 105 27 47 67 45 52 66 2019 42 19 42 53 79 - - — 1900 70 23 8 45 55 70 1 - 78 2080 264 74 22 + 27 55 92 4 45 53 71 2073 57 29 9 57 80 " 1917 16 6 4 43 58 61 - - - - 1920 22 14 3 62 63 68 - - - — 2079 136 18 14 38 66 84 4 52 59 65 2039 87 40 14 44 68 88 4 41 51 75 + Includes one case of male breast cancer. 52 example: Selection of Kindreds Which are Linked to Chromosome 17q and Localization of BRCAl to the Interval Mfdl S - MfrilKK For each sample collected in these 19 kindreds, DNA was extracted from blood (or in two cases from paraffin-embedded 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, four such STR markers on chromosome 17 were developed by screening a chromosome specific cosmid library for CA positive clones. Three of these markers localized to the long arm: (46E6. Easton et al., 1993); (42D6. Easton et al.. 1993); 26C2 (D17S514, Oliphant et al.. 1991), while the other, 12G6 (D17S513, Oliphant et cd., 1991), localized to the short arm near the p53 tumor suppressor locus. Two of these, 42D6 and 46E6, were submitted to the Breast Cancer Linkage Consortium for typing of breast cancer families by investigators worldwide. Oligonucleotide sequences for markers not developed in our laboratory were obtained from published reports, or as part of the Breast Cancer Linkage Consortium, or from other investigators. Ail genotyping films were scored blindly with a standard lane marker used to maintain consistent coding of alleles. Key samples in the four kindreds presented here underwent duplicate typing for all relevant markers. All 19 kindreds have been typed for two polymorphic CA repeat markers: 42D6 (D17S588). a CA repeat isolated in our laboratory, and Mfdl5 (D17S250), a CA repeat provided by J. Weber (Weber et al., 1990). Several sources of probes were used to create genetic markers on chromosome 17. specifically chromosome 17 cosmid and lambda phage libraries created from sorted chromosomes by the Los Alamos National Laboratories (van Dilla et al., 1986).

LOD scores for each kindred with these two markers (42D6, Mfdl 5) and a third marker, Mfdl88 (D17S579, Hall et al, 1992), located roughly midway between these two markers, were calculated for two values of the recombination fraction, 0.001 and 0.1. (For calculation of LOD scores, see Oh. 198S). 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 gene carriers of about 0.80, and population based age-specific risks for breast cancer in non-gene carriers. Allele frequencies for the three markers used for the LOD score calculations were calculated from our own laboratory lypings of unrelated individuals in the CEPH panel (White and Lalouel, 1988). Table 2 shows the results of the pairwise linkage analysis of each kindred with the three markers 42D6, Mfdl88, and Mfdl5.

KIUQ&ED 1910 1001 2035 2027 9018 1925 1927 1911 1929 1901 2082 2019 1900 2080 2073 1917 1920 2079 2039 TABLE 2 Pairwise Linkage Analysis of Kindreds Mfdl5 (D17S250) Mfdl88 (D17S579) 42D6 (D17S588) k.-»rrombinat:ion Recombination Recombination 0.001 0.1 0.001 0.1 0.001 0.1 0.06 0.30 0.06 0.30 0.06 0.30 -0.30 -0.09 NT NT -0.52 -0.19 2.34 1.85 0.94 0.90 2.34 1.82 -1.22 -0.33 -1.20 -0.42 -1.16 -0.33 -0.54 -0.22 -0.17 -0.10 0.11 0.07 1.08 0.79 0.55 0.38 -0.11 -0.07 -0.41 0.01 -0.35 0.07 -0.44 -0.02 -0.27 -0.13 -0.43 -0.49 -0.25 NT 1.50 1.17 0.78 4.25 3.36 6.07 -0.10 -0.01 -0.11 -0.14 -0.11 NT -0.16 -0.04 0.76 -0.41 -0.29 0.63 -0.02 -0.02 NT -0.03 -0.02 NT 0.02 0.01 -0.01 -1.67 -0.83 0.12 -0.23 0.49 0.38 NT -0.49 -0.25 0.57 0.65 0.37 .11 2.00 3.56 -0.05 -0.18 -0.10 NT -0.12 -0.05 0.74 -1.25 -0.58 0.49 -0.23 -0.13 NT -0.01 0.00 NT 0.00 0.00 -0.01 0.01 0.01 0.59 -1.15 0.02 NT - Kindred not typed for Mfdl88.

Using a criterion for linkage to 17q of a LOD score >1.0 for at least one locus under the CASH model (Claus et al, 1991), four of the 19 kindreds appeared to be linked to 17q (K1901, K1925, K2035, K2082). A number of additional kindreds showed some evidence of linkage but at this time could not be definitively assigned to the linked category. These included kindreds K1911, K2073, K2039, and K2080. Three of the 17q-linked kindreds had informative recombinants in this region and these are detailed below.

Kindred 2082 is the largest 17q-linked breast cancer family reported to date by any group. The kindred contains 20 cases of breast cancer, and ten cases of ovarian cancer. Two cases have both ovarian and breast cancer. The evidence of linkage to 17q for this family is overwhelming; the LOD score with the linked haplotype is over 6.0, despite the existence of three cases of breast cancer which appear to be sporadic, i.e., these cases share no part of the linked haplotype between Mfd 15 and 42D6. These three sporadic cases were diagnosed with breast cancer al ages 46,47, and 54. In smaller kindreds, sporadic cancers of this type greatly confound the analysis of linkage and the correct identification of key recombinants. The key recombinant in the 2082 kindred is a woman who developed ovarian cancer at age 45 whose mother and aunt had ovarian cancer at ages 58 and 66, respectively. She inherited the linked portion of the haplotype for both Mfdl 88 and 42D6 while inheriting unlinked alleles al Mfdl5; this recombinant event placed BRCAl distal to Mfdl 5.

K1901 is typical of early-onset breast cancer kindreds. The kindred contains 10 cases of breast cancer with a median age at diagnosis of 43.5 years of age; four cases were diagnosed under age 40. The LOD score for this kindred with the marker 42D6 is 1.5, resulting in a posterior probability of 17q-linkage of 0.96. Examination of haplotypes in this kindred identified a recombinant haplotype in an obligate male carrier and his affected daughter who was diagnosed with breast cancer at age 45. Their linked allele for marker Mfd 15 differs from that found in all other cases in the kindred (except one case which could not be completely inferred from her children). The two haplotypes are identical for Mfdl 88 and 42D6. Accordingly, data from Kindred 1901 would also place the BRCAl locus distal to Mfdl5.

Kindred 2035 is similar to K1901 in disease phenotype. The median age of diagnosis for the eight cases of breast cancer in this kindred is 37. One case also had ovarian cancer at age 60. The breast cancer cases in this family descend from two sisters who were both unaffected with breast cancer until their death in the eighth decade. Each branch contains four cases of breast cancer with at least one case in each branch having markedly early onset. This kindred has a LOD score of 2.34 with Mfdl5. The haplotypes segregating with breast cancer in the two branches share an identical allele at Mfdl 5 but differ for the distal loci Mfdl 88 and NM23 (a marker typed as part of the consortium which is located just distal to 42D6 (Hall et al, 1992)). Although the two > haplotypes are concordant for marker 42D6, it is likely that the alleles are shared identical by state (the same allele but derived from different ancestors), rather than identical by descent (derived from a common ancestor) since the shared allele is the second most common allele observed at this locus. By contrast the linked allele shared at Mfdl 5 has a frequency of 0.04. This is a key recombinant in our dataset as it is the sole recombinant in which BRCAl segregated with the ) proximal portion of the haplotype, thus setting the distal boundary to the BRCAl region. For this event not to be a key recombinant requires that a second mutant BRCAl gene be present in a spouse marrying into the kindred who also shares the rare Mfdl 5 allele segregating with breast cancer in both branches of the kindred. This event has a probability of less than one in a thousand. The evidence from this kindred therefore placed the BRCAl locus proximal to Mfdl 88. example 3 Creation of a Fine Structure Map and Refinement of the BRCAl Region to Mfd 191-Mfd 188 using Additional STR Polymorphisms D In order to improve the characterization of our recombinants and define closer flanking markers, a dense map of this relatively small region on chromosome 17q was required. The chromosome 17 workshop has produced a consensus map of this region (Figure 1) based on a combination of genetic and physical mapping studies (Fain, 1992). This map contains both highly 5 polymorphic STR polymorphisms, and a number of nonpolymorphic expressed genes. Because this map did not give details on the evidence for this order nor give any measure of local support for inversions in the order of adjacent loci, we viewed it as a rough guide for obtaining resources to be used for the development of new markers and construction of our own detailed genetic and physical map of a small region containing BRCAl. Our approach was to analyze existing STR 0 markers provided by other investigators and any newly developed markers from our laboratoiy with respect to both a panel of meiotic (genetic) breakpoints identified using DNA from the CEPH reference families and a panel of somatic cell hybrids (physical breakpoints) constructed for this region. These markers included 26C2 developed in our laboratory which maps proximal to MfdlS, Mfd 191 (provided by James Weber), THRA1 (Futreal et al., 1992a), and three polymorphisms kindly provided to us by Dr. Donald Black, NM23 (Hall et al. 1992), SCG40 (D17S181), and 6C1 (D17S293).

Genetic localization of markers. In order to localize new markers genetically within the region of interest, we have identified a number of key meiotic breakpoints within the region, both in the CEPH reference panel and in our large breast cancer kindred (K2082). Given the small genetic distance in this region, they are likely to be only a relatively small set of recombinants which can be used for this purpose, and they are likely to group markers into sets. The orders of the markers within each set can only be determined by physical mapping. However the number of genotypings necessary to position a new marker is minimized. These breakpoints are illustrated in Tables 3 and 4. Using this approach we were able to genetically order the markers THRA1, 6C1, SCG40, and Mfdl91. As can be seen from Tables 3 and 4, THRA1 and MFD191 both map inside the Mfdl 5-Mfdl 88 region we had previously identified as containing the BRCAl locus. In Tables 3 and 4, M/P indicates a maternal or paternal recombinant A "1" indicates inherited allele is of grandpatemal origin, while a "0" indicates grandmaternal origin, and indicates that the locus was untyped or uninformative.

TABLE 3 Family ID M/E MfdlS THRAl MfdlSl 13292 4 M 1 1 13294 4 M 1 1 13294 6 M 0 0 1334 3 M 1 1 1333 4 M 1 1 1333 6 M 0 0 1333 8 P 1 0 0 1377 8 M 0 - 0 1 1 0 0 0 0 0 1 VO o\ o u> t*J o Mfdl88 SCG4Q 6C1 4?.Pfi 0 0 0 0 0 0 0 0 1 - -11 0 0 0 0 a CD I CA VO c* o K» o w U3 a rs CJ vd o 8 C/] Analvsis of markers MfdlS. Mfd188. Mfdl91. and THRAl in our recombinant families MfdlS, Mfdl88, Mfdl91 and THRAl were typed in our recombinant families and examined for additional information to localize the BRCAl locus. In kindred 1901, the MfdlS recombinant was recombinant for THRAl but uninformative for Mfdl91, thus placing BRCAl distal to THRAl. In K2082, the recombinant with Mfdl5 also was recombinant with Mfdl91, thus placing the BRCAl locus distal to Mfdl91 (Goldgar et al, 1994). Examination of THRAl and Mfdl91 in kindred K2035 yielded no further localization information as the two branches were concordant for both markers. However, SCG40 and 6C1 both displayed the same pattern as Mfdl 88, thus increasing our confidence in the localization information provided by the Mfdl 88 recombinant in this family. The BRCAl locus, or at least a portion of it, therefore lies within an interval bounded by Mfdl91 on the proximal side and Mfdl 88 on the distal side. example 4 Development of Genetic and Physical Resources in the Region of Interest To increase the number of highly polymorphic loci in the Mfdl91-Mfdl88 region, we developed a number of STR markers in our laboratory from cosmids and YACs which physically map to the region. These markers allowed us to further refine the region.

STSs were identified from genes known to be in the desired region to identify YACs which contained these loci, which were then used to identify subclones in cosmids, Pis or BACs. These subclones were then screened for the presence of a CA tandem repeat using a (CA)n oligonucleotide (Pharmacia). Clones with a strong signal were selected preferentially, since they were more likely to represent CA-repeats which have a large number of repeats and/or are of near-perfect fidelity to the (CA)n pattern. Both of these characteristics are known to increase the probability of polymorphism (Weber, 1990). These clones were sequenced directly from the vector to locate the repeat. We obtained a unique sequence on one side of the CA-repeat by using one of a set of possible primers complementaiy to the end of a CA-repeat, such as (GT)10T. 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 the CA-repeat 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 set of 40 unrelated individuals from the Utah and CEPH families to obtain allele frequencies appropriate for the study population. Many of the other markers reported in this study were tested in a smaller group of CEPH unrelated individuals to obtain similarly appropriate 5 allele frequencies.

Using the procedure described above, a total of eight polymorphic STRs was found from these YACS. Of the loci identified in this manner, four were both polymorphic and localized to the BRCAl region. Four markers did not localize to chromosome 17, reflecting the chimeric nature of the YACs used. The four markers which were in the region were denoted AA1, ED2, 4-10 7, and YM29. AA1 and ED2 were developed from YACs positive for the RNU2 gene, 4-7 from an EPB3 YAC and YM29 from a cosmid which localized to the region by the hybrid panel. A description of the number of alleles, heterozygosity and source of these four and all other STR polymorphisms analyzed in the breast cancer kindreds is given below in Table 5.

TABLE 5 Polymorphic Short Tandem Repeat Markers Used for Fine Structure Mapping of the BRCAl Locus Allele* Frequency (%) Hetero- Clone Gene Na** zyaosity 1 2 1 _£ Mfdl5 D17S250 0.82 26 22 7 7 23 THRAl THRAl Mfdl91 D17S776 7 0.55 48 11 7 7 7 ED2 D17S1327 12 0.55 62 9 8 11 AA1 D17S1326 7 0.83 28 28 8 6 CA375 D17S184 0.75 26 11 9 9 4-7 D17S1183 9 0.50 63 8 6 4 4 YM29 9 0.62 42 24 12 7 7 8 Mfdl88 D17S579 12 0.92 33 18 8 8 8 SCG40 D17S181 14 0.90 18 18 8 42D6 D17S588 11 0.86 21 17 11 9 32 6C1 D17S293 7 0.75 11 11 9 9 Z109 D17S750 9 0.70 33 27 7 7 7 19 tdjl475 D17S1321 13 0.84 21 16 11 11 8 33 CF4 D17S1320 6 0.63 50 27 9 7 4 3 tdjl239 D17S1328 0.80 86 9 7 4 14 U5 D17S1325 13 0.83 19 16 12 9 34 Allele codes 1-5 are listed in decreasing frequency; allele numbers do not correspond to fragment sizes. Allele 6 frequency is the joint frequency of all other alleles for each locus.

Number of alleles seen in the genetically independent DNA samples used for calculating allele frequencies.

The four STR polymorphisms which mapped physically to the region (4-7, ED2, AA1, YM29) were analyzed in the meiotic, breakpoint panel shown initially in Tables 3 and 4. Tables 6 and 7 contain the relevant CEPH data and Kindred 2082 data for localization of these four markers. In the tables, M/P indicates a maternal or paternal recombinant. A "1" indicates inherited allele is of grandpaternal origin, while a "0" indicates grandmatemal origin, and indicates that the locus was untyped or uninformative.

• • • • TABT.R fi Key Recombinants Used for Genetic Ordering of New STR Loci Developed in Our Laboratory Within the BRCAl Region of 17q * O \o 0\ fJi u CEPH Family ID M/E MfdlS THRAl Mfdl91 £122. Ml Z1Q9 U YM29 MfdlB8 SCG40 42D6 13292 4 M 1 1 1 13294 4 M 1 0 0 13294 6 M 0 0 1 1333 4 M 1 1 1 1333 6 M 0 0 1 1333 3 M 0 0 1 1 0 1 0 1 0 1 1 0 0 1 0 1 1 0 1 1 I a\ I i t/3 NO Sfi © From CEPH 1333-04, we see that AAl and YM29 must lie distal to Mfdl9l. From 13292, it can be inferred that both AAl and ED2 are proximal to 4-7, YM29, and Mfdl88. The recombinants found in K2082 provide some additional ordering information. Three independent observations (individual numbers 22,40, & 63) place AAl, ED2,4-7, and YM29, and Mfdl88 distal to Mfdl91, while ID 125 places 4-7, YM29, and Mfdl 88 proximal to SCG40. No genetic information on the relative ordering within the two clusters of markers AA1/ED2 and 4-7/YM29/Mfdl 88 was obtained from the genetic recombinant analysis. Although ordering loci with respect to hybrids which are known to contain "holes" in which small pieces of interstitial human DNA may be missing is problematic, the hybrid patterns indicate that 4-7 lies above both YM29 and Mfdl 88. example 5 Genetic Analyses of Breast Cancer Kindreds with Markers AAl. 4-7. ED2. and YM29 In addition to the three kindreds containing key recombinants which have been discussed previously, kindred K2039 was shown through analysis of the newly developed STR markers to be linked to the region and to contain a useful recombinant Table 8 defines the haplotypes (shown in coded form) of the kindreds in terms of specific marker alleles at each locus and their respective frequencies. In Table 8, alleles are listed in descending order of frequency; frequencies of alleles 1-5 for each locus are given in Table 5. Haplotypes coded H are BRCAl associated haplotypes, P designates a partial H haplotype, and an R indicates an observable recombinant haplotype. As evident in Table 8, not all kindreds were typed for all markers; moreover, not all individuals within a kindred were typed for an identical set of markers, especially in K2082. With one exception, only haplotypes inherited from affected or at-risk kindred members are shown; haplotypes from spouses marrying into the kindred are not described. Thus in a given sibship, the appearance of haplotypes X and Y indicates that both haplon-pes from the affected/at risk individual were seen and neither was a breast cancer associated % lABLE_a Breast Cancer Linked Haplotypes Fniind in the Three Kindreds Mfd Mfd tdj Mfd Kin. HAP 15 THRAl 191 1475 ED2 AAl Z109 CA375 4-7 YM29 188 SCG40 6C1 42D6 1901 III 1 5 5 3 1 4 NI NI 1 1 3 NI NI 1 R2 9 2 5 6 1 4 NI NI 11 3 NI NI 1 HI 3 NI 4 6 6 1 NI NI 2 1 4 2 NI 1 PI 3 NI 4 NI NI NI NI NI NI NI 4 2 NI 1 P2 3 NI NI NI NI NI NI NI NI NI 4 NI NI NI R1 6 NI 1 6 1 NI NI 2 1 4 2 NX 1 R2 6 NI 4 6 6 1 NI NI 2 1 4 2 NI 1 R3 3 NI 4 NI 6 1 NI NI 2 1 4 1 NI 7 R4 7 NI 1 NI 1 NI NI 4 6 1 2 NI 1 R5 3 NI 4 NI NI NI NI NI NI 2 1 NI NI NI R6 3 NI 4 3 1 2 NI NI 1 2 2 6 NI 6 R7 3 NI 4 3 7 1 NI NI 1 1 3 7 NI 4 HI 8 2 1 NI 1 1 4 3 H2 8 2 1 NI 1 1 2 1 R2 8 2 1 NI 1 1 2 1 1 6 8 2 4 12 3 14 1 2 3 6 1 In kindred K1901, the new markers showed no observable recombination with breast cancer susceptibility, indicating that the recombination event in this kindred most likely took place between THRAl and ED2. Thus, no new BRCAl localization information was obtained based upon studying the four new markers in this kindred. In kindred 2082 the key recombinant individual has inherited the linked alleles for ED2,4-7, AAl, and YM29, and was recombinant for tdj 1474 indicating that the recombination event occurred in this individual between tdj 1474 and ED2/AA1.

There are three haplotypes of interest in kindred K2035, HI, H2, and R2 shown in Table 8. HI is present in the four cases and one obligate male carrier descendant from individual 17 while H2 is present or inferred in two cases and two obligate male carriers in descendants of individual 10. R2 is identical to H2 for loci between and including Mfd 15 and SCG40, but has recombined between SCG40 and 42D6. Since we have established that BRCAl is proximal to 42D6, this H2/R2 difference adds no further localization information. HI and R2 share an identical allele at Mfdl5, THRAl, AAl, and ED2 but differ for loci presumed distal to ED2, i.e., 4-7, Mfdl88, SCG40, and 6C1. Although the two haplotypes are concordant for the 5th allele for marker YM29, a marker which maps physically between 4-7 and Mfd 188, it is likely that the alleles are shared identical by state rather than identical by descent since this allele is the most common allele at this locus with a frequency estimated in CEPH parents of 0.42. By contrast, the linked alleles shared at the Mfd 15 and ED2 loci have frequencies of 0.04 and 0.09, respectively. They also share more common alleles at Mfdl91 (frequency = 0.52), THRAl, and AAl (frequency = 0.28). This is the key recombinant in the set as it is the sole recombinant in which breast cancer segregated with the proximal portion of the haplotype, thus setting the distal boundary. The evidence from this kindred therefore places the BRCAl locus proximal to 4-7.

The recombination event in kindred 2082 which places BRCAl distal to tdj 1474 is the only one of the four events described which can be directly inferred; that is, the affected mother's genotype can be inferred from her spouse and offspring, and the recombinant haplotype can be seen in her affected daughter. In this family the odds in favor of affected individuals carrying BRCAl susceptibility alleles are extremely high; the only possible interpretations of the data are that BRCAl is distal to Mfdl91 or alternatively that the purported recombinant is a sporadic case of ovarian cancer at age 44. Rather than a directly observable or inferred recombinant, interpretation of kindred 2035 depends on the observation of distinct 17q-haplotypes segregating in PCT/U S95/10203 different and sometimes distantly related branches of the kindred. The observation that portions of these haplotypes have alleles in common for some markers while they differ at other markers places the BRCAl locus in the shared region. The confidence in this placement depends on several factors: the relationship between the individuals carrying the respective haplotypes, the frequency of the shared allele, the certainty with which the haplotypes can be shown to segregate with the BRCAl locus, and the density of the markers in the region which define the haplotype. In the case of kindred 2035, the two branches are closely related, and each branch has a number of early onset cases which carry the respective haplotype. While two of the shared alleles are common, (Mfd 191, THRAl), the estimated frequencies of the shared alleles at Mfdl5, AAl, and ED2 are 0.04, 0.28, and 0.09, respectively. It is therefore highly likely that these alleles are identical by descent (derived from a common ancestor) rather than identical by state (the same allele but derived from the general population). example 6 Refined Physical Mapping Studies Place the BRCAl Gene in a Region Flanked hv tdi 1474 and TJ5R Since its initial localization to chromosome 17q in 1990 (Hall et al, 1990) a great deal of effort has gone into localizing the BRCAl gene to a region small enough to allow implementation of effective positional cloning strategies to isolate the gene. The BRCAl locus was first localized to the interval Mfd 15 (D17S250) - 42D6 (D17S588) by multipoint linkage analysis (Easton et al, 1993) in the collaborative Breast Cancer Linkage Consortium dataset consisting of 214 families collected worldwide. Subsequent refinements of the localization have been based upon individual recombinant events in specific families. The region THRAl - D17S183 was defined by Bowcock et al, 1993; and the region THRAl - D17S78 was defined by Simaid et al, 1993.

We further showed that the BRCAl locus must lie distal to the marker Mfdl 91 (D17S776) (Goldgar et al, 1994). This marker is known to lie distal to THRAl and RARA. The smallest published region for the BRCAl locus is thus between D17S776 and D17S78. This region still contains approximately 1.5 million bases of DNA, making the isolation and testing of all genes in the region a very difficult task. We have therefore undertaken the tasks of constructing a physical map of the region, isolating a set of polymorphic STR markers located in the region, and analyzing these new markers in a set of informative families to refine the location of the BRCAl gene to a manageable interval.

Four families provide important genetic evidence for localization of BRCAl to a sufficiently small region for the application of positional cloning strategies. Two families (K2082, K1901) provide data relating to the proximal boundary for BRCAl and the other two (K2035, K1813) fix the distal boundary. These families are discussed in detail below. A total of 15 Short Tandem Repeat markers assayable by PCR were used to refine this localization in the families studied. These markers include DS17S7654, DS17S975, tdjl474, and tdj 1239. Primer sequences for these markers are provided in SEQ ID NO:3 and SEQ ID NO:4 for DS17S754; in SEQ ID NO:5 and SEQ ID NO:6 for DS17S975; in SEQ ID NO:7 and SEQ ED NO:8 for tdj 1474; and, in SEQ ID NO:9 and SEQ ID N0:10 for tdjl239.

