JP2005124578A - 17q-LINKED BREAST AND OVARIAN CANCER SUSCEPTIBILITY GENE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To carry out diagnosis of germline mutations in the BRCA1 gene and predisposition to breast and ovarian cancer. <P>SOLUTION: The invention relates to the method and material used for isolation and detection of the predisposition gene (BRCA1) to breast and ovarian cancer of human. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates generally to the field of human genetics. Specifically, the present invention relates to a method used by several of its mutant alleles to isolate and detect a human breast and ovarian cancer disease predisposition gene (BRCA1) that causes susceptibility to cancer, particularly breast cancer and ovarian cancer. And material. More particularly, the invention relates to germline mutations in the BRCA1 gene and its use in the diagnosis of predisposition to breast and ovarian cancer. The invention further relates to somatic mutations in the BRCA1 gene in human breast cancer and ovarian cancer, and its use in the diagnosis and prognosis of human breast cancer and ovarian cancer. In addition, the present invention relates to somatic mutations of the BRCA1 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The present invention also relates to the therapy of human cancers having mutations in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The present invention further relates to screening for drugs for cancer therapy. Finally, the present invention relates to the screening of the BRCA1 gene for mutations useful in the diagnosis of predisposition to breast and ovarian cancer.
Publications and other materials used herein to clarify the background of the invention and, in particular, when providing additional details regarding implementation, are hereby expressly incorporated by reference. For this purpose, they are grouped in the reference list shown and attached by the author and date in the following specification.

  The genetics of cancer, including many dominant positive regulatory genes (oncogenes) in the transformed state, as well as many recessive negative regulatory genes (tumor suppressor genes), is complex. Over a hundred oncogenes have been characterized. Although less than a dozen tumor suppressor genes have been identified, the number is expected to increase beyond 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 obvious 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 common oncogene mutation is present in the H-ras gene and has been 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 nearly 50% of all tumors, and CDKN2, which is homozygously deleted in 46% of the tumor cell lines examined (Kamb et al., 1994). Without a common target for all transformed cells, it is unlikely that there will be a “magic bullet” dream that can destroy or revert cancer cells while leaving intact normal tissue intact. The desire for new drug discovery of specifically targeted anticancer agents may be supported by the ability to identify tumor suppressor genes or oncogenes that play a general role in the control of cell division.

  The tumor suppressor genes that have been cloned and characterized include: 1) retinoblastoma (RB1); 2) Wilms tumor (WT1); 3) Li-Fraumeni syndrome (TP53); 4) familial adenomatous edema ( APC); 5) neurofibromatosis type 1 (NF1); 6) neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau disease (VHL); 8) multiple endocrine adenomatosis type 2A (MEN2A) And 9) affect susceptibility to melanoma (CDKN2).

  The genetically mapped but not yet isolated tumor suppressor loci include: multiple endocrine adenomatosis 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 tumor (RCC); tuberous sclerosis type 1 (TSC1); and tuberous sclerosis type 2 (TSC2) are included. The Tumor suppressor genes that have been characterized to date include DNA-binding protein (WT1), accessory transcription factor (RB1), GTPase-activating protein or GAP (NF1), cytoskeletal factor (NF2), membrane-bound receptor kinase It encodes products similar to various types of proteins, including (MEN2A), cell cycle regulator (CDKN2), and others that do not have any apparent identity to known proteins (APC and VHL).

  In many cases, tumor suppressor genes originally identified through genetic studies have been shown to be deleted or mutated in certain sporadic tumors. This result indicates that the region of chromosomal abnormalities can locate important tumor suppressor genes involved in both genetic predisposition to cancer and sporadic cancer.

  One of the hallmarks of several tumor suppressor genes that have been characterized to date is that it is highly defective in certain tumor types. The deletions often include single allelic deletions, so-called heterozygous deletions (LOH), but can also include homozygous deletions of both alleles. For LOH, it is expected that the remaining alleles will become 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 ratio, US women have a 1/8 risk of developing breast cancer by age 95 (American Cancer Society, 1992). Treatment of late-stage breast cancer is often wasteful and ugly, and early detection is a high priority in medical management of disease. Less frequently 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-specified ratio of breast cancer incidence is estimated at 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: early-onset and older onset, based on the inflection of the age-specific incidence curve near 50 years of age. A mutation in one gene, BRCA1, is thought to cause about 45% of familial breast cancers, but at least 80% of families with both breast and ovarian cancers (Easton et al., 1993).

  Since the BRCA1 gene was first mapped in 1990, intense 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 have a proportion of early-onset breast cancer that is almost equivalent to BRCA1, but contributes to the low risk of ovarian cancer doing. The remaining susceptibility to early-onset breast cancer is divided between unmapped genes for familial cancer and rare germline mutations such as TP53 (Malkin et al., 1990). It has also been suggested that heterozygous carriers of vasodilatory schizophrenia genes are at high risk for breast cancer (Swift et al., 1976; Swift et al., 1991). Older-onset breast cancer is often familial, but the relative risk is not as high as in younger-onset breast cancer (Cannon-Albright et al., 1994; Mettlen et al., 1990). However, the percentage of such cases due to genetic susceptibility is unknown.

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

  Most strategies to clone the 17q-linked breast cancer disease predisposition gene (BRCA1) require accurate genetic location studies. The simplest model for the functional role of BRCA1 is that the BRCA1 allele, which is a predisposition to a cancerous disease, is recessive to the wild-type allele; Supports not to get cancer. However, cells containing one wild type BRCA1 allele and one disease predisposing allele are either wild type either by random mutation or by chromosomal deletions (non-chromosomal segregation) during cell division. Allele deletions can often be encountered. All progeny of such mutant cells lack the BRCA1 wild-type function and can develop into tumors. According to this model, even if the disease predisposing allele of BRCA1 is recessive and inherits susceptibility to cancer in a dominant manner, women with one disease predisposing allele (and one wild type allele) develop cancer Hazardous. This is because her breast epithelial cells can spontaneously delete the wild type BRCA1 allele. This model applies to a group of cancer susceptibility alleles known as classes of genes, including tumor retinoblastoma and neuroblastoma genes, tumor suppressors or anti-oncogenes. By inference, this model can also explain the BRCA1 function as recently shown (Smith et al., 1992).

  A second possibility is that the BRCA1 disease predisposition allele is actually dominant; that is, the wild type allele of BRCA1 cannot overcome the oncogenic role of the disease predisposition allele. Thus, cells carrying both wild type and mutant alleles do not necessarily lack a wild type copy of BRCA1 prior to generating malignant cells. Instead, breast cells in predisposed individuals will have certain other stochastic changes (groups) that lead 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. In contrast, 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 3 of 5 families with both breast and ovarian cancer (Narod et al., 1991). These studies claim that the gene is located in a very extensive region of 15 centmorgan (cM), or about 15 Mbp, on either side of the linked marker pCMM86 (D17S74) . However, genetic studies with markers surrounding pCMMS6 have proven unsuccessful in attempting to further define the region. Subsequent studies indicate that the genes are fairly close (Easton et al., 1993) and the original analysis was defective (Margaritte et al., 1992). Hall et al. (1992) recently located the BRCA1 gene at an interval of approximately 8 cM (approximately 8 Mbp) bordering the proximal Mfdl5 (D17S250) and distal human GIP genes. A slightly narrower interval for the BRCA1 allele based on publicly available data was agreed at the March 1992 “Chromosome 17 Study Group” (Fain, 1992). The size of these regions and the uncertainties associated therewith make it too difficult to design and instrument physical mapping and / or cloning strategies to isolate the BRCA1 gene.

  Identification of breast cancer susceptibility alleles will enable early detection of susceptible individuals and greatly enhance our ability to understand the early steps leading to cancer. Since susceptibility alleles often change during tumor progression, cloning of these genes can also be important in the development of better diagnostic and prognostic products, and better cancer therapy.

  The present invention relates generally to the field of human genetics. Specifically, the present invention relates to methods and materials used to isolate and detect the human breast cancer disease 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 its use in the diagnosis of 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 cancer and ovarian cancer. In addition, the present invention relates to somatic mutations of the BRCA1 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The present invention also relates to the therapy of human cancers with mutations in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The invention further relates to screening for drugs for cancer therapy. Finally, the present invention relates to screening the BRCA1 gene for mutations, which are useful in diagnosing predisposition to breast and ovarian cancer.

  The present invention provides materials and methods used to test an individual's BRCA1 allele and an interpretation of the normal or disease-predisposing nature of the allele. Individuals who are higher than normal risk will appropriately modify their lifestyle. In the case of BRCA1, the most significant non-genetic risk factor is the protective effect of the entire gestation period of younger age. Thus, a risky lady could think of making a child early or could think of a therapy designed to stimulate the hormonal effects of pregnancies throughout the young. High-risk women will also strive for early detection and be a great motivation to learn and practice breast self-diagnosis. Such women will 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 may also be applicable for tumor detection and classification. Sequence analysis could be used to diagnose precursor lesions. With the progress of the method and the accumulation of information about BRCA1 and other causal loci, it will be possible to separate cancer into benign and malignant.

  Women with breast cancer will follow different surgical procedures if they are predisposed to disease and therefore will have more cancer than it is not predisposed to disease. Other therapies will be developed using either peptides or small molecules (rational drug design). The peptide may be the missing gene product itself or part of the missing gene product. Alternatively, the therapeutic agent would be another molecule that mimics a deleterious gene function, a peptide or non-peptide molecule that is required to prevent the deleterious effects of inherited loci. Therapy can also be gene-based through introducing a normal BRCA1 allele into an individual to create a protein that would interfere with the effects of harmful alleles. These gene therapies can take many forms and will be aimed at preventing the tumor from forming, curing the cancer once it has occurred, or stopping the cancer from metastasizing.

  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 disease predisposition gene (BRCA1), a certain allele that is susceptible to cancer, particularly breast cancer and ovarian cancer. About. More particularly, the invention relates to germline mutations in the BRCA1 gene and its use in the diagnosis of 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 cancer and ovarian cancer. In addition, the present invention relates to somatic mutations in the BRCA1 gene in other human cancers and their use in the diagnosis and prognosis of human cancers. The present invention also relates to the therapy of human cancers with mutations in the BRCA1 gene, including gene therapy, protein replacement therapy and protein mimetics. The present invention further relates to screening for drugs for treating cancer. Finally, the present invention relates to screening the BRCA1 gene for mutations, which are useful in diagnosing predisposition to breast and ovarian cancer.

  The present invention provides an isolated polynucleotide consisting of all or part of a BRCA1 locus or a mutated BRCA1 locus, preferably at least 8 bases to about 100 kb in length. Such a polynucleotide may be an antisense polynucleotide. The invention also provides a recombinant construct comprising such an isolated polynucleotide, eg, a recombinant construct suitable for expression in a transformed host cell.

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

  The present 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 present invention also provides a kit for detecting a polynucleotide comprising a portion of the BRCA1 locus in an analyte, the kit being 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 8 adjacent nucleotides of the BRCA1 locus; and a polypeptide comprising a polymerizable amino acid to prepare a BRCA1 gene A method is provided for obtaining a sequence comprising at least 5 amino acids encoded within a locus.

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

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

  In addition, the present invention provides a method for screening drugs for cancer therapy to identify drugs suitable for restoring BRCA1 gene product function.

  Finally, the present invention provides the means necessary for the creation of gene-based therapies directed against cancer cells. These therapeutic agents can be placed on a suitable vector so that the function of the BRCA1 protein is reconstituted, or they can be delivered to target cells in a more direct manner, including all or part of the BRCA1 locus. Nucleotide forms may be taken. The therapeutic agent can also take the form of a polypeptide based on either a partial or complete protein sequence of BRCA1. They can functionally replace the activity of BRCA1 in vivo.

  The BRCA1 gene that causes predisposition to individuals for breast cancer and ovarian cancer is a gene encoding the BRCA1 protein. It is knowledge of the invention. This gene is designated herein as BRCA1. It is a finding of the present invention that mutations in the BRCA1 locus in germline are indicative of disease predisposition to breast and ovarian cancer. Finally, somatic mutations at the BRCA1 locus are also associated with breast cancer, ovarian cancer and other cancers, which may represent an indication of these cancers, or a predisposition to these cancers. This is a finding of the present invention. Mutational events at the BRCA1 locus can include deletions, insertions and point mutations within the coding and non-coding sequences.

  17q starting from the region on the long arm of human chromosome 17 of the human genome has a size estimated to be about 8 Mbp, a region containing the locus, BRCA1, and causes susceptibility to cancers including breast cancer and ovarian cancer Has been located.

  The region containing the BRCA1 locus was identified using various genetic techniques. The gene mapping technique first defined the BRCA1 region by recombination with genetic markers. Based on the study of a large extended family ("family") with multiple cases of breast cancer (and ovarian cancer cases in some families) Indicated the position. Two mitotic breakpoints were found on the distal side of the BRCA1 locus expressed as a recombinant between the genetic marker and the disease and as one recombinant at the base of the BRCA1 locus. Therefore, the region containing the BRCA1 locus physically borders these markers.

  The use of genetic markers provided by the present invention allows the identification of clones that cover regions from human yeast artificial chromosome (YAC) or human bacterial artificial chromosome (BAC) libraries. It also allows the easier manipulation of cosmids, P1 and BAC clones from this region, and the construction of contigs from subset clones. These cosmids, P1, YAC and BAC provide the basis for cloning the BRCA1 locus, for example, the basis for developing reagents effective in the diagnosis and treatment of breast and / or ovarian cancer. The BRCA1 gene and other possible susceptibility genes were isolated from this region. Isolation is a software trapping (possibly containing coding exons from continuous or non-contiguous genomic DNA sequences), along with whole or partial cDNA inserts from cosmids, P1 and BAC in the region of screening a cDNA library. Computational methods to identify unique sequences), hybrid selection techniques and direct screening. Using these methods, sequences of loci expressed in breast and other tissues were obtained. These candidate loci were analyzed to identify sequences that confer cancer susceptibility. The inventors have discovered that a mutation exists in the coding sequence of the BRCA1 locus in a family responsible for 17q-linked cancer susceptibility known as BRCA1. It was not known that this gene is present in this region. The present invention not only facilitates early detection of specific cancers to vitalize patient survival, but also allows sensitive individuals to detect them before they develop cancer.

Population-Sources The well-documented Utah family is particularly important in providing a good source of human genetics research. Each large family independently provides the ability to determine whether the BRCA1 susceptibility allele is segregating in the family. Recombination information about the location and isolation of the BRCA1 locus could only be obtained from a family that was large enough to confirm the presence of a susceptible allele. Large siblings are particularly important in studying breast cancer. This is because the penetrance of the BRCA1 susceptibility allele is reduced by both age and sex, making it difficult to find informational siblings. In addition, large siblings are essential to build the halotypes of individuals who have died due to the effects of their close relatives.

