US20020192220A1

US20020192220A1 - Compositions and methods relating to colon specific genes and proteins

Info

Publication number: US20020192220A1
Application number: US10/016,157
Authority: US
Inventors: Yongming Sun; Herve Recipon; Malavika Ghosh; Chenghua Liu
Original assignee: Diadexus Inc
Current assignee: Diadexus Inc
Priority date: 2000-10-31
Filing date: 2001-10-31
Publication date: 2002-12-19
Also published as: US7361475B2; JP2004528823A; EP1364021A2; US20050208546A1; WO2002048370A2; WO2002048370A3; AU2002243444A1

Abstract

The present invention relates to newly identified nucleic acids and polypeptides present in normal and neoplastic colon cells, including fragments, variants and derivatives of the nucleic acids and polypeptides. The present invention also relates to antibodies to the polypeptides of the invention, as well as agonists and antagonists of the polypeptides of the invention. The invention also relates to compositions comprising the nucleic acids, polypeptides, antibodies, variants, derivatives, agonists and antagonists of the invention and methods for the use of these compositions. These uses include identifying, diagnosing, monitoring, staging, imaging and treating colon cancer and non-cancerous disease states in colon tissue, identifying colon tissue, monitoring and identifying and/or designing agonists and antagonists of polypeptides of the invention. The uses also include gene therapy, production of transgenic animals and cells, and production of engineered colon tissue for treatment and research.

Description

This application claims the benefit of priority from U.S. Provisional Application Serial No. 60/244,717 filed Oct. 31, 2000, which is herein incorporated by reference in its entirety.[0001]

FIELD OF THE INVENTION

The present invention relates to newly identified nucleic acid molecules and polypeptides present in normal and neoplastic colon cells, including fragments, variants and derivatives of the nucleic acids and polypeptides. The present invention also relates to antibodies to the polypeptides of the invention, as well as agonists and antagonists of the polypeptides of the invention. The invention also relates to compositions comprising the nucleic acids, polypeptides, antibodies, variants, derivatives, agonists and antagonists of the invention and methods for the use of these compositions. These uses include identifying, diagnosing, monitoring, staging, imaging and treating colon cancer and non-cancerous disease states in colon tissue, identifying colon tissue and monitoring and identifying and/or designing agonists and antagonists of polypeptides of the invention. The uses also include gene therapy, production of transgenic animals and cells, and production of engineered colon tissue for treatment and research.

BACKGROUND OF THE INVENTION

Colorectal cancer is the second most common cause of cancer death in the United States and the third most prevalent cancer in both men and women. M. L. Davila & A. D. Davila, Screening for Colon and Rectal Cancer, in Colon and Rectal Cancer 47 (Peter S. Edelstein ed., 2000). Approximately 100,000 patients every year suffer from colon cancer and approximately half that number die of the disease. Hannah-Ngoc Ha & Bard C. Cosman, Treatment of Colon Cancer, in Colon and Rectal Cancer 157 (Peter S. Edelstein ed., 2000). Nearly all cases of colorectal cancer arise from adenomatous polyps, some of which mature into large polyps, undergo abnormal growth and development, and ultimately progress into cancer. Davila & Davila, supra at 55-56. This progression would appear to take at least 10 years in most patients, rendering it a readily treatable form of cancer if diagnosed early, when the cancer is localized. Id. at 56; Walter J. Burdette, Cancer: Etiology, Diagnosis, and Treatment 125 (1998).

Although our understanding of the etiology of colon cancer is undergoing continual refinement, extensive research in this area points to a combination of factors, including age, hereditary and nonheriditary conditions, and environmental/dietary factors. Age is a key risk factor in the development of colorectal cancer, Davila & Davila, supra at 48, with men and women over 40 years of age become increasingly susceptible to that cancer, Burdette, supra at 126. Incidence rates increase considerably in each subsequent decade of life. Davila et al., supra at 48. A number of hereditary and nonhereditary conditions have also been linked to a heightened risk of developing colorectal cancer, including familial adenomatous polyposis (FAP), hereditary nonpolyposis colorectal cancer (Lynch syndrome or HNPCC), a personal and/or family history of colorectal cancer or adenomatous polyps, inflammatory bowel disease, diabetes mellitus, and obesity. Id. at 47; Henry T. Lynch & Jane F. Lynch, Hereditary Nonpolyposis Colorectal Cancer (Lynch Syndromes), in Colon and Rectal Cancer 67-68 (Peter S. Edelstein ed., 2000).

In the case of FAP, the tumor suppressor gene APC (adenomatous polyposis coli), located at 5q21, has been either mutationally inactivated or deleted. Alberts et al., Molecular Biology of the Cell 1288 (3d ed. 1994). The APC protein plays a role in a number of functions, including cell adhesion, apoptosis, and repression of the c-myc oncogene. N. R. Hall & R. D. Madoff, Genetics and the Polyp-Cancer Sequence, Colon and Rectal Cancer 8 (Peter S. Edelstein, ed., 2000). Of those patients with colorectal cancer who have normal APC genes, over 65% have such mutations in the cancer cells but not in other tissues. Alberts et al., supra at 1288. In the case of HPNCC, patients manifest abnormalities in the tumor suppressor gene HNPCC, but only about 15% of tumors contain the mutated gene. Id. A host of other genes have also been implicated in colorectal cancer, including the K-ras, N-ras, H-ras and c-myc oncogenes, and the tumor suppressor genes DCC (deleted in colon carcinoma) and p53. Hall & Madoff, supra at 8-9; Alberts et al., supra at 1288.

Environmental/dietary factors associated with an increased risk of colorectal cancer include a high fat diet, intake of high dietary red meat, and sedentary lifestyle. Davila & Davila, supra at 47; Reddy, B. S., Prev. Med. 16(4): 460-7 (1987). Conversely, environmental/dietary factors associated with a reduced risk of colorectal cancer include a diet high in fiber, folic acid, calcium, and hormone-replacement therapy in post-menopausal women. Davila & Davila, supra at 50-55. The effect of antioxidants in reducing the risk of colon cancer is unclear. Id. at 53.

Because colon cancer is highly treatable when detected at an early, localized stage, screening should be a part of routine care for all adults starting at age 50, especially those with first-degree relatives with colorectal cancer. One major advantage of colorectal cancer screening over its counterparts in other types of cancer is its ability to not only detect precancerous lesions, but to remove them as well. Davila & Davila, supra at 56. The key colorectal cancer screening tests in use today are fecal occult blood test, sigmoidoscopy, colonoscopy, double-contrast barium enema, and the carcinoembryonic antigen (CEA) test. Id; Burdette, supra at 125.

The fecal occult blood test (FOBT) screens for colorectal cancer by detecting the amount of blood in the stool, the premise being that neoplastic tissue, particularly malignant tissue, bleeds more than typical mucosa, with the amount of bleeding increasing with polyp size and cancer stage. Davila & Davila, supra at 56-57. While effective at detecting early stage tumors, FOBT is unable to detect adenomatous polyps (premalignant lesions), and, depending on the contents of the fecal sample, is subject to rendering false positives. Id. at 56-59. Sigmoidoscopy and colonoscopy, by contrast, allow direct visualization of the bowel, and enable one to detect, biopsy, and remove adenomatous polyps. Id. at 59-60, 61. Despite the advantages of these procedures, there are accompanying downsides: sigmoidoscopy, by definition, is limited to the sigmoid colon and below, colonoscopy is a relatively expensive procedure, and both share the risk of possible bowel perforation and hemorrhaging. Id. at 59-60. Double-contrast barium enema (DCBE) enables detection of lesions better than FOBT, and almost as well a colonoscopy, but it may be limited in evaluating the winding rectosigmoid region. Id. at 60. The CEA blood test, which involves screening the blood for carcinoembryonic antigen, shares the downside of FOBT, in that it is of limited utility in detecting colorectal cancer at an early stage. Burdette, supra at 125.

Once colon cancer has been diagnosed, treatment decisions are typically made in reference to the stage of cancer progression. A number of techniques are employed to stage the cancer (some of which are also used to screen for colon cancer), including pathologic examination of resected colon, sigmoidoscopy, colonoscopy, and various imaging techniques. AJCC Cancer Staging Handbook 84 (Irvin D. Fleming et al. eds., 5^thed. 1998); Montgomery, R. C. and Ridge, J. A., Semin. Surg. Oncol. 15(3): 143-150 (1998). Moreover, chest films, liver functionality tests, and liver scans are employed to determine the extent of metastasis. Fleming et al. eds., supra at 84. While computerized tomography and magnetic resonance imaging are useful in staging colorectal cancer in its later stages, both have unacceptably low staging accuracy for identifying early stages of the disease, due to the difficulty that both methods have in (1) revealing the depth of bowel wall tumor infiltration and (2) diagnosing malignant adenopathy. Thoeni, R. F., Radiol. Clin. N. Am. 35(2): 457-85 (1997). Rather, techniques such as transrectal ultrasound (TRUS) are preferred in this context, although this technique is inaccurate with respect to detecting small lymph nodes that may contain metastases. David Blumberg & Frank G. Opelka, Neoadjuvant and Adjuvant Therapy for Adenocarcinoma of the Rectum, in Colon and Rectal Cancer 316 (Peter S. Edelstein ed., 2000).

Several classification systems have been devised to stage the extent of colorectal cancer, including the Dukes' system and the more detailed International Union against Cancer-American Joint Committee on Cancer TNM staging system, which is considered by many in the field to be a more useful staging system. Burdette, supra at 126-27. The TNM system, which is used for either clinical or pathological staging, is divided into four stages, each of which evaluates the extent of cancer growth with respect to primary tumor (T), regional lymph nodes (N), and distant metastasis (M). Fleming et al. eds., supra at 84-85. The system focuses on the extent of tumor invasion into the intestinal wall, invasion of adjacent structures, the number of regional lymph nodes that have been affected, and whether distant metastasis has occurred. Id. at 81.

Stage 0 is characterized by in situ carcinoma (Tis), in which the cancer cells are located inside the glandular basement membrane (intraepithelial) or lamina propria (intramucosal). Id. at 84-85; Burdette, supra at 127. In this stage, the cancer has not spread to the regional lymph nodes (NO), and there is no distant metastasis (MO). Fleming et al. eds., supra at 85; Burdette, supra at 127. In stage I, there is still no spread of the cancer to the regional lymph nodes and no distant metastasis, but the tumor has invaded the submucosa (T 1) or has progressed further to invade the muscularis propria (T2). Fleming et al. eds., supra at 84-85; Burdette, supra at 127. Stage II also involves no spread of the cancer to the regional lymph nodes and no distant metastasis, but the tumor has invaded the subserosa, or the nonperitonealized pericolic or perirectal tissues (T3), or has progressed to invade other organs or structures, and/or has perforated the visceral peritoneum (T4). Id. Stage 3 is characterized by any of the T substages, no distant metastasis, and either metastasis in 1 to 3 regional lymph nodes (N1) or metastasis in four or more regional lymph nodes (N2). Fleming et al. eds., supra at 85; Burdette, supra at 127. Lastly, stage 4 involves any of the T or N substages, as well as distant metastasis. Id.

Currently, pathological staging of colon cancer is preferable over clinical staging as pathological staging provides a more accurate prognosis. Pathological staging typically involves examination of the resected colon section, along with surgical examination of the abdominal cavity. Fleming et al. eds., supra at 84. Clinical staging would be a preferred method of staging were it at least as accurate as pathological staging, as it does not depend on the invasive procedures of its counterpart.

Turning to the treatment of colorectal cancer, surgical resection results in a cure for roughly 50% of patients. Burdette, supra at 125. Irradiation is used both preoperatively and postoperatively in treating colorectal cancer. Id. at 125, 132-33. Chemotherapeutic agents, particularly 5-fluorouracil, are also powerful weapons in treating colorectal cancer. Id. at 125, 133. Other agents include irinotecan and floxuridine, cisplatin, levamisole, methotrexate, interferon-alpha, and leucovorin. Id. at 133. Nonetheless, thirty to forty percent of patients will develop a recurrence of colon cancer following surgical resection. Wayne De Vos, Follow-up After Treatment of Colon Cancer, Colon and Rectal Cancer 225 (Peter S. Edelstein ed., 2000), which in many patients is the ultimate cause of death. Accordingly, colon cancer patients must be closely monitored to determine response to therapy and to detect persistent or recurrent disease and metastasis.

From the foregoing, it is clear that procedures used for detecting, diagnosing, monitoring, staging, prognosticating, and preventing the recurrence of colorectal cancer are of critical importance to the outcome of the patient. Moreover, current procedures, while helpful in each of these analyses, are limited by their specificity, sensitivity, invasiveness, and/or their cost. As such, highly specific and sensitive procedures that would operate by way of detecting novel markers in cells, tissues, or bodily fluids, with minimal invasiveness and at a reasonable cost, would be highly desirable.

Accordingly, there is a great need for more sensitive and accurate methods for predicting whether a person is likely to develop colorectal cancer, for diagnosing colorectal cancer, for monitoring the progression of the disease, for staging the colorectal cancer, for determining whether the colorectal cancer has metastasized, and for imaging the colorectal cancer. There is also a need for better treatment of colorectal cancer.

SUMMARY OF THE INVENTION

The present invention solves these and other needs in the art by providing nucleic acid molecules and polypeptides as well as antibodies, agonists and antagonists, thereto that may be used to identify, diagnose, monitor, stage, image and treat colon cancer and non-cancerous disease states in colon; identify and monitor colon tissue; and identify and design agonists and antagonists of polypeptides of the invention. The invention also provides gene therapy, methods for producing transgenic animals and cells, and methods for producing engineered colon tissue for treatment and research.