Kindred 2Q82 Kindred 2082 is the largest BRCAl-linked breast/ovarian cancer family studied to date. It has a LOD score of 8.6, providing unequivocal evidence for 17q linkage. This family has been previously described and shown to contain a critical recombinant placing BRCAl distal to MFD191 (D17S776). This recombinant occurred in a woman diagnosed with ovarian cancer at age 45 whose mother had ovarian cancer at age 63. The affected mother was deceased; however, from her children, she could be inferred to have the linked haplotype present in the 30 other linked cases in the family in the region between Mfdl5 and Mfdl 88. Her affected daughter received the linked allele at the loci ED2,4-7, and Mfdl 88, but received the allele on the non-BRCAl chromosome at Mfdl 5 and Mfdl91. In order to further localize this recombination breakpoint, we tested the key members of this family for the following markers derived from physical mapping resources: tdj 1474, tdj 1239, CF4, D17S855. For the markers tdjl474 and CF4, the affected daughter did not receive the linked allele. For the STR locus tdj 1239, however, the mother could be inferred to be informative and her daughter did receive the BRCAl-associated allele. D17S855 was not informative in this family. Based on this analysis, the order is 17q centromere - Mfdl91 - 17HSD -CF4 - tdjl474 - tdjl239 - D17S855 - ED2 - 4-7 - Mfdl88 - 17q telomere. The recombinant described above therefore places BRCAl distal to tdj 1474, and the breakpoint is localized to the interval between tdj 1474 and tdj 1239. The only alternative explanation for the data in this family other than that of BRCAl being located distal to tdj 1474, is that the ovarian cancer present in the recombinant individual is caused by reasons independent of the BRCAl gene. Given that ovarian cancer diagnosed before age 50 is rare, this alternate explanation is exceedingly unlikely.

Kindred 19Q1 Kindred 1901 is an early-onset breast cancer family with 7 cases of breast cancer diagnosed before 50, 4 of which were diagnosed before age 40. In addition, there were three cases of breast cancer diagnosed between the ages of 50 and 70. One case of breast cancer also had ovarian cancer at age 61. This family currently has a LOD score of 1.5 with D17S855. Given this linkage evidence and the presence of at lease one ovarian cancer case, this family has a posterior probability of being due to BRCAl of over 0.99. In this family, the recombination comes from the fact that an individual who is the brother of the ovarian cancer case from which the majority of the other cases descend, only shares a portion of the haplotype which is cosegregating with the other cases in the family. However, he passed this partial haplotype to his daughter who developed breast cancer at age 44. If this case is due to the BRCAl gene, then only the part of the haplotype shared between this brother and his sister can contain the BRCAl gene. The difficulty in interpretation of this kind of information is that while one can be sure of the markers which are not shared and therefore recombinant, markers which are concordant can either be shared because they are non-recombinant, or because their parent was homozygous. Without the parental genotypic data it is impossible to discriminate between these alternatives. Inspection of the haplotype in K1901, shows that he does not share the linked allele at Mfdl5 (D17S250), THRAl, CF4 (D17S1320), and tdj 1474 (17DS1321). He does share the linked allele at Mfdl91 (D17S776), ED2 (D17S1327), tdj 1239 (D17S1328), and Mfdl88 (D17S579). Although the allele shared at Mfdl91 is relatively rare (0.07), we would presume that the parent was homozygous since they are recombinant with markers located nearby on either side, and a double recombination event in this region would be extremely unlikely. Thus the evidence in this family would also place the BRCAl locus distal to tdj 1474. However, the lower limit of this breakpoint is impossible to determine without parental genotype information. It is intriguing that the key recombinant breakpoint in this family confirms the result in Kindred 2082. As before, the localization information in this family is only meaningful if the breast cancer was due to the BRCAl gene. However, her relatively early age at diagnosis (44) makes this seem very likely since the risk of breast cancer before age 45 in the general population is low (approximately 1%).

Kindred 2035 This family is similar to K1901 in that the information on the critical recombinant events is not directly observed but is inferred from the observation that the two haplotypes which are cosegregating with the early onset breast cancer in the two branches of the family appear identical for markers located in the proximal portion of the 17q BRCAl region but differ at more distal loci. Each of these two haplotypes occurs in at least four cases of early-onset or bilateral breast cancer. The overall LOD score with ED2 in this family is 2.2, and considering that there is a case of ovarian cancer in the family (indicating a prior probability of BRCAl linkage of 80%), the resulting posterior probability that this family is linked to BRCAl is 0.998. The haplotypes are identical for the markers Mfdl5, THRAl, Mfdl91, ED2, AAl, D17S858 and D17S902. The common allele at Mfd 15 and ED2 are both quite rare, indicating that this haplotype is shared identical by descent. The haplotypes are discordant, however, for CA375, 4-7, and Mfdl 88, and several more distal markers. This indicates that the BRCAl locus must lie above the marker CA-375. This marker is located approximately 50 kb below D17S78, so it serves primarily as additional confirmation of this previous lower boundary as reported in Simard et al. (1993).

Kindred 1813 Kindred 1813 is a small family with four cases of breast cancer diagnosed under the age of 40 whose mother had breast cancer diagnosed at age 45 and ovarian cancer at age 61. This situation is somewhat complicated by the fact the four cases appear to have three different fathers, only one of whom has been genotyped. However, by typing a number of different markers in the BRCAl region as well as highly polymorphic markers elsewhere in the genome, the paternity of all children in the family has been determined with a high degree of certainty. This family yields a maximum multipoint LOD score of 0.60 with 17q markers and, given that there is at least one case of ovarian cancer, results in a posterior probability of being a BRCAl linked family of 0.93. This family contains a directly observable recombination event in individual 18 (see Figure 5 in Simard et al, Human Mol. Genet. 2:1193-1199 (1993)), who developed breast cancer at age 34. The genotype of her affected mother at the relevant 17q loci can be inferred from her genotypes, her affected sister's genotypes, and the genotypes of three other unaffected siblings. Individual 18 inherits the BRCAl-linked alleles for the following loci: Mfdl5, THRAl, D17S800, D17S855, AA1, and D17S931. However, for markers below D17S931, i.e., U5R, vrs31, D17S858, and D17S579, she has inherited the alleles located on the non-disease bearing chromosome. The evidence from this family therefore would place the BRCAl locus proximal to the marker U5R Because of her early age at diagnosis (34) it is extremely unlikely that the recombinant individual's cancer is not due to the gene responsible for the other cases of breast/ovarian cancer in this family; the uncertainty in this family comes from our somewhat smaller amount of evidence that breast cancer in this family is due to BRCAl rather than a second, as yet unmapped, breast cancer susceptibility locus.

Size of the region containing BRCAl Based on the genetic data described in detail above, the BRCAl locus must lie in the interval between the markers tdj 1474 and U5R, both of which were isolated in our laboratory. Based upon the physical maps shown in Figures 2 and 3, we can try to estimate the physical distance between these two loci. It takes approximately 14 PI clones with an average insert size of approximately 80 kb to span the region. However, because all of these Pis overlap to some unknown degree, the physical region is most likely much smaller than 14 times 80 kb. Based on restriction maps of the clones covering the region, we estimate the size of the region containing BRCAl to be approximately 650 kb. example 7 Identification of Candidate cDNA Clones for the BRCAl Locus hv Genomic Analysis of the Contig Region 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 cosmids and PI and 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.

Zoo blots. The first method for identifying putative coding sequences was by screening the cosmid and PJ clones for sequences conserved through evolution across several species. This technique is referred to as "zoo blot analysis" and is described by Monaco, 1986. Specifically, DNAs from cow, chicken, pig, mouse and rat were digested with the restriction enzymes EcoRI and Hindlll (8 ng of DNA per enzyme). The digested DNAs were separated overnight on an 0.7% gel at 20 volts for 16 hours (14 cm gel), and the DNA transferred to Nylon membranes using standard Southern blot techniques. For example, the zoo blot filter was treated at 65°C in 0.1 x SSC, 0.5% SDS, and 0.2M Tris, pH 8.0, for 30 minutes and then blocked overnight at 42°C in 5x SSC, 10% PEG 8000,20 mM NaP04 pH 6.8,100 (ig/ml Salmon Sperm DNA, Ix Denhardt's, 50% formamide, 0.1% SDS, and 2 ng/ml C0t-1 DNA.

The cosmid and PI 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 gel wedge at 100 volts for at least two hours in 0.5x Tris Acetate buffer (Maniatis et al, 1982). The eluted Not 1 digested DNA (-15 kb to 25 kb) was then digested with EcoRI restriction enzyme to give smaller fragments (~0.5 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, CaL #1004760). The labeled DNA was spermine precipitated (add 100 ill TE, 5 0.1 M spermine, and 5 ul of 10 mg/ml salmon sperm DNA) to remove unincorporated radionucleotides. The labeled DNA was then resuspended in 100 ^1 TE, 0.5 M NaCl at 65°C for 5 minutes and then blocked with Human C0t-1 DNA for 2-4 hrs. as per the manufacturer's instructions (Gibco/BRL, Cat. 25279SA). The C0t-1 blocked probe was incubated on the zoo blot filters in the blocking solution overnight at 42°C. The filters were 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°C. The filters were then exposed 1 to 3 days at -70°C to Kodak XAR-5 film with an intensifying screen. Thus, the zoo blots were hybridized with either the pool of Eco-Rl fragments from the insert, or each of the fragments individually.

HTF island analysis. The second method for iHentifying cosmids to use as probes on the cDNA libraries was HTF island analysis. Since the pulsed-field map can reveal HTF islands, cosmids that map to these HTF island regions were analyzed with priority. 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 Ascl, Notl, BssHlI. EagI, Sacll, Nael, Narl, Smal, and Mlul (Anand, 1992). A pulsed-field map was created using the enzymes Notl, Nrul, EagI, Sacll, and Sail, and two HTF islands were found. These islands are located in the distal end of the region, one being distal to the GP2B locus, and the other being proximal to the same locus, both outside the BRCAl region. The cosmids derived from the YACs that cover these two locations were analyzed to identify those that contain these restriction sites, and thus the HTF islands. cDNA screening. Those clones that contain HTF islands or show hybridization to other species DNA besides human are likely to contain coding sequences. The human DNA from these clones 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 as the zoo blots except that the cDNA filters undergo a more stringent wash of 0.1 x SSC, 0.1% SDS at 65°C for 30 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 C600Hfi 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 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), human brain (Clonetech Cat. HL11810), human placenta (Clonetech Cat 1075b), and human skeletal muscle (Clonetech Cat. HL1124b).

The cDNA libraries were plated with their host cells on NZCYft' plates, and filter lifts are made in duplicate from each plate as per Maniatis et 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 WO 96/05307 PCT/US95/10203 were picked, replated, and screened again with the labeled clone insert or its derived EcoRJ fragment DNA to verify their positive status. Clones that were positive after this second round of screening were then grown up and their DNA purified for Southern blot analysis and sequencing. Clones were either purified as plasmid through in vivo excision of the plasmid from the Lambda 5 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 DNA as 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 10 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 BRCAl 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 15 clones in the BRCAl 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.

We have screened the normal breast, 8 month pregnant breast and fetal brain cDNA libraries 2 0 with zoo blot-positive Eco R1 fragments from cosmid BAC and PI clones in the region. Potential BRCAl 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 checked by Southern blot hybridization against the probe DNA to verify that they originated from 25 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. For example, the clones 694-65,1240-1 and 1240-33 were obtained independently and subsequently shown to derive from the same contiguous cDNA sequence which has been named EST:489:1. 3 0 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 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 (e.g., BLASTN 5 1.3.BMP), for sequence comparison against both local and remote sequence databases (e.g., GenBank). running on Sun SPARC workstations. Sequences reconstructed from collections of cDNA clones identified with the cosmids and Pis have been generated. All candidate genes that represented new sequences were analyzed further to test their candidacy for the putative BRCAl locus. 0 Mutation screening. To screen for mutations in the affected pedigrees, two different approaches were followed. First, genomic DNA isolated from family members known to cany the susceptibility allele of BRCAl 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 5 amplified mixture. By a combination of such amplification experiments and sequencing of PI, BAC or cosmid 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 pedigrees.

A second approach that is much more rapid if the intron/exon structure of the candidate gene is complex involves sequencing fragments amplified from pedigree lymphocyte cDNA. cDNA 0 synthesized from lymphocyte mRNA extracted from pedigree blood 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 !5 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 BRCAl region that is expressed in breast is considered to be a candidate gene for BRCAl. Compelling evidence that a given candidate gene corresponds to BRCAl comes from a demonstration that pedigree families contain defective alleles of the 0 candidate.

EXAMPLE 8 Identification of BRCA1 Identification of BRCAl. Using several strategies, a detailed map of transcripts was developed for the 600 kb region of 17q21 between D17S1321 and D17S1324. Candidate expressed sequences were defined as DNA sequences obtained from: 1) direct screening of breast, fetal brain, or lymphocyte cDNA libraries, 2) hybrid selection of breast, lymphocyte or ovaiy cDNAs, or 3) random sequencing of genomic DNA and prediction of coding exons by XPOUND (Thomas and Skolnick, 1994). These expressed sequences in many cases were assembled into contigs composed of several independently identified sequences. Candidate genes may comprise more than one of these candidate expressed sequences. Sixty-five candidate expressed sequences within this region were identified by hybrid selection, by direct screening of cDNA libraries, and by random sequencing of PI subclones. Expressed sequences v.ire characterized by transcript size, DNA sequence, database comparison, expression pattern, genomic structure, and, most importantly, DNA sequence analysis in individuals from kindreds segregating 17q-linked breast and ovarian cancer susceptibility.

Three independent contigs of expressed sequence, 1141:1 (649 bp), 694:5 (213 bp) and 754:2 (1079 bp) were isolated and eventually shown to represent portions of BRCAl. When ESTs for these contigs were used as hybridization probes for Northern analysis, a single transcript of approximately 7.8 kb was observed in normal breast mRNA, suggesting that they encode different portions of a single gene. Screens of breast, fetal brain, thymus, testes, lymphocyte and placental cDNA libraries and PCR experiments with breast mRNA linked the 1141:1, 694:5 and 754:2 contigs. 5' RACE experiments with thymus, testes, and breast mRNA extended the contig to the putative 5' end, yielding a composite full length sequence. PCR and direct sequencing of Pis and BACs in the region were used to identify the location of introns and allowed the determination of splice donor and acceptor sites. These three expressed sequences were merged into a single transcription unit that proved in the final analysis to be BRCAl. This transcription unit is located adjacent to D17S855 in the center of the 600 kb region (Fig. 4).

Combination of sequences obtained from cDNA clones, hybrid selection sequences, and amplified PCR products allowed construction of a composite frill length BRCAl cDNA (SEQ ID NO:l). The sequence of the BRCAl cDNA (up through the stop codon) has also been deposited with GenBank and assigned accession number U-14680. This deposited sequence is incorporated herein by reference. The cDNA clone extending farthest in the 3' direction contains a poly(A) tract preceded by a polyadenylation signal. Conceptual translation of the cDNA revealed a single long open reading frame of 208 kilodaltons (amino acid sequence: SEQ ID NO:2) with a potential 5 initiation codon flanked by sequences resembling the Kozak consensus sequence (Kozak, 1987). Smith-Waterman (Smith and Waterman, 1981) and BLAST (Altschul et al., 1990) searches identified a sequence near the amino terminus with considerable homology to zinc-finger domains (Fig. 5). This sequence contains cysteine and histidine residues present in the consensus C3HC4 zinc-finger motif and shares multiple other residues with zinc-finger proteins in the databases. The 0 BRCAl gene is composed of 23 coding exons arrayed over more than 100 kb of genomic DNA (Fig. 6). Northern blots using fragments of the BRCAl cDNA as probes identified a single transcript of about 7.8 kb, present most abundantly in breast, thymus and testis, and also present in ovary (Fig. 7). Four alternatively spliced products were observed as independent cDNA clones; 3 of these were detected in breast and 2 in ovaiy mRNA (Fig. 6). A PCR survey from tissue cDNAs 5 further supports the idea that there is considerable heterogeneity near the 5' end of transcripts from this gene; the molecular basis for the heterogeneity involves differential choice of the first splice donor site, and the changes detected all alter the transcript in the region 5' of the identified start codc .. We have detected six potential alternate splice donors in this 5' untranslated region, with the longest deletion being 1,155 bp. The predominant foim of the BRCAl protein in breast and 0 ovary lacks exon 4. The nucleotide sequence for BRCAl exon 4 is shown in SEQ ID NO:l 1, with the predicted amino acid sequence shown in SEQ ID NO: 12.

Additional 5' sequence of BRCAl genomic DNA is set forth in SEQ ED NO: 13. The G at position 1 represents the potential start site in testis. The A in position 140 represents the potential start site in somatic tissue. There are six alternative splice forms of this 5' sequence as shown in 5 Figure 8. The G at position 356 represents the canonical first splice donor site. The G at position 444 represents the first splice donor site in two clones (testis 1 and testis 2). The G at position 889 represents the first splice donor site in thymus 3. A fourth splice donor site is the G at position 1230. The T at position 1513 represents the splice acceptor site for all of the above splice donors. A fifth alternate splice form has a first splice donor site at position 349 with a first acceptor site at ; 0 position 591 and a second splice donor site at position 889 and a second acceptor site at position 1513. A sixth alternate form is unspliced in this 5' region. The A at position 1532 is the canonical start site, which appears at position 120 of SEQ ID NO:l. Partial genomic DNA seqnrnecs determined for BRCAl are set forth in Figures 10A-10H and SEQ ID Numbers: 14-34. The lower case letters (in figures 10A-10H) denote intron sequence while the upper case letters denote exon sequence. Indefinite intervals within introns are designated with wvwwwww in Figures 10A-10H. The intron/exon junctions are shown in Table 9. The CAG found at the 5* end of exons 8 and 14 is found in some cDNAs but not in others. Known polymorphic sites are shown in Figures 1 OA-1 OH in boldface type and are underlined.

TABLE 9 Base Exon Position* No. 5' 3' Length el 1 100 100 e2 101 199 99 e3 200 253 54 e4 *** *** 111 e5 254 331 78 e6 332 420 89 e7 421 560 140 e8 561 666 106 e9 667 712 46 elO 713 789 77 ell 790 4215 3426 el2 4216 4302 87 el3 4303 4476 174 el4 4477 4603 127 GATAAATTAAAACTGCGACTGCGCGGCGTG35* ATATATATA fGTTTTTCTAATGTGTTAAAG17 TTTCTTTTTCTCCCCCCCCTACCCTGCTAG" AGCTACTTTTTTTTTTTTTTTTTGAGACAG41 AATTGTTCTTTCTTTCTTi ATAATTTATAG4 3 GAGTGTGTTTCTCAAACAATTTAATTTCAG4 5 AAACATAATGTTTTCCCTTGTATTTTACAO4 7 TGCTTGACTGTTCTTTACCATACTGTTTAG4® TGATTTATTTTTTGGGGGGAAATTTTTTAG" TCTTATTAQGACTCTGTCTTTTCCCTATAG53 GAGTACCTTGTTATTTTTGTATATTTTCAGSS ACATCTGAACCTCTGTTTTTGTTATTTAAG 5 7 CATTTTCTTGGTACCATTTATCGTTTTTGA5 ' AGTAGATTTGTTTTCTCATTCCATTTAAAG*1 * Base numbers in SEQ ID NO:l.

** Numbers in superscript refer to SBQ ID NOS. *** e4 from SBQ ID NO:11.

Borders 3' GTAGTAGAGTCCCGGGAAAGGGACAGGGGG3' GTAAGTCAGCACAAGAGTGTATTAATTTGG3' GTAAGTTTGAATGTGTTATGTGGCTCCATT40 GTAAGTGCACACCACCATATCCAGCTAAAT42 GTATATAATTTGGTAATGATGCTAGGTTGG4 4 GTAAGTGTTGAATATCCCAAGAATGACACT4* GTAAAACCATTTG'rrri'CTl'Crrcri'CTTC4' GTAAGGGTCTCAGGTTTTTTAAGTATTTAA50 GTGAGTCAAAGAGAACCTTTGTCTATGAAG" GTAATGGCAAAGTTTGCCAACTTAACAGGC54 GTATTGGAACCAGGTTTTTGTGTTTGCCCC5* AGGTAAAAAGCGTGTGTGTGTGTGCACATG5' GTGTGTATTGTTGGCCAAACACTGATATCT* 0 GTAAGAAACATCAATGTAAAGATGCTGTGG" TABLE 9 (cont'd) Base Exon Position* Intron No. 5' 3' Length 5' el5 4604 4794 191 ATGGTTTTCTCCTTCCATTTATCTTTCTAG"** el6 4795 5105 311 TGTAAATTAAACTTCTCCCATTCCTTTCAG45 el7 5106 5193 88 ATGATAATGGAATATTTGATTTAATTTCAG'7 el8 5194 5271 78 CTAATCCTTTGAGTGTTTTTCATTCTGCAG49 el9 5272 5312 41 TGTAACCTGTCTTTTCTATGATCTCTTTAG11 e21 5397 5451 55 CTGTCCCTCTCTCTTCCTCTCTTCTTCCAG7s e22 5452 5525 74 AGTGATTTTACATGTAAATGTCCATTTTAG77 e23 5526 5586 61 TTGAATGCTCTTTCCTTCCTGGGGATCCAG7' e24 5587 5914 328 CTAATCTCTGCTTGTGTTCTCTGTCTCCAG'1 * Base numbers in SBQ ID NO.-l.

+* Numbers in superscript refer to SBQ ID NOS.

Borders 3 • gtaatatttcatctgctgtattggaacaaa6 4 gtgagtgtatccatatgtatctccctaatg65 gtataccaagaacctttacagaataccttg" gtaagtataatactatttctcccctcctcc70 gtaagtacttgatgttacaaactaaccaga7 2 gtaaagctccctccctcaagttgacaaaaa74 gtaagagcctgggagaaccccagagttcca7* GTAAGTATTGGGTGCCCTGTCAGTGTGGGA7• gtaaggtgcctcgcatgtacctgtgctatt,° Low stringency blots in which genomic DNA from organisms of diverse phylogenetic background were probed with BRCAl sequences that lack the zinc-finger region revealed strongly hybridizing fragments in human, monkey, sheep and pig, and very weak hybridization signals in rodents. This result indicates that, apart from the zinc-finger domain, BRCAl is conserved only at a moderate level through evolution.

Germline BRCAl mutations in 17q-linked kindreds. The most rigorous test for BRCAl candidate genes is to search for potentially disruptive mutations in carrier individuals from kindreds that segregate 17q-linked susceptibility to breast and ovarian cancer. Such individuals must contain BRCAl alleles that differ from the wildtype sequence. The set of DNA samples used in this analysis consisted of DNA from individuals representing 8 different BRCAl kindreds (Table 10).

TABLE 10 KINDRED DESCRIPTIONS AND ASSOCIATED LOD SCORES Sporadic Kindred Cases (n) Cases'in) LOP Marker^ Br Br<50 0v Score 2082 31 22 7 9.49 D17S1327 2099 22 14 2* 0 2.36 D17S800/D17S8552 2035 8 1* 0 2.25 D17S1327 1901 7 I* 0 1.50 D17S855 1925 4 3 0 0 0.55 D17S579 1910 4 0 0 0.36 D17S579/D17S2502 1927 4 0 1 -0.44 D17S250 1911 8 0 2 -0.20 D17S250 1 Number of women with breast cancer (diagnosed under age 50) or ovarian cancer (diagnosed at any age) who do not share the BRCAl-linked haplotype segregating in the remainder of the cases in the kindred. 2 Multipoint LOD score calculated using both markers * kindred contains one individual who had both breast and ovarian cancer, this individual is counted as a breast cancer case and as an ovarian cancer case.

The logarithm of the odds (LOD) scores in these kindreds range from 9.49 to -0.44 for a set of markers in 17q21. Four of the families have convincing LOD scores for linkage, and 4 have low positive or negative LOD scores. The latter kindreds were included because they demonstrate haplotype sharing at chromosome 17q21 for at least 3 affected members. Furthermore, all kindreds in the set display early age of breast cancer onset and 4 of the kindreds include at least one case of ovarian cancer, both hallmarks of BRCAl kindreds. One kindred, 2082, has nearly equal incidence of breast and ovarian cancer, an unusual occurrence given the relative rarity of ovarian cancer in the population. All of the kindreds except two were ascertained in Utah. K2035 is from the midwest. K2099 is an African-American kindred from the southern USA.

In the initial screen for predisposing mutations in BRCAl, DNA from one individual who carries the predisposing haplotype in each kindred was tested. The 23 coding exons and associated splice junctions were amplified either from genomic DNA samples or from cDNA prepared from lymphocyte mRNA. When the amplified DNA sequences were compared to the wildtype sequence, 4 of the 8 kindred samples were found to contain sequence variants (Table 11).

TABLE 11 PREDISPOSING MUTATIONS Kindred Number Mutation Location* 2082 1910 2099 2035 1901 11 bp deletion C-VT extra C T-»G ? Gln->Stop frameshift Met—>Arg loss of transcript frameshift 4056 5385 5443 189 * In Scqi:?ncc ID NO:l All four sequence variants are heterozygous and each appears in only one of the kindreds. Kindred 2082 contains a nonsense mutation in exon 11 (Fig. 9A), Kindred 1910 contains a single nucleotide insertion in ey.on 20 (Fig. 9B), and Kindred 2099 contains a missense mutation in exon 21, resulting in a Met—>Arg substitution. The frameshift and nonsense mutations are likely disruptive to the function of the BRCAl product. The peptide encoded by the frameshift allele in Kindred 1910 would contain an altered amino acid sequence beginning 108 residues from the wildtype C-terminus. The peptide encoded by the frameshift allele in Kindred 1901 would contain an altered amino acid sequence beginning with the 24th residue from the wildtype N-terminus. The mutant allele in Kindred 2082 would encode a protein missing 551 residues from the C-terminus. The missense substitution observed in Kindred 2099 is potentially disruptive as it causes the replacement of a small hydrophobic amino acid (Met), by a large charged residue (Arg). Eleven common polymorphisms were also identified, 8 in coding sequence and 3 in introns.

