JP2004135687A - 17q-LINKED BREAST AND OVARIAN CANCER SUSCEPTIBILITY GENE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a material and method used to test an allele of BRCA1, and an interpretation method for the normality or predisposition of the allele. <P>SOLUTION: The present invention relates to germline mutations in the BRCA1 gene and their use in the diagnosis of predisposition to breast and ovarian cancer, and to methods and materials used to isolate and detect a human breast and ovarian cancer predisposing gene (BRCA1). <P>COPYRIGHT: (C)2004,JPO

Description

The present invention relates generally to the field of human genetics. In particular, the present invention relates to a method for isolating and detecting a human breast cancer and ovarian cancer predisposition gene (BRCA1), some of which mutant alleles cause susceptibility to cancer, especially breast and ovarian cancer. And materials. More particularly, the invention relates to germline mutations in the BRCA1 gene and their use in diagnosing a predisposition to breast and ovarian cancer. The invention further relates to somatic mutations in the BRCA1 gene in human breast and ovarian cancer, and their use in diagnosis and prognosis of human breast and ovarian cancer. In addition, the present invention relates to somatic mutations of the BRCA1 gene in other human cancers and their use in diagnosing and prognosing human cancers. The invention also relates to the therapy of human cancers having a mutation in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to screening for agents for cancer therapy. Finally, the present invention relates to the screening of the BRCA1 gene for mutations, which is useful in diagnosing a predisposition to breast and ovarian cancer.
Publications and other materials used herein to elucidate the background of the invention, and in particular, where it provides further details regarding the practice, are hereby incorporated by reference, are simply considered part of the specification, For the purposes of the present invention, they are indicated by author and date in the following specification and are each grouped in an attached reference list.

遺 伝 The genetics of cancer is complex, including many dominant and positive regulatory genes (oncogenes) in the transformed state, as well as many recessive and negative regulatory genes (tumor suppressor genes). More than a hundred oncogenes have been characterized. Less than a dozen tumor suppressor genes have been identified, but their number is expected to increase by more than 50 (Knudson, 1993).

The involvement of such a large number of genes underscores the complexity of growth control mechanisms that operate in cells to maintain normal tissue integrity. This complexity is evident in other ways. To date, no single gene has been shown to be involved in the growth of all or most human cancers. The most ubiquitous oncogene mutation is present in the H-ras gene and is found in 10-15% of all solid tumors (Anderson et al., 1992). The most frequently mutated tumor suppressor genes are the TP53 gene, which is homozygously deleted in almost 50% of all tumors, and the CDKN2, which is homozygously deleted in 46% of the tumor cell lines examined (Kamb et al., 1994). Without a target common to all transformed cells, it is unlikely that a "magic bullet" dream could destroy or reinstate cancer cells while leaving harmless normal tissue. The desire for new drug discovery of specifically targeted anti-cancer drugs may be supported by the ability to identify tumor suppressor genes or oncogenes that play a general role in controlling cell division.

The cloned and characterized tumor suppressor genes include: 1) Retinoblastoma (RB1); 2) Wilms tumor (WT1); 3) Li-Fraumeni syndrome (TP53); 4) Familial adenomatous polyps. APC); 5) neurofibromatosis type 1 (NF1); 6) neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau disease (VHL); 8) multiple endocrine neoplasia type 2A (MEN2A). And 9) affects sensitivity to melanoma (CDKN2).

Tumor suppressor loci that have been genetically mapped but not yet isolated include: Multiple endocrine neoplasia type 1 (MEN1); Lynch cancer familial syndrome type 2 (LCFS2); Neuroblastoma (NB) Genes for basal cell nevus syndrome (BCNS); Beckwith-Wiedemann syndrome (BWS); renal cell tumors (RCC); tuberous sclerosis type 1 (TSC1); and tuberous sclerosis type 2 (TSC2). You. Tumor suppressor genes that have been characterized to date include DNA binding protein (WT1), auxiliary transcription factor (RB1), GTPase activating protein or GAP (NF1), cytoskeletal factor (NF2), membrane-bound receptor kinase It encodes products analogous to various types of proteins, including (MEN2A), cell cycle regulator (CDKN2) and others (APC and VHL) that have no apparent identity to known proteins.

場合 In many cases, tumor suppressor genes originally identified through genetic studies have been shown to be deleted or mutated in certain sporadic tumors. The results indicate that regions of chromosomal abnormalities may be able to locate key tumor suppressor genes involved in both genetic predisposition to cancer and sporadic cancer.

１ One of the salient features of several tumor suppressor genes characterized to date is that they are highly deleted in certain tumor types. Such deletions often include single allele deletions, so-called heterozygous deletions (LOH), but can also include homozygous deletions of both alleles. For LOH, it is expected that the remaining allele will be non-functional either due to pre-existing mutations or due to secondary sporadic mutations.

Breast cancer is one of the most important diseases affecting women. In a recent proportion, U.S. women have an eighth risk of developing breast cancer by the age of 95 (American Cancer Society, 1992). Treatment of late stage breast cancer is often wasteful and ugly, with early detection a high priority in medical care of the disease. Although less frequent than breast cancer, ovarian cancer often results in rapid death and is the fourth most common cause of cancer mortality in US women. The genetic factor contributing to the disease-defined ratio of breast cancer incidence is estimated to be about 5% of all cases, but about 25% of cases are diagnosed before age 40 (Claus et al., 1991). Breast cancer is divided into two types based on the inflection of the age-specific incidence curve around the age of 50, namely, young-onset and old-onset. Mutations in one gene, BRCA1, are thought to account for about 45% of familial breast cancers, but at least 80% of families with both breast and ovarian cancer (Easton et al., 1993).

Since the BRCA1 gene was first mapped in 1990, strenuous efforts have been made to isolate it (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 be considered to have a similar proportion of young-onset breast cancer as BRCA1, but contributes to a lower risk of ovarian cancer are doing. The remaining susceptibility to young-onset breast cancer is divided between the unmapped gene of familial cancer and rare germline mutations such as TP53 (Malkin et al., 1990). It has also been suggested that heterozygous carriers of the ataxia telangiectasia gene are at high risk for breast cancer (Swift et al., 1976; Swift et al., 1991). Older-onset breast cancer is often familial, but the risk in blood relatedness is not as high as that of younger-onset breast cancer (Cannon-Albright et al., 1994; Mettlin et al., 1990). However, the percentage of such cases due to genetic susceptibility is not known.

Breast cancer has long been recognized in part as a familial disease (Anderson, 1972). A vast number of investigators have examined evidence of genetic succession and have concluded that the data are almost consistent with the dominant inheritance of major susceptibility loci or groups of loci (Bishop and Gardner, 1980; Go et al., 1983; Williams and Anderson, 1984; Bishop et al., 1988; Newman et al., 1988; Claus et al., 1991). Recent results demonstrate that there are at least three loci that carry susceptibility to breast cancer as well as other cancers. These loci are the TP53 locus on chromosome 17p.
(Malkin et al., 1990), the 17q-linked susceptibility locus known as BRCA1 (Hall et al., 1990), and one or more loci that contribute to the rest of the unmapped. Hall et al. (1990) showed that inherited breast cancer susceptibility was linked to chromosome 17q21 in families with a younger onset; further studies by this group using a more appropriate genetic model suggest that The limitation to onset breast cancer was partially refuted (Margaritte et al., 1992).

Most strategies for cloning the 17q-linked breast cancer predisposition gene (BRCA1) require accurate genetic location studies. The simplest model for the functional role of BRCA1 is that the BRCA1 allele predisposing to cancer is recessive to the wild-type allele; that is, cells containing at least one wild-type BRCA1 allele are He supports not having cancer. However, cells containing one wild-type BRCA1 allele and one disease-predisposing allele were wild-type, either by random mutation or by chromosomal deletion during cell division (non-chromosomal segregation). Allelic deletions can often be encountered. All progeny of such mutant cells lack the wild-type function of BRCA1 and can develop into tumors. According to this model, the predisposition allele of BRCA1 is recessive, and women with one predisposition allele (and one wild-type allele) develop cancer, even if they dominantly inherit susceptibility to cancer. Has danger. Because her breast epithelial cells can spontaneously lack the wild-type BRCA1 allele. This model applies to a class of genes including retinoblastoma and neuroblastoma genes, a group of cancer susceptibility alleles known as tumor suppressors or anti-oncogenes. By inference, this model can also account for BRCA1 function, as recently shown (Smith et al., 1992).

The second possibility is that the BRCA1 disease predisposing allele is indeed dominant; that is, the wild type allele of BRCA1 cannot overcome the tumorigenic role of the disease predisposing allele. Thus, cells carrying both wild-type and mutant alleles are not necessarily deficient in the wild-type copy of BRCA1 before giving rise to malignant cells. Instead, breast cells in predisposed individuals will have undergone some other stochastic change (s) leading to cancer.

If the BRCA1 disease predisposing allele is recessive, the BRCA1 gene is expected to be expressed in normal breast tissue but not functionally in breast cancer. Conversely, if the BRCA1 disease predisposition allele is dominant, the wild-type BRCA1 gene may or may not be expressed in normal breast tissue. However, the disease predisposing allele appears to be expressed in breast cancer cells.

The 17q linkage of BRCA1 was independently confirmed in three of five families with both breast and ovarian cancer (Narod et al., 1991). These studies claim that the gene is located within a very large region of 15 centiMorgans (cM), or about 15 Mbp, on either side of the linked marker pCMM86 (D17S74). . However, genetic studies using markers surrounding pCMMS6 have proven that attempts to further define the region have been unsuccessful. Subsequent studies indicate that the genes were quite close (Easton et al., 1993) and that the original analysis was defective (Margaritte et al., 1992). Hall et al. (1992) recently localized the BRCA1 gene at an interval of about 8 cM (about 8 Mbp) bordering the proximal Mfdl5 (D17S250) and the distal human GIP gene. A slightly narrower interval for the BRCA1 allele based on publicly available data was agreed in March 1992 at the "Chromosome 17 Study Group" (Fain, 1992). The size of these regions and the associated uncertainties make the design and instrumentation of physical mapping and / or cloning strategies to isolate the BRCA1 gene overly difficult.

Identification of breast cancer susceptibility alleles will allow early detection of susceptible individuals and will greatly enhance our ability to understand the initial steps leading to cancer. Because susceptibility alleles often change during tumor progression, cloning of these genes may also be important in developing better diagnostic and diagnostic products, and in better cancer therapy.

The present invention relates generally to the field of human genetics. In particular, the invention relates to methods and materials used to isolate and detect the human breast cancer predisposition gene (BRCA1), a certain allele that produces susceptibility to cancer, particularly breast and ovarian cancer. More particularly, the invention relates to germline mutations in the BRCA1 gene and their use in diagnosing a predisposition to breast and ovarian cancer. The invention further relates to somatic mutations of the BRCA1 gene in human breast cancer and its use in the diagnosis and prognosis of human breast and ovarian cancer. In addition, the present invention relates to somatic mutations of the BRCA1 gene in other human cancers and their use in diagnosing and prognosing human cancers. The present invention also relates to therapies for human cancers having a mutation in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to screening for agents for cancer therapy. Finally, the invention relates to the screening of the BRCA1 gene for mutations, which is useful for diagnosing a predisposition to breast and ovarian cancer.

The present invention provides materials and methods used to test an individual's BRCA1 allele and the interpretation of the normal or disease predisposing properties of the allele. Individuals who are above normal risk will modify their lifestyle appropriately. In the case of BRCA1, the most significant non-genetic risk factor is the protective effect over the entire gestation period of the young. Thus, at-risk women could consider having children early or could consider therapies designed to stimulate the hormonal effects of pregnancy throughout the young. High-risk women will also strive for early detection and will be a great incentive to learn and practice breast self-diagnosis. Such women would be motivated to take regular mammograms starting at an earlier age than the general population. Ovarian screening will also be done more frequently. Diagnostic methods based on sequence analysis of the BRCA1 locus could also be applied to tumor detection and classification. Precursor foci could be diagnosed using sequence analysis. With advances in the method and accumulation of information about BRCA1 and other causative loci, it will be possible to classify cancer into benign and malignant.

Women with breast cancer will follow a different surgical procedure if it is predisposed to disease and therefore will have more cancer than if it is not predisposed to disease. Other therapies using either peptides or small molecules (rational drug design) will be developed. The peptide may be the missing gene product itself or a part of the missing gene product. Alternatively, the therapeutic agent will be another molecule that mimics the deleterious gene function, a peptide or non-peptide molecule that is sought to prevent the deleterious effects of the inherited locus. Also, the therapy may be gene-based, through introducing a normal BRCA1 allele into the individual and creating a protein that will counteract the effects of the deleterious allele. These gene therapies can take many forms and will be directed to preventing tumors from forming, curing cancer once it has occurred, or stopping cancer from metastasis.

The present invention relates generally to the field of human genetics. In particular, the present invention relates to methods and materials used to isolate and detect the human breast cancer predisposition gene (BRCA1), a certain allele that confers susceptibility to cancer, particularly breast and ovarian cancer. About. More particularly, the invention relates to germline mutations in the BRCA1 gene and their use in diagnosing a predisposition to breast and ovarian cancer. The invention further relates to somatic mutations in the BRCA1 gene in human breast cancer and its use in the diagnosis and prognosis of human breast and ovarian cancer. In addition, the present invention relates to somatic mutations in the BRCA1 gene in other human cancers and their use in diagnosing and prognosing human cancers. The invention also relates to therapies for human cancers having a mutation in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to screening for agents for treating cancer. Finally, the invention relates to the screening of the BRCA1 gene for mutations, which is useful for diagnosing a predisposition to breast and ovarian cancer.

The present invention provides an isolated polynucleotide consisting of all or a part of the BRCA1 locus or a mutant BRCA1 locus, preferably at least 8 bases in length to about 100 kb in length. Such a polynucleotide can be an antisense polynucleotide. 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.

The present invention also provides a method for detecting a polynucleotide containing a part of the BRCA1 locus or its expression product in an analyte. Such a method may further include amplifying a portion of the BRCA1 locus, and may further include providing a set of polynucleotides that are primers for amplifying the portion of the BRCA1 locus. The method is useful for either diagnosing a predisposition to cancer or diagnosing or prognosing cancer.

The invention also provides an isolated antibody, preferably a monoclonal antibody, that specifically binds to an isolated polypeptide consisting of at least 5 amino acid residues encoded by the BRCA1 locus.

The invention also provides a kit for detecting a polynucleotide comprising a portion of the BRCA1 locus in an analyte, the kit comprising a polynucleotide complementary to a portion of the BRCA1 locus sealed in a suitable container. And instructions for using it.

The present invention further provides a method of preparing a polynucleotide comprising a polymerizable nucleotide and obtaining a sequence consisting of at least eight adjacent nucleotides of the BRCA1 locus; and preparing a polypeptide comprising a polymerizable amino acid to obtain a BRCA1 gene. A method is provided for obtaining a sequence comprising at least 5 amino acids encoded in a locus.

The present invention further provides a method of screening the BRCA1 gene to identify a mutation. Such methods can further include amplifying a portion of the BRCA1 locus, and can include providing a set of polynucleotides that are primers for amplifying the portion of the BRCA1 locus. The method is useful for diagnosing a predisposition to cancer or identifying mutations for use in either diagnosing or prognosing cancer.

The present invention further provides a method of screening for a predicted BRCA1 mutant allele to identify a mutation in the BRCA1 gene.

Additionally, the present invention provides a method of screening a drug for cancer therapy to identify a drug suitable for restoring BRCA1 gene product function.

Finally, the present invention provides the tools needed to create a gene-based therapy directed against cancer cells. These therapeutic agents may be placed in a suitable vector so that the function of the BRCA1 protein is reconstituted, or delivered to the target cell in a more direct manner, including the entire or partial BRCA1 locus. It can take the form of nucleotides. Therapeutic agents can also take the form of a polypeptide based on either the partial or complete protein sequence of BRCA1. They can functionally replace the activity of BRCA1 in vivo.

The BRCA1 gene, which causes an individual to have a predisposition to breast and ovarian cancer, is a gene that encodes a BRCA1 protein. It is a finding of the invention. This gene is designated herein as BRCA1. It is an observation of the present invention that mutations in the BRCA1 locus in the germline are indicative of a predisposition to breast and ovarian cancer. Finally, somatic mutations at the BRCA1 locus have also been associated with breast, ovarian and other cancers, which may be indicative of these cancers or predisposing them to disease. It is a finding of the present invention. Mutational events at the BRCA1 locus can include deletions, insertions, and point mutations in coding and non-coding sequences.

Starting from the region on the long arm of human chromosome 17 of the human genome, 17q has a size estimated to be about 8 Mbp, the region containing the locus, BRCA1, and is susceptible to cancer, including breast and ovarian cancer. Is located.

The region containing the BRCA1 locus was identified using various genetic techniques. Genetic mapping techniques first defined the BRCA1 region by recombination with genetic markers. Based on a study of a large spread family ("family") with multiple cases of breast cancer (and ovarian cancer cases in some families), the precise location of the chromosomal region including the BRCA1 gene and other possible susceptibility alleles at the BRCA1 locus. Position. Two mitotic breakpoints were found as recombination between the genetic marker and the disease and at the end of the BRCA1 locus, which is expressed as one recombination proximal to the BRCA1 locus. Thus, the region containing the BRCA1 locus physically borders these markers.

使用 The use of the genetic markers provided by the present invention allows the identification of clones covering regions from human yeast artificial chromosome (YAC) or human bacterial artificial chromosome (BAC) libraries. It also allows the identification and preparation of more easily manipulated cosmid, P1 and BAC clones from this region, and the construction of contigs from subsets of clones. These cosmids, P1, YAC and BAC, provide the basis for the cloning of the BRCA1 locus, for example, to develop effective reagents for the diagnosis and treatment of breast and / or ovarian cancer. The BRCA1 gene and other potential susceptibility genes were isolated from this region. Isolation is performed by software trapping (from a continuous or non-contiguous genomic DNA sequence, likely to include coding exons, along with whole or partial cDNA inserts from cosmids, P1 and BACs in the region where the cDNA library is screened. Computer method to identify unique sequences), hybrid selection techniques and direct screening. Using these methods, the sequences of loci expressed in breast and other tissues were obtained. These candidate loci were analyzed to identify sequences that confer cancer susceptibility. The present inventors have found that there is a mutation in the coding sequence of the BRCA1 locus in a family responsible for 17q-linked cancer susceptibility known as BRCA1. This gene was not known to be present in this region. The present invention not only facilitates the early detection of certain cancers and energizes the survival of patients, but can also detect susceptible individuals before they develop cancer.

Population-Sources Widely documented Utah families are particularly important in providing a good source of human genetics research. Each large pedigree independently provides the ability to determine whether the BRCA1 susceptibility allele is segregated in its family. Recombinational informa- tion for the location and isolation of the BRCAl locus could only be obtained from families large enough to confirm the presence of the susceptible allele. Large siblings are particularly important in studying breast cancer. This is because the penetrance of the BRCA1 sensitive allele is reduced by both age and gender, making it difficult to find informative siblings. In addition, large siblings are essential to constructing haplotypes for deceased individuals due to the effects of their close relative haplotypes.

Other populations can provide useful information, but such studies generally require more effort, and families are usually much smaller, and therefore have less information. Utah's age-adjusted breast cancer incidence rate is 20%, lower than the average US rate. The low incidence in Utah is probably due largely to the young age of the first pregnancy, increasing the likelihood of finding the case in Utah kindreds who carry a genetic predisposition.

Genetic Mapping Given an informative family set, genetic markers are essential for linking disease to chromosomal regions. Such markers include restriction fragment length polymorphism (RELP) (Botstein et al., 1980), markers with a variable number of tandem repeats (VNTR) (Jeffreys et al., 1985; Nakamura et al., 1987), and short tandem repeats (STR). ), Especially a rich class of DNA polymorphisms based on CpA repeats (Weber and May, 1989; Litt et al., 1989). To generate a genetic map, potential genetic markers are selected and tested using DNA extracted from the members of the family to be studied.

遺 伝 子 Genetic markers useful for locating loci associated with disease can either cover specific chromosomes densely, or be selected based on this problem in particular by detailed analysis of specific regions of the chromosomes. A preferred method of selecting genetic markers linked to disease involves assessing the degree of informativeness of a family to determine the ideal distance between genetic markers of a given degree of polymorphism, and then maximizing efficiency. And selecting a marker from a known genetic map that is ideally distant. The informativeness of a family is measured by the probability that the marker is heterozygous in unrelated individuals. Also, it is most efficient to use STR markers detected by amplification of the target nucleic acid sequence using PCR; such markers are very informative, easy to assay (Weber and May, 1989), and allow for complex strategies. And can be assayed simultaneously (Skolnick and Wallace, 1988), which can significantly reduce the number of experiments required.

Once linkage has been established, it is necessary to find a marker adjacent to the disease locus, ie, one or more markers close to the disease locus and one or more markers proximal to the disease locus. is there. If possible, candidate markers can be selected from a known genetic map. If none is known, new markers can be identified by the STR technique shown in the examples.

Gene mapping is usually an iterative process. In the present invention, one begins by defining flanking genetic markers around the BRCA1 locus and then replacing those flanking markers with other markers that are successfully close to the BRCA1 locus. The first step, a recombination event, is defined by a broad pedigree and assists in specifically locating the BRCA1 locus as being away from or near a specific genetic marker (Goldgar et al., 1994). .

ま で Until the present invention was disclosed, the region surrounding BRCA1 was poorly mapped and had few markers. Therefore, a new genetic marker was developed by analyzing short repetitive sequences on cosmids subcloned from physically mapped YACs. Using this approach, one marker of the invention, 42D6, was discovered, which was replaced with pCMM86 as the terminal flanking marker for the BRCA1 region. Since 42D6 is located approximately 14 cM from pCMM86, the BRCA1 region has therefore been reduced by approximately 14 cM (Easton et al., 1993). Thus, the present invention has begun by finding a more closely linked distal flanking marker of the BRCA1 region. Next, BRCA1 was found to be located on the terminal side with respect to the genetic marker Mfdl5. Therefore, it was shown that BRCA1 exists in a region of 6 Mbp to 10 Mbp bordering on Mfdl5 and 42D6. Subsequently, the marker Mfdl91 was found to be distal to Mfdl5 and proximal to BRCA1. Thus, Mdfl5 was replaced with Mfdl91 as the most proximal gene marker. Similarly, it has been discovered that the genetic marker Mfdl88 can replace the genetic marker 42D6, which narrows the region containing the BRCA1 locus to approximately 1.5 Mb. Then, using the techniques known in the art and described herein, the marker Mfdl91 was replaced with tdj1474 as a proximal marker and Mfdl88 was replaced with U5R as a terminal marker, resulting in a BRCA1 locus. Was isolated and the BRCA1 region narrowed to a region small enough to obtain characterization (see FIG. 3).

Physical Mapping The area was physically mapped using three different methods. The first was the use of a yeast artificial chromosome (YAC) to clone the region flanked by tdj1474 and U5R. The second was the generation of a set of P1, BAC and cosmid clones covering the region containing the BRCA1 locus.

Yeast artificial chromosome (YAC)
Once a sufficiently small region containing the BRCA1 locus was identified, the physical isolation of DNA in the region was continued by identifying the set of overlapping YACs covering the region. Useful YACs can be isolated from known libraries, such as the St. Louis and CEPH YAC libraries, each of which is widely distributed and contains approximately 50,000 YACs. YACs can be isolated from these publicly accessible libraries and obtained from a number of sources, including the Michigan Genome Center. Obviously, those accessing these YACs would not have understood the value of the specific YACs we selected without disclosing the present invention. Because they would not have understood which YACs were in the smallest region containing the BRCA1 locus and which were outside.

Cosmid, P1 and BAC clones In the present invention, it is advantageous to continue with this region by cosmid, P1 and BAC clones. These inserts, which are smaller in size compared to the YAC insert, make it more useful as a specific hybridization probe. In addition, having the cloned DNA in bacterial cells rather than yeast cells greatly increases the ease with which the DNA of interest can be manipulated and improves the signal to noise ratio of the hybridization assay. For the cosmid subclone of YAC, the DNA is partially digested with the restriction enzyme Sau3A and cloned into the BamHI site of the pWE15 cosmid vector (Stratagene, catalog number 1251201). Cosmids containing human sequences, as will be detailed in Examples, human repetitive DNA (e.g., Gibco / BRL, Inc., human C _{0 t-1} DNA, Catalog No. 5279A) was screened by hybridization with, then various Fingerprinting by technology.

P1 and BAC clones are obtained by screening libraries constructed from the entire human genome with YACs, cosmids or specific sequence tag sites (STSs) derived from P1 and BAC, isolated as described above.

These P1, BAC and cosmid clones were compared by interspersed repeat (IRS) PCR and / or gel electrophoresis following restriction enzyme digestion and comparison of the resulting DNA fragments (fingerprint) (Maniatis et al., 1982). obtain. The clone may also be characterized by the presence of STS. The fingerprint is used to define an overlapping contiguous set of clones that cover the region but are not too redundant (herein referred to as "minimum tiling path"). Such minimal tile tracts form the basis for subsequent experiments and identify cDNAs that could be derived from the BRCA1 locus.

Coverage of gaps in P1 and BAC clones To cover any gaps in the BRCA1 contig between identified cosmids with genomic clones, it is almost twice as large as the cosmid for P1, and even more so for BAC. A BAC vector containing a large genomic DNA insert and a clone in P1 (Sternberg, 1990; Sternberg et al., 1990; Pierce et al., 1992; Shizuya et al., 1992) were used. The P1 clone was isolated by Genome Sciences using the PCR primers provided by the present inventors for screening. BAC was obtained by a hybridization technique in Dr. Mel Simon's laboratory. The strategy using P1 clones also allows to cover the genomic region with an independent set of clones not derived from YAC. This protects against possible other deletions in the YAC that have not been detected. These new sequences from the P1 clone provide material for further screening for candidate genes, as described below.

Gene Isolation There are a number of techniques for testing genomic clones for the presence of sequences that are likely to be candidate coding sequences for the locus to be isolated, including but not limited to:
a. Zoo blot
b. Identification of HTF island
c. Exon trapping
d. Hybridization of cDNA to cosmid or YAC
e. Screening of a cDNA library is included.
(a) Zoo-blot The first technique is to hybridize a cosmid to a Southern blot and preserve it during evolution, and thus have a varying degree of relationship to humans (monkeys, cows, chickens, pigs, mice). And a DNA sequence that gives a positive hybridization signal to DNA from DNA (such as rat). Southern blots containing such DNA from various species are commercially available (Clonetech, Cat. No. 7753-1).
(b) HTF Island Identification A second technique involves finding a region rich in nucleotides C and G, which often occurs near or in the coding sequence. Such sequences are referred to as HTFs (HpaI small fragments) or CpG islands as restriction enzymes specific for sites containing CpG dimers that are frequently cleaved in these regions (Lindsay et al., 1987).
(c) Exon trapping The third technique is exon trapping, a method for identifying sequences in genomic DNA that contain splice junctions and therefore likely contain the coding sequence of a gene. Using exon amplification (Buckler et al., 1991), exons are selected from the above DNA clones and amplified. Exon amplification is based on the selection of RNA sequences flanked by functional 5 'and / or 3' splice sites. The product of the exon amplification is used to screen a breast cancer cDNA library to identify a processable number of candidate genes for further study. Exon trapping may be performed on small segments of sequencing DNA using a computer program or by software capture.
(d) cDNA hybridizing to cosmid, P1, BAC or YAC A fourth technique utilizes hybridization of cDNA to cosmid, P1, BAC or YAC, and the transcribed sequence is cloned into cloned genomic DNA. It is a modification of a selective enrichment technique that allows it to be identified and recovered from it (Kandpal et al., 1990). The selective enrichment technique modified for this purpose involves binding DNA from the BRCA1 region present in the YAC to a column matrix, then selecting cDNA from a suitable library that hybridizes with the bound DNA, followed by And amplifying and purifying the bound DNA, thereby greatly increasing the cDNA in the region represented by the cloned genomic DNA.
(e) Identification of cDNA The fifth technique is to identify the cDNA corresponding to the BRCA1 locus. Using hybridization probes containing the predicted coding sequence selected using any of the techniques described above, a variety of libraries, including breast tissue cDNA libraries, ovarian cDNA libraries, and any other necessary libraries, may be used. Screen the library.