Accordingly, one object of the invention is to provide nucleic acid molecules that are specific to colon cells and/or colon tissue. These colon specific nucleic acids (CSNAs) may be a naturally-occurring cDNA, genomic DNA, RNA, or a fragment of one of these nucleic acids, or may be a non-naturally-occurring nucleic acid molecule. If the CSNA is genomic DNA, then the CSNA is a colon specific gene (CSG). In a preferred embodiment, the nucleic acid molecule encodes a polypeptide that is specific to colon. In a more preferred embodiment, the nucleic acid molecule encodes a polypeptide that comprises an amino acid sequence of SEQ ID NO: 148 through 250. In another highly preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 1 through 147. By nucleic acid molecule, it is also meant to be inclusive of sequences that selectively hybridize or exhibit substantial sequence similarity to a nucleic acid molecule encoding a CSP, or that selectively hybridize or exhibit substantial sequence similarity to a CSNA, as well as allelic variants of a nucleic acid molecule encoding a CSP, and allelic variants of a CSNA. Nucleic acid molecules comprising a part of a nucleic acid sequence that encodes a CSP or that comprises a part of a nucleic acid sequence of a CSNA are also provided.

A related object of the present invention is to provide a nucleic acid molecule comprising one or more expression control sequences controlling the transcription and/or translation of all or a part of a CSNA. In a preferred embodiment, the nucleic acid molecule comprises one or more expression control sequences controlling the transcription and/or translation of a nucleic acid molecule that encodes all or a fragment of a CSP.

Another object of the invention is to provide vectors and/or host cells comprising a nucleic acid molecule of the instant invention. In a preferred embodiment, the nucleic acid molecule encodes all or a fragment of a CSP. In another preferred embodiment, the nucleic acid molecule comprises all or a part of a CSNA.

Another object of the invention is to provided methods for using the vectors and host cells comprising a nucleic acid molecule of the instant invention to recombinantly produce polypeptides of the invention.

Another object of the invention is to provide a polypeptide encoded by a nucleic acid molecule of the invention. In a preferred embodiment, the polypeptide is a CSP. The polypeptide may comprise either a fragment or a full-length protein as well as a mutant protein (mutein), fusion protein, homologous protein or a polypeptide encoded by an allelic variant of a CSP.

Another object of the invention is to provide an antibody that specifically binds to a polypeptide of the instant invention.

Another object of the invention is to provide agonists and antagonists of the nucleic acid molecules and polypeptides of the instant invention.

Another object of the invention is to provide methods for using the nucleic acid molecules to detect or amplify nucleic acid molecules that have similar or identical nucleic acid sequences compared to the nucleic acid molecules described herein. In a preferred embodiment, the invention provides methods of using the nucleic acid molecules of the invention for identifying, diagnosing, monitoring, staging, imaging and treating colon cancer and non-cancerous disease states in colon. In another preferred embodiment, the invention provides methods of using the nucleic acid molecules of the invention for identifying and/or monitoring colon tissue. The nucleic acid molecules of the instant invention may also be used in gene therapy, for producing transgenic animals and cells, and for producing engineered colon tissue for treatment and research.

The polypeptides and/or antibodies of the instant invention may also be used to identify, diagnose, monitor, stage, image and treat colon cancer and non-cancerous disease states in colon. The invention provides methods of using the polypeptides of the invention to identify and/or monitor colon tissue, and to produce engineered colon tissue.

The agonists and antagonists of the instant invention may be used to treat colon cancer and non-cancerous disease states in colon and to produce engineered colon tissue.

Yet another object of the invention is to provide a computer readable means of storing the nucleic acid and amino acid sequences of the invention. The records of the computer readable means can be accessed for reading and displaying of sequences for comparison, alignment and ordering of the sequences of the invention to other sequences.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press (1989) and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press (2001); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology—4^thEd., Wiley & Sons (1999); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1990); and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1999); each of which is incorporated herein by reference in its entirety.

Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

A “nucleic acid molecule” of this invention refers to a polymeric form of nucleotides and includes both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. A “nucleic acid molecule” as used herein is synonymous with “nucleic acid” and “polynucleotide.” The term “nucleic acid molecule” usually refers to a molecule of at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. In addition, a polynucleotide may include either or both naturally-occurring and modified nucleotides linked together by naturally-occurring and/or non-naturally occurring nucleotide linkages.

The nucleic acid molecules may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular and padlocked conformations. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

A “gene” is defined as a nucleic acid molecule that comprises a nucleic acid sequence that encodes a polypeptide and the expression control sequences that surround the nucleic acid sequence that encodes the polypeptide. For instance, a gene may comprise a promoter, one or more enhancers, a nucleic acid sequence that encodes a polypeptide, downstream regulatory sequences and, possibly, other nucleic acid sequences involved in regulation of the expression of an RNA. As is well-known in the art, eukaryotic genes usually contain both exons and introns. The term “exon” refers to a nucleic acid sequence found in genomic DNA that is bioinformatically predicted and/or experimentally confirmed to contribute a contiguous sequence to a mature mRNA transcript. The term “intron” refers to a nucleic acid sequence found in genomic DNA that is predicted and/or confirmed to not contribute to a mature mRNA transcript, but rather to be “spliced out” during processing of the transcript.

A nucleic acid molecule or polypeptide is “derived” from a particular species if the nucleic acid molecule or polypeptide has been isolated from the particular species, or if the nucleic acid molecule or polypeptide is homologous to a nucleic acid molecule or polypeptide isolated from a particular species.

An “isolated” or “substantially pure” nucleic acid or polynucleotide (e.g., an RNA, DNA or a mixed polymer) is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases, or genomic sequences with which it is naturally associated. The term embraces a nucleic acid or polynucleotide that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the “isolated polynucleotide” is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, (4) does not occur in nature as part of a larger sequence or (5) includes nucleotides or internucleoside bonds that are not found in nature. The term “isolated” or “substantially pure” also can be used in reference to recombinant or cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems. The term “isolated nucleic acid molecule” includes nucleic acid molecules that are integrated into a host cell chromosome at a heterologous site, recombinant fusions of a native fragment to a heterologous sequence, recombinant vectors present as episomes or as integrated into a host cell chromosome.

A “part” of a nucleic acid molecule refers to a nucleic acid molecule that comprises a partial contiguous sequence of at least 10 bases of the reference nucleic acid molecule. Preferably, a part comprises at least 15 to 20 bases of a reference nucleic acid molecule. In theory, a nucleic acid sequence of 17 nucleotides is of sufficient length to occur at random less frequently than once in the three gigabase human genome, and thus to provide a nucleic acid probe that can uniquely identify the reference sequence in a nucleic acid mixture of genomic complexity. A preferred part is one that comprises a nucleic acid sequence that can encode at least 6 contiguous amino acid sequences (fragments of at least 18 nucleotides) because they are useful in directing the expression or synthesis of peptides that are useful in mapping the epitopes of the polypeptide encoded by the reference nucleic acid. See, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); and U.S. Pat. Nos. 4,708,871 and 5,595,915, the disclosures of which are incorporated herein by reference in their entireties. A part may also comprise at least 25, 30, 35 or 40 nucleotides of a reference nucleic acid molecule, or at least 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 or 500 nucleotides of a reference nucleic acid molecule. A part of a nucleic acid molecule may comprise no other nucleic acid sequences. Alternatively, a part of a nucleic acid may comprise other nucleic acid sequences from other nucleic acid molecules.

The term “oligonucleotide” refers to a nucleic acid molecule generally comprising a length of 200 bases or fewer. The term often refers to single-stranded deoxyribonucleotides, but it can refer as well to single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs, among others. Preferably, oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases in length. Other preferred oligonucleotides are 25, 30, 35, 40, 45, 50, 55 or 60 bases in length. Oligonucleotides may be single-stranded, e.g. for use as probes or primers, or may be double-stranded, e.g. for use in the construction of a mutant gene. Oligonucleotides of the invention can be either sense or antisense oligonucleotides. An oligonucleotide can be derivatized or modified as discussed above for nucleic acid molecules.

Oligonucleotides, such as single-stranded DNA probe oligonucleotides, often are synthesized by chemical methods, such as those implemented on automated oligonucleotide synthesizers. However, oligonucleotides can be made by a variety of other methods, including in vitro recombinant DNA-mediated techniques and by expression of DNAs in cells and organisms. Initially, chemically synthesized DNAs typically are obtained without a 5′ phosphate. The 5′ ends of such oligonucleotides are not substrates for phosphodiester bond formation by ligation reactions that employ DNA ligases typically used to form recombinant DNA molecules. Where ligation of such oligonucleotides is desired, a phosphate can be added by standard techniques, such as those that employ a kinase and ATP. The 3′ end of a chemically synthesized oligonucleotide generally has a free hydroxyl group and, in the presence of a ligase, such as T4 DNA ligase, readily will form a phosphodiester bond with a 5′ phosphate of another polynucleotide, such as another oligonucleotide. As is well-known, this reaction can be prevented selectively, where desired, by removing the 5′ phosphates of the other polynucleotide(s) prior to ligation.

The term “naturally-occurring nucleotide” referred to herein includes naturally-occurring deoxyribonucleotides and ribonucleotides. The term “modified nucleotides” referred to herein includes nucleotides with modified or substituted sugar groups and the like. The term “nucleotide linkages” referred to herein includes nucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081-9093 (1986); Stein et al. Nucl. Acids Res. 16:3209-3221 (1988); Zon et al. Anti-Cancer Drug Design 6:539-568 (1991); Zon et al., in Eckstein (ed.) Oligonucleotides and Analogues: A Practical Approach, pp. 87-108, Oxford University Press (1991); U.S. Pat. No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures of which are hereby incorporated by reference.

Unless specified otherwise, the left hand end of a polynucleotide sequence in sense orientation is the 5′ end and the right hand end of the sequence is the 3′ end. In addition, the left hand direction of a polynucleotide sequence in sense orientation is referred to as the 5′ direction, while the right hand direction of the polynucleotide sequence is referred to as the 3′ direction. Further, unless otherwise indicated, each nucleotide sequence is set forth herein as a sequence of deoxyribonucleotides. It is intended, however, that the given sequence be interpreted as would be appropriate to the polynucleotide composition: for example, if the isolated nucleic acid is composed of RNA, the given sequence intends ribonucleotides, with uridine substituted for thymidine.

The term “allelic variant” refers to one of two or more alternative naturally-occurring forms of a gene, wherein each gene possesses a unique nucleotide sequence. In a preferred embodiment, different alleles of a given gene have similar or identical biological properties.

The term “percent sequence identity” in the context of nucleic acid sequences refers to the residues in two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g., the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183: 63-98 (1990); Pearson, Methods Mol. Biol. 132: 185-219 (2000); Pearson, Methods Enzymol. 266: 227-258 (1996); Pearson, J. Mol. Biol. 276: 71-84 (1998); herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. The complementary strand is also useful, e.g., for antisense therapy, hybridization probes and PCR primers.

In the molecular biology art, researchers use the terms “percent sequence identity”, “percent sequence similarity” and “percent sequence homology” interchangeably. In this application, these terms shall have the same meaning with respect to nucleic acid sequences only.

The term “substantial similarity” or “substantial sequence similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 50%, more preferably 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

Alternatively, substantial similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under selective hybridization conditions. Typically, selective hybridization will occur when there is at least about 55% sequence identity, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90% sequence identity, over a stretch of at least about 14 nucleotides, more preferably at least 17 nucleotides, even more preferably at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100 nucleotides.

Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. The most important parameters include temperature of hybridization, base composition of the nucleic acids, salt concentration and length of the nucleic acid. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization. In general, “stringent hybridization” is performed at about 25° C. below the thermal melting point (T _m) for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5° C. lower than the T_mfor the specific DNA hybrid under a particular set of conditions. The T_mis the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook (1989), supra, p. 9.51, hereby incorporated by reference.

The T _mfor a particular DNA-DNA hybrid can be estimated by the formula:

T _m=81.5° C.+16.6(log₁₀[Na⁺])+0.41(fraction G+C)−0.63(% formamide)−(600/l)

where 1 is the length of the hybrid in base pairs.

The T _mfor a particular RNA—RNA hybrid can be estimated by the formula:

T _m=79.8° C.+18.5(log₁₀[Na⁺])+0.58(fraction G+C)+11.8(fraction G+C)²−0.35(% formamide)−(820/l).

The T _mfor a particular RNA-DNA hybrid can be estimated by the formula:

T _m=79.8° C.+18.5(log₁₀[Na⁺])+0.58(fraction G+C)+11.8(fraction G+C)²−0.50(% formamide)−(820/l).

In general, the T _mdecreases by 1-1.5° C. for each 1% of mismatch between two nucleic acid sequences. Thus, one having ordinary skill in the art can alter hybridization and/or washing conditions to obtain sequences that have higher or lower degrees of sequence identity to the target nucleic acid. For instance, to obtain hybridizing nucleic acids that contain up to 10% mismatch from the target nucleic acid sequence, 10-15° C. would be subtracted from the calculated Tm of a perfectly matched hybrid, and then the hybridization and washing temperatures adjusted accordingly. Probe sequences may also hybridize specifically to duplex DNA under certain conditions to form triplex or other higher order DNA complexes. The preparation of such probes and suitable hybridization conditions are well-known in the art.

An example of stringent hybridization conditions for hybridization of complementary nucleic acid sequences having more than 100 complementary residues on a filter in a Southern or Northern blot or for screening a library is 50% formamide/6× SSC at 42° C. for at least ten hours and preferably overnight (approximately 16 hours). Another example of stringent hybridization conditions is 6× SSC at 68° C. without formamide for at least ten hours and preferably overnight. An example of moderate stringency hybridization conditions is 6× SSC at 55° C. without formamide for at least ten hours and preferably overnight. An example of low stringency hybridization conditions for hybridization of complementary nucleic acid sequences having more than 100 complementary residues on a filter in a Southern or Northern blot or for screening a library is 6× SSC at 42° C. for at least ten hours. Hybridization conditions to identify nucleic acid sequences that are similar but not identical can be identified by experimentally changing the hybridization temperature from 68° C. to 42° C. while keeping the salt concentration constant (6× SSC), or keeping the hybridization temperature and salt concentration constant (e.g. 42° C. and 6× SSC) and varying the formamide concentration from 50% to 0%. Hybridization buffers may also include blocking agents to lower background. These agents are well-known in the art. See Sambrook et al. (1989), supra, pages 8.46 and 9.46-9.58, herein incorporated by reference. See also Ausubel (1992), supra, Ausubel (1999), supra, and Sambrook (2001), supra.