  Other populations can also provide useful information, but such studies generally require more effort and families are usually much smaller and therefore less accessible. Utah's age-adjusted breast cancer incidence rate is 20%, lower than the average US rate. The low incidence in Utah is probably due mostly to the early age of the first pregnancy, and the probability that the case will be found in Utah families carrying a genetic predisposition is increased.

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

  Genetic markers useful for searching for genetic loci associated with disease can be selected based on this problem, in particular by dense coverage of specific chromosomes or by detailed analysis of specific regions of the chromosome. The preferred method of selecting genetic markers linked to disease is to assess the degree of information in the family and determine the ideal distance between genetic markers of a given degree of polymorphism, followed by maximum efficiency. And selecting markers from known genetic maps that are ideally separated. Family information is measured by the probability that the marker is heterozygous in an unrelated individual. It is also most efficient to use STR markers detected by amplification of target nucleic acid sequences using PCR; such markers are very informative, easy to assay (Weber and May, 1989), and complex strategies Can be used and assayed simultaneously (Skolnick and Wallace, 1988), significantly reducing 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 known genetic maps. If nothing is known, a new marker can be identified by the STR technique shown in the examples.

  Gene mapping is usually an iterative process. In the present invention, we define adjacent gene markers around the BRCA1 locus and then begin by replacing these adjacent markers with other markers that are successfully close to the BRCA1 locus. The first step, a recombination event, is defined by a wide range of families and helps to specifically locate the BRCA1 locus as being away from or close to specific genetic markers (Goldgar et al., 1994). .

  Until the present invention was disclosed, the region surrounding BRCA1 was not well mapped and there were few markers. Therefore, short repeats on cosmids subcloned from physically mapped YACs were analyzed to develop new genetic markers. Using this approach, one marker of the invention, 42D6, was discovered, which was replaced with pCMM86 as a terminal flanking marker for the BRCA1 region. Since 42D6 is present at approximately 14 cM from pCMM86, the BRCA1 region was therefore reduced by approximately 14 centmorgans (Easton et al., 1993). Thus, the present invention began by finding more closely linked terminal flanking markers in the BRCA1 region. Next, it was discovered that BRCA1 is present on the terminal side with respect to the gene marker Mfdl5. Therefore, it was shown that BRCA1 exists in the region of 6 Mbp to 10 Mbp bordering on Mfdl5 and 42D6. Subsequently, the marker Mfdl9l was found to be distal to Mfdl5 and proximal to BRCA1. Thus, Mdfl5 was replaced with Mfdl9l as the most proximal genetic marker. Similarly, it was 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 Mfdl9l is replaced with tdj1474 as the proximal marker, and Mfdl88 is replaced with U5R as the distal marker, and the BRCA1 locus And the BRCA1 region was 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 a region flanked by tdj1474 and U5R. The second was the creation 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, physical isolation of DNA in the region was continued by identifying a 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 Michigan Genome Center. Clearly, those accessing these YACs would not have understood the value of the specific YACs we chose without disclosing the present invention. This is because they would not have understood which YAC is in the minimal region containing the BRCA1 locus and which YAC is outside of it.

Cosmid, P1 and BAC clones In the present invention, it is advantageous to continue with this region by cosmid, P1 and BAC clones. These smaller sized inserts compared to YAC inserts make it more useful as a specific hybridization probe. Furthermore, having cloned DNA in bacterial cells rather than yeast cells greatly increases the ease with which the target DNA can be manipulated and improves the signal to noise ratio of the hybridization assay. For the YAC cosmid subclone, 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 were screened by hybridization with human repetitive DNA (eg, Gibco / BRL, human C ₀ t-1 DNA, catalog number 5279A), as detailed in the Examples, followed by various Fingerprint by technology.

  P1 and BAC clones are obtained by screening a library constructed from the entire human genome with a specific sequence labeling site (STS) derived from YAC, cosmid or P1 and BAC, isolated as described above.

  These P1, BAC and cosmid clones were compared by scattered repeat (IRS) PCR and / or gel electrophoresis following restriction enzyme digestion and comparison of the resulting DNA fragments (fingerprints) (Maniatis et al., 1982). obtain. The clone can 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 (referred to herein as “minimum tiling paths”). Such a minimal tile path forms the basis for subsequent experiments and identifies cDNAs that can 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, almost twice as large as the cosmids for P1 and even more for BAC BAC vectors containing large genomic DNA inserts and clones 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 PCR primers provided by the present inventors for screening. BAC was obtained by the hybridization technique in Dr. Mel Simon's laboratory. The strategy using P1 clones also allows the genomic region to be covered with an independent set of clones not derived from YAC. This protects against the possibility of other deletions in the YAC that have not been detected. These new sequences from the P1 clone provide material for further screening of candidate genes as described below.

Gene Isolation There are many techniques to test genomic clones for the presence of sequences that are likely candidates for the coding sequence of a 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. Includes screening of cDNA libraries.
(a) Zoo blotting The first technique is to preserve species during evolution by hybridizing cosmids to Southern blots, and thus species with varying degrees of relation to humans (monkeys, cows, chickens, pigs, mice). DNA sequences that give positive hybridization signals with DNA from (such as and rats). Southern blots containing such DNA from various species are commercially available (Clonetech, catalog number 7753-1).
(b) Identification of HTF islands The 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 HTF (HpaI small fragment) 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 A third technique is exon trapping, a method for identifying sequences in genomic DNA that contain a splice junction and therefore are likely to contain the coding sequence of a gene. Using exon amplification (Buckler et al., 1991), exons are selected from the DNA clones described above and amplified. Exon amplification is based on the selection of RNA sequences flanked by functional 5 ′ and / or 3 ′ splice sites. To identify a processable number of candidate genes for further study, exon amplification products are used to screen a breast cancer cDNA library. Exon trapping can also be performed on small segments of sequencing DNA using a computer program or by software capture.
(d) cDNA that hybridizes to cosmid, P1, BAC, or YAC The fourth technique utilizes hybridization of cDNA to cosmid, P1, BAC, or YAC, and the transcribed sequence is incorporated into cloned genomic DNA. A modification of the selective enrichment technique that allows it to be identified and recovered from it (Kandpal et al., 1990). For the selective enrichment technique modified for this purpose, DNA from the region of BRCA1 present in the YAC is bound to a column matrix and then cDNA is selected from an appropriate library that hybridizes with the bound DNA, followed by Amplification of the bound DNA and purification, greatly increasing the cDNA in the region represented by the cloned genomic DNA.
(e) Identification of cDNA A fifth technique is to identify a cDNA corresponding to the BRCA1 locus. A variety of probes, including breast tissue cDNA libraries, ovarian cDNA libraries and any other required libraries, using hybridization probes containing the predicted coding sequences, selected using any of the techniques described above. Screen the library.

In addition, candidate variants of BRCA1 were also found using other variants of direct selection of the subject cDNA (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 subsequent PCR amplification reactions using biotinylated primers. Target cDNA is generated from tissue samples, eg, mRNA from breast tissue, by second strand synthesis followed by either first-strand random origin synthesis or oligo (dT) origin synthesis. The cDNA ends are blunted and ligated to a double stranded adapter. These adapters act as amplification sites for PCR. Target and probe sequences are denatured and mixed with human C ₀ t-1 DNA to block repetitive sequences. 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 a cDNA for Candidates A cDNA is a BRCA1 locus obtained by finding a sequence in DNA extracted from a diseased family member that produces an abnormal BRCA1 gene product or an abnormal level of BRCA1 gene product. Prove that. Such BRCA1 susceptibility alleles will be co-segregated for the disease in a wide range of families. It will also be present more frequently in non-family individuals with breast and ovarian cancer, and then in individuals in the general population. Finally, because tumors are often somatically mutated at loci that mutate in the germline in other examples, we are identical to BRCA1-sensitive alleles in DNA extracted from tumor tissue or It is expected to find a normal germline BRCA1 allele mutated to a sequence that is similar. Either the BRCA1 sequence from the tumor tissue is compared with the BRCA1 allele from the germline of the same individual or the germline BRCA1 allele from the cancer case is compared with that from a non-affected individual, The key is to find mutations that are significant enough to clearly disrupt the normal function of the gene product. These mutations can take many forms. The most critical form is a frameshift mutation or extensive deletion, which will cause the gene to encode an abnormal protein or to express a significantly altered protein. Less severe destructive mutations include small in-frame deletions and non-conservative base pair substitutions that change to or from cysteine residues, basic amino acids to acidic amino acids To the production protein, such as a change to or vice versa, a change 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. It will have a serious effect. Those that result in silent mutations or conservative amino acid substitutions are generally not expected to disrupt protein function.

  According to the diagnosis and prognosis 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 a deletion of a predisposition to cancer at the BRCA1 locus. “Wild-type gene alterations” include all forms of mutations, including deletions, insertions and point mutations in coding and non-coding regions. Deletions can be for the entire gene or only for 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 knowledge of the BRCA1 mutation provides both diagnostic and prognostic information. Non-deleted BRCA1 alleles (eg found on sister chromosomes of chromosomes carrying BRCA1 deletions from sister chromosomes) can be screened for other mutations such as insertions, small deletions and point mutations . It is believed that many mutations found in tumor tissue may lead to decreased 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 the regulatory region, such as at the promoter of a gene, leading to deletion or reduction of mRNA expression. Point mutations can also disrupt proper RNA processing, leading to loss of expression of the BRCA1 gene product, or reduced mRNA stability or translation efficiency.

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

  Cancer predisposition, such as breast and ovarian cancer and other cancers identified herein, can be confirmed by examining any human tissue for mutations in the BRCA1 gene. For example, individuals who have inherited germline BRCA1 mutations will be prone to developing cancer. This can be determined by examining 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 can also be made by examining fetal, placental or amniotic cells for mutations in the BRCA1 gene. Whether by point mutation or deletion, changes in the wild type BRCA1 allele can be detected by any means discussed herein.

  There are several methods that can be used to detect DNA sequence variations. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing, can detect sequence variations. For genes as large as BRCA1, it is a very hard work, but rarely misses mutations in the coding sequence of the gene under optimal conditions. Another approach is the single strand structural polymorphism assay (SSCA) (Orita et al., 1989). This method does not detect all sequence changes, especially if the DNA fragment is larger than 200 bp, but can be optimized to detect most DNA sequence variations. Although the reduced detection sensitivity is disadvantageous, the increased throughput possible with SSCA makes it an attractive and possible alternative to direct sequencing for research-based mutation detection. Fragments with shifted mobility on the SSCA gel are then sequenced to determine the nature of the exact DNA sequence variation. Other approaches based on 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 detect large deletions, duplex formations or insertions, or 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 not missense mutations. A review of recently available methods for detecting DNA sequence variations can be found in a recent review by Grompe (1993). Once the mutation is known, an allele-specific detection approach such as allele-specific oligonucleotide (ASO) hybridization can be used to rapidly screen a large number of other samples for the mutation. .

  In order to detect a change in the wild-type BRCA1 gene in a tissue, it is useful to isolate a tissue released from the surrounding normal tissue. Methods for increasing tumor cell tissue preparations 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. Mutation detection is more difficult when the tumor tissue is very contaminated with normal tissue.

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

  Point mutation detection 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 can be amplified directly from a genomic DNA preparation 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 structure 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 E. coli mutS protein (Modrich, 1991); and 6) Allele-specific PCR ( Rano and Kidd, 1989). Allele-specific PCR uses a primer that hybridizes at its 3 ′ end to a particular BRCA1 mutation. In the absence of a particular BRCA1 mutation, no amplification product is observed. Amplification Refractory Mutation System (ARMS) disclosed in EP 0332435 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 genes or peripheral marker genes can also be used to record allelic changes or insertions in polymorphic fragments. Such a method is particularly useful for screening relatives of an individual for the presence of a BRCA1 mutation found in the affected individual. Other techniques for detecting insertions and deletions known in the art can be used.

In the first three methods (SSCA, DGGE and RAase protection assay), a new electrophoretic band appears. SSCA detects bands that move differently. This is because sequence changes cause differences in single strands, ie intramolecular base pairing. RAase protection includes 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 mutant sequences compared to wild type sequences. In an allele-specific oligonucleotide assay, the oligonucleotide is designed to detect a specific sequence, and the assay is performed by detecting the presence or absence of a hybridization signal.
In the mutS assay, the protein binds only to sequences that contain nucleotide mismatches in the heteroduplex between the mutation and the wild-type sequence.

  Mismatches according to the present invention are hybridized nucleic acid duplexes where the two strands are not 100% complementary. Fully complementary deletions 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 do with vast numbers of tumor samples. An example of a mispaired cutting technique is the RAase protection law. In the practice of the present invention, the method involves the use of a labeled riboprobe complementary to the human wild type BRCA1 gene coding sequence. The enzyme RNase A that anneals (hybridizes) together with either the riboprobe and either mRNA or DNA isolated from tumor tissue, followed by the detection of certain mismatches in the duplex RNA structure. Digest. When mispairing is detected by RAase A, it cleaves at the mispairing site. Thus, when isolating annealed RNA preparations on an electrophoresis gel matrix, if mismatches are detected and cleaved by RNase A, RNA smaller than the full-length duplex RNA of riboprobes and 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 riboprobe contains only BRCA1 mRNA or gene segments, 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, mispairing can be detected by shifting the electrophoretic mobility of the mispairing duplex to the mating 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) prior to hybridization. Changes in the DNA of the BRCA1 gene can also be detected using Southern hybridization, particularly when the change is a global rearrangement, such as deletions and insertions.

  The DNA sequence of the BRCA1 gene amplified by using PCR can also be screened using allele-specific probes. These probes are nucleic acid oligomers, each containing a region of the BRCA1 gene sequence that conceals known mutations. For example, one oligomer can 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 previously identified mutations in the BRCA1 gene. Hybridization of the amplified BRCA1 sequence with the allele-specific probe can be performed, for example, on a nylon filter. Hybridization to a specific probe under stringent hybridization conditions indicates that the mutation is present in the tumor tissue as in an allele-specific probe. The most obvious test for mutations in a candidate locus is to directly compare genomic BRCA1 sequences from a control population with those from cancer patients. Alternatively, mRNA may be sequenced after amplification, for example by PCR, thereby eliminating the need to determine the exon structure of the candidate gene.

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

  Changes in BRCA1 mRNA expression can 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, a tissue can be screened with a monoclonal antibody that is immunoreactive with BRCA1. Lack of a cognate antigen will indicate a BRCA1 mutation. Mutant BRCA1 gene products can also be detected using antibodies 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 altered BRCA1 protein can be used to detect changes 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 the mutant BRCA1 gene product indicates a change in the wild type BRCA1 gene.

  The 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 mutant BRCA1 genes or gene products in tissues can be applied to other body samples. Cancer cells shed 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 even 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, the progress of chemotherapy or radiation therapy can be more easily monitored by testing such body samples for mutant BRCA1 genes or gene products.