Other variants of direct selection of the subject cDNA have also been used to find candidate genes for BRCA1 (Lovett et al., 1991; Futreal, 1993). This method uses cosmid, P1 or BAC DNA as a probe. The probe DNA is digested with a blunt restriction enzyme such as HaeIII. A double-stranded adapter is then ligated to the DNA, which acts as a primer binding site in a subsequent PCR amplification reaction using biotinylated primers. Target cDNA is generated from a tissue sample, eg, mRNA from breast tissue, by either first-strand random or oligo (dT) -origin synthesis followed by second-strand synthesis. The ends of the cDNA are made blunt and ligated to a double-stranded adapter. These adapters act as amplification sites for PCR. Denaturing the target and probe sequences, blocking the repetitive sequences was mixed with human C _{0 t-1} DNA. Solution hybridization is set to a high C ₀ t-1 / 2 value to ensure hybridization of rare target cDNA molecules. The annealed material is then captured on avidin beads, washed with high stringency, and the retained cDNA is eluted and amplified by PCR. The selected cDNA is subjected to a further round of expansion before cloning into a plasmid vector for analysis.

Testing the cDNA for Candidateness The cDNA is the BRCA1 locus obtained by finding sequences in DNA extracted from affected family members that produce abnormal BRCA1 gene products or abnormal levels of BRCA1 gene products. Prove that. Such BRCA1 susceptibility alleles will be co-segregating for the disease in a wide variety of kindreds. It will also be more common in non-family individuals with breast and ovarian cancer, then in individuals in the general population. Finally, since tumors are often somatically mutated in other instances at germline mutated loci, we have identified the same or the same as the BRCA1 sensitive allele in DNA extracted from tumor tissue. It is expected to find normal germline BRCA1 alleles that have been mutagenized to similar sequences. Whether comparing BRCA1 sequences from tumor tissue to BRCA1 alleles from the germline of the same individual, or comparing germline BRCA1 alleles from cancer cases to those from non-affected individuals, The key is to find mutations that are severe enough to disrupt the normal function of the gene product. These mutations can take many forms. The most serious forms are frameshift mutations or widespread deletions, which will cause the gene to encode an abnormal protein or to express a significantly altered protein. Less severe disruptive mutations include small in-frame deletions and non-conservative base pair substitutions, which include changes to or from cysteine residues, from basic amino acids to acidic amino acids. To or from a hydrophobic amino acid to a hydrophilic amino acid, or vice versa, or other mutations that may affect the secondary, tertiary or quaternary structure of the protein. Will have a significant impact. Those that produce silent mutations or conservative amino acid substitutions are generally not expected to disrupt protein function.

According to the diagnosis and the preliminary diagnosis of the present invention, a change in the wild-type BRCA1 locus is detected. In addition, the method can be performed by detecting the wild-type BRCA1 locus and confirming the lack of a predisposition to cancer at the BRCA1 locus. "Wild-type gene alterations" includes all forms of mutations, including deletions, insertions and point mutations in coding and non-coding regions. Deletions can be of the entire gene or only a portion of the gene. Point mutations can result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations occur only in certain tissues, such as tumor tissues, and are not inherited in the germline. Germline mutations can be found in any tissue of the body and are inherited. If only a single allele is somatically mutated, an early neoplastic state is indicated. However, if both alleles are somatically mutated, a late neoplastic state is indicated. Thus, the finding of a BRCA1 mutation provides both diagnostic and prognostic information. Undeleted BRCA1 alleles (eg, found on the sister chromosome of a chromosome carrying a BRCA1 deletion from a sister chromosome) can be screened for other mutations such as insertions, small deletions, and point mutations. . It is believed that many mutations found in tumor tissue will lead to reduced expression of the BRCA1 gene product. However, mutations that lead to non-functional gene products will also lead to cancerous conditions. Point mutation events can occur in regulatory regions, such as at the promoter of a gene, leading to a loss or decrease in mRNA expression. Point mutations can also disrupt proper RNA processing, leading to a loss of expression of the BRCA1 gene product or a decrease in mRNA stability or translation efficiency.

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

癌 Cancer predisposition, such as breast and ovarian cancer and other cancers identified herein, can be confirmed by testing any human tissue for a mutation in the BRCA1 gene. For example, individuals who have inherited the germline BRCA1 mutation will be predisposed to developing cancer. This can be determined by testing DNA from any tissue of the individual's body. Most simply, blood is collected and DNA is extracted from cells of the blood. In addition, prenatal diagnosis may be made by testing fetal, placental or amniotic cells for BRCA1 gene mutations. Changes in the wild-type BRCA1 allele, whether due to point mutations or deletions, 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 variations. For genes as large as BRCA1, it is very hard work, but under optimal conditions mutations in the coding sequence of the gene are rarely missed. Another approach is the single-stranded structural polymorphism assay (SSCA) (Orita et al., 1989). This method does not detect all sequence changes, especially when the DNA fragment is larger than 200 bp, but can be optimized to detect most DNA sequence variations. The disadvantage of reduced detection sensitivity is disadvantageous, but the increased throughput possible with SSCA makes it an attractive, possible alternative to direct sequencing for study-based mutation detection. Fragments with shifted mobility on SSCA gels are then sequenced to determine the nature of the exact DNA sequence variation. Other approaches based on the detection of mismatches between two complementary DNA strands include clamped denaturing denaturing gel elctrophoresis (CDGE) (Sheffield et al., 1991), heteroduplex analysis. (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, duplex formations or insertions, or will not detect regulatory mutations that affect protein transcription or translation. Other methods that can detect these classes of mutations, such as protein truncation assays or asymmetric assays, will detect only specific types of mutations and will not detect missense mutations. A review of currently available methods for detecting DNA sequence variation can be found in a recent review by Grompe (1993). Once the mutation is identified, an allele-specific detection approach, such as allele-specific oligonucleotide (ASO) hybridization, can be used to rapidly screen a vast number of other samples for the mutation. .

In order to detect changes in the wild-type BRCA1 gene in tissues, it is useful to isolate tissues released from surrounding normal tissues. Methods for expanding tissue preparations of tumor cells are known in the art. For example, the tissue can be isolated from paraffin or cryostat sections. Cancer cells can 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 heavily contaminated with normal tissue, detection of the mutation is more difficult.

Rapid preliminary analysis to detect polymorphisms in DNA sequences can be performed by viewing a series of Southern blots of DNA cut with one or more restriction enzymes, preferably a number of restriction enzymes. Each blot contains a series of normal individuals and a series of cancer cases, tumors, or both. Southern blots showing hybridized fragments (different in length than the control when probed at or near the BRCA1 locus) show possible mutations. When using a restriction enzyme that generates a very large restriction fragment, pulse field electrophoresis (PFGE) is used.

Detection of point mutations can be accomplished by molecular cloning of the BRCA1 allele (s) and sequencing the allele (s) using techniques well known in the art. Alternatively, the gene sequence may be amplified directly from genomic DNA preparations from tumor tissue using known techniques. The DNA sequence of the amplified sequence can then be determined.

There are six well-known methods of testing that are more complete and still indirect to confirm the presence of susceptibility alleles: 1) Single-strand formation structural analysis (SSCA) (Orita et al., 1989). 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989); 3) RAase protection assay (Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specific oligonucleotides (AOS) (Conner et al., 1983); 5) Use of proteins that recognize nucleotide mismatches, such as the E. coli mutS protein (Modrich, 1991); and 6) Allele-specific PCR ( Rano and Kidd, 1989). Allele-specific PCR uses primers that hybridize to a particular BRCA1 mutation at its 3 'end. If no particular BRCA1 mutation is present, no amplification product is found. The Amplification Resistance Mutation System (ARMS) disclosed in EP 0 332 435 and Newton et al., 1989 may also be used.

Gene insertions and deletions can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polymorphism (RFLP) probes of the gene or surrounding marker gene may be used to record allelic changes or insertions in polymorphic fragments. Such methods are particularly useful for screening relatives of the affected individual for the presence of the BRCA1 mutation found in the individual. Other techniques for detecting insertions and deletions known in the art may be used.

In the first three methods (SSCA, DGGE and RAase protection assays), new electrophoretic bands appear. SSCA detects bands that move differently. This is because a change in sequence causes a difference in single-stranded, ie, intramolecular base pairing. RAase protection involves cleavage of the mutated polynucleotide into two or more smaller fragments. DGGE uses a denaturing gradient gel to detect differences in the migration rate of the mutated sequence compared to the wild-type sequence. In an allele-specific oligonucleotide assay, the oligonucleotide is designed to detect a specific sequence and is performed by detecting the presence or absence of a hybridization signal.
In the mutS assay, the protein binds only to sequences that contain nucleotide mismatches in the heteroduplex between the mutant and wild-type sequences.

誤 Mismatch according to the invention is a hybridized nucleic acid double helix where the two strands are not 100% complementary. Deletions of total complementarity can result from deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in a gene or its mRNA product. Although these techniques are less sensitive than sequencing, they are simpler to perform on large numbers of tumor samples. One example of a mismatch cutting technique is the RAase protection law. In the practice of the present invention, the method involves the use of a labeled riboprobe that is complementary to the human wild-type BRCA1 gene coding sequence. The riboprobe is annealed (hybridized) together with either mRNA or DNA isolated from tumor tissue, followed by the enzyme RNase A, which can detect certain mismatches in the duplex RNA structure. Digest. If a mismatch is detected by RAase A, it cleaves at the site of the mismatch. Thus, if the annealed RNA preparations are separated on an electrophoretic gel matrix, if mismatches are detected and cleaved by RNase A, the riboprobes and the RNA are smaller than the full-length duplex RNA of mRNA or DNA. The product will be shown. The riboprobe need not be a full-length BRCA1 mRNA or gene, but can be any segment thereof. If the riboprobes contain only BRCA1 mRNA or a segment of the gene, it may be desirable to use a large number of these probes to screen the entire mRNA sequence for mismatches.

Similarly, DNA probes can be used to detect mismatches through enzymatic or chemical cleavage (see, eg, Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986). Alternatively, mismatches can be detected by a shift in the electrophoretic mobility of the mismatched duplex to the matched duplex (see, eg, Cariello, 1988). Whether using riboprobes or DNA probes, cellular mRNA or DNA that may contain mutations can be amplified using PCR (see below) before hybridization. Changes in the DNA of the BRCA1 gene can also be detected using Southern hybridizations, especially if the change is an entire rearrangement, such as a deletion or insertion.

ＤＮＡ The DNA sequence of the BRCA1 gene amplified by using PCR can also be screened using an allele-specific probe. These probes are nucleic acid oligomers, each of which contains a region of the BRCA1 gene sequence that harbors a known mutation. For example, one oligomer may be about 30 nucleotides in length, corresponding to a portion of the BRCA1 gene sequence. By using such a set of allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in the BRCA1 gene. Hybridization of the amplified BRCA1 sequence with an allele-specific probe can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the mutation in tumor tissue as in an allele-specific probe.最 も The clearest test for mutations in candidate loci is to directly compare genomic BRCA1 sequences from a control population with those from cancer patients. Alternatively, the mRNA can be sequenced after amplification, for example by PCR, thereby eliminating the need to determine the exon structure of the candidate gene.

突然 変 異 Mutations from cancer patients that fall outside of the BRCA1 coding region can be detected by examining non-coding regions, such as introns and regulatory sequences near or in the BRCA1 gene. Early suggestions that mutations in non-coding regions are important may come from Northern blot experiments that reveal abnormally sized or abundant mRNA molecules in cancer patients compared to control individuals.

変 化 A change in BRCA1 mRNA expression may be detected by any technique known in the art. These include Northern blot analysis, PCR amplification and RAase protection. A decrease in mRNA expression indicates a change in the wild-type BRCA1 gene. Changes in the wild-type BRCA1 gene can also be detected by screening for changes in the wild-type BRCA1 protein. For example, tissues can be screened using a monoclonal antibody that is immunoreactive with BRCA1. Lack of cognate antigen would indicate a BRCA1 mutation. The mutant BRCA1 gene product can also be detected using an antibody specific for the product of the mutant allele. Such immunological assays can be performed in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA assays. Any means of detecting an altered BRCA1 protein can be used to detect a change in the wild-type BRCA1 gene. Functional assays such as protein binding measurements can be used. In addition, assays that detect BRCA1 biochemical function can be used. The discovery of a mutated BRCA1 gene product indicates an alteration in the wild-type BRCA1 gene.

A mutant BRCA1 gene or gene product can also be detected in other human body samples, such as serum, stool, urine and saliva. The same techniques discussed above for detecting a mutant BRCA1 gene or gene product in a tissue may be applied to other body samples. Cancer cells molt from the tumor and appear in such body samples. In addition, the BRCA1 gene product itself can be secreted into the extracellular space and can be found in these body samples in the absence of cancer cells. By screening such body samples, a simple and early diagnosis can be achieved for many types of cancer. In addition, by testing such body samples for a mutated BRCA1 gene or gene product, the progress of chemotherapy or radiation therapy can be more easily monitored.

診断 The diagnostic method of the present invention can be applied to any tumor in which BRCA1 has a role in tumor development. The diagnostic methods of the present invention are useful to the clinician, who can make decisions during the appropriate course of treatment.

プ ラ イ マ ー The primer pair of the present invention is useful for determining the nucleotide sequence of a specific BRCA1 allele using PCR. The pair of single-stranded DNA primers can anneal to sequences in or around the BRCA1 gene on chromosome 17q21 to initiate amplified DNA synthesis of the BRCA1 gene itself. The complete set of these primers permits the synthesis of all nucleotides of the BRCA1 gene coding sequence, ie, exons. The primer set preferably allows for the synthesis of both intron and exon sequences. Allele-specific primers may also be used. Such primers will only anneal to a particular BRCA1 mutant allele and will therefore only amplify the product in the presence of the mutant allele as a template.

プ ラ イ マ ー To facilitate subsequent cloning of the amplified sequence, the primer may have a restriction enzyme site sequence added to its 5 'end. Thus, all nucleotides of the primer are derived from the BRCA1 sequence or a sequence near BRCA1, except for a few nucleotides required to form a restriction enzyme site. The primers themselves can be synthesized using techniques well known in the art. Generally, the primers can be made using commercially available oligonucleotide synthesizers. The sequence of the BRCA1 open reading frame is shown in SEQ ID NO: 1. The design of a particular primer is well within the skill of the art.

核酸 The nucleic acid probe provided by the present invention is useful for many purposes. It can be used in Southern hybridization to genomic DNA, and in RAase protection methods to detect point mutations already discussed above. The probe can be used to detect PCR amplification products. It can also be used to detect mismatches with the BRCA1 gene or mRNA using other techniques.

個人 Individuals with the wild-type BRCA1 gene were found to have no cancer arising from the BRCA1 allele. However, mutations that disrupt BRCA1 protein function have been implicated in cancer pathology. Thus, the presence of an altered (or mutated) BRCA1 gene that has lost function or produces a protein with altered function is directly related to an increased risk of cancer. To detect a BRCA1 gene mutation, a biological sample is prepared and the difference between the sequence of the BRCA1 allele to be analyzed and the sequence of the wild-type BRCA1 allele is analyzed. Mutant BRCA1 alleles can be initially identified by any of the techniques described above. The mutant allele is then sequenced to identify specific mutations of the particular mutant allele. Alternatively, a mutated BRCA1 allele may be identified initially by identifying the mutated (altered) BRCA1 protein using conventional techniques. The mutant alleles are then sequenced to identify specific mutations in each allele. Mutations, particularly those that lead to altered function of the BRCA1 protein, are then used in the diagnostic and prognostic methods of the invention.

Definitions The present invention uses the following definitions:
For "polynucleotide amplification", methods such as polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction (LCR)) and amplification methods based on the use of Q-beta replicase are used. These methods are well known and widely practiced in the art (eg, US Pat. Nos. 4,683,195 and 4,683,202, and Innis et al., 1990 (for PCR); and Wu (for LCR). Et al., 1989a). Reagents and hardware for performing PCR are commercially available. Primers useful for amplifying sequences from the BRCA1 region are preferably complementary to and specifically hybridize to sequences in the BRCA1 region, or regions in the region adjacent to the target region therein. I do. BRCA1 sequences generated by amplification can be sequenced directly. Alternatively, but less preferably, the amplified sequence (s) can be cloned prior to sequence analysis. Direct cloning and sequence analysis of enzymatically amplified genomic segments has been described by Scharf, 1986.

"Analytical polynucleotide" and "analytical strand" refer to single- or double-stranded polynucleotides that are expected to contain a target sequence and may be present in various types of samples, including biological samples.

"Antibodies"; the invention also provides polyclonal and / or monoclonal antibodies and fragments thereof, and immunological binding equivalents thereof, which include BRCA1 polypeptides and fragments thereof, or, particularly, from the BRCA1 region. It can specifically bind to a polynucleotide sequence from the BRCA1 locus or a portion thereof. The term "antibody" is used to refer to a homogeneous molecular group or a mixture, such as a serum product composed of a plurality of different molecular groups. Polypeptides can be prepared synthetically in a peptide synthesizer, coupled to a carrier molecule (such as keyhole-adsorbing hemocyanin), and injected into rabbits for several months. Rabbit sera is tested for immunoreactivity to the BRCA1 polypeptide or fragment. Monoclonal antibodies can be made by injecting mice with a protein polypeptide, a fusion protein, or a fragment thereof. Monoclonal antibodies will be screened by ELISA and tested for specific immunoreactivity with the BRCA1 polypeptide or fragment thereof (see Harlow and Lane, 1988). These antibodies will be useful in assays as well as in medicine.

を 得 Once a sufficient amount of the desired polypeptide has been obtained, it can be used for various purposes. A typical application is the production of antibodies specific for binding. These antibodies, either polyclonal or monoclonal, can be made by in vitro or in vivo techniques well known in the art. For the production of polyclonal antibodies, a suitable target immune system is selected, typically a mouse or rabbit. Substantially pure antigen is presented to the immune system in a manner determined by animals appropriate methods or by other indicators familiar to immunologists. Typical sites for injection are foot pads intramuscularly, intraperitoneally or intradermally. Of course, other species may be substituted for mice or rabbits. The polyclonal antibody is then purified using techniques known in the art and adjusted to the desired specificity.

Immunological responsiveness is usually assayed in an immunoassay. Usually, such immunoassays include, for example, certain purifications of the antigen source, produced in the same cell as the antigen and by a similar formula. Various immunoassays are well known in the art (see, for example, Harlow and Lane, 1988 or Goding, 1986).
Monoclonal antibodies having 10 ⁻⁸ M ⁻¹ , or preferably 10 ⁻⁹ to 10 ⁻¹⁰ M ⁻¹ or higher affinity, are typically described, for example, in Harlow and Lane, 1988 or Goding, 1986. Will be made by standard methods. Briefly, appropriate animals are selected and the desired immunization protocol is followed. After an appropriate time, the spleen of such animal is excised and individual spleen cells are fused to immortalized melanoma cells under appropriate selection conditions. Thereafter, the cells are clonally separated and the supernatant of each clone is tested for their production of appropriate antibodies specific for the desired region of the antigen.

Other suitable techniques include in vitro exposure of lymphocytes to antigenic polypeptides or, alternatively, to a selected library of antibodies in phage or similar vectors (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, either covalently or non-covalently, by attaching a substance that provides a detectable signal. A wide variety of labeling and conjugate technologies are known and have been widely reported 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. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins can be produced (see US Pat. No. 4,816,567).

"Binding partner" refers to a molecule that has the ability to bind a ligand molecule with high specificity, such as, for example, antigens and antigen-specific antibodies or enzymes and inhibitors thereof. Generally, the specific binding partner must bind with sufficient affinity under isolation conditions to immobilize the analyte copy / (in the case of polynucleotide hybridization) the complementary duplex. Must. Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, a vast array of known receptor-ligand couplings and complementary polynucleotide chains. In the case of a complementary polynucleotide binding partner, the partner will typically be at least about 15 bases in length, at least 40 bases in length. The polynucleotide may be composed of DNA, RNA or synthetic nucleotide analogs.

"Biological sample" refers to a sample of tissue or fluid that is expected to contain an analyte polynucleotide or polypeptide from an individual, including, but not limited to, plasma, serum, cerebrospinal fluid, lymph, skin , External respiratory, intestinal and genitourinary tracts, tears, saliva, erythrocytes, tumors, organs, tissues and samples of in vitro cell culture constructs.

As used herein, the term "diagnosis" or "pre-diagnosis" in the context of neoplasms includes, before, during and after treatment, 1) classification of lesions as neoplasms, 2) measurement of neoplastic extent, Or 3) Used to indicate monitoring of disease progression.

A polynucleotide that "encodes"; when a polynucleotide "encodes" a polypeptide is manipulated in its natural state or by methods well known to those of skill in the art, it is capable of encoding mRNA and / or the polypeptide or a fragment thereof. It can be transcribed and / or translated. The antisense strand is the complement of such a nucleic acid, and the sequence code predictable therefrom.

"Isolated" or "substantially pure"; "isolated" or "substantially pure" nucleic acid (e.g., RNA, DNA or mixed polymer) is a native human sequence or protein (e.g., Ribosomes, polymerases, and many other human genomic sequences and proteins) are substantially isolated from other cellular components with which they naturally accompany. The term encompasses nucleic acid sequences and proteins removed from the naturally occurring environment and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by foreign systems. included.

"BRCA1 alleles" are normal alleles at the BRCA1 locus, as well as alleles carrying variants that cause a predisposed individual to develop cancer at many sites, including, for example, breast, ovarian, colorectal and prostate cancer. Say. Such predisposition alleles are also referred to as "BRCA1 susceptibility alleles."

"BRCA1 locus", "BRCA1 gene", "BRCA1 nucleic acid" or "BRCA1 polynucleotide" each refer to a polynucleotide, all of which are present in the BRCA1 region, which can be used to treat an individual with breast cancer, ovarian cancer, It appears that normal tissues express certain alleles predisposing to colorectal and prostate cancer. Mutations in BRCA1 may be involved in the development and / or progression of other types of tumors. The locus is indicated in part by a mutation that causes cancer in a predisposed individual. These mutations fall within the BRCA1 region described below. The BRCA1 locus is intended to contain coding sequences, intervening sequences and regulatory elements that control transcription and / or translation. The BRCA1 locus is intended to include all allelic variations of the DNA sequence.

As applied to nucleic acids, these terms refer to nucleic acids that encode a BRCA1 polypeptide, fragment, equivalent, or variation, and include, for example, protein fusions or deletions. The nucleic acids of the invention can be derived from, or have substantially the same sequence as, a native BRCA1-encoding gene, or those that have substantial homology to a native BRCA1-encoding gene or a portion thereof. Will have. The coding sequence of a BRCA1 polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1.

The polynucleotide compositions of the present invention include RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both sense or antisense, as will be readily understood by those of skill in the art. Can be modified chemically or biochemically, or can include non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylates, substitutions of one or more naturally occurring nucleotide analogs (e.g., such as methyl phosphonate, phosphotriester, phosphoamidate, carbamate, and the like). Internucleotide modifiers such as uncharged linkages, charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant groups (e.g., polypeptides), (e.g., acridine, psoralen, etc.) ) Intercalators, chelators, alkylating agents, and modified linkages (such as, for example, alpha anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic the polynucleotides with the ability to bind to the specified sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those that use peptide bonds instead of phosphate bonds in the molecular backbone.

The present invention provides a recombinant nucleic acid containing the whole or a part of the BRCA1 region. The recombinant construct can replicate autonomously in the host cell. Alternatively, the recombinant construct may become integrated into the chromosomal DNA of the host cell. Such a recombinant polynucleotide may, by its source or operation, 1) not bind to all or a portion of the polynucleotide to which it naturally binds; 2) bind to a polypeptide other than that to which it binds naturally; Does not occur, including polynucleotides of genomic, cDNA, semi-synthetic or synthetic origin.

Thus, there is provided by the present invention a recombinant nucleic acid comprising other sequences which do not occur in nature. The wild-type sequence can be used, but will often have been modified, for example, by deletion, substitution or insertion.

Various types of cDNA or genomic libraries may be screened as the natural source of the nucleic acids of the invention, or such nucleic acids may be provided by amplification of genomic DNA or other naturally occurring sequences (e.g., by PCR). obtain. The selection of a cDNA library usually corresponds to a tissue source rich in mRNA for the desired protein. Although phage libraries are usually preferred, other types of libraries can be used. Library clones are spread on plates, transferred to screening substrates, denatured, and picked for the presence of the desired sequence.

ＤＮＡ The DNA sequence used in the present invention usually contains at least about 5 codons (15 nucleotides), more usually at least 7-15 codons, and most preferably at least about 35 codons. Also, one or more introns may be present. This number of nucleotides is usually about the minimum length required for a successful probe to hybridize specifically with the BRCA1-coding sequence.

技術 Techniques for manipulating nucleic acids are generally described, for example, in Sambrook et al., 1989 or Ausbel et al., 1992. Reagents that apply to such techniques, such as restriction enzymes, are widely known in the art and include New England BioLabs, Boehringer Mannheim, Amersham, Promega Biotec, US Biochemicals, New England Nuclear and others. Are commercially available from vendors, such as many sources. Recombinant nucleic acid sequences used to make the fusion proteins of the invention can be derived from natural or synthetic sequences. Using the appropriate probes, many natural gene sequences can be obtained from various cDNA or genomic libraries (see GenBank, National Institute of Health).

"BRCA1 region" refers to the portion of human chromosome 17q21 bound by markers tdj1474 and U5R. This region includes the BRCA1 locus containing the BRCA1 gene.

As used herein, “BRCA1 locus”, “BRCA allele” and “BRCA region” all include a locus, a double-stranded DNA containing an allele or region, and a locus, allele or region. Refers to any single-stranded DNA.

As used herein, a “portion” of a BRCA1 locus or region or allele is defined as having a minimum size of at least about 8 nucleotides, or preferably about 15 nucleotides, or more preferably at least about 25 nucleotides. , And a minimum size of at least about 40 nucleotides.

"BRCA1 protein" or "BRCA1 polypeptide" refers to a protein or polypeptide encoded by the BRCA1 locus, a variant or fragment thereof.