The individual studied in Kindred 2035 evidently contains a regulatory mutation in BRCAl. In her cDNA, a polymorphic site (A—>G at base 3667) appeared homozygous, whereas her genomic DNA revealed heterozygosity at this position (Fig. 9C). A possible explanation for this observation is that mRNA from her mutated BRCAl allele is absent due to a mutation that affects its production or stability. This possibility was explored further by examining 5 polymorphic sites in the BRCAl coding region, which are separated by as much as 3.5 kb in the BRCAl transcript In all cases where her genomic DNA appeared heterozygous for a polymorphism, cDNA appeared homozygous. In individuals from other kindreds and in non-haplotype carriers in Kindred 2035, these polymorphic sites could be observed as heterozygous in cDNA, implying that amplification from cDNA was not biased in favor of one allele. This analysis indicates that a BRCAl mutation in Kindred 2035 either prevents transcription or causes instability or aberrant splicing of the BRCAl transcript.

Congregation of BRCAl mutations with BRCAl haplotypes and population frequency analysis. In addition to potentially disrupting protein function, two criteria must be met for a sequence variant to qualify as a candidate predisposing mutation. The variant must: 1) be present in individuals from the kindred who carry the predisposing BRCAl haplotype and absent in other members of the kindred, and 2) be rare in the general population.

Each mutation was tested for cosegregation with BRCAl. For the frameshift mutation in Kindred 1910, two other haplotype carriers and one non-carrier were sequenced (Fig. 9B). Only the carriers exhibited the frameshift mutation. The C to T change in Kindred 2082 created a new Avrll restriction site. Other carriers and non-carriers in the kindred were tested for the presence of the restriction site (Fig. 9A). An allele-specific oligonucleotide (ASO) was designed to detect the presence of the sequence variant in Kindred 2099. Several individuals from the kindred, some known to carry the haplotype associated with the predisposing allele, and others known not to cany the associated haplotype, were screened by ASO for the mutation previously detected in the kindred. In each kindred, the corresponding mutant allele was detected in individuals carrying the BRCAl-associated haplotype, and was not detected in noncairiers. In the case of the potential regulatory mutation observed in the individual from Kindred 2035, cDNA and genomic DNA from carriers in the kindred were compared for heterozygosity at polymorphic sites. In every instance, the extinguished allele in the cDNA sample was shown to lie on the chromosome that carries the BRCAl predisposing allele (Fig. 9C).

To exclude the possibility that the mutations were simply common polymorphisms in the population, ASOs for each mutation were used to screen a set of normal DNA samples. Gene frequency estimates in Caucasians were based on random samples from the Utah population. Gene frequency estimates in African-Americans were based on 39 samples provided by M. Peracek-Vance which originate from African-Americans used in her linkage studies and 20 newborn Utah African-Americans. None of the 4 potential predisposing mutations was found in the appropriate control population, indicating that they are rare in the general population. Thus, two important requirements for BRCAl susceptibility alleles were fulfilled by the candidate predisposing mutations: 1) cosegregation of the mutant allele with disease, and 2) absence of the mutant allele in controls, indicating a low gene frequency in the general population.

Phenotvpic Expression of BRCAl Mutations. The effect of the mutations on the BRCAl protein correlated with differences in the observed phenotypic expression in the BRCAl kindreds. Most BRCAl kindreds have a moderately increased ovarian cancer risk, and a smaller subset have high risks of ovarian cancer, comparable to those for breast cancer (Easton et al, 1993). Three of the four kindreds in which BRCAl mutations were detected fall into the former category, while the fourth (K2082) falls into the high ovarian cancer risk group. Since the BRCAl nonsense mutation found in K2082 lies closer to the amino terminus than the other mutations detected, it might be expected to have a different phenotype. In fact, Kindred K2082 mutation has a high incidence of ovarian cancer, and a later mean age at diagnosis of breast cancer cases than the other kindreds WO 96/05307 PCT/US95/10203 (Goldgar et al, 1994). This difference in age of onset could be due to an ascertainment bias in the smaller, more highly penetrant families, or it could reflect tissue-specific differences in the behavior of BRCAl mutations. The other 3 kindreds that segregate known BRCAl mutations have, on average, one ovarian cancer for every 10 cases of breast cancer, but have a high 5 proportion of breast cancer cases diagnosed in their late 20's or early 30's. Kindred 1910, which has a frameshift mutation, is noteworthy because three of the four affected individuals had bilateral breast cancer, and in each case the second tumor was diagnosed within a year of the first occurrence. Kindred 2035, which segregates a potential regulatory BRCAl mutation, might also be expected to have a dramatic phenotype. Eighty percent of breast cancer cases in this kindred occur 10 under age 50. This figure is as high as any in the set, suggesting a BRCAl mutant allele of high penetrance (Table 10).

Although the mutations described above clearly are deleterious, causing breast cancer in women at very young ages, each of the four kindreds with mutations includes at least one woman who carries the mutation who lived until age 80 without developing a malignancy. It will be of 15 utmost importance in the studies that follow to identify other genetic or environmental factors that may ameliorate the effects of BRCAl mutations.

In four of the eight putative BRCAl-linked kindreds, potential predisposing mutations were not found. Three of these four have LOD scores for BRCAl-linked markers of less than 0.55. Thus, these kindreds may not in reality segregate BRCAl predisposing alleles. Alternatively, the 2 0 mutations in these four kindreds may lie in regions of BRCAl that, for example, affect the level of transcript and therefore have thus far escaped detection.

Role of BRCAl in Cancer. Most tumor suppressor genes identified to dale give rise to protein products that are absent, nonfunctional, or reduced in function. The majority of TP53 25 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). 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 (WT1) that alter DNA binding of the protein (Little et al., 3 0 1993). The nature of the mutations observed in the BRCAl coding sequence is consistent with production of either dominant negative proteins or nonfunctional proteins. The regulatory mutation inferred in Kindred 2035 cannot be a dominant negative; rather, this mutation likely causes reduction or complete loss of BRCAl expression from the affected allele.

The BRCAl protein contains a C3HC4 zinc-finger domain, similar to those found in numerous DNA binding proteins and implicated in zinc-dependent binding to nucleic acids. The first 180 amino acids of BRCAl contain five more basic residues than acidic residues. In contrast, the remainder of the molecule is very acidic, with a net excess of 70 acidic residues. The excess negative charge is particularly concentrated near the C-terminus. Thus, one possibility is that BRCAl encodes a transcription factor with an N-terminal DNA binding domain and a C-terminal transactivational "acidic blob" domain. Interestingly, another familial tumor suppressor gene, WT1, also contains a zinc-finger motif (Haber et al., 1990). Many cancer predisposing mutations in WT1 alter zinc-finger domains (Little et al, 1993; Haber et al., 1990; Little et al, 1992). WT1 encodes a transcription factor, and alternative splicing of exons that encode parts of the zinc-finger domain alter the DNA binding properties of WT1 (Bickmore et al, 1992). Some alternatively spliced forms of WT1 mRNA generate molecules that act as transcriptional repressors (Drummond et al, 1994). Some BRCA1 splicing variants may alter the zinc-finger motifj raising the possibility that a regulatory mechanism similar to that which occurs in WT1 may apply to BRCAl.

EXAMPLE 9 Analysis of Tumors for BRCAl Mutations To focus the analysis on tumors most likely to contain BRCAl mutations, primary breast and ovarian carcinomas were typed for LOH in the BRCAl region. Three highly polymorphic, simple tandem repeat markers were used to assess LOH: D17S1323 and D17S855, which are intragenic to BRCAl, and D17S1327, which lies approximately 100 kb distal to BRCAl. The combined LOH frequency in inforaiative cases (i.e., where the germline was heterozygous) was 32/72 (44%) for the breast carcinomas and 12/21 (57%) for the ovarian carcinomas, consistent with previous measurements of LOH in the region (Futreal et al., 1992b; Jacobs et al, 1993; Sato et al, 1990; Eccles et al, 1990; Cropp et al, 1994). The analysis thus defined a panel of 32 breast tumors and 12 ovarian tumors of mixed race and age of onset to be examined for BRCA mutations.

The complete 5,589 bp coding region and intron/exon boundary sequences of the gene were VCT/US95/10203 screened in this tumor set by direct sequencing alone or by a combination of single-strand conformation analysis (SSCA) and direct sequencing.

A total of six mutations (of which two are identical) w;ts found, one an ovarian tumor, four in breast tumors and one in a male unaffected l:ap!otyp* carrier (Table 12). One mutation, Glul541Ter, introduced a stop codon that would create a truncated protein missing 323 amino acids at the carboxy terminus. In addition, two missense mutations were identified. These are Alal708Glu and Metl775Arg and involve substitutions of small, hydrophobic residues by charged residues. Patients 17764 and 19964 are from the same family. In patient OV24 nucleotide 2575 is deleted and in patients 17764 and 19964 nucleotides 2993-2996 are deleted.

TABLE 12 Predisposing Mutations Nucleotide Amino Acid Age of Family Patient £odon Change Change Onset History BT098 1541 GAG -► IAG Glu —► Stop 39 OV24 819 1 bp deletion frameshift 44 - BT106 1708 G£G —> GAG Ala -> Glu 24 + MC44 1775 AIG —► AQG Met -> Arg 42 + 17764 958 4 bp deletion frameshift 31 + 19964 95S 4 bp deletion frameshift +♦ 2 5 * Unaffected haplotype carrier, male Several lines of evidence suggest that all five mutations represent BRCAl susceptibility alleles: 3 0 (i) all mutations are present in the germline; (ii) all are absent in appropriate control populations, suggesting they are not common polymorphisms; (iii) each mutant allele is retained in the tumor, as is the case in tumors from patients belonging to kindreds that segregate BRCAl susceptibility alleles (Smith et al., 1992; Kelsell et al, 1993) (if the mutations represented neutral polymorphisms, they should be retained in only 50% of the cases); (iv) the age of onset in the four breast cancer cases with mutations varied between 24 and 42 years of age, consistent with the early age of onset of breast cancer in individuals with BRCAl susceptibility; similarly, the ovarian cancer case was diagnosed at 44, an age that falls in the youngest 13% of all ovarian cancer cases; and finally, (v) three of the five cases have positive family histories of breast or ovarian cancer found retrospectively in their medical records, although the tumor set was not selected with regard to this criterion.

BT106 was diagnosed at age 24 with breast cancer. Her mother had ovarian cancer, her father had melanoma, and her paternal grandmother also had breast cancer. Patient MC44, an African-American, had bilateral breast cancer at age 42. This patient had a sister who died of breast cancer at age 34, another sister who died of lymphoma, and a brother who died of lung cancer. Her mutation (Metl 775Arg) had been detected previously in Kindred 2099, an African-American family that segregates a BRCAl susceptibility allele, and was absent in African* American and Caucasian controls. Patient MC44, to our knowledge, is unrelated to Kindred 2099. The detection of a rare mutant allele, once in a BRCAl kindred and once in the germline of an apparently unrelated early-onset breast cancer case, suggests that the Metl 775Arg change may be a common predisposing mutation in African-Americans. Collectively, these observations indicate that all four BRCAl mutations in tumors represent susceptibility alleles; no somatic mutations were detected in the samples analyzed.

The paucity of somatic BRCAl mutations is unexpected, given the frequency of LOH on 17q. and the usual role of susceptibility genes as tumor suppressors in cancer progression. There are three possible explanations for this result: (i) some BRCAl mutations in coding sequences were missed by our screening procedure; (ii) BRCAl somatic mutations fall primarily outside the coding exons; and (iii) LOH events in 17q do not reflect BRCAl somatic mutations.

If somatic BRCAl mutations truly are rare in breast and ovary carcinomas, this would have strong implications for the biology of BRCAl. The apparent lack of somatic BRCAl mutations implies that there may be some fundamental difference in the genesis of tumors in genetically predisposed BRCAl carriers, compared with tumors in the general population. For example, mutations in BRCAl may have an effect only on tumor formation at a specific stage early in breast and ovarian development. This possibility is consistent with a primary function for BRCAl in premenopausal breast cancer. Such a model for the role of BRCAl in breast and ovarian cancer predicts an interaction between reproductive hormones and BRCAl function. However, no clinical or pathological differences in familial versus sporadic breast and ovary tumors, other than age of onset, have been described (Lynch et al., 1990). On the other hand, the recent finding of increased TP53 mutation and microsatellite instability in breast tumors from patients with a family history of breast cancer (Glebov et al, 1994) may reflect some difference in tumors that arise in genetically predisposed persons. The involvement of BRCAl in this phenomenon can now be addressed directly. Alternatively, the lack of somatic BRCAl mutations may result from the existence of multiple genes that function in the same pathway of tumor suppression as BRCAl, but which collectively represent a more favored target for mutation in sporadic tumors. Since mutation of a single element in a genetic pathway is generally sufficient to disrupt the pathway, BRCAl might mutate at a rate that is far lower than the sum of the mutational rates of the other elements.

EXAMPLE 10 Analysis of the BRCAl Gene The structure and function of BRCA 1 gene are determined according to the following methods.

Biological Studies. Mammalian expression vectors containing BRCAl cDNA are constructed and transfected into appropriate breast carcinoma cells with lesions in the gene. Wild-type BRCAl cDNA as well as altered BRCAl cDNA are utilized. The altered BRCAl cDNA can be obtained from altered BRCAl alleles or produced as described below. Phenotypic reversion in cultures (e.g.. cell morphology, doubling time, anchorage-independent growth) and in animals (e.g., tumorigenicity) is examined. The studies will employ both wild-type and mutant forms (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 BRCAl 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 K 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 11 Two Step Assay to Detect the Presence of BRCAl 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 ISO mJ using a GS Gene Linker (Bio-Rad). BRCAl probe corresponding to nucleotide positions 3631-3930 of SEQ ID NO:l 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 mill at 65CC in 7% sodium dodecyl sulfate (SDS) in 0.5 M NaP04. The methods follow those described by Nguyen et al, 1992. The blots are hybridized overnight at 65°C in 7% SDS, 0.5 M NaP04 with 25-50 ng/ml single stranded probe DNA. Post-hybridization washes consist of two 30 min washes in 5% SDS, 40 mM NaP04 at 65°C, followed by two 30 min washes in 1% SDS, 40 mM NaP04 at 65°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 45°C with hybridization buffer consisting of 6 M urea, 0.3 M NaCl, and 5X Denhardt's solution (see Sambrook, et al., 1989). The buffer is removed and replaced with 50-75 (il/cm2 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 45°C as two 10 min washes in 6 M urea, lx standard saline citrate (SSC), 0.1% SDS and one 10 min wash in lx SSC, 0.1% Triton®X-100. The blots are rinsed for 10 min at room temperature with lx SSC.

Blots are incubated for 10 min at room temperature with shaking in the substrate buffer consisting of 0.1 M diethanolamine, 1 mM MgCl2,0.02% sodium azide, pH 10.0. Individual blots are placed in heat sealable bags with substrate buffer and 0.2 mM AMPPD (3<2-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 bards indicate the presence of BRCAl.

EXAMPLE 12 Generation of Polyclonal Antibody against BRCAl Segments of BRCAl coding sequence were expressed as fusion protein in E. coli. The overexpressed protein was 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 BRCAl coding sequence was cloned as a fusion protein in plasmid PET5A (Novagen, Inc., Madison, WI). The BRCAl incorporated sequence includes the amino acids corresponding to #1361-1554 of SEQ ID NO:2. After induction with IPTG, the overexpression of a fusion protein with the expected molecular weight was verified by SDS/PAGE. Fusion protein was purified from the gel by electroelution. The identification of the protein as the BRCAl fusion product was verified by protein sequencing at the N-terminus. Next, the purified protein was used as immunogen in rabbits. Rabbits were immunized with 100 ng 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 jig of immunogen in PBS. Antibody containing serum is collected two weeks thereafter. *-94- This procedure is repeated to generate antibodies against the mutant forms of the BRCAl gene. These antibodies, in conjunction with antibodies to wild type BRCAl, are used to detect the presence and the relative level of the mutant forms in various tissues and biological fluids.

EXAMPLE 13 Generation of Monoclonal Antibodies Specific for BRCAl Monoclonal antibodies are generated according to the following protocol. Mice are immunized with immunogen comprising intact BRCAl or BRCAl 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 jag of immunogen and after the fourth injection blood samples are taken from the mice to determine if the serum contains antibody to the immunogen. Serum titer is determined by ELISA or RIA. Mice with sera indicating the presence of antibody to the immunogen are selected for hybridoma production.

Spleens are removed from immune mice and a single cell suspension is prepared (see Harlow and Lane. 1988). Cell 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 immione spleen cells using polyethylene glycol as described by Harlow and Lane, 1988. Cells are plated at a density of 2x10s cells/well in 96 well tissue culture plates. Individual wells are examined for growth and the supernatants of wells with growth are tested for the presence of BRCAl specific antibodies by ELISA or RIA using wild type or mutant BRCAl target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonality.

Clones with the desired specificities are expanded and grown as ascites in mice or in a hollow fiber system to produce sufficient quantities of antibody for characterization and assay development. -95-EXAMPLE 14 Sandwich Assay for BRCAl 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 p.1 sample (e.g.. serum, urine, tissue cytosol) containing the BRCAl 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 jil of a second monoclonal antibody (to a different determinant on the BRCAl peptide/protein) is added to the solid phase. This antibody is labeled with a detector molecule (e.g., 125I, 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 BRCAl peptide/protein present in the sample, is quantitated. Separate assays are performed using monoclonal antibodies which are specific for the wild-type BRCAl as well as monoclonal antibodies specific for each of the mutations identified in BRCAl.

Industrial Utility As prev iously described above, the present invention provides materials and methods for use in testing BRCAl 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 BRCAl, 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 scrcenii. could also be undertaken at greater frequency. Diagnostic methods based on sequence analysis 01 the BRCAl 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 BRCAl 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 BRCAl 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 invention. Thus, the described embodiments are illustrative and should not be construed as LISTOF RF.FFRRNCF.S Altschul, S.F. etal. (1990). J Mol. Biol. 215: 195-197.

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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 U.S. Patent No. 4,868,105 U.S. Patent No. 5,252,479 EPO Publication No. 225,807 European Patent Application Publication No. 0332435 Geysen, H., PCT published application WO 84/03564, published 13 September 1984 Hitzeman et al., EP 73,675A PCT published application WO 93/07282 PCT/US9S/10203 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Skolnick, Mark H.

Goldgar, David E.

Miki, Yoshio Swenson, Jeff Kamb, Alexander Harshman, Keith D.

Shattuck-Eidens, Donna M.

Tavtigian, Sean V.

Wiseman, Roger W.

Futreal, P. Andrew (ii) TITLE OF INVENTION: 17q-Linked Breast and Ovarian Cimcer Susceptibility Gene (iii) NUMBER OF SEQUENCES: 85 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Venable, Baetjer, Howard & Civiletti, LLP (B) STREET: 1201 New York Avenue, N.W., Suite 1000 (C) CITY: Washington (D) STATE: DC (E) COUNTRY: USA (F) ZIP: 20005 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: 07-JUN-199S (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/409,305 (B) FILING DATE: 24-MAR-1995 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/348,824 (B) FILING DATE: 29-NOV-1994 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/308,104 (B) FILING DATE: 16-SEP-1994 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/300,266 (B) FILING DATE: 02-SEP-1994 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/289,221 (B) FILING DATE: 12-AUG-1994 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Ihnen, Jeffrey L.