  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 clinicians, who can determine during the appropriate course of treatment.

  The primer pairs of the present invention are useful for the determination of the nucleotide sequence of a particular BRCA1 allele using PCR. Single stranded DNA primer pairs can anneal to sequences in or around the BRCA1 gene on chromosome 17q21 to initiate amplified DNA synthesis of the BRCA1 gene itself. A complete set of these primers allows 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 can also be used. Such primers will only anneal to a specific BRCA1 mutant allele and will therefore only amplify the product in the presence of the mutant allele as a template.

  In order 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 sequences near BRCA1 except for some nucleotides necessary to form a restriction enzyme site. The primer itself can be synthesized using techniques well known in the art. In general, 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 specific primers is well within the scope of those skilled in the art.

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

  It has been discovered that individuals with the wild type BRCA1 gene do not have cancers arising from the BRCA1 allele. However, mutations that interfere with the function of the BRCA1 protein are involved in cancer pathology. Thus, the presence of altered (or mutated) BRCA1 gene that produces a protein that has lost or altered function is directly related to an increased risk of cancer. To detect BRCA1 gene mutations, a biological sample is prepared and analyzed for differences between the BRCA1 allele sequence to be analyzed and the wild-type BRCA1 allele sequence. 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 mutant BRCA1 allele can be initially identified by identifying a mutated (changed) BRCA1 protein using conventional techniques. The mutant alleles are then sequenced to identify specific mutations for each allele. Mutations, particularly those that lead to altered functions of the BRCA1 protein, are then used in the diagnostic and prognostic methods of the present invention.

Definitions The present invention uses the following definitions:
“Polynucleotide amplification” uses 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. These methods are well known and widely practiced in the art (eg, US Pat. Nos. 4,683,195 and 4,683,202, and (for PCR) Innis et al., 1990; and (for LCR) Wu. 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 in regions adjacent to the target region therein. To do. The BRCA1 sequence generated by amplification can be directly sequenced. Alternatively, although less desirable, the amplified sequence (s) can be cloned prior to sequence analysis. Direct cloning and sequence analysis of enzymatically amplified genomic segments is 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.

  The present invention also provides polyclonal and / or monoclonal antibodies and fragments thereof, and immunological binding equivalents thereof, which are derived from BRCA1 polypeptides and fragments thereof, or specifically from BRCA1 regions. 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 uniform 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 over several months. Rabbit serum is tested for immunoreactivity against the BRCA1 polypeptide or fragment. Monoclonal antibodies can be produced by injecting mice with protein polypeptides, fusion proteins or fragments 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.

  Once a sufficient amount of the desired polypeptide is obtained, it can be used for various purposes. A typical use is the production of antibodies specific for binding. These antibodies are either polyclonal or monoclonal and can be generated by in vitro or in vivo techniques well known in the art. For the production of polyclonal antibodies, an appropriate target immune system, typically a mouse or rabbit, is selected. Substantially pure antigen is presented to the immune system in a manner determined by methods appropriate for the animal or by other indicators well known to immunologists. A typical site of injection is an intramuscular, intraperitoneal or intradermal footpad. Of course, other species can be substituted for mice or rabbits. The polyclonal antibody is then purified and adjusted to the desired specificity using techniques known in the art.

Immunological responsiveness is usually assayed with an immunoassay. Such immunoassays typically include some purification of the antigen source generated, for example, by the same cell and formula as the antigen. Various immunoassay methods are well known in the art (see, eg, Harlow and Lane, 1988 or Goding, 1986).
Monoclonal antibodies with 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 an animal is excised and individual spleen cells are fused to immortalized melanoma cells under appropriate selection conditions. Thereafter, the cells are clonally isolated 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 can be used with or without modification. Often polypeptides and antibodies will be labeled by binding substances that provide a detectable signal, either covalently or non-covalently. A wide range of labeling and conjugation techniques are known and have been extensively reported in both 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 US Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Recombinant immunoglobulins can also 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. In general, the specific binding partner must bind with sufficient affinity under isolation conditions to immobilize analyte copy / complementary strand duplex (in the case of polynucleotide hybridization). I must. Specific binding partners are known in the art and include, for example, biotin and avidin or streptavidin, IgG and protein A, a vast number of known receptor-ligand couplings and complementary polynucleotide strands. In the case of a complementary polynucleotide binding partner, the partner can usually be at least about 15 bases long and at least 40 bases long. The polynucleotide can be composed of DNA, RNA or synthetic nucleotide analogs.

  “Biological sample” refers to a sample of tissue or fluid expected to contain an analyte polynucleotide or polypeptide from an individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph, skin Samples of external sections, respiratory, intestinal and urogenital organs, tears, saliva, red blood cells, tumors, organs, tissues, and in vitro cell culture constructs.

  As used herein, the term “diagnosis” or “preliminary diagnosis” as used in the context of a neoplasm refers to 1) classification of a lesion as a neoplasm, 2) measurement of the extent of the neoplasm, before, during and after treatment. Or 3) used to indicate a monitor of disease progression.

  A polynucleotide that “encodes” a polypeptide that “encodes” a polypeptide when it is engineered in its native state or by methods well known to those of skill in the art, can be found in mRNA and / or the polypeptide or fragment thereof. It can be transcribed and / or translated. The antisense strand is the complement of such a nucleic acid and the sequence code that can be predicted therefrom.

  “Isolated” or “substantially pure”; “isolated” or “substantially pure” nucleic acid (eg, RNA, DNA or mixed polymers) is a natural human sequence or protein (eg, Ribosomes, polymerases, and many other human genomic sequences and proteins) have been substantially isolated from other cellular components that naturally accompany them. The term includes nucleic acid sequences and proteins removed from the naturally occurring environment, including recombinant or cloned DNA isolates, and chemically synthesized analogs or analogs biologically synthesized by foreign systems. included.

  “BRCA1 allele” refers to the normal allele of the BRCA1 locus and alleles that carry variants that predispose individuals to develop cancer at many sites, including, for example, breast, ovarian, colorectal and prostate cancer Say. Such a predisposing allele is also referred to as a “BRCA1 susceptibility allele”.

  “BRCA1 locus”, “BRCA1 gene”, “BRCA1 nucleic acid” or “BRCA1 polynucleotide” each refers to a polynucleotide, all of which are present in the BRCA1 region, which can be used to treat breast cancer, ovarian cancer, It appears that specific alleles predisposed to developing colorectal and prostate cancers are expressed in normal tissues. Mutations in BRCA1 may be involved in the development and / or progression of other types of tumors. The locus is indicated in part by mutations that cause cancer in predisposed individuals. These mutations fall within the BRCA1 region described below. The BRCA1 locus is intended to include 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.

  When applied to nucleic acids, these terms refer to nucleic acids encoding BRCA1 polypeptides, fragments, equivalents or variants, and include, for example, protein fusions or deletions. The nucleic acids of the invention have a sequence that is either derived from or substantially identical to a native BRCA1-encoding gene, or a native BRCA1-encoding gene or a portion thereof. Would have. The coding sequence for the BRCA1 polypeptide having the amino acid sequence shown in SEQ ID NO: 2 is shown in SEQ ID NO: 1.

  Polynucleotide compositions of the present invention include both sense and antisense strand RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, as will be readily understood by those skilled in the art, May be chemically or biochemically modified, or may 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., methyl phosphonate, phosphotriester, phosphoamidate, carbamate, etc. Internucleotide modifications such as uncharged linkages, charged linkages (such as phosphorothioates, phosphorodithioates, etc.), pendant groups (such as polypeptides), acridines, psoralens, etc. ) Intercalators, chelating agents, alkylating agents and modified linkages (such as alpha anomeric nucleic acids etc.). Also encompassed are synthetic molecules that mimic polynucleotides capable of binding to a specified sequence through 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 comprising all or part of the BRCA1 region. The recombinant construct can replicate autonomously in the host cell. Alternatively, the recombinant construct can become integrated into the chromosomal DNA of the host cell. Such recombinant polynucleotides, depending on their origin or manipulation, 1) do not bind to all or part of a naturally binding polynucleotide; 2) bind to a polypeptide other than those that bind naturally; or 3) naturally Including polynucleotides of genomic, cDNA, semi-synthetic or synthetic origin.

  Accordingly, recombinant nucleic acids comprising other sequences that do not occur in nature are provided by the present invention. Wild type sequences can be used, but they will often have been modified, for example, by deletion, substitution or insertion.

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

  The DNA sequences used in the present invention usually comprise at least about 5 codons (15 nucleotides), more usually at least 7-15 codons, most preferably at least about 35 codons. There can also be one or more introns. This number of nucleotides is usually about the minimum length required for a successful probe that will specifically hybridize to the BRCA1-coding sequence.

  Techniques for nucleic acid manipulation are generally described, for example, in Sambrook et al., 1989 or Ausbel et al., 1992. Reagents applicable to such technologies, 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. Commercially available from many sources such as: The recombinant nucleic acid sequences used to make the fusion proteins of the invention can be derived from natural or synthetic sequences. Many natural gene sequences can be obtained from various cDNA or genomic libraries (see GenBank, National Institute of Health) using appropriate probes.

  “BRCA1 region” refers to the portion of human chromosome 17q21 that is 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 double stranded DNA comprising a locus, allele or region, as well as a locus, allele or region. It refers to any single-stranded DNA.

  As used herein, a “part” 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, variants or fragments thereof.

  The term “polypeptide” refers to an amino acid polymer or its equivalent and not a product of a specific length; thus, peptides, oligopeptides and proteins are included within the definition of polypeptide. The term also does not refer to, or excludes, for example, glucosylated, acetylated, phosphorylated and other polypeptide modifications. Included within the definition are polypeptides comprising one or more analog amino acids, naturally occurring and non-naturally occurring (eg, including non-natural amino acids, etc.), polypeptides having substitution bonds, and the art And other modifications known in the art. Usually, such polypeptides are at least about 50% homologous to the native BRCA1 sequence, preferably greater than about 90%, more preferably at least about 95% homologous. Also included are very similar polypeptides or proteins that hybridize to a BRCA1-encoding nucleic acid under high or low stringency conditions and are recovered by antisera against the BRCA1 protein (s).

  The length of polypeptide sequences compared for homology is generally at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues. Will preferably exceed about 35 residues.

  “Operably linked” refers to a juxtaposition where the components thus described are in a relationship that allows their intended mode of function. For example, a promoter is operably linked to a sequence if the promoter affects the transcription or expression of the coding sequence.

  “Probe”; a predisposition or most of a particular cancer that can be detected by hybridization with a polynucleotide probe that forms a stable hybrid with a polypeptide of the target sequence under appropriate stringent hybridization and wash conditions. Polynucleotide polymorphism associated with the BRCA1 allele associated with cancer. If the probe is expected to be perfectly complementary to the target sequence, stringent conditions will be used. For example, the deformation may reduce hybridization stringency if some mismatch is expected, such as when the probe is expected to have a result that is not perfectly complementary. Select 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 demonstrate the detection of BRCA1-sensitive alleles.

  The BRCA1 allele probe may be derived from the BRCA1 region or the sequence of its cDNA. The probe can be of any suitable length that extends throughout or a portion of the BRCA1 region and allows specific hybridization to the BRCA1 region. If the target sequence comprises the same sequence as the probe, the probe can be shortened, for example, in the range of about 8-30 base pairs. This is because the hybrid will be relatively stable even under stringent conditions. If some mispairing with the probe is expected, ie if the probe is expected to hybridize to various regions, longer probes that hybridize to the target sequence with the required specificity can be used.

  A probe includes an isolated polynucleotide that binds to a label or reporter molecule, which could be used to isolate other polynucleotide sequences having sequence similarity by standard methods. See, for example, Sambrook et al., 1989 or Ausbel et al., 1992 for probe preparation and labeling techniques. Other similar polynucleotides can be selected by using homologous polynucleotides. Alternatively, polynucleotides encoding these or similar polypeptides can be synthesized or selected by using duplications in the genetic code. Various codon substitutions can be introduced, for example, by silent changes (thus creating various restriction sites) or by optimizing specific systems. Mutations can be introduced to modify the properties of the polypeptide (possibly a change in ligand-binding affinity, interchain affinity or polypeptide degradation or turnover rate).

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

  A portion of a polynucleotide sequence from a polynucleotide sequence encoding BRCA1 having at least about 8 nucleotides, usually at least about 15 nucleotides, and shorter than about 6 kb, usually shorter than about 1.0 kb, is preferred as a probe. The probe can also 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, eg, in vivo or in vitro in vitro) chemical and biochemical modifications or those incorporating unusual amino acids. Such modifications will be readily understood by those skilled in the art, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling (eg, with radionuclides), and various enzymatic modifications. Is included. Various methods of labeling polypeptides useful for such purposes and their substitutions or labels are well known in the art and include radioisotopes, such as ³² P, which contain specific binding pairs of labeled ligands. It binds to 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, eg, Sambrook et al., 1989 or Ausbel et al., 1992).

  In addition to substantially full-length polypeptides, the present invention provides biologically active fragments of pseudopolypeptides. Important biological activities include ligand-binding properties, immunological activity and other biological activity properties of the BRCA1 polypeptide. Immunological activity includes both the immunogenic function of the target immune system, as well as sharing the immunological epitope for binding, and acting as either a competing or replacement antigen for the epitope of the BRCA1 protein. It is. As used herein, “epitope” refers to an antigenic determinant of a polypeptide. An epitope can comprise 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, a tandem repeat polypeptide segment can be used as an immunogen, thereby producing highly antigenic proteins. Alternatively, such polypeptides will also act as highly efficient competitors for specific binding. Production of antibodies specific for BRCA1 polypeptide or fragments thereof is described below.

  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 may be constructed that will exhibit a combination of derived protein properties or activities. For example, ligand-binding or other domains can be “exchanged” between different new fusion polypeptides or fragments. Such homo- or hetero-fusion polypeptides can exhibit, for example, altered strength or specificity of 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).

  The fusion protein can typically be made by any recombinant nucleic acid method, as described below, or can be a chemical synthesis method. Techniques for synthesizing polypeptides are described, for example, in Merrifield, 1963.

  “Protein purification” is well known in the art as well as various methods for isolating BRCA1 polypeptides from other biological materials, such as from cells transformed with a recombinant nucleic acid encoding BRCA1. Say the method. For example, such polypeptides can be purified, for example, by immunoaffinity chromatography using the antibodies provided by the present invention. Various methods of protein purification are well known in the art and include those described in Deutscher, 1990 and Scopes, 1982.