The term "polypeptide" refers to an amino acid polymer or its equivalent, and not to products of a specific length; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. The term also does not refer to or exclude, for example, glycosylation, acetylation, phosphorylation, or other modifications of the polypeptide. Included within the definition are polypeptides containing one or more analog amino acids, naturally occurring and non-naturally occurring (including, for example, unnatural amino acids, etc.), polypeptides having substitution bonds, and And other modifications known. Usually, such polypeptides are at least about 50% homologous, preferably greater than about 90%, and more preferably at least about 95% homologous to the native BRCA1 sequence. Also included are highly similar polypeptides or proteins that hybridize to BRCA1-encoding nucleic acids under high or low stringency conditions and are recovered by antisera to the BRCA1 protein (s).

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

"Operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting their intended mode of function. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

"Probe"; a predisposition to or predisposition to a particular cancer that can be detected by hybridization of a polynucleotide probe that forms a stable hybrid with the polypeptide of the target sequence under appropriate stringent hybridization and washing conditions. Refers to a polynucleotide polymorphism associated with the BRCA1 allele associated with cancer. If the probe is expected to be completely complementary to the target sequence, stringent conditions will be used. For example, hybridization stringency may be reduced if some mismatches are expected, such as when the variants are expected to have results that are not perfectly complementary. Choose conditions that eliminate non-specific / accidental binding, ie, minimize noise. Such suggestions identify natural DNA polymorphisms as well as mutations, and these suggestions need to be further analyzed to establish the detection of a BRCA1 sensitive allele.

The プ ロ ー ブ BRCA1 allele probe may be derived from the sequence of the BRCA1 region or its cDNA. The probe may span any or all of the BRCA1 region and may be of any suitable length to allow specific hybridization to the BRCA1 region. If the target sequence comprises the same sequence as the probe, the probe may be shortened, for example, to a range of about 8-30 base pairs. Since the hybrid will be relatively stable even under stringent conditions. If some degree of mismatch with the probe is expected, ie, the probe is expected to hybridize to various regions, a longer probe that hybridizes to the target sequence with the required specificity may be used.

Probes include isolated polynucleotides that bind to a label or reporter molecule and may be used to isolate other polynucleotide sequences having sequence similarity by standard methods. See, eg, Sambrook et al., 1989 or Ausbel et al., 1992 for techniques for probe preparation and labeling. Other similar polynucleotides can be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides may be synthesized or selected by using duplication in the genetic code. For example, various codon substitutions may be introduced by silent changes (thus creating various restriction sites) or by optimizing the particular system. Mutations can be introduced to modify the properties of the polypeptide, possibly altering ligand-binding affinity, interchain affinity or altering polypeptide degradation or turnover rates.

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

A portion of the polynucleotide sequence from the polynucleotide sequence encoding BRCA1 having at least about 8 nucleotides, usually at least about 15 nucleotides, and less than about 6 kb, usually less than about 1.0 kb, is preferred as a probe. In addition, the probe can be used to determine whether mRNA encoding BRCA1 is present in a cell or tissue.

A "protein modification or fragment" provided by the present invention for a BRCA1 polypeptide or fragment thereof is substantially homologous to the primary structural sequence, for example, in vivo or in vitro. (in vitro) chemical and biochemical modifications or those incorporating unusual amino acids. Such modifications will be readily appreciated by those skilled in the art, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling (e.g., with radionuclides), and various enzymatic modifications Is included. Various methods of labeling polypeptides useful for such purpose and their substitution or labeling are well known in the art and include radioisotopes such as ³² P, which are specific binding partners for labeled ligands. Binds labeled anti-ligands (eg, antibodies), fluorophores, chemical chromogenic agents, enzymes and anti-ligands that can act as members. The choice of label depends on the sensitivity required, ease of primer and conjugation, stability requirements and available equipment. Methods for labeling polypeptides are well known in the art (see, for example, Sambrook et al., 1989 or Ausbel et al., 1992).

に In addition to substantially full-length polypeptides, the present invention provides biologically active fragments of pseudopolypeptides. Important biological activities include BRCA1 polypeptide ligand-binding, immunological activity and other biologically active properties. Immunological activity includes both the immunogenic function of the target immune system, as well as sharing immunological epitopes for binding and acting as either a competition or replacement antigen for epitopes on the BRCA1 protein. It is. As used herein, “epitope” refers to an antigenic determinant of a polypeptide. An epitope may include three amino acids in a spatial configuration unique to the epitope. Generally, an epitope consists of at least 5 such amino acids, and more usually, at least 8-10 such amino acids. Methods for determining the spatial configuration of such amino acids are known in the art.

For immunological purposes, tandemly repeated polypeptide segments can be used as immunogens, thereby producing highly antigenic proteins. Alternatively, such polypeptides will also act as highly efficient competitors for specific binding. The generation of antibodies specific for a BRCA1 polypeptide or a fragment thereof is described below.

本 The present invention also provides fusion polypeptides, including BRCA1 polypeptides and fragments. A homopolypeptide may be a fusion between two or more BRCA1 polypeptide sequences, or between a BRCA1 sequence and a related protein. Similarly, heterofusions can be constructed that will exhibit a combination of properties or activities of the inducible protein. For example, ligand-binding or other domains may "swap" between different new fusion polypeptides or fragments. Such homo- or hetero-fusion polypeptides can exhibit, for example, altered strength or specificity binding. Fusion partners include immunoglobulins, bacterial β-galactosidase, trpE, protein A, β-lactamase, α-amylase, alcohol dehydrogenase and yeast alpha mating factor (see, eg, Godowski et al., 1988).

Fusion proteins can typically be prepared by any of the recombinant nucleic acid methods described below, or can be chemically synthesized. Techniques for synthesizing polypeptides are described, for example, in Merrifield, 1963.

"Protein purification" refers to various methods for isolating a BRCA1 polypeptide from other biological materials, such as from cells transformed with a recombinant nucleic acid encoding BRCA1, and is well known in the art. Method. For example, such polypeptides can be purified, for example, by immunoaffinity chromatography using the antibodies provided by the invention. Various methods for 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 interchangeable to describe the separation of proteins or polypeptides from their associated components in nature. Used for A monomeric protein is substantially pure if at least about 60-75% of the sample exhibits a single polypeptide sequence. A substantially pure protein will typically include a protein sample of about 60% to 90% W / W, more usually about 95%, and preferably more than about 99% pure. Protein purity or homogeneity can be indicated by a number of means well known in the art, such as visualization of a single polypeptide band upon staining of the gel following polyacrylamide gel electrophoresis of a protein sample. For certain purposes, high resolution may be provided by using HPLC or other means well known in the art to utilize for purification.

The BRCA1 protein is substantially free of naturally related components when it is separated from its natural contaminants in its natural state. Thus, a polypeptide that is chemically synthesized or synthesized in a cell line different from the cell from which it naturally originates will be substantially free of its naturally associated components. Proteins can be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

ポ リ A polypeptide that is produced as an expression product of an isolated and engineered gene sequence is an “isolated polypeptide” as used herein, even when expressed in a homologous cell type. Synthetic production forms or molecules expressed by heterologous cells are naturally isolated molecules.

"Recombinant nucleic acid" is a nucleic acid that is not naturally occurring or that has been created by the artificial combination of two other discrete segments of a sequence. This artificial combination is often made by any chemical synthetic means or by artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. Such is usually done by replacing the codon with an extra codon encoding the same or a conserved amino acid, but typically introducing or removing a sequence recognition site. Alternatively, it is performed to combine nucleic acid segments of a desired function together to produce a desired combination of functions.

“Regulatory sequence” usually refers to a sequence within 100 kb of the coding region of a locus, but it may be further away from the coding region, which may include (gene transcription and mRNA translation, splicing, stabilization, (Including gender).

"Substantially homologous or similar"; when optimally aligned (with appropriate nucleotide insertions or deletions) with another nucleic acid (or its complement), at least about 60% of the nucleotide bases, usually at least A nucleic acid or fragment thereof is another if there is nucleotide sequence identity of about 70%, more usually at least about 80%, preferably at least about 90%, more preferably at least about 95-98% nucleotide bases. "Substantially homologous" ("or substantially similar").

Alternatively, if the nucleic acid or fragment thereof hybridizes to another nucleic acid (or its complement) under hybridization conditions selective for the strand or its complement, substantial complementarity or (identical) Sex) exists. Hybridization selectivity exists when hybridization occurs that is substantially more selective than the overall lack of specificity. Typically, selective hybridization is performed when at least about 55% homology exists over at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% of the stretch. (See Kanehisa, 1984). The length of the described homology comparisons can also be longer stretches, and in certain embodiments, often will be at least about 9 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically Will span a stretch of at least about 28 nucleotides, more typically at least about 32 nucleotides, preferably at least about 36 or more nucleotides.

Nucleic acid hybridization is well understood by those skilled in the art, as well as base composition, complementary strand length, and the number of nucleotide base mismatches between hybridizing nucleic acids, as well as salt concentration, temperature, Or it will be affected by conditions such as organic solvents. Stringent temperature conditions will generally include temperatures above 30 ° C, typically above 37 ° C, preferably above 45 ° C. Stringent salt conditions will usually be less than 1000 mM, typically less than 500 mM, preferably less than 200 mM. However, the combination of indicators is much more important than the measurement of any single indicator (see, eg, Wetmur and Davidson, 1968). The probe sequence may also specifically hybridize to the duplex DNA under certain conditions to form a triple helix or other higher order DNA complex. Preparation of such probes and appropriate hybridization conditions are well known in the art.

The terms "substantial homology" or "substantial identity" when referring to a polypeptide, are at least about 30% identical, usually at least about 30%, identical to the naturally occurring whole protein or a portion thereof, in which the polypeptide or protein in question is naturally occurring. Indicates 70% identity, preferably at least about 95% identity.

"Substantially the same function" refers to a function of a modified nucleic acid or a modified protein with respect to a wild-type BRCA1 nucleic acid or a wild-type BRCA1 polypeptide. The modified polypeptide will be substantially homologous to the wild-type BRCA1 polypeptide and will have substantially the same function. Modified polypeptides have an altered amino acid sequence and / or can include modified amino acids. In addition to functional identity, modified polypeptides may have other useful properties, such as a longer half-life. The identity of the function (activity) of the modified polypeptide can be substantially the same as the activity of the wild-type BRCA1 polypeptide. Alternatively, the identity of the function (activity) of the modified polypeptide may be higher than the activity of the wild-type BRCA1 polypeptide. Modified polypeptides are synthesized using conventional techniques, or are encoded by modified nucleic acids and are produced using conventional techniques. Modified nucleic acids are prepared by conventional techniques. A nucleic acid having a function substantially similar to the wild-type BRCA1 gene function produces the modified protein described above.

O Polypeptide homology is typically measured using sequence analysis software (see, for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705). Protein analysis software pairs similar sequences using similarity measures, specifying various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions in the following group: 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 at least about 5 to 7 contiguous amino acids, often at least about 7 to 9 contiguous amino acids, typically at least about 9 to 13 contiguous amino acids. Stretches of amino acid residues of contiguous amino acids, most preferably at least about 20-30 or more contiguous amino acids.

The polypeptides of the present invention, when soluble, such as, for example, nitrocellulose, nylon, column packing (eg, Sepharose beads), magnetic beads, glass wool, plastics, metals, polymer gels, cells or other substrates. It can be coupled to a solid support. Such a support may take the form of, for example, beads, wells, dipsticks or membranes.

"Target region" refers to a region of a nucleic acid to be amplified and / or detected. The term "target sequence" refers to a sequence that will form a stable hybrid with a probe or primer under desired conditions.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics and immunology (eg, Maniatis et al., 1982; Sambrook et al., 1989; Ausubel). Glover, 1985; Anand, 1992; Guthrie and Fink, 1991). A general discussion of techniques and materials for human gene mapping, including mapping of human chromosome 17q, is described in, for example, White and Lalouel, 1988.

Preparation of recombinant or chemically synthesized nucleic acids; vectors, transformations, host cells Large quantities of the polynucleotides of the invention may be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments encoding the desired fragments are introduced into prokaryotic or eukaryotic cells and introduced into a recombinant polynucleotide construct, usually a DNA construct, capable of replicating. Usually, the polynucleotide construct will be suitable for replication in a unicellular host, such as a yeast or bacterium, but will be cultivated (integrated or not integrated into the genome) in a mammalian or plant or other eukaryotic state. It can also be intended for introduction into biological cell lines. Purification of nucleic acids produced by the method of the invention is described, for example, in Sambrook et al., 1989 or Ausubel et al., 1992.

The polynucleotides of the present invention can be made, for example, by chemical synthesis by the phosphoramidite method described by Beaucage and Carruthers, 1981, or by the triester method of Matteucci and Carruthers, 1981, and are commercially available on an automated oligonucleotide synthesizer. You can do it too. Double-stranded fragments are synthesized by synthesizing complementary strands and annealing the strands together under appropriate conditions, or by adding the complementary strands using a DNA polymerase with appropriate primer sequences. , Can be obtained from a single strand of chemical synthesis.

A polynucleotide construct prepared for introduction into a prokaryotic or eukaryotic host can include a replication system recognized by the host, including the polynucleotide fragment of interest encoding the desired polynucleotide, preferably the polynucleotide. It will also include transcriptional and translational initiation regulatory sequences operably linked to the coding segment. Expression vectors include, for example, an origin of replication or an autonomously replicating sequence (ARS), and expression control sequences, promoters, such as ribosome-binding sites, RNA splicing sites, polyadenylation sites, transcription terminator sequences and mRNA stabilizing sequences. Enhancers and essential processing information sites may be included. The secretion signal, suitably from either the native BRCA1 protein or from another receptor, or from a secreted polypeptide of the same or related species, transduces and / or binds the protein. And thus achieve those functional topologies or may be secreted from cells. Such vectors can be prepared by standard recombinant techniques well known in the art and discussed, for example, in Sambrook et al., 1989 or Ausbel et al., 1992.

Appropriate promoters and other essential vector sequences are selected to function in the host and may suitably include those naturally associated with the BRCA1 gene. Examples of operable combinations of cell lines and expression vectors are described in Sambrook et al., 1989 or Ausubel et al., 1992; see also, for example, Metzger et al., 1988. Many useful vectors are known in the art and can be obtained from commercial sources such as Stratagene, New England Biolabs, Promega Biotech, and others.
Promoters such as trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters can be used for prokaryotic hosts. Useful yeast promoters include metallothionein, 3-phosphoglycerate kinase, or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes that contribute to maltose and galactose utilization, and other promoter regions. Is included. Vectors and promoters suitable for use in yeast expression are further described in Hitzeman et al., EP 73,675A. Suitable non-natural mammalian promoters include the early and late promoters from SV40 (Fiers et al., 1978) or those derived from mouse Moloney leukemia virus, mouse tumor virus, chicken sarcoma virus, type II adenovirus, bovine papilloma virus or polyoma. A promoter may be included. In addition, the construct can be linked to an amplifiable gene (eg, DHFR) so that multiple copies of the gene can be made. For suitable enhancers and other expression control sequences, see, for example, Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York (1983).

Although such expression vectors can replicate autonomously, they can also replicate by inserting into the genome of a host cell, by methods well known in the art.

Expression and cloning vectors are likely to contain a selectable marker which is a gene encoding a protein essential for the survival or growth of the host cells transformed with the vector. The presence of this gene ensures that only host cells that express the insert will grow. Typical selection genes a) confer resistance to antibiotics or other toxic substances such as ampicillin, neomycin, methotrexate, etc .; b) compensate for auxotrophic deficiencies, or c) are not available from complex media It encodes a protein that supplies important nutrients (eg, the gene encoding D-alanine racemase in Bacilli). Selection of the appropriate selectable marker will depend on the host cell, and appropriate markers for different hosts are well known in the art.

A vector containing the nucleic acid of interest can be transcribed in vitro, and the resulting RNA can be introduced into host cells by well-known methods (eg, injection (Kubo et al., 1988)), or The vector may vary depending on the type of cell host, well known in the art (electroporation; transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectors Il bombardment; lipofection infection (the vector is an infectious agent such as the retroviral genome); and other methods) can be introduced directly into host cells (see generally, Sambrook et al., 1989 and Ausubel et al., 1992). Introduction of a polynucleotide into a host cell by any method known in the art, including, inter alia, those described above, is referred to herein as "transformation." Cells into which the nucleic acid has been introduced are meant to include the progeny of such cells.

Large quantities of the nucleic acids and polynucleotides of the invention can be prepared by expressing the BRCA1 nucleic acid or a portion thereof in a prokaryotic or eukaryotic host cell with a vector or other expression vehicle that is compatible. The most commonly used prokaryotic host is Escherichia coli, but other prokaryotes such as Bacillus subtilis or Pseudomonas may be used.

Mammalian host cells, or other eukaryotic host cells such as yeast, filamentous fungi, plants, insects, or amphibians or birds, may also be useful for producing the proteins of the invention. The growth of mammalian cells in culture is well known per se (see Jakoby and Pastan, 1979). Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK and COS cell lines, although other cell lines, such as higher expression, desirable glycosyl It will be appreciated by those skilled in the art that the pattern may be suitable for providing a pattern, or other features.

ク ロ ー ン Clones can be selected by using a marker that depends on the mode of the vector construct. The markers can be on the same or different DNA molecules, but are preferably on the same DNA molecule. In prokaryotic hosts, transformants can be selected by, for example, resistance to ampicillin, tetracycline, or other antibiotics. The production of a particular product based on temperature sensitivity may also serve as a suitable marker.

Prokaryotic or eukaryotic cells transformed with a polynucleotide of the present invention will be useful not only in producing the nucleic acids and polypeptides of the present invention, but also in, for example, studying the properties of a BRCA1 polypeptide.
Antisense polynucleotide sequences are useful in preventing or reducing the expression of the BRCA1 locus, as will be appreciated by those skilled in the art. For example, a polynucleoside vector containing all or part of the BRCA1 locus or other sequences from the BRCA1 region (particularly those adjacent to the BRCA1 locus) can be placed under the control of the promoter in the antisense orientation and introduced into the cell. Can be. Expression of such an antisense construct in a cell will interfere with BRCA1 transcription and / or translation and / or replication.

プ ロ ー ブ Identify homologous BRCA1 gene sequences and proteins in other species using probes and primers based on the BRCA1 gene sequences disclosed herein. These BRCA1 gene sequences and proteins are used in the diagnostic / prognostic, therapeutic and drug screening methods described herein for the species from which they were isolated.

Methods of Use: Nucleic Acid Diagnostics and Diagnostic Kits To detect the presence of the BRCA1 allele, which predisposes an individual to cancer, prepare a biological sample, such as blood, and analyze for the presence or absence of the susceptibility allele of BRCA1. . In order to detect the presence of a neoplasm, a biological sample of a lesion is prepared as a predictive indicator of the progression of a precursor lesion toward a malignant tumor, or the presence or absence of a mutant allele of BRCA1. Is analyzed. The results of these tests and interpretive information are returned to the healthcare provider for communication to the individuals tested. Such diagnosis may be performed by a diagnostic laboratory or, alternatively, a diagnostic kit may be manufactured and sold to a healthcare provider or private individual for self-diagnosis.

Initially, the screening method involves amplification of the appropriate BRCA1 sequence. In another preferred embodiment of the invention, the screening method comprises 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.

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

If the probe is used to detect the presence of the target sequence (eg, in a screen for cancer susceptibility), the biological sample to be analyzed, such as blood or serum, can be processed to extract nucleic acids, if desired. The sample nucleic acid can be prepared in various ways to facilitate detection of the target sequence; for example, denaturation, restriction digestion, electrophoresis or dot blot. The targeting region of the assay nucleic acid usually must be at least partially single-stranded and hybridize with the target sequence of the probe. If the sequence is naturally single-stranded, no denaturation will be necessary. However, if the sequence is double-stranded, it will probably need to be denatured. Denaturation can be performed by various techniques known in the art.

The assay nucleic acid and probe are incubated under conditions that promote stable hybridization of the expected targeting sequence in the analyte with the target sequence in the probe. The region of the probe used to bind the analyte may be completely complementary to the targeted region of human chromosome 17q. Therefore, high stringency conditions are desirable to prevent false positives. However, high stringency conditions are used only if the probe is complementary to a chromosomal region that is unique in the genome. The stringency of hybridization is determined by a number of factors during hybridization and during the washing step, including temperature, ionic strength, base composition, probe length and formamide concentration. These factors are reviewed, for example, in Maniatis et al., 1982 and Sambrook et al., 1989. Under certain circumstances, it may be desirable to allow higher order hybrids to form, such as triple helices, quadruplexes, etc., to provide a means to detect the target sequence.

In any case, detection of the obtained hybrid is usually performed by using a target probe. Alternatively, the probe may be unlabeled, but detected directly or indirectly by specific binding to a labeled ligand. Suitable labels and methods of labeling probes and ligands are known in the art, including, for example, known methods (e.g., nick translation, random priming or kinasing), biotin, Includes fluorescent groups, chemiluminescent groups (eg, dioxetanes, especially triggered dioxetanes), radioactive labels that can be taken up by enzymes, antibodies, and the like. Variations on this basic scheme are known in the art and include those that facilitate the separation of the hybrids to be detected from foreign substances and / or that amplify the signal from the labeling group. Many of these variations are reviewed, for example, in Matthews and Kricka, 1988; Landegren et al., 1988; Mittlin, 1989; U.S. Patent No. 4,868,105 and EPO Publication No. 225,807.

As noted above, non-PCR-based screening assays are also contemplated by the present invention. An exemplary non-PCR based method is provided in Example 11. This method hybridizes a nucleic acid probe (or an analog, such as replacing the methyl phosphonate backbone with a normal phosphodiester) to low levels of a DNA target. The probe may have an enzyme covalently attached to the probe such that the covalent bond does not interfere with the specificity of the hybridization. The enzyme-probe-conjugate-target nucleic acid complex can then be isolated from the free probe enzyme conjugate, and the substrate is added for enzyme detection. Enzyme activity is observed by a change in color development or luminescence output that results in an increase of 10 ³ -10 ⁶ in sensitivity. For an example of the preparation of oligodeoxynucleotide-alkaline phosphatase conjugates and their use as hybridization probes, see Jablonski et al., 1986.

Two-step label amplification methodology is known in the art. These assays are performed on the principle that a small ligand (such as digoxigenin, biotin, etc.) binds to a nucleic acid probe capable of binding BRCA1 specifically. Exemplary probes are provided in Tables 9 and 10 herein, and further include nucleic acid probes corresponding to nucleotide positions 3631-930 of SEQ ID NO: 1. Allele-specific probes are also intended to be within the scope of this example, and exemplary allele-specific probes include the predisposition mutations summarized in Tables 12 and 13 herein. Probe to be included.

In one example, the small probe that binds to the nucleic acid probe is specifically recognized by the antibody-enzyme conjugate. In one embodiment of this example, digoxigenin binds to the nucleic acid probe. Hybridization is detected by an antibody-alkaline phosphatase conjugate that becomes a chemiluminescent substrate. For a method of labeling a nucleic acid probe according to this embodiment, see Martin et al., 1990. In a second embodiment, the small ligand is recognized by a second ligand-enzyme conjugate capable of specifically forming a complex with the first ligand. A well-known example of this example is the biotin-avidin type interaction. For methods of 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 the present invention that the nucleic acid probe assay of the present invention will use a cocktail of nucleic acid probes capable of detecting BRCA1. Thus, in one example of detecting the presence of BRCA1 in a cell sample, two or more probes complementary to BRCA1 are used, and in particular, many different probes can be used, alternatively 2, 3, or 5 different nucleic acid probe sequences. Used. In another example, to detect the presence of a mutation in the BRCA1 gene sequence in a patient, two or more probes that are complementary to BRCA1 are used, wherein the cocktail comprises a patient having a change in BRCA1. Includes probes that can bind to allele-specific mutations identified in the population. In this embodiment, any number of probes can be used, but will preferably include a probe corresponding to a major gene mutation identified as predisposing an individual to breast cancer. Some candidate probes contemplated within the scope of the present invention include probes containing allele-specific mutations identified in Tables 12 and 13, as well as both 5 'and 3' of SEQ ID NO: 1. Probes having a BRCA1 region corresponding to the mutation site are included.

Methods of Use: Peptide Diagnosis and Diagnostic Kits The neoplastic state of a lesion can also be detected based on alterations in wild-type BCA1 polypeptide. Such changes can be determined by sequence analysis by conventional techniques. More preferably, antibodies (polyclonal or monoclonal antibodies) are used to detect differences or absence in the BRCA1 peptide. The antibodies can be prepared as discussed previously under the heading "Antibodies" and further shown in Examples 12 and 13. Other techniques for raising and purifying antibodies are well known in the art, and any such technique may be used to make the preparations claimed in the present invention. In a preferred embodiment of the invention, the antibody will immunoprecipitate the BRCA1 protein from solution and react with the BRCA1 protein on a Western or immunoblot of a polyacrylamide gel. In another preferred embodiment, the antibody will detect BRCA1 protein in paraffin or frozen tissue sections using immunocytochemical techniques.

Preferred specific examples of the method for detecting BRCA1 or a mutation thereof include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunoradiometric assay, including a sandwich assay using a monoclonal antibody and / or a polyclonal antibody. (IRMA) and immunoenzymatic assays (IEMA). Exemplary sandwich assays are described by David et al. In US Pat.

Methods of Use: Drug Screening The present invention is particularly useful for screening compounds by using BRCA1 polypeptides or binding fragments thereof in any of a variety of drug screening techniques.

Ｂ The BRCA1 polypeptide or fragment used in such a test can be released in solution, immobilized on a solid support, or supported on a cell surface. One method of drug screening uses eukaryotic or prokaryotic host cells stably transformed with recombinant polynucleotides expressing the polypeptide or fragment, preferably in a competitive binding assay. Such cells, either viable or in fixed form, can be used in standard binding assays. For example, the formation of a complex between a BRCA1 polypeptide or fragment and an agent to be tested can be measured, or the formation of a complex between a BRCA1 polypeptide or fragment and a known ligand is blocked by the agent to be tested. You can inspect the extent to which it is done.

Thus, the present invention provides for contacting an agent with a BRCA1 polypeptide or fragment, by methods well known in the art, for (i) the presence of a complex between the agent and the BRCA1 polypeptide or fragment, Or (ii) a method of screening for an agent comprising assaying for the presence of a complex between a BRCA1 polypeptide or a fragment thereof and a ligand. In such a competitive binding assay, the BRCA1 polypeptide or fragment is typically labeled. Free BRCA1 polypeptide or fragment is isolated from that present in the protein: protein complex, and the amount of free (ie, non-complex forming) label is determined by measuring the binding of the agent to be tested to BRCA1, respectively. Alternatively, BRCA1: a value hindered by ligand binding.

Another technique for drug screening provides high-throughput screening for compounds with appropriate binding affinity for BRCA1 polypeptides, which is described in Geysen, PCT International Publication WO84, published September 13, 1984. / 03564. Briefly, a number of different small peptide test compounds are synthesized on a solid support, such as a plastic pin or some other surface. The peptide test compound is reacted with the BRCA1 polypeptide and washed. Bound BRCA1 polypeptide is then detected by methods well known in the art.

Purified BRCA1 can be coated directly on a plate for use in the drug screening techniques described above. However, non-neutralizing antibodies to the polypeptide can be used to capture the antibody and immobilize the BRCA1 polypeptide on the solid phase.