(B) REGISTRATION DUMBER: 28,957 (C) REFERENCE/DOCKET NUMBER: 24884-109347 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 202-962-4810 (B) TELEFAX: 202-962-8300 (2) INFORMATION FOR SEQ ID NO:l: (i! I3QUENCE CHARACTERISTICS: (A) LENGTH: 5914 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 120..5711 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AGCTCGCTGA GACTTCCTGG ACCCCGCACC AGGCTGTGGG GTTTCTCAGA TAACTGGGCC 60 CCTGCGCTCA GGAGGCCTTC ACCCTCTGCT CTGGGTAAAG TTCATTGGAA CAGAAAOAA 119 ATG GAT TTA TCT GCT CTT CGC GTT GAA GAA GTA CAA AAT GTC ATT AAT 167 Met Asp Leu Ser Ala Leu Arg Val Glu Glu Val Gin Asn Val lie Asn 1 5 10 15 GCT ATG CAG AAA ATC TTA GAG TGT CCC ATC TGT CTG GAG TTG ATC AAG 215 Ala Met Gin Lvs lie Leu Glu Cys Pro lie Cya Leu Glu Leu lie Lys 20 25 30 GAA CCT GTC TCC ACA AAG TGT GAC CAC ATA TTT TGC AAA TTT TGC ATG 263 Glu Pro Val Ser Thr Lys Cys Asp His lie Phe Cys Lys Phe Cys Met 35 40 45 CTG AAA CTT CTC AAC CAG AAG AAA GGG CCT TCA CAG TGT CCT TTA TGT 311 Leu Lys Leu Leu Asn Gin Lys Lys Gly Pro Ser Gin Cys Pro Leu Cys 50 55 60 AAG AAT GAT ATA ACC AAA AGG AGC CTA CAA GAA AGT ACG AGA TTT AGT 359 Lys Asn Asp lie Thr Lys Arg Ser Leu Gin Glu Ser Thr Arg Phe Ser 65 70 75 80 CAA CTT GTT GAA GAG CTA TTG AAA ATC ATT TGT GCT TTT CAG CTT GAC 407 Gin Leu Val Glu Glu Leu Leu Lys lie lie Cys Ala Phe Gin Leu Asp 85 90 95 ACA GGT TTG GAG TAT GCA AAC AGC TAT AAT TTT GCA AAA AAG GAA AAT 455 Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Phe Ala Lys Lys Glu Asn 100 105 110 AAC TCT CCT GAA CAT CTA AAA GAT GAA GTT TCT ATC ATC CAA AGT ATG 503 Asn Ser Pro Glu His Leu Lys Asp Glu Val Ser lie lie Gin Ser Met 115 120 125 GGC TAC AGA AAC CGT GCC AAA AGA CTT CTA CAG AGT GAA CCC GAA AAT 551 Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gin Ser Glu Pro Glu Asn 130 135 140 CCT TCC TTG CAG GAA ACC AGT CTC AGT GTC CAA CTC TCT AAC CTT GGA 599 Pro Ser Leu Gin Glu Thr Ser Leu Ser Val Gin Leu Ser Asn Leu Gly 145 150 155 160 ACT GTG AGA ACT CTG AGG ACA AAG CAG CGG ATA CAA CCT CAA AAG ACG 647 Thr Val Arg Thr Leu Arg Thr Lys Gin Arg lie Gin Pro Gin Lys Thr 165 170 175 TCT GTC TAC ATT GAA TTG GGA TCT GAT TCT TCT GAA GAT ACC GTT AAT 695 Ser Val Tyr lie Glu Leu Gly Ser Asp Ser Ser Glu Asp Thr Val Asn 180 185 190 AAG GCA ACT TAT TGC AGT GTG GGA GAT CAA GAA TTG TTA CAA ATC ACC 743 Lys Ala Thr Tyr Cys Ser Val Gly Asp Gin Glu Leu Leu Gin He Thr 195 200 205 CCT CAA GGA ACC AGG GAT GAA ATC AGT TTG GAT TCT GCA AAA AAG GCT 791 Pro Gin Gly Thr Arg Asp Glu lie Ser Leu Asp Ser Ala Lys Lys Ala 210 215 220 GCT TGT GAA TTT TCT GAG ACG GAT GTA ACA AAT ACT GAA CAT CAT CAA 839 Ala Cys Glu Phe Ser Glu Thr Asp Val Thr Asn Thr Glu His His Gin 225 230 235 240 CCC AGT AAT AAT GAT TTG AAC ACC ACT GAG AAG CGT GCA GCT GAG AGG 887 Pro Ser Asn Asn Asp Leu Asn Thr Thr Glu Lys Arg Ala Ala Glu Arg 245 250 255 PCIVUS95/10203 CAT CCA GAA AAG TAT CAG GGT AGT TCT GTT TCA AAC TTG CAT GTG GAG 935 His Pro Glu Lys Tyr Gin Gly Ser Ser Val Ser Asn Leu His Val Glu 260 265 270 CCA TGT GGC ACA AAT ACT CAT GCC AGC TCA TTA CAG CAT GAG AAC AGC 983 Pro Cys Gly Thr Asn Thr His Ala Ser Ser Leu Gin His Glu Asn Ser 275 260 285 AGT TTA TTA CTC ACT AAA GAC AGA ATG AAT GTA GAA AAG GCT GAA TTC 1031 Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Val Glu Lys Ala Glu Phe 290 295 300 TGT AAT AAA AGC AAA CAG CCT GGC TTA GCA AGG AGC CAA CAT AAC AGA 1079 Cys Asn Lys Ser Lys Gin Pro Gly Leu Ala Arg Ser Gin His Asn Arg 305 310 315 320 TGG GCT GGA AGT AAG GAA ACA TGT AAT GAT AGG CGG ACT CCC AGC ACA 1127 Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Arg Arg Thr Px'o Ser Thr 325 330 335 GAA AAA AAG GTA GAT CTG AAT GCT GAT CCC CTG TGT GAG AGA AAA GAA 1175 Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Leu Cys Glu Arg Lys Glu 340 345 350 TGG AAT AAG CAG AAA CTG CCA TGC TCA GAG AAT CCT AGA GAT ACT GAA 1223 Trp Asn Lys Gin Lys Leu Pro Cys Ser Glu Asn Pro Arg Asp Thr Glu 355 360 365 GAT GTT CCT TGG ATA ACA CTA AAT AGC AGC ATT CAG AAA GTT AAT GAG 1271 Asp Val Pro Trp lie Thr Leu Asn Ser Ser lie Gin Lys Val Asn Glu 370 375 380 TGG TTT TCC AGA AGT GAT GAA CTG TTA GGT TCT GAT GAC TCA CAT GAT 1319 Trp Phe Ser Arg Ser Asp Glu Leu Leu Gly Ser Asp Asp Ser His Asp 385 390 395 400 GGG GAG TCT GAA TCA AAT GCC AAA GTA GCT GAT GTA TTG GAC GTT CTA 1367 Gly Glu Ser Glu Ser Asn Ala Lys Val Ala Asp Val Leu Asp Val Leu 405 410 415 AAT GAG GTA GAT GAA TAT TCT GGT TCT TCA GAG AAA ATA GAC TTA CTG 1415 Asn Glu Val Asp Glu Tyr Ser Gly Ser Ser Glu Lys lie Asp Leu Leu 420 425 430 GCC AGT GAT CCT CAT GAG G_T TTA ATA TGT AAA AGT GAA AGA GTT CAC 1463 Ala Ser Asp Pro His Glu Ala Leu lie Cys Lys Ser Glu Arg Val His 435 440 445 TCC AAA TCA GTA GAG AGT AAT ATT GAA GAC AAA ATA TTT GGG AAA ACC 1511 Ser Lys Ser Val Glu Ser Asn lie Glu Asp Lys lie Phe Gly Lys Thr 450 455 460 TAT CGG AAG AAG GCA AGC CTC CCC AAC TTA AGC CAT GTA ACT GAA AAT 1SS9 Tyr Arg Lys Lys Ala Ser Leu Pro Asn Leu Ser His Val Thr Glu Asn 465 470 475 480 CTA ATT ATA GGA GCA TTT GTT ACT GAG CCA CAG ATA ATA CAA GAG CGT 1607 Leu lie lie Gly Ala Phe Val Thr Glu Pro Gin lie lie Gin Glu Arg 485 490 495 CCC CTC ACA AAT AAA TTA AAG CGT AAA AQG AGA CCT ACA TCA GGC CTT 1655 Pro Leu Thr Asn Lys Leu Lys Arg Lys Arg Arg Pro Thr Ser Gly Leu 500 505 510 CAT CCT GAG GAT TTT ATC AAG AAA GCA GAT TTG GCA GTT CAA AAG ACT 1703 His Pro Glu Asp Phe lie Lys Lys Ala Asp Leu Ala Val Gin Lys Thr 515 520 525 CCT GAA ATG ATA AAT CAG GGA ACT AAC CAA ACG GAG CAG AAT GGT CAA 1751 Pro CJlu Met lie Asn Gin Gly Thr Asn Gin Thr Glu Gin Asn Gly Gin '530 535 540 GTG ATG AAT ATT ACT AAT AGT GGT CAT GAG AAT AAA ACA AAA GGT GAT 1799 Val Met Asn lie Thr Asn Ser Gly His Glu Asn Lys Thr Lys Gly Asp 545 550 555 560 TCT ATT CAG AAT GAG AAA AAT CCT AAC CCA ATA GAA TCA CTC GAA AAA 1847 Ser lie Gin Asn Glu Lys Asn Pro Asn Pro lie Glu Ser Leu Glu Lys 565 570 575 GAA TCT GCT TTC AAA ACG AAA GCT GAA CCT ATA AGC AGC AGT ATA AGC IB95 Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro lie Ser Ser Ser lie Ser 580 585 590 AAT ATG GAA CTC GAA TTA AAT ATC CAC AAT TCA AAA GCA CCT AAA AAG 1943 Asn Met Glu Leu Glu Leu Asn lie His Asn Ser Lys Ala Pro Lys Lys 595 600 605 AAT AGG CTG AGG AGG AAG TCT TCT ACC AGG CAT ATT CAT GCG CTT GAA 1991 Asn Arg Leu Arg Arg Lys Ser Ser Tbr Arg His lie His Ala Leu Glu 610 615 620 CTA GTA GTC AGT AGA AAT CTA AGC CCA CCT AAT TGT ACT GAA TTG CAA 2039 Leu Val Val Ser Arg Asn Leu Ser Pro Pro Asn Cys Thr Glu Leu Gin 625 630 635 640 ATT GAT AGT TGT TCT AGC AGT GAA GAG ATA AAG AAA AAA AAG TAC AAC 2087 lie Asp Ser Cys Ser Ser Ser Glu Glu lie Lys Lys Lys Lys Tyr Asn 645 650 655 CAA ATG CCA GTC AGG CAC AGC AGA AAC CTA CAA CTC ATG GAA GGT AAA 2135 Gin Met Pro Val Arg His Ser Arg Asn Leu Gin Leu Met Glu Gly Lys 660 665 670 GAA CCT GCA ACT GGA GCC AAG AAG AGT AAC AAG CCA AAT GAA CAG ACA 2If?3 Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Lys Pro Asn Glu Gin Thr 675 680 685 AGT AAA AGA CAT GAC AGC GAT ACT TTC CCA GAG CTG AAG TTA ACA AAT 2231 Ser Lys Arg His Asp Ser Asp Thr Phe Pro Glu Leu Lys Leu Thr Asn 690 695 700 GCA CCT GGT TCT TTT ACT AAG TGT TCA AAT ACC AGT GAA CTT AAA GAA 2279 Ala Pro Gly Ser Phe Thr Lys Cys Ser Asn Thr Ser Glu Leu Lys Glu 705 710 715 720 TTT GTC AAT CCT AGC CTT CCA AGA GAA GAA AAA GAA GAG AAA CTA GAA 2327 Phe Val Asn Pro Ser Leu Pro Arg Glu Glu Lys Glu Glu Lys Leu Glu 725 730 735 ACA GTT AAA GTG TCT AAT AAT GCT GAA GAC CCC AAA GAT CTC ATG TTA 2375 Thr Val Lys Val Ser Asn Asn Ala Glu Asp Pro Lys Asp Leu Met Leu 740 745 750 AGT GGA GAA AGG GTT TTG CAA ACT GAA AGA TCT GTA GAG AGT AGC AGT 2423 Ser Gly Glu Arg Val Leu Gin Thr Glu Arg Ser Val Glu Ser Ser Ser 755 760 765 ATT TCA TTG GTA CCT GGT ACT GAT TAT GGC ACT CAG GAA AGT ATC TCG 2471 lie Ser Leu Val Pro Gly Thr Asp Tyr Gly Thr Gin Glu Ser lie Ser 770 775 780 TTA CTG GAA GTT AGC ACT CTA GGG AAG GCA AAA ACA GAA CCA AAT AAA 2519 Leu Leu Glu Val Ser Thr Leu Gly Lys Ala Lys Thr Glu Pro Asn Lys 785 790 795 800 TGT GTG AGT CAG TGT GCA GCA TTT GAA AAC CCC AAG GGA CTA ATT CAT 2567 Cys Val Ser Gin Cys Ala Ala Phe Glu Asn Pro Lys Gly Leu lie His 805 810 815 GGT TGT TCC AAA GAT AAT AGA AAT GAC ACA GAA GGC TTT AAG TAT CCA 2615 Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Glu Gly Phe Lys Tyr Pro 820 625 830 TTG GGA CAT GAA GTT AAC CAC AGT CGG GAA ACA AGC ATA GAA ATG GAA 2663 Leu Gly His Glu Val Asn His Ser Arg Glu Thr Ser lie Glu Met Glu 835 840 845 GAA AGT GAA CTT GAT GCT CAG TAT TTG CAG AAT ACA TTC AAG GTT TCA 2711 Glu Ser Glu Leu Asp Ala Gin Tyr Leu Gin Asn Thr Phe Lys Val Ser 850 855 860 AAG CGC CAG TCA TTT GCT CCG TTT TCA AAT CCA GGA AAT GCA GAA GAG 2759 Lys Arg Gin Ser Phe Ala Pro Phe Ser Asn Pro Gly Asn Ala Glu Glu 865 870 875 880 GAA TGT GCA ACA TTC TCT GCC CAC TCT GGG TCC TTA AAG AAA CAA AGT 2807 Glu Cys Ala Thr Phe Ser Ala His Ser Gly Ser Leu Lys Lys Gin Ser 885 890 895 CCA AAA GTC ACT TTT GAA TGT GAA CAA AAG GAA GAA AAT CAA GGA AAG 2855 Pro Lys Val Thr Phe Glu Cys Glu Gin Lys Glu Glu Asn Gin Gly Lys 900 905 910 AAT GAG TCT AAT ATC AAG CCT GTA CAG ACA GTT AAT ATC ACT GCA GGC 2903 Asn Glu Ser Asn lie Lys Pro Val Gin Thr Val Asn lie Thr Ala Gly 915 920 925 TTT CCT GTG GTT GGT CAG AAA GAT AAG CCA GTT GAT AAT GCC AAA TGT 2951 Phe Pro Val Val Gly Gin Lys Asp Lys Pro Val Asp Asn Ala Lys Cys 930 935 940 AGT ATC AAA GGA GGC TCT AGG TTT TGT CTA TCA TCT CAG TTC AGA GGC 2999 Ser lie Lys Gly Gly Ser Arg Phe Cys Leu Ser Ser Gin Phe Arg Gly 945 950 955 960 AAC GAA ACT GGA CTC ATT ACT CCA AAT AAA CAT GGA CTT TTA CAA AAC 3047 Asn Glu Thr Gly Leu lie Thr Pro Asn Lys His Gly Leu Leu Gin Asn 965 970 975 CCA TAT CGT ATA CCA CCA CTT TTT CCC ATC AAG TCA TTT GTT AAA ACT 3095 Pro Tyr Arg lie Pro Pro Leu Phe Pro lie Lys Ser Phe Val Lys Thr 980 985 990 AAA TGT AAG AAA AAT CTG CTA GAG GAA AAC TTT GAG GAA CAT TCA ATG 3143 Lys Cys Lys Lys Asn Leu Leu Glu Glu Asn Phe Glu Glu His Ser Met 995 1000 1005 TCA CCT GAA AGA GAA ATG GGA AAT GAG AAC ATT CCA AGT ACA GTG AGC 3191 Ser Pro Glu Arg Glu Met Gly Asn Glu Asn lie Pro Ser Thr Val Ser 1010 1015 1020 ACA ATT AGC CGT AAT AAC ATT AGA GAA AAT GTT TTT AAA GAA GCC AGC 3239 Thr lie Ser Arg Asn Asn lie Arg Glu Asn Val Phe Lys Glu Ala Ser 1025 1030 1035 1040 TCA AGC AAT ATT AAT GAA GTA GGT TCC AGT ACT AAT GAA GTG GGC TCC 3287 Ser Ser Asn lie Asn Glu Val Gly Ser Ser Thr Asn Glu Val Gly Ser 1045 1050 1055 AGT ATT AAT GAA ATA GGT TCC AGT GAT GAA AAC ATT CAA GCA GAA CTA 3335 Ser lie Asn Glu lie Gly Ser Ser Asp Glu Asn lie Gin Ala Glu Leu 1060 1065 1070 GGT AGA AAC AGA GGG CCA AAA TTG AAT GCT ATG CTT AGA TTA GGG GTT 3383 Gly Arg Asn Arg Gly Pro Lys Leu Asn Ala Met Leu Arg Leu Gly Val 1075 1080 1085 TTG CAA CCT GAG GTC TAT AAA CAA AGT CTT CCT GGA AGT AAT TGT AAG 3431 Leu Gin Pro Glu Val Tyr Lys Gin Ser Leu Pro Gly Ser Asn Cys Lys 1090 1095 1100 CAT CCT GAA ATA AAA AAG CAA GAA TAT GAA GAA GTA GTT CAG ACT GTT His Pro Glu lie Lys Lys Gin Glu Tyr Glu Glu Val Val Gin Thr Val 1105 1110 1115 1120 3479 PCT/US9S/10203 AAT ACA GAT TTC TCT CCA TAT CTG ATT TCA GAT AAC TTA GAA CAG CCT 3527 Asn Thr Asp Phe Ser Pro Tyr Leu lie Ser Asp Asn Leu Glu Gin Pro 1125 1130 1135 ATG GGA AGT AGT CAT GCA TCT CAG GTT TGT TCT GAG ACA CCT GAT GAC 3575 Met Gly Ser Ser His Ala Ser Gin Val Cys Ser Glu Thr Pro Asp Asp 1140 1145 1150 CTG TTA GAT GAT GGT GAA ATA AAG GAA GAT ACT AGT TTT GCT GAA AAT 3623 Leu Leu Asp Asp Gly Glu lie Lys Glu Asp Thr Ser Phe Ala Glu Asn 1155 1160 1165 GAC ATT AAG GAA AGT TCT GCT GTT TTT AGC AAA AGC GTC CAG AAA GGA 3671 Asp lie Lys Glu Ser Ser Ala Val Phe Ser Lys Ser Val Gin Lys Gly 1170 1175 1180 GAG CTT AGC AGG AGT CCT AGC CCT TTC ACC CAT ACA CAT TTG GCT CAG 3719 Glu Leu Ser Arg Ser Pro Ser Pro Phe Thr His Thr His Leu Ala Gin 1185 1190 1195 1200 GGT TAC CGA AGA GGG GCC AAG AAA TTA GAG TCC TCA GAA GAG AAC TTA 3767 Gly Tyr Arg Arg Gly Ala Lys Lys Leu Glu Ser Ser Glu Glu Asn Leu 1205 1210 1215 TCT AGT GAG GAT GAA GAG CTT CCC TGC TTC CAA CAC TTG TTA TTT GGT 3815 Ser Ser Glu Asp Glu Glu Leu Pro Cys Phe Gin His Leu Leu Phe Gly 1220 1225 1230 AAA GTA AAC AAT ATA CCT TCT CAG TCT ACT AGG CAT AGC ACC GTT GCT 3863 Lys Val Asn Asn lie Pro Ser Gin Ser Thr Arg His Ser Thr Val Ala 1235 1240 1245 ACC GAG TGT CTG TCT AAG AAC ACA GAG GAG AAT TTA TTA TCA TTG AAG 3911 Thr Glu Cys Leu Ser Lys Asn Thr Glu Glu Asn Leu Leu Ser Leu Lys 1250 1255 1260 AAT AGC TTA AAT GAC TGC AGT AAC CAG GTA ATA TTG GCA AAG GCA TCT 3959 Asn Ser Leu Asn Asp Cys Ser Asn Gin Val lie Leu Ala Lys Ala Ser 1265 1270 1275 1280 CAG GAA CAT CAC CTT AGT GAG GAA ACA AAA TGT TCT GCT AGC TTG TTT 4007 Gin Glu His His Leu Ser Glu Glu Thr Lys Cys Ser Ala Ser Leu Phe 1285 1290 1295 TCT TCA CAG TGC AGT GAA TTG GAA GAC TTG ACT GCA AAT ACA AAC ACC 4055 Ser Ser Gin Cys Ser Glu Leu Glu Asp Leu Thr Ala Asn Thr Asn Thr 1300 1305 1310 CAG GAT CCT TTC TTG ATT GGT TCT TCC AAA CAA ATG AGG CAT CAG TCT 4103 Gin Asp Pro Phe Leu lie Gly Ser Ser Lys Gin Met Arg His Gin Ser 1315 1320 1325 GAA AGC CAG GGA GTT GGT CTG AGT GAC AAG GAA TTG GTT TCA GAT GAT Glu Ser Gin Gly \ 1 Gly Leu Ser Asp Lys Glu Leu Val Ser Asp Asp 1330 1335 1340 4151 GAA GAA AGA GGA ACG GGC TTG GAA GAA AAT AAT CAA GAA GAG CAA AGC Glu Glu Arg Gly Thr Gly Leu Glu Glu Asn Asn Gin Glu Glu Gin Ser 1345 1350 1355 1360 4199 ATG GAT TCA AAC TTA GGT GAA GCA GCA TCT GGG TGT GAG AGT GAA ACA Met Asp Ser Asn Leu Gly Glu Ala Ala Ser Gly Cys Glu Ser Glu Thr 1365 1370 1375 4247 AGC GTC TCT GAA GAC TGC TCA GGG CTA TCC TCT CAG AGT GAC ATT TTA Ser Val Ser Glu Asp Cys Ser Gly Leu Ser Ser Gin Ser Asp lie Leu 1380 1385 1390 4295 ACC ACT CAG CAG AGG GAT ACC ATG CAA CAT AAC CTG ATA AAG CTC CAG Thr Thr Gin Gin Arg Asp Thr Met Gin His Asn Leu lie Lys Leu Gin 1395 1400 1405 4343 CAG GAA ATG GCT GAA CTA GAA GCT GTG TTA GAA CAG . CAT GGG AGC CAG Gin Glu Met Ala Glu Leu Glu Ala Val Leu Glu Gin His Gly Ser Gin 1410 1415 1420 4391 CCT TCT AAC AGC TAC CCT TCC ATC ATA AGT GAC TCT TCT GCC CTT GAG Pro Ser Asn Ser Tyr Pro Ser lie lie Ser Asp Ser Ser Ala Leu Glu 1425 1430 1435 1440 4439 GAC CTG CGA AAT CCA GAA CAA AGC ACA TCA GAA AAA GCA GTA TTA ACT Asp Leu Arg Asn Pro Glu Gin Ser Thr Ser Glu Lys Ala Val Leu Thr 1445 1450 1455 4487 TCA CAG AAA AGT AGT GAA TAC CCT ATA AGC CAG AAT CCA GAA GGC CTT Ser Gin Lys Ser Ser Glu Tyr Pro lie Ser Gin Asn Pro Glu Gly Leu 1460 1465 1470 4535 TCT GCT GAC AAG TTT GAG GTG TCT GCA GAT AGT TCT ACC AGT AAA AAT Ser Ala Asp Lys Phe Glu Val Ser Ala Asp Ser Ser Thr Ser Lys Asn 1475 1480 1485 4583 AAA GAA CCA GGA GTG GAA AGG TCA TCC CCT TCT AAA TGC CCA TCA TTA Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Ser Lys Cys Pro Ser Leu 1490 1495 1500 4631 GAT GAT AGG TGG TAC ATG CAC AGT TGC TCT GGG AGT CTT CAG AAT AGA Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gly Ser Leu Gin Ann Arg 1505 1510 1515 1520 4679 AAC TAC CCA TCT CAA GAG GAG CTC ATT AAG GTT GTT GAT GTG GAG GAG Asn Tyr Pro Ser Gin Glu Glu Leu lie Lys Val Val Asp Val Glu Glu 1525 1530 1535 4727 CAA CAG CTG GAA GAG TCT GGG CCA CAC GAT TTG ACG GAA ACA TCT TAC Gin Gin Leu Glu Glu Ser Gly Pro His Asp Leu Thr Glu Thr Ser Tyr 1540 1545 1550 4775 TTG CCA AGG CAA GAT CTA GAG GGA ACC CCT TAC CTG GAA TCT GGA ATC 4823 Leu Pro Arg Gin Asp Leu Glu Gly Thr Pro Tyr Leu Glu Ser Gly lie 1555 1560 1565 AGC CTC TTC TCT GAT GAC CCT GAA TCT GAT CCT TCT GAA GAC AGA GCC 4871 Ser Leu Phe Ser Asp Asp Pro Glu Ser Asp Pro Ser Glu Asp Arg Ala 1570 1575 1580 CCA GAG TCA GCT CGT GTT GGC AAC ATA CCA TCT TCA ACC TCT GCA TTG 4919 Pro Glu Ser Ala Arg Val Gly Asn lie Pro Ser Ser Thr Ser Ala Leu 1585 1590 1595 1600 AAA GTT CCC CAA TTG AAA GTT GCA GAA TCT GCC CAG AGT CCA GCT GCT 4967 Lys Val Pro Gin Leu Lys Val Ala Glu Ser Ala Gin Sex Pro Ala Ala 1605 1610 1615 GCT CAT ACT ACT GAT ACT GCT GGG TA1 AAT GCA ATG GAA GAA AGT GTG 5015 Ala His Thr Thr Asp Thr Ala Gly Tyr Asn Ala Met Glu Glu Ser Val 1620 1625 1630 AGC AGG GAG AAG CCA GAA TTG ACA GCT TCA ACA GAA AGG GTC AAC AAA 5063 Ser Arg Glu Lys Pro Glu Leu Thr Ala Ser Thr Glu Arg Val Asn Lys 1635 1640 1645 AGA ATG TCC ATG GTG GTG TCT GGC CTG ACC CCA GAA GAA TTT ATG CTC 5111 Arg Met Ser Met Val Val Ser Gly Leu Thr Pro Glu Glu Phe Met Leu 1650 1655 1660 GTG TAC AAG TTT GCC AGA AAA CAC CAC ATC ACT TTA ACT AAT CTA *TT 5159 Val Tyr Lys Phe Ala Arg Lys His His lie Thr Leu Thr Asn Leu lie 1665 1670 1675 1680 ACT GAA GAG ACT ACT CAT GTT GTT ATG AAA ACA GAT GCT GAG TTT GTG 5207 Thr Glu Glu Thr Thr His Val Val Met Lys Thr Asp Ala Glu Phe Val 1685 1690 1695 TGT GAA CGG ACA CTG AAA TAT TTT CTA GGA ATT GCG GGA GGA AAA TGG 5255 Cys Glu Arg Thr Leu Lys Tyr Phe Leu Gly lie Ala Gly Gly Lys Trp 1700 1705 1710 GTA GTT AGC TAT TTC TGG GTG ACC CAG TCT ATT AAA GAA AGA AAA ATG 5303 Val Val Ser Tyr Phe Trp Val Thr Gin Ser lie Lys Glu Arg Lys Met 1715 1720 1725 CTG AAT GAG CAT GAT TTT GAA GTC AGA GGA GAT GTG GTC AAT GGA AGA 5351 Leu Asn Glu His Asp Phe Glu Val Arg Gly Asp Val Val Asn Gly Arg 1730 1735 1740 AAC CAC CAA GGT CCA AAG CGA GCA AGA GAA TCC CAG GAC AGA AAG ATC 5399 Asn His Gin Gly Pro Lys Arg Ala Arg Glu Ser Gin Asp Arg Lys lie 1745 1750 1755 1760 TTC AGG GGG CTA GAA ATC TGT TGC TAT GGG CCC TTC ACC AAC ATG CCC 5447 Phe Arg Gly Leu Glu lie Cys Cys Tyr Gly Pro Phe Thr Asn Met Pro 1765 1770 1775 ACA GAT CAA CTG GAA TGG ATG GTA CAG CTG TGT GGT GCT TCT GTG GTG 5495 Thr Asp Gin Leu Glu Trp Met Val Gin Leu Cys Gly Ala Ser Val Val 1780 1785 1790 AAG GAG CTT TCA TCA TTC ACC CTT GGC ACA GGT GTC CAC CCA ATT GTG 5543 Lys Glu Leu Ser Ser Phe Thr Leu Gly Thr Gly Val His Pro lie Val 1795 1800 1805 GTT GTG CAG CCA GAT GCC TGG ACA GAG GAC AAT GGC TTC CAT GCA ATT 5591 Val Val Gin Pro Asp Ala Trp Thr Glu Asp Asn Gly Phe His Ala lie 1810 1815 1820 GGG CAG ATG TGT GAG GCA CCT GTG GTG ACC CGA GAG TGG GTG TTG GAC 5639 Gly Gin » Cys Glu Ala Pro Val Val Thr Arg Glu Trp Val Leu Asp 1825 1830 1835 1840 AGT GTA GCA CTC TAC CAG TGC CAG GAG CTG GAC ACC TAC CTG ATA CCC 5687 Ser Val Ala Leu Tyr Gin Cys Gin Glu Leu Asp Thr Tyr Leu lie Pro 1845 1850 1855 CAG ATC CCC CAC AGC CAC TAC TGA CTGCAGCCAG CCACAGGTAC AGAGCCACAG 5741 Gin lie Pro His Ser His Tyr * 1860 GACCCCAAGA ATGAGCTTAC AAAGTGGCCT TTCCAGGCCC TGGGAGCTCC TCTCACTCTT 5801 CAGTCCTTCT ACTGTCCTGG CTACTAAATA TTTTATGTAC ATCAGCCTGA AAAGGACTTC 5861 TGGCTATGCA AGGGTCCCTT AAAGATTTTC TGCTTGAAGT CTCCCTTGGA AAT 5914 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1864 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met . —p i. Ala Leu Arg Val Glu Glu Val Gin Asn Val lie Asn 10 15 Ala Met Gin Lys lie Lt31u Cys Pro lie Cys Leu flu Leu lie Lys 20 25 30 Glu co v'al Ser Th.