Wash conditions also can be altered to change stringency conditions. An example of stringent wash conditions is a 0.2× SSC wash at 65° C. for 15 minutes (see Sambrook (1989), supra, for SSC buffer). Often the high stringency wash is preceded by a low stringency wash to remove excess probe. An exemplary medium stringency wash for duplex DNA of more than 100 base pairs is 1× SSC at 45° C. for 15 minutes. An exemplary low stringency wash for such a duplex is 4× SSC at 40° C. for 15 minutes. In general, signal-to-noise ratio of 2× or higher than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

As defined herein, nucleic acid molecules that do not hybridize to each other under stringent conditions are still substantially similar to one another if they encode polypeptides that are substantially identical to each other. This occurs, for example, when a nucleic acid molecule is created synthetically or recombinantly using high codon degeneracy as permitted by the redundancy of the genetic code.

Hybridization conditions for nucleic acid molecules that are shorter than 100 nucleotides in length (e.g., for oligonucleotide probes) may be calculated by the formula:

T _m=81.5° C.+16.6(log₁₀[Na⁺])+0.41(fraction G+C)−(600/N),

wherein N is change length and the [Na ⁺] is 1 M or less. See Sambrook (1989), supra, p. 11.46. For hybridization of probes shorter than 100 nucleotides, hybridization is usually performed under stringent conditions (5-10° C. below the Tm) using high concentrations (0.1-1.0 pmol/ml) of probe. Id. at p. 11.45. Determination of hybridization using mismatched probes, pools of degenerate probes or “guessmers,” as well as hybridization solutions and methods for empirically determining hybridization conditions are well-known in the art. See, e.g., Ausubel (1999), supra; Sambrook (1989), supra, pp. 11.45-11.57.

The term “digestion” or “digestion of DNA” refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes referred to herein are commercially available and their reaction conditions, cofactors and other requirements for use are known and routine to the skilled artisan. For analytical purposes, typically, 1 μg of plasmid or DNA fragment is digested with about 2 units of enzyme in about 20 μl of reaction buffer. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in proportionately larger volumes. Appropriate buffers and substrate amounts for particular restriction enzymes are described in standard laboratory manuals, such as those referenced below, and they are specified by commercial suppliers. Incubation times of about 1 hour at 37° C. are ordinarily used, but conditions may vary in accordance with standard procedures, the supplier's instructions and the particulars of the reaction. After digestion, reactions may be analyzed, and fragments may be purified by electrophoresis through an agarose or polyacrylamide gel, using well-known methods that are routine for those skilled in the art.

The term “ligation” refers to the process of forming phosphodiester bonds between two or more polynucleotides, which most often are double-stranded DNAS. Techniques for ligation are well-known to the art and protocols for ligation are described in standard laboratory manuals and references, such as, e.g. Sambrook (1989), supra.

Genome-derived “single exon probes,” are probes that comprise at least part of an exon (“reference exon”) and can hybridize detectably under high stringency conditions to transcript-derived nucleic acids that include the reference exon but do not hybridize detectably under high stringency conditions to nucleic acids that lack the reference exon. Single exon probes typically further comprise, contiguous to a first end of the exon portion, a first intronic and/or intergenic sequence that is identically contiguous to the exon in the genome, and may contain a second intronic and/or intergenic sequence that is identically contiguous to the exon in the genome. The minimum length of genome-derived single exon probes is defined by the requirement that the exonic portion be of sufficient length to hybridize under high stringency conditions to transcript-derived nucleic acids, as discussed above. The maximum length of genome-derived single exon probes is defined by the requirement that the probes contain portions of no more than one exon. The single exon probes may contain priming sequences not found in contiguity with the rest of the probe sequence in the genome, which priming sequences are useful for PCR and other amplification-based technologies.

The term “microarray” or “nucleic acid microarray” refers to a substrate-bound collection of plural nucleic acids, hybridization to each of the plurality of bound nucleic acids being separately detectable. The substrate can be solid or porous, planar or non-planar, unitary or distributed. Microarrays or nucleic acid microarrays include all the devices so called in Schena (ed.), DNA Microarrays: A Practical Approach (Practical Approach Series), Oxford University Press (1999); Nature Genet. 21(1)(suppl.):1-60 (1999); Schena (ed.), Microarray Biochip: Tools and Technology, Eaton Publishing Company/BioTechniques Books Division (2000). These microarrays include substrate-bound collections of plural nucleic acids in which the plurality of nucleic acids are disposed on a plurality of beads, rather than on a unitary planar substrate, as is described, inter alia, in Brenner et al., Proc. Natl. Acad. Sci. USA 97(4):1665-1670 (2000).

The term “mutated” when applied to nucleic acid molecules means that nucleotides in the nucleic acid sequence of the nucleic acid molecule may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. In a preferred embodiment, the nucleic acid molecule comprises the wild type nucleic acid sequence encoding a CSP or is a CSNA. The nucleic acid molecule may be mutated by any method known in the art including those mutagenesis techniques described infra.

The term “error-prone PCR” refers to a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product. See, e.g., Leung et al., Technique 1: 11-15 (1989) and Caldwell et al., PCR Methods Applic. 2: 28-33 (1992).

The term “oligonucleotide-directed mutagenesis” refers to a process which enables the generation of site-specific mutations in any cloned DNA segment of interest. See, e.g., Reidhaar-Olson et al., Science 241: 53-57 (1988).

The term “assembly PCR” refers to a process which involves the assembly of a PCR product from a mixture of small DNA fragments. A large number of different PCR reactions occur in parallel in the same vial, with the products of one reaction priming the products of another reaction.

The term “sexual PCR mutagenesis” or “DNA shuffling” refers to a method of error-prone PCR coupled with forced homologous recombination between DNA molecules of different but highly related DNA sequence in vitro, caused by random fragmentation of the DNA molecule based on sequence similarity, followed by fixation of the crossover by primer extension in an error-prone PCR reaction. See, e.g., Stemmer, Proc. Natl. Acad. Sci. U.S.A. 91: 10747-10751 (1994). DNA shuffling can be carried out between several related genes (“Family shuffling”).

The term “in vivo mutagenesis” refers to a process of generating random mutations in any cloned DNA of interest which involves the propagation of the DNA in a strain of bacteria such as E. coli that carries mutations in one or more of the DNA repair pathways. These “mutator” strains have a higher random mutation rate than that of a wild-type parent. Propagating the DNA in a mutator strain will eventually generate random mutations within the DNA.

The term “cassette mutagenesis” refers to any process for replacing a small region of a double-stranded DNA molecule with a synthetic oligonucleotide “cassette” that differs from the native sequence. The oligonucleotide often contains completely and/or partially randomized native sequence.

The term “recursive ensemble mutagenesis” refers to an algorithm for protein engineering (protein mutagenesis) developed to produce diverse populations of phenotypically related mutants whose members differ in amino acid sequence. This method uses a feedback mechanism to control successive rounds of combinatorial cassette mutagenesis. See, e.g., Arkin et al., Proc. Natl. Acad. Sci. U.S.A. 89: 7811-7815 (1992).

The term “exponential ensemble mutagenesis” refers to a process for generating combinatorial libraries with a high percentage of unique and functional mutants, wherein small groups of residues are randomized in parallel to identify, at each altered position, amino acids which lead to functional proteins. See, e.g., Delegrave et al., Biotechnology Research 11: 1548-1552 (1993); Arnold, Current Opinion in Biotechnology 4: 450-455 (1993). Each of the references mentioned above are hereby incorporated by reference in its entirety.

“Operatively linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include the promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

The term “vector,” as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments may be ligated. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Viral vectors that infect bacterial cells are referred to as bacteriophages. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors that serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

As used herein, the phrase “open reading frame” and the equivalent acronym “ORF” refer to that portion of a transcript-derived nucleic acid that can be translated in its entirety into a sequence of contiguous amino acids. As so defined, an ORF has length, measured in nucleotides, exactly divisible by 3. As so defined, an ORF need not encode the entirety of a natural protein.

As used herein, the phrase “ORF-encoded peptide” refers to the predicted or actual translation of an ORF.

As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence intends all nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence.

The term “polypeptide” encompasses both naturally-occurring and non-naturally-occurring proteins and polypeptides, polypeptide fragments and polypeptide mutants, derivatives and analogs. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different modules within a single polypeptide each of which has one or more distinct activities. A preferred polypeptide in accordance with the invention comprises a CSP encoded by a nucleic acid molecule of the instant invention, as well as a fragment, mutant, analog and derivative thereof.

The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is free of other proteins from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well-known in the art.

A protein or polypeptide is “substantially pure,” “substantially homogeneous” or “substantially purified” when at least about 60% to 75% of a sample exhibits a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/W of a protein sample, more usually about 95%, and preferably will be over 99% pure. Protein purity or homogeneity may be indicated by a number of means well-known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well-known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well-known in the art for purification.

The term “polypeptide fragment” as used herein refers to a polypeptide of the instant invention that has an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

A “derivative” refers to polypeptides or fragments thereof that are substantially similar in primary structural sequence but which include, e.g., in vivo or in vitro chemical and biochemical modifications that are not found in the native polypeptide. Such modifications include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Other modification include, e.g., labeling with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well-known in the art, and include radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well-known in the art. See Ausubel (1992), supra; Ausubel (1999), supra, herein incorporated by reference.

The term “fusion protein” refers to polypeptides of the instant invention comprising polypeptides or fragments coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein. Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.

The term “analog” refers to both polypeptide analogs and non-peptide analogs. The term “polypeptide analog” as used herein refers to a polypeptide of the instant invention that is comprised of a segment of at least 25 amino acids that has substantial identity to a portion of an amino acid sequence but which contains non-natural amino acids or non-natural inter-residue bonds. In a preferred embodiment, the analog has the same or similar biological activity as the native polypeptide. Typically, polypeptide analogs comprise a conservative amino acid substitution (or insertion or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.

The term “non-peptide analog” refers to a compound with properties that are analogous to those of a reference polypeptide of the instant invention. A non-peptide compound may also be termed a “peptide mimetic” or a “peptidomimetic.” Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides may be used to produce an equivalent effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a desired biochemical property or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: —CH ₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—, —CH(OH)CH₂—, and —CH₂SO—, by methods well-known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L-lysine) may also be used to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art (Rizo et al., Ann. Rev. Biochem. 61:387-418 (1992), incorporated herein by reference). For example, one may add internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

A “polypeptide mutant” or “mutein” refers to a polypeptide of the instant invention whose sequence contains substitutions, insertions or deletions of one or more amino acids compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and/or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and/or truncations of the amino acid sequence at either or both the amino or carboxy termini. Further, a mutein may have the same or different biological activity as the naturally-occurring protein. For instance, a mutein may have an increased or decreased biological activity. A mutein has at least 50% sequence similarity to the wild type protein, preferred is 60% sequence similarity, more preferred is 70% sequence similarity. Even more preferred are muteins having 80%, 85% or 90% sequence similarity to the wild type protein. In an even more preferred embodiment, a mutein exhibits 95% sequence identity, even more preferably 97%, even more preferably 98% and even more preferably 99%. Sequence similarity may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.

Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs. For example, single or multiple amino acid substitutions preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. In a preferred embodiment, the amino acid substitutions are moderately conservative substitutions or conservative substitutions. In a more preferred embodiment, the amino acid substitutions are conservative substitutions. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to disrupt a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of art-recognized polypeptide secondary and tertiary structures are described in Creighton (ed.), Proteins, Structures and Molecular Principles, W. H. Freeman and Company (1984); Branden et al. (ed.), Introduction to Protein Structure, Garland Publishing (1991); Thornton et al., Nature 354:105-106 (1991), each of which are incorporated herein by reference.

As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Golub et al. (eds.), Immunology—A Synthesis 2^ndEd., Sinauer Associates (1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as −, -disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, —N,N,N-trimethyllysine, —N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, s-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the lefthand direction is the amino terminal direction and the right hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.

A protein has “homology” or is “homologous” to a protein from another organism if the encoded amino acid sequence of the protein has a similar sequence to the encoded amino acid sequence of a protein of a different organism and has a similar biological activity or function. Alternatively, a protein may have homology or be homologous to another protein if the two proteins have similar amino acid sequences and have similar biological activities or functions. Although two proteins are said to be “homologous,” this does not imply that there is necessarily an evolutionary relationship between the proteins. Instead, the term “homologous” is defined to mean that the two proteins have similar amino acid sequences and similar biological activities or functions. In a preferred embodiment, a homologous protein is one that exhibits 50% sequence similarity to the wild type protein, preferred is 60% sequence similarity, more preferred is 70% sequence similarity. Even more preferred are homologous proteins that exhibit 80%, 85% or 90% sequence similarity to the wild type protein. In a yet more preferred embodiment, a homologous protein exhibits 95%, 97%, 98% or 99% sequence similarity.

When “sequence similarity” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. In a preferred embodiment, a polypeptide that has “sequence similarity” comprises conservative or moderately conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 24: 307-31 (1994), herein incorporated by reference.

For instance, the following six groups each contain amino acids that are conservative substitutions for one another:

1) Serine (S), Threonine (T);

2) Aspartic Acid (D), Glutamic Acid (E);

3) Asparagine (N), Glutamine (O);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science 256: 1443-45 (1992), herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

Sequence similarity for polypeptides, which is also referred to as sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Other programs include FASTA, discussed supra.

A preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn. See, e.g., Altschul et al., J. Mol. Biol. 215: 403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997); herein incorporated by reference. Preferred parameters for blastp are:



Expectation value:	10 (default)
Filter:	seg (default)
Cost to open a gap:	11 (default)
Cost to extend a gap:	1 (default
Max. alignments:	100 (default)
Word size:	11 (default)
No. of descriptions:	100 (default)
Penalty Matrix:	BLOSUM62

The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

Database searching using amino acid sequences can be measured by algorithms other than blastp are known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (1990), supra; Pearson (2000), supra. For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default or recommended parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.

An “antibody” refers to an intact immunoglobulin, or to an antigen-binding portion thereof that competes with the intact antibody for specific binding to a molecular species, e.g., a polypeptide of the instant invention. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab′, F(ab′) ₂, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. An Fab fragment is a monovalent fragment consisting of the VL, VH, CL and CH1 domains; an F(ab′)₂fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; an Fd fragment consists of the VH and CH1 domains; an Fv fragment consists of the VL and VH domains of a single arm of an antibody; and a dAb fragment consists of a VH domain. See, e.g., Ward et al., Nature 341: 544-546 (1989).

By “bind specifically” and “specific binding” is here intended the ability of the antibody to bind to a first molecular species in preference to binding to other molecular species with which the antibody and first molecular species are admixed. An antibody is said specifically to “recognize” a first molecular species when it can bind specifically to that first molecular species.

A single-chain antibody (scFv) is an antibody in which a VL and VH region are paired to form a monovalent molecule via a synthetic linker that enables them to be made as a single protein chain. See, e.g., Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988). Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites. See e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al., Structure 2: 1121-1123 (1994). One or more CDRs may be incorporated into a molecule either covalently or noncovalently to make it an immunoadhesin. An immunoadhesin may incorporate the CDR(s) as part of a larger polypeptide chain, may covalently link the CDR(s) to another polypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesin to specifically bind to a particular antigen of interest. A chimeric antibody is an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies.

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a “bispecific” or “bifunctional” antibody has two different binding sites.

An “isolated antibody” is an antibody that (1) is not associated with naturally-associated components, including other naturally-associated antibodies, that accompany it in its native state, (2) is free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. It is known that purified proteins, including purified antibodies, may be stabilized with non-naturally-associated components. The non-naturally-associated component may be a protein, such as albumin (e.g., BSA) or a chemical such as polyethylene glycol (PEG).

A “neutralizing antibody” or “an inhibitory antibody” is an antibody that inhibits the activity of a polypeptide or blocks the binding of a polypeptide to a ligand that normally binds to it. An “activating antibody” is an antibody that increases the activity of a polypeptide.

The term “epitope” includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is less than 1 μM, preferably less than 100 nM and most preferably less than 10 nM.

The term “patient” as used herein includes human and veterinary subjects.

Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

The term “colon specific” refers to a nucleic acid molecule or polypeptide that is expressed predominantly in the colon as compared to other tissues in the body. In a preferred embodiment, a “colon specific” nucleic acid molecule or polypeptide is expressed at a level that is 5-fold higher than any other tissue in the body. In a more preferred embodiment, the “colon specific” nucleic acid molecule or polypeptide is expressed at a level that is 10-fold higher than any other tissue in the body, more preferably at least 15-fold, 20-fold, 25-fold, 50-fold or 100-fold higher than any other tissue in the body. Nucleic acid molecule levels may be measured by nucleic acid hybridization, such as Northern blot hybridization, or quantitative PCR. Polypeptide levels may be measured by any method known to accurately quantitate protein levels, such as Western blot analysis.

Nucleic Acid Molecules, Regulatory Sequences Vectors, Host Cells and Recombinant Methods of Making Polypeptides

Nucleic Acid Molecules

One aspect of the invention provides isolated nucleic acid molecules that are specific to the colon or to colon cells or tissue or that are derived from such nucleic acid molecules. These isolated colon specific nucleic acids (CSNAs) may comprise a cDNA, a genomic DNA, RNA, or a fragment of one of these nucleic acids, or may be a non-naturally-occurring nucleic acid molecule. In a preferred embodiment, the nucleic acid molecule encodes a polypeptide that is specific to colon, a colon-specific polypeptide (CSP). In a more preferred embodiment, the nucleic acid molecule encodes a polypeptide that comprises an amino acid sequence of SEQ ID NO: 148 through 250. In another highly preferred embodiment, the nucleic acid molecule comprises a nucleic acid sequence of SEQ ID NO: 1 through 147.

A CSNA may be derived from a human or from another animal. In a preferred embodiment, the CSNA is derived from a human or other mammal. In a more preferred embodiment, the CSNA is derived from a human or other primate. In an even more preferred embodiment, the CSNA is derived from a human.

By “nucleic acid molecule” for purposes of the present invention, it is also meant to be inclusive of nucleic acid sequences that selectively hybridize to a nucleic acid molecule encoding a CSNA or a complement thereof. The hybridizing nucleic acid molecule may or may not encode a polypeptide or may not encode a CSP. However, in a preferred embodiment, the hybridizing nucleic acid molecule encodes a CSP. In a more preferred embodiment, the invention provides a nucleic acid molecule that selectively hybridizes to a nucleic acid molecule that encodes a polypeptide comprising an amino acid sequence of SEQ ID NO: 148 through 250. In an even more preferred embodiment, the invention provides a nucleic acid molecule that selectively hybridizes to a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 1 through 147.

In a preferred embodiment, the nucleic acid molecule selectively hybridizes to a nucleic acid molecule encoding a CSP under low stringency conditions. In a more preferred embodiment, the nucleic acid molecule selectively hybridizes to a nucleic acid molecule encoding a CSP under moderate stringency conditions. In a more preferred embodiment, the nucleic acid molecule selectively hybridizes to a nucleic acid molecule encoding a CSP under high stringency conditions. In an even more preferred embodiment, the nucleic acid molecule hybridizes under low, moderate or high stringency conditions to a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of SEQ ID NO: 148 through 250. In a yet more preferred embodiment, the nucleic acid molecule hybridizes under low, moderate or high stringency conditions to a nucleic acid molecule comprising a nucleic acid sequence selected from SEQ ID NO: 1 through 147. In a preferred embodiment of the invention, the hybridizing nucleic acid molecule may be used to express recombinantly a polypeptide of the invention.

By “nucleic acid molecule” as used herein it is also meant to be inclusive of sequences that exhibits substantial sequence similarity to a nucleic acid encoding a CSP or a complement of the encoding nucleic acid molecule. In a preferred embodiment, the nucleic acid molecule exhibits substantial sequence similarity to a nucleic acid molecule encoding human CSP. In a more preferred embodiment, the nucleic acid molecule exhibits substantial sequence similarity to a nucleic acid molecule encoding a polypeptide having an amino acid sequence of SEQ ID NO: 148 through 250. In a preferred embodiment, the similar nucleic acid molecule is one that has at least 60% sequence identity with a nucleic acid molecule encoding a CSP, such as a polypeptide having an amino acid sequence of SEQ ID NO: 148 through 250, more preferably at least 70%, even more preferably at least 80% and even more preferably at least 85%. In a more preferred embodiment, the similar nucleic acid molecule is one that has at least 90% sequence identity with a nucleic acid molecule encoding a CSP, more preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, and still more preferably at least 99%. In another highly preferred embodiment, the nucleic acid molecule is one that has at least 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity with a nucleic acid molecule encoding a CSP.

In another preferred embodiment, the nucleic acid molecule exhibits substantial sequence similarity to a CSNA or its complement. In a more preferred embodiment, the nucleic acid molecule exhibits substantial sequence similarity to a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 1 through 147. In a preferred embodiment, the nucleic acid molecule is one that has at least 60% sequence identity with a CSNA, such as one having a nucleic acid sequence of SEQ ID NO: 1 through 147, more preferably at least 70%, even more preferably at least 80% and even more preferably at least 85%. In a more preferred embodiment, the nucleic acid molecule is one that has at least 90% sequence identity with a CSNA, more preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, and still more preferably at least 99%. In another highly preferred embodiment, the nucleic acid molecule is one that has at least 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity with a CSNA.

A nucleic acid molecule that exhibits substantial sequence similarity may be one that exhibits sequence identity over its entire length to a CSNA or to a nucleic acid molecule encoding a CSP, or may be one that is similar over only a part of its length. In this case, the part is at least 50 nucleotides of the CSNA or the nucleic acid molecule encoding a CSP, preferably at least 100 nucleotides, more preferably at least 150 or 200 nucleotides, even more preferably at least 250 or 300 nucleotides, still more preferably at least 400 or 500 nucleotides.

The substantially similar nucleic acid molecule may be a naturally-occurring one that is derived from another species, especially one derived from another primate, wherein the similar nucleic acid molecule encodes an amino acid sequence that exhibits significant sequence identity to that of SEQ ID NO: 148 through 250 or demonstrates significant sequence identity to the nucleotide sequence of SEQ ID NO: 1 through 147. The similar nucleic acid molecule may also be a naturally-occurring nucleic acid molecule from a human, when the CSNA is a member of a gene family. The similar nucleic acid molecule may also be a naturally-occurring nucleic acid molecule derived from a non-primate, mammalian species, including without limitation, domesticated species, e.g., dog, cat, mouse, rat, rabbit, hamster, cow, horse and pig; and wild animals, e.g., monkey, fox, lions, tigers, bears, giraffes, zebras, etc. The substantially similar nucleic acid molecule may also be a naturally-occurring nucleic acid molecule derived from a non-mammalian species, such as birds or reptiles. The naturally-occurring substantially similar nucleic acid molecule may be isolated directly from humans or other species. In another embodiment, the substantially similar nucleic acid molecule may be one that is experimentally produced by random mutation of a nucleic acid molecule. In another embodiment, the substantially similar nucleic acid molecule may be one that is experimentally produced by directed mutation of a CSNA. Further, the substantially similar nucleic acid molecule may or may not be a CSNA. However, in a preferred embodiment, the substantially similar nucleic acid molecule is a CSNA.

By “nucleic acid molecule” it is also meant to be inclusive of allelic variants of a CSNA or a nucleic acid encoding a CSP. For instance, single nucleotide polymorphisms (SNPs) occur frequently in eukaryotic genomes. In fact, more than 1.4 million SNPs have already identified in the human genome, International Human Genome Sequencing Consortium, Nature 409: 860-921 (2001). Thus, the sequence determined from one individual of a species may differ from other allelic forms present within the population. Additionally, small deletions and insertions, rather than single nucleotide polymorphisms, are not uncommon in the general population, and often do not alter the function of the protein. Further, amino acid substitutions occur frequently among natural allelic variants, and often do not substantially change protein function.

In a preferred embodiment, the nucleic acid molecule comprising an allelic variant is a variant of a gene, wherein the gene is transcribed into an mRNA that encodes a CSP. In a more preferred embodiment, the gene is transcribed into an mRNA that encodes a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250. In another preferred embodiment, the allelic variant is a variant of a gene, wherein the gene is transcribed into an mRNA that is a CSNA. In a more preferred embodiment, the gene is transcribed into an mRNA that comprises the nucleic acid sequence of SEQ ID NO: 1 through 147. In a preferred embodiment, the allelic variant is a naturally-occurring allelic variant in the species of interest. In a more preferred embodiment, the species of interest is human.

By “nucleic acid molecule” it is also meant to be inclusive of a part of a nucleic acid sequence of the instant invention. The part may or may not encode a polypeptide, and may or may not encode a polypeptide that is a CSP. However, in a preferred embodiment, the part encodes a CSP. In one aspect, the invention comprises a part of a CSNA. In a second aspect, the invention comprises a part of a nucleic acid molecule that hybridizes or exhibits substantial sequence similarity to a CSNA. In a third aspect, the invention comprises a part of a nucleic acid molecule that is an allelic variant of a CSNA. In a fourth aspect, the invention comprises a part of a nucleic acid molecule that encodes a CSP. A part comprises at least 10 nucleotides, more preferably at least 15, 17, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 or 500 nucleotides. The maximum size of a nucleic acid part is one nucleotide shorter than the sequence of the nucleic acid molecule encoding the full-length protein.

By “nucleic acid molecule” it is also meant to be inclusive of sequence that encoding a fusion protein, a homologous protein, a polypeptide fragment, a mutein or a polypeptide analog, as described below.

Nucleotide sequences of the instantly-described nucleic acids were determined by sequencing a DNA molecule that had resulted, directly or indirectly, from at least one enzymatic polymerization reaction (e.g., reverse transcription and/or polymerase chain reaction) using an automated sequencer (such as the MegaBACE™ 1000, Molecular Dynamics, Sunnyvale, Calif., USA). Further, all amino acid sequences of the polypeptides of the present invention were predicted by translation from the nucleic acid sequences so determined, unless otherwise specified.

In a preferred embodiment of the invention, the nucleic acid molecule contains modifications of the native nucleic acid molecule. These modifications include nonnative internucleoside bonds, post-synthetic modifications or altered nucleotide analogues. One having ordinary skill in the art would recognize that the type of modification that can be made will depend upon the intended use of the nucleic acid molecule. For instance, when the nucleic acid molecule is used as a hybridization probe, the range of such modifications will be limited to those that permit sequence-discriminating base pairing of the resulting nucleic acid. When used to direct expression of RNA or protein in vitro or in vivo, the range of such modifications will be limited to those that permit the nucleic acid to function properly as a polymerization substrate. When the isolated nucleic acid is used as a therapeutic agent, the modifications will be limited to those that do not confer toxicity upon the isolated nucleic acid.

In a preferred embodiment, isolated nucleic acid molecules can include nucleotide analogues that incorporate labels that are directly detectable, such as radiolabels or fluorophores, or nucleotide analogues that incorporate labels that can be visualized in a subsequent reaction, such as biotin or various haptens. In a more preferred embodiment, the labeled nucleic acid molecule may be used as a hybridization probe.