The present invention also contemplates the use of competitive drug screening assays, wherein a neutralizing antibody capable of specifically binding to a BRCA1 polypeptide is combined with a test compound for binding to a BRCA polypeptide or fragment thereof. Let them compete. In this manner, antibodies can be used to detect the presence of any peptide having an antigenic determinant of one or more BRCA1 polypeptides.

Additional techniques for drug screening include the use of host eukaryotic cell lines or cells (as described above) that have a non-functional BRCA1 gene. These host cell lines or cells are deleted at the BRCA1 polypeptide level. The host cell line or cell grows in the presence of the drug compound. The growth rate of the host cell is measured to determine whether the compound can regulate the growth of BRCA1-deficient cells.

Uses: rational drug design The goal of rational drug design is, for example, a more active or stable form of the polypeptide, or an agent that enhances or interferes with the function of the polypeptide in vivo. To design a biologically active polypeptide of interest or a structural analog of a small molecule with which the agent interacts (eg, agonists, antagonists, inhibitors) (eg, Hodgson, 1991). In one approach, the three-dimensional structure of a protein of interest (eg, a BRCA1 polypeptide) or, for example, a BRCA1-receptor or ligand complex is first determined by X-ray crystallography, computer modeling, or most typically, , Determined by a combined approach. Less often, modeling based on the structure of homologous proteins can provide useful information about the structure of the polypeptide. One example of a rational drug design is the development of HIV protease inhibitors (Erickson et al., 1990). In addition, peptides (eg, BRCA1 polypeptides) are analyzed by alanine scanning (Wells, 1991). In this technique, an amino acid residue is replaced with Ala and its effect on peptide activity is measured. Each amino acid residue of the peptide is analyzed in this way to determine important regions of the peptide.

標的 It is also possible to isolate the target-specific antibody selected by the functional assay and then elucidate its crystal structure. In principle, this approach results in a pharmacore that can be based on subsequent drug design. By generating anti-idiotypic antibodies (anti-ids) to functional pharmacologically active antibodies, protein crystallography can be avoided altogether. As a mirror image of the mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The peptides can then be identified and isolated from a bank of chemically or biologically generated banks of peptides using anti-ids. The selected peptide will then act as a pharmacore.

Thus, agents can be designed that have, for example, improved BRCA1 polypeptide activity or stability, or that act, for example, as inhibitors, agonists, antagonists, etc., of BRCA1 polypeptide activity. Depending on the effectiveness of the cloned BRCA1 sequence, an analytical experiment, such as x-ray crystallography, can be performed utilizing a sufficient amount of the BRCA1 polypeptide. In addition, the knowledge of the BRCA1 protein sequence provided herein will lead to the use of computer modeling techniques instead of or in addition to x-ray crystallography.

Method of Use: Gene Therapy The present invention provides a method of providing wild-type BRCA1 function to cells carrying a mutant BRCA allele. Provision of such a function must inhibit neoplastic growth of the recipient cell. The wild-type BRCA1 gene or a portion of the gene can be introduced into cells with a vector such that the gene remains extrachromosomal. In such a situation, the gene will be expressed by the cell from an extrachromosomal location. When the gene fragment is introduced into a cell carrying a mutant BRCA1 allele and expressed, the gene fragment must encode a portion of the BRCA1 protein required for non-neoplastic growth of the cell.

More preferably, a situation in which a wild-type BRCA1 gene or a part thereof is introduced into a mutant cell such that it replaces the endogenous mutant BRCA1 gene present in the cell. Such recombination requires a double recombination event, which results in a correction of the BRCA1 gene mutation. Vectors that introduce both recombinant and extrachromosomal maintenance genes 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 well known in the art, and the choice of method is within the abilities of routine workers. Cells transformed with the wild-type BRCA1 gene can be used as a model system to study cancer remission and drug treatments that promote such remission.

As generally discussed above, the BRCA1 gene or fragment, when applicable, can be used in gene therapy to increase the amount of the expression product of such a gene in cancer cells. Such gene therapy is particularly suitable for use in both cancerous and pre-cancerous cells, in which the level of BRCA1 polypeptide is eliminated or reduced compared to normal cells. Also, although the mutant gene is expressed at "normal" levels, the gene product may be useful in increasing the expression level of a given BRCA1 gene, even in tumor cells that are not fully functional.

O Gene therapy will be performed by generally accepted methods (eg, as described by Friedman, 1991). Cells from the patient's tumor will first be analyzed by the diagnostic methods described above to confirm the production of BRCA1 polypeptide in the tumor cells. A virus or plasmid vector containing a copy of the BRCA1 gene linked to an expression control element and capable of replicating inside tumor cells (see further details below) is prepared. Suitable vectors are known, as disclosed in US Pat. No. 5,252,479 and PCT Publication WO 93/07282. The vector is then injected into the patient, either locally at the tumor site or systemically (to reach any tumor cells that can metastasize to other sites). The process can be repeated periodically if the transfected gene is permanently integrated into the genome of each targeted tumor cell.

遺 伝 子 Gene transfer systems known in the art can be useful for performing the gene therapy of the present invention. These include viral and non-viral transfer methods. 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 Viruses (Moss, 1992), Papovaviridae including adeno-associated virus (Muzyczka, 1992; Ohi et al., 1992), HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freeese) Herpesviridae family, including chickens (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), rats (Miller, 1992; Miler et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1990). A number of viruses are inherited, including retroviruses of human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). It has been used as a child transfer vector. Most human gene therapy protocols are based on inactivated murine retrovirus.

Non-viral gene transfer methods known in the art include chemical techniques such as calcium phosphate co-precipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); for example, microinjection (Anderson et al., 1980; Godon et al., 1980). Mechanical methods such as Brinster et al., 1981; Constantini and Lacy, 1981); liposome-mediated membrane fusion-mediated transfer (Felgner et al., 1987; Wang and Haung, 1989; Kaneda et al., 1989; Stewart et al., 1992; Nabel). 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). Virus-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery to direct the viral vector to tumor cells rather than to surrounding undivided cells. Alternatively, a retroviral vector producing cell line can be injected into the tumor (Culver et al., 1992). The injection of producer cells then provides a continuous source of vector particles. This technology has been approved for use in humans with inoperable brain tumors.

In an approach that combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a polylysine-conjugated antibody specific for adenovirus hexon protein, and the resulting complex is transformed into an adenovirus vector. Join. The cells are then infected using the trimolecular complex. Adenovirus vectors allow efficient binding, internalization and digestion of endosomes before the coupled DNA is damaged.

It has been shown that liposome / DNA complexes can directly mediate in vivo gene transfer. Although the gene transfer process is non-specific in standard liposome preparations, for example, after direct in situ administration, localized in vivo uptake and expression has been reported in tumor deposition. (Nabel, 1992). Gene transfer techniques that target the DNA directly to breast and ovarian tissues (eg, breast or ovarian epithelial cells) are preferred. Receptor-mediated gene transfer is accomplished, for example, by conjugating DNA (usually in the form of a covalently closed supercoiled plasmid) to a protein ligand via polylysine. Ligands are selected based on the presence of the corresponding ligand receptor on the cell surface of the target cell / tissue type. One suitable receptor / ligand pair may include the estrogen receptor and its ligand, estrogen (and estrogen analogs). These ligand-DNA conjugates can, if desired, be injected directly into the blood and directed to target tissues where receptor binding and internalization of the DNA-protein complex occur. To overcome the problem of intracellular destruction of DNA, co-infection with adenovirus may be involved to disrupt endosomal function.

療法 The therapy includes two steps that can be performed alone or in combination. In the first step, prepubertal women carrying the BRCA1 susceptibility allele are subjected to gene delivery such that some or all of their ductal epithelial progenitor cells receive at least one additional copy of the functional BRCA1 allele. Treat with vehicle. In this step, the treated individual has a reduced risk of breast cancer to the extent that the presence of the normal allele suppresses the effects of the susceptible allele. In the second step of prophylactic therapy, pre-treated young women, particularly those who have undergone the proposed gene therapy treatment, are subjected to hormonal therapy to minimize the effects of full-term pregnancy on the breast.

Method of Use: Peptide Therapy A peptide having BRCA1 activity can be provided to cells carrying a mutated or deleted BRCA1 allele. Discloses the sequence of the BRCA1 protein (SEQ ID NO: 2). The protein can be produced, for example, by expressing the cDNA sequence in bacteria using a known expression vector. Alternatively, BRCA1 polypeptides can be extracted from BRCA1 producing-mammalian cells. In addition, BRCA1 proteins can be synthesized using synthetic chemistry techniques. Any such technique can provide a preparation of the present invention that includes the BRCA1 protein. The preparation is substantially free of other human proteins. This is most easily accomplished by microbial or in vitro synthesis.

Active BRCA1 molecules can be introduced into cells by microinjection or by using, for example, liposomes. Alternatively, certain active molecules may be taken up by cells, either actively or by diffusion. Extracellular application of the BRCA1 gene product can significantly affect tumor growth. Application of molecules with BRCA1 activity should lead to a partial reversal of the neoplastic state. The use of other molecules having BRCA1 activity (eg, peptides, drugs or organic compounds) can also affect such reversal. Modified peptides having substantially similar functions are also used in peptide therapy.

Methods of Use: Transformed Hosts Similarly, cells and animals carrying mutant BRCA1 alleles can be used as model systems to experiment and test for substances with therapeutic potential. The cells are typically cultured epithelial cells. These can be isolated from individuals with BRCA1 mutations, either somatic or germline. Alternatively, the cell line may be designed to carry a mutation in the BRCA1 allele, as described above. After applying a test substance to the cells, the neoplasticly transformed phenotype of the cells is determined. Any feature of neoplastic transformed cells can be assessed, including anchorage independent growth, tumorigenicity in nude mice, non-invasiveness of cells, and growth factor dependence. Assays for each of these features are known in the art.

動物 Animals to test for therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatments usually include the insertion of a mutant BRCA1 allele from a second animal species, as well as the insertion of a disrupted homologous gene. Alternatively, the animal's endogenous BRCA1 gene (s) can be disrupted by insertion or deletion mutations or other genetic changes 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 the test substance has been administered to the animals, the growth of the tumor must be assessed. If the test substance prevents or suppresses the growth of the tumor, the test substance is a candidate therapeutic for the treatment of cancer identified herein. These animal models provide a crucial test vehicle for potential therapeutic products.

The invention will be described by reference to the following examples, which are provided for illustrative purposes and do not limit the invention in any way. Standard techniques well known in the art or techniques specifically described below were utilized.

Example 1
Confirmed and experimental pedigrees with 17q-linked breast cancer susceptibility loci Widespread cancer-prone pedigrees provide a large set of widespread families with many cases of breast cancer and relatives available for experimentation. Was confirmed from the group. The large number of meiosis present in these large families provides the ability to detect whether the BRCA1 locus is segregating and provides an opportunity for informative recombinants occurring within the small region to be investigated. Reduced. This greatly improves the opportunity to establish linkage to the BRCA1 region, facilitates reduction of the BRCA1 region to a manageable size, and allows identification of the BRCA1 locus itself.

Expanded families to all probands or all informative first-degree relatives in cancer cases through all available relatives. For these families, additional breast cancer cases and individuals with cancer at other sites of interest (eg, ovaries) that appeared in the family were located through tumor registry-related files. We investigated all breast cancers reported in families not identified in the Utah Cancer Registry. Medical records or death certificates were obtained for confirmation of all cancers. Each key individual and all informative individuals were involved by providing a blood sample from which DNA had been extracted. We also sampled spouses and relatives of the deceased case so that the genotype of the deceased case could be inferred from the genotype of the relative.

Ten families with three or more cancer cases with a deducible genotype were used for linkage experiments against the 17q marker from a set of 29 families originally identified from relevant data beta for the study of proliferative breast and breast cancer. (Skolnick et al., 1990). The criterion for selection of these families was the presence of two sisters or mothers with breast cancer and her daughter. In addition, two kindreds (K1001, K9018), which have been studied as part of our breast cancer linkage study since 1980, and six kindreds (K2019, K2073) identified from relevant databases for the presence of breast and / or ovarian cancer clusters. , K2079, K2080, K2039, K2082) and self-estimated families with early-onset breast cancer (K2035). These families were investigated and expanded in our clinic as described above. Table 1 shows the characteristics of these 19 families, which are the subject of subsequent examples. Table 1 reports the total number of individuals in our database, the number of individuals typed, and the minimum, median, and maximum age at diagnosis of breast / ovarian cancer for each kindred. The families are sorted in order of increasing median age at diagnosis of breast cancer. Four women diagnosed with both ovarian and breast cancer count in both categories.

Example 2
Selection of kindreds linked to chromosome 17q and positioning of BRCA1 on the interval Mfd15-Mfd188 For each sample collected in these 19 kindreds, blood (paraffin embedded tissue in two cases) was used using standard experimental protocols. DNA was extracted from the block). Genotyping in this experiment was restricted to short tandem repeat (STR) markers. Because, in general, they are highly heterozygous and the PCR method provides very rapid rotation while using very small amounts of DNA. To assist 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 are located on the long arm: (46E6. Easton et al., 1993); (42D6, Easton et al., 1993); 26C2 (D17S514, Oliphant et al., 1991), while the outer 12G6 (D17S513). Oliphant et al., 1991) have been 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 researchers worldwide. Oligonucleotide sequences for markers not developed at our laboratory were obtained from published reports, as part of the Breast Cancer Linkage Consortium, or from other researchers.

All genotyping films were blindly scored with standard lane markers used to maintain consistent coding of alleles. Key samples from the four families shown here were typed in duplicate for all relevant markers. All 19 kindreds were provided by two polymorphic CA repeat markers: 42D6 (D17S588), a CA repeat isolated in our laboratory, and Mdf15 (D17S250), J. Weber (Weber et al., 1990). Each CA repeat was typed. Genetic markers were created on chromosome 17, especially on chromosome 17 cosmids, using probes from several sources, and a lambda phage library was generated from chromosomes sorted by Los Alamos National Laboratories (van Dilla et al., 1986). Produced.

The LOD score for each pedigree with these two markers (42D6, Mfd15) and a third marker, Mfd188 (D17S579, Hall et al., 1992), roughly located between these two markers, was Calculations were made for two values of the fractions, 0.001 and 0.1. (For calculation of the LOD score, see Oh, 1985). Likelihood was calculated with a model derived by Claus et al., 1991, which estimated gene frequency of 0.003, lifetime risk in approximately 0.80 gene carriers, and breast cancer in non-gene carriers. It estimates population-based age-specific risks. Allele frequencies for the three markers used in the LOD score calculation were calculated from the typing of unrelated individuals in our own laboratory in the CEPH panel (White and Lalouel, 1988). Table 2 shows the results of linkage analysis on pairs of each kindred with the three markers 42D6, Mfd188, and Mfd15.

Using the criteria for 17q linkage with a LOD score> 1.0 for at least one locus under the CASH model (Claus et al., 1991), four out of 19 families appeared to be linked to 17q. (K1901, K1925, K2035, K2082). Many additional families showed some evidence of linkage, but could not be explicitly assigned to linkage categories at this time. These included the families K1911, K2073, K2039 and K2080. Three of the 17q-linked families have informative recombinants in this region, which are described in detail below.

Kindred 2082 is the largest 17q-linked breast cancer family currently reported by several groups. The family has 20 cases of breast cancer and 10 cases of ovarian cancer. The second case has ovarian and breast cancer. The evidence of linkage to 17q for this family is overwhelming: Despite the presence of three cases of breast cancer that are sporadic, LOD scores with linked haplotypes exceed 6.0. That is, these cases do not share either part of the haplotype linked between Mfd15 and 42D6. These three scattered cases were diagnosed with breast cancer at ages 46, 47 and 54. In smaller families, this type of sporadic cancer greatly disrupts linkage analysis and the correct identification of key recombinants. A key recombinant in the 2082 kindred is a woman who developed ovarian cancer at the age of 45 whose mother and aunt developed ovarian cancer at the ages of 58 and 66, respectively. She inherited a linked portion of the haplotype for both Mfd188 and 42D6, while inheriting the unlinked allele at Mfd15; this recombination event caused BRCA1 to be located distal to Mfd15.

K1901 is typical of an early-onset breast cancer kindred. The family had 10 cases of breast cancer and a median break age of 43.5 years; 4 cases were diagnosed before age 40 years. The LOD score for this family at marker 42D6 is 1.5, which is a posterior probability of 0.96 17q-linkage. Examination of the haplotypes in this kindred identified recombinant haplotypes in an authentic male carrier and his affected daughter diagnosed with breast cancer at the age of 45. Those linked alleles for the marker Mfd15 are different from those found in all other cases in the family (except in one case, which could not be completely inferred from her child). The two haplotypes are identical for Mfd188 and 42D6. Therefore, the BRCA1 locus is also located distal to Mfd15 by data from pedigree 1901.

Family 2035 is similar in disease phenotype to K1901. The median age at diagnosis for eight cases of breast cancer in this family is 37 years. One case was 60 years old and also had ovarian cancer. The breast cancer cases in this family are inherited from two sisters who did not both have breast cancer until their death in their eighties. Each branch had four cases of breast cancer and each branch with significant early onset had at least one case. This kindred has a LOD score of 2.34 per Mfd15. The haplotypes separating breast cancer in the two branches have both identical alleles at Mfd15, but as part of a consortium located just distal to the terminal loci Mfd188 and NM23 (42D6). (Typed markers). Although the two haplotypes are identical for marker 42D6, the alleles are shared identically by state rather than identically (derived from a common ancestor) by pedigree (with the same allele). But from different ancestors). Because the shared allele is the second most common allele observed at this locus. In contrast, linked alleles shared by Mfd15 have a frequency of 0.04. This is a key recombinant in our database. Because BRCA1 is the only recombination that segregates proximal to the haplotype, thus providing an end that borders the BRCA1 region. For this event, not being a key recombination, the second mutant BRCA1 gene was present in a wife married to a family that also had a rare Mfd15 allele segregating for breast cancer in both families of the family. Need to be done. This event has a probability of less than one thousandth. Thus, evidence from this kindred located the BRCA1 locus proximal to Mfd188.

Example 3
Fine structure mapping and refinement of the BRCA1 region to Mfd191-Mfd188 using additional STR polymorphisms To improve the characterization of our recombinants and to define the flanking markers, A dense map of a relatively small area was needed. A study of chromosome 17 produced a consensus map of this region based on a combination of genetic and physical mapping experiments (Fain, 1992) (FIG. 1). This map contains both highly polymorphic STR polymorphisms and a number of polymorphically expressed genes. We use it in the development of new markers, as this map gives no details on the evidence for this order and does not give any measure of local support for reversals in the order of adjacent loci. It has been summarized as a rough guide to get our detailed genetic and physical mapping of the power source and the small region containing BRCA1. Our approach involves a panel of meiotic (genetic) mitotic breakpoints identified using current STR markers provided by other investigators and DNA from CEPH-related families and the body constructed for this region. The analysis was for all newly developed markers from our laboratory for both panels of cell-cell hybrids (physical breakpoints). These markers include Mfd15, Mfd191 (provided by James Weber), THRA1 (Futreal et al., 1992a), and three polymorphisms provided by Dr. Donald Black, NM23 (Hall et al., 1992), SCG40 (D17S181), and This included 26C2 developed in our lab, mapped proximally to 6C1 (D17S293).

Gene Localization of Markers To genetically localize new markers within the region of interest, we used a number of key reductions in the region in the CEPH literature panel and in our large breast cancer kindred (K2082). A mitotic breakpoint was identified. If there is a small genetic distance in this region, they appear to be only a relatively small set of recombinants that can be used for this purpose, and they appear to be markers that can be grouped into sets. The order of the markers within each set can only be determined by physical mapping. However, the number of genotyping required to locate a new marker is minimized. These breakpoints are shown in Tables 3 and 4. Using this approach, we were able to genetically order the markers THRA1, 6C1, SCG40 and Mfd191. As can be seen from Tables 3 and 4, both THRA1 and Mfd191 map inside the Mfd15-Mfd188 region we previously identified as containing the BRCA1 locus. In Tables 3 and 4, M / P indicates maternal or parental recombinant. "1" indicates that the inherited allele is of grandfather origin, while "0" is of grandmother origin and "-" indicates that the locus is untyped or non-informative. Show.

Analysis of markers Mfd15, Mfd188, Mfd191 and THRA1 in our recombinant family Mfd15, Mfd188, Mfd191 and THRA1 were typed in our recombinant family and examined for further information to locate the BRCA1 locus . In kindred 1901, the Mfd15 recombinant is a recombinant for THRA1, but is non-informative for Mfd191, thus positioning BRCA1 distal to THRA1. In K2082, the recombinant for Mfd15 is also a recombinant for Mfd191, thus positioning the BRCA1 locus distal to Mfd191 (Goldgar et al., 1994). A survey of THRA1 and Mfd191 in kindred K2035 did not provide any further localization information. Because the two branches matched for both markers. However, both SCG40 and 6C1 exhibit the same pattern as Mfd188, thus increasing our confidence in the positioning information provided by Mfd188 recombinants in this family. The BRCA1 locus, or at least a portion thereof, is therefore within an interval bordering proximal Mfd191 and distal Mfd188.

Example 4
Development of Genetic and Physical Sources in the Region of Interest To increase the number of highly polymorphic loci in the Mfd191-Mfd188 region, we have derived from cosmids and YACs that are physically mapped to that region. A number of STR markers have been developed at the Institute. These markers allowed us to further refine the region.
The STR, which contains these loci, was then used to identify cosmids P1 or genes that are known to be in the desired region to identify the YAC that identifies the subclone in the BAC. These subclones were then screened for the presence of CA tandem repeats using (CA) _n oligonucleotides (Pharmacia). Clones with strong signals were preferentially selected. Because they appear to represent a large number of repeats and / or CA-repeats with almost perfect accuracy for (CA) _n patterns. Both of these features are known to increase the probability of polymorphism (Weber, 1990). These clones were sequenced directly from the vector to locate the repeat. We, by using one of the set of complementary possible primer terminus of (GT) 10 _T such as CA- repeated to obtain a unique sequence on one side of CA- iteration. Based on this unique sequence, a primer was created that crossed the repeat in the other direction in reverse, resulting in a unique sequence for the design of a second primer adjacent to the CA-repeat. The STR was then screened for polymorphism for a small group of unrelated individuals and tested against the hybrid panel to confirm its physical location. New markers meeting these criteria were typed in a set of 40 unrelated individuals from the Utah and CEPH families to obtain a suitable allele frequency for the experimental population. Many of the other markers reported in this study were tested in a small group of CEPH unrelated individuals to obtain similarly suitable allele frequencies.

用 い Using the above procedure, a total of 8 polymorphic STRs were found in these YACS. In this way, four of the same loci were both polymorphic and localized to the BRCA1 region. The four markers are not located on chromosome 17 and reflect the chimeric nature of the YAC used. The four markers that were in the region were named AA1, ED2, 4-7 and YM29. AA1 and ED2 were developed from YACs positive for the RNU2 gene, 4-7 from EPB3ΔYAC, and YM29 from cosmids located in that region by the hybrid panel. A description of the number of alleles, heterozygosity and sources of these four and all other STR polymorphisms analyzed in breast cancer kindreds is provided below in Table 5.

４Four STR polymorphisms (4-7, ED2, AA1, YM29) that were physically mapped to the region were analyzed by meiosis and the mitotic breakpoint panels are shown in Tables 3 and 4. Tables 6 and 7 contain relevant CEPH and pedigree 2082 data for the location of these four markers. In the table, M / P indicates maternal or paternal recombinants. A "1" indicates that the inherited allele is of paternal origin, while a "0" indicates grandmother origin and "-" indicates that the locus is not typed or Indicates non-informational.

From CEPH1333-04 we see that AA1 and YM29 must be located distal to Mfd191. From 13292, it can be estimated that AA1 and ED2 are both 4-7, and are on the base side relative to YM29 and Mfd188. The recombinant found in K2082 provides some additional ordering information. In three independent observations (personal numbers 22, 40 and 63), AA1, ED2, 4-7, and YM29, and Mf188 were located distal to Mfd191, while ID125 showed 4-7. , YM29 and Mfd188 are located proximal to the SGC 40. Genetic information on the relative ordering within the two clusters of the markers AA1 / ED2 and 4-7 / YM29 / Mfd188 was not obtained from genetic engineering analysis. Locus ordering for hybrids known to contain "holes" where interstitial human DNA fragments are likely to be missing is questionable, but the hybrid pattern shows 4-7 above YM29 and Mfd188 To do so.

Example 5
Genetic analysis of breast cancer pedigrees with markers AA1, 4-7, ED2 and YM29 In addition to the three pedigrees containing key recombinants discussed above, pedigree 2039 is an analysis of newly developed STR markers. Through the above, it was shown to be linked to the region and to contain useful recombinants.

Table 8 defines the haplotypes of the pedigrees (shown in codon form) by specific marker alleles at each locus and their respective frequencies. In Table 8, the alleles are listed in decreasing order of frequency; the frequencies of alleles 1-5 for each locus are shown in Table 5. The haplotype encoding H is a BRCA1-associated haplotype, P indicates a partial H haplotype, and R indicates an observable recombinant haplotype. As is evident in Table 8, not all families were typed for all markers, and particularly for family K2082, not all individuals in the family were typed for the same set of markers. With one exception, only haplotypes inherited from affected or at-risk family members are shown; haplotypes from spouses married to the family are not listed. Thus, in a given sibling, the appearance of haplotypes X and Y was observed for both haplotypes from affected / at-risk individuals, none of which were breast cancer-associated haplotypes.

に お い て In kindred K1901, the new marker shows no observable recombinants with breast cancer susceptibility, indicating that recombination events in this kindred are most likely to occur between THRA1 and ED2. Thus, no new BRCA1 localization information was obtained based on examining four new markers in this kindred. In pedigree 2082, the key recombinant individuals took over the linked alleles for ED2, 4-7, AA1, and YM29, which are recombinants for tdj1474, which indicate that the recombination events were tdj1474 and tdj1474. Indicates what happened to this individual during ED2 / AA1.

家 There are three haplotypes of interest in families K2035, H1, H2 and R2 shown in Table 8. H1 is present in four cases and one true male carrier successor from individual 17, while H2 is present in two cases and two true male carriers in individual 10 successors. R2 is identical to H2 for the locus between and including Mfd15 and SCG40, but has a recombinant between SCG40 and 42D6. Since we have established that BRCA1 is proximal to 42D6, this H2 / R2 difference does not add additional positioning information. H1 and R2 share the same allele in Mfd15, THRA1, AA1 and ED2, but differ for putative loci for ED2, ie, 4-7, Mfd188, SCG40 and 6C1. Although the haplotype of 2 matches for the fifth allele at marker YM29 (a marker physically mapped between 4-7 and Mfd188), the allele is retained identically by the pedigree. It seems that they are held the same depending on the situation rather than being there. This is because this allele is the most common allele at this locus and the estimated frequency in CEPH parents is 0.42. In contrast, linked alleles carried together at the Mfd15 and ED2 loci have frequencies of 0.04 and 0.09, respectively. Also, they carry more common alleles in Mfd191 (frequency = 0.52), THRA1, and AA1 (frequency = 0.28). This is the key recombinant in the set. Because breast cancer is the only recombinant that separates with respect to the base portion of the haplotype, and thus defines the terminal border. Thus, evidence from this kindred places the BRCA1 locus proximal to 4-7.