- _ys Cys Asp His lie Phe Cys Lys Phe Cys Met 35 40 45 118- Leu Lys Lau Leu Asn Gin Lya Lys Gly Fro Ser Gin Cys Pro Leu Cy» 50 55 60 Lys Asn Asp lie Thr Lys Arg Ser Leu Gin Glu Ser Thr Arg Phe Ser 65 70 75 60 Gin Leu Val Glu Glu Leu Leu Lys lie lie Cys Ala Phe Gin Leu Asp 65 90 95 Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Phe Ala Lys Lys Olu Asn 100 105 110 Asn Ser Pro Glu His Leu Lys Asp Glu Val Ser lie lie Gin Ser Met 115 120 125 Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gin Ser Glu Pro Olu Asn 130 135 140 Pro Ser Leu Gin Glu Thr Ser Leu Ser Val Gin lieu Ser Asn Leu Gly 145 150 155 160 Thr Val Arg Thr Leu Arg Thr Lys Gin Arg lie Gin Pro Gin Lys Thr 165 170 175 Ser Val Tyr lie Glu Leu Gly Ser Asp Ser Ser Glu Asp Thr Val Asn 180 185 190 Lys Ala Thr Tyr Cys Ser Val Gly Asp Gin Olu Leu Leu Gin lie Thr 195 200 205 Pro Gin Gly Thr Arg Asp Glu lie Ser Leu Asp Ser Ala Lys Lys Ala 210 215 220 Ala Cys Glu Phe Ser Glu Thr Asp Val Thr Aan Thr Glu His His Gin 225 230 235 240 Pro Ser Asn Asn Asp Leu Asn Thr Thr Glu Lys Arg Ala Ala Glu Arg 245 250 255 His Pro Glu Lys Tyr Gin Gly Ser Ser Val Ser Asn Leu His Val Glu 260 265 270 Pro Cys Gly Thr Asn Thr His Ala Ser Ser Leu Gin His Glu Asn Ser 275 280 265 Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Val Glu Lys Ala Olu Phe 290 295 300 Cys Asn Lys Ser Lys Gin Pro Gly Leu Ala Arg Ser Gin His Aan Arg 305 310 315 320 Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Arg Arg Thr Pro Ser Thr 325 330 335 PCT/l)S95/10203 Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Leu Cys Olu Arg Lys Olu 340 345 350 Trp Asn Lys Qln Lys Leu Pro Cys Ser Olu Asn Pro Arg Asp Thr Qlu 355 360 365 Asp Val Pro Trp lie Thr Leu Asn Ser Ser lie Qln Lys Val Asn Olu 370 375 380 Trp Phe Ser Arg Ser Asp Olu Leu Leu Oly Ser Asp Asp ser His Asp 385 390 395 400 Oly Olu Ser Olu Ser Asn Ala Lys Val Ala Asp Val Leu Asp Val Leu 405 410 415 Asn Olu Val Asp Olu Tyr Ser Oly Ser Ser Qlu Lys lie Asp Leu Leu 420 425 430 Ala Ser Asp Pro His Olu Ala Leu lie Cys Lys Ser Glu Arg Val His 435 440 445 Ser Lys Ser Val Olu Ser Asn lie Olu Asp Lys lie Phe Oly Lys Thr 450 455 460 Tyr Arg Lys Lys Ala Ser Leu Pro Asn Leu Ser His Val Thr Glu Asn 465 470 475 480 Leu lie lie Gly Ala Phe Val Thr Olu Pro Oln lie lie Oln Glu Arg 485 490 495 Pro Leu Thr Asn Lys Leu Lys Arg Lys Arg Arg Pro Thr Ser Oly Leu 500 505 510 His Pro Olu Asp Phe lie Lys Lys Ala Asp Leu Ala Val Oln Lys Thr 515 520 525 Pro Olu Met lie Asn Oln Gly Thr Asn Gin Thr Glu Gin Asn Gly Gin 530 535 540 Val Met Asn lie Thr Asn Ser Gly His Glu Aau Lys Thr Lys Gly Asp 545 550 555 560 Ser lie Gin Asn Olu Lys Asn Pro Asn Pro He Olu Ser Leu Olu Lys 565 570 575 Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro lie Ser Ser Ser lie Ser 580 585 590 Asn Met Glu Leu Qlu Leu Asn lie Uia Asn Ser Lys Ala Pro Lys Lys 595 600 605 Asn Arg Leu Arg Arg Lys Ser Ser Thr Arg His He His Ala Leu Olu 610 615 620 WO 96/05307 PCT/US95/10203 Leu Val Val Ser Arg Asn Leu Ser Pro Pro Asn Cys Thr Qlu Leu Qln 625 630 635 640 lie Asp Set Cys Ser Ser Ser Qlu Qlu lie Lys Lys Lys Lys Tyr Asn 645 650 655 Gin Met Pro Val Arg His Ser Arg Asn Leu Gin Leu Met Qlu Qly Lys 660 665 670 Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Lys Pro Asn Qlu Qln Thr 675 680 685 Ser Lys Arg His Asp Ser Asp Thr Phe Pro Glu Leu Lys Leu Thr Asn 690 695 700 Ala Pro Gly Ser Phe Thr Lys Cys Ser Asn Thr Ser Glu Leu Lys Glu 705 710 715 720 Phe Val Asn Pro Ser Leu Pro Arg Glu Glu Lys Glu Glu Lys Leu Glu 725 730 735 Thr Val Lys Val Ser Asn Asn Ala Qlu Asp Pro Lys Asp Leu Met Leu 740 - 745 750 Ser Gly Glu Arg Val Leu Gin Thr Qlu Arg Ser Val Qlu Ser Ser Ser 755 760 765 lie Ser Leu Val Pro Gly Thr Asp Tyr Qly Thr Qln Glu Ser lie Ser 770 775 780 Leu Leu Glu Val Ser Thr Leu Gly Lys Ala Lys Thr Glu Pro Asn Lys 785 790 795 800 Cys Val Ser Gin Cys Ala Ala Phe Glu Asn Pro Lys Gly Leu lie Bis 805 810 815 Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Glu Qly Phe Lys Tyr Pro 820 825 830 Leu Gly His Qlu Val Asn His Ser Arg Glu Thr Ser lie Glu Met Glu 835 840 545 Glu Ser Glu Lew Asp Ala Gin Tyr Leu Gin Asn Thr Phe Lys Val Ser 850 855 860 Lys Arg Gin Ser Phe Ala Pro Phe Ser Asn Pro Gly Asn Ala Qlu Glu 865 870 875 880 Glu Cys Ala Thr Phe Ser Ala His Ser Gly Ser Leu Lys Lys Gin Ser 885 890 895 Pro Lys Val Thr Phe Glu Cys Glu Gin Lys Glu Glu Asn Qln Gly Lys 900 905 910 Asn Olu Ser Asn lie Lys Pro Val Gin Thr Val Asn lie Thr Ala Gly 915 920 925 Phe Pro Val Val Gly Gin Lys Asp Lys Pro Val Asp Asn Ala Lys Cys 930 935 940 Ser lie Lys Gly Gly Ser Arg Phe Cys Leu Ser Ser Qln Phe Arg Qly 945 950 955 960 Asn Glu Thr G?y Leu lie Thr Pro Asn Lys His Gly Leu Leu Gin Pan 965 970 975 Pro Tyr Arg lie Pro Pro Leu Phe Pro He Lys Ser Phe Val Lys Thr 960 985 990 Lys Cys Lys Lys Asn Leu Leu Qlu Glu Asn Phe Glu Glu His Ser Met 995 1000 1005 Ser Pro Glu Arg Glu Met Gly Asn Glu Asn lie Pro Ser Thr Val Ser 1010 1015 1020 Thr lie Ser Arg Asn Asn lie Arg Glu Asn Val Phe Lys Glu Ala Ser 1025 1030 1035 1040 Ser Ser Asn lie Asn Glu Val Gly Ser Ser Thr Asn Qlu Val Qly Ser 1045 1050 1055 Ser lie Asn Glu lie Gly Ser Ser Asp Glu Asn lie Gin Ala Glu Leu 1060 1065 1070 Gly Arg Asn Arg Gly Pro Lys Leu Asn Ala Met Leu Arg Leu Gly Val 1075 1080 1085 Leu Gin Pro Glu Val Tyr Lys Gin Ser Leu Pro Gly Ser Asn Cys Lys 1090 1095 1100 His Pro Glu lie Lys Lys Gin Glu Tyr Glu Glu Val Val Gin Thr Val 1105 1110 1115 1120 Asn Thr Asp Phe Ser Pro Tyr Leu lie Ser Asp Asn Leu Glu Gin Pro 1125 1130 1135 Met Gly Ser Ser His Ala Ser Gin Val Cys Ser Glu Thr Pro Asp Asp 1140 1145 1150 Leu Leu Asp Asp Gly Glu He Lys Glu Asp Thr Ser Phe Ala Glu Asn 1155 1160 1165 Asp He Lys Glu Ser Ser Ala Val Phe Ser Lys Ser Val Gin Lys Gly 1170 1175 1180 Qlu Leu Ser Arg Ser Pro Ser Pro Phe Thy His Thr His Leu Ala Gin 1185 1190 1195 1200 Gly Tyr Arg Arg Gly Ala Lys Lys Leu Glu Ser Ser Glu Glu Asn Leu 1205 1210 1215 Ser Ser Glu Asp Glu Glu Leu Pro Cys Phe Gin His Leu Leu Phe Gly 1220 1225 1230 Lys Val Asn Asn lie Pro Ser Gin Ser Thr Arg His Ser Thr Val Ala 1235 1240 1245 Thr Glu Cys Leu Ser Lys Asn Thr Glu Glu Asn Leu Leu Ser Leu Lys 1250 1255 1260 Asn Ser Leu Asn Asp Cys Ser Asn Gin Val lie Leu Ala Lys Ala Ser 1265 1270 1275 1280 Gin Glu His His Leu Ser Glu Glu Thr Lys Cys Ser Ala Ser ^Jeu Phe 1285 1290 1295 Ser Ser Gin Cys Ser Glu Leu Glu Asp Leu Thr Ala Asn Thr Asn Thr 1300 1305 1310 Gin Asp Pro Phe Leu He Gly Ser Ser Lys Gin Met Arg His Gin Ser 1315 1320 1325 Glu Ser Gin Gly Val Gly Leu Ser Asp Lys Glu Leu Val Ser Asp Asp 1330 1335 1340 Glu Glu Arg Gly Thr Gly Leu Glu Glu Asn Asn Gin Glu Glu Gin Ser 1345 1350 1355 1360 Met Asp Ser Asn Leu Gly Glu Ala Ala Ser Gly Cys Glu Ser Glu Thr 1365 1370 1375 Ser Val Ser Glu Asp Cys Ser Gly Leu Ser Ser Gin Ser Asp He Leu 1380 1385 1390 Thr Thr Gin Gin Arg Asp Thr Met Gin His Asn Leu lie Lys Leu Gin 1395 1400 1405 Gin Glu Met Ala Glu Leu Glu Ala Val Leu Glu Gin His Gly Ser Gin 1410 1415 1420 Pro Ser Asn Ser Tyr Pro Ser lie lie Ser Asp Ser Ser Ala Leu Glu 1425 1430 1435 1440 Asp Leu Arg Asn Pro Glu Gin Ser Thr Ser Glu Lys Ala Val Leu Thr 1445 1450 1455 Ser Gin Lys Ser Ser Glu Tyr Pro lie Ser Gin Asn Pro Glu Gly Leu 1460 1465 1470 Ser Ala Asp Lys Phe Glu Val Ser Ala Asp Ser Ser Thr Ser Lys Asn 1475 1480 1485 Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Ser Lys Cys Pro Ser Leu 1490 1495 1500 Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gly Ser Leu Gin Asn Arg 1505 1510 1515 . 1520 Asn Tyr Pro Ser Gin Glu Glu Leu lie Lys Val Val Asp Val Glu Glu 1525 1530 1535 Gin Gin Leu Glu Glu Ser Gly Pro His Asp Leu Thr Glu Thr Ser Tyr 1540 1545 1550 Leu Pro Arg Gin Asp Leu Glu Gly Thr Pro Tyr Leu Glu Ser Gly lie 1555 1560 1565 Ser Leu Phe Ser Asp Asp Pro Glu Ser Asp Pro Ser Glu Asp Arg Ala 1570 1575 1580 \ Pro Glu Ser Ala Arg Val Gly Asn lie Pro Ser Ser Thr Ser Ala Leu 1585 1590 1595 1600 Lys Val Pro Gin Leu Lys Val Ala Glu Ser Ala Gin Ser Pro Ala Ala 1605 1610 16ic Ala His Thr Thr Asp Thr Ala Qly Tyr Asn Ala Met Glu Glu- Ser Val 1620 1625 1630 Ser Arg Glu Lys Pro Glu Leu Thr Ala Ser Thr Glu Arg Val Asn Lys 1635 1640 1645 Arg Met Ser Met Val Val Ser Gly Leu Thr Pro Glu Glu Phe Met Leu 1650 1655 1660 Val Tyr Lys Phe Ala Arg Lys His His He Thr Leu Thr Asn Leu lie 1665 1670 1675 1680 Thr Glu Glu Thr Thr His Val Val Met Lys Thr Asp Ala Glu Phe $al 1685 1690 1695 Cys Glu Arg Thr Leu Lys Tyr Phe Leu Gly lie Ala Gly Gly Lys Trp 1700 1705 1710 Val Val Ser Tyr Phe Trp Val Thr Gin Ser lie Lys Glu Arg Lys Met 1715 1720 1725 Leu Asn Glu His Asp Phe Glu Val Arg Gly Asp Val Val Asn Gly Arg 1730 1735 1740 Asn His Gin Gly Pro Lys Arg Ala Arg Glu Ser Gin Asp Arg Lys lie 1745 1750 1755 1760 Phe Arg Gly Leu Qlu lie Cys Cys Tyr Gly Pro Phe Thr Asn Met Pro 1765 1770 1775 Thr Asp Qln Leu Glu Trp 1780 Met Val Gin Leu 1785 Cys Gly Ala Ser Val Val 1790 Lys Glu Leu Ser 1795 Ser Phe Thr Leu Gly Thr 1800 Gly Val His Pro lie Val 1805 Val Val i 1810 Gin Pro Asp Ala Trp Thr Glu Asp I 1815 Asn Gly Phe His Ala lie 1820 Gly Gin Met Cys 1825 Glu Ala Pro Val Val Thr Arg Glu Trp Val Leu Asp 1830 1835 1840 Ser Val Ala Leu Tyr Gin Cys Gin Glu 1845 Leu ; 1850 Asp Tht Tyr Leu lie Pro l 1855 Gin lie Pro His Ser His Tyr * 1860 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Bingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: S754 A (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CTAGCCTGGQ CAACAAACGA 20 (2) INFORMATION FOR SEQ ID N0:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: s754 B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: QCAGGAAQCA GGAATGGAAC (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: s975 A (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TAGGAGATGG ATTATTGGTG (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: S975 B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGGCAACTTT GCAATGAGTG (2) INFORMATION FOR SEQ ID NO:"7: (i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: tdj1474 A (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CAGAGTGAGA CCTTGTCTCA AA (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: B) CLONE: tdj1474 B (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTCTGCAAAC ACCTTAAACT CAG (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS; (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: tdj1239 A (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AACCTGGAAG GCAGAGGTTG (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL! NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: tdjl239 B (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:10 TCTGTACCTG CTAAGCAGTG G (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: ill base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 2..Ill (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: G GKC TTA CTC TOT TGT CCC AGC TGG AGT ACA GWG TGC GAT CAT GAG 46 Xaa Leu Leu Cys Cys Pro Ser Trp Ser Thr Xaa Cys Asp His Glu 1865 1870 1875 GCT TAC TGT TGC TTG ACT CCT AGG CTC AAG CGA TCC TAT CAC CTC AGT 94 Ala Tyr Cys Cys Leu Thr Pro Arg Leu Lys Arg Ser Tyr His Leu Ser 1880 1885 1890 1895 CTC CAA GTA GCT GGA CT 111 Leu Gin Val Ala Gly 1900 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPEs protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Xaa Leu Leu Cys Cys Pro Ser Trp Ser Thr Xaa Cys Asp His Glu Ala 15 10 15 Tyr Cys Cys Leu Thr Pro Arg Leu Lys Arg Ser Tyr His Leu Ser Leu 20 25 30 Gin Val Ala Gly 35 (2) INFORMATION FOR SEQ ID NO: 13: (i) SE(. iJNCE CHARACTERISTICS: (A) LENGTH: 1534 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear PCTAJS95/10203 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GAGGCTAGAG GGCAGGCACT TTATGGCAAA CTCAGGTAGA ATTCTTCCTC TTC03TCTCT 60 TTCCTTTTAC GTCATCGGGG AGACTGGGTG GCAATCGCAG CCCGAGAGAC GCATG1GCTCT 120 TTCTGCCCTC CATCCTCTQA TGTACCTTGA TTTCGTATTC TGAGAGGCTG CTGCTTAGCG 180 GTAGCCCCTT GGTTTCCGTG QCAACQGAAA AGCGCGGGAA TTACAGATAA ATTAAAACTG 240 CGACTGCGCG GCGTGAGCTC GCTGAGACTT CCTGGACCCC GCACCAGGCT GTGGGGTTTC 300 TCAGATAACT GGGCCCCTGC GCTCAGGAGG CCTTCACCCT CTGCTCTGGG TAAAGGl'AGT 360 AGAGTCCCGG GAAAGGGACA GGGGGCCCAA GTGATGCTCT GGGGTACTGG CGTGGGAGAG 420 TGGATTTCCG AAGCTGACAG ATGGGTATTC TTTGACGGGG GGTAGGGGCG GAACCTGAGA 480 GGCGTAAGGC GTTGTGAACC CTGGGGAGGG GGGCAGTTTG TAGGTCGCGA GGGAAGCGCT 540 GAGGATCAGG AAGGGGGCAC TGAGTGTCCG TQGQGGAATC CTCGTGATAG GAACTGGAAT bOO ATGCCTTGAG GGGGACACTA TGTCTTTAAA AACGTCGGCT GGTCATGAGG TCAGGAGTTC 660 CAGACCAGCC TGACCAACGT GGTGAAACTC CGTCTCTACT AAAAATACNA AAATTAGCCG 720 GGCGTGGTGC CGCTCCAGCT ACTCAGGAQG CTGAGGCAGG AGAATCGCTA GAACCCGGGA 780 GGCGGAGGTT GCAGTGAGCC GAGATCGCGC CATTGCACTC CAGCCTGGGC GACAGAGCGA 840 GACTGTCTCA AAACAAAACA AAACAAAACA AAACAAAAAA CACCGGCTGG TATGTATGAG 900 AGGATGGGAC CTTGTGGAAG AAQAGGTQCC AGGAATATQT CTGGGAAGGG GAGGAGACAQ 960 GATTTTGTGG GAGGGAGAAC TTAAGAACTG GATCCATTTG CGCCATTGAG AAAGCGCAAG 1020 AGGGAAGTAG AGGAGCGTCA GTAGTAACAG ATGCTGCCGG CAGGGATGTG CTTGAGGAGG 1080 ATCCAGAGAT GAGAGCAGQT CACTGGGAAA GGTTAGGGGC GGGGAGGCCT TGATTGGTGT 1140 TGGTTTGGTC GTTGTTGATT TTGQTTTTAT QCAAGAAAAA GAAAACAACC AGAAACATTG 1200 GAGAAAGCTA AGGCTACCAC CACCTACCCG GTCAGTCACT CCTCTGTAGC TTTCTCTTTC 1260 TTGGAGAAAG GAAAAGACCC AAGGGGTTGG CAGCGATATG TGAAAAAATT CAGAATTTAT 1320 GTTGTCTAAT TACAAAAAGC AAC'* TCTAGA ATCTTTAAAA ATAAAGGACG TTGTCATTAG 1380 TTCTTCTGGT TTGTATTATT CTAAAACCTT CCAAATCTTC AAATTTACTT TATTTTAAAA 1440 TGATAAAATG AAGTTGTCAT TTTATAAACC TTTTAAAAAG ATATATATAT ATGTTTTTCT 1500 AATGTGTTAA AGTTCATTGG AACAGAAAGA AATG 1534 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1924 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SBQ ID NO:14: GAGGCTAGAG GGCAGGCACT TTATGGCAAA CTCAGGTAGA ATTCTTCCTC TTCCGTCTCT 60 TTCCTTTTAC GTCATCGGGG AGACTGGGTG GCAATCGCAG CCCGAGAGAC GCATGGCTCT 120 TTCTGCCCTC CATCCTCTGA TGTACCTTGA TTTCGTATTC TGAGAGGCTG CTGCTTAGCG 180 GTAGCCCCTT GGTTTCCGTG GCAACGGAAA AGCGCGGGAA TTACAGATAA ATTAAAACTG 240 CGACTGCGCG GCGTGAGCTC GCTGAGACTT CCTGGACCCC GCACCAGGCT GTGGGGTTTC 300 TCAGATAACT GGGCCCCTGC GCTCAGGAGG CCTTCACCCT CTGCTCTGGG TAAAGGTAGT 360 AGAGTCCCGG GAAAGGGACA GGGGGCCCAA GTGATGCTCT GGGGTACTGG CGTGGGAGAG 420 TGGATTTCCG AAGCTGACAG ATGGGTATTC TTTGACGGGG GGTAGGGGCG GAACCTGAGA 480 GGCGTAAGGC GTTGTGAACC CTGGGGAGGG GGGCAGTTTG TAGGTCGCGA GGGAAGCGCT 540 GAGGATCAGG AAGGGGGCAC TGAGTGTCCG TGGGQGAATC CTCGTGATAG GAACTGGAAT 600 ATGCCTTGAG GGGGACACTA TGTCTTTAAA AACGTCGGCT GGTCATGAGG TCAGGAGTTC 660 CAGACCAGCC TGACCAACGT GGTGAAACTC CGTCTCTACT AAAAATACNA AAATTAGCCG 720 GGCQTGGTGC CGCTCCAGCT ACTCAGGAGG CTGAGGCAGG AGAATCGCTA GAACCCGGGA 780 GGCGGAGGTT GCAGTGAGCC GAGATCGCGC CATTGCACTC CAGCCTGGGC GACAGAGCGA 840 GACTGTCTCA AAACAAAACA AAACAAAACA AAACAAAAAA CACCGGCTGG TATGTATGAG 900 AGGATGGGAC CTTGTGGAAG AAGAGGTGCC AGGAATATGT CTGGGAAGGG GAGGAGACAG 960 GATTTTGTGG GAGGGAGAAC TTAAGAACTG GATCCATTTG CGCCATTGAG AAAGCGCAAG 1020 AGGGAAGTAG AGGAGCGTCA GTAGTAACAG ATGCTGCCGG CAQGGATGTG CTTGAGGAGG 1080 ATCCAGAGAT GAGAGCAGGT CACTGGGAAA GGTTAGGGGC GGGGAGGCCT TGATTGGTGT 1140 TGGTTTGGTC GTTGTTGATT TTGGTTTTAT GCAAGAAAAA GAAAACAACC AGAAACATTG 1200 GAGAAAGCTA AGGCTACCAC CACCTACCCG GTCAGTCACT CCTCTGTAGC TTTCTCTTTC 1260 TTGGAGAAAG GAAAAGACCC AAGGGGTTGG CAGCGATATG TGAAAAAATT CAGAATTTAT 1320 GTTGTCTAAT TACAAAAAGC AACTTCTAGA ATCTTTAAAA ATAAAGGACG TTGTCATTAG 1380 TTCTTCTGGT TTGTATTATT CTAAAACCTT CCAAATCTTC AAATTTACTT TATTTTAAAA 1440 TGATAAAATG AAGTTGTCAT TTTATAAACC TTTTAAAAAG ATATATATAT ATGTTTTTCT 1500 AATGTGTTAA AGTTCATTGG AACAGAAAGA AATGGATTTA TCTGCTCTTC GCGTTGAAGA 1560 AGTACAAAAT GTCATTAATG CTATGCAGAA AATCTTAGAG TGTCCCATCT GGTAAGTCAG 1620 CACAAGAGTG TATTAATTTG GGATTCCTAT GATTATCTCC TATGCAAATG AACAGAATTG 1680 ACCTTACATA CTAGGGAAGA AAAGACATGT CTAGTAAGAT TAGGCTATTG TAATTGCTGA 1740 TTTTCTTAAC TGAAGAACTT TAAAAATATA GAAAATGATT CCTTGTTCTC CATCCACTCT 1800 GCCTCTCCCA CTCCTCTCCT TTTCAACACA ATCCTGTGGT CCGGGAAAGA CAGGGCTCTG 1860 TCTTGATTGG TTCTGCACTG GGCAGGATCT GTTAGATACT GCATTTGCTT TCTCCAGCTC 1920 TAAA 1924 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 631 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: AAATGCTGAT GATAGTATAG AGTATTGAAG GGATCAATAT AATTCTGTTT T3ATATCTGA 60 AAGCTCACTG AAGGTAAGGA TCGTATTCTC TGCTGTATTC TCAGTTCCTG ACACAGCAGA 120 CATTTAATAA ATATTGAACG AACTTGAGGC CTTATGTTQA CTCAGTCATA ACAGCTCAAA 180 GTTGAACTTA TTCACTAAGA ATAGCTTTAT TTTTAAATAA ATTATTGAGC CTCATTTATT 240 TTCTTTTTCT CCCCCCCCTA CCCTGCTAGT CTGGAGTTGA TCAAGGAACC TGTCTCCACA 300 AAGTGTGACC ACATATTTTG CAAGTAAGTT TGAATGTGTT ATGTGGCTCC ATTATTAGCT 360 TTTGTTTTTG TCCTTCATAA CCCAGGAAAC ACCTAACTTT ATAGAAfiCTT TACTTTCTTC 420 AATTAAGTGA GAACGAAAAT CCAACTCCAT TTCATTCTTT CTCAGAGAGT ATATAGTTAT 480 CAAAAGTTGG TTGTAATCAT AGTTCCTGGT AAAGTTTTGA CATATATTAT CTTTTTTTTT 540 TTTTGAGACA AGTCTCGCTC TGTCGCCCAG GCTGGAGTGC AGTGGCATGA GGCTTGCTCA 600 CTGCACCTCC GCCCCCGAGT TCAGCGACTC T 631 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 481 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TGAGATCTAG ACCACATGGT CAAAGAGATA GAATGTGAGC AATAAATGAA CCTTAAATTT 60 TTCAACAGCT ACTTTTTTTT TTTTTTTTTG AGACAGGGKC TTACTCTGTT GTCCCAGCTG 120 PCT/US9S/1Q203 GAGTACAGWG TGCGATCATG AGGCTTACTG TTGCTTGACT CCTAQGCTCA AGCGATCCTA 180 TCACCTCAGT CTCCAAGTAG CTGGACTGTA AGTOCACACC ACCATATCCA GCTAAATTTT 240 GTGTTTTCTG TAGAGACGGG GTTTCGCCAT GTTTCCCAGG CTGGTCTTGA ACTTTGGGCT 300 TAACCCGTCT GCCCACCTAG OCATCCCAAA GTGCTAGGAT TACAGGTGTG AGTCATCATG 360 CCTGGCCAGT ATTTTAGTTA GCTCTGTCTT TTCAAGTCAT ATACAAGTTC ATTTTCTTTT 420 AAGTTTAGTT AACAACCTTA TATCATGTAT TCTTTTCTAG CATAAAGAAA GATTCGAGGC 480 C 481 (2) INFORMATION FOR SEQ ID NO:17; (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 522 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double <D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TGTGATCATA ACAGTAAGCC ATATGCATGT AAGTTCAGTT TTCATAGATC ATTGCTTATG 60 TAGTTTAGGT TTTTGCTTAT GCAGCATCCA AAAACAATTA GGAAACTATT GCTTGTAATT 