Common radiolabeled analogues include those labeled with ³³P, ³²P, and ³⁵S, such as -³²P-dATP, -³²P-dCTP, -³²P-dGTP, -³²P-dTTP, -³²P-3′dATP, -³²P-ATP, -³²P-CTP, -³²P-GTP, -³²P-UTP, -³⁵S-dATP, α-³⁵S-GTP, α-³³P-dATP, and the like.

Commercially available fluorescent nucleotide analogues readily incorporated into the nucleic acids of the present invention include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy3-dUTP (Amersham Pharmacia Biotech, Piscataway, N.J., USA), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, Texas Red®-5-dUTP, Cascade Blue®-7-dUTP, BODIPY® FL-14-dUTP, BODIPY® TMR-14-dUTP, BODIPY® TR-14-dUTP, Rhodamine Green™-5-dUTP, Oregon Green® 488-5-dUTP, Texas Red®-12-dUTP, BODIPY® 630/650-14-dUTP, BODIPY® 650/665-14-dUTP, Alexa Fluor® 488-5-dUTP, Alexa Fluor® 532-5-dUTP, Alexa Fluor® 568-5-dUTP, Alexa Fluor® 594-5-dUTP, Alexa Fluor® 546-14-dUTP, fluorescein-12-UTP, tetramethylrhodamine-6-UTP, Texas Red®-5-UTP, Cascade Blue®-7-UTP, BODIPY® FL-14-UTP, BODIPY® TMR-14-UTP, BODIPY® TR-14-UTP, Rhodamine Green™-5-UTP, Alexa Fluor® 488-5-UTP, Alexa Fluor® 546-14-UTP (Molecular Probes, Inc. Eugene, Oreg., USA). One may also custom synthesize nucleotides having other fluorophores. See Henegariu et al., Nature Biotechnol. 18: 345-348 (2000), the disclosure of which is incorporated herein by reference in its entirety.

Haptens that are commonly conjugated to nucleotides for subsequent labeling include biotin (biotin-11-dUTP, Molecular Probes, Inc., Eugene, Oreg., USA; biotin-21-UTP, biotin-21-dUTP, Clontech Laboratories, Inc., Palo Alto, Calif., USA), digoxigenin (DIG-11-dUTP, alkali labile, DIG-11-UTP, Roche Diagnostics Corp., Indianapolis, Ind., USA), and dinitrophenyl (dinitrophenyl-11-dUTP, Molecular Probes, Inc., Eugene, Oreg., USA).

Nucleic acid molecules can be labeled by incorporation of labeled nucleotide analogues into the nucleic acid. Such analogues can be incorporated by enzymatic polymerization, such as by nick translation, random priming, polymerase chain reaction (PCR), terminal transferase tailing, and end-filling of overhangs, for DNA molecules, and in vitro transcription driven, e.g., from phage promoters, such as T7, T3, and SP6, for RNA molecules. Commercial kits are readily available for each such labeling approach. Analogues can also be incorporated during automated solid phase chemical synthesis. Labels can also be incorporated after nucleic acid synthesis, with the 5′ phosphate and 3′ hydroxyl providing convenient sites for post-synthetic covalent attachment of detectable labels.

Other post-synthetic approaches also permit internal labeling of nucleic acids. For example, fluorophores can be attached using a cisplatin reagent that reacts with the N7 of guanine residues (and, to a lesser extent, adenine bases) in DNA, RNA, and PNA to provide a stable coordination complex between the nucleic acid and fluorophore label (Universal Linkage System) (available from Molecular Probes, Inc., Eugene, Oreg., USA and Amersham Pharmacia Biotech, Piscataway, N.J., USA); see Alers et al., Genes, Chromosomes & Cancer 25: 301-305 (1999); Jelsma et al., J. NIH Res. 5: 82 (1994); Van Belkum et al., BioTechniques 16: 148-153 (1994), incorporated herein by reference. As another example, nucleic acids can be labeled using a disulfide-containing linker (FastTag™ Reagent, Vector Laboratories, Inc., Burlingame, Calif., USA) that is photo- or thermally-coupled to the target nucleic acid using aryl azide chemistry; after reduction, a free thiol is available for coupling to a hapten, fluorophore, sugar, affinity ligand, or other marker.

One or more independent or interacting labels can be incorporated into the nucleic acid molecules of the present invention. For example, both a fluorophore and a moiety that in proximity thereto acts to quench fluorescence can be included to report specific hybridization through release of fluorescence quenching or to report exonucleotidic excision. See, e.g., Tyagi et al., Nature Biotechnol. 14: 303-308 (1996); Tyagi et al., Nature Biotechnol. 16: 49-53 (1998); Sokol et al., Proc. Natl. Acad. Sci. USA 95: 11538-11543 (1998); Kostrikis et al., Science 279: 1228-1229 (1998); Marras et al., Genet. Anal. 14: 151-156 (1999); U.S. Pat. Nos. 5,846,726; 5,925,517; 5,925,517; 5,723,591 and 5,538,848; Holland et al., Proc. Natl. Acad. Sci. USA 88: 7276-7280 (1991); Heid et al., Genome Res. 6(10): 986-94 (1996); Kuimelis et al., Nucleic Acids Symp. Ser. (37): 255-6 (1997); the disclosures of which are incorporated herein by reference in their entireties.

Nucleic acid molecules of the invention may be modified by altering one or more native phosphodiester internucleoside bonds to more nuclease-resistant, internucleoside bonds. See Hartmann et al. (eds.), Manual of Antisense Methodology: Perspectives in Antisense Science, Kluwer Law International (1999); Stein et al. (eds.), Applied Antisense Oligonucleotide Technology, Wiley-Liss (1998); Chadwick et al. (eds.), Oligonucleotides as Therapeutic Agents—Symposium No. 209, John Wiley & Son Ltd (1997); the disclosures of which are incorporated herein by reference in their entireties. Such altered internucleoside bonds are often desired for antisense techniques or for targeted gene correction. See Gamper et al., Nucl. Acids Res. 28(21): 4332-4339 (2000), the disclosure of which is incorporated herein by reference in its entirety.

Modified oligonucleotide backbones include, without limitation, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, the disclosures of which are incorporated herein by reference in their entireties. In a preferred embodiment, the modified internucleoside linkages may be used for antisense techniques.

Other modified oligonucleotide backbones do not include a phosphorus atom, but have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH ₂component parts. Representative U.S. patents that teach the preparation of the above backbones include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437 and 5,677,439; the disclosures of which are incorporated herein by reference in their entireties.

In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage are replaced with novel groups, such as peptide nucleic acids (PNA). In PNA compounds, the phosphodiester backbone of the nucleic acid is replaced with an amide-containing backbone, in particular by repeating N-(2-aminoethyl) glycine units linked by amide bonds. Nucleobases are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone, typically by methylene carbonyl linkages. PNA can be synthesized using a modified peptide synthesis protocol. PNA oligomers can be synthesized by both Fmoc and tBoc methods. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Automated PNA synthesis is readily achievable on commercial synthesizers (see, e.g., “PNA User's Guide,” Rev. 2, February 1998, Perseptive Biosystems Part No. 60138, Applied Biosystems, Inc., Foster City, Calif.).

PNA molecules are advantageous for a number of reasons. First, because the PNA backbone is uncharged, PNA/DNA and PNA/RNA duplexes have a higher thermal stability than is found in DNA/DNA and DNA/RNA duplexes. The Tm of a PNA/DNA or PNA/RNA duplex is generally 1° C. higher per base pair than the Tm of the corresponding DNA/DNA or DNA/RNA duplex (in 100 mM NaCl). Second, PNA molecules can also form stable PNA/DNA complexes at low ionic strength, under conditions in which DNA/DNA duplex formation does not occur. Third, PNA also demonstrates greater specificity in binding to complementary DNA because a PNA/DNA mismatch is more destabilizing than DNA/DNA mismatch. A single mismatch in mixed a PNA/DNA 15-mer lowers the Tm by 8-20° C. (15° C. on average). In the corresponding DNA/DNA duplexes, a single mismatch lowers the Tm by 4-16° C. (11° C. on average). Because PNA probes can be significantly shorter than DNA probes, their specificity is greater. Fourth, PNA oligomers are resistant to degradation by enzymes, and the lifetime of these compounds is extended both in vivo and in vitro because nucleases and proteases do not recognize the PNA polyamide backbone with nucleobase sidechains. See, e.g., Ray et al., FASEB J. 14(9): 1041-60 (2000); Nielsen et al., Pharmacol Toxicol. 86(1): 3-7 (2000); Larsen et al., Biochim Biophys Acta. 1489(1): 159-66 (1999); Nielsen, Curr. Opin. Struct. Biol. 9(3): 353-7 (1999), and Nielsen, Curr. Opin. Biotechnol. 10(1): 71-5 (1999), the disclosures of which are incorporated herein by reference in their entireties.

Nucleic acid molecules may be modified compared to their native structure throughout the length of the nucleic acid molecule or can be localized to discrete portions thereof. As an example of the latter, chimeric nucleic acids can be synthesized that have discrete DNA and RNA domains and that can be used for targeted gene repair and modified PCR reactions, as further described in U.S. Pat. Nos. 5,760,012 and 5,731,181, Misra et al., Biochem. 37: 1917-1925 (1998); and Finn et al., Nucl. Acids Res. 24: 3357-3363 (1996), the disclosures of which are incorporated herein by reference in their entireties.

Unless otherwise specified, nucleic acids of the present invention can include any topological conformation appropriate to the desired use; the term thus explicitly comprehends, among others, single-stranded, double-stranded, triplexed, quadruplexed, partially double-stranded, partially-triplexed, partially-quadruplexed, branched, hairpinned, circular, and padlocked conformations. Padlock conformations and their utilities are further described in Baner et al., Curr. Opin. BiotechnoL 12: 11-15 (2001); Escude et al., Proc. Natl. Acad. Sci. USA 14: 96(19):10603-7 (1999); Nilsson et al., Science 265(5181): 2085-8 (1994), the disclosures of which are incorporated herein by reference in their entireties. Triplex and quadruplex conformations, and their utilities, are reviewed in Praseuth et al., Biochim. Biophys. Acta. 1489(1): 181-206 (1999); Fox, Curr. Med. Chem. 7(1): 17-37 (2000); Kochetkova et al., Methods Mol. Biol. 130: 189-201 (2000); Chan et al., J. Mol. Med. 75(4): 267-82 (1997), the disclosures of which are incorporated herein by reference in their entireties.

Methods for Using Nucleic Acid Molecules as Probes and Primers

The isolated nucleic acid molecules of the present invention can be used as hybridization probes to detect, characterize, and quantify hybridizing nucleic acids in, and isolate hybridizing nucleic acids from, both genomic and transcript-derived nucleic acid samples. When free in solution, such probes are typically, but not invariably, detectably labeled; bound to a substrate, as in a microarray, such probes are typically, but not invariably unlabeled.

In one embodiment, the isolated nucleic acids of the present invention can be used as probes to detect and characterize gross alterations in the gene of a CSNA, such as deletions, insertions, translocations, and duplications of the CSNA genomic locus through fluorescence in situ hybridization (FISH) to chromosome spreads. See, e.g., Andreeff et al. (eds.), Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications, John Wiley & Sons (1999), the disclosure of which is incorporated herein by reference in its entirety. The isolated nucleic acids of the present invention can be used as probes to assess smaller genomic alterations using, e.g., Southern blot detection of restriction fragment length polymorphisms. The isolated nucleic acid molecules of the present invention can be used as probes to isolate genomic clones that include the nucleic acid molecules of the present invention, which thereafter can be restriction mapped and sequenced to identify deletions, insertions, translocations, and substitutions (single nucleotide polymorphisms, SNPs) at the sequence level.

In another embodiment, the isolated nucleic acid molecules of the present invention can be used as probes to detect, characterize, and quantify CSNA in, and isolate CSNA from, transcript-derived nucleic acid samples. In one aspect, the isolated nucleic acid molecules of the present invention can be used as hybridization probes to detect, characterize by length, and quantify mRNA by Northern blot of total or poly-A ⁺-selected RNA samples. In another aspect, the isolated nucleic acid molecules of the present invention can be used as hybridization probes to detect, characterize by location, and quantify mRNA by in situ hybridization to tissue sections. See, e.g., Schwarchzacher et al, In Situ Hybridization, Springer-Verlag New York (2000), the disclosure of which is incorporated herein by reference in its entirety. In another preferred embodiment, the isolated nucleic acid molecules of the present invention can be used as hybridization probes to measure the representation of clones in a cDNA library or to isolate hybridizing nucleic acid molecules acids from cDNA libraries, permitting sequence level characterization of mRNAs that hybridize to CSNAs, including, without limitations, identification of deletions, insertions, substitutions, truncations, alternatively spliced forms and single nucleotide polymorphisms. In yet another preferred embodiment, the nucleic acid molecules of the instant invention may be used in microarrays.

All of the aforementioned probe techniques are well within the skill in the art, and are described at greater length in standard texts such as Sambrook (2001), supra; Ausubel (1999), supra; and Walker et al. (eds.), The Nucleic Acids Protocols Handbook, Humana Press (2000), the disclosures of which are incorporated herein by reference in their entirety.

Thus, in one embodiment, a nucleic acid molecule of the invention may be used as a probe or primer to identify or amplify a second nucleic acid molecule that selectively hybridizes to the nucleic acid molecule of the invention. In a preferred embodiment, the probe or primer is derived from a nucleic acid molecule encoding a CSP. In a more preferred embodiment, the probe or primer is derived from a nucleic acid molecule encoding a polypeptide having an amino acid sequence of SEQ ID NO: 148 through 250. In another preferred embodiment, the probe or primer is derived from a CSNA. In a more preferred embodiment, the probe or primer is derived from a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 through 147.