The recombination event in pedigree 2082 that positions BRCA1 distal to tdj1474 is only one of the four events described that can be directly estimated; that is, the genotype of the affected mother is her spouse and offspring. And the recombinant haplotype is observable in her affected daughter. In this family, it is very likely to benefit an affected individual carrying the BRCA1 susceptibility allele; the only possible interpretation of the data is that BRCA1 is distal to Mfd191, or the expressed recombinant Is a sporadic case of 44-year-old ovarian cancer. Rather than directly observable or putative recombinants, the interpretation of pedigree 2033 relies on the observation of distinct 17q-haplotypes, segregating in different and sometimes distantly related branches of the pedigree. . Some of these haplotypes have common alleles for some markers, while they position the BRCA1 locus in other markers on a shared region. The reliability of this localization determines several factors; the relationship between individuals carrying each haplotype, the frequency of shared alleles, the certainty that haplotypes have been shown to segregate for the BRCA1 locus, and the haplotype Depends on the density of the marker in the area. In the case of ancestry 2035, the two detached houses are closely related, with each separated house having a number of early onset cases responsible for each haplotype. Two of the shared alleles are common (Mfd191, THRA1), while the estimated frequencies of the shared alleles in Mfd15, AA1, and ED2 are 0.04, 0.28, and 0.09, respectively. . Thus, these alleles are identical by pedigree (derived from a common ancestor) rather than identical by state (identical alleles but derived from the general population).

Example 6
Extensive physical mapping studies have located BRCA1 in the region flanked by tdj1474 and U5R.
Since the first localization to chromosome 17q in 1990 (Hall et al., 1990), the BRCA1 gene was located in a small enough region to allow for an effective position cloning strategy and to isolate the gene. A great deal of effort has been made. In a collective Breast Cancer Linkage Consortium database of 214 families collected worldwide, multipoint linkage analysis (Easton et al., 1993) first identified the BRCA1 locus at the interval Mfd15 (D17S250) -42D6 (D17S588). Was located. Subsequent fine positioning was based on individual recombination events in specific families. The region THRA1-D17S183 was defined by Bowcock et al., 1993; the region THRA1-D17S28 was defined by Simard et al., 1993.

We have further shown that the BRCA1 locus must be distal to the marker Mfd191 (D17S776) (Goldgar et al., 1994). This marker is known to be distal to THRA1 and RARA. The smallest published region for the BRCA1 locus is thus between D17S776 and D17S78. This region still contains about 1.5 million bases of DNA, making the isolation and testing of all genes in the region a very difficult task. We can therefore perform and work on constructing a physical map of the region, isolating the set of polymorphic STR markers located in the region, and analyzing these new markers in the informative family set. The accuracy of positioning the BRCA1 gene in the interval was increased.

Four families provide important informative genetic evidence for the positioning of BRCA1 in a region small enough for the application of a positional cloning strategy. Two families (K2082, K1901) provide data on the proximal border for BRCA1, and the other two (K2035, K1813) define a distal border. These families are described in detail below. A total of 15 short tandem repeat markers that could be assayed by PCR were used to refine this localization in the studied families. These markers include DS17S7654, DS17S975, tgj1474 and tgj1239. The primer sequences for these markers are SEQ ID NO: 3, SEQ ID NO: 4 for DS17S754, SEQ ID NO: 5 and SEQ ID NO: 6 for DS17S975, SEQ ID NO: 7 and SEQ ID NO: 8 for tgj1474, and SEQ ID NO: 8 for tgj1239. 9 and SEQ ID NO: 10.

Family 2082
Kindred 2082 is the largest BRCA1-linked breast / ovarian cancer family studied to date. It has a LOD score of 8.6 and provides clear evidence for 17q linkage. This family has been described previously and has been shown to contain critical recombinants that position BRCA1 distal to MFD191 (D17S776). This recombinant occurred in a woman diagnosed with ovarian cancer at the age of 45 whose mother had ovarian cancer at the age of 63. The affected mother died; however, from her child she could be inferred to have a linked haplotype present in 30 other cases of the family in the region between Mfd15 and Mfd188. Her affected daughter inherited linked alleles at loci ED2,4-7 and Mfd188, but inherited alleles on non-BRCA1 chromosomes at Mfd15 and Mfd191. To further map this recombination breakpoint, we tested key members of the family for the following markers from physical mapping sources: tdj1474, tdj1239, CF4, D17S855. For markers tdj1474 and CF4, affected daughters did not inherit the linked allele. However, for the STR locus tdj1239, it could be assumed that the mother was informative and her daughter inherited the BRCA1-related allele. D17S855 was not informative in this family. Based on this analysis, the order is 17q centromere-Mfd191-17HSD-CF4-tdj1474-tdj1239-D17S855-ED2-4-7-Mfd188-17q telomere. Thus, the recombinant described above positioned BRCA1 distal to tdj1474 and the breakpoint was positioned at the interval between tdj1475 and tdj1239. The only other explanation for data in this family other than that of BRCA1 located distal to tdj1474 is that ovarian cancer present in recombinant individuals is caused by reasons independent of the BRCA1 gene. is there. If ovarian cancer diagnosed before the age of 50 is rare, this alternative interpretation is unlikely.

Family tree 1901
Pedigree 1901 is an early-onset breast cancer family in which 7 cases were diagnosed before age 50, of which 4 were diagnosed before age 40. In addition, there were three cases diagnosed with breast cancer between the ages of 50 and 70. One case of breast cancer also had ovarian cancer at the age of 61. This family currently has a LOD score of 1.5 for D17S855. Given this linkage evidence and the presence of at least one ovarian eye case, the family has a posterior probability with a BRDA1 of greater than 0.99. In this family, recombination results from the fact that individuals who are siblings of ovarian cancer cases that were inherited in most of the other cases then share some of the haplotypes that co-segregate with other cases in the family. However, he handed this partial haplotype to his daughter, who developed breast cancer at the age of 44. If this case is due to the BRDA1 gene, only some of the haplotypes shared between this brother and his sister can contain the BRDA1 gene. Difficulties in interpreting this type of information can identify non-shared markers, and thus recombinants, but matching markers are likely because they are non-recombinant or when their parents That is, they would be shared because they were homozygous. Without parental genotype data, these alternative interpretations cannot be distinguished. The haplotype in K1901 indicates that he does not have any linked alleles at Mfd15 (D17S250), THRA1, CF4 (S17S1320), and tdj1474 (17S1321). He has both linked alleles at Mfd191 (D17S776), ED2 (S17S1327), tdj1239 (D17S1328) and Mfd188 (D17S579). Although the alleles shared by Mfd191 are relatively rare (0.07), we believe that because the parents are recombinant at a marker where they are located near either side It is homozygous, presuming that double recombination events in this region are extremely unlikely. Thus, evidence in this family positions the BRCA1 locus distal to tdj1474. However, this lower limit of the mitotic breakpoint cannot be determined without parental genotype information. The key recombination breakpoint in this family is disconcerting to confirm the results in pedigree 2082. As before, the positioning information in this family is only meaningful if the breast cancer is due to the BRCA1 gene. However, her relatively early age at diagnosis (44) makes this very likely. The risk of breast cancer before age 45 is low in the general population (almost 1%).

Family 2035
Although information about critical recombination events is not directly observed in this family, observations indicate that two haplotypes co-segregating for early-onset breast cancer in two separate families of the family are located proximal to the 17qBRCA1 region. Similar to K1901 in that it is the same for the located marker, but presumably different at the 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 at ED2 in this family is 2.2 and the family has a case of breast cancer (indicating an 80% prior probability of BRCA1 linkage), and this family is linked to BRCA1 The obtained posterior probability is 0.998. Haplotypes are identical for markers Mfd15, THRA1, Mfd191, ED2, AA1, D17S858 and D17S902. Both common alleles in Mfd15 and ED2 are fairly rare, indicating that this haplotype is shared identically by progeny. However, the haplotypes are inconsistent with CA375, 4-7, and Mfd188, and some more distal markers. This indicates that the BRCA1 locus must be above marker CA375. This marker is located 50 kb below D17S78, and thus serves primarily as a further confirmation of this previous lower boundary, as reported by Simard et al. (1993).

Family 1813
Family 1813 is a small family with four cases whose mother was diagnosed with breast cancer at age 45 and diagnosed with ovarian cancer at age 61 at age 40 before the age of 40. This situation is somewhat complicated by the fact that the four cases have three different fathers, of which only one has been genotyped. However, by typing a number of different markers in the BRCA1 region as well as highly polymorphic markers elsewhere in the genome, it is determined with considerable certainty who is the father of every child in the family. Was done. This family produces a maximum multipoint LOD score of 0.60 for the 17q marker, and if there is at least one case of ovarian cancer, the posterior probability of being a BRCA1-linked family of 0.93. This family contains recombination events that are directly observable in individual 18, who developed breast cancer at the age of 34 (see FIG. 5 in Simard et al., Human Mol. Genet. 2: 1193-1199 (1993)). The genotype of the affected mother at the relevant 17q locus can be estimated from her genotype, her affected sister's genotype, and the genotypes of three other unaffected siblings. Individual 18 inherits the BRCA1 linked allele at the following loci: Mfd15, THRA1, D17S800, D17S855, AA1, and D17S931. However, for the markers below D17S931, namely U5R, vrs31, D17S858 and D17S579, she inherited alleles located on disease-free chromosomes. Evidence from this family therefore maps the BRCA1 locus proximal to marker US5. Because of the early age of her diagnosis (34), it is very unlikely that the recombinant individual's cancer is due to a gene responsible for other cases of breast / ovarian cancer in this family: this family Uncertainty comes from our somewhat less evidence that breast cancer in this family is due to BRCA1 rather than a second, unmapped, breast cancer susceptibility locus.

Size of the Region Containing BRCA1 Based on the genetic data detailed above, the BRCA1 locus must be located in the interval between the markers tdj474 and U5R, both of which were isolated in our laboratory. Based on the physical maps shown in FIGS. 2 and 3, we can attempt to estimate the physical distance between these two loci. It gives an approximately 14 P1 clone with an average insert size of approximately 80 kb over this region. However, due to some unknown degree of all these P1 duplications, the physical area seems to be much smaller than 14 times 80 kb. Based on the restriction map of the clone covering the region, we estimate that the size of the region containing BRCA1 is approximately 650 bp.

Example 7
Genomic analysis of the Contig region identifies candidate cDNA clones for the BRCA1 locus Complete Screening of Possible Regions The first method of identifying candidate cDNA required considerable effort but used known techniques. The method consisted of screening cosmids and P1 and BAC clones in the contig to identify putative coding sequences. The clone containing the putative coding sequence was then used as a probe on a filter of a cDNA library to identify candidate cDNA clones for future analysis. The clones were screened for putative coding sequences by one of two methods.

Zoo Blots The first method to identify putative coding sequences was by screening cosmid and P1 clones for sequences conserved through evolution across several species. This technique is called "zooblot analysis" and is described by Monaco, 1986. In particular, DNA from cows, chickens, pigs, mice and rats was digested with restriction enzymes EcoRI and HinDIII (8 μg DNA per enzyme). The digested DNA was separated overnight (14 cm gel) on a 0.7% gel at 20 volts for 16 hours, and the DNA was transferred to a nylon membrane using standard Southern blot techniques. For example, treatment at 65 ° C. in 0.1 × SSC, 0.5% SDS and 0.2 M Tris, pH 8.0 for 30 minutes, then at 42 ° C., 5 × SSC, 10% PEG 8000, 20 mM NaPO _4, pH6.8,100μg / ml salmon sperm DNA, 1 × Denhardt's, 50 % formamide, and blocked with 0.1% SDS and 2 [mu] g / _ml C 0 in t-1 DNA.

The cosmid and P1 clone to be analyzed were digested with restriction enzymes to release human DNA from vector DNA. The DNA was separated on a 14 cm 0.5% agarose gel electrophoresis at 20 volts for 16 hours overnight. The human DNA band was excised from the gel and electroeluted from the gel wedge in 0.5 x Tris acetate buffer at 100 volts for at least 2 hours (Maniatis et al., 1982). The eluted Not I digested DNA (〜15 kb to 25 kb) was then digested with EcoRI restriction enzyme to give a small fragment (〜0.5 kb to 5.0 kb), which was then used to label the DNA with radionucleotides. It melted easily for the next step. The DNA fragment was labeled by the hexamer random priming labeling method (Boehringer-Mannhein, catalog number 100460). Labeled DNA was spermine precipitated (100 μl TE, 5 μl 0.1 M spermine, and 5 μl 10 mg / ml salmon sperm DNA added) to remove unincorporated radionucleotides. The labeled DNA was then resuspended in 100 μl TE, 0.5 M NaCl at 65 ° C. for 5 minutes, and then reconstituted with human C ₀ t-1 DNA according to the manufacturer's instructions (Gibco / BRL, catalog number 5279SA). Blocked for 2-4 hours. Blocking solution the C _{0 t-1} blocked probe, at 42 ° C., and incubated on Zoo blot filter. The filters were washed in 2 × SSC, 0.1% SDS at room temperature for 30 minutes, then at 55 ° C. in the same buffer for 30 minutes. The filters were then exposed to Kodak XAR-5 film with sensitizer at -70 ° C for 1-3 days. Thus, the Zooblot was hybridized individually with a pool of Eco-R1 fragments from the insert, or with each fragment.

HTF island analysis A second method of identifying cosmids for use as probes for cDNA libraries was HTF island analysis. Since pulse field maps can reveal HTF islands, cosmids mapped to these HTF island regions were analyzed preferentially. HTF islands are segments of DNA that contain a very high frequency of unmethylated CpG dinucleotides (Tonolio et al., 1990) whose recognition sequence is due to clustering of restriction sites of enzymes containing CpG dinucleotides. Will be revealed. Enzymes known to be useful in HTF-island analysis are AscI, NotI, BssHII, EagI, ScaII, NaeI, NarI, SmaI and MluI (Andand, 1992). A pulse field map was created using the enzymes NotI, NruI, EagI, SacII and SalI, and two HTF islands were found. These islands are located at the end of the region, one at the end of the GP2B locus and the other at the base of the locus, both outside the BRCA1 region. Cosmids derived from TAC covering these two positions were analyzed to identify those containing these restriction sites, and thus HTF islands.

cDNA Screening Clones that contain HTF islands or that hybridize to DNA of other species than human are likely to contain coding sequences. Human DNA from these clones was isolated as a total insert or as an EcoRI fragment and labeled as described above. Using the labeled DNA, various cDNAs were obtained under the same conditions as the Zooblot, except that the cDNA filter was subjected to two more 30 minute washes of 0.1 × SSC, 0.1% SDS at 65 ° C. for 30 minutes. The library filters were screened.
Most of the cDNA libraries used to date in our experiments (normal breast tissue, libraries from breast tissue from women with malignant malignancy at 8 months of gestation) are available from Clonetech, Inc. Prepared from A cDNA library generated from breast tissue of 8-month-old pregnant women is available from Clonetech (Cat. No. HL1037a) in the lambda gt-10 vector and is propagated in C600Hfl bacterial host cells. Normal and malignant breast tissue samples were isolated from a 37 year old Caucasian woman and 1 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, to select the size of the final product, which was then cloned into a lambda ZapII vector, and the bacterial library was described as described by the manufacturer. The XL1-blue strain was grown. Additional tissue-specific cDNA libraries include human fetal brain (Stratagene, catalog number 936206), human testis (Clonetech, catalog HL3024), human thymus (Clonetech, catalog HL1127n), human brain (Clonetech, catalog HL11810), human placenta. (Clonetech, catalog 1075b), and human skeletal muscle (Clonetech, catalog HL1124b).

平板 Plate the cDNA library with its host cells on NZCYM plates and make filter lifts from each plate in duplicate as in Maniatis et al. (1982). Insert (human) DNA from candidate genomic clones was purified and radiolabeled to high specific activity. Then, the radioactive DNA was hybridized to the cDNA filter to identify the cDNA corresponding to the gene located in the candidate cosmid clone. The cDNAs identified by this method were picked, re-plated, and screened again with the labeled clone insert or EcoRI fragment DNA derived therefrom to confirm their positive status. Clones that were positive after the second round of screening were then expanded and their DNA was purified for Southern blot analysis and sequencing. Either purify the clone as a plasmid through in vivo excision of the plasmid from the lambda phage vector, as described in the protocol from the manufacturer, or isolate the clone from lambda phage as a restriction fragment and subtype the plasmid vector. Cloned.

Southern blot analysis was performed in duplicate, and one confirmed that the cDNA insert contained a hybridizing sequence using the original genomic insert DNA as a probe. A second blot was hybridized with the cDNA insert DNA from the largest cDNA insert to determine which clone represented the same gene. All cDNAs that hybridized and were unique with the genomic clone were sequenced and DNA analysis was performed to determine if the sequence represented a known or unique gene. All cDNA clones that appeared to be unique were further analyzed as candidate BRCA1 loci. Specifically, the clones are hybridized to a Northern blot to look for breast specific expression and differential expression in normal-breast tumor RNA. They were also analyzed by PCR for clones in the BRCA1 region to confirm their location. To map the locus spread, full-length cDNAs are isolated and their sequences are used as PCR probes around the YAC and the clone containing and containing the same original clone. The intron-exon boundaries were then further defined by sequence analysis.

We screened normal breast, 8 month gestational breast and fetal brain cDNA libraries with zooblot positive EcoRI fragments from cosmid BAC and P1 clones in this region. Potential BRCA1 cDNA clones were identified among the three libraries. Clones were picked, replated, and rescreened with the original probe to confirm that they were positive.

Analysis of Hybrid-Selected cDNA cDNA fragments obtained from direct selection were checked by Southern blot hybridization to probe DNA to confirm that they were derived from contig. Those that passed this test were sequenced in their entirety. The set of DNA sequences thus obtained was then checked against each other and duplicate independent clones were found. For example, clones 694-65, 1240-1 and 1240-33 were obtained independently and were subsequently shown to be derived from the same contiguous cDNA sequence designated EST: 489: 1.

Analysis of Candidate Clones One or more of the candidate genes from above were sequenced and the information was used to identify and classify each expressed gene. DNA sequences were compared to known genes by nucleotide sequence comparison and by translation in all frames, followed by comparison to known amino acid sequences. This is the Genetic Data Environment (GDE) version 2.2 software and Basic software from a client / server software package (eg, BLASTN 1.3.13MP) for sequence comparisons against both local and remote sequence data banks (eg, GenBank). Achieved using the Local Alignment Search Tool (Blast) series, running on a Sun SPARC workstation. The reconstructed sequence was obtained from a collection of cDNA clones identified by cosmid and P1. All candidate genes representing the new sequence were further analyzed to test them for the putative BRCA1 locus.

Mutation Screening To screen for mutations in affected pedigrees, two different approaches were taken. First, genomic DNA isolated from a family member known to carry the susceptibility allele of BRCA1 was used as a template for amplifying candidate gene sequences by PCR. If the PCR primers flank or overlap the intron / exon boundaries, the amplified fragment will be larger than expected from the cDNA sequence or will not be present in the amplification mixture. By combining such amplification experiments and sequencing of P1, BAC or cosmid clones with designed primers, an intron / exon structure can be established and ultimately the DNA sequence of genomic DNA from the pedigree can be obtained. .

と If the intron / exon structure of the candidate gene is complex, a much faster second approach involves sequencing fragments amplified from pedigree lymphocyte cDNA. CDNA synthesized from lymphocyte mRNA extracted from pedigree blood was used as a substrate for PCR amplification using the designed primer set. If the candidate gene is expressed to a significant extent in lymphocytes, such experiments usually result in amplified fragments, which can be sequenced directly without knowledge of intron / exon binding.

産物 The products of such sequencing reactions were analyzed by gel electrophoresis to determine positions in the sequence containing mutations such as deletions or insertions, or base pair substitutions causing amino acid changes or other deleterious effects.

配 列 Any sequence within the BRCA1 region expressed in the breast is considered a candidate gene for BRCA1. Strong evidence that a given candidate gene corresponds to BRCA1 comes from evidence that pedigree families contain the candidate defective allele.

Example 8
Identification of BRCA1 Identification of BRCA1 A number of strategies were used to develop a detailed map of the transcript for the 600 kb region of 17q21 between D17S1321 and D17S1324. Sequences expressing candidates include: 1) direct screening of breast, fetal brain, or lymphocyte cDNA libraries, 2) hybrid selection of breast, lymphocyte or ovarian cDNA, or 3) random sequencing of genomic DNA and XPOUUND ( Production of coding exons by Thomas and Skolnick, 1994). These expressed sequences in many cases were assembled into a contig consisting of several independently identified sequences. Candidate genes may consist of more than one of these candidate expression sequences. 65 candidate expressed sequences within this region were identified by hybrid selection, by direct screening of a cDNA library, and by random sequencing of the P1 subclone. The expressed sequences were 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 the expressed sequences, 1141: 1 (649 bp), 694: 5 (213 bp) and 754: 2 (1079 bp), were isolated and shown to eventually represent part of BRCA1. 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, which encodes different parts of a single gene. Suggest that. Screening of breast, fetal brain, thymus, testis, lymphocyte and placental cDNA and PCR experiments with breast mRNA linked 1141: 1, 694: 5 and 754: 2 contig. 5 'RACE experiments with thymus, testis and breast mRNA extended contig to the putative 5' end, resulting in a composite full-length sequence. Direct sequencing of PCR and P1 and BACs in the region were used to locate introns and to determine splice donor and acceptor sites. These three expressed sequences were merged into a single transcriptional transfer unit, which was found to be BTCA1 by final analysis. This transcription unit was located adjacent to D17S855 at the center of the 600 kb region (FIG. 4).

The combination of the sequence obtained from the cDNA clone, the hybrid selection sequence, and the amplified PCR product allowed the construction of a composite full-length BTCA1 cDNA (SEQ ID NO: 1). The sequence of the BRCA1 cDNA (up to the stop codon) has also been deposited with GenBank and given accession number U-14680. The deposited sequence is referred to and integrated. The cDNA clone that extends furthest in the 3 'direction contains a poly (A) tract preceded by a polyadenylation signal. The conceptual translation of the cDNA resulted in a single long open reading frame of 208 kilodaltons (amino acid sequence, SEQ ID NO: 2) with potential start codons flanked by sequences similar to the Kozak consensus sequence (Kozak, 1987). It was made clear. A search of Smith-Waterman (Smith and Waterman, 1981) and BLAST (Altschyl et al., 1990) identified a near amino-terminal sequence with considerable homology to the zinc-finger domain (FIG. 5). This sequence, a consensus C ₃ HC ₄ zinc - containing cysteine and histidine residues present in the finger motif, zinc in the database - having both a number of other residues regarding finger proteins. The BRCA1 gene consists of 23 coding exons located over 100 kb of genomic DNA (FIG. 6). Northern blots using a fragment of the BRCA1 cDNA as a probe identified a single transcript of approximately 7.8 kb, which was most abundantly present in the breast, thymus and testis, and also in the ovaries (FIG. 7). Four separately spliced products were detected in independent cDNA clones; three of these were found in breast and two in ovarian mRNA (FIG. 6). The PCR overview from the tissue cDNA further supports the notion that there is considerable heterozygosity near the 5 'end of the transcript from this gene; All the changes detected alter the transcript in the region 5 'to the identified start codon. We have detected six potential alternative splice donors in this 5 'untranslated region, with the longest deletion being 1,155 bp. The predominant form of BRCA1 protein in the breast and ovary lacks exon 4. The nucleotide sequence of BRCA1 exon 4 is shown in SEQ ID NO: 11, and the predicted amino acid sequence is shown in SEQ ID NO: 12.

An additional 5 'sequence of' BRCA1 genomic DNA is shown in SEQ ID NO: 13. G at position 1 represents a potential start site in the testis. A at position 140 represents a potential start site in somatic tissue. There are six alternative splice forms of the 5 'sequence shown in FIG. G at position 356 represents the first recognized splice donor site. G at position 444 represents the first splice donor site in two clones (testis 1 and testis 2). G at position 889 represents the first splice donor site in thymus 3. The fourth splice donor site is G at position 1230. T at position 1513 represents a splice acceptor site for all of the splice donors. A fifth alternative splice form has a first splice donor site at position 349 with a first splice donor site at position 591 and a second splice donor site at position 889 and a second acceptor site at position 1513. Six other forms are not spliced in this 5 'region. A at position 1532 is the recognized start site, which appears at position 120 in SEQ ID NO: 1. The partial genomic DNA sequence determined for BRCA1 is shown in FIGS. 12-19 and SEQ ID NOs: 14-34. The lower case letters (Figures 12-19) represent intron sequences, while the upper case letters represent exon sequences. In FIGS. 12-19, the undefined intervals in the intron are denoted by vvvvvvvv. Intron / exon binding is shown in Tables 9 and 10. CAG found at the 5 'end of exons 8 and 14 was found in some cDNAs, but not others. Known polymorphic sites are shown in boldface type in FIGS. 12-19 and are underlined.

Low stringency blots probing genomic DNA from divergent phylogenetic background organisms with a BRCA1 sequence lacking the zinc-finger region reveals strongly hybridizing fragments in humans, monkeys, sheep and pigs. Very weak hybridization signals were revealed in rodents. This result indicates that, apart from zinc-finger dometon, BRCA1 is only conserved at moderate levels throughout evolution.

Germline BRCA1 Mutations in 17q-Linked Pedigrees The most stringent test for BRCA1 candidate genes is for potentially disruptive mutations in carrier individuals from a pedigree that isolate 17q-linked susceptibility to breast and ovarian cancer. Is to find out. Such individuals must contain a different BRCA1 allele than the wild-type sequence. The set of DNA samples used in this analysis consisted of DNA from individuals representing eight different BRCA1 kindreds (Table 11).

対 The logarithm of the likelihood (LOD) score in these families ranges from 9.49 to -0.44 for the set of markers at 17q21. Four families have a satisfactory LOD score per linkage, and four have a low positive or negative LOD score. The latter ancestry was included. Since they exhibit a haplotype shared on chromosome 17q21 for at least three affected members. Furthermore, all families in the set show early age of onset of breast cancer, four of the families include at least one case of ovarian cancer, and both are characteristic of the BRCA1 family. One kindred 2082 has almost the same degree of breast and ovarian cancer, which is an abnormal occurrence given that ovarian cancer is relatively rare in the population. All but two families have been identified in Utah. K2035 is from the Midwest. K2099 is an African-American-Family from South America.

In an initial screen for disease predisposing mutations in BRCA1, DNA from one individual carrying a predisposing haplotype in each kindred was tested. Twenty-three coding exons and related splice junctions were amplified from genomic DNA samples or from cDNA prepared from lymphocyte mRNA. Comparison of the amplified DNA sequence with the wild-type sequence revealed that four out of eight pedigree samples contained sequence variants (Table 12).

All four sequence variants are heterozygous, and each appears only in one of the families. Kindred 2082 contains a nonsense mutation in exon 11 (FIG. 9). Kindred 1910 contains a single nucleotide insertion at exon 20 (FIG. 10). Kindred 2099 contains a missense mutation in exon 21 resulting in a Met → Arg substitution. Frameshift and nonsense mutations appear to be disruptive to the function of the BRCA1 product. The peptide encoded by the frameshift allele in kindred 1910 contains a modified amino acid sequence beginning 108 residues from the wild-type C-terminus. The peptide encoded by the frameshift allele in kindred 1901 contains an amino acid sequence beginning with the 24th residue from the wild-type N-terminus. The mutant allele in kindred 2082 encodes a protein that loses 551 residues from the C-terminus. The missense substitution observed in pedigree 2099 is potentially destructive as it replaces small hydrophobic amino acids (Met) with large charged residues (Arg). Eleven common polymorphisms were also identified, eight in the coding sequence and three in the intron.

