Patent Publication Number: US-2002009738-A1

Title: Methods, compositions and kits for the detection and monitoring of breast cancer

Description:
REFERENCE TO RELATED APPLICATIONS  
     [0001] This application claims the benefit of U.S. Provisional Application No. 60/194,241, filed Apr. 3, 2000; U.S. Provisional Application No. 60/219,862, filed Jul. 20, 2000; U.S. Provisional Application No. 60/221,300, filed Jul. 27, 2000; and U.S. Provisional Application No. 60/256,592, filed Dec. 18, 2000, each of which applications are incorporated herein by reference in their entirety. 
    
    
     GOVERNMENTAL SUPPORT  
     [0002] This work was supported in part by Grants CA-75794 and CA-80518 from the National Cancer Institute. The government may have certain rights in the invention. 
    
    
     
       TECHNICAL FIELD OF THE INVENTION  
       [0003] The present invention relates generally to the field of cancer diagnostics. More specifically, the present invention relates to methods, compositions and kits for the detection of cancer that employ oligonucleotide hybridization and/or amplification to simultaneously detect two or more tissue-specific polynucleotides in a biological sample suspected of containing cancer cells.  
       BACKGROUND OF THE INVENTION  
       [0004] Cancer remains a significant health problem throughout the world. The failure of conventional cancer treatment regimens can commonly be attributed, in part, to delayed disease diagnosis. Although significant advances have been made in the area of cancer diagnosis, there still remains a need for improved detection methodologies that permit early, reliable and sensitive determination of the presence of cancer cells.  
       [0005] Breast cancer is second only to lung cancer in mortality among women in the U.S., affecting more than 180,000 women each year and resulting in approximately 40,000-50,000 deaths annually. For women in North America, the life-time odds of getting breast cancer are one in eight.  
       [0006] Management of the disease currently relies on a combination of early diagnosis (through routine breast screening procedures) and aggressive treatment, which may include one or more of a variety of treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. The course of treatment for a particular breast cancer is often selected based on a variety of prognostic parameters, including analysis of specific tumor markers. See, e.g., Porter-Jordan et al.,  Breast Cancer  8:73-100 (1994). The use of established markers often leads, however, to a result that is difficult to interpret; and the high mortality observed in breast cancer patients indicates that improvements are needed in the diagnosis of the disease.  
       [0007] The recent introduction of immunotherapeutic approaches to breast cancer treatment which are targeted to Her2/neu have provided significant motivation to identify additional breast cancer specific genes as targets for therapeutic antibodies and T-cell vaccines as well as for diagnosis of the disease. To this end, mammaglobin, has been identified as one of the most breast-specific genes discovered to date, being expressed in approximately 70-80% of breast cancers. Because of its highly tissue-specific distribution, detection of mammaglobin gene expression has been used to identify micrometastatic lesions in lymph node tissues and, more recently, to detect circulating breast cancer cells in peripheral blood of breast cancer patients with known primary and metastatic lesions.  
       [0008] Mammaglobin is a homologue of a rabbit uteroglobin and the rat steroid binding protein subunit C3 and is a low molecular weight protein that is highly glycosylated. Watson et al.,  Cancer Res.  56:860-5 (1996); Watson et al.,  Cancer Res.  59:3028-3031 (1999); Watson et al.,  Oncogene  16:817-24 (1998). In contrast to its homologs, mammaglobin has been reported to be breast specific and overexpression has been described in breast tumor biopsies (23%), primary and metastatic breast tumors (˜75%) with reports of the detection of mammaglobin mRNA expression in 91% of lymph nodes from metastatic breast cancer patients. Leygue et al.,  J. Pathol.  189:28-33 (1999) and Min et al.,  Cancer Res.  58:4581-4584 (1998).  
       [0009] Since mammaglobin gene expression is not a universal feature of breast cancer, the detection of this gene alone may be insufficient to permit the reliable detection of all breast cancers. Accordingly, what is needed in the art is a methodology that employs the detection of two or more breast cancer specific genes in order to improve the sensitivity and reliability of detection of micrometastases, for example, in lymph nodes and bone marrow and/or for recognition of anchorage-independent cells in the peripheral circulation.  
       [0010] The present invention achieves these and other related objectives by providing methods that are useful for the identification of tissue-specific polynucleotides, in particular tumor-specific polynucleotides, as well as methods, compositions and kits for the detection and monitoring of cancer cells in a patient afflicted with the disease.  
       SUMMARY OF THE INVENTION  
       [0011] By certain embodiments, the present invention provides methods for identifying one or more tissue-specific polynucleotides which methods comprise the steps of: (a) performing a genetic subtraction to identify a pool of polynucleotides from a tissue of interest; (b) performing a DNA microarray analysis to identify a first subset of said pool of polynucleotides of interest wherein each member polynucleotide of said first subset is at least two-fold over-expressed in said tissue of interest as compared to a control tissue; and (c) performing a quantitative polymerase chain reaction analysis on polynucleotides within said first subset to identify a second subset of polynucleotides that are at least two-fold over-expressed as compared to the control tissue. Preferred genetic subtractions are selected from the group consisting of differential display and cDNA subtraction and are described in further detail herein below.  
       [0012] Alternate embodiments of the present invention provide methods of identifying a subset of polynucleotides showing concordant and/or complementary tissue-specific expression profiles in a tissue of interest. Such methods comprise the steps of, (a) performing an expression analysis selected from the group consisting of DNA microarray and quantitative PCR to identify a first polynucleotides that is at least two-fold over-expressed in a tissue of interest as compared to a control tissue; and (b) performing an expression analysis selected from the group consisting of DNA microarray and quantitative PCR to identify a first polynucleotides that is at least two-fold over-expressed in a tissue of interest as compared to a control tissue.  
       [0013] Further embodiments of the present invention provide methods for detecting the presence of a cancer cell in a patient. Such methods comprise the steps of: (a) obtaining a biological sample from the patient; (b) contacting the biological sample with a first oligonucleotide pair wherein the members of the first oligonucleotide pair hybridize, under moderately stringent conditions, to a first polynucleotide and the complement thereof, respectively; (c) contacting the biological sample with a second oligonucleotide pair wherein the members of the second oligonucleotide pair hybridize, under moderately stringent conditions, to a second polynucleotide and the complement thereof, respectively and wherein the first polynucleotide is unrelated in nucleotide sequence to the second polynucleotide; (d) amplifying the first polynucleotide and the second polynucleotide; and (e) detecting the amplified first polynucleotide and the amplified second polynucleotide; wherein the presence of the amplified first polynucleotide or amplified second polynucleotide indicates the presence of a cancer cell in the patient.  
       [0014] By some embodiments, detection of the amplified first and/or second polynucleotides may be preceded by a fractionation step such as, for example, gel electrophoresis. Alternatively or additionally, detection of the amplified first and/or second polynucleotides may be achieved by hybridization of a labeled oligonucleotide probe that hybridizes specifically, under moderately stringent conditions, to the first or second polynucleotide. Oligonucleotide labeling may be achieved by incorporating a radiolabeled nucleotide or by incorporating a fluorescent label.  
       [0015] In certain preferred embodiments, cells of a specific tissue type may be enriched from the biological sample prior to the steps of detection. Enrichment may be achieved by a methodology selected from the group consisting of cell capture and cell depletion. Exemplary cell capture methods include immunocapture and comprise the steps of: (a) adsorbing an antibody to a tissue-specific cell surface to cells said biological sample; (b) separating the antibody adsorbed tissue-specific cells from the remainder of the biological sample. Exemplary cell depletion may be achieved by cross-linking red cells and white cells followed by a subsequent fractionation step to remove the cross-linked cells.  
       [0016] Alternative embodiments of the present invention provide methods for determining the presence or absence of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from the patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a breast tumor protein; (b) detecting in the sample a level of a polynucleotide (such as, for example, mRNA) that hybridizes to the oligonucleotide; and (c) comparing the level of polynucleotide that hybridizes to the oligonucleotide with a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient. Within certain embodiments, the amount of mRNA is detected via polymerase chain reaction using, for example, at least one oligonucleotide primer that hybridizes to a polynucleotide encoding a polypeptide as recited above, or a complement of such a polynucleotide. Within other embodiments, the amount of mRNA is detected using a hybridization technique, employing an oligonucleotide probe that hybridizes to a polynucleotide that encodes a polypeptide as recited above, or a complement of such a polynucleotide.  
       [0017] In related aspects, methods are provided for monitoring the progression of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide that encodes a breast tumor protein; (b) detecting in the sample an amount of a polynucleotide that hybridizes to the oligonucleotide; (c) repeating steps (a) and (b) using a biological sample obtained from the patient at a subsequent point in time; and (d) comparing the amount of polynucleotide detected in step (c) with the amount detected in step (b) and therefrom monitoring the progression of the cancer in the patient.  
       [0018] Certain embodiments of the present invention provide that the step of amplifying said first polynucleotide and said second polynucleotide is achieved by the polymerase chain reaction (PCR).  
       [0019] Within certain embodiments, the cancer cell to be detected may be selected from the group consisting of prostate cancer, breast cancer, colon cancer, ovarian cancer, lung cancer head &amp; neck cancer, lymphoma, leukemia, melanoma, liver cancer, gastric cancer, kidney cancer, bladder cancer, pancreatic cancer and endometrial cancer. Still further embodiments of the present invention provide that the biological sample is selected from the group consisting of blood, a lymph node and bone marrow. The lymph node may be a sentinel lymph node.  
       [0020] Within specific embodiments of present invention it is provided that the first polynucleotide is selected from the group consisting of mammaglobin, lipophilin B, GABAπ (B899P), B726P, B511S, B533S, B305D and B311D. Other embodiments provide that the second polynucleotide is selected from the group consisting of mammaglobin, lipophilin B, GABAπ (B899P), B726P, B511S, B533S, B305D and B311D.  
       [0021] Alternate embodiments of the present invention provide methods for detecting the presence or absence of a cancer in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with a first oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of mammaglobin and lipophilin B; (b) contacting the biological sample with a second oligonucleotide that hybridizes to a polynucleotide sequence selected from the group consisting of GABAπ (B899P), B726P, B511S, B533S, B305D and B311D; (c) detecting in the sample an amount of a polynucleotide that hybridizes to at least one of the oligonucleotides; and (d) comparing the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.  
       [0022] According to certain embodiments, oligonucleotides may be selected from those disclosed herein such as those presented in SEQ ID Nos:33-72. By other embodiments, the amount of polynucleotide that hybridizes to the oligonucleotide is determined using a polymerase chain reaction. Alternatively, the amount of polynucleotide that hybridizes to the oligonucleotide may be determined using a hybridization assay.  
       [0023] Still other embodiments of the present invention provide methods for determining the presence or absence of a cancer cell in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with a first oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of a polynucleotide depicted in SEQ ID NO:73 and SEQ ID NO:74 or complement thereof; (b) contacting the biological sample with a second oligonucleotide that hybridizes to a polynucleotide depicted in SEQ ID NO:75 or complement thereof; (c) contacting the biological sample with a third oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of a polynucleotide depicted in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 or complement thereof; (d) contacting the biological sample with a fourth oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of a polynucleotide depicted in SEQ ID NO:11 or complement thereof; (e) contacting the biological sample with a fifth oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of a polynucleotide depicted in SEQ ID NO:13, 15 and 17 or complement thereof; (f) contacting the biological sample with a sixth oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of a polynucleotide depicted in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24 or complement thereof; (g) contacting the biological sample with a seventh oligonucleotide that hybridizes to a polynucleotide depicted in SEQ ID NO:30 or complement thereof; (h) contacting the biological sample with an eighth oligonucleotide that hybridizes to a polynucleotide depicted in SEQ ID NO:32 or complement thereof; (i) contacting the biological sample with a ninth oligonucleotide that hybridizes to a polynucleotide depicted in SEQ ID NO:76 or complement thereof; (j) detecting in the sample a hybridized oligonucleotide of any one of steps (a) through (i); and (j) comparing the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, wherein the presence of a hybridized oligonucleotide in any one of steps (a) through (i) in excess of the pre-determined cut-off value indicates the presence of a cancer cell in the biological sample of said patient.  
       [0024] Other related embodiments of the present invention provide methods for determining the presence or absence of a cancer cell in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with a first oligonucleotide and a second oligonucleotide wherein said first and second oligonucleotides hybridize under moderately stringent conditions to a first and a second polynucleotide selected from the group selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:76 and wherein said first polynucleotide is unrelated structurally to said second polynucleotide; (b) detecting in the sample said first and said second hybridized oligonucleotides; and (c) comparing the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, wherein the presence of a hybridized first oligonucleotide or a hybridized second oligonucleotide in excess of the pre-determined cut-off value indicates the presence of a cancer cell in the biological sample of said patient.  
       [0025] Other related embodiments of the present invention provide methods for determining the presence or absence of a cancer cell in a patient, comprising the steps of: (a) contacting a biological sample obtained from a patient with a first oligonucleotide and a second oligonucleotide wherein said first and second oligonucleotides hybridize under moderately stringent conditions to a first and a second polynucleotide are both tissue-specific polynucleotides of the cancer to be detected and wherein said first polynucleotide is unrelated structurally to said second polynucleotide; (b) detecting in the sample said first and said second hybridized oligonucleotides; and (c) comparing the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, wherein the presence of a hybridized first oligonucleotide or a hybridized second oligonucleotide in excess of the pre-determined cut-off value indicates the presence of a cancer cell in the biological sample of said patient.  
       [0026] In other related aspects, the present invention further provides compositions useful in the methods disclosed herein. Exemplary compositions comprise two or more oligonucleotide primer pairs each one of which specifically hybridizes to a distinct polynucleotide. Exemplary oligonucleotide primers suitable for compositions of the present invention are disclosed herein by SEQ ID NOs: 33-71. Exemplary polynucleotides suitable for compositions of the present invention are disclosed in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:76.  
       [0027] The present invention also provides kits that are suitable for performing the detection methods of the present invention. Exemplary kits comprise oligonucleotide primer pairs each one of which specifically hybridizes to a distinct polynucleotide. Within certain embodiments, kits according to the present invention may also comprise a nucleic acid polymerase and suitable buffer. Exemplary oligonucleotide primers suitable for kits of the present invention are disclosed herein by SEQ ID NOs: 33-71. Exemplary polynucleotides suitable for kits of the present invention are disclosed in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:76.  
       [0028] These and other aspects of the present invention will become apparent upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.  
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS  
     [0029]FIG. 1 shows the mRNA expression profiles for B311D, B533S and B726P as determined using quantitative PCR (Taqman™). Abbreviations: B.T.: Breast tumor; B.M.: Bone marrow; B.R.: Breast reduction.  
     [0030]FIG. 2 shows the relationship of B533S expression to pathological stage of tumor. Tissues from normal breast (8), benign breast disorders (3), and breast tumors stage I (5), stage II (6), stage III (7), stage IV (3) and metastases (1 lymph node and 3 pleural effusions) were tested in real-time PCR. The data is expressed as the mean copies/ng β-actin for each group tested and the line is the calculated trend line.  
     [0031]FIGS. 3A and 3B show the gene complementation of B305D C-form, B726P, GABAπ and mammaglobin in metastases and primary tumors, respectively. The cut-off for each of the genes was 6.57, 1.65, 4.58 and 3.56 copies/ng β-Actin based on the mean of the negative normal tissues plus 3 standard deviations.  
     [0032]FIG. 4 shows the full-length cDNA sequence for mammaglobin.  
     [0033]FIG. 5 shows the determined cDNA sequence of the open reading frame encoding a mammaglobin recombinant polypeptide expressed in  E. coli.    
     [0034]FIG. 6 shows the full-length cDNA sequence for GABAπ.  
     [0035]FIG. 7 shows the mRNA expression levels for mammaglobin, GABAπ, B305D (C form) and B726P in breast tumor and normal samples determined using real-time PCR and the SYBR detection system. Abbreviations: BT: Breast tumor; BR: Breast reduction; A. PBMC: Activated peripheral blood mononuclear cells; R. PBMC: resting PBMC; T. Gland: Thyroid gland; S. Cord: Spinal Cord; A. Gland: Adrenal gland; B. Marrow: Bone marrow; S. Muscle: Skeletal muscle.  
     [0036]FIG. 8 is a bar graph showing a comparison between the LipophilinB alone and the LipophilinB-B899P-B305D-C-B726 multiplex assays tested on a panel of breast tumor samples. Abbreviations: BT: Breast tumor; BR: Breast reduction; SCID: severe combined immunodeficiency.  
     [0037]FIG. 9 is a gel showing the unique band length of four amplification products of tumor genes of interest (mammaglobin, B305D, B899P, B726P) tested in a multiplex Real-time PCR assay.  
     [0038]FIG. 10 shows a comparison of a multiplex assay using intron-exon border spanning primers (bottom panel) and those using non-optimized primers (top panel), to detect breast cancer cells in a panel of lymph node tissues. 
    