  The terms “isolated”, “substantially pure” and “substantially uniform” are interchangeable to describe the separation of proteins or polypeptides from their associated components in the natural state. Used for. A monomeric protein is substantially pure when at least about 60-75% of the sample exhibits a single polypeptide sequence. Substantially pure protein will typically comprise about 60% to 90% W / W protein sample, more usually about 95%, preferably greater than about 99% pure protein sample. Protein purity or homogeneity can be demonstrated 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 utilized for purification.

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

  A polypeptide that is generated as an expression product of an isolated and engineered gene sequence is an “isolated polypeptide” as used herein, even when expressed in a homocellular form. A synthetic production form or molecule expressed by a heterologous cell is essentially an isolated molecule.

  A “recombinant nucleic acid” is a nucleic acid that is not naturally occurring or made by the artificial combination of two other separated segments of sequence. This artificial combination is often made by any chemical synthesis means or by the 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 introduces or removes a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of the desired function to produce the desired combination of functions.

  A “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 includes (gene transcription and mRNA translation, splicing, stability Affects gene expression (including sex).

  “Substantially homologous or similar”; at least about 60% of the nucleotide bases, usually at least about 60% when aligned optimally with other nucleic acids (or their complementary strands) (with appropriate nucleotide insertions or deletions) A nucleic acid or fragment thereof is another when there is a 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 Are “substantially homologous” (or “substantially similar”).

  Alternatively, when a nucleic acid or fragment thereof hybridizes to another nucleic acid (or its complementary strand) under hybridization conditions selective for the strand or its complementary strand, substantial complementarity or (identical) Sex) exists. Hybridization selectivity exists when substantially more selective hybridization occurs than an overall lack of specificity. Typically, selective hybridization is where there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least 65%, more preferably at least about 75%, and most preferably at least about 90%. (See Kanehisa, 1984). The length of the homology comparison described can also be a longer stretch, and in certain embodiments, often 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, as will be readily understood by those skilled in the art, in addition to base composition, complementary strand length, and number of nucleotide base mismatches between hybridizing nucleic acids, 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. Stoint 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). Probe sequences can also specifically hybridize to duplex DNA under certain conditions to form triple helices or other higher order DNA complexes. The preparation of such probes and appropriate hybridization conditions are well known in the art.

  The term “substantial homology” or “substantial identity” when referring to a polypeptide refers to at least about 30% identity, usually at least about about 30%, with the whole protein or portion thereof in which the polypeptide or protein in question naturally occurs. It represents 70% identity, preferably at least about 95% identity.

  “Substantially the same function” refers to the function of the modified nucleic acid or protein with respect to the wild-type BRCA1 nucleic acid or wild-type BRCA1 polypeptide. The modified polypeptide will be substantially homologous to the wild type BRCA1 polypeptide and will have substantially the same function. A modified polypeptide has 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 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 function (activity) identity 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 generated using conventional techniques. Modified nucleic acids are prepared by conventional techniques. A nucleic acid having substantially the same function as the wild-type BRCA1 gene function produces the modified protein described above.

  Polypeptide homology is typically measured using sequence analysis software (see, eg, 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 homology measurements specifying various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; , Tyrosine.

  A polypeptide “fragment”, “part” or “segment” refers to at least about 5-7 contiguous amino acids, often at least about 7-9 contiguous amino acids, typically at least about 9-13. A stretch of amino acid residues of adjacent amino acids, most preferably at least about 20-30 or more adjacent amino acids.

  When the polypeptide of the present invention is soluble, such as, for example, nitrocellulose, nylon, column packing (eg, Sepharose beads), magnetic beads, glass wool, plastic, metal, polymer gel, cells or other substrates It can be coupled to a solid support. Such a support may take the form of, for example, a bead, well, dipstick or membrane.

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

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

Recombinant or chemically synthesized nucleic acids; vector, transformation, host cell preparation Large quantities of the polynucleotides of the invention can be produced by replication in a suitable host cell. Natural or synthetic polynucleotide fragments encoding the desired fragment are introduced into a recombinant polynucleotide construct, usually a DNA construct, which has the ability to introduce and replicate into prokaryotic or eukaryotic cells. Typically, the polynucleotide construct will be suitable for replication in a unicellular host, such as yeast or bacteria, but in cultured mammals or plants or other eukaryotic cells (integrated or not integrated into the genome). It can also be intended for introduction into a biological cell line. Purification of nucleic acids produced by the methods of the present 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 by Matteucci and Caruthers, 1981, using commercially available automated oligonucleotide synthesizers. It can also be done. Double-stranded fragments are synthesized by synthesizing complementary strands and annealing the strands together under appropriate conditions, or by adding complementary strands using DNA polymerase together with appropriate primer sequences. Can be obtained from a single chain 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 contain regulatory sequences for transcription and translation initiation operably linked to the coding segment. Expression vectors include, for example, origins of replication or autonomously replicating sequences (ARS), and expression control sequences such as ribosome-binding sites, RNA splicing sites, polyadenylation sites, transcription terminator sequences and mRNA stabilizing sequences, promoters, Enhancers and essential processing information sites can be included. The secretory signal is suitably from either the native BRCA1 protein or from another receptor, or from a secreted polypeptide of the same or related species, and the protein is transmembrane and / or coveted. Thus, it can include those that achieve their functional topology or are secreted from the cell. Such vectors are well known in the art and can be prepared by standard recombinant techniques as 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 are available from vendors such as Stratagene, New England Biolabs, Promega Biotech and others.
Promoters such as trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may 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, other promoter regions that contribute to maltose and galactose utilization Is included. Suitable vectors and promoters for use in yeast expression are further described in Hitzeman et al., EP 73,675A. Suitable non-natural mammalian promoters include early and late promoters from SV40 (Fiers et al., 1978) or murine moloney leukemia virus, murine tumor virus, chicken sarcoma virus, type II adenovirus, bovine papilloma virus or polyoma. A promoter can be included. In addition, the construct can be linked to an amplifiable gene (eg, DHFR) so that multiple copies of the gene can be generated. 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).

  Such expression vectors can replicate autonomously, but they can also be replicated by insertion into the genome of a host cell by methods well known in the art.

  Expression and cloning vectors appear to contain a selectable marker that is a gene encoding a protein essential for the survival or growth of host cells transformed with the vector. The presence of this gene ensures the growth of only host cells that express the insert. Typical selection genes confer resistance to a) antibiotics or other toxic substances such as ampicillin, neomycin, methotrexate, etc .; b) supplement auxotrophic deficiencies, or c) not available from complex media It encodes a protein that supplies important nutrients (for example, the gene encoding D-alanine racemase for Bacilli). The selection of the appropriate selectable marker depends on the host cell, and appropriate markers for various 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 a host cell by well-known methods (eg, injection (Kubo et al., 1988)), or The vectors vary depending on the type of cell host, well-known methods in the art (electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectors; Can be directly introduced into host cells by lipofection infection (vector is an infectious agent such as a retroviral genome); and other methods) (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 those specifically mentioned above, is referred to herein as “transformation”. A cell into which the nucleic acid has been introduced is meant to include the progeny of such a cell.

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

  Mammalian host cells or other eukaryotic host cells such as yeast, filamentous fungi, plants, insects, or amphibians or birds can also be useful for the production of 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 also have higher expression, desirable glycosyls It will be appreciated by those skilled in the art that it may be appropriate to provide a pattern, or other features.

  Clones can be selected by using markers that depend on the mode of the vector construct. The marker may be on the same or different DNA molecule, but is preferably on the same DNA molecule. In prokaryotic hosts, transformants can be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics. The production of specific products based on temperature sensitivity can also act as a suitable marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides of the invention will be useful not only in the production of nucleic acids and polypeptides of the invention, but also in, for example, studying the properties of BRCA1 polypeptides.
Antisense polynucleotide sequences are useful to prevent or reduce 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 (especially those adjacent to the BRCA1 locus) should be placed under the control of the promoter in the antisense orientation and introduced into the cell. Can do. Expression of such an antisense construct in the cell will interfere with BRCA1 transcription and / or translation and / or replication.

  Probes and primers based on the BRCA1 gene sequence disclosed herein are used to identify homologous BRCA1 gene sequences and proteins in other species. These BRCA1 gene sequences and proteins are used in the diagnostic / predictive, 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 a BRCA1 allele that predisposes an individual to cancer, a biological sample such as blood is prepared and analyzed for the presence or absence of a sensitive allele of BRCA1 . In order to detect the presence of neoplasms, the progression of precursor lesions towards malignant tumors, or as a predictive indicator, a biological sample of lesions is prepared and the presence or absence of a mutant allele of BRCA1 Analyze about. The results of these trials and interpretation information are returned to the healthcare provider for communication to the individuals tested. Such a diagnosis may be performed by a diagnostic laboratory, or alternatively, a diagnostic kit may be manufactured and sold to healthcare providers or private individuals 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 2-step label amplification methodologies well known in the art. Both PCR and non-PCR based screening strategies can detect target sequences with a high level of sensitivity.

  The most popular method used today is target amplification. Here, the target nucleic acid sequence is amplified with a polymerase. One particularly preferred method using polymerase-actuated amplification is the polymerase chain reaction (PCR). Polymerase chain reactions and other polymerase-actuated amplification assays can achieve over a million-fold increase in copy number through the use of polymerase-actuated 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 a target sequence (eg, in screening for cancer susceptibility), a biological sample to be analyzed, such as blood or serum, can be processed to extract nucleic acids, if desired. Sample nucleic acids can be prepared in a variety of ways to facilitate detection of the target sequence; eg, denaturation, restriction digestion, electrophoresis or dot blot. The targeting region of the analytical nucleic acid is usually at least partially single stranded and must hybridize to the target sequence of the probe. If the sequence is naturally single stranded, denaturation will not be necessary. However, if the sequence is double stranded, the sequence probably needs to be denatured. Denaturation can be performed by various techniques known in the art.

  The analytical 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 to the analyte can be completely complementary to the targeting region of human chromosome 17q. Therefore, high stringency conditions are desirable to prevent false positives. However, high stringency conditions are used only when the probe is complementary to a chromosomal region that is unique in the genome. Hybridization stringency is determined by a number of factors during the hybridization and wash steps, 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 provide a means of detecting the target sequence by forming higher order hybrids such as triple helices, quadruple helices and others.

  In any case, detection of the resulting hybrid is usually done by using a target probe. Alternatively, the probe may be unlabeled but can be detected by specific binding with a directly or indirectly labeled ligand. Appropriate labels and methods for labeling probes and ligands are known in the art and include, for example, known methods (e.g., nick translation, random priming or kinasing), biotin, Radioactive labels that can be incorporated by fluorescent groups, chemiluminescent groups (eg, dioxetanes, especially triggered dioxetanes), enzymes, antibodies, etc. are included. Variations on this basic scheme are known in the art and include variations that facilitate separation of hybrids to be detected from foreign materials and / or variations that amplify signals from labeling groups. Many of these variations are reviewed, for example, in Matthews and Kricka, 1988; Landegren et al., 1988; Mittlin, 1989; US Pat. 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 a methyl phosphonate backbone replaced with a normal phosphodiester) to a low level DNA target. The probe can have an enzyme covalently attached to the probe so that the covalent bond does not interfere with the specificity of the hybridization. This enzyme-probe-conjugate-target nucleic acid complex can then be isolated from the free probe enzyme conjugate and a substrate is added for enzyme detection. Enzymatic activity is observed with a change in color development or luminescence output that results in a 10 ³ -10 ⁶ increase 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 methodologies are known in the art. These assays are performed on the principle that small ligands (such as digoxigenin, biotin, etc.) bind to nucleic acid probes that can specifically bind to BRCA1. Exemplary probes are provided in Tables 9 and 10 herein, and further include nucleic acid probes corresponding to nucleotide positions 3631 to 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 predisposing mutations summarized in Tables 12 and 13 herein. Probes to be included.

  In one example, a small probe that binds to a nucleic acid probe is specifically recognized by an 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. See Martin et al., 1990 for a method for labeling nucleic acid probes according to this embodiment. In the second embodiment, small ligands are recognized by a second ligand-enzyme conjugate that can specifically form a complex with the first ligand. A well-known example of this example is the biotin-avidin type interaction. See Rigby et al., 1977 and Nguyen et al., 1992 for methods of labeling nucleic acid probes and their use in biotin-avidin based assays.

  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, especially many different probes, alternatively two, three or five different nucleic acid probe sequences. Use. In another example, in order to detect the presence of a mutation in the BRCA1 gene sequence in a patient, two or more probes complementary to BRCA1 are used, where the cocktail is used in patients with changes in BRCA1. Includes probes capable of binding to allele-specific mutations identified in the population. In this embodiment, any number of probes can be used, but preferably will include probes corresponding to major gene mutations that have been identified as predisposing individuals 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, and 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 Diagnostics and Diagnostic Kits Neoplastic status of lesions can also be detected based on changes 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 may be prepared as discussed previously under the heading “antibodies” and as further illustrated in Examples 12 and 13. Other techniques for raising and purifying antibodies are well known in the art, and any such technique can be used to make the preparation 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 embodiments relating to methods for detecting BRCA1 or mutations thereof include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradioanalytical assay, including sandwich assays using monoclonal and / or polyclonal antibodies. (IRMA) and immunoenzyme assay (IEMA). An exemplary sandwich assay is described by David et al. In US Pat. Nos. 4,376,110 and 4,486,530 (which are hereby expressly incorporated by reference and illustrated in Example 14).

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.

  The BRCA1 polypeptide or fragment used in such tests can be released in solution, immobilized on a solid support, or supported on the cell surface. One method of drug screening uses eukaryotic or prokaryotic host organism cells stably transformed with a recombinant polynucleotide expressing the polypeptide or fragment, preferably in a competitive binding assay. Such cells that are 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 can be interfered with by the agent to be tested. The degree to which it is done can be inspected.

  Thus, the present invention provides for contacting a drug with a BRCA1 polypeptide or fragment and, by methods well known in the art, (i) for the presence of a complex between the agent and the BRCA1 polypeptide or fragment, Or (ii) a method of screening for a drug, characterized by assaying for the presence of a complex between a BRCA1 polypeptide or fragment thereof and a ligand. In such competitive binding assays, 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 uncomplexed) label is a measure of the binding of the agent to be tested to BRCA1, respectively. Alternatively, BRCA1: a value that is disturbed by ligand binding.

  Another technique for drug screening provides high throughput screening of compounds with appropriate binding affinity for BRCA1 polypeptide, which is published in Geysen, 13 September 1984, PCT International Publication WO 84 It is described in detail in / 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. Peptide test compounds are reacted with BRCA1 polypeptide and washed. Bound BRCA1 polypeptide is then detected by methods well known in the art.

  Purified BRCA1 can be coated directly onto the plate for use in the drug screening techniques described above. However, a non-neutralizing antibody against 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, in which neutralizing antibodies capable of specifically binding to BRCA1 polypeptide are combined with test compounds for binding to BRCA polypeptide or fragments thereof. Make it conflict. In this way, 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 that have a non-functional BRCA1 gene (as described above). 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 modulate the growth of BRCA1-deficient cells.