個人 Individuals studied in kindred 2035 clearly contain a regulatory mutation at BRCA1. In her cDNA, the polymorphic site (A → G at base 3667) appeared heterozygous, while her genomic DNA revealed heterozygosity at this site (FIG. 11). A possible explanation for this observation is that mRNA from her mutated BRCA1 allele is absent due to mutations that affect its production or stability. This possibility was further investigated by examining five polymorphic sites in the BRCA1 coding region, which were separated by as much as 3.5 kb in the BRCA1 transcript. In cases where her genomic DNA is heterozygous for the polymorphism, the cDNA is heterozygous. In individuals recruited to non-haplotype carriers from other kindreds and in kindred 2035, these polymorphic sites were observed as heterozygous in the cDNA, indicating that amplification from the cDNA did not favor one allele. Indicates that This analysis indicates that the BRCA1 mutation in kindred 2035 prevents transcription or causes instability or abnormal splicing of the BRCA1 transcript.

Co-segregation and population frequency analysis of BRCA1 mutations with BRCA1 haplotypes Besides potentially disrupting protein function, two criteria must be met with sequence variants that qualify as candidate disease predisposing mutations. Mutants must be consistent with being 1) present in individuals from a family carrying the predisposing BRCA1 haplotype, not present in other members of the family, and 2) rare in the general population.

Each mutation was tested for cosegregation for BRCA1. For the frameshift mutation in kindred 1910, two other haplotype carriers and one non-carrier were sequenced (FIG. 10). Only carriers exhibited frameshift mutations. The change from C to T in kindred 2082 created a new AvrII restriction site. Other carriers and non-carriers in the family were tested for the presence of restriction sites (FIG. 9). Allele-specific oligonucleotides (ASO) were designed to detect the presence of sequence variants in pedigree 2099. Some individuals from the pedigree have previously been detected in the pedigree, some known to carry the haplotype associated with the predisposition allele, and others known not to carry the associated haplotype. Mutations that have been screened by ASO. In each kindred, the corresponding mutant allele was detected in individuals carrying the BRCA1-associated haplotype, but not in non-carriers. In the case of potential regulatory mutations observed in individuals from kindred 2035, cDNA and genomic DNA from carriers in that kindred were compared for heterozygosity at polymorphic sites. In each case, the distinguished allele in the cDNA sample was shown to be on the chromosome carrying the BRCA1 disease predisposition allele (FIG. 11).

組 A set of normal DNA samples was screened using the ASO for each mutation to exclude the possibility that the mutation was simply a common polymorphism in the population. Gene frequency estimates in Caucasians were based on random samples from the Utah population. Gene frequency estimates in African-American were derived from African Americans used in her linkage studies and M. A.M. from 20 newly born African Americans in Utah. Based on 39 samples provided by Peracek-Vance. None of the four potential disease predisposing mutations were found in the appropriate control population, indicating that they are rare in the general population. Thus, two important requirements for the BRCA1 susceptibility allele were met by the candidate disease predisposing mutation: 1) co-segregation of the mutant allele with the disease, and 2) absence of the mutant allele in the control ( This indicates a low gene frequency in the general population)

Phenotypic expression of the BRCA1 mutation The effect of the mutation on the BRCA1 protein correlated with the observed differences in phenotypic expression in BRCA1 kindreds. Most BRCA1 families have a moderately increased risk of ovarian cancer and a smaller subset have a higher risk of ovarian cancer compared to those for breast cancer (Easton et al., 1993). Three of the four families in which the BRCA1 mutation was detected fall into the former category, while the fourth (K2082) falls into the high ovarian cancer risk group. Since the BRCA1 nonsense mutation found in K2082 is located closer to the amino terminus than the other mutations detected, it is predicted to have a different phenotype. In fact, the family K2082 mutation has a higher likelihood of ovarian cancer and has a later average age of diagnosis of breast cancer cases than other families (Goldgar et al., 1994). This difference in onset age is due to certainty bias in smaller, more penetrant families, or that reflects tissue-specific differences in the behavior of the BRCA1 mutation Will. The other three kindreds segregating the known BRCA1 mutations have, on average, one ovarian cancer every 10 breast cancers, but have a high population of breast cancer cases diagnosed in their late 20s or early 30s. Have. Family 1910 with a frameshift mutation is notable. This is because three of the four affected individuals had bilateral breast cancer, and in each case a second tumor was diagnosed within one of the first appearance. Kindred 2035 segregating the potential regulatory BRCA1 mutation would be expected to have a dramatic phenotype. Eighty percent of breast cancer cases in this kindred occur before the age of 50. This figure is as high as any in the set, indicating a highly penetrating BRCA1 mutant allele (Table 11).

Although the mutation is clearly harmful and causes breast cancer in very young women, each of the four families with the mutation has at least one mutation that has lived to age 80 without developing malignancy. Including women. Subsequent identification of other genetic or environmental factors that could reduce the effects of the BRCA1 mutation would be of paramount importance in the study.

No potential disease-predisposing mutations were found in four of the $ 8 putative BRCA1-linked families. Three of these four have LOD scores for BRCA1-linked markers of less than 0.55. Thus, these families do not appear to actually segregate the BRCA1 disease predisposition allele. Alternatively, mutations in these four kindreds, for example, appear to be present in regions of BRCA1 that affect transcript levels and thus divert detection.

Role of BRCA1 in Cancer Most currently identified tumor suppressor genes result in non-existent, non-functional or impaired protein products. Most of the TP53 mutations are missense; some of them have been shown to produce abnormal p53 molecules that interfere with the function of the wild-type product (Shaulian et al., 1992; Srivastava et al., 1993). A similar dominant negative mechanism of action is for several adenoma polybosis coli (APC) alleles producing truncated molecules (Su et al., 1993), and Wilms' tumor that alters DNA binding of proteins A point mutation in the gene (WT1) has been proposed (Little et al., 1993). The nature of the mutation observed in the BRCA1 coding sequence is consistent with the production of a dominant negative or non-functional protein. The putative regulatory mutation in kindred 2035 cannot be dominant negative; rather, this mutation appears to cause a reduction or complete loss of BRCA1 expression from the affected allele.

BRCA1 protein was found in a number of DNA-binding proteins, zinc to nucleic acids - similar to that deeply as dependent binding, C ₃ HC ₄ zinc - containing finger domain. The first 180 amino acids of BRCA1 contain five more basic residues than acidic residues. In contrast, the rest 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 BRCA1 encodes a transcription factor with an N-terminal DNA binding domain and a C-terminal transactivating "acid blob" domain. Interestingly, another familial tumor suppressor gene, WT1, also contains a zinc-finger motif (Haber et al., 1990). Many cancer predisposition mutations in WT1 alter the zinc-finger domain (Little et al., 1993; Haber et al., 1990; Little et al., 1992). WT1 encodes an alternative splicing exon that encodes a transcription factor and part of the zinc-finger domain that alters the DNA binding properties of WT1 (Bickmore et al., 1992). Some alternatively spliced forms of WT1 mRNA create molecules that act as transcriptional repressors (Drummond et al., 1994). Some BRCA1 splice variants alter the zinc-finger motif, raising the possibility that a similar regulatory mechanism as occurs in WT1 could be applied to BRCA1.

Example 9
Analysis of Tumors for BRCA1 Mutations To focus on the analysis for tumors that most likely contain the BRCA1 mutation, primary breast and ovarian cancers were typed for LOH in the BRCA1 region. Access LOH: D17S1323 and D17S855 (which is within the gene for BRCA1) and D17S1327 (which is approximately 100 kb distal to BRCA1) using three highly polymorphic simple tandem repeat markers. did. The total LOH frequency in the informative case (ie, the germline is heterozygous) was 32/72 (44%) for breast cancer and 12/21 (57%) for ovarian cancer, and the LOH Consistent with previous measurements (Futreal et al., 1992b; Jacobs et al., 1993; Sato et al., 1990; Eccles et al., 1990; Cropp et al., 1994). Thus, the analysis defined a panel of 32 breast and 12 ovarian tumors of mixed race and the starting age to be examined for BRCA mutations. The complete 5,589 bp coding region of the gene and intron / exon border sequences were screened in this tumor set by a combination of single-stranded conformational analysis (SSCA) and direct sequencing.

A total of 6 mutations (two of which are identical) were found, one in ovarian tumors, four in breast tumors and one in male unaffected haplotype carriers (Table 13). One mutant Glu1541 Ter introduced a stop codon at the carboxyl terminus that would result in a truncated protein missing 323 amino acids. In addition, two missense mutations were identified. These are Ala1708Glu and Met1775Arg, which involve the replacement of small hydrophobic residues by charged residues. Patients 17764 and 19964 are from the same family. In patient OV24, nucleotide 2575 is missing and in patients 17764 and 19964 nucleotides 2993-2996 are missing.

Several lines of evidence suggest that all five mutations represent a BRCA1 sensitive allele.
(I) all mutations are in the germline;
(Ii) all are absent from the appropriate control population, suggesting that they are not common polymorphs;
(Iii) also applies to tumors from patients belonging to kindreds segregating the BRCA1 sensitive allele (Smith et al., 1992; Kelcell et al., 1993) (if the mutations represent a neutral polymorphism, they are 50% of cases). %), Each mutant allele is carried in tumors;
(Iv) The age of onset in the four breast cancer cases with mutations varies between 24 and 42 years, consistent with the early onset of breast cancer in individuals with BRCA1; Diagnosed, which falls into the youngest 13% of all ovarian cancers; and finally,
(V) Despite the tumor set not being selected for this criterion, three of the five cases have a positive family history of breast and ovarian cancer found in their medical records as far back as the past.

BT106 was diagnosed with breast cancer at the age of 24. Her mother had ovarian cancer, her father had melanoma, and her paternal grandmother also had breast cancer. Patient MC44 (African-American) was 42 years old and had bilateral breast cancer. This patient had a sister who died of breast cancer at the age of 34, another sister who died of lymphoma, and a brother who died of lung cancer. Her mutation (Met1775Arg) has previously been detected in kindred 2099 (African-American, which segregates the BRCA1 sensitive allele and is absent in African-American and Caucasian controls). To our knowledge, patient MC44 is not associated with family 2099. Detection of rare mutant alleles (once in BRCA1 kindreds and once in apparently unrelated early onset breast cancer cases) suggests that Met1775Arg alterations may be a common disease predisposition in African-American. Taken together, these observations indicated that all four BRCA1 mutations in the tumor represented susceptibility alleles; no somatic mutations were detected in the samples analyzed.

少 The low number of somatic BRCA1 mutations was not expected given the frequency of LOH for 17q and the normal role of susceptibility genes as tumor suppressors in cancer progression. There are three possible explanations for this result: (i) some BRCA1 mutations were missing in the coding sequence according to our screening approach; (ii) BRCA1 somatic mutations were mainly outside of the coding exons. And (iii) the LOH event at 17q does not reflect a BRCA1 somatic mutation.

で あ れ ば If somatic BRCA1 mutations are truly rare in breast and ovarian cancers, this will have a strong implication for BRCA1 biology. The apparent absence of somatic BRCA1 mutations implies that there may be some fundamental differences in the development of tumors in genetically predisposed BRCA1 carriers as compared to tumors in the general population. I do. For example, mutations in BRCA1 may only have an effect on tumor formation at certain early stages in breast and ovarian pathogenesis. This probability is consistent with a major function for BRCA1 in premenopausal breast cancer. Such a model for the role of BRCA1 in breast and ovarian cancer predicts the interaction between reproductive hormones and BRCA1 function. However, no clinical or pathological differences (other than age of onset) of familial-sporadic breast and ovarian cancers have been described (Lynch et al., 1990). On the other hand, recent findings of increased TP53 mutations and micro-accompanying instability in breast cancer from patients with a family history of breast cancer (Glevov et al., 1994) indicate that some of the tumors genetically live in disease predisposing individuals Will reflect the differences. Now, the involvement of BRCA1 in this phenomenon can be addressed directly. Alternatively, the lack of somatic BRCA1 mutations may result from the presence of multiple genes that function in the same tumor suppressor pathway as BRCA1 but collectively represent a more favorable target for mutations in sporadic tumors. . Since a single element mutation in the genetic pathway is generally sufficient to disrupt the pathway, BRCA1 will mutate at a rate much lower than the sum of the mutation rates of the other elements.

Example 10
Analysis of BRCA1 gene The structure and function of the BRCA1 gene are measured by the following method.
Biological studies Mammalian expression vectors containing the BRCA1 cDNA are constructed and transfected into appropriate breast cancer cells that have a defect in the gene. The wild-type BRCA1 cDNA as well as the modified BRCA1 cDNA are used. The modified BRCA1 cDNA can be obtained from a modified BRCA1 allele or can be produced as described below. The phenotypic reversion in culture (eg, cell morphology, doubling time, anchorage-dependent growth) and the phenotype reversion in animals (eg, tumorigenicity) are examined. The study uses both wild-type and mutant forms of the gene (Section B).

Molecular Genetics Studies In vitro mutagenesis is performed to construct absent and missense mutants (by single base pair substitution at individual codons and cluster charge → alanine scanning mutagenesis). The mutant is used in biological, biochemical and biophysical studies.

Mechanism studies The ability of the BRCA1 protein to bind to known and unknown DNA sequences is examined. Its ability to transactivate a promoter is analyzed by a transient reporter expression system in mammalian cells. Any functional partner is found and identified using conventional techniques, such as a particle-capture and yeast two-hybrid system. Characterize the nature and function of the partner. These partners are targets for drug discovery.

Structural studies Recombinant proteins are produced in E. coli, yeast, insect and / or mammalian cells and used in crystallography and NMR experiments. It also uses protein molecule making. These studies facilitate structure-driven drug design.

Example 11
Two-Step Assay for Detecting the Presence of BRCA1 in a Sample Patient samples are processed according to the method disclosed by Antonarakis et al. (1985), separated through a 1% agarose gel, and transferred to a nylon membrane for Southern blot analysis. The membrane is UV cross-linked at 150 mJ using a GS Gene linker (Bio-Rad). The BRCA1 probe corresponding to nucleotide positions 3631-3930 of SEQ ID NO: 1 is subcloned into pTZ18U. E. coli MV1190 infected with M13KO7 helper phage (Bio-Rad, Richmond, CA) is transformed with phagemid. Single-stranded DNA is isolated by standard techniques (see Sambrook et al., 1989).

Blots are prehybridized in 7% sodium dodecyl sulfate (SDS) in 0.5 M NaPO ₄ at 65 ° C for 14-30 minutes. The method follows that described in Nguyen et al., 1992. The blot is hybridized overnight at 65 ° C. in 7% SDS, 0.5 M NaPO ₄ containing 25-50 mg / ml single-stranded probe DNA. The post-hybridization wash was two 30 minute washes in 5% SDS, 40 mM NaPO ₄ at 65 ° C., followed by two 30 minute 1% SDS, 40 mM NaPO ₄ washes at 65 ° C. Become.

ブ ロ ッ ト The blot is then rinsed with phosphate buffered saline (pH 6.8) for 5 minutes at room temperature, incubated with 0.2% casein in PBS for 30-60 minutes at room temperature, and rinsed for 5 minutes in PBS. The blot was then purified with 6M urea, 0.3M.

Preincubate with a hybridization buffer consisting of NaCl and 5 × Denhardt's solution (see Sambrook et al., 1989) at 45 ° C. in a shaking water bath for 5-10 minutes. The buffer was removed and the nucleotide sequence complementary to the universal primer site (UP-AP, Bio-Rad) of 50-75 μl / cm ² of fresh hybridization buffer + covalently cross-linked oligonucleotide-alkaline phosphatase With 2.5 nM of the conjugate. The blot was hybridized at 45 ° C. for 20-30 minutes, and the post-hybridization wash was washed twice at 45 ° C. for 10 minutes in 6 M urea, 1 × standard quenched saline (SSC), Incubate as a single wash in 1% SDS and 1 × SSC, 0.1% Triton X-100. The blot is rinsed for 10 minutes with 1 × SSC at room temperature.

The blot is incubated for 10 minutes at room temperature with shaking in a substrate buffer consisting of 0.1 M diethanolamine, 1 mM MgCl ₂ , 0.02% sodium azide, pH 10.0. Individually with substrate buffer and 0.2 mM AMPPD (3- (2'-spiroadamantane) -4-methoxy-4- (3'-phosphoryloxy) phenyl-1,2-dioxetane, disodium salt, Bio-Rad) Put the blots in a heat-sealable bag. After a 20 minute incubation at room temperature with shaking, the excess AMPPD solution is removed. The blot is exposed to X-rays overnight. A positive band indicates the presence of BRCA1.

Example 12
Preparation of Polyclonal Antibodies to BRCA1 A segment of the BRCA1 coding sequence was expressed as a fusion protein in E. coli. The overproduced protein was purified by gel elution and used to immunize rabbits and mice using a procedure similar to that described by Harlow and Lane, 1988. This approach has been shown to generate Abs for a variety of other proteins (eg, Kraemer et al., 1993).

ス ト レ ッ チ Briefly, a stretch of the BRCA1 coding sequence was cloned as a fusion protein in plasmid PET5A (Novagen, Inc., Madison, WI). The sequence incorporating BRCA1 contains the amino acid corresponding to # 1361-1554 of SEQ ID NO: 2. After induction with IPTG, overproduction of the fusion protein with the expected molecular weight was confirmed by SDS / PAGE. The fusion protein was purified from the gel by electroelution. Identification of the protein as a BRCA1 fusion product was confirmed by protein sequencing at the N-terminus. The purified protein was then used as an immunogen in Ukiza. Rabbits were immunized with 100 μg protein in Freund's complete adjuvant and boosted twice at three week intervals (first with 100 μg of immunogen in Freund's incomplete adjuvant, followed by 100 μg of immunogen in PBS). Thereafter, antibodies containing serum are collected for two weeks.

Repeat this procedure to obtain antibodies against mutant forms of the BRCA1 gene. In combination with antibodies to wild-type BRCA1, these antibodies are used to detect the presence and relative levels of mutant forms in various tissues and biological fluids.

Example 13
Preparation of Monoclonal Antibody Specific to BRCA1 A monoclonal antibody is prepared according to the following protocol. As is well known, mice are immunized with glutaraldehyde or EDC with an immunogen consisting of intact BRCA1 or BRCA1 peptide (wild-type or mutant) conjugated to keyhole limpet hemocyanin.

混合 Mix the immunogen with an adjuvant. Each mouse receives four injections of 10-100 μg of immunogen, and after the fourth injection, a blood sample is taken from the mouse to determine whether the serum contains antibodies to the immunogen. . Serum titers are measured by ELISA or RIA. Mice with sera indicating the presence of antibodies to the immunogen are selected for hybridoma generation.

The spleen is excised from the immunized mouse and a single cell suspension is prepared (see Harlow and Lane, 1988). Cell fusion is performed substantially as described by Kohler and Milstein, 1975. Briefly, P3.65.3 myeloma cells (American Type Culture Collection, Rockville, Md.) Are fused with immune spleen cells using polyethylene glycol as described by Harlowe and Lane, 1988. Cells are plated at a density of 2 × 10 ⁵ cells / well in 96-well tissue culture plates. Individual wells are examined for growth and the supernatants of the grown wells are tested for the presence of BRCA1-specific antibodies by ELISA or RIA using wild-type or mutant BRCA1 target protein. Cells in positive wells are expanded and subcloned to establish and confirm monoclonal antibodies.

ク ロ ー ン Grow clones with the desired specificity and grow as ascites in mice or hollow fibers to obtain sufficient antibodies for characterization and assay development.

Example 14
Sandwich assay for BRCA1 A monoclonal antibody is bound to a solid surface, such as a plate, tube, bead, or particle. Preferably, the antibodies are bound to the well surfaces of a 96-well ELISA plate. A 100 μl sample (eg, serum, urine, tissue cytosol) containing the BRCA1 peptide / protein (wild type or mutant) is bound to the solid phase antibody. Incubate the sample at room temperature for 2 hours. The sample fluid is then decanted and the solid phase washed with buffer to remove unbound material. 100 μl of a second monoclonal antibody (for a different antigenic determinant on the BRCA1 peptide / protein) is added to the solid phase. The antibody is labeled with a detector molecule (eg, ¹²⁵ I, enzyme, fluorophore, or chromophore) and the solid phase is incubated with the second antibody for 2 hours at room temperature. The second antibody is decanted and the solid phase is washed with buffer to remove unbound material.

定量 Quantify the amount of bound label, which is proportional to the amount of BRCA1 peptide / protein present in the sample. Separate assays are performed using a monoclonal antibody specific for wild-type BRCA1 and a monoclonal antibody specific for each of the mutants identified with BRCA1.

It will be appreciated that the methods and compositions of the present invention can be embodied in various illustrative forms, only a few of which have been disclosed herein. It will be apparent to those skilled in the art that other embodiments exist without departing from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as limiting.

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List of Patents and Patent Applications US Patent No. 3,817,837 US Patent No. 3,850,752 US Patent No. 3,939,350 US Patent No. 3,996,345 US Patent No. 4,275,149 U.S. Pat. No. 4,277,437 U.S. Pat. No. 4,366,241 U.S. Pat. No. 4,376,110 U.S. Pat. No. 4,486,530 U.S. Pat. No. 4,683,195 U.S. Pat. U.S. Pat. No. 4,816,567 U.S. Pat. No. 4,868,105 U.S. Pat. No. 5,252,479 EP 225,807 EP U.S. Pat. PCT Application Publication WO 84/03564, published September 13, 1984 Hitzeman et al., EP 73,675A.
PCT Application Publication WO 93/07282

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 Cancer
Susceptibility Gene

(iii) NUMBER OF SEQUENCES: 85

(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Venable, Baetjer, Howard & Civiletti, LLP
(B) STREET: 1201 New York Avenue, NW, 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: PatentIn 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-1995

(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 NUMBER: 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: 1:

(i) SEQUENCE 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..5708

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

agctcgctga gacttcctgg accccgcacc aggctgtggg gtttctcaga taactgggcc 60

CCTGCGCTCA GGAGGCCTTC ACCCTCTGCT CTGGGTAAAG TTCATTGGAA CAGAAAGAA 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 Gln Asn Val Ile Asn
1 5 10 15

GCT ATG CAG AAA ATC TTA GAG TGT CCC ATC TGT CTG GAG TTG ATC AAG 215
Ala Met Gln Lys Ile Leu Glu Cys Pro Ile Cys Leu Glu Leu Ile 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 Ile 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 Gln Lys Lys Gly Pro Ser Gln 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 Ile Thr Lys Arg Ser Leu Gln 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
Gln Leu Val Glu Glu Leu Leu Lys Ile Ile Cys Ala Phe Gln 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 Ile Ile Gln 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 Gln 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 Gln Glu Thr Ser Leu Ser Val Gln 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 Gln Arg Ile Gln Pro Gln 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 Ile 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 Gln Glu Leu Leu Gln Ile Thr
195 200 205

CCT CAA GGA ACC AGG GAT GAA ATC AGT TTG GAT TCT GCA AAA AAG GCT 791
Pro Gln Gly Thr Arg Asp Glu Ile 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 Gln
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

CAT CCA GAA AAG TAT CAG GGT AGT TCT GTT TCA AAC TTG CAT GTG GAG 935
His Pro Glu Lys Tyr Gln 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 Gln His Glu Asn Ser
275 280 285

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 Gln Pro Gly Leu Ala Arg Ser Gln 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 Pro 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 Gln 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 Ile Thr Leu Asn Ser Ser Ile Gln 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 Ile Asp Leu Leu
420 425 430

GCC AGT GAT CCT CAT GAG GCT TTA ATA TGT AAA AGT GAA AGA GTT CAC 1463
Ala Ser Asp Pro His Glu Ala Leu Ile 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 Ile Glu Asp Lys Ile Phe Gly Lys Thr
450 455 460

TAT CGG AAG AAG GCA AGC CTC CCC AAC TTA AGC CAT GTA ACT GAA AAT 1559
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 Ile Ile Gly Ala Phe Val Thr Glu Pro Gln Ile Ile Gln Glu Arg
485 490 495

CCC CTC ACA AAT AAA TTA AAG CGT AAA AGG 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 Ile Lys Lys Ala Asp Leu Ala Val Gln Lys Thr
515 520 525

CCT GAA ATG ATA AAT CAG GGA ACT AAC CAA ACG GAG CAG AAT GGT CAA 1751
Pro Glu Met Ile Asn Gln Gly Thr Asn Gln Thr Glu Gln Asn Gly Gln
530 535 540

GTG ATG AAT ATT ACT AAT AGT GGT CAT GAG AAT AAA ACA AAA GGT GAT 1799
Val Met Asn Ile 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 Ile Gln Asn Glu Lys Asn Pro Asn Pro Ile Glu Ser Leu Glu Lys
565 570 575

GAA TCT GCT TTC AAA ACG AAA GCT GAA CCT ATA AGC AGC AGT ATA AGC 1895
Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro Ile Ser Ser Ser Ile 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 Ile 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 Thr Arg His Ile 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 Gln
625 630 635 640

ATT GAT AGT TGT TCT AGC AGT GAA GAG ATA AAG AAA AAA AAG TAC AAC 2087
Ile Asp Ser Cys Ser Ser Ser Glu Glu Ile 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
Gln Met Pro Val Arg His Ser Arg Asn Leu Gln Leu Met Glu Gly Lys
660 665 670

GAA CCT GCA ACT GGA GCC AAG AAG AGT AAC AAG CCA AAT GAA CAG ACA 2183
Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Lys Pro Asn Glu Gln 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 Gln 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
Ile Ser Leu Val Pro Gly Thr Asp Tyr Gly Thr Gln Glu Ser Ile 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 Gln Cys Ala Ala Phe Glu Asn Pro Lys Gly Leu Ile 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
820 825 830

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 Ile 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 Gln Tyr Leu Gln 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 Gln 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 Gln 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 Gln Lys Glu Glu Asn Gln 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 Ile Lys Pro Val Gln Thr Val Asn Ile 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 Gln 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 Ile Lys Gly Gly Ser Arg Phe Cys Leu Ser Ser Gln 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 Ile Thr Pro Asn Lys His Gly Leu Leu Gln Asn
965 970 975

CCA TAT CGT ATA CCA CCA CTT TTT CCC ATC AAG TCA TTT GTT AAA ACT 3095
Pro Tyr Arg Ile Pro Pro Leu Phe Pro Ile 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 Ile 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 Ile Ser Arg Asn Asn Ile 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 Ile 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 Ile Asn Glu Ile Gly Ser Ser Asp Glu Asn Ile Gln 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 Gln Pro Glu Val Tyr Lys Gln 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 3479
His Pro Glu Ile Lys Lys Gln Glu Tyr Glu Glu Val Val Gln Thr Val
1105 1110 1115 1120