120 CACCTGCCAT TACTTTTTAA ATGGCTCTTA AGGGCAGTTG TGAGATTATC TTTTCATGGC 180 TATTTGCCTT TTGAGTATTC TTTCTACAAA AGGAAGTAAA TTAAATTGTT CTTTCTTTCT 240 TTATAATTTA TAGATTTTGC ATGCTGAAAC TTCTCAACCA GAAGAAAGGG CCTTCACAGT 300 GTCCTTTATG TAAGAATGAT ATAACCAAAA GGTATATAAT TTGGTAATGA TGCTAGGTTG 360 GAAQCAACCA CAGTAGGAAA AAGTAGAAAT TATTTAATAA CATAGCGTTC CTATAAAACC 420 ATTCATCAGA AAAATTTATA AAAGAGTTTT TAGCACACAG TAAATTATTT CCAAAGTTAT 480 TTTCCTGAAA GTTTTATGGG CATCTGCCTT ATACAGGTAT TG 522 (2) INFORMATION FOR SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 465 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:IB: GGTAGGCTTA AATGAATGAC AAAAAGTTAC TAAATCACTG CCATCACACG GTTTATACAG 60 ATGTCAATGA TGTATTGATT ATAGAGGTTT TCTACTGTTG CTGCATCTTA TTTTTATTTG 120 TTTACATGTC TTTTCTTATT TTAGTGTCCT TAAAAGGTTG ATAATCACTT GCTGAGTGTG 180 TTTCTCAAAC AATTTAATTT CAGGAGCCTA CAAGAAAGTA CGAGATTTAG TCAACTTGTT 240 GAAGAGCTAT TGAAAATCAT TTGTGCTTTT CAGCTTOACA CAGGTTTGGA GTGTAAGTGT 300 TOAATATCCC AAQAATGACA CTCAAGTGCT GTCCATOAAA ACTCAGGAAG TTTGCACAAT 360 TACTTTCTAT GACGTGGTGA TAAQACCTTT TAGTCTAGGT TAATTTTA3T TCTGTATCTG 420 TAATCTATTT TAAAAAATTA CTCCCACTOO TCTCACACCT TATTT 45P (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 513 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO]19t AAAAAATCAC AGGTAACCTT AATGCATTOT CTTAACACAA CAAAGAGCAT ACATAGGKaTT 60 TCTCTTGGTT TCTTTOATTA TAATTCATAC ATTTTTCTCT AACTGCAAAC ATAATOTTTT 120 CCCTTGTATT TTACAGATGC AAACAOCTAT AATTTTGCAA AAAAGGAAAA TAACTCTCCT ISO OAACATCTAA AAQATOAAGT TTCTATCATC CAAAGTATGG GCTACAGAAA CCQTGCCAAA 240 AGACTTCTAC AQAQTQAACC COAAAATCCT TCCTTOGTAA AACCATTTQT TTTCTTCTTC 300 TTCTTCTTCT TCTTTTCTTT TTTTTTTCTT TTTTTTTTTO AGATGGAQTC TTGCTCTGTG 360 aCCCAOOCTA OAAQCAOTCC TCCTQCCTTA OCCNCCTTAQ TAGCTGQGAT TACAOOCACQ 420 CQCACCATQC CAQQCTAATT TTTQTATTTT TAGTAQAQAC GGGGTTTCAT CATGTTGGCC 480 AGGCTGGTCT CGAACTCCTA ACCTCAGGTG ATC 513 (2) INFORMATION FOR SBQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6769 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: ATGATGGAGA TCTTAAAAAG TAATCATTCT GGGGCTGGGC GTAGTAGCTT GCACCTGTAA 60 TCCCAGCACT TCGGGAGGCT GAGGCAGGCA GATAATTTGA GGTCAGOAGT TTGAGACCAO 120 CCTGGCCAAC ATGGTGAAAC CCATCTCTAC TAAAAATACA AAAATTAGCT GGGTGTGGTG 180 GCACGTACCT GTAATCCCAG CTACTCGGGA GGCGGAGGCA CAAGAATTGC TTGAACCTAG 240 GACGCGGAGG TTGCAGCGAG CCAAOATCGC GCCACTGCAC TCCAGCCTGG GCCGTAGAGT 300 GAOACTCTGT CTCAAAAAAG AAAAAAAAGT AATTOTTCTA GCTGGGCGCA GTOGCTCTTG 360 CCTOTAATCC CAGCACTTTG GGAGGCCAAG GCGGGTGGAT CTCGAGTCCT AGAGTTCAAG 420 ACCAGCCTAG GCAATGTGGT QAAACCCCAT COCTACAAAA AATACAAAAA TTAGCCAGGC 480 ATGGTGQCGT GCGCATGTAG TCCCAQCTCC TTGGOAGGCT GAGGTGGGAG GATCACTTGA 540 ACCCAGGAGA CAGAGGTTGC AGTGAACCGA GATCACGCCA CCACGCTCCA GCCTGGGCAA 600 CAGAACAAGA CTCTGTCTAA AAAAATACAA ATAAAATAAA AOTAGTTCTC ACAGTACCAG 660 CATTCATTTT TCAAAAGATA TAGAGCTAAA AA9GAAGGAA AAAAAAAGTA ATGTTGGGCT 720 TTTAAATACT CGTTCCTATA CTAAATGTTC TTAGGAGTGC TGGGGTTTTA TTGTCATCAT 780 TTATCCTTTT TAAAAATGTT ATTGGCCAGG CACaGTGGCT CATOGCTOTA ATCCCAGCAC 840 TTTGGGAGGC CGAGGCAGGC AGATCACCTG AGGTCAGGAG TGTGAGACCA GCCTGGCCAA 900 CATGGCGAAA CCTGTCTCTA CTAAAAATAC AAAAATTAAC TAGGCGTGGT GGTGTACGCC 960 TGTAGTCCCA GCTACTCGGG AGGCTGAGGC AGGAGAATCA ACTGAACCAG GGAGGTGOAG 1020 GTTGCAGTGT GCCGAGATCA CGCCACTGCA CTCTAGCCTG GCAACAGAGC AAGATTCTGT 1080 CTCAAAAAAA AAAAACATAT ATACACATAT ATCCCAAAOT GCTGGGATTA CATATATATA 1140 TATATATATA TATTATATAT ATATATATAT ATATATGTGA TATATATGTG ATATATATAT 1200 AACATATATA TATGTAATAT ATATGTQATA TATATATAAT ATATATATGT AATATATATG 1260 TGATATATAT ATATACACAC ACACACACAT ATATATGTAT GTGTGTOTAC ACACACACAC 1320 ACA'ATTAGC CAGGCATAGT TGCACACGCT TGGTAGACCC AGCTACTCAG GAGGCTGAGG 1380 GAGGAGAATC TCTTGAACTT AGGAGGCGGA GGTTGCAGTG AGCTGAGATT GCGCCACTGC 1440 ACTCCA3CCT GGGTGACAGA GCAGGACTCT GTACACCCCC CAAAACAAAA AAAAAAGTTA 1500 TCAGATGTGA TTGGAATGTA TATCAAGTAT CAGCTTCAAA ATATGCTATA TTAATACTTC 1560 AAAAATTACA CAAATAATAC ATAATCAGGT TTGAAAAATT TAAGACAACM SAARAAAAAA 1620 WYCMAATCAC AMATATCCCA CACATTTTAT TATTMCTMCT MCWATTATTT TGVfAGAGMCT 1680 GGGTCTCACY CYKTTGCTWA TGCTGGTCTT TOAACYCCYK GCCYCAARCA RTCCTSCTCC 1740 ABCCTCCCAA RGTGCTGGGG ATWATAGGCA TGARCTAACC GCACCCAGCC CCAGACATTT 1800 TAGTGTGTAA ATTCCTGGGC ATTTTTTCAA GGCATCATAC ATGTTAGCTG ACTGATGATG 1860 GTCAATTTAT TTTGTCCATG GTGTCAAGTT TCTCTTCAGG AGGAAAAGCA CAGAACTGGC 1920 CAACAATTGC TTGACTGTTC TTTACCATAC TGTTTAGCAG GAAACCAGTC TCAGTGTCCA 1980 ACTCTCTAAC CTTGGAACTG TGAGAACTCT OAGGACAAAG CAGCGGATAC AACCTCAAAA 2040 * GACQTCTGTC TACATTGAAT TGGQTAAGGG TCTCAGGTTT TTTAAGTATT TAATAATAAT 2100 TGCTGGATTC CTTATCTTAT AGTTTTGCCA AAAATCTTGG TCATAATTTG TATTTGTGGT 2160 AGQCAGCTTT GQGAAGTGAA TTTTATGAGC CCTATQGTGA GTTATAAAAA ATGTAAAAGA 2220 CGCAGTTCCC ACCTTGAAGA ATCTTACTTT AAAAAGOGAG CAAAAGAGGC CAGGCATGGT 2280 GGCTCACACC TGTAATCCCA GCACTTTGGG AGGCCAAAGT GGGTGGATCA CCTGAGGTCG 2340 GGAQTTCGAG ACCAGCCTAG CCAACATGGA GAAACTCTGT CTGTACCAAA AAATAAAAAA 2400 TTAGCCAGGT GTGGTGGCAC ATAACTGTAA TCCCAGCTAC TCGGGAGGCT GAGGCAGGAG 2460 AATCACTTGA ACCCGGGAGG TGGA3GTTGC GGTGAACCGA GATCGCACCA TTGCACTCCA 2520 GCCTGGGCAA AAATAGCGAA ACTCCATCTA AAAAAAAAAA AGAQAGCAAA AGAAAGAMTM 2580 TCTGGTTTTA AMTMTGTGTA AATATGTTTT TGGAAAGATG GAGAGTAGCA ATAAOAAAAA 2640 ACATGATGGA TTGCTACAGT ATTTAGTTCC AAGATAAATT OTACTAGATG AGGAAGCCTT 2700 TTAAQAAGAG CTGAATTGCC AGGCGCAGTG GCTCACGCCT 3TAATCCCAG CACTTTGGGA 2760 GGCCGAGGTG GGCGC3ATCAC CTGAGGTCGG GAGTTCAAQA CCAGCCTGAC CAACATGGAG 2820 AAACCCCATC TCTACTAAAA AAAAAAAAAA AAAAATTAGC CGGGGTGGTG GCTTATGCCT 2880 GTAATCCCAG CTACTCAGGA GGCTGAGGCA GGAGAATCGC TTGAACCCAG GAAGCAQAGG 2940 TTGCAGTGAG CCAAGATCGC ACCATTGCAC TCCAGCCTAG GCAACAAGAG TGAAACTCCA 3000 TCTCAAAAAA AAAAAAAAAG AGCTGAATCT TGGCTGGGCA GGATGGCTCG TGCCTGTAAT 3060 CCTAACGCTT TGGAAGACCG AGGCAGAAGG ATTGGTTGAO TCCACGAGTT TAAGACCAGC 3120 CTGGCCAACA TAGGGGAACC CTGTCTCTAT TTTTAAAATA ATAATACATT TTTGGCCGGT 3180 GCGGTGGCTC ATGCCTGTAA TCCCAATACT TTGGOAGGCT GAGGCAGGTA GATCACCTGA 3240 GGTCAQAGTT CGAGACCAGC CTGOATAACC TGGTGAAACC CCTCTTTACT AAAAATACAA 3300 AAAAAAAAAA AAATTAGCTG GGTGTGGTAG CACATGCTTG TAATCCCAGC TACTTGGGAG 3360 GCTGAGGCAG GAGAATCGCT TGAACCAGGG AGGCGGAGGT TACAATGAGC CAACACTACA 3420 CCACTQCACT CCAGCCTGGG CAATAGAGTG AGACTGCATC TCAAAAAAAT AATAATTTTT 3480 AAAAATAATA AATTTTTTTA AGCTTATAAA AAGA7AAGTT GAGGCCAGCA TAGTAGCTCA 3540 CATCTGTAAT CTCAGCAGTG GCAGAGGATT GCTTG>AGCC AGGAGTTTGA GACCAGCCTG 3600 GGCAACATAG CAAGACCTCA TCTCTACAAA AAAATiTCTT TTTTAAATTA GCTGGGTGTG 3660 GTGGTGTGCA TCTGTAGTCC CAGCTACTCA GGAOGCAGAG GTGAGTGGAT ACATTGAACC 3720 CAGGAGTTTG AGGCTGTAGT GAGCTATGAT CATGCCACTG CACTCCAACC TG3QTGACAG 3780 AGCAAGACCT CCAAAAAAAA AAAAAAAAGA GCTGCTGAGC TCAGAATTCA AACTGGGCTC 3840 TCAAATTGGA TTTTCTTTTA GAATATATTT ATAATTAAAA AGGATAGCCA TCTTTTGAGC 3900 TCCCAGGCAC CACCATCTAT TTATCATAAC ACTTACTGTT TTCCCCCCTT ATGATCATAA 3960 ATTCCTAGAC AACAGGCATT GTAAAAATAG TTATAGTAGT TGATATTTAG GAGCACTTAA 4020 CTATATTCCA GGCACTATTG TGCTTTTCTT GTATAACTCA TTAGATGCTT GTCAGACCTC 4080 TGAGATTGTT CCTATTATAC TTATTTTACA GATGAGAAAA TTAAGGCACA GAGAAGTTAT 4140 GAAATTTTTC CAAGGTATTA AACCTAGTAA GTGGCTGAGC CATGATTCAA ACCTAGGAAG 4200 TTAGATGi-CA GAGCCTGTGC TTTTTTTTTG TTTTTGTTTT TGTTTTCAGT AGAAACGGGG 4260 GTCTCACTTT GTTGGCCAGG CTGGTCTTGA ACTCCTAACC TCAAATAATC CACCCATCTC 4320 GGCCTCCTCA AGTGCTGGGA TTACAGGTGA GAGCCACTGT GCCTGGCGAA GCCCATGCCT 4380 TTAACCACTT CTCTGTATTA CATACTAOCT TAACTAGCAT TGTACCTGCC ACAGTAGATG 4440 CTCAGTAAAT ATTTCTAGTT GAATATCTGT TTTTCAACAA GTACATTTTT TTAACCCTTT 4500 TAATTAAGAA AACTTTTATT GATTTATTTT TTGGGGGGAA ATTTTTTAGG ATCTGATTCT 4560 TCTGAAGATA CCGTTAATAA GGCAACTTAT TGCAGGTGAG TCAAAGAGAA CCTTTGTCTA 4620 TGAAGCTGGT ATTTTCCTAT TTAGTTAATA TTAAGGATTG ATGTTTCTCT CTTTTTAAAA 4680 ATATTTTAAC TTTTATTTTA GGTTCAGGGA TGTATGTGCA GTTTGTTATA TAGGTAAACA 4740 CACGACTTGG GATTTGGTGT ATAGATTTTT TTCATCATCC GGGTACTAAG CATACCCCAC 4800 AGTTTTTTGT TTGCTTTCTT TCTGAATTTC TCCCTCTTCC CACCTTCCTC CCTCAAGTAG 4860 GCTGGTGTTT CTCCAGACTA GAATCATGGT ATTGGAAGAA ACCTTAGAGA TCATCTAGTT 4920 TAGTTCTCTC ATTTTATAGT GGAGGAAATA CCCTTTTTGT TTGTTGGATT TAGTTATTAG 4980 CACTGTCCAA AGGAATTTAG GATAACAGTA GAACTCTGCA CATGCTTGCT TCTAGCAGAT 5040 TGTTCTCTAA GTTCCTCATA TACAGTAATA TTGACACAGC AGTAATTGTG ACTGATGAAA 5100 ATGTTCAAGG ACTTCATTTT CAACTCTTTC TTTCCTCTGT TCCTTATTTC CACATATCTC 5160 TCAAGCTTTG TCTGTATGTT ATATAATAAA CTACAAGCAA CCCCAACTAT GTTACCTACC 5220 TTCCTTAGGA ATTATTGCTT GACCCAGGTT TTTTTTTTTT TTTTTTTGGA GACGGGGTCT 5280 TGCCCTGTTG CCAGGATGGA GTGTAGTGGC GCCATCTCGG CTCACTGCAA TCTCCAACTC 5340 CCTGGTTCAA GCGATTCTCC TGTCTCAATC TCACGAGTAG CTGGGACTAC AGGTATACAC 5400 CACCACGCCC GGTTAATTGA CCATTCCATT TCTTTCTTTC TCTCTTTTTT TTTTTTTTTT 5460 TTGAGACAGA GTCTTGCTCT GTTGCCCAGG CTGGAGTACA GAGGTGTGAT CTCACCTCTC 5520 CGCAACGTCT GCCTCCCAGG TTGAAGCCAT ACTCCTGCCT CAGCCTCTCT AGTAGCTGGG 5580 ACTACAGGCG CGCGCCACCA 2ACCCGGCTA ATTTTTGTAT TTTTAGTAGA GATGGQGTTT 5640 CACCATGTTG GCCAGGCTGG TCTTGAACTC ATGACCTCAA GTGGTCCACC CGCCTCAGCC 5700 TCCCAAAGTG CTGGAATTAC AGGCTTGAGC CACCGTGCCC A3CAACCAT' TCATTTCAAC 5760 TAGAAGTTTC TAAAGGAGAG AGCAGCTTTC ACTAACTAAA TAAGATTGGT CAGCTTTCTG 5820 TAATCGAAAG AGCTAAAATG TTTGATCTTG GTCATTTGAC AGTTCTGCAT ACATGTAACT 5880 AGTGTTTCTT ATTAGGACTC TGTCTTTTCC CTATAGTGTG GGAGATCAAG AATTGTTACA 5940 AATCACCCCT CAAGGAACCA GGGATGAAAT CAGTTTGGAT TCTGCAAAAA AGGGTAATGG 6000 CAAAGTTTGC CAACTTAACA GGCACTGAAA AGAGAGTGGG TAGATACAGT ACTGTAATTA 6060 GATTATTCTG AAGACCATTT GGGACCTTTA CAACCCACAA AATCTCTTGG CAGAGTTAGA 6120 GTATCATTCT CTGTCAAATG TCGTGGTATG GTCTGATAGA TTTAAATGGT ACTAGACTAA 6180 TGTACCTATA ATAAGACCTT CTTGTAACTG ATTGTTGCCC TTTCGCTTTT TTTTTTGTTT 6240 GTTTGTTTGT TTTTTTTTGA GATGGGGTCT CACTCTGTTG CCCAGGCTGG AGTGCAGTGA 6300 TGCAATCTTG GCTCACTGCA ACCTCCACCT CCAAAGGCTC AAGCTATCCT CCCACTTCAG 6360 CCTCCTGAGT AGCTGGGACT ACAGGCGCAT GCCACCACAC CCGGTTAATT TTTTGTGGTT 6420 TTATAGAG?,T GGGGTTTCAC CATGTTACCG AGGCTGGTCT CAAACTCCTG GACTCAAGCA 6480 GTCTGCCCAC TTCAGCCTCC CAAAGTGCTG CAGTTACAGG CTTGAGCCAC TGTGCCTGGC 6540 CTGCCCTTTA CTTTTAATTG GTGTATTTGT GTTTCATCTT TTACCTACTG GTTTTTAAAT 6600 ATAGGGAGTG GTAAGTCTGT AGATAGAACA GAGTATTAAG TAGACTTAAT GGCCAGTAAT 6660 CTTTAGAGTA CATCAGAACC AGTTTTCTGA .TGGCCAATCT GCTTTTAATT CACTCTTAGA 6720 CGTTAGAGAA ATAGGTGTGG TTTCTGCATA GGGAAAATTC TGAAATTAA 6769 (2) INFORMATION FOR SEQ ID NO:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4249 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear -MO- di) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL; NO (iv) ANTI-SENSE; NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homft sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: QATCCTAAGT GGAAATAATC TAGGTAAATA GGAATTAAAT GAAAGAGTAT GAGCTACATC 60 TTCAGTATAC TTGGTAGTTT ATGAGGTTAG TTTCTCTAAT ATAGCCAGTT GGTTGATTTC 120 CACCTCCAAG GTGTATGAAG TATGTATTTT TTTAATGACA ATTCAQTTTT TGAGTACCTT 180 GTTATTTTTG TATATTTTCA GCTGCTTGTG AATTTTCTGA GACGGATGTA ACAAATACTG 240 AACATCATCA ACCCAGTAAT AATGATTTGA ACACCACTGA GAAGCGTGCA GCTGAGAGGC 300 ATCCAGAAAA GTATCAGGGT AGTTCTGTTT CAAACTTSCA TGTGGAGCCA TGTGGCACAA 360 ATACTCATGC CAGCTCATTA CAGCATGAGA ACAGCAGTTT ATTACTCACT AAAGACAGAA 420 TGAATGTAGA AAAGGCTGAA TTCTGTAATA AAAGCAAACA GCCTGGCTTA GCAAGGAGCC 480 AACATAACAG ATGGGCTGGA AGTAAGGAAA CATGTAATGA TAGGCOGACT CCCAGCACAG 540 AAAAAAAGGT AGATCTGAAT GCTGATCCCC TGTGTGAGAG AAAAGAATGG AATAAGCAGA 600 AACTGCCATG CTCAGAGAAT CCTAGAGATA CTGAAGATGT TCCTTGGATA ACACTAAATA 660 GCAGCATTCA GAAAGTTAAT GAGTGGTTTT CCAGAAGTGA TGAACTGTTA GGTTCTGATG 720 ACTCACATGA TGGGGAGTCT GAATCAAATG CCAAAGTAGC TGATGTATTG GACGTTCTAA 780 ATGAGGTAGA TGAATATTCT GGTTCTTCAG AGAAAATAGA CTTACTGGCC AGTGATCCTC 840 ATGAGGCTTT AATATGTAAA AGTGAAAGAG TTCACTCCAA ATCAGTAGAG AGTAATATTG 900 AAGGCCAAAT ATTTGGGAAA ACCTATCGGA AGAAGGCAAG CCTCCCCAAC TTAAGCCATG 960 TAACTGAAAA TCTAATTATA GGAGCATTTG TTACTGAGCC ACAGATAATA CAAGAGCGTC 1020 CCCTCACAAA TAAATTAAAG CGTAAiAGGA GACCTACATC AGGCCTTCAT CCTGAGGATT 1080 TTATCAAGAA AGCAGATTTG GCAGTTCAAA AQACTCCTGA AATGATAAAT CAGGGAACTA 1140 ACCAAACGGA GCAGAATGGT CAAGTGATGA ATATTACTAA TAGTGGTCAT GAGAATAAAA 1200 CAAAAGGTGA TTCTATTCAG AATGAGAAAA ATCCTAACCC AATAGAATCA CTCGAAAAAG 1260 AATCTGCTTT CAAAACGAAA GCTGAACCTA TAAGCAQCAO TATAAGCAAT ATGGAACTCG 1320 AATTAAATAT CCACAATTCA AAAGCACCTA AAAAGAATAG GCTGAGGAGG AAGTCTTCTA 1380 CCAGGCATAT TCATGCGCTT GAACTAGTAG TCAGTAGAAA TCTAAGCCCA CCTAATTGTA 1440 CTGAATTGCA AATTGATAGT TGTTCTAGCA GTGAAGAGAT AAAGAAAAAA AAGTACAACC 1500 AAATGCCAGT CAGGCACAGC AOAAACCTAC AACTCATGGA AGGTAAAGAA CCTGCAACTG 1560 GAGCCAAGAA GAGTAACAAG CCAAATGAAC AGACAAGTAA AAGACATGAC AGCGATACTT 1620 TCCCAGAGCT GAAGTTAACA AATGC^CCTG GTTCTTTTAC TAAGTGTTCA AATACCAGTG 1680 AACTTAAAGA ATTTGTCAAT CCTAGCCTTC CAAGAGAAGA AAAAGAAGAG AACTAGAAAC 1740 AGTTAAAGTG TCTAATAATG CTGAAGACCC CAAAGATCTC ATGTTAAGTG GAGAAAGGGT 1800 TTTGCAAACT GAAAGATCTG TAGAGAGTAG CAGTATTTCA TTGGTACCTG GTACTGATTA 1860 TGGCACTCAG GAAAGTATCT CGTTACTGGA AGTTAGCACT CTAGGCAAGG CAAAAACAGA 1920 ACCAAATAAA TGTGTGAGTC AGTGTGCAGC ATTTGAAAAC CCCAAGGGAC TAATTCATGG 1980 TTGTTCCAAA GATAATAGAA ATGACACAGA AGGCTTTAAG TATCCATTGG GACATGAAGT 2040 TAACCACAGT CGGGAAACAA GCATAGAAAT GGAAGAAAGT GAACTTGATG CTCAGTATTT 2100 GCAGAATACA TTCAAGGTTT CAAAGCGCCA GTCATTTGCT CCGTTTTCAA ATCCAGGAAA 2160 TGCAGAAGAG GAATGTGCAA CATTCTCTGC CCACTCTGGG TCCTTAAAGA AACAAAGTCC 2220 AAAAGTCACT TTTGAAT3TG AACAAAAGGA AGAAAATCAA GGAAAGAATG AGTCTAATAT 2280 CAAGCCTGTA CAGACA3TTA ATATCACTGC AGGCTTTCCT GTGGTTGGTC AGAAAGATAA 2340 GCCAGTTGAT AATGCCAAAT GTAGTATCAA AGGAGGCTCT AGGTTTTGTC TATCATCTCA 2400 GTTCAGAGGC AACGAAACTG GACTCATTAC TCCAAATAAA CATGGACTTT TACAAAACCC 2460 ATATCGTATA CCACCACTTT TTCCCATCAA GTCATTTGTT AAAACTAAAT GTAAGAAAAA 2520 TCTGCTAGAG GAAAACTTTG AGGAACATTC AATGTCACCT GAAAGAGAAA TGGGAAATGA 2580 GAACATTCCA AGTACAGTGA GCACAATTAG CCGTAATAAC ATTAGAGAAA ATGTTTTTAA 2640 AGAAGCCAGC TCAAGCAATA TTAATGAAGT AGGTTCCAGT ACTAATGAAG TGGGCTCCAG 2700 TATTAATGAA ATAGGTTCCA GTGATGAAAA CATTCAAGCA GAACTAGGTA GAAACAGAGG 2760 GCCAAAATTG AATGCTATGC TTAGATTAGG GGTTTTGCAA CCTGAGGTCT ATAAACAAAG 2820 TCTTCCTGGA AGTAATTGTA AGCA1CCTGA AATAAAAAAG CAAGAATATG AAGAAGTAGT 2880 TCAGACTGTT AATACAGATT TCTCTCCATA TCTGATTTCA GATAACTTAG AACAGCCTAT 2940 GGGAAGTAGT CATGCATCTC AGGTTTGTTC TGAGACACCT GATGACCTGT TAGATGATGG 3000 TGAAATAAAG GAAGATACTA GTTTTGCTGA AAATGACATT AAGGAAAGTT CTGCTGTTTT 3060 TAGCAAAAGC GTCCAGAAAG GAGAGCTTAG CAGGAGTCCT AGCCCTTTCA CCCATACACA 3X20 TTTGGCTCAG GGTTACCGAA GAGGOGCCAA GAAATTAGAG TCCTCAGAAG AGAACTTATC 3180 TAGTGAGGAT GAAGAGCTTC CCTGCTTCCA ACACTTGTTA TTTGGTAAAG TAAACAATAT 3240 ACCTTCTCAG TCTACTAGGC ATAGCACCGT TGCTACCGAG TGTCTGTCTA AGAACACAGA 3300 GGAGAATTTA TTATCATTGA AGAATAGCTT AAATGACTGC AGTAACCAGG TAATATTGGC 3360 ^AAGGCATCT CAGGAACATC ACCTTAGTGA GGAAACAAAA TGTTCTGCTA GCTTGTTTTC 3420 TTCACAGTGC AGTGAATTGG AAGACTTGAC TGCAAATACA AACACCCAGG ATCCTTTCTT 3480 GATTGGTTCT TCCAAACAAA TGAGGCATCA GTCTGAAAGC CAGGGAGTTG GTCTGAGTGA 3540 CAAGGAATTG GTTTCAGATG ATGAAGAAAG AGGAACGGGC TTGGAAGAAA ATAATCAAGA 3600 AGAGCAAAGC ATGGATTCAA ACTTAGGTAT TGGAACCAGG TTTTTGTGTT TGCCCCAGTC 3660 TATTTATAGA AGTGAGCTAA ATGTTTATGC TTTTGGGGAG CACATTTTAC AAATTTCCAA 3720 GTATAGTTAA AGGAACTGCT TCTTAAACTT GAAACATGTT CCTCCTAAGG TGCTTTTCAT 3780 AGAAAAAAGT CCTTCACACA GCTAGGACGT CATCTTTGAC TGAATGAGCT TTAACATCCT 3840 AATTACTGGT GGACTTACTT CTGGTTTCAT TTTATAAAGC AAATCCCGGT GTCCCAAAGC 3900 AAGGAATTTA ATCATTTTGT GT3ACATGAA AGTAAATCCA GTCCTGCCAA TGAGAAGAAA 3960 AAGACACAGC AAGTTGCAGC GTTTATAGTC TGCTTTTACA TCTGAACCTC TGTTTTTGTT 4020 ATTTAAGGTG AAGCAGCATC TGGGTGTGAG AGTGAAACAA GCGTCTCTGA AGACTGCTCA 4080 GGGCTATCCT CTCAGAGTGA CATTTTAACC ACTCAGGTAA AAAGCGTGTG TGTGTGTGCA 4140 CATGCGTGTG TGTGGTGTCC TTTGCATTCA GTAGTATGTA TCCCACATTC TTAGGTTTGC 4200 TGACATCATC TCTTTGAATT AATGGCACAA TTGTTTGTGG TTCATTGTC 4249 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARAC^-Y L^'ICS: (A) LENGTH: 710 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: NGNGAATGTA ATCCTAATAT TTCNCNCCNA CTTAAAAGAA TACCACTCCA ANGGCATCNC 60 AATACATCAA TCAATTGGGG AATTGGGA.TT TTCCCTCNCT AACATCANTG GAATAATTTC 120 ATGGCATTAA TTGCATGAAT GTGGTTAOAT TAAAAGGTGT TCATGCTAGA ACTTGTAGTT 180 CCATACTAGG TGATTTCAAT TCCTGTGCTA AAATTAATTT GTATGATATA TTNTCATTTA 240 ATGGAAAGCT TCTCAAAGTA TTTCATTTTC TTQGTACCAT TTAT.CGTTTT TGAAGCAGAG 300 GGA^ACCATG CAACATAACC TGATAAAGCT CCAGCAGGAA ATGGCTGAAC TAGAAGCTGT 360 GTTAGAACAG CATGGGAGCC AGCCTTCTAA CAGCTACCCT TCCATCATAA GTGACTCTTC 420 TGCCCTTGAG GACCTGCGAA ATCCAGAACA AAGCACATCA GAAAAAGGTG TGTATTGTTG 480 GCCAAACACT QATATCTTAA GCAAAATTCT TTCCTTCCCC TTTATCTCCT TCTGAAGAGT 540 AAGGACCTAG CTCCAACATT TTATGATCCT TGCTCAGCAC ATGGGTAATT ATGGAGCCTT 600 GGTTCTTGTC CCTGCTCACA ACTAATATAC CAGTCAGAGG GACCCAAGGC AGTCATTCAT 660 GTTGTCATCT GAGATACCTA CAACAAGTAG ATGCTATGGG GAGCCCATGG 710 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 473 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens 144- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CCATTGGTGC TAGCATCTGT CTQTTOCATT GCTTQTGTTT ATAAAATTCT GCCTGATATA 60 CTTGTTAAAA ACCAATTTGT GTATCATAGA TTGATGCTTT TGAAAAAAAT CAGTATTCTA 120 ACCTGAATTA TCACTATCAG AACAAAGCAG TAAAGTAGAT TTGTTTTCTC ATTCCATTTA 180 AAGCAGTATT AACTTCACAG AAAAGTAGTG AATACCCTAT AAGCCAGAAT CCAGAAGGCC 240 TTTCTGCTGA CAAGTTTGAG GTGTCTGCAG ATAGTTCTAC CAGTAAAAAT AAAGAACCAG 300 GAGTGGAAAG GTAAGAAACA TCAATGTAAA GATGCTGTGG TATCTGACAT CTTTATTTAT 360 ATTGAACTCT GATTGTTAAT TTTTTTCACC ATACTTTCTC CAGTTTTTTT GCATACAGGC 420 ATTTATACAC TTTTATTGCT CTAGGATACT TCTTTTGTTT AATCCTATAT AGG 473 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 421 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: GGATAAGNTC AAGAGATATI TTGATAGGTG ATGCAGTGAT NAATTGNGAA AATTTNCTGC 60 CTGCTTTTAA TCTTCCCCCG TTCTTTCTTC CTNCCTCCCT CCCTTCCTNC CTCCCGTCCT 120 TNCCTTTCCT TTCCCTCCCT TCCNCCTTCT TTCCNTCTNT CTTTCCTTTC TTTCCTGTCT 180 ACCTTTCTTT CCTTCCTCCC TTCCTTTTCT TTTCTTTCTT TCCTTTCCTT TTCTTTCCTT 240 TCTTTCCTTT CCTTTCTTTC TTGACAGAGT CTTGCTCTGT CACTCAGGCT GGAGTGCAGT 300 GGCGTGATCT CGNCTCACTG CAACCTCTGT CTCCCAGGTT CAAGCAATTT TCCTGCCTCA 360 GCCTCCCGAG TAGCTGAGAT TACAGGCGCC AGCCACCACA CCCAGCTACT GACCTGCTTT 420 T 421 (2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 997 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A< ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: AAACAGCTGG GAGATATGGT GCCTCAGACC AACCCCATGT TATATGTCAA CCCTGACATA 60 TTGGCAGGCA ACATGAATCC AGACTTCTAG