    
     [0039] SEQ ID NO:1 is the determined cDNA sequence for a first splice variant of B305D isoform A.  
     [0040] SEQ ID NO:2 is the amino acid sequence encoded by the sequence of SEQ ID NO:1.  
     [0041] SEQ ID NO:3 is the determined cDNA sequence for a second splice variant of B305D isoform A.  
     [0042] SEQ ID NO:4 is the amino acid sequence encoded by the sequence of SEQ ID NO:3.  
     [0043] SEQ ID NO:5-7 are the determined cDNA sequences for three splice variants of B305D isoform C.  
     [0044] SEQ ID NO:8-10 are the amino acid sequences encoded by the sequence of SEQ ID NO:5-7, respectively.  
     [0045] SEQ ID NO:11 is the determined cDNA sequence for B311D.  
     [0046] SEQ ID NO:12 is the amino acid sequence encoded by the sequence of SEQ ID NO:11.  
     [0047] SEQ ID NO:13 is the determined cDNA sequence of a first splice variant of B726P.  
     [0048] SEQ ID NO:14 is the amino acid sequence encoded by the sequence of SEQ ID NO:13.  
     [0049] SEQ ID NO:15 is the determined cDNA sequence of a second splice variant of B726P.  
     [0050] SEQ ID NO:16 is the amino acid sequence encoded by the sequence of SEQ ID NO:15.  
     [0051] SEQ ID NO:17 is the determined cDNA sequence of a third splice variant of B726P.  
     [0052] SEQ ID NO:18 is the amino acid sequence encoded by the sequence of SEQ ID NO:17.  
     [0053] SEQ ID NO:19-24 are the determined cDNA sequences of further splice variants of B726P.  
     [0054] SEQ ID NO:25-29 are the amino acid sequences encoded by SEQ ID NO: 19-24, respectively.  
     [0055] SEQ ID NO:30 is the determined cDNA sequence for B511S.  
     [0056] SEQ ID NO:31 is the amino acid sequence encoded by SEQ ID NO:30.  
     [0057] SEQ ID NO:32 is the determined cDNA sequence for B533S.  
     [0058] SEQ ID NO:33 is the DNA sequence of Lipophilin B forward primer.  
     [0059] SEQ ID NO:34 is the DNA sequence of Lipophilin B reverse primer.  
     [0060] SEQ ID NO:35 is the DNA sequence of Lipophilin B probe.  
     [0061] SEQ ID NO:36 is the DNA sequence of GABA (B899P) forward primer.  
     [0062] SEQ ID NO:37 is the DNA sequence of GABA (B899P) reverse primer.  
     [0063] SEQ ID NO:38 is the DNA sequence of GABA (B899P) probe.  
     [0064] SEQ ID NO:39 is the DNA sequence of B305D (C form) forward primer.  
     [0065] SEQ ID NO:40 is the DNA sequence of B305D (C form) reverse primer.  
     [0066] SEQ ID NO:41 is the DNA sequence of B305D (C form) probe.  
     [0067] SEQ ID NO:42 is the DNA sequence of B726P forward primer.  
     [0068] SEQ ID NO:43 is the DNA sequence of B726P reverse primer.  
     [0069] SEQ ID NO:44 is the DNA sequence of B726P probe.  
     [0070] SEQ ID NO:45 is the DNA sequence of Actin forward primer.  
     [0071] SEQ ID NO:46 is the DNA sequence of Actin reverse primer.  
     [0072] SEQ ID NO:47 is the DNA sequence of Actin probe.  
     [0073] SEQ ID NO:48 is the DNA sequence of Mammaglobin forward primer.  
     [0074] SEQ ID NO:49 is the DNA sequence of Mammaglobin reverse primer.  
     [0075] SEQ ID NO:50 is the DNA sequence of Mammaglobin probe.  
     [0076] SEQ ID NO:51 is the DNA sequence of a second GABA (B899P) reverse primer.  
     [0077] SEQ ID NO:52 is the DNA sequence of a second B726P forward primer.  
     [0078] SEQ ID NO:53 is the DNA sequence of a GABA B899P-INT forward primer.  
     [0079] SEQ ID NO:54 is the DNA sequence of a GABA B899P-INT reverse primer.  
     [0080] SEQ ID NO:55 is the DNA sequence of a GABA B899P-INT Taqman probe.  
     [0081] SEQ ID NO:56 is the DNA sequence of a B305D-INT forward primer.  
     [0082] SEQ ID NO:57 is the DNA sequence of a B305D-INT reverse primer.  
     [0083] SEQ ID NO:58 is the DNA sequence of a B305D-INT Taqman probe.  
     [0084] SEQ ID NO:59 is the DNA sequence of a B726-INT forward primer.  
     [0085] SEQ ID NO:60 is the DNA sequence of a B726-INT reverse primer.  
     [0086] SEQ ID NO:61 is the DNA sequence of a B726-INT Taqman probe.  
     [0087] SEQ ID NO:62 is the DNA sequence of a GABA B899P Taqman probe.  
     [0088] SEQ ID NO:63 is the DNA sequence of a B311D forward primer.  
     [0089] SEQ ID NO:64 is the DNA sequence of a B311D reverse primer.  
     [0090] SEQ ID NO:65 is the DNA sequence of a B311D Taqman probe.  
     [0091] SEQ ID NO:66 is the DNA sequence of a B533S forward primer.  
     [0092] SEQ ID NO:67 is the DNA sequence of a B533S reverse primer.  
     [0093] SEQ ID NO:68 is the DNA sequence of a B533S Taqman probe.  
     [0094] SEQ ID NO:69 is the DNA sequence of a B511S forward primer.  
     [0095] SEQ ID NO:70 is the DNA sequence of a B511S reverse primer.  
     [0096] SEQ ID NO:71 is the DNA sequence of a B511S Taqman probe.  
     [0097] SEQ ID NO:72 is the DNA sequence of a GABAπ reverse primer.  
     [0098] SEQ ID NO:73 is the full-length cDNA sequence for mammaglobin.  
     [0099] SEQ ID NO:74 is the determined cDNA sequence of the open reading frame encoding a mammaglobin recombinant polypeptide expressed in  E. coli.    
     [0100] SEQ ID NO:75 is the full-length cDNA sequence for GABAπ.  
     [0101] SEQ ID NO:76 is the full-length cDNA sequence for lipophilin B.  
     [0102] SEQ ID NO:77 is the amino acid sequence encoded by the sequence of SEQ ID NO:76.  
     DETAILED DESCRIPTION OF THE INVENTION  
     [0103] As noted above, the present invention is directed generally to methods that are suitable for the identification of tissue-specific polynucleotides as well as to methods, compositions and kits that are suitable for the diagnosis and monitoring of cancer. While certain exemplary methods, compositions and kits disclosed herein are directed to the identification, detection and monitoring of breast cancer, in particular breast cancer-specific polynucleotides, it will be understood by those of skill in the art that the present invention is generally applicable to the identification, detection and monitoring of a wide variety of cancers, and the associated over-expressed polynucleotides, including, for example, prostate cancer, breast cancer, colon cancer, ovarian cancer, lung cancer, head &amp; neck cancer, lymphoma, leukemia, melanoma, liver cancer, gastric cancer, kidney cancer, bladder cancer, pancreatic cancer and endometrial cancer. Thus, it will be apparent that the present invention is not limited solely to the identification of breast cancer-specific polynucleotides or to the detection and monitoring of breast cancer.  
     [0104] Identification of Tissue-specific Polynucleotides  
     [0105] Certain embodiments of the present invention provide methods, compositions and kits for the detection of a cancer cell within a biological sample. These methods comprise the step of detecting one or more tissue-specific polynucleotide(s) from a patient&#39;s biological sample the over-expression of which polynucleotides indicates the presence of a cancer cell within the patient&#39;s biological sample. Accordingly, the present invention also provides methods that are suitable for the identification of tissue-specific polynucleotides. As used herein, the phrases “tissue-specific polynucleotides” or “tumor-specific polynucleotides” are meant to include all polynucleotides that are at least two-fold over-expressed as compared to one or more control tissues. As discussed in further detail herein below, over-expression of a given polynucleotide may be assessed, for example, by microarray and/or quantitative real-time polymerase chain reaction (Real-time PCR™) methodologies.  
     [0106] Exemplary methods for detecting tissue-specific polynucleotides may comprise the steps of: (a) performing a genetic subtraction to identify a pool of polynucleotides from a tissue of interest; (b) performing a DNA microarray analysis to identify a first subset of said pool of polynucleotides of interest wherein each member polynucleotide of said first subset is at least two-fold over-expressed in said tissue of interest as compared to a control tissue; and (c) performing a quantitative polymerase chain reaction analysis on polynucleotides within said first subset to identify a second subset of polynucleotides that are at least two-fold over-expressed as compared to said control tissue.  
     [0107] Polynucleotides Generally  
     [0108] As used herein, the term “polynucleotide” refers generally to either DNA or RNA molecules. Polynucleotides may be naturally occurring as normally found in a biological sample such as blood, serum, lymph node, bone marrow, sputum, urine and tumor biopsy samples. Alternatively, polynucleotides may be derived synthetically by, for example, a nucleic acid polymerization reaction. As will be recognized by the skilled artisan, polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.  
     [0109] Polynucleotides may comprise a native sequence (i.e. an endogenous sequence that encodes a tumor protein, such as a breast tumor protein, or a portion thereof) or may comprise a variant, or a biological or antigenic functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below. The term “variants” also encompasses homologous genes of xenogenic origin.  
     [0110] When comparing polynucleotide or polypeptide sequences, two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.  
     [0111] Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins—Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645  Methods in Enzymology  vol. 183, Academic Press, Inc., San Diego, Calif.; Higgins, D. G. and Sharp, P. M. (1989)  CABIOS  5:151-153; Myers, E. W. and Muller W. (1988)  CABIOS  4:11-17; Robinson, E. D. (1971)  Comb. Theor  11:105; Santou, N. Nes, M. (1987)  Mol. Biol. Evol.  4:406-425; Sneath, P. H. A. and Sokal, R. R. (1973)  Numerical Taxonomy—the Principles and Practice of Numerical Taxonomy,  Freeman Press, San Francisco, Calif.; Wilbur, W. J. and Lipman, D. J. (1983)  Proc. Natl. Acad., Sci. USA  80:726-730.  
     [0112] Alternatively, optimal alignment of sequences for comparison may be conducted by the local identity algorithm of Smith and Watennan (1981)  Add. APL. Math  2:482, by the identity alignment algorithm of Needleman and Wunsch (1970)  J. Mol. Biol.  48:443, by the search for similarity methods of Pearson and Lipman (1988)  Proc. Natl. Acad. Sci. USA  85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.  
     [0113] One preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977)  Nucl. Acids Res.  25:3389-3402 and Altschul et al. (1990)  J. Mol. Biol.  215:403-410, respectively. BLAST and BLAST 2.0 can be used, for example with the parameters described herein, to determine percent sequence identity for the polynucleotides and polypeptides of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In one illustrative example, cumulative scores can be calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always &gt;0) and N (penalty score for mismatching residues; always &lt;0). For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)  Proc. Natl. Acad. Sci. USA  89:10915) alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparison of both strands.  
     [0114] Preferably, the “percentage of sequence identity” is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.  
     [0115] Therefore, the present invention encompasses polynucleotide and polypeptide sequences having substantial identity to the sequences disclosed herein, for example those comprising at least 50% sequence identity, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher, sequence identity compared to a polynucleotide or polypeptide sequence of this invention using the methods described herein, (e.g., BLAST analysis using standard parameters, as described below). One skilled in this art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.  
     [0116] In additional embodiments, the present invention provides isolated polynucleotides and polypeptides comprising various lengths of contiguous stretches of sequence identical to or complementary to one or more of the sequences disclosed herein. For example, polynucleotides are provided by this invention that comprise at least about 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one or more of the sequences disclosed herein as well as all intermediate lengths there between. It will be readily understood that “intermediate lengths”, in this context, means any length between the quoted values, such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including all integers through 200-500; 500-1,000, and the like.  
     [0117] The polynucleotides of the present invention, or fragments thereof, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol. For example, illustrative DNA segments with total lengths of about 10,000, about 5000, about 3000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length, and the like, (including all intermediate lengths) are contemplated to be useful in many implementations of this invention.  
     [0118] In other embodiments, the present invention is directed to polynucleotides that are capable of hybridizing under moderately stringent conditions to a polynucleotide sequence provided herein, or a fragment thereof, or a complementary sequence thereof. Hybridization techniques are well known in the art of molecular biology. For purposes of illustration, suitable moderately stringent conditions for testing the hybridization of a polynucleotide of this invention with other polynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50° C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.  
     [0119] Moreover, it will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and/or database sequence comparison).  
     [0120] Microarray Analyses  
     [0121] Polynucleotides that are suitable for detection according to the methods of the present invention may be identified, as described in more detail below, by screening a microarray of cDNAs for tissue and/or tumor-associated expression (e.g., expression that is at least two-fold greater in a tumor than in normal tissue, as determined using a representative assay provided herein). Such screens may be performed, for example, using a Synteni microarray (Palo Alto, Calif.) according to the manufacturer&#39;s instructions (and essentially as described by Schena et al.,  Proc. Natl. Acad. Sci. USA  93:10614-10619 (1996) and Heller et al.,  Proc. Natl. Acad. Sci. USA  94:2150-2155 (1997)).  
     [0122] Microarray is an effective method for evaluating large numbers of genes but due to its limited sensitivity it may not accurately determine the absolute tissue distribution of low abundance genes or may underestimate the degree of overexpression of more abundant genes due to signal saturation. For those genes showing overexpression by microarray expression profiling, further analysis was performed using quantitative RT-PCR based on Taqman™ probe detection, which comprises a greater dynamic range of sensitivity. Several different panels of normal and tumor tissues, distant metastases and cell lines were used for this purpose.  
     [0123] Quantitative Real-time Polymerase Chain Reaction  
     [0124] Suitable polynucleotides according to the present invention may be further characterized or, alternatively, originally identified by employing a quantitative PCR methodology such as, for example, the Real-time PCR methodology. By this methodology, tissue and/or tumor samples, such as, e.g., metastatic tumor samples, may be tested along side the corresponding normal tissue sample and/or a panel of unrelated normal tissue samples.  
     [0125] Real-time PCR (see Gibson et al.,  Genome Research  6:995-1001, 1996; Heid et al.,  Genome Research  6:986-994, 1996) is a technique that evaluates the level of PCR product accumulation during amplification. This technique permits quantitative evaluation of mRNA levels in multiple samples. Briefly, mRNA is extracted from tumor and normal tissue and cDNA is prepared using standard techniques.  
     [0126] Real-time PCR may, for example, be performed either on the ABI 7700 Prism or on a GeneAmp® 5700 sequence detection system (PE Biosystems, Foster City, Calif.). The 7700 system uses a forward and a reverse primer in combination with a specific probe with a 5′ fluorescent reporter dye at one end and a 3′ quencher dye at the other end (Taqman T). When the Real-time PCR is performed using Taq DNA polymerase with 5′-3′ nuclease activity, the probe is cleaved and begins to fluoresce allowing the reaction to be monitored by the increase in fluorescence (Real-time). The 5700 system uses SYBR® green, a fluorescent dye, that only binds to double stranded DNA, and the same forward and reverse primers as the 7700 instrument. Matching primers and fluorescent probes may be designed according to the primer express program (PE Biosystems, Foster City, Calif.). Optimal concentrations of primers and probes are initially determined by those of ordinary skill in the art. Control (e.g., β-actin) primers and probes may be obtained commercially from, for example, Perkin Elmer/Applied Biosystems (Foster City, Calif.).  
     [0127] To quantitate the amount of specific RNA in a sample, a standard curve is generated using a plasmid containing the gene of interest. Standard curves are generated using the Ct values determined in the real-time PCR, which are related to the initial cDNA concentration used in the assay. Standard dilutions ranging from 10-10 6  copies of the gene of interest are generally sufficient. In addition, a standard curve is generated for the control sequence. This permits standardization of initial RNA content of a tissue sample to the amount of control for comparison purposes.  
     [0128] In accordance with the above, and as described further below, the present invention provides the illustrative breast tissue- and/or tumor-specific polynucleotides mammaglobin, lipophilin B, GABAπ (B899P), B726P, B511S, B533S, B305D and B311D having sequences set forth in SEQ ID NO: 1, 3, 5-7, 11, 13, 15, 17, 19-24, 30, 32, and 73-76 illustrative polypeptides encoded thereby having amino acid sequences set forth in SEQ ID NO: 2, 4, 8-10, 12, 14, 16,18, 25-29 and 31 and 77 that may be suitably employed in the detection of cancer, more specifically, breast cancer.  
     [0129] The methods disclosed herein will also permit the identification of additional and/or alternative polynucleotides that are suitable for the detection of a wide range of cancers including, but not limited to, prostate cancer, breast cancer, colon cancer, ovarian cancer, lung cancer head &amp; neck cancer, lymphoma, leukemia, melanoma, liver cancer, gastric cancer, kidney cancer, bladder cancer, pancreatic cancer and endometrial cancer.  
     [0130] Methodologies for the Detection of Cancer  
     [0131] In general, a cancer cell may be detected in a patient based on the presence of one or more polynucleotides within cells of a biological sample (for example, blood, lymph nodes, bone marrow, sera, sputum, urine and/or tumor biopsies) obtained from the patient. In other words, such polynucleotides may be used as markers to indicate the presence or absence of a cancer such as, e.g., breast cancer.  
     [0132] As discussed in further detail herein, the present invention achieves these and other related objectives by providing a methodology for the simultaneous detection of more than one polynucleotide, the presence of which is diagnostic of the presence of cancer cells in a biological sample. Each of the various cancer detection methodologies disclosed herein have in common a step of hybridizing one or more oligonucleotide primers and/or probes, the hybridization of which is demonstrative of the presence of a tumor- and/or tissue-specific polynucleotide. Depending on the precise application contemplated, it may be preferred to employ one or more intron-spanning oligonucleotides that are inoperative against polynucleotide of genomic DNA and, thus, these oligonucleotides are effective in substantially reducing and/or eliminating the detection of genomic DNA in the biological sample.  
     [0133] Further disclosed herein are methods for enhancing the sensitivity of these detection methodologies by subjecting the biological samples to be tested to one or more cell capture and/or cell depletion methodologies.  
     [0134] By certain embodiments of the present invention, the presence of a cancer cell in a patient may be determined by employing the following steps: (a) obtaining a biological sample from said patient; (b) contacting said biological sample with a first oligonucleotide that hybridizes to a first polynucleotide said first polynucleotide selected from the group consisting of polynucleotides depicted in SEQ ID NO:73 and SEQ ID NO:74; (c) contacting said biological sample with a second oligonucleotide that hybridizes to a second polynucleotide selected from the group consisting of SEQ ID NO: 1, 3, 5-7, 11, 13, 15, 17, 19-24, 30, 32, and 75; (d) detecting in said sample an amount of a polynucleotide that hybridizes to at least one of the oligonucleotides; and (e) comparing the amount of the polynucleotide that hybridizes to said oligonucleotide to a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.  
     [0135] Alternative embodiments of the present invention provide methods wherein the presence of a cancer cell in a patient is determined by employing the steps of: (a) obtaining a biological sample from said patient; (b) contacting said biological sample with a first oligonucleotide that hybridizes to a first polynucleotide said first polynucleotide depicted in SEQ ID NO:76; (c) contacting said biological sample with a second oligonucleotide that hybridizes to a second polynucleotide selected from the group consisting of SEQ ID NO: 1, 3, 5-7, 11, 13, 15, 17, 19-24, 30, 32, and 75; (d) detecting in said sample an amount of a polynucleotide that hybridizes to at least one of the oligonucleotides; and (e) comparing the amount of the polynucleotide that hybridizes to said oligonucleotide to a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.  
     [0136] Other embodiments of the present invention provide methods for determining the presence or absence of a cancer in a patient. Such methods comprise the steps of: (a) obtaining a biological sample from said patient; (b) contacting said biological sample obtained from a patient with a first oligonucleotide that hybridizes to a polynucleotide sequence selected from the group consisting of polynucleotides depicted in SEQ ID NO:73, SEQ ID NO:74 and SEQ ID NO:76; (c) contacting said biological sample with a second oligonucleotide that hybridizes to a polynucleotide as depicted in SEQ ID NO:75; (d) contacting said biological sample with a third oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of polynucleotides depicted in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; (e) contacting said biological sample with a fourth oligonucleotide that hybridizes to a polynucleotide selected from the group consisting of polynucleotides depicted in SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; (f) detecting in said biological sample an amount of a polynucleotide that hybridizes to at least one of said oligonucleotides; and (g) comparing the amount of polynucleotide that hybridizes to the oligonucleotide to a predetermined cut-off value, and therefrom determining the presence or absence of a cancer in the patient.  
     [0137] To permit hybridization under assay conditions, oligonucleotide primers and probes should comprise an oligonucleotide sequence that has at least about 60%, preferably at least about 75% and more preferably at least about 90%, identity to a portion of a polynucleotide encoding a breast tumor protein that is at least 10 nucleotides, and preferably at least 20 nucleotides, in length. Preferably, oligonucleotide primers hybridize to a polynucleotide encoding a polypeptide described herein under moderately stringent conditions, as defined above. Oligonucleotide primers which may be usefully employed in the diagnostic methods described herein preferably are at least 10-40 nucleotides in length. In a preferred embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides, of a DNA molecule having a sequence recited in SEQ ID NO: 1, 3, 5-7, 11, 13, 15, 17, 19-24, 30, 32 and 73-76. Techniques for both PCR based assays and hybridization assays are well known in the art (see, for example, Mullis et al.,  Cold Spring Harbor Symp. Quant. Biol.,  51:263, 1987; Erlich ed.,  PCR Technology,  Stockton Press, NY, 1989).  
     [0138] The present invention also provides amplification-based methods for detecting the presence of a cancer cell in a patient. Exemplary methods comprise the steps of (a) obtaining a biological sample from a patient; (b) contacting the biological sample with a first oligonucleotide pair the first pair comprising a first oligonucleotide and a second oligonucleotide wherein the first oligonucleotide and the second oligonucleotide hybridize to a first polynucleotide and the complement thereof, respectively; (c) contacting the biological sample with a second oligonucleotide pair the second pair comprising a third oligonucleotide and a fourth oligonucleotide wherein the third and the fourth oligonucleotide hybridize to a second polynucleotide and the complement thereof, respectively, and wherein the first polynucleotide is unrelated in nucleotide sequence to the second polynucleotide; (d) amplifying the first polynucleotide and the second polynucleotide; and (e) detecting the amplified first polynucleotide and the amplified second polynucleotide; wherein the presence of the amplified first polynucleotide or the amplified second polynucleotide indicates the presence of a cancer cell in the patient.  
     [0139] Methods according to the present invention are suitable for identifying polynucleotides obtained from a wide variety of biological sample such as, for example, blood, serum, lymph node, bone marrow, sputum, urine and tumor biopsy sample. In certain preferred embodiments, the biological sample is either blood, a lymph node or bone marrow. In other embodiments of the present invention, the lymph node may be a sentinel lymph node.  
     [0140] It will be apparent that the present methods may be employed in the detection of a wide variety of cancers. Exemplary cancers include, but are not limited to, prostate cancer, breast cancer, colon cancer, ovarian cancer, lung cancer head &amp; neck cancer, lymphoma, leukemia, melanoma, liver cancer, gastric cancer, kidney cancer, bladder cancer, pancreatic cancer and endometrial cancer.  
     [0141] Certain exemplary embodiments of the present invention provide methods wherein the polynucleotides to be detected are selected from the group consisting of mammaglobin, lipophilin B, GABAπ (B899P), B726P, B511S, B533S, B305D and B311D. Alternatively and/or additionally, polynucleotides to be detected may be selected from the group consisting of those depicted in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, and SEQ ID NO:76.  
     [0142] Suitable exemplary oligonucleotide probes and/or primers that may be used according to the methods of the present invention are disclosed herein by SEQ ID NOs:33-35 and 63-72. In certain preferred embodiments that eliminate the background detection of genomic DNA, the oligonucleotides may be intron spanning oligonucleotides. Exemplary intron spanning oligonucleotides suitable for the detection of various polynucleotides disclosed herein are depicted in SEQ ID NOs:36-62.  
     [0143] Depending on the precise application contemplated, the artisan may prefer to detect the tissue- and/or tumor-specific polynucleotides by detecting a radiolabel and detecting a fluorophore. More specifically, the oligonucleotide probe and/or primer may comprises a detectable moiety such as, for example, a radiolabel and/or a fluorophore.  
     [0144] Alternatively or additionally, methods of the present invention may also comprise a step of fractionation prior to detection of the tissue- and/or tumor-specific polynucleotides such as, for example, by gel electrophoresis.  
     [0145] In other embodiments, methods described herein may be used as to monitor the progression of cancer. By these embodiments, assays as provided for the diagnosis of a cancer may be performed over time, and the change in the level of reactive polypeptide(s) or polynucleotide(s) evaluated. For example, the assays may be performed every 24-72 hours for a period of 6 months to 1 year, and thereafter performed as needed. In general, a cancer is progressing in those patients in whom the level of polypeptide or polynucleotide detected increases over time. In contrast, the cancer is not progressing when the level of reactive polypeptide or polynucleotide either remains constant or decreases with time.  
     [0146] Certain in vivo diagnostic assays may be performed directly on a tumor. One such assay involves contacting tumor cells with a binding agent. The bound binding agent may then be detected directly or indirectly via a reporter group. Such binding agents may also be used in histological applications. Alternatively, polynucleotide probes may be used within such applications.  
     [0147] As noted above, to improve sensitivity, multiple breast tumor protein markers may be assayed within a given sample. It will be apparent that binding agents specific for different proteins provided herein may be combined within a single assay. Further, multiple primers or probes may be used concurrently. The selection of tumor protein markers may be based on routine experiments to determine combinations that results in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.  
     [0148] Cell Enrichment  
     [0149] In other aspects of the present invention, cell capture technologies may be used prior to polynucleotide detection to improve the sensitivity of the various detection methodologies disclosed herein.  
     [0150] Exemplary cell enrichment methodologies employ immunomagnetic beads that are coated with specific monoclonal antibodies to surface cell markers, or tetrameric antibody complexes, may be used to first enrich or positively select cancer cells in a sample. Various commercially available kits may be used, including Dynabeads® Epithelial Enrich (Dynal Biotech, Oslo, Norway), StemSep™ (StemCell Technologies, Inc., Vancouver, BC), and RosetteSep (StemCell Technologies). The skilled artisan will recognize that other readily available methodologies and kits may also be suitably employed to enrich or positively select desired cell populations.  
     [0151] Dynabeads® Epithelial Enrich contains magnetic beads coated with mAbs specific for two glycoprotein membrane antigens expressed on normal and neoplastic epithelial tissues. The coated beads may be added to a sample and the sample then applied to a magnet, thereby capturing the cells bound to the beads. The unwanted cells are washed away and the magnetically isolated cells eluted from the beads and used in further analyses.  
     [0152] RosetteSep can be used to enrich cells directly from a blood sample and consists of a cocktail of tetrameric antibodies that target a variety of unwanted cells and crosslinks them to glycophorin A on red blood cells (RBC) present in the sample, forming rosettes. When centrifuged over Ficoll, targeted cells pellet along with the free RBC.  
     [0153] The combination of antibodies in the depletion cocktail determines which cells will be removed and consequently which cells will be recovered. Antibodies that are available include, but are not limited to: CD2, CD3, CD4, CD5, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD24, CD25, CD29, CD33, CD34, CD36, CD38, CD41, CD45, CD45RA, CD45RO, CD56, CD66B, CD66e, HLA-DR, IgE, and TCRαβ. Additionally, it is contemplated in the present invention that mAbs specific for breast tumor antigens, can be developed and used in a similar manner. For example, mAbs that bind to tumor-specific cell surface antigens may be conjugated to magnetic beads, or formulated in a tetrameric antibody complex, and used to enrich or positively select metastatic breast tumor cells from a sample.  
     [0154] Once a sample is enriched or positively selected, cells may be further analysed. For example, the cells may be lysed and RNA isolated. RNA may then be subjected to RT-PCR analysis using breast tumor-specific multiplex primers in a Real-time PCR assay as described herein.  
     [0155] In another aspect of the present invention, cell capture technologies may be used in conjunction with Real-time PCR to provide a more sensitive tool for detection of metastatic cells expressing breast tumor antigens. Detection of breast cancer cells in bone marrow samples, peripheral blood, and small needle aspiration samples is desirable for diagnosis and prognosis in breast cancer patients.  
     [0156] Probes and Primers  
     [0157] As noted above and as described in further detail herein, certain methods, compositions and kits according to the present invention utilize two or more oligonucleotide primer pairs for the detection of cancer. The ability of such nucleic acid probes to specifically hybridize to a sequence of interest will enable them to be of use in detecting the presence of complementary sequences in a biological sample.  
     [0158] Alternatively, in other embodiments, the probes and/or primers of the present invention may be employed for detection via nucleic acid hybridization. As such, it is contemplated that nucleic acid segments that comprise a sequence region of at least about 15 nucleotide long contiguous sequence that has the same sequence as, or is complementary to, a 15 nucleotide long contiguous sequence of a polynucleotide to be detected will find particular utility. Longer contiguous identical or complementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths) and even up to full length sequences will also be of use in certain embodiments.  
     [0159] Oligonucleotide primers having sequence regions consisting of contiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200 nucleotides or so (including intermediate lengths as well), identical or complementary to a polynucleotide to be detected , are particularly contemplated as primers for use in amplification reactions such as, e.g., the polymerase chain reaction (PCR™). This would allow a polynucleotide to be analyzed, both in diverse biological samples such as, for example, blood, lymph nodes and bone marrow.  
     [0160] The use of a primer of about 15-25 nucleotides in length allows the formation of a duplex molecule that is both stable and selective. Molecules having contiguous complementary sequences over stretches greater than 15 bases in length are generally preferred, though, in order to increase stability and selectivity of the hybrid, and thereby improve the quality and degree of specific hybrid molecules obtained. One will generally prefer to design primers having gene-complementary stretches of 15 to 25 contiguous nucleotides, or even longer where desired.  
     [0161] Primers may be selected from any portion of the polynucleotide to be detected. All that is required is to review the sequence, such as those exemplary polynucleotides set forth in SEQ ID NO: 1, 3, 5-7, 11, 13, 15, 17, 19-24, 30, 32, 73-75 (FIGS.  3 - 6 , respectively) and SEQ ID NO:76 (lipophilin B) or to any continuous portion of the sequence, from about 15-25 nucleotides in length up to and including the full length sequence, that one wishes to utilize as a primer. The choice of primer sequences may be governed by various factors. For example, one may wish to employ primers from towards the termini of the total sequence. The exemplary primers disclosed herein may optionally be used for their ability to selectively form duplex molecules with complementary stretches of the entire polynucleotide of interest such as those set forth in SEQ ID NO: 1, 3, 5-7, 11, 13, 15, 17, 19-24, 30, 32, 73-75 (FIGS.  3 - 6 , respectively), and SEQ ID NO:76 (lipophilin B).  
     [0162] The present invention further provides the nucleotide sequence of various exemplary oligonucleotide primers and probes, set forth in SEQ ID NOs: 33-71, that may be used, as described in further detail herein, according to the methods of the present invention for the detection of cancer.  
     [0163] Oligonucleotide primers according to the present invention may be readily prepared routinely by methods commonly available to the skilled artisan including, for example, directly synthesizing the primers by chemical means, as is commonly practiced using an automated oligonucleotide synthesizer. Depending on the application envisioned, one will typically desire to employ varying conditions of hybridization to achieve varying degrees of selectivity of probe towards target sequence. For applications requiring high selectivity, one will typically desire to employ relatively stringent conditions to form the hybrids, e.g., one will select relatively low salt and/or high temperature conditions, such as provided by a salt concentration of from about 0.02 M to about 0.15 M salt at temperatures of from about 50° C. to about 70° C. Such selective conditions tolerate little, if any, mismatch between the probe and the template or target strand, and would be particularly suitable for isolating related sequences.  
     [0164] Polynucleotide Amplification Techniques  
     [0165] Each of the specific embodiments outlined herein for the detection of cancer has in common the detection of a tissue- and/or tumor-specific polynucleotide via the hybridization of one or more oligonucleotide primers and/or probes. Depending on such factors as the relative number of cancer cells present in the biological sample and/or the level of polynucleotide expression within each cancer cell, it may be preferred to perform an amplification step prior to performing the steps of detection. For example, at least two oligonucleotide primers may be employed in a polymerase chain reaction (PCR) based assay to amplify a portion of a breast tumor cDNA derived from a biological sample, wherein at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) a polynucleotide encoding the breast tumor protein. The amplified cDNA may optionally be subjected to a fractionation step such as, for example, gel electrophoresis.  
     [0166] A number of template dependent processes are available to amplify the target sequences of interest present in a sample. One of the best known amplification methods is the polymerase chain reaction (PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. Briefly, in PCR™, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates is added to a reaction mixture along with a DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers will dissociate from the target to form reaction products, excess primers will bind to the target and to the reaction product and the process is repeated. Preferably reverse transcription and PCR™ amplification procedure may be performed in order to quantify the amount of mRNA amplified. Polymerase chain reaction methodologies are well known in the art.  
     [0167] One preferred methodology for polynucleotide amplification employs RT-PCR, in which PCR is applied in conjunction with reverse transcription. Typically, RNA is extracted from a biological sample, such as blood, serum, lymph node, bone marrow, sputum, urine and tumor biopsy samples, and is reverse transcribed to produce cDNA molecules. PCR amplification using at least one specific primer generates a cDNA molecule, which may be separated and visualized using, for example, gel electrophoresis. Amplification may be performed on biological samples taken from a patient and from an individual who is not afflicted with a cancer. The amplification reaction may be performed on several dilutions of cDNA spanning two orders of magnitude. A two-fold or greater increase in expression in several dilutions of the test patient sample as compared to the same dilutions of the non-cancerous sample is typically considered positive.  
     [0168] Any of a variety of commercially available kits may be used to perform the amplification step. One such amplification technique is inverse PCR (see Triglia et al.,  Nucl. Acids Res.  16:8186, 1988), which uses restriction enzymes to generate a fragment in the known region of the gene. The fragment is then circularized by intramolecular ligation and used as a template for PCR with divergent primers derived from the known region. Within an alternative approach, sequences adjacent to a partial sequence may be retrieved by amplification with a primer to a linker sequence and a primer specific to a known region. The amplified sequences are typically subjected to a second round of amplification with the same linker primer and a second primer specific to the known region. A variation on this procedure, which employs two primers that initiate extension in opposite directions from the known sequence, is described in WO 96/38591. Another such technique is known as “rapid amplification of cDNA ends” or RACE. This technique involves the use of an internal primer and an external primer, which hybridizes to a polyA region or vector sequence, to identify sequences that are 5′ and 3′ of a known sequence. Additional techniques include capture PCR (Lagerstrom et al.,  PCR Methods Applic.  1:111-19, 1991) and walking PCR (Parker et al.,  Nucl. Acids. Res.  19:3055-60, 1991). Other methods employing amplification may also be employed to obtain a full length cDNA sequence.  
     [0169] Another method for amplification is the ligase chain reaction (referred to as LCR), disclosed in Eur. Pat. Appl. Publ. No. 320,308 (specifically incorporated herein by reference in its entirety). In LCR, two complementary probe pairs are prepared, and in the presence of the target sequence, each pair will bind to opposite complementary strands of the target such that they abut. In the presence of a ligase, the two probe pairs will link to form a single unit. By temperature cycling, as in PCR™, bound ligated units dissociate from the target and then serve as “target sequences” for ligation of excess probe pairs. U.S. Pat. No. 4,883,750, incorporated herein by reference in its entirety, describes an alternative method of amplification similar to LCR for binding probe pairs to a target sequence.  
     [0170] Qbeta Replicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880, incorporated herein by reference in its entirety, may also be used as still another amplification method in the present invention. In this method, a replicative sequence of RNA that has a region complementary to that of a target is added to a sample in the presence of an RNA polymerase. The polymerase will copy the replicative sequence that can then be detected.  
     [0171] An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5′-[α-thio]triphosphates in one strand of a restriction site (Walker et al., 1992, incorporated herein by reference in its entirety), may also be useful in the amplification of nucleic acids in the present invention.  
     [0172] Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e. nick translation. A similar method, called Repair Chain Reaction (RCR) is another method of amplification which may be useful in the present invention and is involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection. A similar approach is used in SDA.  
     [0173] Sequences can also be detected using a cyclic probe reaction (CPR). In CPR, a probe having a 3′ and 5′ sequences of non-target DNA and an internal or “middle” sequence of the target protein specific RNA is hybridized to DNA which is present in a sample. Upon hybridization, the reaction is treated with RNaseH, and the products of the probe are identified as distinctive products by generating a signal that is released after digestion. The original template is annealed to another cycling probe and the reaction is repeated. Thus, CPR involves amplifying a signal generated by hybridization of a probe to a target gene specific expressed nucleic acid.  
     [0174] Still other amplification methods described in Great Britain Pat. Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No. PCT/US89/01025, each of which is incorporated herein by reference in its entirety, may be used in accordance with the present invention. In the former application, “modified” primers are used in a PCR-like, template and enzyme dependent synthesis. The primers may be modified by labeling with a capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latter application, an excess of labeled probes is added to a sample. In the presence of the target sequence, the probe binds and is cleaved catalytically. After cleavage, the target sequence is released intact to be bound by excess probe. Cleavage of the labeled probe signals the presence of the target sequence.  
     [0175] Other nucleic acid amplification procedures include transcription-based amplification systems (TAS) (Kwoh et al., 1989; PCT Intl. Pat. Appl. Publ. No. WO 88/10315, incorporated herein by reference in its entirety), including nucleic acid sequence based amplification (NASBA) and 3SR. In NASBA, the nucleic acids can be prepared for amplification by standard phenol/chloroform extraction, heat denaturation of a sample, treatment with lysis buffer and minispin columns for isolation of DNA and RNA or guanidinium chloride extraction of RNA. These amplification techniques involve annealing a primer that has sequences specific to the target sequence. Following polymerization, DNA/RNA hybrids are digested with RNase H while double stranded DNA molecules are heat-denatured again. In either case the single stranded DNA is made fully double stranded by addition of second target-specific primer, followed by polymerization. The double stranded DNA molecules are then multiply transcribed by a polymerase such as T7 or SP6. In an isothermal cyclic reaction, the RNAs are reverse transcribed into DNA, and transcribed once again with a polymerase such as T7 or SP6. The resulting products, whether truncated or complete, indicate target-specific sequences.  
     [0176] Eur. Pat. Appl. Publ. No. 329,822, incorporated herein by reference in its entirety, disclose a nucleic acid amplification process involving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention. The ssRNA is a first template for a first primer oligonucleotide, which is elongated by reverse transcriptase (RNA-dependent DNA polymerase). The RNA is then removed from resulting DNA:RNA duplex by the action of ribonuclease H (RNase H, an RNase specific for RNA in a duplex with either DNA or RNA). The resultant ssDNA is a second template for a second primer, which also includes the sequences of an RNA polymerase promoter (exemplified by T7 RNA polymerase) 5′ to its homology to its template. This primer is then extended by DNA polymerase (exemplified by the large “Klenow” fragment of  E. coli  DNA polymerase I), resulting as a double-stranded DNA (“dsDNA”) molecule, having a sequence identical to that of the original RNA between the primers and having additionally, at one end, a promoter sequence. This promoter sequence can be used by the appropriate RNA polymerase to make many RNA copies of the DNA. These copies can then re-enter the cycle leading to very swift amplification. With proper choice of enzymes, this amplification can be done isothermally without addition of enzymes at each cycle. Because of the cyclical nature of this process, the starting sequence can be chosen to be in the form of either DNA or RNA.  
     [0177] PCT Intl. Pat. Appl. Publ. No. WO 89/06700, incorporated herein by reference in its entirety, disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. This scheme is not cyclic; i.e. new templates are not produced from the resultant RNA transcripts. Other amplification methods include “RACE” (Frohman, 1990), and “one-sided PCR” (Ohara, 1989) which are well-known to those of skill in the art.  
     [0178] Compositions and Kits for the Detection of Cancer  
     [0179] The present invention further provides kits for use within any of the above diagnostic methods. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, reagents, containers and/or equipment. For example, one container within a kit may contain a monoclonal antibody or fragment thereof that specifically binds to a breast tumor protein. Such antibodies or fragments may be provided attached to a support material, as described above. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay. Such kits may also, or alternatively, contain a detection reagent as described above that contains a reporter group suitable for direct or indirect detection of antibody binding.  
     [0180] The present invention also provides kits that are suitable for performing the detection methods of the present invention. Exemplary kits comprise oligonucleotide primer pairs each one of which specifically hybridizes to a distinct polynucleotide. Within certain embodiments, kits according to the present invention may also comprise a nucleic acid polymerase and suitable buffer. Exemplary oligonucleotide primers suitable for kits of the present invention are disclosed herein by SEQ ID NOs: 33-71. Exemplary polynucleotides suitable for kits of the present invention are disclosed in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, and lipophilin B.  
     [0181] Alternatively, a kit may be designed to detect the level of mRNA encoding a breast tumor protein in a biological sample. Such kits generally comprise at least one oligonucleotide probe or primer, as described above, that hybridizes to a polynucleotide encoding a breast tumor protein. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a second oligonucleotide and/or a diagnostic reagent or container to facilitate the detection of a polynucleotide encoding a breast tumor protein.  
     [0182] In other related aspects, the present invention further provides compositions useful in the methods disclosed herein. Exemplary compositions comprise two or more oligonucleotide primer pairs each one of which specifically hybridizes to a distinct polynucleotide. Exemplary oligonucleotide primers suitable for compositions of the present invention are disclosed herein by SEQ ID NOs: 33-71. Exemplary polynucleotides suitable for compositions of the present invention are disclosed in SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:32, and lipophilin B.  
     [0183] The following Examples are offered by way of illustration and not by way of limitation.  
     EXAMPLES  
     Example 1  
     Differential Display  
     [0184] This example discloses the use of differential display to enrich for polynucleotides that are over-expressed in breast tumor tissues.  
     [0185] Differential display was performed as described in the literature (see, e.g., Liang, P. et al.,  Science  257:967-971 (1993), incorporated herein by reference in its entirety) with the following modifications: (a) PCR amplification products were visualized on silver stained gels (b) genetically matched pairs of tissues were used to eliminate polymorphic variation (c) two different dilutions of cDNA were used as template to eliminate any dilutional effects (see, Mou, E. et al.,  Biochem Biophy Res Commun.  199:564-569 (1994), incorporated herein by reference in its entirety).  
     Example 2  
     Preparation of cDNA Subtraction library  
     [0186] This example discloses the preparation of a breast tumor cDNA subtraction library enriched in breast tumor specific polynucleotides.  
     [0187] cDNA library subtraction was performed as described with some modification. See, Hara, T. et al.,  Blood  84: 189-199 (1994), incorporated herein by reference in its entirety. The breast tumor library (tracer) that was made from a pool of three breast tumors was subtracted with normal breast library (driver) to identify breast tumor specific genes. More recent subtractions utilized 6-10 normal tissues as driver to subtract out common genes more efficiently, with an emphasis on essential tissues along with one “immunological” tissue (e.g., spleen, lymph node, or PBMC), to assist in the removal of cDNAs derived from lymphocyte infiltration in tumors. The breast tumor specific subtracted cDNA library was generated as follows: driver cDNA library was digested with EcoRI, NotI, and SfuI (SfuI cleaves the vector), filled in with DNA polymerase klenow fragment. After phenol-chloroform extraction and ethanol precipitation, the DNA was labeled with Photoprobe biotin and dissolved in H 2 0. Tracer cDNA library was digested with BamHI and XhoI, phenol chloroform extracted, passed through Chroma spin-400 columns, ethanol precipitated, and mixed with driver DNA for hybridization at 68° C. for 20 hours [long hybridization (LH)]. The reaction mixture was then subjected to the streptavidin treatment followed by phenol/chloroform extraction for a total of four times. Subtracted DNA was precipitated and subjected to a hybridization at 68° C. for 2 hours with driver DNA again [short hybridization (SH)]. After removal of biotinylated double-stranded DNA, subtracted cDNA was ligated into BamHI/XhoI site of Chloramphenicol resistant pBCSK+and transformed into ElectroMax  E. coli  DH10B cells by electroporation to generate subtracted cDNA library. To clone less abundant breast tumor specific genes, cDNA library subtraction was repeated by subtracting the tracer cDNA library with the driver cDNA library plus abundant cDNAs from primary subtractions. This resulted in the depletion of these abundant sequences and the generation of subtraction libraries that contain less abundant sequences.  
     [0188] To analyze the subtracted cDNA library, plasmid DNA was prepared from 100-200 independent clones, which were randomly picked from the subtracted library, and characterized by DNA sequencing. The determined cDNA and expected amino acid sequences for the isolated cDNAs were compared to known sequences using the most recent Genbank and human EST databases.  
     Example 3  
     PCR-subtraction  
     [0189] This example discloses PCR subtraction to enrich for breast tumor specific polynucleotides.  
     [0190] PCR-subtraction was performed essentially as described in the literature. See, Diatchenko, L. et al.,  Proc Natl Acad Sci USA.  93:6025-6030 (1996) and Yang, G. P. et al.,  Nucleic acids Res.  27:1517-23 (1999), incorporated herein by reference in their entirety. Briefly, this type of subtraction works by ligating two different adapters to different aliquots of a restriction enzyme digested tester (breast tumor) cDNA sample, followed by mixing of the testers separately with excess driver (without adapters). This first hybridization results in normalization of single stranded tester specific cDNA due to the second order kinetics of hybridization. These separate hybridization reactions are then mixed without denaturation, and a second hybridization performed which produces the target molecules; double stranded cDNA fragments containing both of the different adapters. Two rounds of PCR were performed, which results in the exponential amplification of the target population molecules (normalized tester specific cDNAs), while other fragments were either unamplified or only amplified in a linear manner. The subtractions performed included a pool of breast tumors subtracted with a pool of normal breast and a pool of breast tumors subtracted with a pool of normal tissues including PBMC, brain, pancreas, liver, small intestine, stomach, heart and kidney.  
     [0191] Prior to cDNA synthesis RNA was treated with DNase I (Ambion) in the presence of RNasin (Promega Biotech, Madison, Wis.) to remove DNA contamination. The cDNA for use in real-time PCR tissue panels was prepared using 25 μl Oligo dT (Boehringer-Mannheim) primer with superscript II reverse transcriptase (Gibco BRL, Bethesda, Md.).  
     Example 4  
     Detection of Breast Cancer using Breast-specific Antigens  
     [0192] The isolation and characterization of the breast-specific antigens B511S and B533S is described in U.S. patent application Ser. No. 09/346,327, filed Jul. 2, 1999, the disclosure of which is hereby incorporated by reference in its entirety. The determined cDNA sequence for B511S is provided in SEQ ID NO: 30, with the corresponding amino acid sequence being provided in SEQ ID NO: 31. The determined cDNA sequence for B533S is provided in SEQ ID NO: 32. The isolation and characterization of the breast-specific antigen B726P is described in U.S. patent application Ser. No. 09/285,480, filed Apr. 2, 1999, and Ser. No. 09/433,826, filed Nov. 3, 1999, the disclosures of which are hereby incorporated by reference in their entirety.  
     [0193] The determined cDNA sequences for splice variants of B726P are provided in SEQ ID NO: 13, 15, 17 and 19-24, with the corresponding amino acid sequences being provided in SEQ ID NO: 14, 16, 18 and 25-29.  
     [0194] The isolation and characterization of the breast-specific antigen B305D forms A and C has been described in U.S. patent application Ser. No. 09/429,755, filed Oct. 28, 1999, the disclosure of which is hereby incorporated by reference in its entirety. Determined cDNA sequences for B305D isoforms A and C are provided in SEQ ID NO: 1, 3 and 5-7, with the corresponding amino acid sequences being provided in SEQ ID NO: 2, 4 and 8-10.  
     [0195] The isolation and characterization of the breast-specific antigen B31 1 D has been described in U.S. patent application Ser. No. 09/289,198, filed Apr. 9, 1999, the disclosure of which is hereby incorporated by reference in its entirety. The determined cDNA sequence for B311D is provided in SEQ ID NO:11, with the corresponding amino acid sequence being provided in SEQ ID NO:12.  
     [0196] cDNA sequences for mammaglobin are provided in FIGS. 4 and 5, with the cDNA sequence for GABAπ being provided in FIG. 6 and are disclosed in SEQ ID NOs: 73-75, respectively.  
     [0197] The isolation and characteization of the breast-specific antigen lipophilin B has been described in U.S. patent application Ser. No. 09/780,842, filed Feb. 8, 2001, the disclosure of which is hereby incorporated by reference in its entirety. The determined cDNA sequence for lipophilin B is provided in SEQ ID NO:76, with the corresponding amino acid sequence being provided in SEQ ID NO:77. The nucleotide sequences of several sequence variants of lipophilin B are also described in the Ser. No. 09/780,842 application.  
     Example 5  
     Microarray Analysis  
     [0198] This example discloses the use of microarray analyses to identify polynucleotides that are at least two-fold overexpressed in breast tumor tissue samples as compared to normal breast tissue samples.  
     [0199] mRNA expression of the polynucleotides of interest was performed as follows. cDNA for the different genes was prepared as described above and arrayed on a glass slide (Incyte, Palo Alto, Calif.). The arrayed cDNA was then hybridized with a 1:1 mixture of Cy3 or Cy5 fluorescent labeled first strand cDNAs obtained from polyA+RNA from breast tumors, normal breast and normal tissues and other tumors as described in Shalon, D. et al.,  Genome Res.  6:639-45 (1996), incorporated herein by reference in its entirety. Typically Cy3 (Probe 1) was attached to cDNAs from breast tumors and Cy5 (Probe 2) to normal breast tissue or other normal tissues. Both probes were allowed to compete with the immobilized gene specific cDNAs on the chip, washed then scanned for fluorescence intensity of the individual Cy3 and Cy5 fluorescence to determine extent of hybridization. Data were analyzed using GEMTOOLS software (Incyte, Palo Alto, Calif.) which enabled the overexpression patterns of breast tumors to be compared with normal tissues by the ratios of Cy3/Cy5. The fluorescence intensity was also related to the expression level of the individual genes. DNA microarray analyses was used primarily as a screening tool to determine tissue/tumor specificity of cDNA&#39;s recovered from the differential display, cDNA library and PCR subtractions, prior to more rigorous analysis by quantitative RT-PCR, northern blotting, and immunohistochemistry. Microarray analysis was performed on two microchips. A total of 3603 subtracted cDNA&#39;s and 197 differential display templates were evaluated to identify 40 candidates for further analysis by quantitative PCR.  
     [0200] From these candidates, several were chosen on the basis of favorable tissue specificity profiles, including B305D, B311D, B726P, B511S and B533S, indicating their overexpression profiles in breast tumors and/or normal breast versus other normal tissues. It was evident that the expression of these genes showed a high degree of specificity for breast tumors and/or breast tissue. In addition, these genes have in many cases complementary expression profiles.  
     [0201] The two known breast-specific genes, mammaglobin and γ-aminobutyrate type A receptor π subunit (GABAπ) were also subjected to microarray analysis. mRNA expression of mammaglobin has been previously described to be upregulated in proliferating breast tissue, including breast tumors. See, (Watson et al.,  Cancer Res.,  56: 860-5 (1996); Watson et al., Cancer Res., 59: 3028-3031 (1999); Watson et al., Oncogene. 16:817-24 (1998), incorporated herein by reference in their entirety). The GABAπ mRNA levels were over-expressed in breast tumors. Previous studies had demonstrated its overexpression in uterus and to some degree in prostate and lung (Hedblom et al., J Biol. Chem. 272:15346-15350 (1997)) but no previous study had indicated elevated levels in breast tumors.  
     Example 6  
     Quantitative Real-time PCR Analysis  
     [0202] This example discloses the use of quantitative Real-time PCR to confirm the microarray identification polynucleotide that are at least two-fold overexpressed in breast tumor tissue samples as compared to normal breast tissue samples.  
     [0203] The tumor- and/or tissue-specificity of the polynucleotides identified by the microarray analyses disclosed herein in Example 5, were confirmed by quantitative PCR analyses. Breast metastases, breast tumors, benign breast disorders and normal breast tissue along with other normal tissues and tumors were tested in quantitative (Real time) PCR. This was performed either on the ABI 7700 Prism or on a GeneAmp® 5700 sequence detection system (PE Biosystems, Foster City, Calif.). The 7700 system uses a forward and a reverse primer in combination with a specific probe designed to anneal to sequence between the forward and reverse primer. This probe was conjugated at the 5′end with a fluorescent reporter dye and a quencher dye at the other 3′ end (Taqman™). During PCR the Taq DNA polymerase with it&#39;s 5′-3′ nuclease activity cleaved the probe which began to fluoresce, allowing the reaction to be monitored by the increase in fluorescence (Real-time). Holland et al.,  Proc Natl Acad Sci U S A.  88:7276-7280 (1991). The 5700 system used SYBR® green, a fluorescent dye, that only binds to double stranded DNA (Schneeberger et al., PCR Methods Appl. 4:234-8 (1995)), and the same forward and reverse primers as the 7700 instrument. No probe was needed. Matching primers and fluorescent probes were designed for each of the genes according to the Primer Express program (PE Biosystems, Foster City, Calif.).  
               TABLE 1                          Primer and Probe Sequences for the Genes of Interest                                 Forward Primer   Reverse primer   Probe                                         Mammaglobin   TGCCATAGATGA   TGTCATATATTAATT   TCTTAACCAAACGG           ATTGAAGGAATG   GCATAAACACCTCA   ATGAAACTCTGAGC           (SEQ ID NO: 48)   (SEQ ID NO: 49)   AATG (SEQ ID                   NO: 50)       B305D-C form   AAAGCAGATGGT   CCTGAGACCAAATG   ATTCCATGCCGGCT           GGTTGAGGTT   GCTTCTTC (SEQ ID   GCTTCTTCTG (SEQ           (SEQ ID NO: 39)   NO: 40)   ID NO: 41)       B311D   CCGCTTCTGACAA   CCTATAAAGATGTT   CCCCTCCCTCAGGG           CACTAGAGATC   ATGTACCAAAAATG   TATGGCCC (SEQ ID           (SEQ ID NO: 63)   AAGT (SEQ ID NO: 64)   NO: 65)       B726P   TCTGGTTTTCTCA   TGCCAAGGAGCGGA   CAACCACGTGACA           TTCTTTATTCATT   TTATCT (SEQ ID   AACACTGGAATTAC           TATT (SEQ ID   NO: 43)   AGG (SEQ ID NO: 44)           NO: 42)       B533S   CCCTTTCTCACCC   TGCATTCTCTCATAT   CCGGGCCTCAGGC           ACACACTGT (SEQ   GTGGAAGCT (SEQ ID   ATATACTATTCTAC           ID NO: 66)   NO: 67)   TGTCTG (SEQ ID                   NO: 68)       GABAπ   AAGCCTCAGAGT   AAATATAAGTGAAG   AATCCATTGTATCT           CCTTCCAGTATG   AAAAAAATTAGTAG   TAGAACCGAGGGA           (SEQ ID NO: 36)   AT (SEQ ID NO: 72)   TTTGTTTAGA (SEQ                   ID NO: 38)       B511S   GACATTCCAGTTT   TGCAGAAGACTCAA   TCTCAGGGACACAC           TACCCAAATGG   GCTGATTCC (SEQ ID   TCTACCATTCGGGA           (SEQ ID NO: 69)   NO: 70)   (SEQ ID NO: 71)                  
 