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 Ile Ser Asp Asn Leu Glu Gln 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 Gln 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 Ile 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 Ile Lys Glu Ser Ser Ala Val Phe Ser Lys Ser Val Gln 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 Gln
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 Gln 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 Ile Pro Ser Gln 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 Gln Val Ile 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
Gln 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 Gln 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
Gln Asp Pro Phe Leu Ile Gly Ser Ser Lys Gln Met Arg His Gln Ser
1315 1320 1325

GAA AGC CAG GGA GTT GGT CTG AGT GAC AAG GAA TTG GTT TCA GAT GAT 4151
Glu Ser Gln Gly Val Gly Leu Ser Asp Lys Glu Leu Val Ser Asp Asp
1330 1335 1340

GAA GAA AGA GGA ACG GGC TTG GAA GAA AAT AAT CAA GAA GAG CAA AGC 4199
Glu Glu Arg Gly Thr Gly Leu Glu Glu Asn Asn Gln Glu Glu Gln Ser
1345 1350 1355 1360

ATG GAT TCA AAC TTA GGT GAA GCA GCA TCT GGG TGT GAG AGT GAA ACA 4247
Met Asp Ser Asn Leu Gly Glu Ala Ala Ser Gly Cys Glu Ser Glu Thr
1365 1370 1375

AGC GTC TCT GAA GAC TGC TCA GGG CTA TCC TCT CAG AGT GAC ATT TTA 4295
Ser Val Ser Glu Asp Cys Ser Gly Leu Ser Ser Gln Ser Asp Ile Leu
1380 1385 1390

ACC ACT CAG CAG AGG GAT ACC ATG CAA CAT AAC CTG ATA AAG CTC CAG 4343
Thr Thr Gln Gln Arg Asp Thr Met Gln His Asn Leu Ile Lys Leu Gln
1395 1400 1405

CAG GAA ATG GCT GAA CTA GAA GCT GTG TTA GAA CAG CAT GGG AGC CAG 4391
Gln Glu Met Ala Glu Leu Glu Ala Val Leu Glu Gln His Gly Ser Gln
1410 1415 1420

CCT TCT AAC AGC TAC CCT TCC ATC ATA AGT GAC TCT TCT GCC CTT GAG 4439
Pro Ser Asn Ser Tyr Pro Ser Ile Ile Ser Asp Ser Ser Ala Leu Glu
1425 1430 1435 1440

GAC CTG CGA AAT CCA GAA CAA AGC ACA TCA GAA AAA GCA GTA TTA ACT 4487
Asp Leu Arg Asn Pro Glu Gln Ser Thr Ser Glu Lys Ala Val Leu Thr
1445 1450 1455

TCA CAG AAA AGT AGT GAA TAC CCT ATA AGC CAG AAT CCA GAA GGC CTT 4535
Ser Gln Lys Ser Ser Glu Tyr Pro Ile Ser Gln Asn Pro Glu Gly Leu
1460 1465 1470

TCT GCT GAC AAG TTT GAG GTG TCT GCA GAT AGT TCT ACC AGT AAA AAT 4583
Ser Ala Asp Lys Phe Glu Val Ser Ala Asp Ser Ser Thr Ser Lys Asn
1475 1480 1485

AAA GAA CCA GGA GTG GAA AGG TCA TCC CCT TCT AAA TGC CCA TCA TTA 4631
Lys Glu Pro Gly Val Glu Arg Ser Ser Pro Ser Lys Cys Pro Ser Leu
1490 1495 1500

GAT GAT AGG TGG TAC ATG CAC AGT TGC TCT GGG AGT CTT CAG AAT AGA 4679
Asp Asp Arg Trp Tyr Met His Ser Cys Ser Gly Ser Leu Gln Asn Arg
1505 1510 1515 1520

AAC TAC CCA TCT CAA GAG GAG CTC ATT AAG GTT GTT GAT GTG GAG GAG 4727
Asn Tyr Pro Ser Gln Glu Glu Leu Ile Lys Val Val Asp Val Glu Glu
1525 1530 1535

CAA CAG CTG GAA GAG TCT GGG CCA CAC GAT TTG ACG GAA ACA TCT TAC 4775
Gln Gln Leu Glu Glu Ser Gly Pro His Asp Leu Thr Glu Thr Ser Tyr
1540 1545 1550

TTG CCA AGG CAA GAT CTA GAG GGA ACC CCT TAC CTG GAA TCT GGA ATC 4823
Leu Pro Arg Gln Asp Leu Glu Gly Thr Pro Tyr Leu Glu Ser Gly Ile
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 Ile 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 Gln Leu Lys Val Ala Glu Ser Ala Gln Ser Pro Ala Ala
1605 1610 1615

GCT CAT ACT ACT GAT ACT GCT GGG TAT 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 ATT 5159
Val Tyr Lys Phe Ala Arg Lys His His Ile Thr Leu Thr Asn Leu Ile
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 Ile 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 Gln Ser Ile 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 Gln Gly Pro Lys Arg Ala Arg Glu Ser Gln Asp Arg Lys Ile
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 Ile 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 Gln Leu Glu Trp Met Val Gln 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 Ile Val
1795 1800 1805

GTT GTG CAG CCA GAT GCC TGG ACA GAG GAC AAT GGC TTC CAT GCA ATT 5591
Val Val Gln Pro Asp Ala Trp Thr Glu Asp Asn Gly Phe His Ala Ile
1810 1815 1820

GGG CAG ATG TGT GAG GCA CCT GTG GTG ACC CGA GAG TGG GTG TTG GAC 5639
Gly Gln Met 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 Gln Cys Gln Glu Leu Asp Thr Tyr Leu Ile Pro
1845 1850 1855

CAG ATC CCC CAC AGC CAC TAC TGA CTGCAGCCAG CCACAGGTAC AGAGCCACAG 5741
Gln Ile 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: 1863 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Asp Leu Ser Ala Leu Arg Val Glu Glu Val Gln Asn Val Ile Asn
1 5 10 15

Ala Met Gln Lys Ile Leu Glu Cys Pro Ile Cys Leu Glu Leu Ile Lys
20 25 30

Glu Pro Val Ser Thr Lys Cys Asp His Ile Phe Cys Lys Phe Cys Met
35 40 45

Leu Lys Leu Leu Asn Gln Lys Lys Gly Pro Ser Gln Cys Pro Leu Cys
50 55 60

Lys Asn Asp Ile Thr Lys Arg Ser Leu Gln Glu Ser Thr Arg Phe Ser
65 70 75 80

Gln Leu Val Glu Glu Leu Leu Lys Ile Ile Cys Ala Phe Gln Leu Asp
85 90 95

Thr Gly Leu Glu Tyr Ala Asn Ser Tyr Asn Phe Ala Lys Lys Glu Asn
100 105 110

Asn Ser Pro Glu His Leu Lys Asp Glu Val Ser Ile Ile Gln Ser Met
115 120 125

Gly Tyr Arg Asn Arg Ala Lys Arg Leu Leu Gln Ser Glu Pro Glu Asn
130 135 140

Pro Ser Leu Gln Glu Thr Ser Leu Ser Val Gln Leu Ser Asn Leu Gly
145 150 155 160

Thr Val Arg Thr Leu Arg Thr Lys Gln Arg Ile Gln Pro Gln Lys Thr
165 170 175

Ser Val Tyr Ile Glu Leu Gly Ser Asp Ser Ser Glu Asp Thr Val Asn
180 185 190

Lys Ala Thr Tyr Cys Ser Val Gly Asp Gln Glu Leu Leu Gln Ile Thr
195 200 205

Pro Gln Gly Thr Arg Asp Glu Ile Ser Leu Asp Ser Ala Lys Lys Ala
210 215 220

Ala Cys Glu Phe Ser Glu Thr Asp Val Thr Asn Thr Glu His His Gln
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 Gln Gly Ser Ser Val Ser Asn Leu His Val Glu
260 265 270

Pro Cys Gly Thr Asn Thr His Ala Ser Ser Leu Gln His Glu Asn Ser
275 280 285

Ser Leu Leu Leu Thr Lys Asp Arg Met Asn Val Glu Lys Ala Glu Phe
290 295 300

Cys Asn Lys Ser Lys Gln Pro Gly Leu Ala Arg Ser Gln His Asn Arg
305 310 315 320

Trp Ala Gly Ser Lys Glu Thr Cys Asn Asp Arg Arg Thr Pro Ser Thr
325 330 335

Glu Lys Lys Val Asp Leu Asn Ala Asp Pro Leu Cys Glu Arg Lys Glu
340 345 350

Trp Asn Lys Gln Lys Leu Pro Cys Ser Glu Asn Pro Arg Asp Thr Glu
355 360 365

Asp Val Pro Trp Ile Thr Leu Asn Ser Ser Ile Gln Lys Val Asn Glu
370 375 380

Trp Phe Ser Arg Ser Asp Glu Leu Leu Gly Ser Asp Asp Ser His Asp
385 390 395 400

Gly Glu Ser Glu Ser Asn Ala Lys Val Ala Asp Val Leu Asp Val Leu
405 410 415

Asn Glu Val Asp Glu Tyr Ser Gly Ser Ser Glu Lys Ile Asp Leu Leu
420 425 430

Ala Ser Asp Pro His Glu Ala Leu Ile Cys Lys Ser Glu Arg Val His
435 440 445

Ser Lys Ser Val Glu Ser Asn Ile Glu Asp Lys Ile Phe Gly 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 Ile Ile Gly Ala Phe Val Thr Glu Pro Gln Ile Ile Gln Glu Arg
485 490 495

Pro Leu Thr Asn Lys Leu Lys Arg Lys Arg Arg Pro Thr Ser Gly Leu
500 505 510

His Pro Glu Asp Phe Ile Lys Lys Ala Asp Leu Ala Val Gln Lys Thr
515 520 525

Pro Glu Met Ile Asn Gln Gly Thr Asn Gln Thr Glu Gln Asn Gly Gln
530 535 540

Val Met Asn Ile Thr Asn Ser Gly His Glu Asn Lys Thr Lys Gly Asp
545 550 555 560

Ser Ile Gln Asn Glu Lys Asn Pro Asn Pro Ile Glu Ser Leu Glu Lys
565 570 575

Glu Ser Ala Phe Lys Thr Lys Ala Glu Pro Ile Ser Ser Ser Ile Ser
580 585 590

Asn Met Glu Leu Glu Leu Asn Ile His Asn Ser Lys Ala Pro Lys Lys
595 600 605

Asn Arg Leu Arg Arg Lys Ser Ser Thr Arg His Ile His Ala Leu Glu
610 615 620

Leu Val Val Ser Arg Asn Leu Ser Pro Pro Asn Cys Thr Glu Leu Gln
625 630 635 640

Ile Asp Ser Cys Ser Ser Ser Glu Glu Ile Lys Lys Lys Lys Tyr Asn
645 650 655

Gln Met Pro Val Arg His Ser Arg Asn Leu Gln Leu Met Glu Gly Lys
660 665 670

Glu Pro Ala Thr Gly Ala Lys Lys Ser Asn Lys Pro Asn Glu Gln 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 Glu Asp Pro Lys Asp Leu Met Leu
740 745 750

Ser Gly Glu Arg Val Leu Gln Thr Glu Arg Ser Val Glu Ser Ser Ser
755 760 765

Ile Ser Leu Val Pro Gly Thr Asp Tyr Gly Thr Gln Glu Ser Ile 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 Gln Cys Ala Ala Phe Glu Asn Pro Lys Gly Leu Ile His
805 810 815

Gly Cys Ser Lys Asp Asn Arg Asn Asp Thr Glu Gly Phe Lys Tyr Pro
820 825 830

Leu Gly His Glu Val Asn His Ser Arg Glu Thr Ser Ile Glu Met Glu
835 840 845

Glu Ser Glu Leu Asp Ala Gln Tyr Leu Gln Asn Thr Phe Lys Val Ser
850 855 860

Lys Arg Gln Ser Phe Ala Pro Phe Ser Asn Pro Gly Asn Ala Glu Glu
865 870 875 880

Glu Cys Ala Thr Phe Ser Ala His Ser Gly Ser Leu Lys Lys Gln Ser
885 890 895

Pro Lys Val Thr Phe Glu Cys Glu Gln Lys Glu Glu Asn Gln Gly Lys
900 905 910

Asn Glu Ser Asn Ile Lys Pro Val Gln Thr Val Asn Ile Thr Ala Gly
915 920 925

Phe Pro Val Val Gly Gln Lys Asp Lys Pro Val Asp Asn Ala Lys Cys
930 935 940

Ser Ile Lys Gly Gly Ser Arg Phe Cys Leu Ser Ser Gln Phe Arg Gly
945 950 955 960

Asn Glu Thr Gly Leu Ile Thr Pro Asn Lys His Gly Leu Leu Gln Asn
965 970 975

Pro Tyr Arg Ile Pro Pro Leu Phe Pro Ile Lys Ser Phe Val Lys Thr
980 985 990

Lys Cys Lys Lys Asn Leu Leu Glu Glu Asn Phe Glu Glu His Ser Met
995 1000 1005

Ser Pro Glu Arg Glu Met Gly Asn Glu Asn Ile Pro Ser Thr Val Ser
1010 1015 1020

Thr Ile Ser Arg Asn Asn Ile Arg Glu Asn Val Phe Lys Glu Ala Ser
1025 1030 1035 1040

Ser Ser Asn Ile Asn Glu Val Gly Ser Ser Thr Asn Glu Val Gly Ser
1045 1050 1055

Ser Ile Asn Glu Ile Gly Ser Ser Asp Glu Asn Ile Gln 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 Gln Pro Glu Val Tyr Lys Gln Ser Leu Pro Gly Ser Asn Cys Lys
1090 1095 1100

His Pro Glu Ile Lys Lys Gln Glu Tyr Glu Glu Val Val Gln Thr Val
1105 1110 1115 1120

Asn Thr Asp Phe Ser Pro Tyr Leu Ile Ser Asp Asn Leu Glu Gln Pro
1125 1130 1135

Met Gly Ser Ser His Ala Ser Gln Val Cys Ser Glu Thr Pro Asp Asp
1140 1145 1150

Leu Leu Asp Asp Gly Glu Ile Lys Glu Asp Thr Ser Phe Ala Glu Asn
1155 1160 1165

Asp Ile Lys Glu Ser Ser Ala Val Phe Ser Lys Ser Val Gln Lys Gly
1170 1175 1180

Glu Leu Ser Arg Ser Pro Ser Pro Phe Thr His Thr His Leu Ala Gln
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 Gln His Leu Leu Phe Gly
1220 1225 1230

Lys Val Asn Asn Ile Pro Ser Gln 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 Gln Val Ile Leu Ala Lys Ala Ser
1265 1270 1275 1280

Gln Glu His His Leu Ser Glu Glu Thr Lys Cys Ser Ala Ser Leu Phe
1285 1290 1295

Ser Ser Gln Cys Ser Glu Leu Glu Asp Leu Thr Ala Asn Thr Asn Thr
1300 1305 1310

Gln Asp Pro Phe Leu Ile Gly Ser Ser Lys Gln Met Arg His Gln Ser
1315 1320 1325

Glu Ser Gln 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 Gln Glu Glu Gln 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 Gln Ser Asp Ile Leu
1380 1385 1390

Thr Thr Gln Gln Arg Asp Thr Met Gln His Asn Leu Ile Lys Leu Gln
1395 1400 1405

Gln Glu Met Ala Glu Leu Glu Ala Val Leu Glu Gln His Gly Ser Gln
1410 1415 1420

Pro Ser Asn Ser Tyr Pro Ser Ile Ile Ser Asp Ser Ser Ala Leu Glu
1425 1430 1435 1440

Asp Leu Arg Asn Pro Glu Gln Ser Thr Ser Glu Lys Ala Val Leu Thr
1445 1450 1455

Ser Gln Lys Ser Ser Glu Tyr Pro Ile Ser Gln 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 Gln Asn Arg
1505 1510 1515 1520

Asn Tyr Pro Ser Gln Glu Glu Leu Ile Lys Val Val Asp Val Glu Glu
1525 1530 1535

Gln Gln Leu Glu Glu Ser Gly Pro His Asp Leu Thr Glu Thr Ser Tyr
1540 1545 1550

Leu Pro Arg Gln Asp Leu Glu Gly Thr Pro Tyr Leu Glu Ser Gly Ile
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 Ile Pro Ser Ser Thr Ser Ala Leu
1585 1590 1595 1600

Lys Val Pro Gln Leu Lys Val Ala Glu Ser Ala Gln Ser Pro Ala Ala
1605 1610 1615

Ala His Thr Thr Asp Thr Ala Gly 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 Ile Thr Leu Thr Asn Leu Ile
1665 1670 1675 1680

Thr Glu Glu Thr Thr His Val Val Met Lys Thr Asp Ala Glu Phe Val
1685 1690 1695

Cys Glu Arg Thr Leu Lys Tyr Phe Leu Gly Ile Ala Gly Gly Lys Trp
1700 1705 1710

Val Val Ser Tyr Phe Trp Val Thr Gln Ser Ile 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 Gln Gly Pro Lys Arg Ala Arg Glu Ser Gln Asp Arg Lys Ile
1745 1750 1755 1760

Phe Arg Gly Leu Glu Ile Cys Cys Tyr Gly Pro Phe Thr Asn Met Pro
1765 1770 1775

Thr Asp Gln Leu Glu Trp Met Val Gln Leu Cys Gly Ala Ser Val Val
1780 1785 1790

Lys Glu Leu Ser Ser Phe Thr Leu Gly Thr Gly Val His Pro Ile Val
1795 1800 1805

Val Val Gln Pro Asp Ala Trp Thr Glu Asp Asn Gly Phe His Ala Ile
1810 1815 1820

Gly Gln Met Cys Glu Ala Pro Val Val Thr Arg Glu Trp Val Leu Asp
1825 1830 1835 1840

Ser Val Ala Leu Tyr Gln Cys Gln Glu Leu Asp Thr Tyr Leu Ile Pro
1845 1850 1855

Gln Ile 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: 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 A

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

CTAGCCTGGG CAACAAACGA 20

(2) INFORMATION FOR SEQ ID NO: 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:

GCAGGAAGCA GGAATGGAAC 20

(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 20

(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 20

(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 22

(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 23

(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 20

(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: tdj1239 B

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

TCTGTACCTG CTAAGCAGTG G 21

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 111 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..111

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

G GKC TTA CTC TGT 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 Gln 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 TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

Xaa Leu Leu Cys Cys Pro Ser Trp Ser Thr Xaa Cys Asp His Glu Ala
1 5 10 15

Tyr Cys Cys Leu Thr Pro Arg Leu Lys Arg Ser Tyr His Leu Ser Leu
20 25 30

Gln Val Ala Gly
35

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1534 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: 13:

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 TGGGGGAATC CTCGTGATAG GAACTGGAAT 600

ATGCCTTGAG GGGGACACTA TGTCTTTAAA AACGTCGGCT GGTCATGAGG TCAGGAGTTC 660

CAGACCAGCC TGACCAACGT GGTGAAACTC CGTCTCTACT AAAAATACNA AAATTAGCCG 720

GGCGTGGTGC 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 CAGGGATGTG 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 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: SEQ 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 TGGGGGAATC CTCGTGATAG GAACTGGAAT 600

ATGCCTTGAG GGGGACACTA TGTCTTTAAA AACGTCGGCT GGTCATGAGG TCAGGAGTTC 660

CAGACCAGCC TGACCAACGT GGTGAAACTC CGTCTCTACT AAAAATACNA AAATTAGCCG 720

GGCGTGGTGC 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 CAGGGATGTG 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 TGATATCTGA 60

AAGCTCACTG AAGGTAAGGA TCGTATTCTC TGCTGTATTC TCAGTTCCTG ACACAGCAGA 120

CATTTAATAA ATATTGAACG AACTTGAGGC CTTATGTTGA 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 ATAGAAGCTT 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

GAGTACAGWG TGCGATCATG AGGCTTACTG TTGCTTGACT CCTAGGCTCA AGCGATCCTA 180

TCACCTCAGT CTCCAAGTAG CTGGACTGTA AGTGCACACC ACCATATCCA GCTAAATTTT 240

GTGTTTTCTG TAGAGACGGG GTTTCGCCAT GTTTCCCAGG CTGGTCTTGA ACTTTGGGCT 300

TAACCCGTCT GCCCACCTAG GCATCCCAAA 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

GAAGCAACCA 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: 18:

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 CAGCTTGACA CAGGTTTGGA GTGTAAGTGT 300

TGAATATCCC AAGAATGACA CTCAAGTGCT GTCCATGAAA ACTCAGGAAG TTTGCACAAT 360

TACTTTCTAT GACGTGGTGA TAAGACCTTT TAGTCTAGGT TAATTTTAGT TCTGTATCTG 420

TAATCTATTT TAAAAAATTA CTCCCACTGG TCTCACACCT TATTT 465

(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: 19:

AAAAAATCAC AGGTAACCTT AATGCATTGT CTTAACACAA CAAAGAGCAT ACATAGGGTT 60

TCTCTTGGTT TCTTTGATTA TAATTCATAC ATTTTTCTCT AACTGCAAAC ATAATGTTTT 120

CCCTTGTATT TTACAGATGC AAACAGCTAT AATTTTGCAA AAAAGGAAAA TAACTCTCCT 180

GAACATCTAA AAGATGAAGT TTCTATCATC CAAAGTATGG GCTACAGAAA CCGTGCCAAA 240

AGACTTCTAC AGAGTGAACC CGAAAATCCT TCCTTGGTAA AACCATTTGT TTTCTTCTTC 300

TTCTTCTTCT TCTTTTCTTT TTTTTTTCTT TTTTTTTTTG AGATGGAGTC TTGCTCTGTG 360

GCCCAGGCTA GAAGCAGTCC TCCTGCCTTA GCCNCCTTAG TAGCTGGGAT TACAGGCACG 420

CGCACCATGC CAGGCTAATT TTTGTATTTT TAGTAGAGAC GGGGTTTCAT CATGTTGGCC 480

AGGCTGGTCT CGAACTCCTA ACCTCAGGTG ATC 513

(2) INFORMATION FOR SEQ 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 GGTCAGGAGT TTGAGACCAG 120

CCTGGCCAAC ATGGTGAAAC CCATCTCTAC TAAAAATACA AAAATTAGCT GGGTGTGGTG 180

GCACGTACCT GTAATCCCAG CTACTCGGGA GGCGGAGGCA CAAGAATTGC TTGAACCTAG 240

GACGCGGAGG TTGCAGCGAG CCAAGATCGC GCCACTGCAC TCCAGCCTGG GCCGTAGAGT 300

GAGACTCTGT CTCAAAAAAG AAAAAAAAGT AATTGTTCTA GCTGGGCGCA GTGGCTCTTG 360

CCTGTAATCC CAGCACTTTG GGAGGCCAAG GCGGGTGGAT CTCGAGTCCT AGAGTTCAAG 420

ACCAGCCTAG GCAATGTGGT GAAACCCCAT CGCTACAAAA AATACAAAAA TTAGCCAGGC 480

ATGGTGGCGT GCGCATGTAG TCCCAGCTCC TTGGGAGGCT GAGGTGGGAG GATCACTTGA 540

ACCCAGGAGA CAGAGGTTGC AGTGAACCGA GATCACGCCA CCACGCTCCA GCCTGGGCAA 600

CAGAACAAGA CTCTGTCTAA AAAAATACAA ATAAAATAAA AGTAGTTCTC ACAGTACCAG 660

CATTCATTTT TCAAAAGATA TAGAGCTAAA AAGGAAGGAA AAAAAAAGTA ATGTTGGGCT 720

TTTAAATACT CGTTCCTATA CTAAATGTTC TTAGGAGTGC TGGGGTTTTA TTGTCATCAT 780

TTATCCTTTT TAAAAATGTT ATTGGCCAGG CACGGTGGCT CATGGCTGTA ATCCCAGCAC 840

TTTGGGAGGC CGAGGCAGGC AGATCACCTG AGGTCAGGAG TGTGAGACCA GCCTGGCCAA 900

CATGGCGAAA CCTGTCTCTA CTAAAAATAC AAAAATTAAC TAGGCGTGGT GGTGTACGCC 960

TGTAGTCCCA GCTACTCGGG AGGCTGAGGC AGGAGAATCA ACTGAACCAG GGAGGTGGAG 1020

GTTGCAGTGT GCCGAGATCA CGCCACTGCA CTCTAGCCTG GCAACAGAGC AAGATTCTGT 1080

CTCAAAAAAA AAAAACATAT ATACACATAT ATCCCAAAGT GCTGGGATTA CATATATATA 1140

TATATATATA TATTATATAT ATATATATAT ATATATGTGA TATATATGTG ATATATATAT 1200

AACATATATA TATGTAATAT ATATGTGATA TATATATAAT ATATATATGT AATATATATG 1260

TGATATATAT ATATACACAC ACACACACAT ATATATGTAT GTGTGTGTAC ACACACACAC 1320

ACAAATTAGC CAGGCATAGT TGCACACGCT TGGTAGACCC AGCTACTCAG GAGGCTGAGG 1380

GAGGAGAATC TCTTGAACTT AGGAGGCGGA GGTTGCAGTG AGCTGAGATT GCGCCACTGC 1440

ACTCCAGCCT 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 TGWAGAGMCT 1680

GGGTCTCACY CYKTTGCTWA TGCTGGTCTT TGAACYCCYK 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 GAGGACAAAG CAGCGGATAC AACCTCAAAA 2040