In general, a probe or primer is at least 10 nucleotides in length, more preferably at least 12, more preferably at least 14 and even more preferably at least 16 or 17 nucleotides in length. In an even more preferred embodiment, the probe or primer is at least 18 nucleotides in length, even more preferably at least 20 nucleotides and even more preferably at least 22 nucleotides in length. Primers and probes may also be longer in length. For instance, a probe or primer may be 25 nucleotides in length, or may be 30, 40 or 50 nucleotides in length. Methods of performing nucleic acid hybridization using oligonucleotide probes are well-known in the art. See, e.g., Sambrook et al., 1989, supra, Chapter 11 and pp. 11.31-11.32 and 11.40-11.44, which describes radiolabeling of short probes, and pp. 11.45-11.53, which describe hybridization conditions for oligonucleotide probes, including specific conditions for probe hybridization (pp. 11.50-11.51).

Methods of performing primer-directed amplification are also well-known in the art. Methods for performing the polymerase chain reaction (PCR) are compiled, inter alia, in McPherson, PCR Basics: From Background to Bench, Springer Verlag (2000); Innis et al. (eds.), PCR Applications: Protocols for Functional Genomics, Academic Press (1999); Gelfand et al. (eds.), PCR Strategies, Academic Press (1998); Newton et al., PCR, Springer-Verlag New York (1997); Burke (ed.), PCR: Essential Techniques, John Wiley & Son Ltd (1996); White (ed.), PCR Cloning Protocols: From Molecular Cloning to Genetic Engineering, Vol. 67, Humana Press (1996); McPherson et al. (eds.), PCR 2: A Practical Approach, Oxford University Press, Inc. (1995); the disclosures of which are incorporated herein by reference in their entireties. Methods for performing RT-PCR are collected, e.g., in Siebert et al. (eds.), Gene Cloning and Analysis by RT-PCR, Eaton Publishing Company/Bio Techniques Books Division, 1998; Siebert (ed.), PCR Technique:RT-PCR, Eaton Publishing Company/BioTechniques Books (1995); the disclosure of which is incorporated herein by reference in its entirety.

PCR and hybridization methods may be used to identify and/or isolate allelic variants, homologous nucleic acid molecules and fragments of the nucleic acid molecules of the invention. PCR and hybridization methods may also be used to identify, amplify and/or isolate nucleic acid molecules that encode homologous proteins, analogs, fusion protein or muteins of the invention. The nucleic acid primers of the present invention can be used to prime amplification of nucleic acid molecules of the invention, using transcript-derived or genomic DNA as template.

The nucleic acid primers of the present invention can also be used, for example, to prime single base extension (SBE) for SNP detection (See, e.g., U.S. Pat. No. 6,004,744, the disclosure of which is incorporated herein by reference in its entirety).

Isothermal amplification approaches, such as rolling circle amplification, are also now well-described. See, e.g., Schweitzer et al., Curr. Opin. Biotechnol. 12(1): 21-7 (2001); U.S. Pat. Nos. 5,854,033 and 5,714,320; and international patent publications WO 97/19193 and WO 00/15779, the disclosures of which are incorporated herein by reference in their entireties. Rolling circle amplification can be combined with other techniques to facilitate SNP detection. See, e.g., Lizardi et al., Nature Genet. 19(3): 225-32 (1998).

Nucleic acid molecules of the present invention may be bound to a substrate either covalently or noncovalently. The substrate can be porous or solid, planar or non-planar, unitary or distributed. The bound nucleic acid molecules may be used as hybridization probes, and may be labeled or unlabeled. In a preferred embodiment, the bound nucleic acid molecules are unlabeled.

In one embodiment, the nucleic acid molecule of the present invention is bound to a porous substrate, e.g., a membrane, typically comprising nitrocellulose, nylon, or positively-charged derivatized nylon. The nucleic acid molecule of the present invention can be used to detect a hybridizing nucleic acid molecule that is present within a labeled nucleic acid sample, e.g., a sample of transcript-derived nucleic acids. In another embodiment, the nucleic acid molecule is bound to a solid substrate, including, without limitation, glass, amorphous silicon, crystalline silicon or plastics. Examples of plastics include, without limitation, polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereof. The solid substrate may be any shape, including rectangular, disk-like and spherical. In a preferred embodiment, the solid substrate is a microscope slide or slide-shaped substrate.

The nucleic acid molecule of the present invention can be attached covalently to a surface of the support substrate or applied to a derivatized surface in a chaotropic agent that facilitates denaturation and adherence by presumed noncovalent interactions, or some combination thereof. The nucleic acid molecule of the present invention can be bound to a substrate to which a plurality of other nucleic acids are concurrently bound, hybridization to each of the plurality of bound nucleic acids being separately detectable. At low density, e.g. on a porous membrane, these substrate-bound collections are typically denominated macroarrays; at higher density, typically on a solid support, such as glass, these substrate bound collections of plural nucleic acids are colloquially termed microarrays. As used herein, the term microarray includes arrays of all densities. It is, therefore, another aspect of the invention to provide microarrays that include the nucleic acids of the present invention.

Expression Vectors, Host Cells and Recombinant Methods of Producing Polypeptides

Another aspect of the present invention relates to vectors that comprise one or more of the isolated nucleic acid molecules of the present invention, and host cells in which such vectors have been introduced.

The vectors can be used, inter alia, for propagating the nucleic acids of the present invention in host cells (cloning vectors), for shuttling the nucleic acids of the present invention between host cells derived from disparate organisms (shuttle vectors), for inserting the nucleic acids of the present invention into host cell chromosomes (insertion vectors), for expressing sense or antisense RNA transcripts of the nucleic acids of the present invention in vitro or within a host cell, and for expressing polypeptides encoded by the nucleic acids of the present invention, alone or as fusions to heterologous polypeptides (expression vectors). Vectors of the present invention will often be suitable for several such uses.

Vectors are by now well-known in the art, and are described, inter alia, in Jones et al. (eds.), Vectors: Cloning Applications: Essential Techniques (Essential Techniques Series), John Wiley & Son Ltd. (1998); Jones et al. (eds.), Vectors: Expression Systems: Essential Techniques (Essential Techniques Series), John Wiley & Son Ltd. (1998); Gacesa et al., Vectors: Essential Data, John Wiley & Sons Ltd. (1995); Cid-Arregui (eds.), Viral Vectors: Basic Science and Gene Therapy, Eaton Publishing Co. (2000); Sambrook (2001), supra; Ausubel (1999), supra; the disclosures of which are incorporated herein by reference in their entireties. Furthermore, an enormous variety of vectors are available commercially. Use of existing vectors and modifications thereof being well within the skill in the art, only basic features need be described here.

Nucleic acid sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Such operative linking of a nucleic sequence of this invention to an expression control sequence, of course, includes, if not already part of the nucleic acid sequence, the provision of a translation initiation codon, ATG or GTG, in the correct reading frame upstream of the nucleic acid sequence.

A wide variety of host/expression vector combinations may be employed in expressing the nucleic acid sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic nucleic acid sequences.

In one embodiment, prokaryotic cells may be used with an appropriate vector. Prokaryotic host cells are often used for cloning and expression. In a preferred embodiment, prokaryotic host cells include E. coli, Pseudomonas, Bacillus and Streptomyces. In a preferred embodiment, bacterial host cells are used to express the nucleic acid molecules of the instant invention. Useful expression vectors for bacterial hosts include bacterial plasmids, such as those from E. coli, Bacillus or Streptomyces, including pBluescript, pGEX-2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, λGT10 and λGT11, and other phages, e.g., M13 and filamentous single-stranded phage DNA. Where E. coli is used as host, selectable markers are, analogously, chosen for selectivity in gram negative bacteria: e.g., typical markers confer resistance to antibiotics, such as ampicillin, tetracycline, chloramphenicol, kanamycin, streptomycin and zeocin; auxotrophic markers can also be used.

In other embodiments, eukaryotic host cells, such as yeast, insect, mammalian or plant cells, may be used. Yeast cells, typically S. cerevisiae, are useful for eukaryotic genetic studies, due to the ease of targeting genetic changes by homologous recombination and the ability to easily complement genetic defects using recombinantly expressed proteins. Yeast cells are useful for identifying interacting protein components, e.g. through use of a two-hybrid system. In a preferred embodiment, yeast cells are useful for protein expression. Vectors of the present invention for use in yeast will typically, but not invariably, contain an origin of replication suitable for use in yeast and a selectable marker that is functional in yeast. Yeast vectors include Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp and YEp series plasmids), Yeast Centromere plasmids (the YCp series plasmids), Yeast Artificial Chromosomes (YACs) which are based on yeast linear plasmids, denoted YLp, pGPD-2, 2 μ plasmids and derivatives thereof, and improved shuttle vectors such as those described in Gietz et al., Gene, 74: 527-34 (1988) (YIplac, YEplac and YCplac). Selectable markers in yeast vectors include a variety of auxotrophic markers, the most common of which are (in Saccharomyces cerevisiae) URA3, HIS3, LEU2, TRP1 and LYS2, which complement specific auxotrophic mutations, such as ura3-52, his3-D1, leu2-D1, trp1-D1 and lys2-201.

Insect cells are often chosen for high efficiency protein expression. Where the host cells are from Spodoptera frugiperda, e.g., Sf9 and Sf21 cell lines, and expresSF™ cells (Protein Sciences Corp., Meriden, Conn., USA)), the vector replicative strategy is typically based upon the baculovirus life cycle. Typically, baculovirus transfer vectors are used to replace the wild-type AcMNPV polyhedrin gene with a heterologous gene of interest. Sequences that flank the polyhedrin gene in the wild-type genome are positioned 5′ and 3′ of the expression cassette on the transfer vectors. Following co-transfection with AcMNPV DNA, a homologous recombination event occurs between these sequences resulting in a recombinant virus carrying the gene of interest and the polyhedrin or p10 promoter. Selection can be based upon visual screening for lacZ fusion activity.

In another embodiment, the host cells may be mammalian cells, which are particularly useful for expression of proteins intended as pharmaceutical agents, and for screening of potential agonists and antagonists of a protein or a physiological pathway. Mammalian vectors intended for autonomous extrachromosomal replication will typically include a viral origin, such as the SV40 origin (for replication in cell lines expressing the large T-antigen, such as COS1 and COS7 cells), the papillomavirus origin, or the EBV origin for long term episomal replication (for use, e.g., in 293-EBNA cells, which constitutively express the EBV EBNA-1 gene product and adenovirus E1A). Vectors intended for integration, and thus replication as part of the mammalian chromosome, can, but need not, include an origin of replication functional in mammalian cells, such as the SV40 origin. Vectors based upon viruses, such as adenovirus, adeno-associated virus, vaccinia virus, and various mammalian retroviruses, will typically replicate according to the viral replicative strategy. Selectable markers for use in mammalian cells include resistance to neomycin (G418), blasticidin, hygromycin and to zeocin, and selection based upon the purine salvage pathway using HAT medium.

Expression in mammalian cells can be achieved using a variety of plasmids, including pSV2, pBC12BI, and p91023, as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retroviruses). Useful vectors for insect cells include baculoviral vectors and pVL 941.

Plant cells can also be used for expression, with the vector replicon typically derived from a plant virus (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) and selectable markers chosen for suitability in plants.

It is known that codon usage of different host cells may be different. For example, a plant cell and a human cell may exhibit a difference in codon preference for encoding a particular amino acid. As a result, human mRNA may not be efficiently translated in a plant, bacteria or insect host cell. Therefore, another embodiment of this invention is directed to codon optimization. The codons of the nucleic acid molecules of the invention may be modified to resemble, as much as possible, genes naturally contained within the host cell without altering the amino acid sequence encoded by the nucleic acid molecule.

Any of a wide variety of expression control sequences may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include the expression control sequences associated with structural genes of the foregoing expression vectors. Expression control sequences that control transcription include, e.g., promoters, enhancers and transcription termination sites. Expression control sequences in eukaryotic cells that control post-transcriptional events include splice donor and acceptor sites and sequences that modify the half-life of the transcribed RNA, e.g., sequences that direct poly(A) addition or binding sites for RNA-binding proteins. Expression control sequences that control translation include ribosome binding sites, sequences which direct targeted expression of the polypeptide to or within particular cellular compartments, and sequences in the 5′ and 3′ untranslated regions that modify the rate or efficiency of translation.

Examples of useful expression control sequences for a prokaryote, e.g., E. coli, will include a promoter, often a phage promoter, such as phage lambda pL promoter, the trc promoter, a hybrid derived from the trp and lac promoters, the bacteriophage T7 promoter (in E. coli cells engineered to express the T7 polymerase), the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, or the araBAD operon. Prokaryotic expression vectors may further include transcription terminators, such as the aspA terminator, and elements that facilitate translation, such as a consensus ribosome binding site and translation termination codon, Schomer et al., Proc. Natl. Acad. Sci. USA 83: 8506-8510 (1986).

Expression control sequences for yeast cells, typically S. cerevisiae, will include a yeast promoter, such as the CYC1 promoter, the GAL1 promoter, the GAL10 promoter, ADH1 promoter, the promoters of the yeast_-mating system, or the GPD promoter, and will typically have elements that facilitate transcription termination, such as the transcription termination signals from the CYC1 or ADH1 gene.

Expression vectors useful for expressing proteins in mammalian cells will include a promoter active in mammalian cells. These promoters include those derived from mammalian viruses, such as the enhancer-promoter sequences from the immediate early gene of the human cytomegalovirus (CMV), the enhancer-promoter sequences from the Rous sarcoma virus long terminal repeat (RSV LTR), the enhancer-promoter from SV40 or the early and late promoters of adenovirus. Other expression control sequences include the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase. Other expression control sequences include those from the gene comprising the CSNA of interest. Often, expression is enhanced by incorporation of polyadenylation sites, such as the late SV40 polyadenylation site and the polyadenylation signal and transcription termination sequences from the bovine growth hormone (BGH) gene, and ribosome binding sites. Furthermore, vectors can include introns, such as intron II of rabbit β-globin gene and the SV40 splice elements.