     [0204] The concentrations used in the quantitative PCR for the forward primers for mammaglobin, GABAπ, B305D C form, B311D, B511S, B533S and B726P were 900, 900, 300, 900, 900, 300 and 300nM respectively. For the reverse primers they were 300, 900, 900, 900, 300, 900 and 900 nM respectively. Primers and probes so produced were used in the universal thermal cycling program in real-time PCR. They were titrated to determine the optimal concentrations using a checkerboard approach. A pool of cDNA from target tumors was used in this optimization process. The reaction was performed in 25 μl volumes. The final probe concentration in all cases was 160 nM. dATP, dCTP and dGTP were at 0.2 mM and dUTP at 0.4 mM. Amplitaq gold and Amperase UNG (PE Biosystems, Foster City, Calif.) were used at 0.625 units and 0.25 units per reaction. MgCl 2  was at a final concentration of 5 mM. Trace amounts of glycerol, gelatin and Tween 20 (Sigma Chem Co, St Louis, Mo.) were added to stabilize the reaction. Each reaction contained 2 μl of diluted template. The cDNA from RT reactions prepared as above was diluted 1:10 for the gene of interest and 1:100 for P-Actin. Primers and probes for β-Actin (PE Biosystems, Foster City, Calif.) were used in a similar manner to quantitate the presence of β-actin in the samples. In the case of the SYBR® green assay, the reaction mix (25μl) included 2.5 μl of SYBR green buffer, 2 μl of cDNA template and 2.5 μl each of the forward and reverse primers for the gene of interest. This mix also contained 3 mM MgCl 2 , 0.25units of AmpErase UNG, 0.625 units of Amplitaq gold, 0.08% glycerol, 0.05% gelatin, 0.0001% Tween 20 and 1 mM dNTP mix. In both formats, 40 cycles of amplification were performed.  
     [0205] In order to quantitate the amount of specific cDNA (and hence initial mRNA) in the sample, a standard curve was generated for each run using the plasmid containing the gene of interest. Standard curves were generated using the Ct values determined in the real-time PCR which were related to the initial cDNA concentration used in the assay. Standard dilutions ranging from 20-2×10 6  copies of the gene of interest were used for this purpose. In addition, a standard curve was generated for the housekeeping gene β-actin ranging from 200 fg-200 pg to enable normalization to a constant amount of β-Actin. This allowed the evaluation of the over-expression levels seen with each of the genes.  
     [0206] The genes B311D, B533S and B726P were evaluated in quantitative PCR as described above on two different panels consisting of: (a) breast tumor, breast normal and normal tissues; and (b) breast tumor metastases (primarily lymph nodes), using the primers and probes shown above in Table 1. The data for panel (a) is shown in FIG. 1 for all three genes. The three genes showed identical breast tissue expression profiles. However, the relative level of gene expression was very different in each case. B311D in general was expressed at lower levels than B533S and both less than B726P, but all three were restricted to breast tissue. The quantitative PCR thus confirmed there was a differential expression between normal breast tissue and breast tumors for all three genes, and that approximately 50% of breast tumors over-expressed these genes. When tested on a panel of distant metastases derived from breast cancers all three genes reacted with 14/21 metastases and presented similar profiles. All three genes also exhibited increasing levels of expression as a function of pathological stage of the tumor, as shown for B533S in FIG. 2.  
     [0207] Mammaglobin is a homologue of a rabbit uteroglobin and the rat steroid binding protein subunit C3 and is a low molecular weight protein that is highly glycosylated. In contrast to its homologs, mammaglobin has been reported to be breast specific and over-expression has been described in breast tumor biopsies (23%) and primary and metastatic breast tumors (˜75%) with reports of the detection of mammaglobin mRNA expression in 91% of lymph nodes from metastatic breast cancer patients. However, more rigorous analysis of mammaglobin gene expression by microarray and quantitative PCR as described above (panels (a) and (b) and a panel of other tumors and normal tissues and additional breast tumors), showed expression at significant levels in skin and salivary gland with much lower levels in esophagus and trachea, as shown in Table 2 below.  
               TABLE 2                          Normalized Distribution of Mammaglobin and B511S mRNA in Various Tissues                                         Mean Copies       Mean Copies                   Mammaglobin       B511S       PCR Positive           /ngβ-Actin   PCR   /ngβ-Actin   PCR   (Mammaglobin/       Tissue   ± SD   Positive   ± SD   Positive   B511S)               Breast   1233.88 ± 3612    31/42   1800.40 ± 3893.24   33/42   38/42       Tumors   .74       Breast   1912.54 ± 4625    14/24    3329.50 ± 10820.71   14/24   17/24       tumor   .85       Metastases       Benign   121.87 ± 78.63   3/3   524.66 ± 609.43   2/3   3/3       Breast       disorders       Normal   114.19 ± 94.40   11/11   517.64 ± 376.83   8/9   11/11       breast       Breast   231.50 ± 276.6   2/3   482.54 ± 680.28   ½   2/3       reduction   8       Other    0.13 ± 0.65    1/39   24.17 ± 36.00    5/23       tumors       Salivary   435.65 ± 705.1   2/3   45766.61 ± 44342.43   3/3       gland   1       Skin   415.74 ± 376.1   7/9   7039.05 ± 7774.24   9/9           4       Esophagus    4.45 ± 3.86   2/3   1.02 ± 0.14   0/3       Bronchia   0.16   0/1   84.44 ± 53.31   2/2       Other    0.33 ± 1.07    0/85    5.49 ± 10.65    3/75       normal       tissues                  
 
     [0208] The breast-specific gene B511S, while having a different profile of reactivity on breast tumors and normal breast tissue to mammaglobin, reacted with the same subset of normal tissues as mammaglobin. B511S by PSORT analysis is indicated to have an ORF of 90 aa and to be a secreted protein as is the case for mammaglobin. B511S has no evidence of a transmembrane domain but may harbor a cleavable signal sequence. Mammaglobin detected 14/24 of distant metastatic breast tumors, 31/42 breast tumors and exhibited ten-fold over-expression in tumors and metastases as compared to normal breast tissue. There was at least 300-fold over-expression in normal breast tissue versus other negative normal tissues and tumors tested, which were essentially negative for mammaglobin expression. B511S detected 33/42 breast tumors and 14/24 distant metastases, while a combination of B511S with mammaglobin would be predicted to detect 38/42 breast tumors and 17/24 metastatic lesions (Table 2 above). The quantitative level of expression of B511S and mammaglobin were also in similar ranges, in concordance with the microarray profiles observed for these two genes. Other genes that were additive with mammaglobin are shown in Table 3.  
               TABLE 3                          mRNA Complementation of Mammaglobin with Other Genes                         Mammaglobin Negative                                             Mammaglobin               B305D +   B305D + GABAπ +           Positive   B305D   GABAπ   B726P   GABAπ   B726P                                                     Breast   13/21   2/8   5/8   3/8   7/8   8/8       Metastases       Breast   18/25   3/7   4/7   5/7   7/7   7/7       tumors                  
 
     [0209] B305D was shown to be highly over-expressed in breast tumors, prostate tumors, normal prostate tissue and testis compared to normal tissues, including normal breast tissue. Different splice variants of B305D have been identified with form A and C being the most abundant but all tested have similar tissue profiles in quantitative PCR. The A and C forms contain ORF&#39;s of 320 and 385 aa, respectively. B305D is predicted by PSORT to be a Type II membrane protein that comprises a series of ankyrin repeats. A known gene shown to be complementary with B305D, in breast tumors, was GABAπ. This gene is a member of the GABA A  receptor family and encodes a protein that has 30-40% amino acid homology with other family members, and has been shown by Northern blot analysis to be over-expressed in lung, thymus and prostate at low levels and highly over-expressed in uterus. Its expression in breast tissue has not been previously described. This is in contrast to other GABA A  receptors that have appreciable expression in neuronal tissues. Tissue expression profiling of this gene showed it to be over-expressed in breast tumors in an inverse relationship to the B305D gene (Table 3). GABAπ detected 15/25 tumors and 6/21 metastases including 4 tumors and 5 metastases missed by mammaglobin. In contrast, B305D detected 13/25 breast tumors and 8/21 metastases, again including 3 tumors and 2 metastases missed by mammaglobin. A combination of just B305D and the GABAπ would be predicted to identify 22/25 breast tumors and 14/21 metastases. The combination of B305D and GABAπ with mammaglobin in detecting breast metastases is shown in Table 3 above and FIGS. 3A and 3B. This combination detected 20/21 of the breast metastases as well as 25/25 breast tumors that were evaluated on the same panels for all three genes. The one breast metastasis that was negative for these three genes was strongly positive for B726P (FIGS. 3A and 3B).  
     [0210] To evaluate the presence of circulating tumor cells, an immunocapture (cell capture) method was employed to first enrich for epithelial cells prior to RT-PCR analysis. Immunomagnetic polystyrene beads coated with specific monoclonal antibodies to two glycoproteins on the surface of epithelial cells were used for this purpose. Such an enrichment procedure increased the sensitivity of detection (˜100 fold) as compared to direct isolation of poly A +  RNA, as shown in Table 4.  
               TABLE 4                          Extraction of Mammaglobin Positive Cells (MB415) Spiked       into Whole Blood and Detection by Real-time PCR                             Epithelial cell extraction   Direct Extraction           (Poly A +  RNA)   (Poly A +  RNA)                     MB415 cells/ml Blood   Copies Mammaglobin/ng β-Actin                                 100000   54303.2   58527.1        10000   45761.9   925.9        1000   15421.2   61.6         100   368.0   5.1         10   282.0   1.1          1   110.2   0          0   0   0                  
 
     [0211] Mammaglobin-positive cells (MB415) were spiked into whole blood at various concentrations and then extracted using either epithelial cell enrichment or direct isolation from blood. Using enrichment procedures, mammaglobin mRNA was found to be detectable at much lower levels than when direct isolation was used. Whole blood samples from patients with metatastic breast cancer were subsequently treated with the immunomagnetic beads. Poly A +  RNA was then isolated, cDNA prepared and run in quantitative PCR using two gene specific primers (Table 1) and a fluorescent probe (Taqman™). As observed in breast cancer tissues, complementation was also seen in the detection of circulating tumor cells derived from breast cancers. Again, mammaglobin PCR detected circulating tumor cells in a high percentage of blood samples, albeit at low levels, from metastatic breast cancer patients (20/32) when compared to the normal blood samples (Table 5) but several of the other genes tested to date further increased this detection rate. This included B726P, B305D, B311D, B533S and GABAπ. The detection level of mammaglobin in blood samples from metastatic breast cancer patients is higher than described previously (62 vs. 49%), despite testing smaller blood volumes, probably because of the use of epithelial marker-specific enrichment in our study. A combination of all the genes tested indicate that 27/32 samples were positive by one or more of these genes.  
               TABLE 5                          Gene Complementation in Epithelial Cells Isolated from Blood of Normal Individuals       and Metastatic Breast Cancer Patients                                             Sample ID   Mammaglobin   B305D   B311D   B533S   B726P   GABAπ   Combo                2   +   −   −   +   −   −   +        3   +   −   −   +   −   −   +        5   +   +   −   −   +   −   +        6   +   −   −   +   +   −   +        8   −   −   +   −   −   −   +        9   +   +   +   −   +   −   +       10   +   −   +   −   +   −   +       11   −   −   −   −   −   −   −       12   −   +   +   −   −   −   +       13   −   −   −   +   −   −   +       15   −   −   −   −   −   −   −       18   +   −   −   −   −   −   +       19   +   −   −   −   −   +   +       21   +   −   −   −   −   −   +       22   −   −   −   −   −   −   −       23   +   −   −   −   −   −   +       24   +   −   −   −   −   −   +       25   −   +   −   −   −   −   +       26   −   −   −   −   −   −   −       29   +   −   +   +   +   −   +       31   +   −   −   +   −   −   +       32   −   −   −   −   −   ±   ±       33   −   −   −   −   +   −   +       34   +   −   −   −   −   +   +       35   +   −   −   −   +   −   +       36   −   −   −   −   −   +   +       37   +   −   −   +   −   −   +       38   −   −   −   −   −   −   −       40   +   −   −   −   −   −   +       41   +   −   −   +   −   −   +       42   +   −   −   −   −   −   +       43   −   −   −   −   −   +   +       Donor 104   −   −   −   −   −   +   +       Donor 348   −   −   −   −   −   Nd   −       Donor 392   −   −   −   −   −   Nd   −       Donor 408   −   −   −   −   −   Nd   −       Donor 244   −   −   −   −   −   −   −       Donor 355   −   −   −   −   −   −   −       Donor 264   −   −   −   −   −   −   −       Donor 232   −   −   −   −   −   Nd   −       Donor 12    −   −   −   −   −   −   −       Donor 415   −   −   −   −   −   Nd   −       Donor 35    −   −   −   −   −   −   −       Donor 415   −   −   −   −   −   Nd   −       Donor 35    −   −   −   −   −   −   −       Sensitivity   20/32   4/32    7/32   9/32   7/32   4/32   27/32                  
 