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

  It is also possible to isolate a target-specific antibody selected by a functional assay and then elucidate its crystal structure. In principle, this approach provides a pharmacore that can be based on subsequent drug design. By generating anti-idiotype antibodies (anti-ids) against functional pharmacologically active antibodies, protein crystallography can be avoided altogether. Like the mirror image, the anti-ids binding site is expected to be an analog of the original receptor. The anti-ids can then be used to identify and isolate peptides from banks of chemically or biologically generated banks of peptides. 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, a sufficient amount of BRCA1 polypeptide can be utilized to perform analytical experiments such as x-ray crystallography. In addition, the knowledge of the BRCA1 protein sequence provided herein will lead to using computer modeling techniques instead of or in addition to x-ray crystallography.

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

  More preferably, the situation is such that the wild type BRCA1 gene or a part thereof is introduced into the mutant cell so that it recombines with the endogenous mutant BRCA1 gene present in the cell. Such recombination requires a double recombination event, which results in correction of the BRCA1 gene mutation. Vectors for introducing 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 coprecipitation, and viral transduction, are known in the art, and the choice of method is within the capabilities of the daily worker. 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 previously, the BRCA1 gene or fragment, if applicable, can be used in gene therapy to increase the amount of expression product of such 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 mutated gene is expressed at a “normal” level, the gene product can also be useful in increasing the expression level of a given BRCA1 gene, even in tumor cells that are not fully functional.

  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 viral or plasmid vector (see further details below) is prepared that contains a copy of the BRCA1 gene linked to an expression regulator and has the replication ability inside the tumor cell. Suitable vectors are known, such as those disclosed in US Pat. No. 5,252,479 and PCT International Publication No. WO 93/07282. The vector is then injected into the patient either locally at the tumor site or systemically (to reach any tumor cell that can metastasize to other sites). If the transfected gene is permanently incorporated into the genome of each targeted tumor cell, the treatment can be repeated periodically.

  Gene transfer systems known in the art can be useful in the practice of 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 Virus (Moss, 1992), papoviridae including adeno-associated virus (Muzyczka, 1992; Ohi et al., 1992), HSV and EBV (Margorskee, 1992; Johson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese 1990), and chickens (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), mice (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1988) and numerous viruses, including retroviruses of human origin (Shimada et al., 1991; Helseth et al., 1990; Page et al., 1990; Buchschacher and Panganiba, 1992) Used as a child transfer vector. Most human gene therapy protocols are based on inactivated murine retroviruses.

  Non-viral gene transfer methods known in the art include chemical techniques such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); for example, microinjection (Anderson et al., 1980; Godon et al., 1980). Mechanical methods such as Brinster et al., 1981; Constantanti and Lacy, 1981); Membrane fusion-mediated transfer via liposomes (Felgner et al., 1987; Wang and Haung, 1989; Kaneda et al., 1989; Stewart et al., 1992; Nabel; 1990; Lim et al., 1992); and direct DNA uptake and receptor-mediated DNA transfer (Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989b; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b). Virus-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery to direct viral vectors to tumor cells rather than surrounding non-dividing cells. Alternatively, retroviral vector-producing cell lines can be injected into tumors (Culver et al., 1992). The injection of production cells then provides a continuous source of vector particles. This technique is approved for use in humans with inoperable brain tumors.

  In an approach that combines biological and physical gene transfer methods, plasmid DNA of either size is combined with a polylysine-conjugated antibody specific for adenovirus hexon protein and the resulting complex is converted to an adenovirus vector. Join. The trimolecular complex is then used to infect cells. Adenoviral vectors allow efficient binding, internalization and digestion of endosomes before the coupled DNA is damaged.

  It has been shown that liposome / DNA complexes can mediate direct in vivo gene transfer. Although the gene transfer process is nonspecific in standard liposome preparations, for example, localized in vivo uptake and expression has been reported in tumor deposition after direct in situ administration. (Nabel, 1992). Gene transfer techniques that target 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. The ligand is 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 can include the estrogen receptor and its ligand, estrogen (and estrogen analogs). These ligand-DNA conjugates can be injected directly into the blood, if desired, and can be directed to target tissues where receptor binding and internalization of the DNA-protein complex occurs. In order to overcome the problem of intracellular destruction of DNA, co-infection with adenovirus can be involved to disrupt endosomal function.

  The therapy includes two steps that can be performed alone or in combination. In the first step, prepubertal women carrying a BRCA1 sensitive allele are treated with gene delivery so that some or all of their mammary epithelial progenitor cells receive at least one additional copy of a functional normal BRCA1 allele. Process with vehicle. In this process, the treated individual has a risk of breast cancer that is reduced to the extent that the presence of the normal allele precludes the action of the susceptible allele. In the second step of prophylactic therapy, pre-treated young women, especially those who have received the proposed gene therapy treatment, are subjected to hormone therapy to minimize the impact on the breasts of the full-term pregnancy.

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

  Active BRCA1 molecules can be introduced into cells by microinjection or by using, for example, liposomes. Alternatively, certain active molecules can be taken up by cells, either actively or by diffusion. Extracellular application of the BRCA1 gene product can fully affect tumor growth. Application of molecules with BRCA1 activity must lead to partial reversal of the neoplastic state. Other molecules with BRCA1 activity (eg, peptides, drugs or organic compounds) can also be used to 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 test and test for substances with potential as therapeutic agents. The cell is typically a cultured epithelial cell. They can be isolated from individuals with either somatic or germline BRCA1 mutations. Alternatively, the cell line can be designed to carry mutations in the BRCA1 allele as described above. After the test substance is applied to the cells, the neoplastically transformed phenotype of the cells is determined. Any characteristics of neoplastically transformed cells can be assessed, including anchorage independent growth, tumorigenicity in nude mice, cell non-invasiveness, and growth factor dependence. Each of these feature assays is known in the art.

  Animals to be tested for therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. Such treatment usually includes 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). Tumor growth must be assessed after administration of the test substance to the animal. If the test substance prevents or suppresses tumor growth, the test substance is a candidate therapeutic for the treatment of cancer identified herein. These animal models provide critical test vehicles for potential therapeutic products.

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

Example 1
Confirmed and experimental families with 17q-linked breast cancer susceptibility loci A wide range of cancer-susceptible families provides a large set of relatives available for a wide range of families and experiments with many cases of breast cancer. Confirmed from the group. The vast number of meiosis present in these large families provides the ability to detect whether the BRCA1 locus is segregating and provides an opportunity for informational recombinants that occur within the small region to be investigated. Decreased. This greatly improves the opportunity to establish linkage to the BRCA1 region, facilitates the reduction of the BRCA1 region to a manageable size, and allows identification of the BRCA1 locus itself.

  Each family was expanded through all available related relatives to all probable relatives in each proband or cancer case. For these families, additional breast cancer cases and individuals with cancer at other sites of interest that appeared in the family (eg, ovaries) were sought through tumor registry-related files. We examined all breast cancers reported in families that were not identified in the Utah Cancer Registry. Medical records or death certificates were obtained for confirmation of all cancers. Each key individual and all informational individuals were allowed to participate by providing a blood sample from which DNA was extracted. We also sampled from the deceased case's spouse and relatives so that the deceased case's genotype could be inferred from the relative's genotype.

  Ten families with more than three cancer cases with putative genotypes were linked to 17q markers from a set of 29 families originally identified from relevant data beta for studies of proliferative breast disease and breast cancer (Skolnick et al., 1990). The criteria for selection of these families was the presence of two sisters or mothers with breast cancer and her daughter. In addition, two families (K1001, K9018) that have been studied as part of our breast cancer chain study since 1980, six families (K2019, K2073) identified from the relevant database about the presence of breast and / or ovarian cancer clusters. , K2079, K2080, K2039, K2082) and self-estimated families with early-onset breast cancer (K2035) were included. These families were investigated and expanded in our clinical practice as described above. Table 1 shows the characteristics of these 19 families that are the subject of subsequent examples. Table 1 reports for each family, 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. Sort families by increasing median age at the time of breast cancer diagnosis. Four women diagnosed with both ovary and breast cancer count in both categories.

Example 2
Selection of family linked to chromosome 17q and positioning of BRCA1 in interval Mfd15-Mfd188 For each sample collected in these 19 families, blood (in 2 cases, paraffin-embedded tissue) was used. DNA was extracted from (block). Genotyping in this experiment was limited to short tandem repeat (STR) markers. This is because, in general, they are highly heterozygous and PCR methods provide rapid rotation while using very small amounts of DNA. To aid 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 in the long arm: (46E6. Easton et al., 1993); (42D6, Easton et al., 1993); 26C2 (D17S514, Oliphant et al., 1991), while the other 12G6 (D17S513). Oliphant et al., 1991) was located in the short arm near the p53 tumor suppressor locus. Two of these, 42D6 and 46E6, were submitted to the Breast Cancer Linkage Consortium for type classification of breast cancer families by researchers worldwide. Oligonucleotide sequences for markers not developed in our laboratory were obtained from published reports, as part of the Breast Cancer Linkage Consortium, or from other researchers.

  All genotyping films were standard lane markers used to maintain consistent allele coding and scored blindly. The key samples in the four families shown here were typed in duplicate for all relevant markers. All 19 families were served by two polymorphic CA repeat markers: 42D6 (D17S588), CA repeats isolated in our laboratory, and Mdf15 (D17S250), J. Weber (Weber et al., 1990). Each CA repeat was typed. Using probes from several sources, create genetic markers on chromosome 17, especially on chromosome 17 cosmids, and create lambda phage libraries from classified chromosomes by Los Alamos National Laboratories (van Dilla et al., 1986). Produced.

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

  Under the CASH model (Claus et al., 1991), using the criterion for 17q linkage with LOD score> 1.0 per at least one locus, 4 out of 19 families appeared to be linked to 17q. (K1901, K1925, K2035, K2082). A number of additional families showed some evidence of linkage, but at this point they could not be clearly assigned to the linkage category. These included the families K1911, K2073, K2039 and K2080. Three of the 17q-linked families have informational recombinants in this region, which are detailed below.

  Family 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. Two cases have ovarian cancer 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 scattered, the LOD score with linked haplotypes is over 6.0. That is, these cases do not have both of the haplotypes linked between Mfd15 and 42D6. These three scattered cases were diagnosed with breast cancer at ages 46, 47 and 54 years. In smaller families, this type of sporadic cancer greatly confounds linkage analysis and correct identification of key recombinants. The key recombinant in the 2082 family is the woman who developed ovarian cancer at age 45 when her mother and aunt became ovarian cancer at the age of 58 and 66 years, respectively. She inherited the linked part of the haplotype for both Mfd188 and 42D6, inheriting the unlinked allele in Mfd15; this recombination event resulted in BRCA1 being located distal to Mfd15.

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

  Family 2035 is similar to K1901 in disease phenotype. The median age of diagnosis for 8 cases of breast cancer in this family is 37 years. One case was 60 years old and also had ovarian cancer. Breast cancer cases in this family are inherited from two sisters who both had not had breast cancer until their death in their 80s. Each branch had 4 cases of breast cancer, and each branch with a marked early onset had at least one case. This family has an LOD score of 2.34 per Mfd15. The haplotypes that segregate breast cancer in the two branch have both identical alleles at Mfd15, but as part of a consortium located just distal to loci Mfd188 and NM23 (42D6) Different for typed markers). The two haplotypes are identical for marker 42D6, but the alleles are identically shared by the state (same alleles) rather than identical by the pedigree (derived from a common ancestor). But from different ancestors). This is because the shared allele is the second most common allele observed at this locus. In contrast, the linked allele shared by Mfd15 has a frequency of 0.04. This is a key recombinant in our database. This is because BRCA1 is the only recombination that segregates at the base side of the haplotype, thus providing an end that borders the BRCA1 region. For this event, there is a second mutant BRCA1 gene in a wife who is married to a family that also has a rare Mfd15 allele that segregates for breast cancer in both ancestry families, which is not a key recombination. It is necessary to do. This event has a probability less than 1/1000. Thus, with evidence from this family, the BRCA1 locus was located proximal to Mfd188.

Example 3
Refinement mapping and further determination of the BRCA1 region for Mfd191-Mfd188 using additional STR polymorphisms To improve the characterization of our recombinants and clarify the flanking markers, this on chromosome 17q 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 the highly polymorphic STR polymorphism and many polymorphic expressed genes. Since this map does not give details about the evidence for this order, nor does it give any measure of local support for reversals in the order of adjacent loci, we use it in the development of new markers. Summarized as a rough guide to obtain our detailed genetic and physical maps of the power region and subregions containing BRCA1. Our approach is based on current STR markers provided by other investigators, and a panel of meiotic (genetic) cessation points identified using DNA from CEPH-related families and bodies constructed for this region It was to analyze all newly developed markers from our laboratory for both panels of cell-cell hybrids (physical breakpoints). These markers are 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 It included 26C2 developed in our laboratory, mapped on the base side to 6C1 (D17S293).

Gene localization of markers To genetically locate new markers within a region of interest, we have a number of key reductions within the region in the CEPH literature panel and in our large breast cancer family (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 markers within each set can only be determined by physical mapping. However, the number of genotyping required to locate the new marker is minimized. These disruption points 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 family 1901, the Mfd15 recombinant is a recombinant for THRA1, but Mfd191 is non-informative, 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). No further positioning information was obtained from investigation of THRA1 and Mfd191 in family line K2035. This is because the two split houses 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 thus in an interval bordering the proximal Mfd191 and the 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 the cosmids and YACs physically mapped to the region A number of STR markers have been developed at laboratories. These markers allowed us to further refine the region.
STR contains these loci, which were then used to identify from genes known to be in the desired region to identify YACs that identify subclones in cosmid P1 or BAC. These subclones were then screened for the presence of CA tandem repeats using (CA) _n oligonucleotide (Pharmacia). A clone with a strong signal was preferentially selected. This is because they appear to represent CA-repeats with a very large number of iterations and / or 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 determine the location of the repeats. We have obtained a unique sequence on one side of the CA-repeat by using one of the possible primer sets complementary to the end of the CA-repeat, such as (GT) ₁₀ T. Based on this unique sequence, a primer was created that crossed the repeat in the other direction, resulting in a unique sequence for the design of the second primer adjacent to the CA-repeat. The STR was then screened for polymorphisms for a small group of unrelated individuals and tested against a 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 allelic frequencies suitable 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.

  Using the approach, a total of 8 polymorphic STRs were found from these YACS. Thus, four of the same loci were both polymorphic and located relative 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 a YAC positive for the RNU2 gene, 4-7 from an EPB3 YAC, and YM29 from a cosmid located in the region by a hybrid panel. A description of the number of alleles analyzed in breast cancer families, heterozygosity and the source of these four and all other STR polymorphisms is provided below in Table 5.