GCTGTCATGC GGGCTCTTTT TTGCCAGTCA 120 TTTCTGATCT CTCTGACATG AGCTGTTTCA TTTATGCTTT GGCTGCCCAG CAAGTATGAT 180 TTGTCCTTTC ACAATTGGTG GCGATGGTTT TCTCCTTCCA TTTATCTTTC TAGGTCATCC 240 CCTTCTAAAT GCCCATCATT AGATGATAGG TGGTACATGC ACAGTTGCTC TGGGAGTCTT 300 CAGAATAGAA ACTACCCATC TCAAGAGGAG CTCATTAAGG TTGTTGATGT GGAGGAGCAA 360 CAGCTGGAAG AGTCTGGGCC ACACGATTTG ACGGAAACAT CTTACTTGCC AAGGCAAGAT 420 CTAGGTAATA TTTCATCTGC TGTATTGGAA CAAACACTYT GATTTTACTC TGAATCCTAC 480 ATAAAGATAT TCTGGTTAAC CAACTTTTAG ATGTACTAGT CTATCATGGA CACTTTTGTT 540 ATACTTAATT AAGCCCACTT TAGAAAAATA GCTCAAGTGT TAATCAAGGT TTACTTGAAA 600 ATTATTGAAA CTGTTAATCC ATCTATATTT TAATTAATGG TTTAACTAAT GATTTTGAGG 660 ATGWGGGAGT CKTGGTGTAC TCTAMATGTA TTATTTCAGG CCAGGCATAG TGGCTCACGC 720 CTGGTAATCC CAGTAYYCMR GAGCCCGAGG CAGGTGGAGC CAGCTGAGGT CAGGAGTTCA 780 AGACCTGTCT TGGCCAACAT GGGNGAAACC CTGTCTTCTT CTTAAAAAAN ACAAAAAAAA 840 TTAACTGGGT TGTGCTTAGG TGNATGCCCC GNATCCTAGT TOTTCTTGNG GGTTGAGGGA 900 GGAGATCACN TTGGACCCCG GAGGGGNGGG TGGGGGNGBG CAGGNCAAAA CACNGACCCA 960 GCTGGGGTGG AAGGGAAGCC CACTCNAAAA AANNTTN 997 PCT/US95/10:!03 (2) INFORMATION FOR SEQ ID NO 126: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 639 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TTTTTAGGAA ACAAGCTACT TTGGATTTCC ACCAACACCT GTATTCATGT ACCCATTTTT 60 CTCTTAACCT AACTTTATTG GTCTTTTTAA TTCTTAACAG AGACCAGAAC TTTGTAATTC 120 AACATTCATC GTTGTGTAAA TTAAACTTCT CCCATTCCTT TCAGAGGGAA CCCCTTACCT 180 GGAATCTGGA ATCAGCCTCT TCTCTGATGA CCCTGAATCT OATCCTTCTG AAGACAGAGC 240 CCCAGAGTCA GCTCGTGTTG GCAACATACC ATCTTCAACC TCTGCATTGA AAGTTCCCCA 300 ATTGAAAGTT GCAGAATCTG CCCASAGTCC AGCTGCTGCT CATACTACTG ATACTGCTGG 360 GTATAATGCA ATGGAAGAAA GTGTGAGCAG GGAGAAGCCA GAATTGACAG CTTCAACAGA 420 AAGGGTCAAC AAAAGAATGT CCATOGTGGT GTCTGGCCTG ACCCCAGAAG AATTTGTGAG 480 TGTATCCATA TGTATCTCCC TAATGACTAA GACTTAACAA CATTCTGGAA AGAGTTTTAT 540 GTAGGTATTG TCAATTAATA ACCTAGAGGA AGAAATCTAG AAAACAATCA CAGTTCTOTG 600 TAATTTAATT TCGATTACTA ATTTCTGAAA ATTTAGAAY 639 (2) INFORMATION FOR SEQ IDNO:27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 922 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO M WO 96/05307 PCIYUS95/10203 (iv) ANTI-SENSE» NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: NCCCNNCCCC CNAATCTGAA ATGGGGGTAA CCCCCCCCCA ACCGANACNT GGGTNGCNTA 60 GAGANTTTAA TGGCCCNTTC TGAGGNACAN AAG CTT AAG C CAGGNGACGT GGANCNATGN 120 GTTGTTTNTT GTTTGGTTAC CTCCAGCCTO GGTGACAGAG CAAGACTCTG TCTAAAAAAA 180 AAAAAAAAAA AAATCGACTT TAAATAGTTC CAGGACACGT GTAGAACGTG CAGGATTGCT 240 ACGTAGGTAA ACATATGCCA TGGTGGGATA ACTAGTATTC TGAGCTGTGT GCTAGAGGTA 300 ACTCATGATA ATGGAATATT TGATTTAATT TCAGATGCTC GTGTACAAGT TTGCCAGAAA 360 ACACCACATC ACTTTAACTA ATCTAATTAC TGAAGAGACT ACTCATGTTG TTATGAAAAC 420 AGGTATACCA AGAACCTTTA CAGAATACCT TGCATCTGCT GCATAAAACC ACATGAGGCG 480 AGGCACGGTG GCGCATGCCI1 GTAATCGCAG CACTTTGGGA GGCCGAGGCG GGCAGATCAC 540 GAGATTAGGA GATCGAGACC ATCCTGGCCA GCATGGTGAA ACCCCGTCTC TACTANNAAA 600 TGGNAAAATT ANCTGGGTGT GGTCGCGTGC NCCTGTAGTC CCAGCTACTC GTGAGGCTGA 660 GGCAGGAGAA TCACTTGAAC CGGGOAAATG GAGGTTTCAG TGAGCAGAGA TCATNCCCCT 720 NCATTCCAGC CTGGCGACAG AGCAAGGCTC CGTCNCCNAA AAAATAAAAA AAAACGTGAA 780 CAAATAAGAA TATTTGTTGA GCATAGCATG GATGATAGTC TTCTAATAGT CAATCAATTA 840 CTTTATGAAA GACAAATAAT AGTTTTGCTG CTTCCTTACC TCCTTTTGTT TTGGGTTAAG 900 ATTTGGAGTG TGGGCCAGGC AC 922 (2) INFORMATION FOR SEQ ID NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 867 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO PCTAJS95/10203 (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: GATCTATAGC TAQCCTTQGC GTCTAGAAGA TGGGTGTTGA GAAGAGGGAG TGGAAAGATA 60 TTTCCTCTGG TCTTAACTTC ATATCAGCCT CCCCTAGACT TCCAAATATC CATACCTGCT 120 GQTTATAATT AQTGGTGTTT TCAGCCTCTG ATTCTGTCAC CAGGGGTTTT AGAATCATAA 180 ATCCAGATTG ATCTTGGGAG TGTAAAAAAC TGAGGCTCTT TAGCTTCTTA GGACAGCACT 240 TCCTGATTTT GTTTTCAACT TCTAATCCTT TGAGTGTTTT TCATTCTGCA GATGCT3AGT 300 TTGTGTGTGA ACGGACACTG AAATATTTTC TAGGAATTGC (3QGAGQAAAA TGGGTAGTTA 360 GCTATTTCTG TAAGTATAAT ACTATTTCTC CCCTCCTCCC TTTAACACCT CAGAATTGCA 420 TTTTTACACC TAACATTTAA CACCTAAGGT TTTTGCTGAT GCTGAGTCTG AGTTACCAAA 480 AGGTCTTTAA ATTGTAATAC TAAACTACTT TTATCTTTAA TATCACTTTO TTCAAGATAA 540 GCTGGTGATG CTGGGAAAAT GGGTCTCTTT TATAACTAAT AGGACCTAAT CTGCTCCTAG 600 CAATGTTAGC ATATGAGCTA GGGATTTATT TAATAGTCGG CAGGAATCCA TGTGCARCAG 660 NCAAACTTAT AATGTTTAAA TTAAACATCA ACTCTGTCTC CAGAAGGAAA CTGCTGCTAC 720 AAGCCTTATT AAAGGGCTGT GGCTTTAGAG GGAAGOACCT CTCCTCTGTC ATTCTTCCTG 780 TGCTCTTTTG TGAATCGCTG ACCTCTCTAT CTCCCTGAAA AGAGCACGTT CTTCTGCTGT 840 ATGTAACCTG TCTTTTCTAT GATCTCT 867 (2) INFORMATION FOR SEQ ID NO:29: '.i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 561 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double ID) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens PCT/U895/10203 (Xi) SEQUENCE DESCRIPTION: SBQ ID NO:29: NAAAAACGGG GNNGGGANTO GGCCTTAAAN CCAAAGGbwJ AACTCCCCAA CCATTNAAAA 60 ANTGACNGGG GATTATTAAA ANCGGCGGGA AACATTTCAC NGCCCAACTA ATATTQTTAA 120 ATTAAAACCA CCACCNCTGC NCCAAGOAGG GAAACTGCTG CTACAAGCCT TATTAAAGGG 180 CTGTGOCTTT AGAGGQAAGG ACCTCTCCTC TGTCATTCTT CCTGTGCTCT TTTGTGAATC 240 GCTGACCTCT CTATGTCCGT GAAAAGAGCA CGTTCTTCGT CTGTATGTAA CCTGTCTTTT 300 CTATGATCTC TTTAGQGGTG ACCCAGTCTA TTAAAGAAAG AAAAATGCTG AATGAGGTAA 360 GTACTTGATG TTACAAACTA ACCAGAGATA TTCATTCAGT CATATAGTTA AAAATGTATT 420 TGCTTCCTTC CATCAATGCA CCACTTTCCT TAACAATGCA CAAATTTTCC ATGATAATGA 480 GGATCATCAA GAATTATGCA GGCCTGCACT GTGGCTCATA CCTATAATCC CAGCGCTTTG 540 GGAGGCTGAG GCGCTTGGAT C 561 (2) INFORMATION FOR SEQ ID NO:30: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 567 base pairs (B) TYPE: nucleic a .id (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQ*. NCE DESCRIPTION: SEQ ID NO:30: AATTTTTTGT ATTTTTAGTA GAGATGAGGT TCACCATGTT GGTCTAGATC TGGTGTCGAA 60 CGTCCTGACC TCAAGTGATC TGCCAGCCTC AGTCTCCCAA AGTGCTAGGA TTACAGGGGT 120 GAGCCACTGC GCCTGGCCTG AATGCCTAAA ATATGACGTG TCTGCTCCAC TTCCATTGAA 180 GGAAGCTTCT CTTTCTCTTA TCCTGATGGG TTGTGTTTGG TTTCTTTCAG CATGATTTTG 240 AAGTCAGAGG AGATGTGGTC AATGGAAGAA ACCACCAAGG TCCAAAGCQA GCAAGAGAAT 300 CCCAGGACAG AAAGGTAAAG CTCCCTCCCT CAAGTTGACA AAAATCTCAC CCCACCACTC 360 TG7ATTCCAC TCCCCTTTGC AGAGATGGGC CGCTTCATTT TGTAAGACTT ATTACATACA 420 TACACAGTGC TAGATACTTT CACACAGGTT CTTTTTTCAC TCTTCCATCC CAACCACATA 460 AATAAGTATT GTCTCTACTT TATOAATGAT AAAACTAAGA GATTTAGAGA GGCTGTGTAA 540 TTTGOATTCC CGTCTCGGGT TCAGATC 567 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 633 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY; linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: ?EQ ID NO:31: TTGGCCTGAT TGGTGACAAA AGTGAGATGC TCAGTCCTTG AATGACAAAG AATGCCTGTA 60 GAGTTGCAGG TCCAACTACA TATGCACTTC AAGAAGATCT TCTGAAATCT AGTAGTGTTC 120 TGGACATTGG ACTGCTTGTC CCTGGGAAGT AQCAGCAGAA ATGATCGGTG GTGAACAGAA 180 GAAAAAGAAA AGCTCTTCCT TTTTGAAAGT CTGTTTTTTG AATAAAAGCC AATATTCTTT 240 TATAACTAGA TTTTCCTTCT CTCCATTCCC CTGTCCCTCT CTCTTCCTCT CTTCTTCCAG 300 ATCTTCAGGG GGCTAGAAAT CTGTTGCTAT GGGCCCTTCA CCAACATGCC CACAGQTAAG 360 AGCCTGGGAG AACCCCAGAG TTCCAGCACC AGCCTTTGTC TTACATAGTG GAGTATTATA 420 AGCAAGGTCC CACGATGGGG GTTCCTCAGA TTGCTG-"AAT GTTCTAGAGG CTATTCTATT 480 TCTCTACCAC TCTCCAAACA AAACAGCACC TAAAT3TTAT CCTATGGCAA AAAAAAACTA 540 TACCTTGTCC CCCTTCTCAA GAGCATGAAG GTGGTTAATA GTTAGGATTC AGTATGTTAT 600 GTGTTCAGAT GGCGTTGAGC TGCTGTTAGT GCC 633 WO 96/05307 PCIYUS95/102Q3 (2) INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 470 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SBQ ID NO:33: TTTGAGAGAC TATCAAACCT TATACCAAGT GGCCTTATGG AGACTGATAA CCAGAGTACA 60 TGGCATATCA GTGOCAAATT GACTTAAAAT CCATACCCCT ACTATTTTAA GACCATTGTC 120 CTTTGGAGCA QAGAGACAGA CTCTCCCATT GAOAGGTCTT GCTATAAOCC TTCATCCGOA 180 QAGTGTAGGG TAGAQGGCCT GGGTTAAGTA TGCAGATTAC TGCAGTGATT TTACATGTAA 240 ATOTCCATTT TAGATCAACT GGAATOOATO OTACAOCTGT GTGGTGCTTC TGTGOTOAAG 300 GAGCTTTCAT CATTCACCCT TGGCACAGTA AGTATTGOGT OCCCTOTCAG TGTGGGAGGA 360 CACAATATTC TCTCCTGTGA GCAAGACTGG CACCTGTCAG TCCCTATGGA TGCCCCTACT 420 GTAGCCTCAG AAGTCTTCTC TOCCCACATA CCTGTOCCAA AAOACTCCAT 470 (2) INFORMATION FOR SEQ ID NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 517 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: line audi) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (X.1) SEQUENCE DESCRIPTIONS SEQ ID NO:33i GGTGGTACQT GTCTGTAGTT CCAQCTACTT SOOAOOCTQA OATOQAAOOA TTGCTTOAGC 60 CCAGGAOaCA GAGGTGGNAN NTTACGCTGA GATCACACCA CTOCACTCC.V GCCTGGGTGA 120 CAGAGCAAGA CCCTGTCTCA AAAACAAACA AAAAAAATGA TGAAGTGACA GTTCCAGTAO 180 TCCTACTTTG ACACTTTGAA TGCTCTTTCC TTCCTGGGGA TCCAGGGTGT CCACCCAATT 240 GTGGTTGTGC AGCCAGATGC CTGGACAGAG GACAATGGCT TCCATGGTAA GGTGCCTCGC 300 ATGTACCTGT GCTATTAGTO GGGTCCTTGT GCATGGGTTT QGTTTATCAC TCATTACCTG 360 GTGCTTGAGT AGCACAGTTC TTGGCACATT TTTAAATATT TGTTGAATGA ATGGCTAAAA 420 TGTCTTTTTG ATGTTTTTAT TGTTATTTGT TTTATATTGT AAAAGTAATA CATGAACTGT 480 TTCCATGGGG TGGGAGTAAG ATATGAATGT TCATCAC 517 (2) INFORMATION FOR SBQ ID NO:34: (i) SEQUENCE CHARACTERISTICSt (A) LENGTH: 434 bass pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CAGTAATCCT NAGAACTCAT ACGACCGGGC CCCTGGAOTC GNTGNTTNGA OCCTAOTCCN 60" GGAGAATGAA TTGACACTAA TCTCTGCTTG TGTTCTCTGT CTCCAGCAAT TGGGCAGATG 120 TGTGAGGCAC CTGTGGTGAC CCGAGAGTGG GTOTTOGACA GTGTAGCACT CTACCAGTGC 180 CAGGAGCTGG ACACCTACCT GATACCCCAG ATCCCCCACA GCCACTACTG ACTGCAGCCA 24?' GCCACAGGTA CAGAGCCACA GGACCCCAAG AATGAGCTTA CAAAGTGGCC TTTCCAOGCC 300 CTGGGAGCTC CTCTCACTCT TCAGTCCTTC TACTGTCCTG GCTACTAAAT ATTTTATGTA 360 CATCAGCCTG AAAAGGACTT CTGGCTATGC AAGGGTCCCT TAAAGATTTT CTGCTTGAAG 420 TCTCCCTTGG AAAT (2) INFORMATION FOR SBQ ID NO:35: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 baa* pair* (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SBQ ID NO:35 GATAAATTAA AACTGCOACT GCGCGGCGTG (2) INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO;36 GTAGTAGAGT CCCGGGAAAG GGACAGGGGG (2) INFORMATION FOR SEQ ID NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH- 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) PCTAJS9S/10203 (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: ATATATATAT QTTTTTCTAA TGTGTTAAAG 30 (2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SBQ ID NO:38: GTAAGTCAGC ACAAGAGTGT ATTAATTTGG 30 (2) INFORMATION FOR SEQ ID NO:39: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: TTTCTTTTTC TCCCCCCCCT ACCCTGCTAG (2) INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: GTAAGTTTGA ATGTGTTATG TGGCTCCATT 30 (2) INFORMATION FOR SEQ ID NO:41: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: AGCTACTTTT 'I'l'l'mTlTl' TTTGAGACAG 30 (2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GTAAGTGCAC ACCACCATAT CCAGCTAAAT 30 (2) INFORMATION FOR SEQ ID NO:43: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: AATTGTTCTT TCTTTCTTTA TAATTTATAG 30 (2) INFORMATION FOR SEQ ID NO:44: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: QTATATAATT TGGTAATGAT GCTAGGTTGG (2) INFORMATION FOR SEQ ID NO:45: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRA1JDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (Vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45 GAGTGTQTTT CTCAAACAAT TTAATTTCAG, (2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SBQ ID NO:46 GTAAGTGTTG AATATCCCAA GAATGACACT (2) INFORMATION FOR SEQ ID NO:47: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47 AAACATAATQ TTTTCCCTTG TATTTTACAQ (2) INFORMATION FOR SEQ ID NO:48: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:48 GTAAAACCAT TTOTTTTCTT CTTCTTCTTC (2) INFORMATION FOR SEQ ID NO:49: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:49 TQCTTGACTG TTCTTTACCA TACTGTTTAG (2) INFORMATION FOR SEQ ID NO:50: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: GTAAGGGTCT CAGGTTTTTT AAGTATTTAA 30 (2) INFORMATION FOR SEQ ID NO:51: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE; nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: TGATTTATTT TTTGGGGGGA AATTTTTTAG 30 (2) INFORMATION FOR SEQ ID NO:52: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52 GTQAGTCAAA GAGAACCTTT GTCTATGAAG (2) INFORMATION FOR SEQ ID NO:53: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53 TCTTATTAGG ACTCTGTCTT TTCCCTATAG (2) INFORMATION FOR SEQ ID NO:3-1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54 GTAATGGCAA AGTTTQCCAA CTTAACAGSC (2) INFORMATION FOR SEQ ID NO:55: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55 GAGTACCTTG TTATTTTTGT ATATTTTCAG (2) INFORMATION FOR 3EQ ID NO:56. (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) fTRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56 GTATTGGAAC CAGGTTTTTG TGTTTGCCCC (2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE) (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: ACATCTQAAC CTCTQTTTTT GTTATTTAAG (2) INFORMATION FOR SEQ ID NO:58: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: AGGTAAAAAG CGTGTGTGTG TGTGCACATG (2) INFORMATION FOR SEQ ID NO:59: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CATTTTCTTG GTACCATTTA TCOTTTTTOA (2) INFORMATION FOR SEQ ID NO:60s (i) SEQUENCE CHARACTERISTICSs (A) LENQTHs 30 base pairs (B) TYPEs nuclaic acid (C) STRANDEDNESSs single (D) TOPOLOGY: linear (ii) MOLECULE TYPEs DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTIONS SEQ ID NOs60 GTGTGTATTG TTGGCCAAAC ACTGATATCT (2) INFORMATION FOR SEQ ID NO:61s (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGYs linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICALs NO (vi) ORIGINAL SOURCE: (A) ORGANISMS Homo sapiens (xi) SEQUENCE DESCRIPTIONS SEQ ID NO:61 AGTAGATTTG TTTTCTCATT CCATTTAAAG (2) INFORMATION FOR SEQ ID NO:62: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: Gingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: GTAAGAAACA TCAATGTAAA GATGCTGTGG 30 (2) INFORMATION FOR SEQ ID NO:63: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: ATGGTTTTCT CCTTCCATTT ATCTTTCTAG 30 (2) INFORMATION FOR SEQ ID NO:64: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairB (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (■■■<) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: GTAATATTTC ATCTGCTfSTA TTGGAACAAA (2) INFORMATION FOR SEQ ID 270:65: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65 TGTAAATTAA ACTTCTCCCA TTCCTTTCAO (2) INFORMATION FOR SEQ ID NO:66: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66 GTGAGTGTAT CCATATOTAT CTCCCTAATG (2) INFORMATION FOR SEQ ID NO:67: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: &EQ ID NO:67: ATGATAATGG AATATTTGAT TTAATTTCAG 30 (2) INFORMATION FOR SEQ ID NO:68: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: GTATACCAAG AACCTTTACA GAATACCTTG 30 (2) INFORMATION FOR SBQ ID NO:69: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CTAATCCTTT GAGTGTTTTT CATTCTQCAQ PCT/US9S/10203 (2) INFORMATION FOR SEQ ID NO:70: (i) SEQUBNtt' CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID N0:70: JAAGTATAA TACTATTTCT CCCCTCCTCC 30 (2) INFORMATION FOR SEQ ID NO:71: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (H) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: TGTAACCTQT CTTTTTATG ATCTCTTTAG 30 (2) INFORMATION FOR SEQ ID NO:72: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: GTAAGTACTT GATGTTACAA ACTAACCAGA 30 (2) INFORMATION FOR SEQ ID NO:73: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pair* (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) rOLECULB TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: TCCTOATOGG TTGTGTTTGG TTTCTTTCAG 30 (2) INFORMATION FOR SEQ ID NO: 74: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SBQ ID NO:74: GTAAAGCTCC CTCCCTCAA3 TTGACAAAAA (2) INFORMATION FOR SBQ ID NOi75s (1) SEQUENCE CHARACTERISTICSs (A) LENQTHt 3C base pairs (B) TYPEs nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPEs DNA (ganomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCEs (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTIONS SBQ ID NO:75: CTGTCCCTCT CTCTTCCTCT CTTCTTCCAO (2) INFORMATION FOR SBQ ID NO:76: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUBNCB DESCRIPTION: SBQ ID NO:76: GTAAGAGCCT GGGAGAACCC CAOAGTTCCA (2) INFORMATION FOR SEQ ID NO:77: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE* nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: AGTGATTTTA CATGTAAATG TCCATTTTAG 30 (2) INFORMATION FOR SEQ ID NO:78i (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (?) ORGANISM: Homo sapiens (Xi) SEQUBNCB DESCRIPTION: SBQ ID NO:78: QTAAGTATTO GGTGCCCTGT CAGTGTGGGA 30 (2) INFORMATION FOR SBQ ID NO:79: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:79: TTGAATGCTC TTTCCTTCCT GGGGATCCAG PCT/US95/1020? (2) INFORMATION FOR SEQ ID NO:80: (1) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: GTAAGGTGCC TCGCATOTAC CTGTGCTATT 30 (2) INFORMATION FOR SBQ ID NO:81: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: CTAATCTCTG CTTGTGTTCT CTGTCTCCAG 30 (2) INFORMATION FOR SI? ID NO:82: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO PCT/US95/J0203 (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: Cys Pro lie Cys Lsu Glu Leu lie Lys Glu Pro Val Ser Thr Lys Cys 15 10 15 Asp His lie Phe Cys Lys Phe Cys Met Leu Lys Leu Leu Asn Gin Lys 20 25 30 Lys Gly Pro ser Gin Cys Pro Leu Cys Lys 35 40 (2) INFORMATION FOR SEQ ID NO:83: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: Cys Pro lie Cys Leu Glu Leu Leu Lys Glu Pro Val Ser Ala Asp Cys 15 10 15 Asn His Ser Phe Cys Arg Ala Cys lie Thr Leu Asn Tyr Glu Ser Asn 20 25 30 Arg Asn Thr Asp Gly Lys Gly Asn Cys Pro Val Cys Arg 35 40 45 (2) INFORMATION FOR SEQ ID NO:84: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTIONS SEQ ID NO:84: Cya Pro lie Cys Leu Asp Met Leu Lys Asn Thr Met Thr Thr Lys Glu 1 5 10 15 Cys Leu His Arg Phe Cys Ser Asp Cys lie Val Thr Ala Leu Arg Ser 20 25 30 Gly Asn Lys Glu cys Pro Thr Cys Arg 35 40 (2) INFORMATION FOR SEQ ID NO:85: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (Xi) SEQUENCE DESCRIPTION: SBQ ID NO:85: Cys Pro Val Cys Leu Gin Tyr Phe Ala Glu Pro Met Met Leu Asp Cys 15 10 15 Gly His Asn lie Cys Cys Ala Cys Leu Ala Arg Cya Tip Gly Thr Ala 20 25 30 Cys Thr Asn Val Ser Cys Pro Oln Cys Arg 35 40