     [0212] In further studies, mammaglobin, GABAπ, B305D (C form) and B726P specific primers and specific Taqman probes were employed in different combinations to analyze their combined mRNA expression profile in breast metastases (B. met) and breast tumor (B. tumors) samples using real-time PCR. The forward and reverse primers and probes employed for mammaglobin, B305D (C form) and B726P are shown in Table 1. The forward primer and probe employed for GABAπ are shown in Table 1, with the reverse primer being as follows: TTCAAATATAAGTGAAGAAAAAATTAGTAGATCAA (SEQ ID NO:51). As shown below in Table 6, a combination of mammaglobin, GABAπ, B305D (C form) and B726P was found to detect 22/22 breast tumor samples, with an increase in expression being seen in 5 samples (indicated by ++).  
               TABLE 6                          Real-time PCR Detection of Tumor Samples using Different Primer Combinations                                         Mammaglobin +   Mammaglobin +   Mammaglobin + GABA +       Tumor sample   Mammaglobin   GABA   GABA + B305D   B305D + B726P               B. Met 316A       +   +   +       B. Met 317A   +   +   +   +       B. Met 318A       +   +   +−       B. Met 595A   +   +   +   +       B. Met 611A   +   +   +   +       B. Met 612A   +   +   +   +       B. Met 614A       +   +   +       B. Met 616A       +   +   +       B. Met 618A   +   +   +   +       B. Met 620A   +   +   +   +       B. Met 621A   +   +   +   +       B. Met 624A   +   +   +   +       B. Met 625A           +   +       B. Met 627A   +       +   +       B. Met 629A       +   +   +       B. Met 631A   +   +   +   +       B. Tumor 154A   +   +   +   ++       B. Tumor 155A   +   +   +   ++       B. Tumor 81D           +   ++       B. Tumor 209A       −   +   +       B. Tumor 208A       +   +   ++       B. Tumor 10A   −   +   +   +                  
 
     [0213] The increase of message signals by the addition of specific primers was further demonstrated in a one plate experiment employing the four tumor samples B. met 316A, B. met 317A, B. tumor 81D and B. tumor 209A.  
     [0214] Expression of a combination of mammaglobin, GABAπ, B305D (C form) and B726P in a panel of breast tumor and normal tissue samples was also detected using real-time PCR with a SYBR Green detection system instead of the Taqman probe approach. The results obtained using this system are shown in FIG. 7.  
     Example 7  
     Quantitative PCR in Peripheral Blood of Breast Cancer Patients  
     [0215] The known genes evaluated in this study were mammaglobin and 7 aminobutyrate type A receptor π subunit (GABATπ). In order to identify novel genes which are over-expressed in breast cancer we have used an improved version of the differential display RT-PCR (DDPCR) technique (Liang et al., Science 257:967-971 (1993); Mou et al., Biochem Biophy Res Commun. 199:564-569 (1994)); cDNA library extraction methods (Hara et al., Blood 84:189-199 (1994)) and PCR subtraction (Diatchenko et al.,  Proc Natl Acad Sci USA,  93:6025-6030 (1996); Yang et al., Nucleic Acids Res. 27:1517-23 (1999)).  
     [0216] Differential display resulted in the recovery of two cDNA fragments designated as B305D and B311D (Houghton et al.,  Cancer Res.  40: Abstract #217, 32-33, (1999). B511S and B533S are two cDNA fragments isolated using cDNA library subtraction approach (manuscript in preparation) while the B726P cDNA fragment was derived from PCR subtraction (Jiang et al., Proceedings of the Amer Assoc Cancer Res. 40:Abstract #216, 32 (1999); Xu et al., Proceedings of the Amer Assoc Cancer Res. 40:Abstract #2115, 319 (1999); and Molesh et al., Proceedings of Amer Assoc Cancer Res. 41:Abstract #4330, 681 (2000).  
     [0217] Three of the novel genes, B311D, B533S and B726P, showed identical breast tissue expression profile by quantitative PCR analysis. These genes were evaluated in quantitative PCR on two different panels consisting of (a) breast tumor, breast normal and normal tissues and (b) panel of breast tumor metastases (primarily lymph nodes). Primers and probes used are shown in Table 1. The data for panel (a) is shown in FIG. 2 for all three genes. Overall, the expression profiles are comparable and are in the same rank order, however, the levels of expression are considerably different. B311D in general was expressed at lower levels than B533S and both less than B726P but all three were restricted to breast tissue. All three sequences were used to search against the Genbank database. Both B311D and B533S sequences contain different repetitive sequences and an ORF has not been identified for either. B726P is a novel gene, with mRNA splicing yielding several different putative ORF&#39;s.  
     [0218] The quantitative PCR confirmed there was a differential mRNA expression between normal breast tissue and breast tumors, with approximately 50% of breast tumors overexpressed these genes. When tested on a panel of distant metastases derived from breast cancers all three genes reacted with 14/21 metastases and presented similar profiles (data not shown). Interestingly, when tested on a prostate cancer panel, all three genes identified the same 3/24 prostate tumors but at much lower expression levels than in breast. This group of genes exhibited increasing levels of expression as a function of pathological stage of the tumor as shown for B533S.  
     [0219] More rigorous analysis of mammaglobin gene expression by microarray, and quantitative PCR showed expression at significant levels in skin and salivary gland and much lower levels in esophagus and trachea. B511S had a slightly different profile of reactivity on breast tumors and normal breast tissue when compared to mammaglobin, yet reacted with a similar subset of normal tissues as mammaglobin. Mammaglobin detected 14/24 of distant metastatic breast tumors, 31/42 breast tumors and exhibited ten-fold over-expression in tumors and metastases as compared to normal breast tissue. There was at least 300-fold over-expression of mammaglobin in normal breast tissue versus other negative normal tissues and tumors tested. B511S detected 33/42 breast tumors and 14/24 distant metastases. A combination of B511S with mammaglobin would be predicted to detect 38/42 breast tumors and 17/24 metastatic lesions. The quantitative level of expression of B511S and mammaglobin were also in similar ranges, in concordance with the microarray profiles observed for these two genes.  
     [0220] Certain genes complemented mammglobin&#39;s expression profile, i.e. were shown to express in tumors that mammaglobin did not. B305D was highly over-expressed in breast tumors, prostate tumors, normal prostate tissue and testis compared to normal tissues including normal breast tissue. Different splice variants of B305D were identified with the forms A and C being the most abundant. All forms tested had similar tissue profiles in quantitative PCR. The A and C forms contain ORF&#39;s of 320 and 385 aa, respectively. A known gene shown to be complementary with B305D, in breast tumors, was GABAπ. This tissue expression profile is in contrast to other GABAA receptors that typically have appreciable expression in neuronal tissues. An additional observation was that tissue expression profiling of this gene showed it to be over-expressed in breast tumors in an inverse relationship to the B305D gene (Table 3). GABAπ detected 15/25 tumors and 6/21 metastases including 4 tumors and 5 metastases missed by mammaglobin. In contrast, B305D detected 13/25 breast tumors and 8/21 metastases again including 3 tumors and 2 metastases missed by mammaglobin. A combination of just B305D and the GABAπ would be predicted to identify 22/25 breast tumors and 14/21 metastases. This combination detected 20/21 of the breast metastases as well as 25/25 breast tumors that were evaluated on the same panels for all three genes. The one breast metastasis that was negative for these three genes was strongly positive for B726P.  
     [0221] The use of microarray analysis followed by quantitative PCR provided a methodology to accurately determine the expression of breast cancer genes both in breast tissues (tumor and normal) as well as in normal tissues and to assess their diagnostic and therapeutic potential. Five novel genes and two known genes were evaluated using these techniques. Three of these genes B311D, B533S and B726P exhibited concordant mRNA expression and collectively the data is consistent with coordinated expression of these three loci at the level of transcription control. All three genes showed differential expression in breast tumors versus normal breast tissue and the level of overexpression appeared related to the pathological stage of the tumor. In the case of mammaglobin, expression was found in other tissues apart from breast tissue. Expression was seen in skin, salivary gland and to a much lesser degree in trachea.  
     [0222] Expression of GABAπ in breast tumors was also a novel observation. While the expression of several genes complemented that seen with mammaglobin, two genes in particular, B305D and GABAπ added to the diagnostic sensitivity of mammaglobin detection. A combination of these three genes detected 45/46 (97.8%) breast tumors and metastases evaluated. Inclusion of B726P enabled the detection of all 25 of the breast tumors and 21 distant metastases.  
     Example 8  
     Enrichment of Circulating Breast Cancer Cells by Immunocapture  
     [0223] This example discloses the enhanced sensitivity achieved by use of the immunocapture cell capture methodology for enrichment of circulating breast cancer cells.  
     [0224] To evaluate the presence of circulating tumor cells an immunocapture method was adopted to first enrich for epithelial cells prior to RT-PCR analysis. Epithelial cells were enriched from blood samples with an immunomagnetic bead separation method (Dynal A.S, Oslo, Norway) utilizing magnetic beads coated with monoclonal antibodies specific for glycopolypeptide antigens on the surface of human epithelial cells. (Exemplary suitable cell-surface antigens are described, for example, in Momburg, F. et al.,  Cancer Res.,  41:2883-91 (1997); Naume, B. et al.,  Journal of Hemotherapy.  6:103-113 (1997); Naume, B. et al., Int J Cancer. 78:556-60 (1998); Martin, V. M. et al., Exp Hematol., 26:252-64 (1998); Hildebrandt, M. et al.,  Exp Hematol.  25:57-65 (1997); Eaton, M. C. et al.,  Biotechniques  22:100-5 (1997); Brandt, B. et al.,  Clin Exp Metastases  14:399-408 (1996), each of which are incorporated herein by reference in their entirety. Cells isolated this way were lysed and the magnetic beads removed. The lysate was then processed for poly A +  mRNA isolation using magnetic beads (Dynabeads) coated with Oligo (dT) 25 After washing the beads in the kit buffer bead/polyA + RNA samples were finally suspended in 10 mM Tris HCl pH 8 and subjected to reverse transcription. The RNA was then subjected to Real time PCR using gene specific primers and probes with reaction conditions as outlined herein above. β-Actin content was also determined and used for normalization. Samples with gene of interest copies/ng β-actin greater than the mean of the normal samples+3 standard deviations were considered positive. Real time PCR on blood samples was performed exclusively using the Taqman™ procedure but extending to 50 cycles.  
     [0225] Mammaglobin mRNA using enrichment procedures was found to be detectable at much lower levels than when direct isolation was used. Whole blood samples from patients with metatastic breast cancer were subsequently treated with the immunomagnetic beads, polyA +  RNA was then isolated, cDNA made and run in quantitative PCR using two gene specific primers to mammaglobin and a fluorescent probe (Taqman™). As observed in breast cancer tissues, complementation was also seen in the detection of circulating tumor cells derived from breast cancers. Again, mammaglobin PCR detected circulating tumor cells in a high percentage of bloods, albeit at low levels, from metastatic breast cancer (20/32) when compared to the normal blood samples. Several of the other genes tested to date could further increase this detection rate; this includes B726P, B305D, B311D, B533S and GABAπ. A combination of all the genes tested indicates that 27/32 samples were positive by one or more of these genes.  
     Example 9  
     Multiplex Detection of Breast Tumors  
     [0226] Additional Multiplex Real-time PCR assays were established in order to simultaneously detect the expression of four breast cancer-specific genes: LipophilinB, Gaba (B899P), B305D-C and B726P. In contrast to detection approaches relying on expression analysis of single breast cancer-specific genes, this Multiplex assay was able to detect all breast tumor samples tested.  
     [0227] This Multiplex assay was designed to detect LipophilinB expression instead of Mammaglobin. Due to their similar expression profiles, LipophilinB can replace Mammaglobin in this Multiplex PCR assay for breast cancer detection. The assay was carried out as follows: LipophilinB, B899P (Gaba), B305D, and B726P specific primers, and specific Taqman probes, were used to analyze their combined mRNA expression profile in breast tumors. The primers and probes are shown below:  
     [0228] LipophilinB: Forward Primer (SEQ ID NO:33): 5′ TGCCCCTCCGGAAGCT. Reverse Primer (SEQ ID NO:34): 5′ CGTTTCTGAAGGGACATCTGATC. Probe (SEQ ID NO:35) (FAM-5′-3′-TAMRA): TTGCAGCCAAGTTAGGAGTGAAGAGATGCA.  
     [0229] GABA (B899P): Forward Primer (SEQ ID NO:36): 5′ AAGCCTCAGAGTCCTTCCAGTATG. Reverse Primer (SEQ ID NO:37): 5′ TTCAAATATAAGTGAAGAAAAAATTAGTAGATCAA. Probe (SEQ ID NO:38) (FAM-5′-3′-TAMRA): AATCCATTGTATCTTAGAACCGAGGGATTTGTTTAGA.  
     [0230] B305D (C form): Forward Primer (SEQ ID NO:39): 5′ AAAGCAGATGGTGGTTGAGGTT. Reverse Primer (SEQ ID NO:40): 5′ CCTGAGACCAAATGGCTTCTTC. Probe (SEQ ID NO:41) (FAM-5′-3′-TAMRA) ATTCCATGCCGGCTGCTTCTTCTG.  
     [0231] B726P: Forward Primer (SEQ ID NO:42): 5′ TCTGGTTTTCTCATTCTTTATTCATTTATT. Reverse Primer (SEQ ID NO:43): 5′ TGCCAAGGAGCGGATTATCT. Probe (SEQ ID NO:44) (FAM-5′-3′-TAMRA): CAACCACGTGACAAACACTGGAATTACAGG.  
     [0232] Actin: Forward Primer (SEQ ID NO:45): 5′ ACTGGAACGGTGAAGGTGACA. Reverse Primer (SEQ ID NO 46): 5′ CGGCCACATTGTGAACTTTG. Probe (SEQ ID NO:47): (FAM-5′-3′-TAMRA): CAGTCGGTTGGAGCGAGCATCCC.  
     [0233] The assay conditions were:  
     [0234] Taqman protocol (7700 Perkin Elmer):  
     [0235] In 25 μl final volume: lx Buffer A, 5 mM MgCl, 0.2 mM dCTP, 0.2 mM dATP, 0.4 mM dUTP, 0.2 mM dGTP, 0.01 U/μl AmpErase UNG, 0.025 u/μl TaqGold, 8% (v/v) Glycerol, 0.05% (v/v) Gelatin, 0.01% (v/v) Tween20, 4 pmol of each gene specific Taqman probe (LipophilinB+Gaba+B305D+B726P), 100 nM of B726P-F+B726P-R, 300 nM of Gaba-R, and 50 nM of LipophilinB-F+LipophilinB-R +B305D-R+Gaba-R, template cDNA (originating from 0.02 μg polyA+RNA).  
     [0236] LipophilinB expression was detected in 14 out of 27 breast tumor samples. However, the Multiplex assay for LipophilinB, B899P, B305D-C and B726P detected an expression signal in 27 out of 27 tumors with the detection level above 10 mRNA copies/1000 pg actin in the majority of samples and above 100 mRNA copies/1000 pg actin in 5 out of the 27 samples tested (FIG. 8).  
     Example 10  
     Multiplex Detection Optimization  
     [0237] The Multiplex Real-time PCR assay described above was used to detect the expression of Mammaglobin (or LipophilinB), Gaba (B899P), B305D-C and B726P simultaneously. According to this Example, assay conditions and primer sequences were optimized to achieve parallel amplification of four PCR products with different lengths. Positive samples of this assay can be further characterized by gel electrophoresis and the expressed gene(s) of interest can be determined according to the detected amplicon size(s).  
     [0238] Mammaglobin (or LipophilinB), Gaba (B899P), B305D and B726P specific primers and specific Taqman probes were used to simultaneously detect their expression. The primers and probes used in this example are shown below.  
     [0239] Mammaglobin: Forward Primer (SEQ ID NO:48): 5′ TGCCATAGATGAATTGAAGGAATG. Reverse Primer (SEQ ID NO:49): 5′ TGTCATATATTAATTGCATAAACACCTCA. Probe (SEQ ID NO:50): (FAM-5′-3′-TAMRA): TCTTAACCAAACGGATGAAACTCTGAGCAATG.  
     [0240] GABA (B899P): Forward Primer (SEQ ID NO:36): 5′ AAGCCTCAGAGTCCTTCCAGTATG. Reverse Primer (SEQ ID NO:51): 5′ ATCATTGAAAATTCAAATATAAGTGAAG. Probe (SEQ ID NO:38) (FAM-5′-3′-TAMRA) AATCCATTGTATCTTAGAACCGAGGGATTTGTTTAGA.  
     [0241] B305D (C form): Forward Primer (SEQ ID NO:39): 5′ AAAGCAGATGGTGGTTGAGGTT. Reverse Primer (SEQ ID NO:40): 5′ CCTGAGACCAAATGGCTTCTTC. Probe (SEQ ID NO:41): (FAM-5′-3′-TAMRA): ATTCCATGCCGGCTGCTTCTTCTG.  
     [0242] B726P: Forward Primer (SEQ ID NO:52): 5′ GTAGTTGTGCATTGAAATAATTATCATTAT. Reverse Primer (SEQ ID NO:43): 5′ TGCCAAGGAGCGGATTATCT. Probe (SEQ ID NO:44) (FAM-5′-3′-TAMRA): CAACCACGTGACAAACACTGGAATTACAGG.  
     [0243] Primer locations and assay conditions were optimized to achieve parallel amplification of four PCR products with different sizes. The assay conditions were:  
     [0244] Tagman protocol (7700 Perkin Elmer):  
     [0245] In 25 μl final volume: lx Buffer A, 5 mM MgCl, 0.2 mM dCTP, 0.2 mM dATP, 0.4 mM dUTP, 0.2 mM dGTP, 0.01 U/μl AmpErase UNG, 0.0375 U/μl TaqGold, 8% (v/v) Glycerol, 0.05% (v/v) Gelatin, 0.01% (v/v) Tween20, 4 pmol of each gene specific Taqman probe (Mammaglobin+Gaba+B305D+B726P), 300 nM of Gaba-R+Gaba-F, 100 nM of Mammaglobin-F+R; B726P-F+R, and 50 nM of B305D-F+R template cDNA (originating from 0.02 (μg polyA+RNA).  
     [0246] PCR protocol:  
     [0247] 50° for 2′: x 1, 95° for 10′: X 1, and 95° for 15″/60° for 1′/68° for 1′: x 50.  
     [0248] Since each primer set in the multiplex assay results in a band of unique length, expression signals of the four genes of interest can be measured individually by agarose gel analysis (see, FIG. 9), or the combined expression signal of all four genes can be measured in real-time on an ABI 7700 Prism sequence detection system (PE Biosystems, Foster City, Calif.). The expression of LipophilinB can also be detected instead of Mammaglobin. Although specific primers have been described herein, different primer sequences, different primer or probe labeling and different detection systems could be used to perform this multiplex assay. For example, a second fluorogenic reporter dye could be incorporated for parallel detection of a reference gene by real-time PCR. Or, for example a SYBR Green detection system could be used instead of the Taqman probe approach.  
     Example 11  
     Design and use of Genomic DNA-excluding, Intron-exon Border Spanning Primer Rairs for Breast Cancer Multiplex Assay  
     [0249] The Multiplex Real-time PCR assay described herein can detect the expression of Mammaglobin, Gaba (B899P), B305D-C and B726P simultaneously. The combined expression levels of these genes is measured in real-time on an ABI 7700 Prism sequence detection system (PE Biosystems, Foster City, Calif.). Individually expressed genes can also be identified due to different amplicon sizes via gel electrophoresis. In order to use this assay with samples derived from non-DNase treated RNAs (e.g. lymph node cDNA) and to avoid DNase-treatment for small RNA-samples (e.g. from blood specimens, tumor and lymph node aspirates), intron-spanning primer pairs have been designed to exclude the amplification of genomic DNA and therefore to eliminate nonspecific and false positive signals. False positive signal is caused by genomic DNA contamination in cDNA specimens. The optimized Multiplex assay described herein excludes the amplification of genomic DNA and allows specific detection of target gene expression without the necessity of prior DNase treatment of RNA samples. Moreover the genomic match and the location of the Intron-Exon border could be verified with these primer sets.  
     [0250] Mammaglobin, Gaba (B899P), B305D and B726P specific primers and specific Taqman probes were used to simultaneously detect their expression (Table 7). Primer locations were optimized (Intron-Exon border spanning) to exclusively detect cDNA and to exclude genomic DNA from amplification. The identity of the expressed gene(s) was determined by gel electrophoresis.  
               TABLE 7                          Intron-Exon border Spanning Primer and Probe Sequences       for Breast Tumor Multiples Assay                                         Taqman probe       Gene   Forward Primer   Reverse Primer   (FAM-5′- 3′TAMRA)               Mammaglobin   tgccatagatgaattgaagga   tgtcatatattaattgcataaacacct   tcttaaccaaacggatgaaactctgagca           atg (SEQ ID NO:48)   ca (SEQ ID NO:49)   atg (SEQ ID NO:50)       B899P   aagcctcagagtccttccagta   ttcaaatataagtgaagaaaaaatta   aatccattgtatcttagaaccgagggattt           tg (SEQ ID NO:36)   gtagatcaa (SEQ ID   gtt (SEQ ID NO:62)               NO:37)       B305D   aaagcagatggtggttgaggt   cctgagaccaaatggcttcttc   attccatgccggctgcttcttctg (SEQ           t (SEQ ID NO:39)   (SEQ ID NO:40)   ID NO:41)       B726P   tctggttttctcattctttattcatt   tgccaaggagcggattatct   caaccacgtgacaaacactggaattaca           tatt (SEQ ID NO:42)   (SEQ ID NO:43)   gg (SEQ ID NO:44)       Actin   actggaacggtgaaggtgac   cggccacattgtgaactttg   cagtcggttggagcgagcatccc           a (SEQ ID NO:45   (SEQ ID NO:46)   (SEQ ID NO:47)       B899P-INT   caattttggtggagaacccg   gctgtcggaggtatatggtg   catttcagagagtaacatggactacaca           (SEQ ID NO:53)   (SEQ ID NO:54)   (SEQ ID NO:55)       B305D-INT   tctgataaaggccgtacaatg   tcacgacttgctgtttttgctc   atcaaaaaacaagcatggcctcacacca           (SEQ ID NO:56)   (SEQ ID NO:57)   ct (SEQ ID NO:58)       B726P-INT   gcaagtgccaatgatcagagg   atatagactcaggtatacacact   tcccatcagaatccaaacaagaggaaga           (SEQ ID NO:59)   (SEQ ID NO:60)   tg (SEQ ID NO:61)                  
 
     [0251] Primer locations and assay conditions were optimized to achieve parallel amplification of the four PCR products. The assay conditions were as follows:  
     [0252] Tagman Protocol (7700 Perkin Elmer)  
     [0253] In 25 μl final volume: 1× Buffer A, 5 mM MgCl, 0.2 mM dCTP, 0.2 mM dATP, 0.4 mM dUTP, 0.2 mM dGTP, 0.01 U/AmpErase UNG, 8% (v/v) Glycerol, 0.05% (v/v) Gelatin, 0.01% (v/v) Tween20, 4 pmol of each gene specific Taqman probe (Mammaglobin+B899P-INT+B305D-INT+B726P-INT), 300 nM of B305D-INT-F; B899P-INT-F, 100 nM of Mammaglobin-F+R; B726P-INT-F +R, 50 nM of B899P-INT-R; B305D-INT-R, template cDNA (originating from 0.02 μg polyA+ RNA).  
     [0254] PCR Cycling Conditions  
     [0255] 1 cycle at 50° C. for 2 minutes, 1 cycle at 95° C. for 10 minutes, 50 cycles of 95° C. for 1 minute and 68° C. for 1 minute.  
     [0256]FIG. 10 shows a comparison of the multiplex assay using intron-exon border spanning primers (bottom panel) and the multiplex assay using non-optimized primers (top panel), to detect breast cancer cells in a panel of lymph node tissues. This experiment shows that reduction in background resulting from genomic DNA contamination in samples is achieved using the intron-exon spanning primers of the present invention.  
     Example 12  
     Multiplex Detection of Metastasized Breast Tumor Cells in Sentinel Lymph Node Biopsy Samples  
     [0257] Lymph node staging is important for determining appropriate adjuvant hormone and chemotherapy. In contrast to conventional axillary dissection a less invasive approach for staging of minimal residual disease is sentinel lymph node biopsy. Sentinel lymph node biopsy (SLNB) has the potential to improve detection of metastases and to provide prognostic values to lead to therapy with minimal morbidity associated with complete lymph node dissection. SLNB implements mapping of the one or two lymph nodes which primarily drain the tumor and therefore are most likely to harbor metastatic disease (the sentinel nodes). Routine pathological analysis of lymph nodes result in a high false-negative rate: one-third of women with pathologically negative lymph nodes develop recurrent disease [Bland: The Breast: Saunders 1991]. A more sensitive detection technique for tumor cells would be RT-PCR but its application is limited by lack of a single specific markers. The multimarker assay described above increases the likelihood of cancer detection across the population without producing false-positive results from normal lymph nodes.  
     [0258] As mentioned above, lymphatic afferents from a primary tumor drain into a single node, the sentinel lymph node, before drainage into the regional lymphatic basin occurs. Sentinel lymph nodes are located with dyes and/or radiolabelled colloid injected in the primary lesion site and sentinel lymph node biopsy allows pathological examination for micrometastatic deposits, staging of the axilla and therefore can avoid unnecessary axillary dissection. Nodal micrometastases can be located with staining (haematoxylin or eosin) or immunohistochemical analysis for cytokeratin proteins. Immunocytochemical staining techniques can produce frequent false-negative results by missing small metastatic foci due to inadequate sectioning of the node. Immunohistochemistry can result in false-positive results due to illegitimate expression of cytokeratins (reticulum cells) or in false-negative results when using the antibody Ber-Ep4 which corresponding antigen is not expressed on all tumor cells.  
     [0259] The multiplex assay described herein could provide a more sensitive detection tool for positive sentinel lymph nodes. Moreover the detection of breast cancer cells in bone marrow samples, peripheral blood and small needle aspiration samples is desirable for diagnosis and prognosis in breast cancer patients.  
     [0260] Twenty-two metastatic lymph node samples, in addition to 15 samples also previously analyzed and shown in FIG. 3A, were analyzed using the intron-exon border spanning multiplex PCR assay described herein. The results from this analysis are summarized in Table 8. Twenty-seven primary tumors were also analyzed and the results shown in Table 9. Twenty normal lymph node samples tested using this assay were all negative.  
               TABLE 8                          Multiples Real-time PCR Analysis of 37 Metastatic Lymph Nodes                                     breast metastatic   Mamma-               Multi-       lymph node samples   globin   B305D   B899P   B726P   plex               B. Met 317A   ++   +       +   +++       B. Met 318A           ++       +++       B. Met 595A   +           +   +++       B. Met 611A   +   +   +++       ++       B. Met 612A   ++   ++       +   ++       B. Met 614A       ++       ++   +++       B. Met 616A           +       ++       B. Met 618A   +++   +           +++       B. Met 620A   ++   ++       ++   +++       B. Met 621A   +   −++       +   +++       B. Met 624A           −+       +++       B. Met 625A       −+       ++   +       B. Met 627A       +       +   +       B. Met 629A   ++               +++       B. Met 631A   +       ++       +       1255   +++   ++       ++   ++       1257   +++   +   +   +   ++       769   +++           +   ++       1258   +   +   +       +       1259       ++   ++       +++       1250   +++   +       +   +++       1726   +++   +       +   +++       786   −++   +   +       +++       281-LI-r   +++               +++       289-L2   −+   +           ++       366-S   +               +       374-S+   +++   ++           +++       376-S   ++           +   ++       381-S   +   +           +       383-Sx   +++   ++           +++       496-M   +++   ++           +++       591-SI-A   +   +           +       652-I       +   ++       +++       772   −               +       777   +   +       ++   ++       778   +++               +++       779   +       ++       ++                  
 
     [0261]               TABLE 9                          Multiplex Real-time PCR Analysis of 27 Primary Breast Tumors                                     breast primary tumor   Mamma-               Multi-       samples   globin   B305D   B899P   B726P   plex               T443   +   ++       +++   +++       T457       +   +       ++       T395           ++       ++       T10A   −   +++       +++   +++       T446       +       ++   ++       T11C   +       +++       +++       T23B   +   ++           +++       T207A       ++           +       T437   +   +       ++   +++       T391   +   ++       +++   +++       T392   +   +           ++       TS76   +   ++           +++       T483   ++   +           +++       T81G   +   +   ++   ++   +++       T430   +       ++       ++       T465   +   +       +   ++       TS80           +       +       T469   +           +   −++       T467   +           ++   +++       T439       +           +       T387   ++       +   +   ++       T318           +       ++       T154A               +   +       T387A   +++       +   +   +++       T155A   +       ++   +   +       T209A       ++           ++       T208A       +       +   ++                    
     [0262] From the foregoing it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.  
    