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

  From CEPH 1333-04 we see that AA1 and YM29 must be located distal to Mfd191. From 13292, it can be estimated that AA1 and ED2 are both on the base side with respect to 4-7, YM29 and Mfd188. The recombinant found in K2082 provides some additional ordering information. In three independent observations (individual numbers 22, 40 and 63), AA1, ED2, 4-7, and YM29, and Mf188 are located distal to Mfd191, whereas by ID125, 4-7 , YM29, and Mfd188 are positioned proximal to SGC40. Genetic information about the relative ordering within the two clusters of markers AA1 / ED2 and 4-7 / YM29 / Mfd188 was not obtained from genetic recombination analysis. The ordering of the loci for hybrids known to contain “holes” that appear to have lost pieces of interstitial human DNA is questionable, but the hybrid pattern is 4-7 above YM29 and Mfd188 Indicates to do.

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

  Table 8 reveals the family haplotypes (shown in codon form) by specific marker alleles at each locus and their respective frequencies. In Table 8, alleles are listed in order of decreasing frequency; the frequency of alleles 1-5 for each locus is shown in Table 5. The haplotype encoding H is a BRCA1-related haplotype, P indicates a partial H haplotype, and R indicates an observable recombinant haplotype. As can be seen in Table 8, not all families were typed for all markers, especially in family K2082, not all individuals in the family were typed for the same set of markers. Only one haplotype inherited from affected or dangerous family members is shown with one exception; 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 / risk individuals, neither of which was a breast cancer-related haplotype.

  In family K1901, the new marker does not show an observable recombinant with breast cancer susceptibility, indicating that recombination events in this family most likely occur between THRA1 and ED2. Thus, no new BRCA1 positioning information was obtained based on examining four new markers in this family. In pedigree 2082, the key recombinant individual takes over the allele linked for ED2, 4-7, AA1, and YM29, which is the recombinant for tdj1474, which is the recombination event tdj1474 and Indicates what happened to this individual during ED2 / AA1.

  There are three haplotypes of interest in the families K2035, H1, H2 and R2 shown in Table 8. H1 is present in 1 genuine male carrier successor from 4 cases and individual 17, while H2 is present in 2 genuine male carriers in 2 cases and successor of individual 10. R2 is identical to H2 at the locus between and including Mfd15 and SCG40, but has a recombinant between SCG40 and 42D6. Since we established that BRCA1 is proximal to 42D6, this H2 / R2 difference does not add any additional positioning information. H1 and R2 have the same allele in Mfd15, THRA1, AA1 and ED2, but differ for loci putative to ED2, ie 4-7, Mfd188, SCG40 and 6C1. Although the two haplotypes match for the fifth allele at marker YM29 (a marker physically mapped between 4-7 and Mfd188), the allele is held identical by the pedigree It seems that they are held identically by state rather than being. This is because this allele is the most common allele at this locus and the estimated frequency in the CEPH parent is 0.42. In contrast, the linked alleles carried together at the Mfd15 and ED2 loci have a frequency of 0.04 and 0.09, respectively. They also carry the more common alleles in Mfd191 (frequency = 0.52), THRA1, and AA1 (frequency = 0.28). This is a key recombinant in the set. This is because breast cancer is the only recombinant that separates with respect to the base part of the haplotype, thus determining the terminal boundary. Thus, with evidence from this family, the BRCA1 locus is located on the 4-7 base side.

  The recombination event in pedigree 2082 that positions BRCA1 distal to tdj1474 is only one of the four described events that can be estimated directly; that is, the genotype of the affected mother is her spouse and offspring The recombinant haplotype can be observed in her affected daughter. In this family, it is very likely that the affected individual carrying the BRCA1 susceptibility allele will be favored; the only possible interpretation of the data is that BRCA1 is at the end of Mfd191 or the expressed recombinant Is a 44-year-old ovarian cancer scattered case. Rather than directly observable or putative recombinants, the interpretation of family 2033 relies on the observation of distinct 17q-haplotypes that segregate in different and sometimes distant related families of the family. . Some of these haplotypes have a common allele for some markers, while they locate the BRCA1 locus in a shared region in other markers. The reliability of this positioning determines several factors; the relationship between individuals bearing each haplotype, the frequency of shared alleles, the certainty that the haplotype has been shown to segregate with respect to the BRCA1 locus, and the haplotype. Depends on the density of markers in the area. In the case of family line 2035, the two branch houses are closely related, and each branch house has a number of early start cases for each haplotype. Two of the shared alleles are common (Mfd191, THRA1), while the estimated frequencies of 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 being identical by state (same allele but from the general population).

Example 6
Precise physical mapping studies position BRCA1 in the region flanked by tdj1474 and U5R.
Since the first localization to chromosome 17q in 1990 (Hall et al., 1990), positioning the BRCA1 gene in a region small enough to allow for an effective positional cloning strategy and to isolate the gene A great deal of effort has been made. According to multipoint linkage analysis (Easton et al., 1993), the BRCA1 locus is first identified as interval Mfd15 (D17S250) -42D6 (D17S588) in the globally collected 214 Breast Cancer Linkage Consortium database. Was located. Subsequent precise positioning was based on individual recombination events in a particular family. Region THRA1-D17S183 was clarified by Bowcock et al., 1993; region THRA1-D17S28 was clarified by Simard et al., 1993.

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

  Four families provide important informational genetic evidence for the positioning of BRCA1 in a sufficiently small region for application of position cloning strategies. Two families (K2082, K1901) provide data on the proximal boundary for BRCA1, and the other two (K2035, K1813) define the distal boundary. These families are detailed below. A total of 15 short tandem repeat markers that can be assayed by PCR were used to increase the accuracy of 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, DS17S754 for SEQ ID NO: 4, DS17S975 for SEQ ID NO: 5 and SEQ ID NO: 6, tgj1474 for SEQ ID NO: 7 and SEQ ID NO: 8, and tgj1239 for SEQ ID NO: 9 and SEQ ID NO: 10.

Family 2082
Family 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 the 17q linkage. This family has been previously described and has been shown to contain critical recombinants that position BRCA1 distal to MFD191 (D17S776). This recombinant occurred in a woman whose mother had been diagnosed with ovarian cancer at age 45 and had ovarian cancer at age 63. The affected mother passed away; however, from her children, it could be presumed that she had a linked haplotype that was present in 30 other cases of the family in the region between Mfd15 and Mfd188. Her affected daughter inherited alleles at loci ED2, 4-7 and Mfd188 but inherited alleles on the non-BRCA1 chromosome at Mfd15 and Mfd191. To further locate this recombination breakpoint, we tested key members of the family for the following markers from the physical mapping source: tdj1474, tdj1239, CF4, D17S855. For markers tdj1474 and CF4, the affected daughter 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. Therefore, the above-mentioned recombinant positioned BRCA1 on the terminal side with respect to tdj1474, and the discontinuation point was positioned in 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 age 50 is rare, this alternative interpretation is unlikely.

Family 1901
Family 1901 is an early-onset breast cancer family in which 7 cases were diagnosed with breast cancer before 50 years of age, 4 of which were diagnosed before 40 years of age. In addition, there were 3 cases diagnosed with breast cancer between the ages of 50 and 70. One case of breast cancer also had ovarian cancer at age 61. This family currently has a LOD score of 1.5 per D17S855. Given this linkage evidence and the presence of at least one ovarian eye case, this family has a posterior probability with BRDA1 greater than 0.99. In this family, recombination stems from the fact that individuals who are siblings of the ovarian cancer case that were then inherited in the majority of other cases have both haplotypes that co-segregate with other cases in the family. However, he gave this partial haplotype to his daughter, who developed breast cancer at age 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. The difficulty in interpreting this type of information can identify unshared markers, and therefore recombinants, but matching markers are not because they are non-recombinant or their parents It will be shared because it was a homozygote. Without the parental genotype data these distinct interpretations cannot be distinguished. In view of the haplotype at K1901, he is shown to have no linked alleles at Mfd15 (D17S250), THRA1, CF4 (S17S1320), and tdj1474 (17S1321). He has both linked alleles in Mfd191 (D17S776), ED2 (S17S1327), tdj1239 (D17S1328) and Mfd188 (D17S579). Despite the relatively rare (0.07) allele shared by Mfd191, we do not know that the parents are recombinant at the marker they are located near either side. It is homozygous and assumes that double recombination events in this region are extremely unlikely. Thus, evidence in this family locates the BRCA1 locus distal to tdj1474. However, the lower limit of this disruption point cannot be determined without parental genotype information. The key recombination breakpoint in this family is embarrassing to confirm the results in family 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 in diagnosis (44) makes this very likely. This is because the general population has a low risk of breast cancer before age 45 (almost 1%).

Family 2035
In this family, information about critical recombination events is not directly observed, but the observation shows that two haplotypes co-segregating for early-onset breast cancer in the two branch of the family are in the proximal part of the 17qBRCA1 region Similar to K1901 in that it is presumed to be the same for the positioned marker but different at the more distal locus. Each of these two haplotypes occurs in at least 4 cases of early-onset or bilateral breast cancer. The overall LOD score for ED2 in this family is 2.2, and there is a case of breast cancer in this family (which indicates an 80% prior probability of BRCA1 linkage), and this family is linked to BRCA1 The obtained posterior probability is 0.998. The haplotype is the same 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 equally by offspring. However, the haplotypes do not match for CA375, 4-7, and Mfd188, and some more distal markers. This indicates that the BRCA1 locus must be above the marker CA375. This marker is located 50 kb below D17S78, so it primarily serves as a further confirmation of this previous low 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 40 and before age 40 was diagnosed with breast cancer at age 61 and ovarian cancer at age 61. This situation is somewhat complicated by the fact that the four cases have three different fathers, of which only one is genotyped. However, by typing many different markers in the BRCA1 region as well as highly polymorphic markers elsewhere in the genome, it is determined with great certainty who is the father of all children in the family. It was. 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, this results in a posterior probability of being a 0.93 BRCA1 linked family. This family contains a recombination event that is directly observable in an individual 18 who developed breast cancer at age 34 (see FIG. 5 in Simard et al., Human Mol. Genet. 2: 1193-1199 (1993)). The genotype of an affected mother at the relevant 17q locus can be inferred from her genotype, her affected sister's genotype, and three other unaffected sibling genotypes. Individual 18 inherits the BRCA1 linked allele for the following loci: Mfd15, THRA1, D17S800, D17S855, AA1, and D17S931. However, for the markers below D17S931, ie U5R, vrs31, D17S858 and D17S579, she inherited alleles located on a chromosome that does not carry disease. Evidence from this family therefore positions the BRCA1 locus proximal to the marker US5. Due to her early age of diagnosis (34), it is very unlikely that the cancer of the recombinant individual is due to a gene responsible for other cases of breast / ovarian cancer in this family: this family The uncertainty in is 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 present in the interval between the markers tdj474 and U5R, both isolated in our laboratory. Based on the physical maps shown in FIGS. 2 and 3, we can try to estimate the physical distance between these two loci. It shall be about 14 P1 clones with an average insert size of about 80 kb over this region. However, due to some unknown degree of all these P1 overlaps, the physical area appears to be much smaller than 14 times 80 kb. Based on the restriction map of the clones covering the region, we estimate that the size of the region containing BRCA1 is approximately 650 bp.

Example 7
Identification of candidate cDNA clones for the BRCA1 locus by genomic analysis of the Contig region Complete screening of possible regions The first method of identifying candidate cDNAs, which is quite labor intensive, used known techniques. The method consisted of screening the cosmid and P1 and BAC clones in the contig to identify the putative coding sequence. The clone containing the putative coding sequence was then used as a probe on the cDNA library filter to identify candidate cDNA clones for future analysis. The clones were screened for putative coding sequences by either of two methods.

Zoo blot The first method for identifying putative coding sequences was by screening cosmids and P1 clones for sequences conserved throughout evolution across several species. This technique is referred to as “zoom analysis” and is described by Monaco, 1986. In particular, DNA from bovine, chicken, pig, mouse and rat was digested with the restriction enzymes EcoRI and HinDIII (8 μg DNA per enzyme). Digested DNA was separated on a 0.7% gel overnight at 20 volts for 16 hours (14 cm gel) and the DNA transferred to a nylon membrane using standard Southern blot techniques. For example, treatment at 65 ° C. in 0.1 × SSC, 0.5% SDS and 0.2M Tris, pH 8.0 for 30 minutes, followed by 5 × SSC, 10% PEG 8000, 20 mM at 42 ° C. Blocked in NaPO ₄ , pH 6.8, 100 μg / ml salmon sperm DNA, 1 × Denhardt's, 50% formamide, 0.1% SDS and 2 μg / ml C ₀ 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 overnight on a 14% 0.5% agarose gel run at 20 volts for 16 hours. The human DNA band was excised from the gel and electroeluted from the gel wedge in 0.5 × 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) is then digested with EcoRI restriction enzyme to give a small fragment (˜0.5 kb to 5.0 kb) which is then used to label the DNA with radioactive nucleotides. It melted easily for the next step. The DNA fragment was labeled by the hexamer random priming labeling method (Boehringer-Mannheim, catalog number 1004760). 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 radionucleotide. The labeled DNA is then resuspended in 100 μl TE, 0.5 M NaCl at 65 ° C. for 5 minutes and then as per manufacturer's instructions (Gibco / BRL, catalog number 5279SA) with human C ₀ t-1 DNA. Blocked in 2-4 hours. The C ₀ t-1 blocked probe was incubated on a zoom blot filter at 42 ° C. in blocking solution. The filter was washed for 30 minutes in 2 × SSC, 0.1% SDS at room temperature, then for 30 minutes in the same buffer at 55 ° C. The filter was then exposed to Kodak XAR-5 film containing sensitizer for 1 to 3 days at -70 ° C. Thus, the zoom blots were hybridized with a pool of Eco-R1 fragments from the insert or individually with each fragment.

HTF island analysis The second method of identifying cosmids for use as probes for cDNA libraries was HTF island analysis. Since the pulse field map can reveal HTF islands, cosmids mapped to these HTF island regions were preferentially analyzed. HTF islands are segments of DNA that contain a very high frequency of unmethylated CpG dinucleotides (Tonolio et al., 1990) and 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 (Anand, 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 is at the end of the GP2B locus, the other is at the base of the locus, and both are outside the BRCA1 region. Cosmids derived from TAC covering these two positions were analyzed to identify those containing restriction sites and thus HTF islands.

cDNA Screening Clones that contain HTF islands or show hybridization to other species of DNA other than humans appear 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. A variety of cDNAs were used under the same conditions as the Zooblot except that the labeled DNA was used and the cDNA filter was subjected to 0.1 × SSC, 0.1% SDS twice more for 30 minutes at 65 ° C. Library filters were screened.
Most of the cDNA libraries used to date in our experiments (normal breast tissue, library from breast tissue from women with breast malignancy at 8 months of gestation) Clonetech, Inc. Prepared from A cDNA library made from mammary tissue of an 8-month pregnant woman 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 female 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, selecting the size of the final product, which was then cloned into the lambda ZapII vector and the bacterial strain as described by the manufacturer. Grown on XL1-blue strain. 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).