Preferred nucleic acid vectors also include a selectable or amplifiable marker gene and means for amplifying the copy number of the gene of interest. Such marker genes are well-known in the art. Nucleic acid vectors may also comprise stabilizing sequences (e.g., ori- or ARS-like sequences and telomere-like sequences), or may alternatively be designed to favor directed or non-directed integration into the host cell genome. In a preferred embodiment, nucleic acid sequences of this invention are inserted in frame into an expression vector that allows high level expression of an RNA which encodes a protein comprising the encoded nucleic acid sequence of interest. Nucleic acid cloning and sequencing methods are well-known to those of skill in the art and are described in an assortment of laboratory manuals, including Sambrook (1989), supra, Sambrook (2000), supra; and Ausubel (1992), supra, Ausubel (1999), supra. Product information from manufacturers of biological, chemical and immunological reagents also provide useful information.

Expression vectors may be either constitutive or inducible. Inducible vectors include either naturally inducible promoters, such as the trc promoter, which is regulated by the lac operon, and the pL promoter, which is regulated by tryptophan, the MMTV-LTR promoter, which is inducible by dexamethasone, or can contain synthetic promoters and/or additional elements that confer inducible control on adjacent promoters. Examples of inducible synthetic promoters are the hybrid Plac/ara-1 promoter and the PLtetO-1 promoter. The PltetO-1 promoter takes advantage of the high expression levels from the PL promoter of phage lambda, but replaces the lambda repressor sites with two copies of operator 2 of the Tn10 tetracycline resistance operon, causing this promoter to be tightly repressed by the Tet repressor protein and induced in response to tetracycline (Tc) and Tc derivatives such as anhydrotetracycline. Vectors may also be inducible because they contain hormone response elements, such as the glucocorticoid response element (GRE) and the estrogen response element (ERE), which can confer hormone inducibility where vectors are used for expression in cells having the respective hormone receptors. To reduce background levels of expression, elements responsive to ecdysone, an insect hormone, can be used instead, with coexpression of the ecdysone receptor.

In one aspect of the invention, expression vectors can be designed to fuse the expressed polypeptide to small protein tags that facilitate purification and/or visualization. Tags that facilitate purification include a polyhistidine tag that facilitates purification of the fusion protein by immobilized metal affinity chromatography, for example using NiNTA resin (Qiagen Inc., Valencia, Calif., USA) or TALON™ resin (cobalt immobilized affinity chromatography medium, Clontech Labs, Palo Alto, Calif., USA). The fusion protein can include a chitin-binding tag and self-excising intein, permitting chitin-based purification with self-removal of the fused tag (IMPACT™ system, New England Biolabs, Inc., Beverley, Mass., USA). Alternatively, the fusion protein can include a calmodulin-binding peptide tag, permitting purification by calmodulin affinity resin (Stratagene, La Jolla, Calif., USA), or a specifically excisable fragment of the biotin carboxylase carrier protein, permitting purification of in vivo biotinylated protein using an avidin resin and subsequent tag removal (Promega, Madison, Wis., USA). As another useful alternative, the proteins of the present invention can be expressed as a fusion protein with glutathione-S-transferase, the affinity and specificity of binding to glutathione permitting purification using glutathione affinity resins, such as Glutathione-Superflow Resin (Clontech Laboratories, Palo Alto, Calif., USA), with subsequent elution with free glutathione. Other tags include, for example, the Xpress epitope, detectable by anti-Xpress antibody (Invitrogen, Carlsbad, Calif., USA), a myc tag, detectable by anti-myc tag antibody, the V5 epitope, detectable by anti-V5 antibody (Invitrogen, Carlsbad, Calif., USA), FLAG® epitope, detectable by anti-FLAG® antibody (Stratagene, La Jolla, Calif., USA), and the HA epitope.

For secretion of expressed proteins, vectors can include appropriate sequences that encode secretion signals, such as leader peptides. For example, the pSecTag2 vectors (Invitrogen, Carlsbad, Calif., USA) are 5.2 kb mammalian expression vectors that carry the secretion signal from the V-J2-C region of the mouse Ig kappa-chain for efficient secretion of recombinant proteins from a variety of mammalian cell lines.

Expression vectors can also be designed to fuse proteins encoded by the heterologous nucleic acid insert to polypeptides that are larger than purification and/or identification tags. Useful fusion proteins include those that permit display of the encoded protein on the surface of a phage or cell, fusion to intrinsically fluorescent proteins, such as those that have a green fluorescent protein (GFP)-like chromophore, fusions to the IgG Fc region, and fusion proteins for use in two hybrid systems.

Vectors for phage display fuse the encoded polypeptide to, e.g., the gene III protein (pIII) or gene VIII protein (pVIII) for display on the surface of filamentous phage, such as M13. See Barbas et al., Phage Display: A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001); Kay et al. (eds.), Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, Inc., (1996); Abelson et al. (eds.), Combinatorial Chemistry (Methods in Enzymology, Vol. 267) Academic Press (1996). Vectors for yeast display, e.g. the pYD1 yeast display vector (Invitrogen, Carlsbad, Calif., USA), use the -agglutinin yeast adhesion receptor to display recombinant protein on the surface of S. cerevisiae. Vectors for mammalian display, e.g., the pDisplay™ vector (Invitrogen, Carlsbad, Calif., USA), target recombinant proteins using an N-terminal cell surface targeting signal and a C-terminal transmembrane anchoring domain of platelet derived growth factor receptor.

A wide variety of vectors now exist that fuse proteins encoded by heterologous nucleic acids to the chromophore of the substrate-independent, intrinsically fluorescent green fluorescent protein from Aequorea victoria (“GFP”) and its variants. The GFP-like chromophore can be selected from GFP-like chromophores found in naturally occurring proteins, such as A. victoria GFP (GenBank accession number AAA27721), Renilla reniformis GFP, FP583 (GenBank accession no. AF168419) (DsRed), FP593 (AF272711), FP483 (AF168420), FP484 (AF168424), FP595 (AF246709), FP486 (AF168421), FP538 (AF168423), and FP506 (AF168422), and need include only so much of the native protein as is needed to retain the chromophore's intrinsic fluorescence. Methods for determining the minimal domain required for fluorescence are known in the art. See Li et al., J. Biol. Chem. 272: 28545-28549 (1997). Alternatively, the GFP-like chromophore can be selected from GFP-like chromophores modified from those found in nature. The methods for engineering such modified GFP-like chromophores and testing them for fluorescence activity, both alone and as part of protein fusions, are well-known in the art. See Heim et al., Curr. Biol 6: 178-182 (1996) and Palm et al., Methods Enzymol. 302: 378-394 (1999), incorporated herein by reference in its entirety. A variety of such modified chromophores are now commercially available and can readily be used in the fusion proteins of the present invention. These include EGFP (“enhanced GFP”), EBFP (“enhanced blue fluorescent protein”), BFP2, EYFP (“enhanced yellow fluorescent protein”), ECFP (“enhanced cyan fluorescent protein”) or Citrine. EGFP (see, e.g., Cormack et al., Gene 173: 33-38 (1996); U.S. Pat. Nos. 6,090,919 and 5,804,387) is found on a variety of vectors, both plasmid and viral, which are available commercially (Clontech Labs, Palo Alto, Calif., USA); EBFP is optimized for expression in mammalian cells whereas BFP2, which retains the original jellyfish codons, can be expressed in bacteria (see, e.g., Heim et al., Curr. Biol. 6: 178-182 (1996) and Cormack et al., Gene 173: 33-38 (1996)). Vectors containing these blue-shifted variants are available from Clontech Labs (Palo Alto, Calif., USA). Vectors containing EYFP, ECFP (see, e.g., Heim et al., Curr. Biol. 6: 178-182 (1996); Miyawaki et al., Nature 388: 882-887 (1997)) and Citrine (see, e.g., Heikal et al., Proc. Natl. Acad. Sci. USA 97: 11996-12001 (2000)) are also available from Clontech Labs. The GFP-like chromophore can also be drawn from other modified GFPs, including those described in U.S. Pat. Nos. 6,124,128; 6,096,865; 6,090,919; 6,066,476; 6,054,321; 6,027,881; 5,968,750; 5,874,304; 5,804,387; 5,777,079; 5,741,668; and 5,625,048, the disclosures of which are incorporated herein by reference in their entireties. See also Conn (ed.), Green Fluorescent Protein (Methods in Enzymology, Vol. 302), Academic Press, Inc. (1999). The GFP-like chromophore of each of these GFP variants can usefully be included in the fusion proteins of the present invention.

Fusions to the IgG Fc region increase serum half life of protein pharmaceutical products through interaction with the FcRn receptor (also denominated the FcRp receptor and the Brambell receptor, FcRb), further described in International Patent Application Nos. WO 97/43316, WO 97/34631, WO 96/32478, WO 96/18412.

For long-term, high-yield recombinant production of the proteins, protein fusions, and protein fragments of the present invention, stable expression is preferred. Stable expression is readily achieved by integration into the host cell genome of vectors having selectable markers, followed by selection of these integrants. Vectors such as pUB6/V5-His A, B, and C (Invitrogen, Carlsbad, Calif., USA) are designed for high-level stable expression of heterologous proteins in a wide range of mammalian tissue types and cell lines. pUB6/V5-His uses the promoter/enhancer sequence from the human ubiquitin C gene to drive expression of recombinant proteins: expression levels in 293, CHO, and NIH3T3 cells are comparable to levels from the CMV and human EF-1a promoters. The bsd gene permits rapid selection of stably transfected mammalian cells with the potent antibiotic blasticidin.

Replication incompetent retroviral vectors, typically derived from Moloney murine leukemia virus, also are useful for creating stable transfectants having integrated provirus. The highly efficient transduction machinery of retroviruses, coupled with the availability of a variety of packaging cell lines such as RetroPack™ PT 67, EcoPack2™-293, AmphoPack-293, and GP2-293 cell lines (all available from Clontech Laboratories, Palo Alto, Calif., USA), allow a wide host range to be infected with high efficiency; varying the multiplicity of infection readily adjusts the copy number of the integrated provirus.

Of course, not all vectors and expression control sequences will function equally well to express the nucleic acid sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences and hosts without undue experimentation and without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must be replicated in it. The vector's copy number, the ability to control that copy number, the ability to control integration, if any, and the expression of any other proteins encoded by the vector, such as antibiotic or other selection markers, should also be considered. The present invention further includes host cells comprising the vectors of the present invention, either present episomally within the cell or integrated, in whole or in part, into the host cell chromosome. Among other considerations, some of which are described above, a host cell strain may be chosen for its ability to process the expressed protein in the desired fashion. Such post-translational modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation, and it is an aspect of the present invention to provide CSPs with such post-translational modifications.

Polypeptides of the invention may be post-translationally modified. Post-translational modifications include phosphorylation of amino acid residues serine, threonine and/or tyrosine, N-linked and/or O-linked glycosylation, methylation, acetylation, prenylation, methylation, acetylation, arginylation, ubiquination and racemization. One may determine whether a polypeptide of the invention is likely to be post-translationally modified by analyzing the sequence of the polypeptide to determine if there are peptide motifs indicative of sites for post-translational modification. There are a number of computer programs that permit prediction of post-translational modifications. See, e.g., www.expasy.org (accessed Aug. 31, 2001), which includes PSORT, for prediction of protein sorting signals and localization sites, SignalP, for prediction of signal peptide cleavage sites, MITOPROT and Predotar, for prediction of mitochondrial targeting sequences, NetOGlyc, for prediction of type O-glycosylation sites in mammalian proteins, big-PI Predictor and DGPI, for prediction of prenylation-anchor and cleavage sites, and NetPhos, for prediction of Ser, Thr and Tyr phosphorylation sites in eukaryotic proteins. Other computer programs, such as those included in GCG, also may be used to determine post-translational modification peptide motifs.

General examples of types of post-translational modifications may be found in web sites such as the Delta Mass database http://www.abrf.org/ABRF/Research Committees/deltamass/deltamass.html (accessed Oct. 19, 2001); “GlycoSuiteDB: a new curated relational database of glycoprotein glycan structures and their biological sources” Cooper et al. Nucleic Acids Res. 29; 332-335 (2001) and http://www.glycosuite.com/ (accessed Oct. 19, 2001); “O-GLYCBASE version 4.0: a revised database of O-glycosylated proteins” Gupta et al. Nucleic Acids Research, 27: 370-372 (1999) and http://www.cbs.dtu.dk/databases/OGLYCBASE/ (accessed Oct. 19, 2001); “PhosphoBase, a database of phosphorylation sites: release 2.0.”, Kreegipuu et al. Nucleic Acids Res 27(1):237-239 (1999) and http://www.cbs.dtu.dk/ databases/PhosphoBase/ (accessed Oct. 19, 2001); or http://pir.georgetown.edu/ pirwww/search/textresid.html (accessed Oct. 19, 2001).

Tumorigenesis is often accompanied by alterations in the post-translational modifications of proteins. Thus, in another embodiment, the invention provides polypeptides from cancerous cells or tissues that have altered post-translational modifications compared to the post-translational modifications of polypeptides from normal cells or tissues. A number of altered post-translational modifications are known. One common alteration is a change in phosphorylation state, wherein the polypeptide from the cancerous cell or tissue is hyperphosphorylated or hypophosphorylated compared to the polypeptide from a normal tissue, or wherein the polypeptide is phosphorylated on different residues than the polypeptide from a normal cell. Another common alteration is a change in glycosylation state, wherein the polypeptide from the cancerous cell or tissue has more or less glycosylation than the polypeptide from a normal tissue, and/or wherein the polypeptide from the cancerous cell or tissue has a different type of glycosylation than the polypeptide from a noncancerous cell or tissue. Changes in glycosylation may be critical because carbohydrate-protein and carbohydrate-carbohydrate interactions are important in cancer cell progression, dissemination and invasion. See, e.g., Barchi, Curr. Pharm. Des. 6: 485-501 (2000), Verma, Cancer Biochem. Biophys. 14: 151-162 (1994) and Dennis et al., Bioessays 5: 412-421 (1999).