     
       
         1 
         
           
             77  
           
           
             1  
             1851  
             DNA  
             Homo sapien  
           
            1 

tcatcaccat tgccagcagc ggcaccgtta gtcaggtttt ctgggaatcc cacatgagta     60 

cttccgtgtt cttcattctt cttcaatagc cataaatctt ctagctctgg ctggctgttt    120 

tcacttcctt taagcctttg tgactcttcc tctgatgtca gctttaagtc ttgttctgga    180 

ttgctgtttt cagaagagat ttttaacatc tgtttttctt tgtagtcaga aagtaactgg    240 

caaattacat gatgatgact agaaacagca tactctctgg ccgtctttcc agatcttgag    300 

aagatacatc aacattttgc tcaagtagag ggctgactat acttgctgat ccacaacata    360 

cagcaagtat gagagcagtt cttccatatc tatccagcgc atttaaattc gcttttttct    420 

tgattaaaaa tttcaccact tgctgttttt gctcatgtat accaagtagc agtggtgtga    480 

ggccatgctt gttttttgat tcgatatcag caccgtataa gagcagtgct ttggccatta    540 

atttatcttc attgtagaca gcatagtgta gagtggtatt tccatactca tctggaatat    600 

ttggatcagt gccatgttcc agcaacatta acgcacattc atcttcctgg cattgtacgg    660 

cctttgtcag agctgtcctc tttttgttgt caaggacatt aagttgacat cgtctgtcca    720 

gcacgagttt tactacttct gaattcccat tggcagaggc cagatgtaga gcagtcctct    780 

tttgcttgtc cctcttgttc acatccgtgt ccctgagcat gacgatgaga tcctttctgg    840 

ggactttacc ccaccaggca gctctgtgga gcttgtccag atcttctcca tggacgtggt    900 

acctgggatc catgaaggcg ctgtcatcgt agtctcccca agcgaccacg ttgctcttgc    960 

cgctcccctg cagcagggga agcagtggca gcaccacttg cacctcttgc tcccaagcgt   1020 

cttcacagag gagtcgttgt ggtctccaga agtgcccacg ttgctcttgc cgctccccct   1080 

gtccatccag ggaggaagaa atgcaggaaa tgaaagatgc atgcacgatg gtatactcct   1140 

cagccatcaa acttctggac agcaggtcac ttccagcaag gtggagaaag ctgtccaccc   1200 

acagaggatg agatccagaa accacaatat ccattcacaa acaaacactt ttcagccaga   1260 

cacaggtact gaaatcatgt catctgcggc aacatggtgg aacctaccca atcacacatc   1320 

aagagatgaa gacactgcag tatatctgca caacgtaata ctcttcatcc ataacaaaat   1380 

aatataattt tcctctggag ccatatggat gaactatgaa ggaagaactc cccgaagaag   1440 

ccagtcgcag agaagccaca ctgaagctct gtcctcagcc atcagcgcca cggacaggar   1500 

tgtgtttctt ccccagtgat gcagcctcaa gttatcccga agctgccgca gcacacggtg   1560 

gctcctgaga aacaccccag ctcttccggt ctaacacagg caagtcaata aatgtgataa   1620 

tcacataaac agaattaaaa gcaaagtcac ataagcatct caacagacac agaaaaggca   1680 

tttgacaaaa tccagcatcc ttgtatttat tgttgcagtt ctcagaggaa atgcttctaa   1740 

cttttcccca tttagtatta tgttggctgt gggcttgtca taggtggttt ttattacttt   1800 

aaggtatgtc ccttctatgc ctgttttgct gagggtttta attctcgtgc c            1851 

 
           
             2  
             329  
             PRT  
             Homo sapien  
           
            2 

Met Asp Ile Val Val Ser Gly Ser His Pro Leu Trp Val Asp Ser Phe 
 1               5                  10                  15 

Leu His Leu Ala Gly Ser Asp Leu Leu Ser Arg Ser Leu Met Ala Glu 
            20                  25                  30 

Glu Tyr Thr Ile Val His Ala Ser Phe Ile Ser Cys Ile Ser Ser Ser 
        35                  40                  45 

Leu Asp Gly Gln Gly Glu Arg Gln Glu Gln Arg Gly His Phe Trp Arg 
    50                  55                  60 

Pro Gln Arg Leu Leu Cys Glu Asp Ala Trp Glu Gln Glu Val Gln Val 
65                  70                  75                  80 

Val Leu Pro Leu Leu Pro Leu Leu Gln Gly Ser Gly Lys Ser Asn Val 
                85                  90                  95 

Val Ala Trp Gly Asp Tyr Asp Asp Ser Ala Phe Met Asp Pro Arg Tyr 
            100                 105                 110 

His Val His Gly Glu Asp Leu Asp Lys Leu His Arg Ala Ala Trp Trp 
        115                 120                 125 

Gly Lys Val Pro Arg Lys Asp Leu Ile Val Met Leu Arg Asp Thr Asp 
    130                 135                 140 

Val Asn Lys Arg Asp Lys Gln Lys Arg Thr Ala Leu His Leu Ala Ser 
145                 150                 155                 160 

Ala Asn Gly Asn Ser Glu Val Val Lys Leu Val Leu Asp Arg Arg Cys 
                165                 170                 175 

Gln Leu Asn Val Leu Asp Asn Lys Lys Arg Thr Ala Leu Thr Lys Ala 
            180                 185                 190 

Val Gln Cys Gln Glu Asp Glu Cys Ala Leu Met Leu Leu Glu His Gly 
        195                 200                 205 

Thr Asp Pro Asn Ile Pro Asp Glu Tyr Gly Asn Thr Thr Leu His Tyr 
    210                 215                 220 

Ala Val Tyr Asn Glu Asp Lys Leu Met Ala Lys Ala Leu Leu Leu Tyr 
225                 230                 235                 240 

Gly Ala Asp Ile Glu Ser Lys Asn Lys His Gly Leu Thr Pro Leu Leu 
                245                 250                 255 

Leu Gly Ile His Glu Gln Lys Gln Gln Val Val Lys Phe Leu Ile Lys 
            260                 265                 270 

Lys Lys Ala Asn Leu Asn Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu 
        275                 280                 285 

Ile Leu Ala Val Cys Cys Gly Ser Ala Ser Ile Val Ser Pro Leu Leu 
    290                 295                 300 

Glu Gln Asn Val Asp Val Ser Ser Gln Asp Leu Glu Arg Arg Pro Glu 
305                 310                 315                 320 

Ser Met Leu Phe Leu Val Ile Ile Met 
                325 

 
           
             3  
             1852  
             DNA  
             Homo sapiens  
           
            3 

ggcacgagaa ttaaaaccct cagcaaaaca ggcatagaag ggacatacct taaagtaata     60 

aaaaccacct atgacaagcc cacagccaac ataatactaa atggggaaaa gttagaagca    120 

tttcctctga gaactgcaac aataaataca aggatgctgg attttgtcaa atgccttttc    180 

tgtgtctgtt gagatgctta tgtgactttg cttttaattc tgtttatgtg attatcacat    240 

ttattgactt gcctgtgtta gaccggaaga gctggggtgt ttctcaggag ccaccgtgtg    300 

ctgcggcagc ttcgggataa cttgaggctg catcactggg gaagaaacac aytcctgtcc    360 

gtggcgctga tggctgagga cagagcttca gtgtggcttc tctgcgactg gcttcttcgg    420 

ggagttcttc cttcatagtt catccatatg gctccagagg aaaattatat tattttgtta    480 

tggatgaaga gtattacgtt gtgcagatat actgcagtgt cttcatctct tgatgtgtga    540 

ttgggtaggt tccaccatgt tgccgcagat gacatgattt cagtacctgt gtctggctga    600 

aaagtgtttg tttgtgaatg gatattgtgg tttctggatc tcatcctctg tgggtggaca    660 

gctttctcca ccttgctgga agtgacctgc tgtccagaag tttgatggct gaggagtata    720 

ccatcgtgca tgcatctttc atttcctgca tttcttcctc cctggatgga cagggggagc    780 

ggcaagagca acgtgggcac ttctggagac cacaacgact cctctgtgaa gacgcttggg    840 

agcaagaggt gcaagtggtg ctgccactgc ttcccctgct gcagggggag cggcaagagc    900 

aacgtggtcg cttggggaga ctacgatgac agcgccttca tggatcccag gtaccacgtc    960 

catggagaag atctggacaa gctccacaga gctgcctggt ggggtaaagt ccccagaaag   1020 

gatctcatcg tcatgctcag ggacacggat gtgaacaaga gggacaagca aaagaggact   1080 

gctctacatc tggcctctgc caatgggaat tcagaagtag taaaactcgt gctggacaga   1140 

cgatgtcaac ttaatgtcct tgacaacaaa aagaggacag ctctgacaaa ggccgtacaa   1200 

tgccaggaag atgaatgtgc gttaatgttg ctggaacatg gcactgatcc aaatattcca   1260 

gatgagtatg gaaataccac tctacactat gctgtctaca atgaagataa attaatggcc   1320 

aaagcactgc tcttatacgg tgctgatatc gaatcaaaaa acaagcatgg cctcacacca   1380 

ctgctacttg gtatacatga gcaaaaacag caagtggtga aatttttaat caagaaaaaa   1440 

gcgaatttaa atgcgctgga tagatatgga agaactgctc tcatacttgc tgtatgttgt   1500 

ggatcagcaa gtatagtcag ccctctactt gagcaaaatg ttgatgtatc ttctcaagat   1560 

ctggaaagac ggccagagag tatgctgttt ctagtcatca tcatgtaatt tgccagttac   1620 

tttctgacta caaagaaaaa cagatgttaa aaatctcttc tgaaaacagc aatccagaac   1680 

aagacttaaa gctgacatca gaggaagagt cacaaaggct taaaggaagt gaaaacagcc   1740 

agccagagct agaagattta tggctattga agaagaatga agaacacgga agtactcatg   1800 

tgggattccc agaaaacctg actaacggtg ccgctgctgg caatggtgat ga           1852 

 
           
             4  
             292  
             PRT  
             Homo sapiens  
           
            4 

Met His Leu Ser Phe Pro Ala Phe Leu Pro Pro Trp Met Asp Arg Gly 
                  5                  10                  15 

Ser Gly Lys Ser Asn Val Gly Thr Ser Gly Asp His Asn Asp Ser Ser 
             20                  25                  30 

Val Lys Thr Leu Gly Ser Lys Arg Cys Lys Trp Cys Cys His Cys Phe 
         35                  40                  45 

Pro Cys Cys Arg Gly Ser Gly Lys Ser Asn Val Val Ala Trp Gly Asp 
     50                  55                  60 

Tyr Asp Asp Ser Ala Phe Met Asp Pro Arg Tyr His Val His Gly Glu 
 65                  70                  75                  80 

Asp Leu Asp Lys Leu His Arg Ala Ala Trp Trp Gly Lys Val Pro Arg 
                 85                  90                  95 

Lys Asp Leu Ile Val Met Leu Arg Asp Thr Asp Val Asn Lys Arg Asp 
            100                 105                 110 

Lys Gln Lys Arg Thr Ala Leu His Leu Ala Ser Ala Asn Gly Asn Ser 
        115                 120                 125 

Glu Val Val Lys Leu Val Leu Asp Arg Arg Cys Gln Leu Asn Val Leu 
    130                 135                 140 

Asp Asn Lys Lys Arg Thr Ala Leu Thr Lys Ala Val Gln Cys Gln Glu 
145                 150                 155                 160 

Asp Glu Cys Ala Leu Met Leu Leu Glu His Gly Thr Asp Pro Asn Ile 
                165                 170                 175 

Pro Asp Glu Tyr Gly Asn Thr Thr Leu His Tyr Ala Val Tyr Asn Glu 
            180                 185                 190 

Asp Lys Leu Met Ala Lys Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu 
        195                 200                 205 

Ser Lys Asn Lys His Gly Leu Thr Pro Leu Leu Leu Gly Ile His Glu 
    210                 215                 220 

Gln Lys Gln Gln Val Val Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu 
225                 230                 235                 240 

Asn Ala Leu Asp Arg Tyr Gly Arg Thr Ala Leu Ile Leu Ala Val Cys 
                245                 250                 255 

Cys Gly Ser Ala Ser Ile Val Ser Pro Leu Leu Glu Gln Asn Val Asp 
            260                 265                 270 

Val Ser Ser Gln Asp Leu Glu Arg Arg Pro Glu Ser Met Leu Phe Leu 
        275                 280                 285 

Val Ile Ile Met 
    290 

 
           
             5  
             1155  
             DNA  
             Homo sapien  
           
            5 

atggtggttg aggttgattc catgccggct gcctcttctg tgaagaagcc atttggtctc     60 

aggagcaaga tgggcaagtg gtgctgccgt tgcttcccct gctgcaggga gagcggcaag    120 

agcaacgtgg gcacttctgg agaccacgac gactctgcta tgaagacact caggagcaag    180 

atgggcaagt ggtgccgcca ctgcttcccc tgctgcaggg ggagtggcaa gagcaacgtg    240 

ggcgcttctg gagaccacga cgactctgct atgaagacac tcaggaacaa gatgggcaag    300 

tggtgctgcc actgcttccc ctgctgcagg gggagcggca agagcaaggt gggcgcttgg    360 

ggagactacg atgacagtgc cttcatggag cccaggtacc acgtccgtgg agaagatctg    420 

gacaagctcc acagagctgc ctggtggggt aaagtcccca gaaaggatct catcgtcatg    480 

ctcagggaca ctgacgtgaa caagaaggac aagcaaaaga ggactgctct acatctggcc    540 

tctgccaatg ggaattcaga agtagtaaaa ctcctgctgg acagacgatg tcaacttaat    600 

gtccttgaca acaaaaagag gacagctctg ataaaggccg tacaatgcca ggaagatgaa    660 

tgtgcgttaa tgttgctgga acatggcact gatccaaata ttccagatga gtatggaaat    720 

accactctgc actacgctat ctataatgaa gataaattaa tggccaaagc actgctctta    780 

tatggtgctg atatcgaatc aaaaaacaag catggcctca caccactgtt acttggtgta    840 

catgagcaaa aacagcaagt cgtgaaattt ttaatcaaga aaaaagcgaa tttaaatgca    900 

ctggatagat atggaaggac tgctctcata cttgctgtat gttgtggatc agcaagtata    960 

gtcagccttc tacttgagca aaatattgat gtatcttctc aagatctatc tggacagacg   1020 

gccagagagt atgctgtttc tagtcatcat catgtaattt gccagttact ttctgactac   1080 

aaagaaaaac agatgctaaa aatctcttct gaaaacagca atccagaaaa tgtctcaaga   1140 

accagaaata aataa                                                    1155 

 
           
             6  
             2000  
             DNA  
             Homo sapien  
           
            6 

atggtggttg aggttgattc catgccggct gcctcttctg tgaagaagcc atttggtctc     60 

aggagcaaga tgggcaagtg gtgctgccgt tgcttcccct gctgcaggga gagcggcaag    120 

agcaacgtgg gcacttctgg agaccacgac gactctgcta tgaagacact caggagcaag    180 

atgggcaagt ggtgccgcca ctgcttcccc tgctgcaggg ggagtggcaa gagcaacgtg    240 

ggcgcttctg gagaccacga cgactctgct atgaagacac tcaggaacaa gatgggcaag    300 

tggtgctgcc actgcttccc ctgctgcagg gggagcggca agagcaaggt gggcgcttgg    360 

ggagactacg atgacagtgc cttcatggag cccaggtacc acgtccgtgg agaagatctg    420 

gacaagctcc acagagctgc ctggtggggt aaagtcccca gaaaggatct catcgtcatg    480 

ctcagggaca ctgacgtgaa caagaaggac aagcaaaaga ggactgctct acatctggcc    540 

tctgccaatg ggaattcaga agtagtaaaa ctcctgctgg acagacgatg tcaacttaat    600 

gtccttgaca acaaaaagag gacagctctg ataaaggccg tacaatgcca ggaagatgaa    660 

tgtgcgttaa tgttgctgga acatggcact gatccaaata ttccagatga gtatggaaat    720 

accactctgc actacgctat ctataatgaa gataaattaa tggccaaagc actgctctta    780 

tatggtgctg atatcgaatc aaaaaacaag catggcctca caccactgtt acttggtgta    840 

catgagcaaa aacagcaagt cgtgaaattt ttaatcaaga aaaaagcgaa tttaaatgca    900 

ctggatagat atggaaggac tgctctcata cttgctgtat gttgtggatc agcaagtata    960 

gtcagccttc tacttgagca aaatattgat gtatcttctc aagatctatc tggacagacg   1020 

gccagagagt atgctgtttc tagtcatcat catgtaattt gccagttact ttctgactac   1080 

aaagaaaaac agatgctaaa aatctcttct gaaaacagca atccagaaca agacttaaag   1140 

ctgacatcag aggaagagtc acaaaggttc aaaggcagtg aaaatagcca gccagagaaa   1200 

atgtctcaag aaccagaaat aaataaggat ggtgatagag aggttgaaga agaaatgaag   1260 

aagcatgaaa gtaataatgt gggattacta gaaaacctga ctaatggtgt cactgctggc   1320 

aatggtgata atggattaat tcctcaaagg aagagcagaa cacctgaaaa tcagcaattt   1380 

cctgacaacg aaagtgaaga gtatcacaga atttgcgaat tagtttctga ctacaaagaa   1440 

aaacagatgc caaaatactc ttctgaaaac agcaacccag aacaagactt aaagctgaca   1500 

tcagaggaag agtcacaaag gcttgagggc agtgaaaatg gccagccaga gctagaaaat   1560 

tttatggcta tcgaagaaat gaagaagcac ggaagtactc atgtcggatt cccagaaaac   1620 

ctgactaatg gtgccactgc tggcaatggt gatgatggat taattcctcc aaggaagagc   1680 

agaacacctg aaagccagca atttcctgac actgagaatg aagagtatca cagtgacgaa   1740 

caaaatgata ctcagaagca attttgtgaa gaacagaaca ctggaatatt acacgatgag   1800 

attctgattc atgaagaaaa gcagatagaa gtggttgaaa aaatgaattc tgagctttct   1860 

cttagttgta agaaagaaaa agacatcttg catgaaaata gtacgttgcg ggaagaaatt   1920 

gccatgctaa gactggagct agacacaatg aaacatcaga gccagctaaa aaaaaaaaaa   1980 

aaaaaaaaaa aaaaaaaaaa                                               2000 

 
           
             7  
             2040  
             DNA  
             Homo sapien  
           
            7 

atggtggttg aggttgattc catgccggct gcctcttctg tgaagaagcc atttggtctc     60 

aggagcaaga tgggcaagtg gtgctgccgt tgcttcccct gctgcaggga gagcggcaag    120 

agcaacgtgg gcacttctgg agaccacgac gactctgcta tgaagacact caggagcaag    180 

atgggcaagt ggtgccgcca ctgcttcccc tgctgcaggg ggagtggcaa gagcaacgtg    240 

ggcgcttctg gagaccacga cgactctgct atgaagacac tcaggaacaa gatgggcaag    300 

tggtgctgcc actgcttccc ctgctgcagg gggagcggca agagcaaggt gggcgcttgg    360 

ggagactacg atgacagtgc cttcatggag cccaggtacc acgtccgtgg agaagatctg    420 

gacaagctcc acagagctgc ctggtggggt aaagtcccca gaaaggatct catcgtcatg    480 

ctcagggaca ctgacgtgaa caagaaggac aagcaaaaga ggactgctct acatctggcc    540 

tctgccaatg ggaattcaga agtagtaaaa ctcctgctgg acagacgatg tcaacttaat    600 

gtccttgaca acaaaaagag gacagctctg ataaaggccg tacaatgcca ggaagatgaa    660 

tgtgcgttaa tgttgctgga acatggcact gatccaaata ttccagatga gtatggaaat    720 

accactctgc actacgctat ctataatgaa gataaattaa tggccaaagc actgctctta    780 

tatggtgctg atatcgaatc aaaaaacaag catggcctca caccactgtt acttggtgta    840 

catgagcaaa aacagcaagt cgtgaaattt ttaatcaaga aaaaagcgaa tttaaatgca    900 

ctggatagat atggaaggac tgctctcata cttgctgtat gttgtggatc agcaagtata    960 

gtcagccttc tacttgagca aaatattgat gtatcttctc aagatctatc tggacagacg   1020 

gccagagagt atgctgtttc tagtcatcat catgtaattt gccagttact ttctgactac   1080 

aaagaaaaac agatgctaaa aatctcttct gaaaacagca atccagaaca agacttaaag   1140 

ctgacatcag aggaagagtc acaaaggttc aaaggcagtg aaaatagcca gccagagaaa   1200 

atgtctcaag aaccagaaat aaataaggat ggtgatagag aggttgaaga agaaatgaag   1260 

aagcatgaaa gtaataatgt gggattacta gaaaacctga ctaatggtgt cactgctggc   1320 

aatggtgata atggattaat tcctcaaagg aagagcagaa cacctgaaaa tcagcaattt   1380 

cctgacaacg aaagtgaaga gtatcacaga atttgcgaat tagtttctga ctacaaagaa   1440 

aaacagatgc caaaatactc ttctgaaaac agcaacccag aacaagactt aaagctgaca   1500 

tcagaggaag agtcacaaag gcttgagggc agtgaaaatg gccagccaga gaaaagatct   1560 

caagaaccag aaataaataa ggatggtgat agagagctag aaaattttat ggctatcgaa   1620 

gaaatgaaga agcacggaag tactcatgtc ggattcccag aaaacctgac taatggtgcc   1680 

actgctggca atggtgatga tggattaatt cctccaagga agagcagaac acctgaaagc   1740 

cagcaatttc ctgacactga gaatgaagag tatcacagtg acgaacaaaa tgatactcag   1800 

aagcaatttt gtgaagaaca gaacactgga atattacacg atgagattct gattcatgaa   1860 

gaaaagcaga tagaagtggt tgaaaaaatg aattctgagc tttctcttag ttgtaagaaa   1920 

gaaaaagaca tcttgcatga aaatagtacg ttgcgggaag aaattgccat gctaagactg   1980 

gagctagaca caatgaaaca tcagagccag ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa   2040 

 
           
             8  
             384  
             PRT  
             Homo sapien  
           
            8 

Met Val Val Glu Val Asp Ser Met Pro Ala Ala Ser Ser Val Lys Lys 
 1               5                  10                  15 

Pro Phe Gly Leu Arg Ser Lys Met Gly Lys Trp Cys Cys Arg Cys Phe 
            20                  25                  30 

Pro Cys Cys Arg Glu Ser Gly Lys Ser Asn Val Gly Thr Ser Gly Asp 
        35                  40                  45 

His Asp Asp Ser Ala Met Lys Thr Leu Arg Ser Lys Met Gly Lys Trp 
    50                  55                  60 

Cys Arg His Cys Phe Pro Cys Cys Arg Gly Ser Gly Lys Ser Asn Val 
65                  70                  75                  80 

Gly Ala Ser Gly Asp His Asp Asp Ser Ala Met Lys Thr Leu Arg Asn 
                85                  90                  95 

Lys Met Gly Lys Trp Cys Cys His Cys Phe Pro Cys Cys Arg Gly Ser 
            100                 105                 110 

Gly Lys Ser Lys Val Gly Ala Trp Gly Asp Tyr Asp Asp Ser Ala Phe 
        115                 120                 125 

Met Glu Pro Arg Tyr His Val Arg Gly Glu Asp Leu Asp Lys Leu His 
    130                 135                 140 

Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys Asp Leu Ile Val Met 
145                 150                 155                 160 

Leu Arg Asp Thr Asp Val Asn Lys Lys Asp Lys Gln Lys Arg Thr Ala 
                165                 170                 175 

Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu Val Val Lys Leu Leu 
            180                 185                 190 

Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp Asn Lys Lys Arg Thr 
        195                 200                 205 

Ala Leu Ile Lys Ala Val Gln Cys Gln Glu Asp Glu Cys Ala Leu Met 
    210                 215                 220 

Leu Leu Glu His Gly Thr Asp Pro Asn Ile Pro Asp Glu Tyr Gly Asn 
225                 230                 235                 240 

Thr Thr Leu His Tyr Ala Ile Tyr Asn Glu Asp Lys Leu Met Ala Lys 
                245                 250                 255 

Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser Lys Asn Lys His Gly 
            260                 265                 270 

Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln Lys Gln Gln Val Val 
        275                 280                 285 

Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn Ala Leu Asp Arg Tyr 
    290                 295                 300 

Gly Arg Thr Ala Leu Ile Leu Ala Val Cys Cys Gly Ser Ala Ser Ile 
305                 310                 315                 320 

Val Ser Leu Leu Leu Glu Gln Asn Ile Asp Val Ser Ser Gln Asp Leu 
                325                 330                 335 

Ser Gly Gln Thr Ala Arg Glu Tyr Ala Val Ser Ser His His His Val 
            340                 345                 350 

Ile Cys Gln Leu Leu Ser Asp Tyr Lys Glu Lys Gln Met Leu Lys Ile 
        355                 360                 365 

Ser Ser Glu Asn Ser Asn Pro Glu Asn Val Ser Arg Thr Arg Asn Lys 
    370                 375                 380 

 
           
             9  
             656  
             PRT  
             Homo sapien  
           
            9 

Met Val Val Glu Val Asp Ser Met Pro Ala Ala Ser Ser Val Lys Lys 
 1               5                  10                  15 

Pro Phe Gly Leu Arg Ser Lys Met Gly Lys Trp Cys Cys Arg Cys Phe 
            20                  25                  30 

Pro Cys Cys Arg Glu Ser Gly Lys Ser Asn Val Gly Thr Ser Gly Asp 
        35                  40                  45 

His Asp Asp Ser Ala Met Lys Thr Leu Arg Ser Lys Met Gly Lys Trp 
    50                  55                  60 

Cys Arg His Cys Phe Pro Cys Cys Arg Gly Ser Gly Lys Ser Asn Val 
65                  70                  75                  80 

Gly Ala Ser Gly Asp His Asp Asp Ser Ala Met Lys Thr Leu Arg Asn 
                85                  90                  95 

Lys Met Gly Lys Trp Cys Cys His Cys Phe Pro Cys Cys Arg Gly Ser 
            100                 105                 110 

Gly Lys Ser Lys Val Gly Ala Trp Gly Asp Tyr Asp Asp Ser Ala Phe 
        115                 120                 125 

Met Glu Pro Arg Tyr His Val Arg Gly Glu Asp Leu Asp Lys Leu His 
    130                 135                 140 

Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys Asp Leu Ile Val Met 
145                 150                 155                 160 

Leu Arg Asp Thr Asp Val Asn Lys Lys Asp Lys Gln Lys Arg Thr Ala 
                165                 170                 175 

Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu Val Val Lys Leu Leu 
            180                 185                 190 

Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp Asn Lys Lys Arg Thr 
        195                 200                 205 

Ala Leu Ile Lys Ala Val Gln Cys Gln Glu Asp Glu Cys Ala Leu Met 
    210                 215                 220 

Leu Leu Glu His Gly Thr Asp Pro Asn Ile Pro Asp Glu Tyr Gly Asn 
225                 230                 235                 240 

Thr Thr Leu His Tyr Ala Ile Tyr Asn Glu Asp Lys Leu Met Ala Lys 
                245                 250                 255 

Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser Lys Asn Lys His Gly 
            260                 265                 270 

Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln Lys Gln Gln Val Val 
        275                 280                 285 

Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn Ala Leu Asp Arg Tyr 
    290                 295                 300 

Gly Arg Thr Ala Leu Ile Leu Ala Val Cys Cys Gly Ser Ala Ser Ile 
305                 310                 315                 320 

Val Ser Leu Leu Leu Glu Gln Asn Ile Asp Val Ser Ser Gln Asp Leu 
                325                 330                 335 

Ser Gly Gln Thr Ala Arg Glu Tyr Ala Val Ser Ser His His His Val 
            340                 345                 350 

Ile Cys Gln Leu Leu Ser Asp Tyr Lys Glu Lys Gln Met Leu Lys Ile 
        355                 360                 365 

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

Glu Glu Ser Gln Arg Phe Lys Gly Ser Glu Asn Ser Gln Pro Glu Lys 
385                 390                 395                 400 

Met Ser Gln Glu Pro Glu Ile Asn Lys Asp Gly Asp Arg Glu Val Glu 
                405                 410                 415 