  The cDNA library is plated with its host cells on NZCYM plates and filter lifts are made in duplicate from each plate as in Maniatis et al. (1982). Insert (human) DNA from candidate genomic clones was purified and radiolabeled to high specific activity. Radioactive DNA was then hybridized to the cDNA filter and the cDNA corresponding to the gene located within the candidate cosmid clone was identified. The cDNAs identified by this method were picked, re-plated and screened again with labeled clone inserts 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 purified for Southern blot analysis and sequencing. As described in the manufacturer's protocol, the clone is purified as a plasmid through in vivo excision of the plasmid from the lambda phage vector, or the clone is isolated from the lambda phage as a restriction fragment and subcultured into the plasmid vector. Cloned.

  Southern blot analysis was performed in duplicate and one confirmed that the cDNA inserter contained a hybridizing sequence using the original genomic insert DNA as a probe. A second blot was hybridized with cDNA insert DNA from the largest cDNA insert to determine which clones represent the same gene. All cDNAs that hybridize with the genomic clone and are unique were sequenced and DNA analysis was performed to determine whether the sequence represents a known or unique gene. All cDNA clones that appeared to be unique were further analyzed as candidate BRCA1 loci. Specifically, the clone is hybridized to a Northern blot looking for differential expression in breast specific expression and 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 used as PCR probes for the YAC and clones around and including the original identical clone. The intron-exon boundary was then further defined by sequence analysis.

  We screened normal breast, 8-month-old breast and fetal brain cDNA libraries with zoomid positive EcoRI fragments from cosmid BAC and P1 clones in the region. Potential BRCA1 cDNA clones were identified among the three libraries. Clones were picked, plated again, and screened again 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 originated 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 to find overlapping independent clones. For example, clones 694-65, 1240-1 and 1240-33 were obtained independently and were subsequently shown to be derived from the same adjacent cDNA sequence designated EST: 489: 1.

Analysis of candidate clones One or more of the candidate genes from above were sequenced and this 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 because of the sequence comparison for both local and remote sequence databanks (eg GenBank), the Client / Server software package (eg BLASTN 1.3.13MP) Genetic Data Environment (GDE) version 2.2 software and Basic This was accomplished using the Local Alignment Search Tool (Blast) series and running on a Sun SPARC workstation. A reconstructed sequence was obtained from a cosmid and a clone of the cDNA clone identified in P1. All candidate genes representing the new sequence were further analyzed and tested for the putative BRCA1 locus.

Mutation Screening Two different approaches were taken to screen for mutations in the affected pedigree. First, genomic DNA isolated from a family member known to bear a sensitive allele of BRCA1 was used as a template for amplification of candidate gene sequences by PCR. If the PCR primer is adjacent to or overlaps the intron / exon boundary, 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 using the designed primers, intron / exon structures can be established and ultimately the DNA sequence of genomic DNA from the pedigree can be obtained .

  A second approach, which is much more rapid if the intron / exon structure of the candidate gene is complex, involves sequencing fragments amplified from lineage 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 yield amplified fragments that can be sequenced directly without knowledge of intron / exon binding.

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

  Any sequence within the BRCA1 region expressed in the breast is considered a candidate gene for BRCA1. The strong evidence that a given candidate gene corresponds to BRCA1 comes from the evidence that the pedigree family contains 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 17q21 600 kb region between D17S1321 and D17S1324. Candidate expressing sequences are: 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 XPOUND ( Defined as the DNA sequence obtained from the production of coding exons by Thomas and Skolnick, 1994). These expressed sequences in many cases were assembled into contigs consisting of several independently identified sequences. A candidate gene may consist of more than one of these candidate expression sequences. 65 candidate expression sequences within this region were identified by hybrid selection, by direct screening of cDNA libraries, and by random sequencing of the P1 subclone. Expression sequences were characterized by DNA sequence analysis in transcript size, DNA sequence, database comparison, expression pattern, genomic structure, and most importantly, individuals from families separating 17q-linked breast and ovarian cancer susceptibility.

  Three independent contigs of the expression sequence, 1141: 1 (649 bp), 694: 5 (213 bp) and 754: 2 (1079 bp) were isolated and were shown to eventually represent a portion 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 I 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 contigs. 5'RACE experiments with thymus, testis and breast mRNA extended contig to the putative 5 'end, resulting in a composite full-length sequence. PCR and direct sequencing of P1 and BAC in the region were used to locate the intron and allow determination of splice donor and acceptor sites. These three expressed sequences were merged into a single transcriptional transfer unit, which was found to be BTCA1 in the final analysis. This transcription unit was positioned 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 BRCA1 cDNA (up to the stop codon) has also been deposited with GenBank and given accession number U-14680. Integrate with reference to this deposited sequence. 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 results in a single long open reading frame of 208 kilodaltons (amino acid sequence, SEQ ID NO: 2) with a potential start codon surrounded by and adjacent to a sequence similar to the Kozak consensus sequence (Kozak, 1987). It was made clear. A search by Smith-Waterman (Smith and Waterman, 1981) and BLAST (Altschyl et al., 1990) identified sequences near the amino terminus with significant 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 BRCA1 cDNA as a probe identified a single transcript of approximately 7.8 kb, most abundant in the breast, thymus and testis, and also in the ovary (FIG. 7). Four separately spliced products were detected in independent cDNA clones; three of these were observed in breast and two in ovarian mRNA (FIG. 6). A PCR overview from tissue cDNA further supports the notion that there is considerable heterozygosity near the 5 'end of transcripts from this gene; the molecular basis for the heterozygosity is the initial splice donor site All detected changes alter the transcript in the region 5 'to the identified start codon. We have detected 6 potential alternative splice donors in this 5 ′ untranslated region, the longest missing 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.

  A further 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 2 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. The fifth alternative splice form has a first acceptor site at position 591 and a first splice donor site at position 349 as well as 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 a recognized start site, which appears at position 120 of 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 (FIGS. 12-19) represent intron sequences, while the upper case letters represent exon sequences. In FIG. 12-19, the unclear interval in the intron is indicated by vvvvvvvv. Intron / exon binding is shown in Tables 9 and 10. CAGs found at the 5 'ends of exons 8 and 14 were found in some cDNAs but not others. Known polymorphic sites are shown in bold type in FIGS. 12-19 and are underlined.

  A low stringency blot that probes genomic DNA from a divergent phylogenetic background organism with a BRCA1 sequence lacking the zinc-finger region reveals fragments that hybridize strongly in humans, monkeys, sheep and pigs. A very weak hybridization signal was revealed in rodents. This result shows that, apart from zinc-fingered methones, BRCA1 is conserved only at moderate levels throughout evolution.

Germline BRCA1 mutations in 17q-linked families The most stringent test for BRCA1 candidate genes is for potentially destructive mutations in carrier individuals from families that isolate 17q-linked susceptibility to breast and ovarian cancer. It is to investigate. Such individuals must contain a BRCA1 allele that differs from the wild type sequence. The set of DNA samples used in this analysis consisted of DNA from individuals representing 8 different BRCA1 families (Table 11).

  The logarithm of the likelihood (LOD) score in these families ranges from 9.49 to -0.44 for the set of markers at 17q21. Family of 4 has a satisfactory LOD score per chain and 4 has a low positive or negative LOD score. The latter family was included. Because they exhibit a haplotype shared on chromosome 17q21 for at least 3 affected members. In addition, all families in the set show an early age of onset of breast cancer, and four of the families contain at least one case of ovarian cancer, both of which are characteristic of the BRCA1 family. One family 2082 has almost comparable degrees of breast and ovarian cancer, which is an unusual occurrence assuming that ovarian cancer in the population is relatively rare. All but two were confirmed in Utah. K2035 is from the Midwest. K2099 is an African-American-family from South America.

  In the initial screen for predisposing mutations in BRCA1, DNA from one individual bearing the predisposing haplotype in each family was tested. Twenty-three coding exons and associated splice junctions were amplified from genomic DNA samples or from cDNA prepared from lymphocyte mRNA. When the amplified DNA sequence was compared with the wild type sequence, it was found that 4 out of 8 family samples contained sequence variants (Table 12).

  All four sequence variants are heterozygous, each appearing in only one of the families. Family 2082 contains a nonsense mutation in exon 11 (FIG. 9). Family 1910 contains a single nucleotide insertion in exon 20 (FIG. 10). Family 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 family 1910 contains a modified amino acid sequence starting at 108 residues from the wild-type C-terminus. The peptide encoded by the frameshift allele in family 1901 contains an amino acid sequence starting at the 24th residue from the wild type N-terminus. The mutant allele in family 2082 encodes a protein that loses 551 residues from the C-terminus. The missense substitution observed in family 2099 is potentially destructive because it replaces a small hydrophobic amino acid (Met) with a large charged residue (Arg). Eleven common polymorphisms have also been identified, eight in the coding sequence and three in the introns.

  Individuals studied in pedigree 2035 clearly contain regulatory mutations in BRCA1. In her cDNA, the polymorphic site (A → G at base 3667) appeared heterozygous, whereas her genomic DNA revealed heterozygosity at this site (FIG. 11). A possible explanation for this observation is that the 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 are separated by as much as 3.5 kb in the BRCA1 transcript. The cDNA is heterozygous when her genomic DNA is heterozygous for the polymorphism. In individuals from other families and in non-haplotype carriers in family 2035, these polymorphic sites are observed as heterozygous in the cDNA, which indicates that amplification from the cDNA does not favor one allele. It shows that. This analysis shows that a BRCA1 mutation in family 2035 prevents transcription or causes instability or abnormal splicing of the BRCA1 transcript.

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

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

  In order to eliminate the possibility that the mutations were simply a common polymorphism in the population, ASO for each mutation was used to screen a set of normal DNA samples. Gene frequency estimates in Caucasians were based on random samples from the Utah population. Gene frequency estimates in African-American are from M. african-American and 20 newly born Utah African-Americans used in her linkage study. 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 candidate disease predisposing mutations: 1) co-segregation of diseased mutant alleles, and 2) absence of mutant alleles in controls ( This indicates a low gene frequency in the general population)

Phenotypic expression of BRCA1 mutation The effect of the mutation on the BRCA1 protein correlated with differences in observed phenotypic expression in the BRCA1 family. Most BRCA1 families have a moderately increased risk of ovarian cancer compared to that for breast cancer (Easton et al., 1993), with a smaller subset having a higher risk of ovarian cancer. Of the four families with BRCA1 mutations detected, three 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 high likelihood of ovarian cancer and has a later mean age of diagnosis of breast cancer cases than other families (Goldgar et al., 1994). This difference in onset age is due to a bias in certainty in the smaller, more penetrant family, or it reflects tissue-specific differences in the behavior of the BRCA1 mutation Will. The other three families that isolate known BRCA1 mutations, on average, have one ovarian cancer per 10 cases of breast cancer, but have a high population of breast cancer cases diagnosed in their late 20s or early 30s Have. It is worth noting the family 1910 with the frameshift mutation. This is because 3 out of 4 affected individuals have bilateral breast cancer and in each case the second tumor was diagnosed within 1 person from the first appearance. A family 2035 that isolates a potential regulatory BRCA1 mutation would be expected to have a dramatic phenotype. 80% of breast cancer cases in this family occur before age 50. This number is as high as any in the set, suggesting 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 carries at least one mutation that lived up to 80 years of age without developing malignancy Including women. It would be of utmost importance in the study to subsequently locate other genetic or environmental factors that could reduce the effects of BRCA1 mutations.

  In 4 of 8 putative BRCA1-linked families, no potential disease predisposing mutations were found. Three of these four have LOD scores for BRCA1-linked markers of less than 0.55. Thus, these families do not appear to segregate the BRCA1 disease predisposing allele in practice. Alternatively, mutations in these four families are likely to be present in the region of BRCA1, which affects, for example, the level of transcripts and thus keeps detection away.

The role of BRCA1 in cancer Most tumor suppressor genes currently identified give rise to non-functional or reduced function protein products. Most of the TP53 mutations are missense; some of these 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 found for several adenoma polyvosis coli (APC) alleles that produce truncated molecules (Su et al., 1993), and Wilms' tumors that alter protein DNA binding 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 dominant negative or nonfunctional proteins. The regulatory mutation putative in family 2035 cannot be dominant negative; rather, this mutation appears to cause a reduction or complete loss of BRCA1 expression from the affected allele.

The BRCA1 protein contains a C ₃ HC ₄ zinc-finger domain similar to that found in many DNA binding proteins, deeply associated with zinc-dependent binding to nucleic acids. The first 180 amino acids of BRCA1 contain 5 more basic residues than acidic residues. In contrast, the rest of the molecule is very acidic with a net excess of 70 acidic residues. 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 transactivated “acid blob” domain. Interestingly, another familial tumor suppressor gene, WT1, also contains a zinc-finger motif (Haber et al., 1990). Many cancer predisposing mutations in WT1 alter the zinc-finger domain (Little et al., 1993; Haber et al., 1990; Little et al., 1992). WT1 encodes an alternative splicing of transcription factors and exons that encode portions of the zinc-finger domain that modify the DNA binding properties of WT1 (Bickmore et al., 1992). Several alternatively spliced forms of WT1 mRNA create molecules that act as transcriptional repressors (Drummond et al., 1994). Some BRCA1 splicing variants modify the zinc-finger motif, giving rise to the possibility that a similar regulatory mechanism can be applied to BRCA1 as occurs in WT1.

Example 9
Tumor Analysis for BRCA1 Mutations To focus on analysis for tumors most likely to contain BRCA1 mutations, primary and ovarian cancers were typed for LOH in the BRCA1 region. Access LOH: D17S1323 and D17S855 (which are within the gene of BRCA1) and D17S1327 (which is approximately 100 kb terminal to BRCA1) using three highly polymorphic simple tandem repeat markers did. The total LOH frequency in the informational case (ie germline is heterozygous) is 32/72 (44%) for breast cancer and 12/21 (57%) for ovarian cancer, and the LOH frequency in the region 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 and intron / exon border sequences of the gene were screened in this tumor set by a combination of single strand conformation analysis (SSCA) and direct sequencing.