GACGTCTGTC TACATTGAAT TGGGTAAGGG TCTCAGGTTT TTTAAGTATT TAATAATAAT 2100

TGCTGGATTC CTTATCTTAT AGTTTTGCCA AAAATCTTGG TCATAATTTG TATTTGTGGT 2160

AGGCAGCTTT GGGAAGTGAA TTTTATGAGC CCTATGGTGA GTTATAAAAA ATGTAAAAGA 2220

CGCAGTTCCC ACCTTGAAGA ATCTTACTTT AAAAAGGGAG CAAAAGAGGC CAGGCATGGT 2280

GGCTCACACC TGTAATCCCA GCACTTTGGG AGGCCAAAGT GGGTGGATCA CCTGAGGTCG 2340

GGAGTTCGAG ACCAGCCTAG CCAACATGGA GAAACTCTGT CTGTACCAAA AAATAAAAAA 2400

TTAGCCAGGT GTGGTGGCAC ATAACTGTAA TCCCAGCTAC TCGGGAGGCT GAGGCAGGAG 2460

AATCACTTGA ACCCGGGAGG TGGAGGTTGC GGTGAACCGA GATCGCACCA TTGCACTCCA 2520

GCCTGGGCAA AAATAGCGAA ACTCCATCTA AAAAAAAAAA AGAGAGCAAA AGAAAGAMTM 2580

TCTGGTTTTA AMTMTGTGTA AATATGTTTT TGGAAAGATG GAGAGTAGCA ATAAGAAAAA 2640

ACATGATGGA TTGCTACAGT ATTTAGTTCC AAGATAAATT GTACTAGATG AGGAAGCCTT 2700

TTAAGAAGAG CTGAATTGCC AGGCGCAGTG GCTCACGCCT GTAATCCCAG CACTTTGGGA 2760

GGCCGAGGTG GGCGGATCAC CTGAGGTCGG GAGTTCAAGA CCAGCCTGAC CAACATGGAG 2820

AAACCCCATC TCTACTAAAA AAAAAAAAAA AAAAATTAGC CGGGGTGGTG GCTTATGCCT 2880

GTAATCCCAG CTACTCAGGA GGCTGAGGCA GGAGAATCGC TTGAACCCAG GAAGCAGAGG 2940

TTGCAGTGAG CCAAGATCGC ACCATTGCAC TCCAGCCTAG GCAACAAGAG TGAAACTCCA 3000

TCTCAAAAAA AAAAAAAAAG AGCTGAATCT TGGCTGGGCA GGATGGCTCG TGCCTGTAAT 3060

CCTAACGCTT TGGAAGACCG AGGCAGAAGG ATTGGTTGAG TCCACGAGTT TAAGACCAGC 3120

CTGGCCAACA TAGGGGAACC CTGTCTCTAT TTTTAAAATA ATAATACATT TTTGGCCGGT 3180

GCGGTGGCTC ATGCCTGTAA TCCCAATACT TTGGGAGGCT GAGGCAGGTA GATCACCTGA 3240

GGTCAGAGTT CGAGACCAGC CTGGATAACC TGGTGAAACC CCTCTTTACT AAAAATACAA 3300

AAAAAAAAAA AAATTAGCTG GGTGTGGTAG CACATGCTTG TAATCCCAGC TACTTGGGAG 3360

GCTGAGGCAG GAGAATCGCT TGAACCAGGG AGGCGGAGGT TACAATGAGC CAACACTACA 3420

CCACTGCACT CCAGCCTGGG CAATAGAGTG AGACTGCATC TCAAAAAAAT AATAATTTTT 3480

AAAAATAATA AATTTTTTTA AGCTTATAAA AAGAAAAGTT GAGGCCAGCA TAGTAGCTCA 3540

CATCTGTAAT CTCAGCAGTG GCAGAGGATT GCTTGAAGCC AGGAGTTTGA GACCAGCCTG 3600

GGCAACATAG CAAGACCTCA TCTCTACAAA AAAATTTCTT TTTTAAATTA GCTGGGTGTG 3660

GTGGTGTGCA TCTGTAGTCC CAGCTACTCA GGAGGCAGAG GTGAGTGGAT ACATTGAACC 3720

CAGGAGTTTG AGGCTGTAGT GAGCTATGAT CATGCCACTG CACTCCAACC TGGGTGACAG 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

TTAGATGTCA GAGCCTGTGC TTTTTTTTTG TTTTTGTTTT TGTTTTCAGT AGAAACGGGG 4260

GTCTCACTTT GTTGGCCAGG CTGGTCTTGA ACTCCTAACC TCAAATAATC CACCCATCTC 4320

GGCCTCCTCA AGTGCTGGGA TTACAGGTGA GAGCCACTGT GCCTGGCGAA GCCCATGCCT 4380

TTAACCACTT CTCTGTATTA CATACTAGCT 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 CACCCGGCTA ATTTTTGTAT TTTTAGTAGA GATGGGGTTT 5640

CACCATGTTG GCCAGGCTGG TCTTGAACTC ATGACCTCAA GTGGTCCACC CGCCTCAGCC 5700

TCCCAAAGTG CTGGAATTAC AGGCTTGAGC CACCGTGCCC AGCAACCATT 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

TTATAGAGAT 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

(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: 21:

GATCCTAAGT GGAAATAATC TAGGTAAATA GGAATTAAAT GAAAGAGTAT GAGCTACATC 60

TTCAGTATAC TTGGTAGTTT ATGAGGTTAG TTTCTCTAAT ATAGCCAGTT GGTTGATTTC 120

CACCTCCAAG GTGTATGAAG TATGTATTTT TTTAATGACA ATTCAGTTTT TGAGTACCTT 180

GTTATTTTTG TATATTTTCA GCTGCTTGTG AATTTTCTGA GACGGATGTA ACAAATACTG 240

AACATCATCA ACCCAGTAAT AATGATTTGA ACACCACTGA GAAGCGTGCA GCTGAGAGGC 300

ATCCAGAAAA GTATCAGGGT AGTTCTGTTT CAAACTTGCA TGTGGAGCCA TGTGGCACAA 360

ATACTCATGC CAGCTCATTA CAGCATGAGA ACAGCAGTTT ATTACTCACT AAAGACAGAA 420

TGAATGTAGA AAAGGCTGAA TTCTGTAATA AAAGCAAACA GCCTGGCTTA GCAAGGAGCC 480

AACATAACAG ATGGGCTGGA AGTAAGGAAA CATGTAATGA TAGGCGGACT 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 CGTAAAAGGA GACCTACATC AGGCCTTCAT CCTGAGGATT 1080

TTATCAAGAA AGCAGATTTG GCAGTTCAAA AGACTCCTGA AATGATAAAT CAGGGAACTA 1140

ACCAAACGGA GCAGAATGGT CAAGTGATGA ATATTACTAA TAGTGGTCAT GAGAATAAAA 1200

CAAAAGGTGA TTCTATTCAG AATGAGAAAA ATCCTAACCC AATAGAATCA CTCGAAAAAG 1260

AATCTGCTTT CAAAACGAAA GCTGAACCTA TAAGCAGCAG 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 AGAAACCTAC AACTCATGGA AGGTAAAGAA CCTGCAACTG 1560

GAGCCAAGAA GAGTAACAAG CCAAATGAAC AGACAAGTAA AAGACATGAC AGCGATACTT 1620

TCCCAGAGCT GAAGTTAACA AATGCACCTG 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 CTAGGGAAGG 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 TTTGAATGTG AACAAAAGGA AGAAAATCAA GGAAAGAATG AGTCTAATAT 2280

CAAGCCTGTA CAGACAGTTA 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 AGCATCCTGA 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 3120

TTTGGCTCAG GGTTACCGAA GAGGGGCCAA 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

AAAGGCATCT 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 GTGACATGAA 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 CHARACTERISTICS:
(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 AATTGGGATT TTCCCTCNCT AACATCANTG GAATAATTTC 120

ATGGCATTAA TTGCATGAAT GTGGTTAGAT TAAAAGGTGT TCATGCTAGA ACTTGTAGTT 180

CCATACTAGG TGATTTCAAT TCCTGTGCTA AAATTAATTT GTATGATATA TTNTCATTTA 240

ATGGAAAGCT TCTCAAAGTA TTTCATTTTC TTGGTACCAT TTATCGTTTT TGAAGCAGAG 300

GGATACCATG CAACATAACC TGATAAAGCT CCAGCAGGAA ATGGCTGAAC TAGAAGCTGT 360

GTTAGAACAG CATGGGAGCC AGCCTTCTAA CAGCTACCCT TCCATCATAA GTGACTCTTC 420

TCCCTTGAG GACCTGCGAA ATCCAGAACA AAGCACATCA GAAAAAGGTG TGTATTGTTG 480

GCCAAACACT GATATCTTAA 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

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

CCATTGGTGC TAGCATCTGT CTGTTGCATT GCTTGTGTTT 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 AAGAGATATT 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 TNTTCTTGNG GGTTGAGGGA 900

GGAGATCACN TTGGACCCCG GAGGGGNGGG TGGGGGNGAG CAGGNCAAAA CACNGACCCA 960

GCTGGGGTGG AAGGGAAGCC CACTCNAAAA AANNTTN 997

(2) INFORMATION FOR SEQ ID NO: 26:

(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 GATCCTTCTG AAGACAGAGC 240

CCCAGAGTCA GCTCGTGTTG GCAACATACC ATCTTCAACC TCTGCATTGA AAGTTCCCCA 300

ATTGAAAGTT GCAGAATCTG CCCAGAGTCC AGCTGCTGCT CATACTACTG ATACTGCTGG 360

GTATAATGCA ATGGAAGAAA GTGTGAGCAG GGAGAAGCCA GAATTGACAG CTTCAACAGA 420

AAGGGTCAAC AAAAGAATGT CCATGGTGGT GTCTGGCCTG ACCCCAGAAG AATTTGTGAG 480

TGTATCCATA TGTATCTCCC TAATGACTAA GACTTAACAA CATTCTGGAA AGAGTTTTAT 540

GTAGGTATTG TCAATTAATA ACCTAGAGGA AGAAATCTAG AAAACAATCA CAGTTCTGTG 600

TAATTTAATT TCGATTACTA ATTTCTGAAA ATTTAGAAY 639

(2) INFORMATION FOR SEQ ID NO: 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

(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 AAGCTTAAGC CAGGNGACGT GGANCNATGN 120

GTTGTTTNTT GTTTGGTTAC CTCCAGCCTG 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 GCGCATGCCT GTAATCGCAG CACTTTGGGA GGCCGAGGCG GGCAGATCAC 540

GAGATTAGGA GATCGAGACC ATCCTGGCCA GCATGGTGAA ACCCCGTCTC TACTANNAAA 600

TGGNAAAATT ANCTGGGTGT GGTCGCGTGC NCCTGTAGTC CCAGCTACTC GTGAGGCTGA 660

GGCAGGAGAA TCACTTGAAC CGGGGAAATG 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

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

GATCTATAGC TAGCCTTGGC GTCTAGAAGA TGGGTGTTGA GAAGAGGGAG TGGAAAGATA 60

TTTCCTCTGG TCTTAACTTC ATATCAGCCT CCCCTAGACT TCCAAATATC CATACCTGCT 120

GGTTATAATT AGTGGTGTTT TCAGCCTCTG ATTCTGTCAC CAGGGGTTTT AGAATCATAA 180

ATCCAGATTG ATCTTGGGAG TGTAAAAAAC TGAGGCTCTT TAGCTTCTTA GGACAGCACT 240

TCCTGATTTT GTTTTCAACT TCTAATCCTT TGAGTGTTTT TCATTCTGCA GATGCTGAGT 300

TTGTGTGTGA ACGGACACTG AAATATTTTC TAGGAATTGC GGGAGGAAAA TGGGTAGTTA 360

GCTATTTCTG TAAGTATAAT ACTATTTCTC CCCTCCTCCC TTTAACACCT CAGAATTGCA 420

TTTTTACACC TAACATTTAA CACCTAAGGT TTTTGCTGAT GCTGAGTCTG AGTTACCAAA 480

AGGTCTTTAA ATTGTAATAC TAAACTACTT TTATCTTTAA TATCACTTTG 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 GGAAGGACCT CTCCTCTGTC ATTCTTCCTG 780

TGCTCTTTTG TGAATCGCTG ACCTCTCTAT CTCCGTGAAA 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
(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: 29:

NAAAAACGGG GNNGGGANTG GGCCTTAAAN CCAAAGGGCN AACTCCCCAA CCATTNAAAA 60

ANTGACNGGG GATTATTAAA ANCGGCGGGA AACATTTCAC NGCCCAACTA ATATTGTTAA 120

ATTAAAACCA CCACCNCTGC NCCAAGGAGG GAAACTGCTG CTACAAGCCT TATTAAAGGG 180

CTGTGGCTTT AGAGGGAAGG ACCTCTCCTC TGTCATTCTT CCTGTGCTCT TTTGTGAATC 240

GCTGACCTCT CTATGTCCGT GAAAAGAGCA CGTTCTTCGT CTGTATGTAA CCTGTCTTTT 300

CTATGATCTC TTTAGGGGTG 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:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 567 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: 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 TCCAAAGCGA GCAAGAGAAT 300

CCCAGGACAG AAAGGTAAAG CTCCCTCCCT CAAGTTGACA AAAATCTCAC CCCACCACTC 360

TGTATTCCAC TCCCCTTTGC AGAGATGGGC CGCTTCATTT TGTAAGACTT ATTACATACA 420

TACACAGTGC TAGATACTTT CACACAGGTT CTTTTTTCAC TCTTCCATCC CAACCACATA 480

AATAAGTATT GTCTCTACTT TATGAATGAT AAAACTAAGA GATTTAGAGA GGCTGTGTAA 540

TTTGGATTCC 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: SEQ ID NO: 31:

TTGGCCTGAT TGGTGACAAA AGTGAGATGC TCAGTCCTTG AATGACAAAG AATGCCTGTA 60

GAGTTGCAGG TCCAACTACA TATGCACTTC AAGAAGATCT TCTGAAATCT AGTAGTGTTC 120

TGGACATTGG ACTGCTTGTC CCTGGGAAGT AGCAGCAGAA ATGATCGGTG GTGAACAGAA 180

GAAAAAGAAA AGCTCTTCCT TTTTGAAAGT CTGTTTTTTG AATAAAAGCC AATATTCTTT 240

TATAACTAGA TTTTCCTTCT CTCCATTCCC CTGTCCCTCT CTCTTCCTCT CTTCTTCCAG 300

ATCTTCAGGG GGCTAGAAAT CTGTTGCTAT GGGCCCTTCA CCAACATGCC CACAGGTAAG 360

AGCCTGGGAG AACCCCAGAG TTCCAGCACC AGCCTTTGTC TTACATAGTG GAGTATTATA 420

AGCAAGGTCC CACGATGGGG GTTCCTCAGA TTGCTGAAAT GTTCTAGAGG CTATTCTATT 480

TCTCTACCAC TCTCCAAACA AAACAGCACC TAAATGTTAT CCTATGGCAA AAAAAAACTA 540

TACCTTGTCC CCCTTCTCAA GAGCATGAAG GTGGTTAATA GTTAGGATTC AGTATGTTAT 600

GTGTTCAGAT GGCGTTGAGC TGCTGTTAGT GCC 633

(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: SEQ ID NO: 32:

TTTGAGAGAC TATCAAACCT TATACCAAGT GGCCTTATGG AGACTGATAA CCAGAGTACA 60

TGGCATATCA GTGGCAAATT GACTTAAAAT CCATACCCCT ACTATTTTAA GACCATTGTC 120

CTTTGGAGCA GAGAGACAGA CTCTCCCATT GAGAGGTCTT GCTATAAGCC TTCATCCGGA 180

GAGTGTAGGG TAGAGGGCCT GGGTTAAGTA TGCAGATTAC TGCAGTGATT TTACATGTAA 240

ATGTCCATTT TAGATCAACT GGAATGGATG GTACAGCTGT GTGGTGCTTC TGTGGTGAAG 300

GAGCTTTCAT CATTCACCCT TGGCACAGTA AGTATTGGGT GCCCTGTCAG TGTGGGAGGA 360

CACAATATTC TCTCCTGTGA GCAAGACTGG CACCTGTCAG TCCCTATGGA TGCCCCTACT 420

GTAGCCTCAG AAGTCTTCTC TGCCCACATA CCTGTGCCAA AAGACTCCAT 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: 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: 33:

GGTGGTACGT GTCTGTAGTT CCAGCTACTT GGGAGGCTGA GATGGAAGGA TTGCTTGAGC 60

CCAGGAGGCA GAGGTGGNAN NTTACGCTGA GATCACACCA CTGCACTCCA GCCTGGGTGA 120

CAGAGCAAGA CCCTGTCTCA AAAACAAACA AAAAAAATGA TGAAGTGACA GTTCCAGTAG 180

TCCTACTTTG ACACTTTGAA TGCTCTTTCC TTCCTGGGGA TCCAGGGTGT CCACCCAATT 240

GTGGTTGTGC AGCCAGATGC CTGGACAGAG GACAATGGCT TCCATGGTAA GGTGCCTCGC 300

ATGTACCTGT GCTATTAGTG GGGTCCTTGT GCATGGGTTT GGTTTATCAC TCATTACCTG 360

GTGCTTGAGT AGCACAGTTC TTGGCACATT TTTAAATATT TGTTGAATGA ATGGCTAAAA 420

TGTCTTTTTG ATGTTTTTAT TGTTATTTGT TTTATATTGT AAAAGTAATA CATGAACTGT 480

TTCCATGGGG TGGGAGTAAG ATATGAATGT TCATCAC 517

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 434 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: 34:

CAGTAATCCT NAGAACTCAT ACGACCGGGC CCCTGGAGTC GNTGNTTNGA GCCTAGTCCN 60

GGAGAATGAA TTGACACTAA TCTCTGCTTG TGTTCTCTGT CTCCAGCAAT TGGGCAGATG 120

TGTGAGGCAC CTGTGGTGAC CCGAGAGTGG GTGTTGGACA GTGTAGCACT CTACCAGTGC 180

CAGGAGCTGG ACACCTACCT GATACCCCAG ATCCCCCACA GCCACTACTG ACTGCAGCCA 240

GCCACAGGTA CAGAGCCACA GGACCCCAAG AATGAGCTTA CAAAGTGGCC TTTCCAGGCC 300

CTGGGAGCTC CTCTCACTCT TCAGTCCTTC TACTGTCCTG GCTACTAAAT ATTTTATGTA 360

CATCAGCCTG AAAAGGACTT CTGGCTATGC AAGGGTCCCT TAAAGATTTT CTGCTTGAAG 420

TCTCCCTTGG AAAT 434

(2) INFORMATION FOR SEQ ID NO: 35:

(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: 35:

GATAAATTAA AACTGCGACT GCGCGGCGTG 30

(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 30

(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)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

ATATATATAT GTTTTTCTAA 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: SEQ 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 30

(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 TTTTTTTTTT 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:

GTATATAATT TGGTAATGAT GCTAGGTTGG 30

(2) INFORMATION FOR SEQ ID NO: 45:

(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: 45:

GAGTGTGTTT CTCAAACAAT TTAATTTCAG 30

(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: SEQ ID NO: 46:

GTAAGTGTTG AATATCCCAA GAATGACACT 30

(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:

AAACATAATG TTTTCCCTTG TATTTTACAG 30

(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 TTGTTTTCTT CTTCTTCTTC 30

(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:

TGCTTGACTG TTCTTTACCA TACTGTTTAG 30

(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: 50:

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:

GTGAGTCAAA GAGAACCTTT GTCTATGAAG 30

(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 30

(2) INFORMATION FOR SEQ ID NO: 54:

(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 AGTTTGCCAA CTTAACAGGC 30

(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 30

(2) INFORMATION FOR SEQ ID NO: 56:

(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: 56:

GTATTGGAAC CAGGTTTTTG TGTTTGCCCC 30

(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:

ACATCTGAAC CTCTGTTTTT GTTATTTAAG 30

(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 30

(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 TCGTTTTTGA 30

(2) INFORMATION FOR SEQ ID NO: 60:

(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: 60:

GTGTGTATTG TTGGCCAAAC ACTGATATCT 30

(2) INFORMATION FOR SEQ ID NO: 61:

(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: 61:

AGTAGATTTG TTTTCTCATT CCATTTAAAG 30

(2) INFORMATION FOR SEQ ID NO: 62:

(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: 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 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: 64:

GTAATATTTC ATCTGCTGTA TTGGAACAAA 30

(2) INFORMATION FOR SEQ ID NO: 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 TTCCTTTCAG 30

(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 CCATATGTAT CTCCCTAATG 30

(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: SEQ 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 SEQ 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 CATTCTGCAG 30

(2) INFORMATION FOR SEQ ID NO: 70:

(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: 70:

GTAAGTATAA 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

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:

TGTAACCTGT CTTTTCTATG 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: 72:

GTAAGTACTT GATGTTACAA ACTAACCAGA 30

(2) INFORMATION FOR SEQ ID NO: 73:

(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:

TCCTGATGGG 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: SEQ ID NO: 74:

GTAAAGCTCC CTCCCTCAAG TTGACAAAAA 30

(2) INFORMATION FOR SEQ ID NO: 75:

(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: 75:

CTGTCCCTCT CTCTTCCTCT CTTCTTCCAG 30

(2) INFORMATION FOR SEQ 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) SEQUENCE DESCRIPTION: SEQ ID NO: 76:

GTAAGAGCCT GGGAGAACCC CAGAGTTCCA 30

(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: 78:

(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: 78:

GTAAGTATTG GGTGCCCTGT CAGTGTGGGA 30

(2) INFORMATION FOR SEQ 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 30

(2) INFORMATION FOR SEQ ID NO: 80:

(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: 80:

GTAAGGTGCC TCGCATGTAC CTGTGCTATT 30

(2) INFORMATION FOR SEQ 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 SEQ 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

(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:

Cys Pro Ile Cys Leu Glu Leu Ile Lys Glu Pro Val Ser Thr Lys Cys
1 5 10 15

Asp His Ile Phe Cys Lys Phe Cys Met Leu Lys Leu Leu Asn Gln Lys
20 25 30

Lys Gly Pro Ser Gln 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 Ile Cys Leu Glu Leu Leu Lys Glu Pro Val Ser Ala Asp Cys
1 5 10 15

Asn His Ser Phe Cys Arg Ala Cys Ile 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 DESCRIPTION: SEQ ID NO: 84:

Cys Pro Ile 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 Ile 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: SEQ ID NO: 85:

Cys Pro Val Cys Leu Gln Tyr Phe Ala Glu Pro Met Met Leu Asp Cys
1 5 10 15

Gly His Asn Ile Cys Cys Ala Cys Leu Ala Arg Cys Trp Gly Thr Ala
20 25 30

Cys Thr Asn Val Ser Cys Pro Gln Cys Arg
35 40

It is a figure which shows the order of the locus near BRCA1 determined by "chromosome 17 workshop". FIG. 1 was reproduced from Fain, 1992. It is a schematic map of YAC which defines the Mfd15-Mfd188 region part. 1 is a schematic map of STS, P1 and BAC in the BRCA1 region. It is a schematic map of human chromosome 17. The directly related region containing BRCA1 has been expanded to show the relative location of the two previously identified genes, CA125 and RNU2, and BRCA1 extends over marker D17S855. Figure 6 shows the sequence of the BRCA1 zinc-finger domain with three other zinc-finger domains best recorded in the Smith-Waterman sequence. RPT1 encodes a protein that appears to be a negative regulator of the IL-2 receptor in mice. RIN1 encodes a DNA-binding protein that contains a zinc-finger related RING-finger motif. RFP1 encodes a putative transcription factor that is the N-terminal domain of the RET oncogene product. The underlined portion contains the C3HC4 consensus zinc-finger sequence indicating the location of cysteine and one histidine to form a zinc ion binding pocket. FIG. 4 is a diagram of BRCA1 mRNA showing the location of introns generated by alternative splicing, and a variation of BRCA1 mRNA. Intron positions are indicated by "solid triangles" and exons are numbered below the cDNA line. The cDNA at the top is a composition used to prepare the peptide sequence of BRCA1. Other forms identified as cDNA clones or hybrid selection clones are shown below. 3 shows the tissue expression pattern of BRCA1. Blots were obtained from Clontech, which contains RNA from the indicated tissues. Hybridization conditions were recommended by the manufacturer and a probe consisting of nucleotide positions 3631-930 of BRCA1 was used. It is noted that both breast and ovary are heterozygous tissues and the percentage of epithelial cells involved can vary. The molecular weight marker is kb. FIG. 4 is a diagram of the position of the intron generated by alternative splicing and the starting point of the 5 ′ untranslated region of BRCA1 + translated region showing the modification of BRCA1 mRNA. The position of the intron is indicated by a dashed line. 6 shows six alternative splicing configurations. The nonsense mutation in Kindred 2082 is shown. P indicates the originally screened individual, b and c are halotype carriers, and a, d, e, f and g do not carry the BRCA1 haplotype. The C to T mutation creates a stop codon, creating a site for the restriction enzyme AvrII. The PCR amplification product is cut with this enzyme. The carrier is heterozygous for the site and therefore shows three bands. Non-carriers remain uncut. Figure 2 shows mutation and co-segregation analysis in BRCA1 kindreds. Carrier individuals are shown as "solid circles" and "solid squares" in the family tree. Frameshift mutation in Kindred 1910. The first three lanes are control, non-carrier samples. Lanes labeled 1-3 contain sequences from individual carriers. Lane 4 contains DNA from family members who do not carry the BRCA1 mutation. Diamonds are used to prevent family identification. Additional frameshifts resulting from C are evident in lanes labeled 1, 2, and 3. Figure 2 shows mutation and co-segregation analysis in BRCA1 kindreds. Carrier individuals are shown as "solid circles" and "solid squares" in the family tree. The inferred regulatory mutation is in Kindred 2035. ASO analysis of carriers and non-carriers of two different polymorphisms (PM1 and PM7) was examined for germline heterozygosity and compared to that of lymphocyte mRNA. The top two rows of each panel contain PCR products amplified from genomic DNA, and the bottom two rows contain PCR products amplified from cDNA. "A" and "G" are the two alleles detected by ASO. Dark spots indicate that a particular allele is present in the sample. The first three lanes of PM7 represent the three genotypes of the general population. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type. 1 shows the genomic sequence of BRCA1. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. Ambiguous intervals within introns are denoted by vvvvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and bold type.

Claims (9)

(I) the nucleotide sequence according to SEQ ID NO: 1 from nucleotide 120 to nucleotide 5708, its complement or the corresponding RNA, or (ii) an allelic variant of the nucleotide sequence according to SEQ ID NO: 1 from nucleotide 120 to nucleotide 5708, (A) a nucleic acid encoding a BRCA1 polypeptide comprising its complement or corresponding RNA and comprising the amino acid sequence set forth in SEQ ID NO: 2; and (b) a stringent strain at a temperature exceeding 45 ° C. and a salt concentration of less than 200 mM. A nucleic acid that hybridizes under hybridization conditions to a BRCA1 nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 1 from nucleotide 120 to nucleotide 5708, encoding BRCA1, an allelic variant thereof, or a naturally occurring mutant thereof. From the nucleic acid An isolated nucleic acid selected from that group. 2. The isolated nucleic acid of claim 1, operably linked to a BRCA1 gene regulatory sequence. (I) a nucleic acid comprising a nucleotide sequence selected from the nucleotide sequence of SEQ ID NO: 1 from nucleotide 120 to nucleotide 5708 of claim 1 or an allelic variant thereof or a naturally occurring mutant form thereof, or (ii) A) a hybridization probe for determining the presence of RNA corresponding to said nucleic acid in a sample, comprising an isolated DNA comprising a portion of the nucleotide sequence of the nucleic acid according to claim 1, wherein the sequence of said probe is Under stringent hybridization conditions at a temperature greater than 45 ° C. and a salt concentration of less than 200 mM, the nucleic acid or RNA can specifically hybridize to a target sequence that is a portion of the BRCA1 polypeptide coding sequence, wherein the portion comprises at least 15 The hybridization probe comprising a nucleotide. 4. A nucleic acid probe suitable for the hybridization probe according to claim 3, wherein the nucleotide sequence of the nucleic acid probe comprises the DNA sequence of SEQ ID NO: 1 at nucleotide positions 3631 to 3930. A vector comprising the isolated nucleic acid according to claim 1. (A) (i) the isolated nucleic acid of claim 1 or the probe of claim 3, and (ii) a replication cloning vector comprising a replicon operable in a host cell of the vector; and (b) the BRCA1 poly. The peptide or a mutated form thereof is selected from the group consisting of an expression vector comprising the isolated nucleic acid of claim 1 operably linked to a promoter sequence capable of directing the expression of the coding sequence in a host cell of the vector. vector. A host cell transformed or transfected with the vector according to claim 5 or 6. The method for identifying a mutant BRCA1 nucleotide sequence according to claim 2, wherein the nucleotide sequence of the predicted mutant BRCA1 allele is the coding sequence according to SEQ ID NO: 1 from nucleotide 120 to nucleotide 5708 and / or an allelic variation thereof. The method comprising comparing to the nucleotide sequence of BRCA1 containing the body. DNA fragments prepared by amplification using the primer pairs listed in Table 9.

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