Glu Glu Met Lys Lys His Glu Ser Asn Asn Val Gly Leu Leu Glu Asn 
            420                 425                 430 

Leu Thr Asn Gly Val Thr Ala Gly Asn Gly Asp Asn Gly Leu Ile Pro 
        435                 440                 445 

Gln Arg Lys Ser Arg Thr Pro Glu Asn Gln Gln Phe Pro Asp Asn Glu 
    450                 455                 460 

Ser Glu Glu Tyr His Arg Ile Cys Glu Leu Val Ser Asp Tyr Lys Glu 
465                 470                 475                 480 

Lys Gln Met Pro Lys Tyr Ser Ser Glu Asn Ser Asn Pro Glu Gln Asp 
                485                 490                 495 

Leu Lys Leu Thr Ser Glu Glu Glu Ser Gln Arg Leu Glu Gly Ser Glu 
            500                 505                 510 

Asn Gly Gln Pro Glu Leu Glu Asn Phe Met Ala Ile Glu Glu Met Lys 
        515                 520                 525 

Lys His Gly Ser Thr His Val Gly Phe Pro Glu Asn Leu Thr Asn Gly 
    530                 535                 540 

Ala Thr Ala Gly Asn Gly Asp Asp Gly Leu Ile Pro Pro Arg Lys Ser 
545                 550                 555                 560 

Arg Thr Pro Glu Ser Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr 
                565                 570                 575 

His Ser Asp Glu Gln Asn Asp Thr Gln Lys Gln Phe Cys Glu Glu Gln 
            580                 585                 590 

Asn Thr Gly Ile Leu His Asp Glu Ile Leu Ile His Glu Glu Lys Gln 
        595                 600                 605 

Ile Glu Val Val Glu Lys Met Asn Ser Glu Leu Ser Leu Ser Cys Lys 
    610                 615                 620 

Lys Glu Lys Asp Ile Leu His Glu Asn Ser Thr Leu Arg Glu Glu Ile 
625                 630                 635                 640 

Ala Met Leu Arg Leu Glu Leu Asp Thr Met Lys His Gln Ser Gln Leu 
                645                 650                 655 

 
           
             10  
             671  
             PRT  
             Homo sapien  
           
            10 

Met Val Val Glu Val Asp Ser Met Pro Ala Ala Ser Ser Val Lys Lys 
 1               5                  10                  15 

Pro Phe Gly Leu Arg Ser Lys Met Gly Lys Trp Cys Cys Arg Cys Phe 
            20                  25                  30 

Pro Cys Cys Arg Glu Ser Gly Lys Ser Asn Val Gly Thr Ser Gly Asp 
        35                  40                  45 

His Asp Asp Ser Ala Met Lys Thr Leu Arg Ser Lys Met Gly Lys Trp 
    50                  55                  60 

Cys Arg His Cys Phe Pro Cys Cys Arg Gly Ser Gly Lys Ser Asn Val 
65                  70                  75                  80 

Gly Ala Ser Gly Asp His Asp Asp Ser Ala Met Lys Thr Leu Arg Asn 
                85                  90                  95 

Lys Met Gly Lys Trp Cys Cys His Cys Phe Pro Cys Cys Arg Gly Ser 
            100                 105                 110 

Gly Lys Ser Lys Val Gly Ala Trp Gly Asp Tyr Asp Asp Ser Ala Phe 
        115                 120                 125 

Met Glu Pro Arg Tyr His Val Arg Gly Glu Asp Leu Asp Lys Leu His 
    130                 135                 140 

Arg Ala Ala Trp Trp Gly Lys Val Pro Arg Lys Asp Leu Ile Val Met 
145                 150                 155                 160 

Leu Arg Asp Thr Asp Val Asn Lys Lys Asp Lys Gln Lys Arg Thr Ala 
                165                 170                 175 

Leu His Leu Ala Ser Ala Asn Gly Asn Ser Glu Val Val Lys Leu Leu 
            180                 185                 190 

Leu Asp Arg Arg Cys Gln Leu Asn Val Leu Asp Asn Lys Lys Arg Thr 
        195                 200                 205 

Ala Leu Ile Lys Ala Val Gln Cys Gln Glu Asp Glu Cys Ala Leu Met 
    210                 215                 220 

Leu Leu Glu His Gly Thr Asp Pro Asn Ile Pro Asp Glu Tyr Gly Asn 
225                 230                 235                 240 

Thr Thr Leu His Tyr Ala Ile Tyr Asn Glu Asp Lys Leu Met Ala Lys 
                245                 250                 255 

Ala Leu Leu Leu Tyr Gly Ala Asp Ile Glu Ser Lys Asn Lys His Gly 
            260                 265                 270 

Leu Thr Pro Leu Leu Leu Gly Val His Glu Gln Lys Gln Gln Val Val 
        275                 280                 285 

Lys Phe Leu Ile Lys Lys Lys Ala Asn Leu Asn Ala Leu Asp Arg Tyr 
    290                 295                 300 

Gly Arg Thr Ala Leu Ile Leu Ala Val Cys Cys Gly Ser Ala Ser Ile 
305                 310                 315                 320 

Val Ser Leu Leu Leu Glu Gln Asn Ile Asp Val Ser Ser Gln Asp Leu 
                325                 330                 335 

Ser Gly Gln Thr Ala Arg Glu Tyr Ala Val Ser Ser His His His Val 
            340                 345                 350 

Ile Cys Gln Leu Leu Ser Asp Tyr Lys Glu Lys Gln Met Leu Lys Ile 
        355                 360                 365 

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

Glu Glu Ser Gln Arg Phe Lys Gly Ser Glu Asn Ser Gln Pro Glu Lys 
385                 390                 395                 400 

Met Ser Gln Glu Pro Glu Ile Asn Lys Asp Gly Asp Arg Glu Val Glu 
                405                 410                 415 

Glu Glu Met Lys Lys His Glu Ser Asn Asn Val Gly Leu Leu Glu Asn 
            420                 425                 430 

Leu Thr Asn Gly Val Thr Ala Gly Asn Gly Asp Asn Gly Leu Ile Pro 
        435                 440                 445 

Gln Arg Lys Ser Arg Thr Pro Glu Asn Gln Gln Phe Pro Asp Asn Glu 
    450                 455                 460 

Ser Glu Glu Tyr His Arg Ile Cys Glu Leu Val Ser Asp Tyr Lys Glu 
465                 470                 475                 480 

Lys Gln Met Pro Lys Tyr Ser Ser Glu Asn Ser Asn Pro Glu Gln Asp 
                485                 490                 495 

Leu Lys Leu Thr Ser Glu Glu Glu Ser Gln Arg Leu Glu Gly Ser Glu 
            500                 505                 510 

Asn Gly Gln Pro Glu Lys Arg Ser Gln Glu Pro Glu Ile Asn Lys Asp 
        515                 520                 525 

Gly Asp Arg Glu Leu Glu Asn Phe Met Ala Ile Glu Glu Met Lys Lys 
    530                 535                 540 

His Gly Ser Thr His Val Gly Phe Pro Glu Asn Leu Thr Asn Gly Ala 
545                 550                 555                 560 

Thr Ala Gly Asn Gly Asp Asp Gly Leu Ile Pro Pro Arg Lys Ser Arg 
                565                 570                 575 

Thr Pro Glu Ser Gln Gln Phe Pro Asp Thr Glu Asn Glu Glu Tyr His 
            580                 585                 590 

Ser Asp Glu Gln Asn Asp Thr Gln Lys Gln Phe Cys Glu Glu Gln Asn 
        595                 600                 605 

Thr Gly Ile Leu His Asp Glu Ile Leu Ile His Glu Glu Lys Gln Ile 
    610                 615                 620 

Glu Val Val Glu Lys Met Asn Ser Glu Leu Ser Leu Ser Cys Lys Lys 
625                 630                 635                 640 

Glu Lys Asp Ile Leu His Glu Asn Ser Thr Leu Arg Glu Glu Ile Ala 
                645                 650                 655 

Met Leu Arg Leu Glu Leu Asp Thr Met Lys His Gln Ser Gln Leu 
            660                 665                 670 

 
           
             11  
             800  
             DNA  
             Homo sapien  
           
            11 

atkagcttcc gcttctgaca acactagaga tccctcccct ccctcagggt atggccctcc     60 

acttcatttt tggtacataa catctttata ggacaggggt aaaatcccaa tactaacagg    120 

agaatgctta ggactctaac aggtttttga gaatgtgttg gtaagggcca ctcaatccaa    180 

tttttcttgg tcctccttgt ggtctaggag gacaggcaag ggtgcagatt ttcaagaatg    240 

catcagtaag ggccactaaa tccgaccttc ctcgttcctc cttgtggtct gggaggaaaa    300 

ctagtgtttc tgttgctgtg tcagtgagca caactattcc gatcagcagg gtccagggac    360 

cactgcaggt tcttgggcag ggggagaaac aaaacaaacc aaaaccatgg gcrgttttgt    420 

ctttcagatg ggaaacactc aggcatcaac aggctcacct ttgaaatgca tcctaagcca    480 

atgggacaaa tttgacccac aaaccctgga aaaagaggtg gctcattttt tttgcactat    540 

ggcttggccc caacattctc tctctgatgg ggaaaaatgg ccacctgagg gaagtacaga    600 

ttacaatact atcctgcagc ttgacctttt ctgtaagagg gaaggcaaat ggagtgaaat    660 

accttatgtc caagctttct tttcattgaa ggagaataca ctatgcaaag cttgaaattt    720 

acatcccaca ggaggacctc tcagcttacc cccatatcct agcctcccta tagctcccct    780 

tcctattagt gataagcctc                                                800 

 
           
             12  
             102  
             PRT  
             Homo sapien  
             
               VARIANT  
               (1)...(102)  
               Xaa = Any Amino Acid  
             
           
            12 

Met Gly Xaa Phe Val Phe Gln Met Gly Asn Thr Gln Ala Ser Thr Gly 
 1               5                  10                  15 

Ser Pro Leu Lys Cys Ile Leu Ser Gln Trp Asp Lys Phe Asp Pro Gln 
            20                  25                  30 

Thr Leu Glu Lys Glu Val Ala His Phe Phe Cys Thr Met Ala Trp Pro 
        35                  40                  45 

Gln His Ser Leu Ser Asp Gly Glu Lys Trp Pro Pro Glu Gly Ser Thr 
    50                  55                  60 

Asp Tyr Asn Thr Ile Leu Gln Leu Asp Leu Phe Cys Lys Arg Glu Gly 
65                  70                  75                  80 

Lys Trp Ser Glu Ile Pro Tyr Val Gln Ala Phe Phe Ser Leu Lys Glu 
                85                  90                  95 

Asn Thr Leu Cys Lys Ala 
            100 

 
           
             13  
             1206  
             DNA  
             Homo sapien  
           
            13 

ggcacgagga agttttgtgt actgaaaaag aaactgtcag aagcaaaaga aataaaatca     60 

cagttagaga accaaaaagt taaatgggaa caagagctct gcagtgtgag gtttctcaca    120 

ctcatgaaaa tgaaaattat ctcttacatg aaaattgcat gttgaaaaag gaaattgcca    180 

tgctaaaact ggaaatagcc acactgaaac accaatacca ggaaaaggaa aataaatact    240 

ttgaggacat taagatttta aaagaaaaga atgctgaact tcagatgacc ctaaaactga    300 

aagaggaatc attaactaaa agggcatctc aatatagtgg gcagcttaaa gttctgatag    360 

ctgagaacac aatgctcact tctaaattga aggaaaaaca agacaaagaa atactagagg    420 

cagaaattga atcacaccat cctagactgg cttctgctgt acaagaccat gatcaaattg    480 

tgacatcaag aaaaagtcaa gaacctgctt tccacattgc aggagatgct tgtttgcaaa    540 

gaaaaatgaa tgttgatgtg agtagtacga tatataacaa tgaggtgctc catcaaccac    600 

tttctgaagc tcaaaggaaa tccaaaagcc taaaaattaa tctcaattat gccggagatg    660 

ctctaagaga aaatacattg gtttcagaac atgcacaaag agaccaacgt gaaacacagt    720 

gtcaaatgaa ggaagctgaa cacatgtatc aaaacgaaca agataatgtg aacaaacaca    780 

ctgaacagca ggagtctcta gatcagaaat tatttcaact acaaagcaaa aatatgtggc    840 

ttcaacagca attagttcat gcacataaga aagctgacaa caaaagcaag ataacaattg    900 

atattcattt tcttgagagg aaaatgcaac atcatctcct aaaagagaaa aatgaggaga    960 

tatttaatta caataaccat ttaaaaaacc gtatatatca atatgaaaaa gagaaagcag   1020 

aaacagaagt tatataatag tataacactg ccaaggagcg gattatctca tcttcatcct   1080 

gtaattccag tgtttgtcac gtggttgttg aataaatgaa taaagaatga gaaaaccaga   1140 

agctctgata cataatcata atgataatta tttcaatgca caactacggg tggtgctgct   1200 

cgtgcc                                                              1206 

 
           
             14  
             317  
             PRT  
             Homo sapien  
           
            14 

Met Gly Thr Arg Ala Leu Gln Cys Glu Val Ser His Thr His Glu Asn 
 1               5                  10                  15 

Glu Asn Tyr Leu Leu His Glu Asn Cys Met Leu Lys Lys Glu Ile Ala 
            20                  25                  30 

Met Leu Lys Leu Glu Ile Ala Thr Leu Lys His Gln Tyr Gln Glu Lys 
        35                  40                  45 

Glu Asn Lys Tyr Phe Glu Asp Ile Lys Ile Leu Lys Glu Lys Asn Ala 
    50                  55                  60 

Glu Leu Gln Met Thr Leu Lys Leu Lys Glu Glu Ser Leu Thr Lys Arg 
65                  70                  75                  80 

Ala Ser Gln Tyr Ser Gly Gln Leu Lys Val Leu Ile Ala Glu Asn Thr 
                85                  90                  95 

Met Leu Thr Ser Lys Leu Lys Glu Lys Gln Asp Lys Glu Ile Leu Glu 
            100                 105                 110 

Ala Glu Ile Glu Ser His His Pro Arg Leu Ala Ser Ala Val Gln Asp 
        115                 120                 125 

His Asp Gln Ile Val Thr Ser Arg Lys Ser Gln Glu Pro Ala Phe His 
    130                 135                 140 

Ile Ala Gly Asp Ala Cys Leu Gln Arg Lys Met Asn Val Asp Val Ser 
145                 150                 155                 160 

Ser Thr Ile Tyr Asn Asn Glu Val Leu His Gln Pro Leu Ser Glu Ala 
                165                 170                 175 

Gln Arg Lys Ser Lys Ser Leu Lys Ile Asn Leu Asn Tyr Ala Gly Asp 
            180                 185                 190 

Ala Leu Arg Glu Asn Thr Leu Val Ser Glu His Ala Gln Arg Asp Gln 
        195                 200                 205 

Arg Glu Thr Gln Cys Gln Met Lys Glu Ala Glu His Met Tyr Gln Asn 
    210                 215                 220 

Glu Gln Asp Asn Val Asn Lys His Thr Glu Gln Gln Glu Ser Leu Asp 
225                 230                 235                 240 

Gln Lys Leu Phe Gln Leu Gln Ser Lys Asn Met Trp Leu Gln Gln Gln 
                245                 250                 255 

Leu Val His Ala His Lys Lys Ala Asp Asn Lys Ser Lys Ile Thr Ile 
            260                 265                 270 

Asp Ile His Phe Leu Glu Arg Lys Met Gln His His Leu Leu Lys Glu 
        275                 280                 285 

Lys Asn Glu Glu Ile Phe Asn Tyr Asn Asn His Leu Lys Asn Arg Ile 
    290                 295                 300 

Tyr Gln Tyr Glu Lys Glu Lys Ala Glu Thr Glu Val Ile 
305                 310                 315 

 
           
             15  
             1665  
             DNA  
             Homo sapien  
           
            15 

gcaaactttc aagcagagcc tcccgagaag ccatctgcct tcgagcctgc cattgaaatg     60 

caaaagtctg ttccaaataa agccttggaa ttgaagaatg aacaaacatt gagagcagat    120 

cagatgttcc cttcagaatc aaaacaaaag aaggttgaag aaaattcttg ggattctgag    180 

agtctccgtg agactgtttc acagaaggat gtgtgtgtac ccaaggctac acatcaaaaa    240 

gaaatggata aaataagtgg aaaattagaa gattcaacta gcctatcaaa aatcttggat    300 

acagttcatt cttgtgaaag agcaagggaa cttcaaaaag atcactgtga acaacgtaca    360 

ggaaaaatgg aacaaatgaa aaagaagttt tgtgtactga aaaagaaact gtcagaagca    420 

aaagaaataa aatcacagtt agagaaccaa aaagttaaat gggaacaaga gctctgcagt    480 

gtgaggtttc tcacactcat gaaaatgaaa attatctctt acatgaaaat tgcatgttga    540 

aaaaggaaat tgccatgcta aaactggaaa tagccacact gaaacaccaa taccaggaaa    600 

aggaaaataa atactttgag gacattaaga ttttaaaaga aaagaatgct gaacttcaga    660 

tgaccctaaa actgaaagag gaatcattaa ctaaaagggc atctcaatat agtgggcagc    720 

ttaaagttct gatagctgag aacacaatgc tcacttctaa attgaaggaa aaacaagaca    780 

aagaaatact agaggcagaa attgaatcac accatcctag actggcttct gctgtacaag    840 

accatgatca aattgtgaca tcaagaaaaa gtcaagaacc tgctttccac attgcaggag    900 

atgcttgttt gcaaagaaaa atgaatgttg atgtgagtag tacgatatat aacaatgagg    960 

tgctccatca accactttct gaagctcaaa ggaaatccaa aagcctaaaa attaatctca   1020 

attatgccgg agatgctcta agagaaaata cattggtttc agaacatgca caaagagacc   1080 

aacgtgaaac acagtgtcaa atgaaggaag ctgaacacat gtatcaaaac gaacaagata   1140 

atgtgaacaa acacactgaa cagcaggagt ctctagatca gaaattattt caactacaaa   1200 

gcaaaaatat gtggcttcaa cagcaattag ttcatgcaca taagaaagct gacaacaaaa   1260 

gcaagataac aattgatatt cattttcttg agaggaaaat gcaacatcat ctcctaaaag   1320 

agaaaaatga ggagatattt aattacaata accatttaaa aaaccgtata tatcaatatg   1380 

aaaaagagaa agcagaaaca gaaaactcat gagagacaag cagtaagaaa cttcttttgg   1440 

agaaacaaca gaccagatct ttactcacaa ctcatgctag gaggccagtc ctagcattac   1500 

cttatgttga aaatcttacc aatagtctgt gtcaacagaa tacttatttt agaagaaaaa   1560 

ttcatgattt cttcctgaag cctgggcgac agagcgagac tctgtctcaa aaaaaaaaaa   1620 

aaaaaaagaa agaaagaaat gcctgtgctt acttcgcttc ccagg                   1665 

 
           
             16  
             179  
             PRT  
             Homo sapien  
           
            16 

Ala Asn Phe Gln Ala Glu Pro Pro Glu Lys Pro Ser Ala Phe Glu Pro 
 1               5                  10                  15 

Ala Ile Glu Met Gln Lys Ser Val Pro Asn Lys Ala Leu Glu Leu Lys 
            20                  25                  30 

Asn Glu Gln Thr Leu Arg Ala Asp Gln Met Phe Pro Ser Glu Ser Lys 
        35                  40                  45 

Gln Lys Lys Val Glu Glu Asn Ser Trp Asp Ser Glu Ser Leu Arg Glu 
    50                  55                  60 

Thr Val Ser Gln Lys Asp Val Cys Val Pro Lys Ala Thr His Gln Lys 
65                  70                  75                  80 

Glu Met Asp Lys Ile Ser Gly Lys Leu Glu Asp Ser Thr Ser Leu Ser 
                85                  90                  95 

Lys Ile Leu Asp Thr Val His Ser Cys Glu Arg Ala Arg Glu Leu Gln 
            100                 105                 110 

Lys Asp His Cys Glu Gln Arg Thr Gly Lys Met Glu Gln Met Lys Lys 
        115                 120                 125 

Lys Phe Cys Val Leu Lys Lys Lys Leu Ser Glu Ala Lys Glu Ile Lys 
    130                 135                 140 

Ser Gln Leu Glu Asn Gln Lys Val Lys Trp Glu Gln Glu Leu Cys Ser 
145                 150                 155                 160 

Val Arg Phe Leu Thr Leu Met Lys Met Lys Ile Ile Ser Tyr Met Lys 
                165                 170                 175 

Ile Ala Cys 

 
           
             17  
             1681  
             DNA  
             Homo sapien  
           
            17 

gatacagtca ttcttgtgaa agagcaaggg aacttcaaaa agatcactgt gaacaacgta     60 

caggaaaaat ggaacaaatg aaaaagaagt tttgtgtact gaaaaagaaa ctgtcagaag    120 

caaaagaaat aaaatcacag ttagagaacc aaaaagttaa atgggaacaa gagctctgca    180 

gtgtgagatt gactttaaac caagaagaag agaagagaag aaatgccgat atattaaatg    240 

aaaaaattag ggaagaatta ggaagaatcg aagagcagca taggaaagag ttagaagtga    300 

aacaacaact tgaacaggct ctcagaatac aagatataga attgaagagt gtagaaagta    360 

atttgaatca ggtttctcac actcatgaaa atgaaaatta tctcttacat gaaaattgca    420 

tgttgaaaaa ggaaattgcc atgctaaaac tggaaatagc cacactgaaa caccaatacc    480 

aggaaaagga aaataaatac tttgaggaca ttaagatttt aaaagaaaag aatgctgaac    540 

ttcagatgac cctaaaactg aaagaggaat cattaactaa aagggcatct caatatagtg    600 

ggcagcttaa agttctgata gctgagaaca caatgctcac ttctaaattg aaggaaaaac    660 

aagacaaaga aatactagag gcagaaattg aatcacacca tcctagactg gcttctgctg    720 

tacaagacca tgatcaaatt gtgacatcaa gaaaaagtca agaacctgct ttccacattg    780 

caggagatgc ttgtttgcaa agaaaaatga atgttgatgt gagtagtacg atatataaca    840 

atgaggtgct ccatcaacca ctttctgaag ctcaaaggaa atccaaaagc ctaaaaatta    900 

atctcaatta tgccggagat gctctaagag aaaatacatt ggtttcagaa catgcacaaa    960 

gagaccaacg tgaaacacag tgtcaaatga aggaagctga acacatgtat caaaacgaac   1020 

aagataatgt gaacaaacac actgaacagc aggagtctct agatcagaaa ttatttcaac   1080 

tacaaagcaa aaatatgtgg cttcaacagc aattagttca tgcacataag aaagctgaca   1140 

acaaaagcaa gataacaatt gatattcatt ttcttgagag gaaaatgcaa catcatctcc   1200 

taaaagagaa aaatgaggag atatttaatt acaataacca tttaaaaaac cgtatatatc   1260 

aatatgaaaa agagaaagca gaaacagaaa actcatgaga gacaagcagt aagaaacttc   1320 

ttttggagaa acaacagacc agatctttac tcacaactca tgctaggagg ccagtcctag   1380 

cattacctta tgttgaaaaa tcttaccaat agtctgtgtc aacagaatac ttattttaga   1440 

agaaaaattc atgatttctt cctgaagcct acagacataa aataacagtg tgaagaatta   1500 

cttgttcacg aattgcataa aagctgccca ggatttccat ctaccctgga tgatgccgga   1560 

gacatcattc aatccaacca gaatctcgct ctgtcactca ggctggagtg cagtgggcgc   1620 

aatctcggct cactgcaact ctgcctccca ggttcacgcc attctctggc acagcctccc   1680 

g                                                                   1681 

 
           
             18  
             432  
             PRT  
             Homo sapien  
           
            18 

Asp Thr Val His Ser Cys Glu Arg Ala Arg Glu Leu Gln Lys Asp His 
 1               5                  10                  15 

Cys Glu Gln Arg Thr Gly Lys Met Glu Gln Met Lys Lys Lys Phe Cys 
            20                  25                  30 

Val Leu Lys Lys Lys Leu Ser Glu Ala Lys Glu Ile Lys Ser Gln Leu 
        35                  40                  45 

Glu Asn Gln Lys Val Lys Trp Glu Gln Glu Leu Cys Ser Val Arg Leu 
    50                  55                  60 

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

Glu Lys Ile Arg Glu Glu Leu Gly Arg Ile Glu Glu Gln His Arg Lys 
                85                  90                  95 

Glu Leu Glu Val Lys Gln Gln Leu Glu Gln Ala Leu Arg Ile Gln Asp 
            100                 105                 110 

Ile Glu Leu Lys Ser Val Glu Ser Asn Leu Asn Gln Val Ser His Thr 
        115                 120                 125 

His Glu Asn Glu Asn Tyr Leu Leu His Glu Asn Cys Met Leu Lys Lys 
    130                 135                 140 

Glu Ile Ala Met Leu Lys Leu Glu Ile Ala Thr Leu Lys His Gln Tyr 
145                 150                 155                 160 

Gln Glu Lys Glu Asn Lys Tyr Phe Glu Asp Ile Lys Ile Leu Lys Glu 
                165                 170                 175 

Lys Asn Ala Glu Leu Gln Met Thr Leu Lys Leu Lys Glu Glu Ser Leu 
            180                 185                 190 

Thr Lys Arg Ala Ser Gln Tyr Ser Gly Gln Leu Lys Val Leu Ile Ala 
        195                 200                 205 

Glu Asn Thr Met Leu Thr Ser Lys Leu Lys Glu Lys Gln Asp Lys Glu 
    210                 215                 220 

Ile Leu Glu Ala Glu Ile Glu Ser His His Pro Arg Leu Ala Ser Ala 
225                 230                 235                 240 

Val Gln Asp His Asp Gln Ile Val Thr Ser Arg Lys Ser Gln Glu Pro 
                245                 250                 255 

Ala Phe His Ile Ala Gly Asp Ala Cys Leu Gln Arg Lys Met Asn Val 
            260                 265                 270 

Asp Val Ser Ser Thr Ile Tyr Asn Asn Glu Val Leu His Gln Pro Leu 
        275                 280                 285 

Ser Glu Ala Gln Arg Lys Ser Lys Ser Leu Lys Ile Asn Leu Asn Tyr 
    290                 295                 300 

Ala Gly Asp Ala Leu Arg Glu Asn Thr Leu Val Ser Glu His Ala Gln 
305                 310                 315                 320 

Arg Asp Gln Arg Glu Thr Gln Cys Gln Met Lys Glu Ala Glu His Met 
                325                 330                 335 

Tyr Gln Asn Glu Gln Asp Asn Val Asn Lys His Thr Glu Gln Gln Glu 
            340                 345                 350 

Ser Leu Asp Gln Lys Leu Phe Gln Leu Gln Ser Lys Asn Met Trp Leu 
        355                 360                 365 

Gln Gln Gln Leu Val His Ala His Lys Lys Ala Asp Asn Lys Ser Lys 
    370                 375                 380 

Ile Thr Ile Asp Ile His Phe Leu Glu Arg Lys Met Gln His His Leu 
385                 390                 395                 400 

Leu Lys Glu Lys Asn Glu Glu Ile Phe Asn Tyr Asn Asn His Leu Lys 
                405                 410                 415 

Asn Arg Ile Tyr Gln Tyr Glu Lys Glu Lys Ala Glu Thr Glu Asn Ser 
            420                 425                 430 

 
           