  A total of 6 mutations (of which 2 were identical) were found, 1 in ovarian tumors, 4 in breast tumors, and 1 in male unaffected haplotype carriers (Table 13). One mutation, Glu1541Ter, introduced a stop codon at the carboxyl terminus that would result in a truncated protein that had lost 323 amino acids. In addition, two missense mutations have been identified. These are Ala1708Glu and Met1775Arg, including substitution 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 5 mutations represent a BRCA1-sensitive allele.
(I) all mutations are present in the germline;
(Ii) All are absent from the appropriate control population, suggesting that they are not common polymorphs;
(Iii) This also applies to tumors from patients belonging to families that isolate BRCA1-sensitive alleles (Smith et al., 1992; Kelsel et al., 1993) (if the mutation represents a neutral polymorphism, they are Each mutant allele is retained in the tumor;
(Iv) The age of onset in the four breast cancer cases with mutations varies between 24 and 42 years, consistent with the early age of onset of breast cancer in individuals with BRCA1; Diagnosed, which falls into the youngest 13% of all ovarian cancers; and finally,
(V) Three of the five cases have a positive family history of breast and ovarian cancer found in their medical records dating back in the past, even though the tumor set was not selected for this criterion.

  BT106 was diagnosed with breast cancer at age 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. The patient had a sister who died of breast cancer at age 34, another sister who died of lymphoma, and a brother who died of lung cancer. Her mutation (Met1775Arg) has been previously detected in family 2099 (African-American that isolates the BRCA1-sensitive allele and is absent in the African-American and Caucasian controls). In our knowledge, patient MC44 is not related to family 2099. Detection of rare mutant alleles (once in the BRCA1 family and once in the unrelated early-onset breast cancer case) suggests that the Met1775Arg change may be a common disease predisposition in African-American. Taken together, these observations show that all four BRCA1 mutations in the tumor represent sensitive alleles; somatic mutations were not detected in the analyzed samples.

  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) Several BRCA1 mutations were lost in the coding sequence according to our screening procedure; (ii) BRCA1 somatic mutations were primarily outside the coding exon. And (iii) the LOH event at 17q does not reflect a BRCA1 somatic mutation.

  If somatic BRCA1 mutations are truly rare in breast and ovarian cancer, this will have a strong close relationship with the biology of BRCA1. The apparent lack of somatic BRCA1 mutations implies that there may be some fundamental differences in the development of tumors in genetically predisposed BRCA1 carriers compared to tumors in the general population To do. For example, mutations in BRCA1 may only have an effect on tumor formation at certain early stages in breast cancer and ovarian pathogenesis. This probability is consistent with the primary function for BRCA1 in premenopausal breast cancer. Such a model for the role of BRCA1 in breast and ovarian cancer predicts an interaction between reproductive hormones and BRCA1 function. However, clinical or pathological differences (other than the onset age) of familial-sporadic breast cancer and ovarian cancer have not been described (Lynch et al., 1990). On the other hand, recent findings of increased TP53 mutations and micro-incidental instability in breast cancer from patients with a family history of breast cancer (Glebov et al., 1994) have revealed some of the genetically viable tumors in predisposed individuals Will reflect the difference. Now, the involvement of BRCA1 in this phenomenon can be handled 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 convenient target for mutations in sporadic tumors. . Since single element mutations in the genetic pathway are generally sufficient to disrupt the pathway, BRCA1 will mutate at a much lower rate 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 A mammalian expression vector containing the BRCA1 cDNA is constructed and transfected into appropriate breast cancer cells that have damage in the gene. Wild type BRCA1 cDNA as well as modified BRCA1 cDNA are used. The modified BRCA1 cDNA can be obtained from the modified BRCA1 allele or can be produced as described below. Phenotypic reversion in culture (eg, cell morphology, doubling time, anchorage dependent growth) and phenotypic 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 missing and missense mutants (by single base pair substitution and cluster charge → alanine scanning mutagenesis at individual codons). The mutant is used in biological, biochemical and biophysical studies.

Mechanism studies The ability of BRCA1 protein to bind to known and unknown DNA sequences is examined. Its ability to transactivate the promoter is analyzed by a transient reporter expression system in mammalian cells. Any functional partner is found and identified using conventional techniques such as particle-capture and yeast two-hybrid systems. 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. Also used to make protein molecules. These studies facilitate drug design derived from the structure.

Example 11
Two-step assay to detect the presence of BRCA1 in a sample Patient samples are processed according to the method disclosed by Antonio Arakis 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 GS Gene linker (Bio-Rad). The BRCA1 probe corresponding to nucleotides 3631-3930 of SEQ ID NO: 1 is subcloned into pTZ18U. Phagemid is transformed into E. coli MV1190 infected with M13KO7 helper phage (Bio-Rad, Richmond, Calif.). Single stranded DNA is isolated by standard techniques (see Sambrook et al., 1989).

The blot is 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 by 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 washes are two 30 min washes in 5% SDS, 40 mM NaPO ₄ at 65 ° C., followed by two 30 min 1% SDS, 40 mM NaPO ₄ washes at 65 ° C. Become.

  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 at room temperature for 30-60 minutes, and rinsed in PBS for 5 minutes. The blot was then analyzed with 6M urea, 0.3M.

Preincubate for 5-10 minutes in a shaking water bath at 45 ° C. with hybridization buffer consisting of NaCl and 5 × Denhardt's solution (see Sambrook et al., 1989). Nucleotide sequence complementary to the universal primer site (UP-AP, Bio-Rad) of 50-75 μl / cm ² fresh hybridization buffer + covalently crosslinked oligonucleotide-alkaline phosphatase, with buffer removed Replace 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 citrated saline (SSC), 0 Incubate as a single wash in 0.1% SDS and 1 × SSC, 0.1% Triton X-100. Rinse the blot for 10 minutes with 1 × SSC at room temperature.

0.1M diethanolamine, 1 mM MgCl _2, 0.02% sodium azide, while shaking in the substrate buffer consisting of pH 10.0, to Inkyobeto 10 minutes at room temperature the blots. Individual with substrate buffer and 0.2 mM AMPPD (3- (2′-spiroadamantane) -4-methoxy-4- (3′-phosphoryloxy) phenyl-1,2-dioxetane, disodium salt, Bio-Rad) In a heat-sealable bag. After 20 minutes incubation at room temperature with shaking, excess AMPPD solution is removed. The blot is exposed to X-rays overnight. A positive band indicates the presence of BRCA1.

Example 12
Production of polyclonal antibody against BRCA1 A segment of the BRCA1 coding sequence was expressed as a fusion protein in E. coli. Overproduced protein was purified by gel elution and used to immunize rabbits and mice using techniques similar to those described by Harlow and Lane, 1988. This approach has been shown to generate Abs against a variety of other proteins (eg, Kraemer et al., 1993).

  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 includes the amino acids 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. The 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 3 week intervals (first with 100 μg of immunogen in Freund's incomplete adjuvant followed by 100 μg of immunogen in PBS). Thereafter, serum-containing antibodies are collected for 2 weeks.

  This procedure is repeated to obtain antibodies against mutant forms of the BRCA1 gene. In combination with antibodies against 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
Production of monoclonal antibody specific for BRCA1 A monoclonal antibody is produced according to the following protocol. As is well known, mice are immunized with an immunogen consisting of intact BRCA1 or BRCA1 peptide (wild type or mutant) conjugated to keyhole limpet hemocyanin using glutaraldehyde or EDC.

  The immunogen is mixed with an adjuvant. Each mouse receives 4 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 showing the presence of antibodies to the immunogen are selected for hybridoma production.

Spleens are removed from immunized mice and single cell suspensions are 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 in 96-well tissue culture plates at a density of 2 × 10 ⁵ cells / well. 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 proteins. Cells in positive wells are expanded and subcloned to establish and confirm monoclonal antibodies.

  Clones with the desired specificity are grown and grown as ascites in mice or hollow fibers to obtain a sufficient amount of antibody for characterization and assay development.

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

  The amount of bound label (which is proportional to the amount of BRCA1 peptide / protein present in the sample) is quantified. Separate assays are performed using a monoclonal antibody specific for wild-type BRCA1 as well as 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 may be embodied in the form of various embodiments, only a few of which are 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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Wolff et al. (1990). Science 247: 1465-1468 Wolff et al. (1991). BioTechniques 11: 474-485
Wooster, R.A. (1994). Science 265: 2088 Wu et al. (1989a). Genomics 4: 560-569
Wu et al. (1989b). J. et al. Biol. Chem 264: 16985-16987
Wu et al. (1991). J. et al. Biol. Chem. 266: 14338-14342
Zenke et al. (19901). Proc. Natl. Acad. Sci. USA 87: 3655-3659

List of Patents and Patent Applications US Pat. No. 3,817,837 US Pat. No. 3,850,752 US Pat. No. 3,939,350 US Pat. No. 3,996,345 US Pat. No. 4,275,149 US Patent No. 4,277,437 US Patent No. 4,366,241 US Patent No. 4,376,110 US Patent No. 4,486,530 US Patent No. 4,683,195 US Patent No. 4 U.S. Pat. No. 4,816,567 U.S. Pat. No. 4,868,105 U.S. Pat. No. 5,252,479 EP-A 225,807 EP-A-0332435 Geysen, H. 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.
Goldagar, 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 GTGTGTGTGACACACACAC 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 gene locus near BRCA1 determined by "Chromosome 17 study group". Figure 1 is reproduced from Fain, 1992. It is a schematic map of YAC demarcating Mfd15-Mfd188 area | region part. It is a schematic map of STS, P1 and BAC in the BRCA1 region. 3 is a schematic map of human chromosome 17. The directly related region containing BRCA1 has been expanded to show the relative position of the two previously identified genes, CA125 and RNU2, and BRCA1 extends across the marker D17S855. The sequence of the BRCA1 zinc-finger domain with the three other zinc-finger domains best recorded in the Smith-Waterman sequence is shown. 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 ring-finger motif associated with a zinc-finger. RFP1 encodes a predicted transcription factor that is the N-terminal domain of the RET oncogene product. The underlined portion contains a C3HC4 common zinc-finger sequence indicating the position of cysteine and one histidine that form the zinc ion binding pocket. FIG. 5 is a BRCA1 mRNA diagram showing the location of introns generated by alternative splicing and the BRCA1 mRNA variant. Intron positions are indicated by “black triangles” and exons are numbered below the line representing the cDNA. The uppermost cDNA is a composition used for preparing the peptide sequence of BRCA1. Another form identified as a cDNA clone or hybrid selected clone is shown below. The tissue expression pattern of BRCA1 is shown. The blot is 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 to 930 of BRCA1 was used. Note that both the breast and ovary are heterozygous tissues and the percentage of associated epithelial cells can vary. The molecular weight marker is kb. FIG. 4 is a diagram of the BRCA1 5 ′ untranslated region + translated region start point showing the location of the intron generated by alternative splicing and the deformation of BRCA1 mRNA. The position of the intron is indicated by a broken line. Six alternative splicing configurations are shown. The nonsense mutation in Kindred2082 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 halotype. The C to T mutation results in a stop codon and creates a site for the restriction enzyme AvrII. The PCR amplification product is cleaved with this enzyme. The carrier is heterozygous for the site and therefore exhibits three bands. Non-carriers remain uncut. Figure 2 shows mutation and cosegregation analysis in a BRCA1 family. Individual carriers are shown as “black circles” and “black squares” in the family tree. Frameshift mutation in Kindred1910. The first three lanes are control, non-carrier samples. Lanes labeled with 1-3 contain sequences from carrier individuals. Lane 4 contains DNA from a family member who does not carry the BRCA1 mutation. The rhombus is used to prevent the family from being identified. Further frame shifts resulting from C are evident in the lanes labeled 1, 2 and 3. Figure 2 shows mutation and cosegregation analysis in a BRCA1 family. Individual carriers are shown as “black circles” and “black squares” in the family tree. There are regulatory mutations inferred in Kindred 2035. Carrier and non-carrier ASO analyzes of two different polymorphs (PM1 and PM7) were examined for heterozygosity in the germline and compared to lymphocyte mRNA heterozygosity. 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. A dark spot indicates that a particular allele is present in the sample. The first three lanes of PM7 represent the three genotypes of the general population. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type. The genomic sequence of BRCA1 is shown. Lower case letters indicate intron sequences, while upper case letters indicate exon sequences. The unclear spacing within the intron is indicated by vvvvvvvvvvvvv. Known polymorphic sites are shown as underlined and meat bold type.

Claims (12)

A total that extends to the genetic marker D17S855 on human chromosome 17q21 under stringent hybridization conditions at temperatures above 45 ° C. and salt concentrations below 200 mM, the N-terminal amino acid is methionine and the C-terminal amino acid is tyrosine An isolated nucleic acid that hybridizes to a BRCA1 cDNA or mRNA derived from a BRCA1 gene defined as a gene encoding 1863 amino acids. The isolated nucleic acid of claim 1 that hybridizes to a probe comprising the sequence of nucleotide positions 3631 to 3930 of SEQ ID NO: 1. Selected from the group consisting of SEQ ID NOs: 35, 38, 41, 42, 47, 57, 62, 66, 67, 72 and 81 under stringent hybridization conditions at temperatures above 45 ° C. and salt concentrations below 200 mM The isolated nucleic acid according to claim 1, which hybridizes to a probe comprising a nucleotide sequence. The isolated nucleic acid according to claim 1 or 3, comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35, 38, 41, 42, 47, 57, 62, 66, 67, 72 and 81. 5. The isolated nucleic acid of claim 1, 3 or 4 encoding BRCA1, an allelic variant thereof or a naturally occurring BRCA1. 6. The isolated nucleic acid according to claim 5, wherein the BRCA1 variant is a BRCA1 protein truncated at the carboxy terminus. A method for identifying a mutant BRCA1 nucleotide sequence encoding a BRCA1 mutant as defined in claim 6 in a tissue sample comprising:
(a) amplifying at least a portion of the coding sequence of the BRCA1 gene in the tissue sample;
(B) determining whether the BRCA1 gene encodes a short amino acid sequence caused by a frameshift mutation or an early stop codon. A method for determining a predisposition for breast cancer and ovarian cancer in a tissue sample derived from a human, comprising determining whether there is a germline change in the BRCA1 gene of claim 1 in the tissue sample, Wherein the change comprises encoding a protein truncated at the carboxy terminus of the altered gene, and the presence of the change comprises indicating the predisposition to cancer. 9. The method of claim 8, wherein the BRCA1 gene hybridizes to a probe comprising a sequence at nucleotide positions 3631 to 3930 of SEQ ID NO: 1. 9. The method of claim 8, wherein the change is selected from the group consisting of a frameshift mutation or an early stop codon. 11. The method according to claim 8 or 10, wherein the change can be detected by a protein tip cleavage assay. The determining step is
(A) amplifying at least part of the coding sequence of the BRCA1 gene;
(B) determining the sequence of the amplified DNA;
(C) determining the total number of amino acid residues encoded by the amplified DNA or the size of the polypeptide encoded by the amplified DNA;
12. The method according to claim 8, 10 or 11, wherein the result that the BRCA1 gene encodes a truncated protein indicates the disease predisposition to cancer.

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