Claims (1)

1. PCT/U3 95/10203 IPEA/US18 OCT 1996 AMKNmF.DCT.ATMS An isolated nucleic acid selected from the group consisting of a DNA coding for a BRCAl polypeptide having the amino acid sequence set forth in SEQ ID NO:2 and a DNA which hybridizes to and is at least 95% complementary to the DNA coding for said BRCAl polypeptide. The isolated nucleic acid as claimed in claim 1 which comprises the nucleotide sequence set forth in SEQ ID NO: 1 or a corresponding RNA. The isolated nucleic acid as claimed in claim 1 which comprises a nucleotide sequence which hybridizes to and is at least 95% complementary to the nucleotide sequence set forth • in SEQ ID NO: 1 or a corresponding RNA. An isolated nucleic acid which is. a DNA comprising a mutated form of the nucleotide sequence set forth in SEQ ID NO: 1 or a corresponding RNA. The isolated nucleic acid as claimed in claim 4, wherein the mutation is selected from the group consisting of a missense mutation in the zinc finger moti£ a deletion, an insertion, a frameshift mutation, a nonsense mutation and a splice site mutation. The isolated nucleic acid as claimed in claim 4, wherein the mutation is a nonsense mutation. The isolated nucleic acid as claimed in claim 4, wherein the mutation is a frameshift mutation. The isolated nucleic acid as claimed in claim 4, wherein the mutation is an insertion. The isolated nucleic acid as claimed in claim 4, wherein the mutation is a deletion. FCT/uC- 95/10203 IPEA/US18 CCT 1996 -175- The isolated nucleic acid as claimed in claim 4 selected from the group consisting of: (a) SEQ ID NO: 1 having T at nucleotide position 4056; (b) SEQ ID NO: 1 having an extra C at nucleotide position 5385; (c) SEQ ID NO:l having G at nucleotide position 5443; and (d) SEQ ID NO:l having 11 base pairs at nucleotide positions 189-199 deleted. The isolated nucleic acid as claimed in any one of claims 1 to 10 which further contains a BRCAl gene regulatory region, wherein said regulatory region corresponds to nucleotides " 1-1531 of SEQ ID NO:13. An isolated nucleic acid as claimed in claim 2 or claim 3 wherein the nucleotide sequence is operably-linked to a BRCAl gene regulatory sequence which hybridizes to the BRCAl gene regulatory region corresponding to nucleotides 1-1531 of SEQ ID NO:13 and which has a mutation which in vivo inhibits or prevents expression of the BRCAl polypeptide. Use of an isolated nucleic acid having a portion of the nucleotide sequence of the nuclcic acid as claimed in any one of claims 1 to 10 as a hybridization probe to detect in a sample a DNA encoding a BRCAl polypeptide or an RNA corresponding to said DNA or to detect a cDNA made from said RNA. A nucleic acid probe suitable for a use as claimed in claim 13 wherein the nucleotide sequence of said probe comprises the DNA sequence set forth in SEQ ID NO:l from nucleotide position 3631 to 3930 or a DNA probe sequence as set forth in Table 9 or an RNA corresponding thereto. A nucleic acid probe suitable for a use as claimed in claim 13 wherein the DNA sequence of said probe is a portion of the nucleic acid as claimed in any one of claims 1 to 10. An isolated nucleic acid having at least 15 contiguous nucleotides of a nucleic acid as claimed in any one of claims 4 to 10 and including a mutation compared to the nucleotide sequence set forth in SEQ ID NO: 1. -176- PCT/U2 95/10203 IPEA/US18 OCT 1996 17. A implicative cloning vector which comprises an isolated nucleic acid as claimed in any one of claims 1 to 12 and 14 to 16 and a replicon operative in a host cell for said vector. 18. An expression vector which comprises an isolated nucleic acid as claimed in any one of claims 1 to 10 wherein the nucleic acid is operably-linked to a promoter sequence capable of directing expression of said nucleic acid in host cells for said vector. 19. Host cells transformed with a vector as claimed in claim 17 or claim 18. 20. A method of producing a polypeptide which comprises: (i) culturing host cells as claimed in claim 19 containing an expression vector encoding said polypeptide under conditions suitable for production of said polypeptide and (ii) recovering said polypeptide. 21. A method as claimed in claim 20 which further comprises labeling the recovered polypeptide. 22. A preparation of human BRCAl polypeptide substantially free of other human proteins, said polypeptide having the amino acid sequence set forth in SEQ ID NO:2. 23. A preparation of a human BRCAl polypeptide substantially free of other human proteins, the amino acid sequence of said polypeptide having an amino acid sequence at least 95% homologous to the amino acid sequence set forth in SEQ ID NO:2. 24. A preparation of a polypeptide substantially free of other proteins, said polypeptide being a mutated human BRCAl polypeptide obtainable by expression of the nucleic acid of claim 4. 25. A preparation of a polypeptide substantially free of other proteins, said polypeptide being an antigenic fragment of the polypeptide as defined in any one of claims 22 to 24. AMENDED SHEET -177- QT/U3 95/1020? IPEA/ 26. 27. 29. 30. 31. 32. 33. 34. A preparation of a polypeptide wherein said polypeptide is in the form of a fusion protein wLi the polypeptide of any one of claims 22 to 25. A preparation as claimed in any one of claims 22 to 24 wherein said polypeptide is labeled. An antibody capable of specifically binding to an epitope of the polypeptide as claimed in any one of claims 22 to 25. An antibody as claimed in claim 28 which is a monoclonal antibody. Use of a polypeptide as defined in any one of claims 22 to 26 as an immunogen for antibody production. A use as claimed in claim 30, wherein one or more antibodies produced are subsequently labeled or bound to a solid support A pair of single-stranded oligonucleotide primers for determination of a nucleotide sequence of a BRCAl gene by a nucleic acid amplification reaction, the sequence of said primers being derived from human chromosome 17q and the use of said primers in a nucleic acid amplification reaction resulting in the synthesis of DNA and/or RNA corresponding to part of the sequence of the BRCAl gene. A pair of primers as claimed in claim 32 for determination of all or part of the sequence of the BRCAl gene having the nucleotide sequence set forth in SEQ ID NO:l and/or a mutated form thereof. A kit for detecting mutations in the BRCAl gene comprising a set of single-stranded oligonucleotide primers for determination of a nucleotide sequence of a BRCAl gene by a nucleic acid amplification reaction and instructions relating to detecting alterations in the BRCAl gene, wherein the sequence of said primers is derived from human chromosome 17q and the use of said primers in a nucleic acid amplification reaction results in the 291330 synthesis of DNA and/or RNA corresponding to • part of the sequence of the BRCAl gene. A kit for detecting mutations in the BRCAl gene comprising at least one allele-specific oligonucleotide probe for a BRCAl gene mittation and instructions relating to detecting mutations in the BRCAl gene. A kit for detecting mutations in the BRCAl gene comprising at least one oligonucleotide primer specific for a BRCAl gene mutation and instructions relating to detecting mutations in the BRCAl gene. * A nucleic acid selected from the group consisting of a wild-type BRCAl gene nucleic acid having the sequence set forth in SEQ ID NO: 1 and a nucleic acid which hybridizes to and is at least 95% complementary to a wild-type BRCAl gene nucleic acid and which has substantially the same function as a wild-type BRCAl gene nucleic acid for use in gene therapy to supply a wild-type BRCAl gene function to a cell which has lost said gene function or has altered gene function by virtue of a mutation in the BRCAl gene. A nucleic acid as claimed in claim 37 which mtains a BRCAl gene regulatory region, wherein said regulatory region corresponds to nucleotides 1-1531 of SEQ ID NO: 13. A nucleic acid as claimed in claim 37 or claim 38 for use in gene therapy wherein said nucleic acid is incorporated into the genome of said cell. A molecule selected from the group consisting of a wild-type BRCAl polypeptide having the sequence set forth in SEQ ID NO: 2, a polypeptide which has an amino acid sequence that is at least 95% homologous to a wild type BRCAl and which has substantially the same function as a wild-type BRCAl polypeptide and a molecule which mimics the function of said wild-type BRCAl polypeptide for use in peptide therapy to supply a wild-type BRAC1 gene function to a cell which has lost said gene function or has altered gene function by virtue of a mutation in the BRCAl gene. % PCT7U8 95/10203 ,179. IPEA/US18 OCT 1990 41. A method for screening potential cancer therapeutics which comprises: combining (i) a BRCAl polypeptide binding partner, (ii) a BRCAl polypeptide selec ed from the group consisting of a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 and a polypeptide having a portion of said amino acid sequence which binds to said binding partner and (iii) a compound suspected of being a cancer therapeutic and determining the amount of binding of the BRCAl polypeptide to its binding partner. 42. A method for screening potential cancer therapeutics which comprises: combining a BRCAl polypeptide binding partner and a compound suspected of being a cancer therapeutic and measuring the biological activity of the binding partner. 43. A method for screening potential cancer therapeutics Which comprises: growing a transformed eukaryotic host cell containing an altered BRCAl gene associated with a predisposition *o cancer in die presence of a compound suspected of being a cancer therapeutic and determining the rate of growth of said host cell. 44. A method for screening potential cancer therapeutics which comprises: administering a compound suspected of being a cancer therapeutic to a transgenic animal which carries in its genome an altered BRCAl gene associated with a predisposition to cancer from a second animal and determining the development or growth of a cancer lesion. 45. A transgenic animal which carries an altered BRCAl allele. 46. A transgenic animal as claimed in claim 45 wherein the alteration is selected from the group consisting of a missense mutation in the zinc finger motif, a deletion, an insertion, a frameshift mutation, a nonsense mutation and a splice site mutation. 47. The transgenic animal as claimed in claim 45 wherein the alteration is a nonsense mutation. 48. The transgenic animal as claimed in claim 45 wherein the alteration is a frameshift mutation. AMENDED SHEET "180" !*» 'I'B 1 <5 49. The transgenic animal as claimed in claim 45 wherein the alteration is an insertion !2 .} '-■] ^ \ ^ 50. The transgenic animal as claimed in claim 45 wherein the alteration is a deletion. 51. The transgenic animal as claimed in claim 45 wherein the altered BRCAl allele is a disrupted allele. 52. A method for supplying a wild-type BRCAl gene function to a cell which has lost said gene function or has altered gene function by virtue of a mutation in the BRCAl gene, comprising: introducing into the cell a nucleic acid which suppresses a transformed state of said cell, said nucleic acid selected from the group consisting of a wild-type BRCAl gene nucleic acid having the sequence set forth in SEQ ID NO: 1 and a nucleic acid which hybridizes to and is at least 95% complementary to a wild-type BRCAl gene nucleic acid and which has substantially the same function as a wild-type BRAC1 gene nucleic acid. 53. The method of claim 52 wherein said nucleic acid is a wild-type BRCAl gene nucleic acid. 54. The method of claim 52 wherein said nucleic acid is a nucleic acid which hybridizes to and is at least 95% complementary to a wild-type BRCAl gene nucleic acid and which has substantially the same function as a wild-type BRCAl gene nucleic acid. 55. The method of any one of claims 52 to 54 wherein said nucleic acid contains a BRCAl gene regulatory region, wherein said regulatory region corresponds to nucleotides 1-1531 of SEQ ID NO: 13. 56. The method of any one of claims 52 to 55 wherein said nucleic acid is incorporated into the genome of said cell. 57. A method for supplying a wild-type BRCAl gene function or a BRCAl function substantially similar to the wild-type to a cell which has lost said gene fruition or has altered gene function by virtue of a mutation in the BRCAl gene, comprising: introducing into the cell a * -181- molecule which suppresses a transformed state of said cell, said molecule selected from the group consisting of a wild-type BRCAl polypeptide having the sequence set forth in SEQ ID NO: 2, a polypeptide which has an amino acid sequence that is at least 95% homologous to a wild-type BRCAl and which has substantially the same function as a wild-type BRCAl polypeptide and a molecule which mimics the function of said wild-type BRCAl polypeptide. 58. The method of claim 57 wherein said molecule is a wild-type BRCAl polypeptide. 59. The method of claim 57 wherein said molecule is a polypeptide which has an amino acid sequence that is at least 95% homologous to a wild-type BRCAl and which has substantially the same function as a wild-type BRCAl polypeptide. 60. The method of claim 57 wherein said molecule is a molecule which mimics the function of said wild-type BRCAl polypeptide. 61. An isolated nucleic acid as defined in claim 1 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 62. Use of an isolated nucleic acid as claimed in claim 13 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 63. A nucleic acid probe as claimed in claim 14 or claim 15 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 64. An isolated nucleic acid as defined in claim 16 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 65. A replicative cloning vector as defined in claim 17 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. <% 29 1 3 3 0 -182- f 66. An expression vector as defined in claim 18 substantially as herein described with ^ reference to : ny example thereof and with or without reference to the accompanying i v drawings. \ ' \ 67. Host cells as claimcd in claim 19 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 68. A method as defined in claim 20 of producing a polypeptide substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 69. A preparation of human BRCAl polypeptide substantially free of other human proteins as defined in claim 22 or claim 23 substantially as herein describ :d with reference to any example thereof and with or without reference to the accompanying drawings. 70. A preparation of a polypeptide free of other proteins as defined in claim 24 or claim 25 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 71. A preparation of a polypeptide as defined in claim 26 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 72. An antibody as defined in claim 28 substantially as herein described with reference to any exampl a thereof and with or without reference to the accompanying drawings. 73. Use of a polypeptide as defined in claim 30 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 74. A pair of single-stranded oligonucleotide primers as defined in claim 32 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 1 .r office - 5 FEB 1997. FuCEIVED JMWWI lliUli'WMWM—i # yrn. .(/ . 183- 75. A kit as claimed in any one of claims 34 to 36 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 76. A nucleic acid as defined in claim 37 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. w 77. A molecule as claimed in claim 40 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 78. A method as claimed in any one of claims 41 to 44 for screening potential cancer therapeutics substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 79. A transgenic animal as defined in claim 45 substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 80. A method as defined in claim 52 for supplying a wild-type BRCAl gene function to a cell which has lost said gene function or has altered gene function by virtue of mutation in the BRCAl substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. 81. A method as defined in claim 57 for supplying a wild-type BRCAl gene function substantially similar to the wild-type to a cell which has lost said gene function or has altered gene function by virtue of a mutation in the BRCAl gene substantially as herein described with reference to any example thereof and with or without reference to the accompanying drawings. OF CLAIM!