             19  
             3681  
             DNA  
             Homo sapiens  
           
            19 

tccgagctga ttacagacac caaggaagat gctgtaaaga gtcagcagcc acagccctgg     60 

ctagctggcc ctgtgggcat ttattagtaa agttttaatg acaaaagctt tgagtcaaca    120 

cacccgtggg taattaacct ggtcatcccc accctggaga gccatcctgc ccatgggtga    180 

tcaaagaagg aacatctgca ggaacacctg atgaggctgc acccttggcg gaaagaacac    240 

ctgacacagc tgaaagcttg gtggaaaaaa cacctgatga ggctgcaccc ttggtggaaa    300 

gaacacctga cacggctgaa agcttggtgg aaaaaacacc tgatgaggct gcatccttgg    360 

tggagggaac atctgacaaa attcaatgtt tggagaaagc gacatctgga aagttcgaac    420 

agtcagcaga agaaacacct agggaaatta cgagtcctgc aaaagaaaca tctgagaaat    480 

ttacgtggcc agcaaaagga agacctagga agatcgcatg ggagaaaaaa gaagacacac    540 

ctagggaaat tatgagtccc gcaaaagaaa catctgagaa atttacgtgg gcagcaaaag    600 

gaagacctag gaagatcgca tgggagaaaa aagaaacacc tgtaaagact ggatgcgtgg    660 

caagagtaac atctaataaa actaaagttt tggaaaaagg aagatctaag atgattgcat    720 

gtcctacaaa agaatcatct acaaaagcaa gtgccaatga tcagaggttc ccatcagaat    780 

ccaaacaaga ggaagatgaa gaatattctt gtgattctcg gagtctcttt gagagttctg    840 

caaagattca agtgtgtata cctgagtcta tatatcaaaa agtaatggag ataaatagag    900 

aagtagaaga gcctcctaag aagccatctg ccttcaagcc tgccattgaa atgcaaaact    960 

ctgttccaaa taaagccttt gaattgaaga atgaacaaac attgagagca gatccgatgt   1020 

tcccaccaga atccaaacaa aaggactatg aagaaaattc ttgggattct gagagtctct   1080 

gtgagactgt ttcacagaag gatgtgtgtt tacccaaggc tacacatcaa aaagaaatag   1140 

ataaaataaa tggaaaatta gaagagtctc ctaataaaga tggtcttctg aaggctacct   1200 

gcggaatgaa agtttctatt ccaactaaag ccttagaatt gaaggacatg caaactttca   1260 

aagcagagcc tccggggaag ccatctgcct tcgagcctgc cactgaaatg caaaagtctg   1320 

tcccaaataa agccttggaa ttgaaaaatg aacaaacatt gagagcagat gagatactcc   1380 

catcagaatc caaacaaaag gactatgaag aaagttcttg ggattctgag agtctctgtg   1440 

agactgtttc acagaaggat gtgtgtttac ccaaggctrc rcatcaaaaa gaaatagata   1500 

aaataaatgg aaaattagaa gggtctcctg ttaaagatgg tcttctgaag gctaactgcg   1560 

gaatgaaagt ttctattcca actaaagcct tagaattgat ggacatgcaa actttcaaag   1620 

cagagcctcc cgagaagcca tctgccttcg agcctgccat tgaaatgcaa aagtctgttc   1680 

caaataaagc cttggaattg aagaatgaac aaacattgag agcagatgag atactcccat   1740 

cagaatccaa acaaaaggac tatgaagaaa gttcttggga ttctgagagt ctctgtgaga   1800 

ctgtttcaca gaaggatgtg tgtttaccca aggctrcrca tcaaaaagaa atagataaaa   1860 

taaatggaaa attagaagag tctcctgata atgatggttt tctgaaggct ccctgcagaa   1920 

tgaaagtttc tattccaact aaagccttag aattgatgga catgcaaact ttcaaagcag   1980 

agcctcccga gaagccatct gccttcgagc ctgccattga aatgcaaaag tctgttccaa   2040 

ataaagcctt ggaattgaag aatgaacaaa cattgagagc agatcagatg ttcccttcag   2100 

aatcaaaaca aaagaasgtt gaagaaaatt cttgggattc tgagagtctc cgtgagactg   2160 

tttcacagaa ggatgtgtgt gtacccaagg ctacacatca aaaagaaatg gataaaataa   2220 

gtggaaaatt agaagattca actagcctat caaaaatctt ggatacagtt cattcttgtg   2280 

aaagagcaag ggaacttcaa aaagatcact gtgaacaacg tacaggaaaa atggaacaaa   2340 

tgaaaaagaa gttttgtgta ctgaaaaaga aactgtcaga agcaaaagaa ataaaatcac   2400 

agttagagaa ccaaaaagtt aaatgggaac aagagctctg cagtgtgagg tttctcacac   2460 

tcatgaaaat gaaaattatc tcttacatga aaattgcatg ttgaaaaagg aaattgccat   2520 

gctaaaactg gaaatagcca cactgaaaca ccaataccag gaaaaggaaa ataaatactt   2580 

tgaggacatt aagattttaa aagaaaagaa tgctgaactt cagatgaccc taaaactgaa   2640 

agaggaatca ttaactaaaa gggcatctca atatagtggg cagcttaaag ttctgatagc   2700 

tgagaacaca atgctcactt ctaaattgaa ggaaaaacaa gacaaagaaa tactagaggc   2760 

agaaattgaa tcacaccatc ctagactggc ttctgctgta caagaccatg atcaaattgt   2820 

gacatcaaga aaaagtcaag aacctgcttt ccacattgca ggagatgctt gtttgcaaag   2880 

aaaaatgaat gttgatgtga gtagtacgat atataacaat gaggtgctcc atcaaccact   2940 

ttctgaagct caaaggaaat ccaaaagcct aaaaattaat ctcaattatg cmggagatgc   3000 

tctaagagaa aatacattgg tttcagaaca tgcacaaaga gaccaacgtg aaacacagtg   3060 

tcaaatgaag gaagctgaac acatgtatca aaacgaacaa gataatgtga acaaacacac   3120 

tgaacagcag gagtctctag atcagaaatt atttcaacta caaagcaaaa atatgtggct   3180 

tcaacagcaa ttagttcatg cacataagaa agctgacaac aaaagcaaga taacaattga   3240 

tattcatttt cttgagagga aaatgcaaca tcatctccta aaagagaaaa atgaggagat   3300 

atttaattac aataaccatt taaaaaaccg tatatatcaa tatgaaaaag agaaagcaga   3360 

aacagaaaac tcatgagaga caagcagtaa gaaacttctt ttggagaaac aacagaccag   3420 

atctttactc acaactcatg ctaggaggcc agtcctagca tcaccttatg ttgaaaatct   3480 

taccaatagt ctgtgtcaac agaatactta ttttagaaga aaaattcatg atttcttcct   3540 

gaagcctaca gacataaaat aacagtgtga agaattactt gttcacgaat tgcataaagc   3600 

tgcacaggat tcccatctac cctgatgatg cagcagacat cattcaatcc aaccagaatc   3660 

tcgctctgtc actcaggctg g                                             3681 

 
           
             20  
             1424  
             DNA  
             Homo sapiens  
           
            20 

tccgagctga ttacagacac caaggaagat gctgtaaaga gtcagcagcc acagccctgg     60 

ctagctggcc ctgtgggcat ttattagtaa agttttaatg acaaaagctt tgagtcaaca    120 

cacccgtggg taattaacct ggtcatcccc accctggaga gccatcctgc ccatgggtga    180 

tcaaagaagg aacatctgca ggaacacctg atgaggctgc acccttggcg gaaagaacac    240 

ctgacacagc tgaaagcttg gtggaaaaaa cacctgatga ggctgcaccc ttggtggaaa    300 

gaacacctga cacggctgaa agcttggtgg aaaaaacacc tgatgaggct gcatccttgg    360 

tggagggaac atctgacaaa attcaatgtt tggagaaagc gacatctgga aagttcgaac    420 

agtcagcaga agaaacacct agggaaatta cgagtcctgc aaaagaaaca tctgagaaat    480 

ttacgtggcc agcaaaagga agacctagga agatcgcatg ggagaaaaaa gaagacacac    540 

ctagggaaat tatgagtccc gcaaaagaaa catctgagaa atttacgtgg gcagcaaaag    600 

gaagacctag gaagatcgca tgggagaaaa aagaaacacc tgtaaagact ggatgcgtgg    660 

caagagtaac atctaataaa actaaagttt tggaaaaagg aagatctaag atgattgcat    720 

gtcctacaaa agaatcatct acaaaagcaa gtgccaatga tcagaggttc ccatcagaat    780 

ccaaacaaga ggaagatgaa gaatattctt gtgattctcg gagtctcttt gagagttctg    840 

caaagattca agtgtgtata cctgagtcta tatatcaaaa agtaatggag ataaatagag    900 

aagtagaaga gcctcctaag aagccatctg ccttcaagcc tgccattgaa atgcaaaact    960 

ctgttccaaa taaagccttt gaattgaaga atgaacaaac attgagagca gatccgatgt   1020 

tcccaccaga atccaaacaa aaggactatg aagaaaattc ttgggattct gagagtctct   1080 

gtgagactgt ttcacagaag gatgtgtgtt tacccaaggc tacacatcaa aaagaaatag   1140 

ataaaataaa tggaaaatta gaaggtaaga accgtttttt atttaaaaat cagttgaccg   1200 

aatatttctc taaactgatg aggagggata tcctctagta gctgaagaaa attacctcct   1260 

aaatgcaaac catggaaaaa aagagaagtg caatggtcgt aagttgtatg tctcatcagg   1320 

tgttggcaac agactatatt gagagtgctg aaaaggagct gaattattag tttgaattca   1380 

agatattgca agacctgaga gaaaaaaaaa aaaaaaaaaa aaaa                    1424 

 
           
             21  
             674  
             DNA  
             Homo sapiens  
           
            21 

attccgagct gattacagac accaaggaag atgctgtaaa gagtcagcag ccacagccct     60 

ggctagctgg ccctgtgggc atttattagt aaagttttaa tgacaaaagc tttgagtcaa    120 

cacacccgtg ggtaattaac ctggtcatcc ccaccctgga gagccatcct gcccatgggt    180 

gatcaaagaa ggaacatctg caggaacacc tgatgaggct gcacccttgg cggaaagaac    240 

acctgacaca gctgaaagct tggtggaaaa aacacctgat gaggctgcac ccttggtgga    300 

aagaacacct gacacggctg aaagcttggt ggaaaaaaca cctgatgagg ctgcatcctt    360 

ggtggaggga acatctgaca aaattcaatg tttggagaaa gcgacatctg gaaagttcga    420 

acagtcagca gaagaaacac ctagggaaat tacgagtcct gcaaaagaaa catctgagaa    480 

atttacgtgg ccagcaaaag gaagacctag gaagatcgca tgggagaaaa aagatgactc    540 

agttaaggca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa    600 

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa    660 

aaaaaaaaaa aaaa                                                      674 

 
           
             22  
             1729  
             DNA  
             Homo sapiens  
             
               unsure  
               (11)  
               n=A,T,C or G  
             
           
            22 

gaaagttcga ncagtcagca gaagaaacac ctagggaaat tacgagtcct gcaaaagaaa     60 

catctgagaa atttacgtgg ccagcaaaag gaagacctag gaagatcgca tgggagaaaa    120 

aagaagacac acctagggaa attatgagtc ccgcaaaaga aacatctgag aaatttacgt    180 

gggcagcaaa aggaagacct aggaagatcg catgggagaa aaaagaaaca cctgtaaaga    240 

ctggatgcgt ggcaagagta acatctaata aaactaaagt tttggaaaaa ggaagatcta    300 

agatgattgc atgtcctaca aaagaatcat ctacaaaagc aagtgccaat gatcagaggt    360 

tcccatcaga atccaaacaa gaggaagatg aagaatattc ttgtgattct cggagtctct    420 

ttgagagttc tgcaaagatt caagtgtgta tacctgagtc tatatatcaa aaagtaatgg    480 

agataaatag agaagtagaa gagcctccta agaagccatc tgccttcaag cctgccattg    540 

aaatgcaaaa ctctgttcca aataaagcct ttgaattgaa gaatgaacaa acattgagag    600 

cagatccgat gttcccacca gaatccaaac aaaaggacta tgaagaaaat tcttgggatt    660 

ctgagagtct ctgtgagact gtttcacaga aggatgtgtg tttacccaag gctacacatc    720 

aaaaagaaat agataaaata aatggaaaat tagaagagtc tcctaataaa gatggtcttc    780 

tgaaggctac ctgcggaatg aaagtttcta ttccaactaa agccttagaa ttgaaggaca    840 

tgcaaacttt caaagcagag cctccgggga agccatctgc cttcgagcct gccactgaaa    900 

tgcaaaagtc tgtcccaaat aaagccttgg aattgaaaaa tgaacaaaca ttgagagcag    960 

atgagatact cccatcagaa tccaaacaaa aggactatga agaaaattct tgggatactg   1020 

agagtctctg tgagactgtt tcacagaagg atgtgtgttt acccaaggct gcgcatcaaa   1080 

aagaaataga taaaataaat ggaaaattag aagggtctcc tggtaaanat ggtcttctga   1140 

aggctaactg cggaatgaaa gtttctattc caactaaagc cttagaattg atggacatgc   1200 

aaactttcaa agcagagcct cccgagaagc catctgcctt cgagcctgcc attgaaatgc   1260 

aaaagtctgt tccaaataaa gccttggaat tgaagaatga acaaacattg agagcagatg   1320 

agatactccc atcagaatcc aaacaaaagg actatgaaga aagttcttgg gattctgaga   1380 

gtctctgtga gactgtttca cagaaggatg tgtgtttacc caaggctgcg catcaaaaag   1440 

aaatagataa aataaatgga aaattagaag gtaagaaccg ttttttattt aaaaatcatt   1500 

tgaccaaata tttctctaaa ttgatgagga aggatatcct ctagtagctg aagaaaatta   1560 

cctcctaaat gcaaaccatg gaaaaaaaga gaagtgcaat ggtcataagc tatgtgtctc   1620 

atcaggcatt ggcaacagac tatattgtga gtgctgaaga ggagctgaat tactagttta   1680 

aattcaagat attccaagac gtgaggaaaa tgagaaaaaa aaaaaaaaa               1729 

 
           
             23  
             1337  
             DNA  
             Homo sapiens  
           
            23 

aaaaagaaat agataaaata aatggaaaat tagaagggtc tcctgttaaa gatggtcttc     60 

tgaaggctaa ctgcggaatg aaagtttcta ttccaactaa agccttagaa ttgatggaca    120 

tgcaaacttt caaagcagag cctcccgaga agccatctgc cttcgagcct gccattgaaa    180 

tgcaaaagtc tgttccaaat aaagccttgg aattgaagaa tgaacaaaca ttgagagcag    240 

atgagatact cccatcagaa tccaaacaaa aggactatga agaaagttct tgggattctg    300 

agagtctctg tgagactgtt tcacagaagg atgtgtgttt acccaaggct gcgcatcaaa    360 

aagaaataga taaaataaat ggaaaattag aagagtctcc tgataatgat ggttttctga    420 

aggctccctg cagaatgaaa gtttctattc caactaaagc cttagaattg atggacatgc    480 

aaactttcaa agcagagcct cccgagaagc catctgcctt cgagcctgcc attgaaatgc    540 

aaaagtctgt tccaaataaa gccttggaat tgaagaatga acaaacattg agagcagatc    600 

agatgttccc ttcagaatca aaacaaaaga aggttgaaga aaattcttgg gattctgaga    660 

gtctccgtga gactgtttca cagaaggatg tgtgtgtacc caaggctaca catcaaaaag    720 

aaatggataa aataagtgga aaattagaag attcaactag cctatcaaaa atcttggata    780 

cagttcattc ttgtgaaaga gcaagggaac ttcaaaaaga tcactgtgaa caacgtacag    840 

gaaaaatgga acaaatgaaa aagaagtttt gtgtactgaa aaagaaactg tcagaagcaa    900 

aagaaataaa atcacagtta gagaaccaaa aagttaaatg ggaacaagag ctctgcagtg    960 

tgagattgac tttaaaccaa gaagaagaga agagaagaaa tgccgatata ttaaatgaaa   1020 

aaattaggga agaattagga agaatcgaag agcagcatag gaaagagtta gaagtgaaac   1080 

aacaacttga acaggctctc agaatacaag atatagaatt gaagagtgta gaaagtaatt   1140 

tgaatcaggt ttctcacact catgaaaatg aaaattatct cttacatgaa aattgcatgt   1200 

tgaaaaagga aattgccatg ctaaaactgg aaatagccac actgaaacac caataccagg   1260 

aaaaggaaaa taaatacttt gaggacatta agattttaaa agaaaagaat gctgaacttc   1320 

agatgacccc tcgtgcc                                                  1337 

 
           
             24  
             2307  
             DNA  
             Homo sapiens  
           
            24 

attgagagca gatgagatac tcccatcaga atccaaacaa aaggactatg aagaaagttc     60 

ttgggattct gagagtctct gtgagactgt ttcacagaag gatgtgtgtt tacccaaggc    120 

tacacatcaa aaagaaatag ataaaataaa tggaaaatta gaagggtctc ctgttaaaga    180 

tggtcttctg aaggctaact gcggaatgaa agtttctatt ccaactaaag ccttagaatt    240 

gatggacatg caaactttca aagcagagcc tcccgagaag ccatctgcct tcgagcctgc    300 

cattgaaatg caaaagtctg ttccaaataa agccttggaa ttgaagaatg aacaaacatt    360 

gagagcagat gagatactcc catcagaatc caaacaaaag gactatgaag aaagttcttg    420 

ggattctgag agtctctgtg agactgtttc acagaaggat gtgtgtttac ccaaggctac    480 

acatcaaaaa gaaatagata aaataaatgg aaaattagaa gagtctcctg ataatgatgg    540 

ttttctgaag tctccctgca gaatgaaagt ttctattcca actaaagcct tagaattgat    600 

ggacatgcaa actttcaaag cagagcctcc cgagaagcca tctgccttcg agcctgccat    660 

tgaaatgcaa aagtctgttc caaataaagc cttggaattg aagaatgaac aaacattgag    720 

agcagatcag atgttccctt cagaatcaaa acaaaagaac gttgaagaaa attcttggga    780 

ttctgagagt ctccgtgaga ctgtttcaca gaaggatgtg tgtgtaccca aggctacaca    840 

tcaaaaagaa atggataaaa taagtggaaa attagaagat tcaactagcc tatcaaaaat    900 

cttggataca gttcattctt gtgaaagagc aagggaactt caaaaagatc actgtgaaca    960 

acgtacagga aaaatggaac aaatgaaaaa gaagttttgt gtactgaaaa agaaactgtc   1020 

agaagcaaaa gaaataaaat cacagttaga gaaccaaaaa gttaaatggg aacaagagct   1080 

ctgcagtgtg aggtttctca cactcatgaa aatgaaaatt atctcttaca tgaaaattgc   1140 

atgttgaaaa aggaaattgc catgctaaaa ctggaaatag ccacactgaa acaccaatac   1200 

caggaaaagg aaaataaata ctttgaggac attaagattt taaaagaaaa gaatgctgaa   1260 

cttcagatga ccctaaaact gaaagaggaa tcattaacta aaagggcatc tcaatatagt   1320 

gggcagctta aagttctgat agctgagaac acaatgctca cttctaaatt gaaggaaaaa   1380 

caagacaaag aaatactaga ggcagaaatt gaatcacacc atcctagact ggcttctgct   1440 

gtacaagacc atgatcaaat tgtgacatca agaaaaagtc aagaacctgc tttccacatt   1500 

gcaggagatg cttgtttgca aagaaaaatg aatgttgatg tgagtagtac gatatataac   1560 

aatgaggtgc tccatcaacc actttctgaa gctcaaagga aatccaaaag cctaaaaatt   1620 

aatctcaatt atgcaggaga tgctctaaga gaaaatacat tggtttcaga acatgcacaa   1680 

agagaccaac gtgaaacaca gtgtcaaatg aaggaagctg aacacatgta tcaaaacgaa   1740 

caagataatg tgaacaaaca cactgaacag caggagtctc tagatcagaa attatttcaa   1800 

ctacaaagca aaaatatgtg gcttcaacag caattagttc atgcacataa gaaagctgac   1860 

aacaaaagca agataacaat tgatattcat tttcttgaga ggaaaatgca acatcatctc   1920 

ctaaaagaga aaaatgagga gatatttaat tacaataacc atttaaaaaa ccgtatatat   1980 

caatatgaaa aagagaaagc agaaacagaa aactcatgag agacaagcag taagaaactt   2040 

cttttggaga aacaacagac cagatcttta ctcacaactc atgctaggag gccagtccta   2100 

gcatcacctt atgttgaaaa tcttaccaat agtctgtgtc aacagaatac ttattttaga   2160 

agaaaaattc atgatttctt cctgaagcct acagacataa aataacagtg tgaagaatta   2220 

cttgttcacg aattgcataa agctgcacag gattcccatc taccctgatg atgcagcaga   2280 

catcattcaa tccaaccaga atctcgc                                       2307 

 
           
             25  
             650  
             PRT  
             Homo sapiens  
             
               unsure  
               (310)  
               Xaa = Any Amino Acid  
             
           
            25 

Met Ser Pro Ala Lys Glu Thr Ser Glu Lys Phe Thr Trp Ala Ala Lys 
                  5                  10                  15 

Gly Arg Pro Arg Lys Ile Ala Trp Glu Lys Lys Glu Thr Pro Val Lys 
             20                  25                  30 

Thr Gly Cys Val Ala Arg Val Thr Ser Asn Lys Thr Lys Val Leu Glu 
         35                  40                  45 

Lys Gly Arg Ser Lys Met Ile Ala Cys Pro Thr Lys Glu Ser Ser Thr 
     50                  55                  60 

Lys Ala Ser Ala Asn Asp Gln Arg Phe Pro Ser Glu Ser Lys Gln Glu 
 65                  70                  75                  80 

Glu Asp Glu Glu Tyr Ser Cys Asp Ser Arg Ser Leu Phe Glu Ser Ser 
                 85                  90                  95 

Ala Lys Ile Gln Val Cys Ile Pro Glu Ser Ile Tyr Gln Lys Val Met 
            100                 105                 110 

Glu Ile Asn Arg Glu Val Glu Glu Pro Pro Lys Lys Pro Ser Ala Phe 
        115                 120                 125 

Lys Pro Ala Ile Glu Met Gln Asn Ser Val Pro Asn Lys Ala Phe Glu 
    130                 135                 140 

Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Pro Met Phe Pro Pro Glu 
145                 150                 155                 160 

Ser Lys Gln Lys Asp Tyr Glu Glu Asn Ser Trp Asp Ser Glu Ser Leu 
                165                 170                 175 

Cys Glu Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Thr His 
            180                 185                 190 

Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu Ser Pro Asn 
        195                 200                 205 

Lys Asp Gly Leu Leu Lys Ala Thr Cys Gly Met Lys Val Ser Ile Pro 
    210                 215                 220 

Thr Lys Ala Leu Glu Leu Lys Asp Met Gln Thr Phe Lys Ala Glu Pro 
225                 230                 235                 240 

Pro Gly Lys Pro Ser Ala Phe Glu Pro Ala Thr Glu Met Gln Lys Ser 
                245                 250                 255 

Val Pro Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala 
            260                 265                 270 

Asp Glu Ile Leu Pro Ser Glu Ser Lys Gln Lys Asp Tyr Glu Glu Ser 
        275                 280                 285 

Ser Trp Asp Ser Glu Ser Leu Cys Glu Thr Val Ser Gln Lys Asp Val 
    290                 295                 300 

Cys Leu Pro Lys Ala Xaa His Gln Lys Glu Ile Asp Lys Ile Asn Gly 
305                 310                 315                 320 

Lys Leu Glu Gly Ser Pro Val Lys Asp Gly Leu Leu Lys Ala Asn Cys 
                325                 330                 335 

Gly Met Lys Val Ser Ile Pro Thr Lys Ala Leu Glu Leu Met Asp Met 
            340                 345                 350 

Gln Thr Phe Lys Ala Glu Pro Pro Glu Lys Pro Ser Ala Phe Glu Pro 
        355                 360                 365 

Ala Ile Glu Met Gln Lys Ser Val Pro Asn Lys Ala Leu Glu Leu Lys 
    370                 375                 380 

Asn Glu Gln Thr Leu Arg Ala Asp Glu Ile Leu Pro Ser Glu Ser Lys 
385                 390                 395                 400 

Gln Lys Asp Tyr Glu Glu Ser Ser Trp Asp Ser Glu Ser Leu Cys Glu 
                405                 410                 415 

Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Xaa His Gln Lys 
            420                 425                 430 

Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu Ser Pro Asp Asn Asp 
        435                 440                 445 

Gly Phe Leu Lys Ala Pro Cys Arg Met Lys Val Ser Ile Pro Thr Lys 
    450                 455                 460 

Ala Leu Glu Leu Met Asp Met Gln Thr Phe Lys Ala Glu Pro Pro Glu 
465                 470                 475                 480 

Lys Pro Ser Ala Phe Glu Pro Ala Ile Glu Met Gln Lys Ser Val Pro 
                485                 490                 495 

Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Gln 
            500                 505                 510 

Met Phe Pro Ser Glu Ser Lys Gln Lys Xaa Val Glu Glu Asn Ser Trp 
        515                 520                 525 

Asp Ser Glu Ser Leu Arg Glu Thr Val Ser Gln Lys Asp Val Cys Val 
    530                 535                 540 

Pro Lys Ala Thr His Gln Lys Glu Met Asp Lys Ile Ser Gly Lys Leu 
545                 550                 555                 560 

Glu Asp Ser Thr Ser Leu Ser Lys Ile Leu Asp Thr Val His Ser Cys 
                565                 570                 575 

Glu Arg Ala Arg Glu Leu Gln Lys Asp His Cys Glu Gln Arg Thr Gly 
            580                 585                 590 

Lys Met Glu Gln Met Lys Lys Lys Phe Cys Val Leu Lys Lys Lys Leu 
        595                 600                 605 

Ser Glu Ala Lys Glu Ile Lys Ser Gln Leu Glu Asn Gln Lys Val Lys 
    610                 615                 620 

Trp Glu Gln Glu Leu Cys Ser Val Arg Phe Leu Thr Leu Met Lys Met 
625                 630                 635                 640 

Lys Ile Ile Ser Tyr Met Lys Ile Ala Cys 
                645                 650 

 
           
             26  
             228  
             PRT  
             Homo sapiens  
           
            26 

Met Ser Pro Ala Lys Glu Thr Ser Glu Lys Phe Thr Trp Ala Ala Lys 
                  5                  10                  15 

Gly Arg Pro Arg Lys Ile Ala Trp Glu Lys Lys Glu Thr Pro Val Lys 
             20                  25                  30 

Thr Gly Cys Val Ala Arg Val Thr Ser Asn Lys Thr Lys Val Leu Glu 
         35                  40                  45 

Lys Gly Arg Ser Lys Met Ile Ala Cys Pro Thr Lys Glu Ser Ser Thr 
     50                  55                  60 

Lys Ala Ser Ala Asn Asp Gln Arg Phe Pro Ser Glu Ser Lys Gln Glu 
 65                  70                  75                  80 

Glu Asp Glu Glu Tyr Ser Cys Asp Ser Arg Ser Leu Phe Glu Ser Ser 
                 85                  90                  95 

Ala Lys Ile Gln Val Cys Ile Pro Glu Ser Ile Tyr Gln Lys Val Met 
            100                 105                 110 

Glu Ile Asn Arg Glu Val Glu Glu Pro Pro Lys Lys Pro Ser Ala Phe 
        115                 120                 125 

Lys Pro Ala Ile Glu Met Gln Asn Ser Val Pro Asn Lys Ala Phe Glu 
    130                 135                 140 

Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Pro Met Phe Pro Pro Glu 
145                 150                 155                 160 

Ser Lys Gln Lys Asp Tyr Glu Glu Asn Ser Trp Asp Ser Glu Ser Leu 
                165                 170                 175 

Cys Glu Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Thr His 
            180                 185                 190 

Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Gly Lys Asn Arg 
        195                 200                 205 

Phe Leu Phe Lys Asn Gln Leu Thr Glu Tyr Phe Ser Lys Leu Met Arg 
    210                 215                 220 

Arg Asp Ile Leu 
225 

 
           
             27  
             154  
             PRT  
             Homo sapiens  
             
               unsure  
               (148)  
               Xaa = Any Amino Acid  
             
           
            27 

Met Arg Leu His Pro Trp Arg Lys Glu His Leu Thr Gln Leu Lys Ala 
                  5                  10                  15 

Trp Trp Lys Lys His Leu Met Arg Leu His Pro Trp Trp Lys Glu His 
             20                  25                  30 

Leu Thr Arg Leu Lys Ala Trp Trp Lys Lys His Leu Met Arg Leu His 
         35                  40                  45 

Pro Trp Trp Arg Glu His Leu Thr Lys Phe Asn Val Trp Arg Lys Arg 
     50                  55                  60 

His Leu Glu Ser Ser Asn Ser Gln Gln Lys Lys His Leu Gly Lys Leu 
 65                  70                  75                  80 

Arg Val Leu Gln Lys Lys His Leu Arg Asn Leu Arg Gly Gln Gln Lys 
                 85                  90                  95 

Glu Asp Leu Gly Arg Ser His Gly Arg Lys Lys Met Thr Gln Leu Arg 
            100                 105                 110 

Gln Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 
        115                 120                 125 

Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 
    130                 135                 140 

Lys Lys Lys Xaa Lys Lys Lys Lys Lys Lys 
145                 150 

 
           
             28  
             466  
             PRT  
             Homo sapiens  
             
               unsure  
               (329)  
               Xaa = Any Amino Acid  
             
           
            28 

Met Ser Pro Ala Lys Glu Thr Ser Glu Lys Phe Thr Trp Ala Ala Lys 
                  5                  10                  15 

Gly Arg Pro Arg Lys Ile Ala Trp Glu Lys Lys Glu Thr Pro Val Lys 
             20                  25                  30 

Thr Gly Cys Val Ala Arg Val Thr Ser Asn Lys Thr Lys Val Leu Glu 
         35                  40                  45 

Lys Gly Arg Ser Lys Met Ile Ala Cys Pro Thr Lys Glu Ser Ser Thr 
     50                  55                  60 

Lys Ala Ser Ala Asn Asp Gln Arg Phe Pro Ser Glu Ser Lys Gln Glu 
 65                  70                  75                  80 

Glu Asp Glu Glu Tyr Ser Cys Asp Ser Arg Ser Leu Phe Glu Ser Ser 
                 85                  90                  95 

Ala Lys Ile Gln Val Cys Ile Pro Glu Ser Ile Tyr Gln Lys Val Met 
            100                 105                 110 

Glu Ile Asn Arg Glu Val Glu Glu Pro Pro Lys Lys Pro Ser Ala Phe 
        115                 120                 125 

Lys Pro Ala Ile Glu Met Gln Asn Ser Val Pro Asn Lys Ala Phe Glu 
    130                 135                 140 

Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp Pro Met Phe Pro Pro Glu 
145                 150                 155                 160 

Ser Lys Gln Lys Asp Tyr Glu Glu Asn Ser Trp Asp Ser Glu Ser Leu 
                165                 170                 175 

Cys Glu Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Thr His 
            180                 185                 190 

Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Glu Ser Pro Asn 
        195                 200                 205 

Lys Asp Gly Leu Leu Lys Ala Thr Cys Gly Met Lys Val Ser Ile Pro 
    210                 215                 220 

Thr Lys Ala Leu Glu Leu Lys Asp Met Gln Thr Phe Lys Ala Glu Pro 
225                 230                 235                 240 

Pro Gly Lys Pro Ser Ala Phe Glu Pro Ala Thr Glu Met Gln Lys Ser 
                245                 250                 255 

Val Pro Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala 
            260                 265                 270 

Asp Glu Ile Leu Pro Ser Glu Ser Lys Gln Lys Asp Tyr Glu Glu Asn 
        275                 280                 285 

Ser Trp Asp Thr Glu Ser Leu Cys Glu Thr Val Ser Gln Lys Asp Val 
    290                 295                 300 

Cys Leu Pro Lys Ala Ala His Gln Lys Glu Ile Asp Lys Ile Asn Gly 
305                 310                 315                 320 

Lys Leu Glu Gly Ser Pro Gly Lys Xaa Gly Leu Leu Lys Ala Asn Cys 
                325                 330                 335 

Gly Met Lys Val Ser Ile Pro Thr Lys Ala Leu Glu Leu Met Asp Met 
            340                 345                 350 

Gln Thr Phe Lys Ala Glu Pro Pro Glu Lys Pro Ser Ala Phe Glu Pro 
        355                 360                 365 

Ala Ile Glu Met Gln Lys Ser Val Pro Asn Lys Ala Leu Glu Leu Lys 
    370                 375                 380 

Asn Glu Gln Thr Leu Arg Ala Asp Glu Ile Leu Pro Ser Glu Ser Lys 
385                 390                 395                 400 

Gln Lys Asp Tyr Glu Glu Ser Ser Trp Asp Ser Glu Ser Leu Cys Glu 
                405                 410                 415 

Thr Val Ser Gln Lys Asp Val Cys Leu Pro Lys Ala Ala His Gln Lys 
            420                 425                 430 

Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Gly Lys Asn Arg Phe Leu 
        435                 440                 445 

Phe Lys Asn His Leu Thr Lys Tyr Phe Ser Lys Leu Met Arg Lys Asp 
    450                 455                 460 

Ile Leu 
465 

 
           
             29  
             445  
             PRT  
             Homo sapiens  
           
            29 

Lys Glu Ile Asp Lys Ile Asn Gly Lys Leu Glu Gly Ser Pro Val Lys 
                  5                  10                  15 

Asp Gly Leu Leu Lys Ala Asn Cys Gly Met Lys Val Ser Ile Pro Thr 
             20                  25                  30 

Lys Ala Leu Glu Leu Met Asp Met Gln Thr Phe Lys Ala Glu Pro Pro 
         35                  40                  45 

Glu Lys Pro Ser Ala Phe Glu Pro Ala Ile Glu Met Gln Lys Ser Val 
     50                  55                  60 

Pro Asn Lys Ala Leu Glu Leu Lys Asn Glu Gln Thr Leu Arg Ala Asp 
 65                  70                  75                  80 

Glu Ile Leu Pro Ser Glu Ser Lys Gln Lys Asp Tyr Glu Glu Ser Ser 
                 85                  90                  95 

Trp Asp Ser Glu Ser Leu Cys Glu Thr Val Ser Gln Lys Asp Val Cys 
            100                 105                 110 

Leu Pro Lys Ala Ala His Gln Lys Glu Ile Asp Lys Ile Asn Gly Lys 
        115                 120                 125 

Leu Glu Glu Ser Pro Asp Asn Asp Gly Phe Leu Lys Ala Pro Cys Arg 
    130                 135                 140 

Met Lys Val Ser Ile Pro Thr Lys Ala Leu Glu Leu Met Asp Met Gln 
145                 150                 155                 160 

Thr Phe Lys Ala Glu Pro Pro Glu Lys Pro Ser Ala Phe Glu Pro Ala 
                165                 170                 175 

Ile Glu Met Gln Lys Ser Val Pro Asn Lys Ala Leu Glu Leu Lys Asn 
            180                 185                 190 

Glu Gln Thr Leu Arg Ala Asp Gln Met Phe Pro Ser Glu Ser Lys Gln 
        195                 200                 205 

Lys Lys Val Glu Glu Asn Ser Trp Asp Ser Glu Ser Leu Arg Glu Thr 
    210                 215                 220 

Val Ser Gln Lys Asp Val Cys Val Pro Lys Ala Thr His Gln Lys Glu 
225                 230                 235                 240 

Met Asp Lys Ile Ser Gly Lys Leu Glu Asp Ser Thr Ser Leu Ser Lys 
                245                 250                 255 

Ile Leu Asp Thr Val His Ser Cys Glu Arg Ala Arg Glu Leu Gln Lys 
            260                 265                 270 

Asp His Cys Glu Gln Arg Thr Gly Lys Met Glu Gln Met Lys Lys Lys 
        275                 280                 285 

Phe Cys Val Leu Lys Lys Lys Leu Ser Glu Ala Lys Glu Ile Lys Ser 
    290                 295                 300 

Gln Leu Glu Asn Gln Lys Val Lys Trp Glu Gln Glu Leu Cys Ser Val 
305                 310                 315                 320 

Arg Leu Thr Leu Asn Gln Glu Glu Glu Lys Arg Arg Asn Ala Asp Ile 
                325                 330                 335 

Leu Asn Glu Lys Ile Arg Glu Glu Leu Gly Arg Ile Glu Glu Gln His 
            340                 345                 350 

Arg Lys Glu Leu Glu Val Lys Gln Gln Leu Glu Gln Ala Leu Arg Ile 
        355                 360                 365 

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

His Thr His Glu Asn Glu Asn Tyr Leu Leu His Glu Asn Cys Met Leu 
385                 390                 395                 400 

Lys Lys Glu Ile Ala Met Leu Lys Leu Glu Ile Ala Thr Leu Lys His 
                405                 410                 415 

Gln Tyr Gln Glu Lys Glu Asn Lys Tyr Phe Glu Asp Ile Lys Ile Leu 
            420                 425                 430 

Lys Glu Lys Asn Ala Glu Leu Gln Met Thr Pro Arg Ala 
        435                 440                 445 

 
           
             30  
             578  
             DNA  
             Human  
           
            30 

cttgccttct cttaggcttt gaagcatttt tgtctgtgct ccctgatctt caggtcacca     60 

ccatgaagtt cttagcagtc ctggtactct tgggagtttc catctttctg gtctctgccc    120 

agaatccgac aacagctgct ccagctgaca cgtatccagc tactggtcct gctgatgatg    180 

aagcccctga tgctgaaacc actgctgctg caaccactgc gaccactgct gctcctacca    240 

ctgcaaccac cgctgcttct accactgctc gtaaagacat tccagtttta cccaaatggg    300 

ttggggatct cccgaatggt agagtgtgtc cctgagatgg aatcagcttg agtcttctgc    360 

aattggtcac aactattcat gcttcctgtg atttcatcca actacttacc ttgcctacga    420 

tatccccttt atctctaatc agtttatttt ctttcaaata aaaaataact atgagcaaca    480 

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa    540 

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa                            578 

 
           
             31  
             90  
             PRT  
             Homo sapien  
           
            31 

Met Lys Phe Leu Ala Val Leu Val Leu Leu Gly Val Ser Ile Phe Leu 
 1               5                  10                  15 

Val Ser Ala Gln Asn Pro Thr Thr Ala Ala Pro Ala Asp Thr Tyr Pro 
            20                  25                  30 

Ala Thr Gly Pro Ala Asp Asp Glu Ala Pro Asp Ala Glu Thr Thr Ala 
        35                  40                  45 

Ala Ala Thr Thr Ala Thr Thr Ala Ala Pro Thr Thr Ala Thr Thr Ala 
    50                  55                  60 

Ala Ser Thr Thr Ala Arg Lys Asp Ile Pro Val Leu Pro Lys Trp Val 
65                  70                  75                  80 

Gly Asp Leu Pro Asn Gly Arg Val Cys Pro 
                85                  90 

 
           
             32  
             3101  
             DNA  
             Homo sapien  
           
            32 

tgttggggcc tcagcctccc aagtagctgg gactacaggt gcctgccacc acgcccagct     60 

aattttttgt atatttttta gtagagacgg ggtttcaccg tggtctcaat ctcctgacct    120 

cgtgatctgc cagccttggc ctcccaaagt gtattctctt tttattatta ttattatttt    180 

tgagatggag tctgtctctg tcgcccaggc tggagtgcag tggtgcgatc tctgctcact    240 

gcaagctccg cctcctgggt tcatgccatt ctcctgcctc agcctcccga gtagctggga    300 

ctacaggccc ctgccaccac acccggctaa ttttttgtat ttttagtaga gacagggttt    360 

caccatgtta gccagggtgg tctctatctt ctgacctcgt gatccgcctg cctcagtctc    420 

tcaaagtgct gggattacag gcgtgagcca ccgcgaccag ccaactattg ctgtttattt    480 

ttaaatatat tttaaagaaa caattagatt tgttttcttt ctcattcttt tacttctact    540 

cttcatgtat gtataattat atttgtgttt tctattacct tttctccttt tactgtattg    600 

gactataata attgtgctca ctaatttctg ttcactaata ttatcagctt agataatact    660 

ttaattttta acttatatat tgagtattaa attgatcagt tttatttgta attatctatc    720 

ttccgcttgg ctgaatataa cttcttaagc ttataacttc ttgttctttc catgttattt    780 

ttttcttttt tttaatgtat tgaatttctt ctgacactca ttctagtaac ttttttctcg    840 

gtgtgcaacg taagttataa tttgtttctc agatttgaga tctgccataa gtttgaggct    900 

ttattttttt tttttatttg ctttatggca agtcggacaa cctgcatgga tttggcatca    960 

atgtagtcac ccatatctaa gagcagcact tgcttcttag catgatgagt tgtttctgga   1020 

ttgtttcttt attttactta tattcctggt agattcttat attttccctt caactctatt   1080 

cagcatttta ggaattctta ggactttctg agaattttag ctttctgtat taaatgtttt   1140 

taatgagtat tgcattttct caaaaagcac aaatatcaat agtgtacaca tgaggaaaac   1200 

tatatatata ttctgttgca gatgacagca tctcataaca aaatcctagt tacttcattt   1260 

aaaagacagc tctcctccaa tatactatga ggtaacaaaa atttgtagtg tgtaattttt   1320 

ttaatattag aaaactcatc ttacattgtg cacaaatttc tgaagtgata atacttcact   1380 

gtttttctat agaagtaact taatattggc aaaattactt atttgaattt aggttttggc   1440 

tttcatcata tacttcctca ttaacatttc cctcaatcca taaatgcaat ctcagtttga   1500 

atcttccatt taacccagaa gttaattttt aaaaccttaa taaaatttga atgtagctag   1560 

atattatttg ttggttacat attagtcaat aatttatatt acttacaatg atcagaaaat   1620 

atgatctgaa tttctgctgt cataaattca ataacgtatt ttaggcctaa acctttccat   1680 

ttcaaatcct tgggtctggt aattgaaaat aatcattatc ttttgttttc tggccaaaaa   1740 

tgctgcccat ttatttctat ccctaattag tcaaactttc taataaatgt atttaacgtt   1800 

aatgatgttt atttgcttgt tgtatactaa aaccattagt ttctataatt taaatgtcac   1860 

ctaatatgag tgaaaatgtg tcagaggctg gggaagaatg tggatggaga aagggaaggt   1920 

gttgatcaaa aagtacccaa gtttcagtta cacaggaggc atgagattga tctagtgcaa   1980 

aaaatgatga gtataataaa taataatgca ctgtatattt tgaaattgct aaaagtagat   2040 

ttaaaattga tttacataat attttacata tttataaagc acatgcaata tgttgttaca   2100 

tgtatagaat gtgcaacgat caagtcaggg tatctgtggt atccaccact ttgagcattt   2160 

atcgattcta tatgtcagga acatttcaag ttatctgttc tagcaaggaa atataaaata   2220 

cattatagtt aactatggcc tatctacagt gcaactaaac actagatttt attcctttcc   2280 

aactgtgggt ttgtattcat ttaccaccct cttttcattc cctttctcac ccacacactg   2340 

tgccgggcct caggcatata ctattctact gtctgtctct gtaaggatta tcattttagc   2400 

ttccacatat gagagaatgc atgcaaagtt tttctttcca tgtctggctt atttcactta   2460 

acaaaatgac ctccgcttcc atccatgtta tttatattac ccaatagtgt tcataaatat   2520 

atatacacac atatatacca cattgcattt gtccaattat tcattgacgg aaactggtta   2580 

atgttatatc gttgctattg tgaatagtgc tgcaataaac acgcaagtgg ggatataatt   2640 

tgaagagttt ttttgttgat gttccataca aattttaaga ttgttttgtc tatgtttgtg   2700 

aaaatggcgt tagtattttc atagagattg cattgaatct gtagattgct ttgggtaagt   2760 

atggttattt tgatggtatt aattttttca ttccatgaag atgagatgtc tttccatttg   2820 

tttgtgtcct ctacattttc tttcatcaaa gttttgttgt atttttgaag tagatgtatt   2880 

tcaccttata gatcaagtgt attccctaaa tattttattt ttgtagctat tgtagatgaa   2940 

attgccttct cgatttcttt ttcacttaat tcattattag tgtatggaaa tgttatggat   3000 

ttttatttgt tggtttttaa tcaaaaactg tattaaactt agagtttttt gtggagtttt   3060 

taagtttttc tagatataag atcatgacat ctaccaaaaa a                       3101 

 
           
             33  
             16  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            33 

tgcccctccg gaagct                                                     16 

 
           
             34  
             23  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            34 

cgtttctgaa gggacatctg atc                                             23 

 
           
             35  
             30  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            35 

ttgcagccaa gttaggagtg aagagatgca                                      30 

 
           
             36  
             24  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            36 

aagcctcaga gtccttccag tatg                                            24 

 
           
             37  
             35  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            37 

ttcaaatata agtgaagaaa aaattagtag atcaa                                35 

 
           
             38  
             37  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            38 

aatccattgt atcttagaac cgagggattt gtttaga                              37 

 
           
             39  
             22  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            39 

aaagcagatg gtggttgagg tt                                              22 

 
           
             40  
             22  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            40 

cctgagacca aatggcttct tc                                              22 

 
           
             41  
             24  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            41 

attccatgcc ggctgcttct tctg                                            24 

 
           
             42  
             30  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            42 

tctggttttc tcattcttta ttcatttatt                                      30 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            43 

tgccaaggag cggattatct                                                 20 

 
           
             44  
             30  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            44 

caaccacgtg acaaacactg gaattacagg                                      30 

 
           
             45  
             21  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            45 

actggaacgg tgaaggtgac a                                               21 

 
           
             46  
             20  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            46 

cggccacatt gtgaactttg                                                 20 

 
           
             47  
             23  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            47 

cagtcggttg gagcgagcat ccc                                             23 

 
           
             48  
             24  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            48 

tgccatagat gaattgaagg aatg                                            24 

 
           
             49  
             29  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            49 

tgtcatatat taattgcata aacacctca                                       29 

 
           
             50  
             32  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            50 

tcttaaccaa acggatgaaa ctctgagcaa tg                                   32 

 
           
             51  
             28  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            51 

atcattgaaa attcaaatat aagtgaag                                        28 

 
           
             52  
             30  
             DNA  
             Artificial Sequence  
             
               PCR primer  
             
           
            52 

gtagttgtgc attgaaataa ttatcattat                                      30 

 
           
             53  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            53 

caattttggt ggagaacccg                                                 20 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            54 

gctgtcggag gtatatggtg                                                 20 

 
           
             55  
             28  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            55 

catttcagag agtaacatgg actacaca                                        28 

 
           
             56  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            56 

tctgataaag gccgtacaat g                                               21 

 
           
             57  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            57 

tcacgacttg ctgtttttgc tc                                              22 

 
           
             58  
             30  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            58 

atcaaaaaac aagcatggcc tcacaccact                                      30 

 
           
             59  
             21  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            59 

gcaagtgcca atgatcagag g                                               21 

 
           
             60  
             23  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            60 

atatagactc aggtatacac act                                             23 

 
           
             61  
             30  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            61 

tcccatcaga atccaaacaa gaggaagatg                                      30 

 
           
             62  
             34  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            62 

aatccattgt atcttagaac cgagggattt gttt                                 34 

 
           
             63  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            63 

ccgcttctga caacactaga gatc                                            24 

 
           
             64  
             32  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            64 

cctataaaga tgttatgtac caaaaatgaa gt                                   32 

 
           
             65  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            65 

cccctccctc agggtatggc cc                                              22 

 
           
             66  
             22  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            66 

ccctttctca cccacacact gt                                              22 

 
           
             67  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            67 

tgcattctct catatgtgga agct                                            24 

 
           
             68  
             33  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            68 

ccgggcctca ggcatatact attctactgt ctg                                  33 

 
           
             69  
             24  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            69 

gacattccag ttttacccaa atgg                                            24 

 
           
             70  
             23  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            70 

tgcagaagac tcaagctgat tcc                                             23 

 
           
             71  
             28  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            71 

tctcagggac acactctacc attcggga                                        28 

 
           
             72  
             30  
             DNA  
             Artificial Sequence  
             
               PCR Primer  
             
           
            72 

aaatataagt gaagaaaaaa attagtagat                                      30 

 
           
             73  
             503  
             DNA  
             Homo sapiens  
           
            73 

gacagcggct tccttgatcc ttgccacccg cgactgaaca ccgacagcag cagcctcacc     60 

atgaagttgc tgatggtcct catgctggcg gccctctccc agcactgcta cgcaggctct    120 

ggctgcccct tattggagaa tgtgatttcc aagacaatca atccacaagt gtctaagact    180 

gaatacaaag aacttcttca agagttcata gacgacaatg ccactacaaa tgccatagat    240 

gaattgaagg aatgttttct taaccaaacg gatgaaactc tgagcaatgt tgaggtgttt    300 

ctgcaattaa tatatgacag cagtctttgt gatttatttt aactttctgc aagacctttg    360 

gctcacagaa ctgcagggta tggtgagaaa ccaactacgg attgctgcaa accacacctt    420 

ctctttctta tgtcttttta ctacaaacta caagacaatt gttgaaacct gctatacatg    480 

tttattttaa taaattgatg gca                                            503 

 
           
             74  
             301  
             DNA  
             Homo sapiens  
           
            74 

cactgctacg caggctctgg ctgcccctta ttggagaatg tgatttccaa gacaatcaat     60 

ccacaagtgt ctaagactga atacaaagaa cttcttcaag agttcataga cgacaatgcc    120 

actacaaatg ccatagatga attgaaggaa tgttttctta accaaacgga tgaaactctg    180 

agcaatgttg aggtgtttat gcaattaata tatgacagca gtctttgtga tttatttggc    240 

ggccatcacc atcaccatca ctaaggtccc gagctcgaat tctgcagata tccatcacac    300 

t                                                                    301 

 
           
             75  
             3282  
             DNA  
             Homo sapiens  
           
            75 

gggacagggc tgaggatgag gagaaccctg gggacccaga agaccgtgcc ttgcccggaa     60 

gtcctgcctg taggcctgaa ggacttgccc taacagagcc tcaacaacta cctggtgatt    120 

cctacttcag ccccttggtg tgagcagctt ctcaacatga actacagcct ccacttggcc    180 

ttcgtgtgtc tgagtctctt cactgagagg atgtgcatcc aggggagtca gttcaacgtc    240 

gaggtcggca gaagtgacaa gctttccctg cctggctttg agaacctcac agcaggatat    300 

aacaaatttc tcaggcccaa ttttggtgga gaacccgtac agatagcgct gactctggac    360 

attgcaagta tctctagcat ttcagagagt aacatggact acacagccac catatacctc    420 

cgacagcgct ggatggacca gcggctggtg tttgaaggca acaagagctt cactctggat    480 

gcccgcctcg tggagttcct ctgggtgcca gatacttaca ttgtggagtc caagaagtcc    540 

ttcctccatg aagtcactgt gggaaacagg ctcatccgcc tcttctccaa tggcacggtc    600 

ctgtatgccc tcagaatcac gacaactgtt gcatgtaaca tggatctgtc taaatacccc    660 

atggacacac agacatgcaa gttgcagctg gaaagctggg gctatgatgg aaatgatgtg    720 

gagttcacct ggctgagagg gaacgactct gtgcgtggac tggaacacct gcggcttgct    780 

cagtacacca tagagcggta tttcacctta gtcaccagat cgcagcagga gacaggaaat    840 

tacactagat tggtcttaca gtttgagctt cggaggaatg ttctgtattt cattttggaa    900 

acctacgttc cttccacttt cctggtggtg ttgtcctggg tttcattttg gatctctctc    960 

gattcagtcc ctgcaagaac ctgcattgga gtgacgaccg tgttatcaat gaccacactg   1020 

atgatcgggt cccgcacttc tcttcccaac accaactgct tcatcaaggc catcgatgtg   1080 

tacctgggga tctgctttag ctttgtgttt ggggccttgc tagaatatgc agttgctcac   1140 

tacagttcct tacagcagat ggcagccaaa gataggggga caacaaagga agtagaagaa   1200 

gtcagtatta ctaatatcat caacagctcc atctccagct ttaaacggaa gatcagcttt   1260 

gccagcattg aaatttccag cgacaacgtt gactacagtg acttgacaat gaaaaccagc   1320 

gacaagttca agtttgtctt ccgagaaaag atgggcagga ttgttgatta tttcacaatt   1380 

caaaacccca gtaatgttga tcactattcc aaactactgt ttcctttgat ttttatgcta   1440 

gccaatgtat tttactgggc atactacatg tatttttgag tcaatgttaa atttcttgca   1500 

tgccataggt cttcaacagg acaagataat gatgtaaatg gtattttagg ccaagtgtgc   1560 

acccacatcc aatggtgcta caagtgactg aaataatatt tgagtctttc tgctcaaaga   1620 

atgaagctcc aaccattgtt ctaagctgtg tagaagtcct agcattatag gatcttgtaa   1680 

tagaaacatc agtccattcc tctttcatct taatcaagga cattcccatg gagcccaaga   1740 

ttacaaatgt actcagggct gtttattcgg tggctccctg gtttgcattt acctcatata   1800 

aagaatggga aggagaccat tgggtaaccc tcaagtgtca gaagttgttt ctaaagtaac   1860 

tatacatgtt ttttactaaa tctctgcagt gcttataaaa tacattgttg cctatttagg   1920 

gagtaacatt ttctagtttt tgtttctggt taaaatgaaa tatgggctta tgtcaattca   1980 

ttggaagtca atgcactaac tcaataccaa gatgagtttt taaataatga atattattta   2040 

ataccacaac agaattatcc ccaatttcca ataagtccta tcattgaaaa ttcaaatata   2100 

agtgaagaaa aaattagtag atcaacaatc taaacaaatc cctcggttct aagatacaat   2160 

ggattcccca tactggaagg actctgaggc tttattcccc cactatgcat atcttatcat   2220 

tttattatta tacacacatc catcctaaac tatactaaag cccttttccc atgcatggat   2280 

ggaaatggaa gatttttttg taacttgttc tagaagtctt aatatgggct gttgccatga   2340 

aggcttgcag aattgagtcc attttctagc tgcctttatt cacatagtga tggggtacta   2400 

aaagtactgg gttgactcag agagtcgctg tcattctgtc attgctgcta ctctaacact   2460 

gagcaacact ctcccagtgg cagatcccct gtatcattcc aagaggagca ttcatccctt   2520 

tgctctaatg atcaggaatg atgcttatta gaaaacaaac tgcttgaccc aggaacaagt   2580 

ggcttagctt aagtaaactt ggctttgctc agatccctga tccttccagc tggtctgctc   2640 

tgagtggctt atcccgcatg agcaggagcg tgctggccct gagtactgaa ctttctgagt   2700 

aacaatgaga cacgttacag aacctatgtt caggttgcgg gtgagctgcc ctctccaaat   2760 

ccagccagag atgcacattc ctcggccagt ctcagccaac agtaccaaaa gtgatttttg   2820 

agtgtgccag ggtaaaggct tccagttcag cctcagttat tttagacaat ctcgccatct   2880 

ttaatttctt agcttcctgt tctaataaat gcacggcttt acctttcctg tcagaaataa   2940 

accaaggctc taaaagatga tttcccttct gtaactccct agagccacag gttctcattc   3000 

cttttcccat tatacttctc acaattcagt ttctatgagt ttgatcacct gattttttta   3060 

acaaaatatt tctaacggga atgggtggga gtgctggtga aaagagatga aatgtggttg   3120 

tatgagccaa tcatatttgt gattttttaa aaaaagttta aaaggaaata tctgttctga   3180 

aaccccactt aagcattgtt tttatataaa aacaatgata aagatgtgaa ctgtgaaata   3240 

aatataccat attagctacc caccaaaaaa aaaaaaaaaa aa                      3282 

 
           
             76  
             463  
             DNA  
             Homo sapiens  
           
            76 

tagaattcag cggccgctta attctagaag tccaaatcac tcattgtttg tgaaagctga     60 

gctcacagca aaacaagcca ccatgaagct gtcggtgtgt ctcctgctgg tcacgctggc    120 

cctctgctgc taccaggcca atgccgagtt ctgcccagct cttgtttctg agctgttaga    180 

cttcttcttc attagtgaac ctctgttcaa gttaagtctt gccaaatttg atgcccctcc    240 

ggaagctgtt gcagccaagt taggagtgaa gagatgcacg gatcagatgt cccttcagaa    300 

acgaagcctc attgcggaag tcctggtgaa aatattgaag aaatgtagtg tgtgacatgt    360 

aaaaactttc atcctggttt ccactgtctt tcaatgacac cctgatcttc actgcagaat    420 

gtaaaggttt caacgtcttg ctttaataaa tcacttgctc tac                      463 

 
           
             77  
             90  
             PRT  
             Homo sapiens  
           
            77 

Met Lys Leu Ser Val Cys Leu Leu Leu Val Thr Leu Ala Leu Cys Cys 
 1               5                  10                  15 

Tyr Gln Ala Asn Ala Glu Phe Cys Pro Ala Leu Val Ser Glu Leu Leu 
            20                  25                  30 

Asp Phe Phe Phe Ile Ser Glu Pro Leu Phe Lys Leu Ser Leu Ala Lys 
        35                  40                  45 

Phe Asp Ala Pro Pro Glu Ala Val Ala Ala Lys Leu Gly Val Lys Arg 
    50                  55                  60 

Cys Thr Asp Gln Met Ser Leu Gln Lys Arg Ser Leu Ile Ala Glu Val 
65                  70                  75                  80 

Leu Val Lys Ile Leu Lys Lys Cys Ser Val 
                85                  90