Another post-translational modification that may be altered in cancer cells is prenylation. Prenylation is the covalent attachment of a hydrophobic prenyl group (either farnesyl or geranylgeranyl) to a polypeptide. Prenylation is required for localizing a protein to a cell membrane and is often required for polypeptide function. For instance, the Ras superfamily of GTPase signaling proteins must be prenylated for function in a cell. See, e.g., Prendergast et al., Semin. Cancer Biol. 10: 443-452 (2000) and Khwaja et al., Lancet 355: 741-744 (2000).

Other post-translation modifications that may be altered in cancer cells include, without limitation, polypeptide methylation, acetylation, arginylation or racemization of amino acid residues. In these cases, the polypeptide from the cancerous cell may exhibit either increased or decreased amounts of the post-translational modification compared to the corresponding polypeptides from noncancerous cells.

Other polypeptide alterations in cancer cells include abnormal polypeptide cleavage of proteins and aberrant protein-protein interactions. Abnormal polypeptide cleavage may be cleavage of a polypeptide in a cancerous cell that does not usually occur in a normal cell, or a lack of cleavage in a cancerous cell, wherein the polypeptide is cleaved in a normal cell. Aberrant protein-protein interactions may be either covalent cross-linking or non-covalent binding between proteins that do not normally bind to each other. Alternatively, in a cancerous cell, a protein may fail to bind to another protein to which it is bound in a noncancerous cell. Alterations in cleavage or in protein-protein interactions may be due to over- or underproduction of a polypeptide in a cancerous cell compared to that in a normal cell, or may be due to alterations in post-translational modifications (see above) of one or more proteins in the cancerous cell. See, e.g., Henschen-Edman, Ann. N.Y. Acad. Sci. 936: 580-593 (2001).

Alterations in polypeptide post-translational modifications, as well as changes in polypeptide cleavage and protein-protein interactions, may be determined by any method known in the art. For instance, alterations in phosphorylation may be determined by using anti-phosphoserine, anti-phosphothreonine or anti-phosphotyrosine antibodies or by amino acid analysis. Glycosylation alterations may be determined using antibodies specific for different sugar residues, by carbohydrate sequencing, or by alterations in the size of the glycoprotein, which can be determined by, e.g., SDS polyacrylamide gel electrophoresis (PAGE). Other alterations of post-translational modifications, such as prenylation, racemization, methylation, acetylation and arginylation, may be determined by chemical analysis, protein sequencing, amino acid analysis, or by using antibodies specific for the particular post-translational modifications. Changes in protein-protein interactions and in polypeptide cleavage may be analyzed by any method known in the art including, without limitation, non-denaturing PAGE (for non-covalent protein-protein interactions), SDS PAGE (for covalent protein-protein interactions and protein cleavage), chemical cleavage, protein sequencing or immunoassays.

In another embodiment, the invention provides polypeptides that have been post-translationally modified. In one embodiment, polypeptides may be modified enzymatically or chemically, by addition or removal of a post-translational modification. For example, a polypeptide may be glycosylated or deglycosylated enzymatically. Similarly, polypeptides may be phosphorylated using a purified kinase, such as a MAP kinase (e.g., p38, ERK, or JNK) or a tyrosine kinase (e.g., Src or erbB2). A polypeptide may also be modified through synthetic chemistry. Alternatively, one may isolate the polypeptide of interest from a cell or tissue that expresses the polypeptide with the desired post-translational modification. In another embodiment, a nucleic acid molecule encoding the polypeptide of interest is introduced into a host cell that is capable of post-translationally modifying the encoded polypeptide in the desired fashion. If the polypeptide does not contain a motif for a desired post-translational modification, one may alter the post-translational modification by mutating the nucleic acid sequence of a nucleic acid molecule encoding the polypeptide so that it contains a site for the desired post-translational modification. Amino acid sequences that may be post-translationally modified are known in the art. See, e.g., the programs described above on the website www.expasy.org. The nucleic acid molecule is then be introduced into a host cell that is capable of post-translationally modifying the encoded polypeptide. Similarly, one may delete sites that are post-translationally modified by either mutating the nucleic acid sequence so that the encoded polypeptide does not contain the post-translational modification motif, or by introducing the native nucleic acid molecule into a host cell that is not capable of post-translationally modifying the encoded polypeptide.

In selecting an expression control sequence, a variety of factors should also be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the nucleic acid sequence of this invention, particularly with regard to potential secondary structures. Unicellular hosts should be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the nucleic acid sequences of this invention, their secretion characteristics, their ability to fold the polypeptide correctly, their fermentation or culture requirements, and the ease of purification from them of the products coded for by the nucleic acid sequences of this invention.

The recombinant nucleic acid molecules and more particularly, the expression vectors of this invention may be used to express the polypeptides of this invention as recombinant polypeptides in a heterologous host cell. The polypeptides of this invention may be full-length or less than full-length polypeptide fragments recombinantly expressed from the nucleic acid sequences according to this invention. Such polypeptides include analogs, derivatives and muteins that may or may not have biological activity.

Vectors of the present invention will also often include elements that permit in vitro transcription of RNA from the inserted heterologous nucleic acid. Such vectors typically include a phage promoter, such as that from T7, T3, or SP6, flanking the nucleic acid insert. Often two different such promoters flank the inserted nucleic acid, permitting separate in vitro production of both sense and antisense strands.

Transformation and other methods of introducing nucleic acids into a host cell (e.g., conjugation, protoplast transformation or fusion, transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion) can be accomplished by a variety of methods which are well-known in the art (See, for instance, Ausubel, supra, and Sambrook et al., supra). Bacterial, yeast, plant or mammalian cells are transformed or transfected with an expression vector, such as a plasmid, a cosmid, or the like, wherein the expression vector comprises the nucleic acid of interest. Alternatively, the cells may be infected by a viral expression vector comprising the nucleic acid of interest. Depending upon the host cell, vector, and method of transformation used, transient or stable expression of the polypeptide will be constitutive or inducible. One having ordinary skill in the art will be able to decide whether to express a polypeptide transiently or stably, and whether to express the protein constitutively or inducibly.

A wide variety of unicellular host cells are useful in expressing the DNA sequences of this invention. These hosts may include well-known eukaryotic and prokaryotic hosts, such as strains of, fungi, yeast, insect cells such as Spodoptera frugiperda (SF9), animal cells such as CHO, as well as plant cells in tissue culture. Representative examples of appropriate host cells include, but are not limited to, bacterial cells, such as E. coli, Caulobacter crescentus, Streptomyces species, and Salmonella typhimurium; yeast cells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichia methanolica; insect cell lines, such as those from Spodoptera frugiperda, e.g., Sf9 and Sf21 cell lines, and expresSF™ cells (Protein Sciences Corp., Meriden, Conn., USA), Drosophila S2 cells, and Trichoplusia ni High Five® Cells (Invitrogen, Carlsbad, Calif., USA); and mammalian cells. Typical mammalian cells include BHK cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, COS1 cells, COS7 cells, Chinese hamster ovary (CHO) cells, 3T3 cells, NIH 3T3 cells, 293 cells, HEPG2 cells, HeLa cells, L cells, MDCK cells, HEK293 cells, W138 cells, murine ES cell lines (e.g., from strains 129/SV, C57/BL6, DBA-1, 129/SVJ), K562 cells, Jurkat cells, and BW5147 cells. Other mammalian cell lines are well-known and readily available from the American Type Culture Collection (ATCC) (Manassas, Va., USA) and the National Institute of General Medical Sciences (NIGMS) Human Genetic Cell Repository at the Coriell Cell Repositories (Camden, N.J., USA). Cells or cell lines derived from colon are particularly preferred because they may provide a more native post-translational processing. Particularly preferred are human colon cells.

Particular details of the transfection, expression and purification of recombinant proteins are well documented and are understood by those of skill in the art. Further details on the various technical aspects of each of the steps used in recombinant production of foreign genes in bacterial cell expression systems can be found in a number of texts and laboratory manuals in the art. See, e.g., Ausubel (1992), supra, Ausubel (1999), supra, Sambrook (1989), supra, and Sambrook (2001), supra, herein incorporated by reference.

Methods for introducing the vectors and nucleic acids of the present invention into the host cells are well-known in the art; the choice of technique will depend primarily upon the specific vector to be introduced and the host cell chosen.

Nucleic acid molecules and vectors may be introduced into prokaryotes, such as E. coli, in a number of ways. For instance, phage lambda vectors will typically be packaged using a packaging extract (e.g., Gigapack® packaging extract, Stratagene, La Jolla, Calif., USA), and the packaged virus used to infect E. coli.

Plasmid vectors will typically be introduced into chemically competent or electrocompetent bacterial cells. E. coli cells can be rendered chemically competent by treatment, e.g., with CaCl₂, or a solution of Mg²⁺, Mn²⁺, Ca²⁺, Rb⁺ or K⁺, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III), Hanahan, J. Mol. Biol. 166(4):557-80 (1983), and vectors introduced by heat shock. A wide variety of chemically competent strains are also available commercially (e.g., Epicurian Coli® XL10-Gold® Ultracompetent Cells (Stratagene, La Jolla, Calif., USA); DH5 competent cells (Clontech Laboratories, Palo Alto, Calif., USA); and TOP10 Chemically Competent E. coli Kit (Invitrogen, Carlsbad, Calif., USA)). Bacterial cells can be rendered electrocompetent, that is, competent to take up exogenous DNA by electroporation, by various pre-pulse treatments; vectors are introduced by electroporation followed by subsequent outgrowth in selected media. An extensive series of protocols is provided online in Electroprotocols (BioRad, Richmond, Calif., USA) (http://www.biorad.com/LifeScience/pdf/ New_Gene_Pulser.pdf).

Vectors can be introduced into yeast cells by spheroplasting, treatment with lithium salts, electroporation, or protoplast fusion. Spheroplasts are prepared by the action of hydrolytic enzymes such as snail-gut extract, usually denoted Glusulase, or Zymolyase, an enzyme from Arthrobacter luteus, to remove portions of the cell wall in the presence of osmotic stabilizers, typically 1 M sorbitol. DNA is added to the spheroplasts, and the mixture is co-precipitated with a solution of polyethylene glycol (PEG) and Ca²⁺. Subsequently, the cells are resuspended in a solution of sorbitol, mixed with molten agar and then layered on the surface of a selective plate containing sorbitol.

For lithium-mediated transformation, yeast cells are treated with lithium acetate, which apparently permeabilizes the cell wall, DNA is added and the cells are co-precipitated with PEG. The cells are exposed to a brief heat shock, washed free of PEG and lithium acetate, and subsequently spread on plates containing ordinary selective medium. Increased frequencies of transformation are obtained by using specially-prepared single-stranded carrier DNA and certain organic solvents. Schiestl et al., Curr. Genet. 16(5-6): 339-46 (1989).

For electroporation, freshly-grown yeast cultures are typically washed, suspended in an osmotic protectant, such as sorbitol, mixed with DNA, and the cell suspension pulsed in an electroporation device. Subsequently, the cells are spread on the surface of plates containing selective media. Becker et al., Methods Enzymol. 194: 182-187 (1991). The efficiency of transformation by electroporation can be increased over 100-fold by using PEG, single-stranded carrier DNA and cells that are in late log-phase of growth. Larger constructs, such as YACs, can be introduced by protoplast fusion.

Mammalian and insect cells can be directly infected by packaged viral vectors, or transfected by chemical or electrical means. For chemical transfection, DNA can be coprecipitated with CaPO ₄or introduced using liposomal and nonliposomal lipid-based agents. Commercial kits are available for CaPO₄transfection (Calphos™ Mammalian Transfection Kit, Clontech Laboratories, Palo Alto, Calif., USA), and lipid-mediated transfection can be practiced using commercial reagents, such as LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ Reagent, CELLFECTIN® Reagent, and LIPOFECTIN™ Reagent (Invitrogen, Carlsbad, Calif., USA), DOTAP Liposomal Transfection Reagent, FuGENE 6, X-tremeGENE Q2, DOSPER, (Roche Molecular Biochemicals, Indianapolis, Ind. USA), Effectene™, PolyFect®, Superfect® (Qiagen, Inc., Valencia, Calif., USA). Protocols for electroporating mammalian cells can be found online in Electroprotocols (Bio-Rad, Richmond, Calif., USA) (http://www.bio-rad.com/LifeScience/pdf/New_Gene_Pulser.pdf); Norton et al. (eds.), Gene Transfer Methods: Introducing DNA into Living Cells and Organisms, BioTechniques Books, Eaton Publishing Co. (2000); incorporated herein by reference in its entirety. Other transfection techniques include transfection by particle bombardment and microinjection. See, e.g., Cheng et al., Proc. Natl. Acad. Sci. USA 90(10): 4455-9 (1993); Yang et al., Proc. Natl. Acad. Sci. USA 87(24): 9568-72 (1990).

Production of the recombinantly produced proteins of the present invention can optionally be followed by purification.

Purification of recombinantly expressed proteins is now well by those skilled in the art. See, e.g., Thorner et al. (eds.), Applications of Chimeric Genes and Hybrid Proteins, Part A: Gene Expression and Protein Purification (Methods in Enzymology, Vol. 326), Academic Press (2000); Harbin (ed.), Cloning, Gene Expression and Protein Purification: Experimental Procedures and Process Rationale, Oxford Univ. Press (2001); Marshak et al., Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Cold Spring Harbor Laboratory Press (1996); and Roe (ed.), Protein Purification Applications, Oxford University Press (2001); the disclosures of which are incorporated herein by reference in their entireties, and thus need not be detailed here.

Briefly, however, if purification tags have been fused through use of an expression vector that appends such tags, purification can be effected, at least in part, by means appropriate to the tag, such as use of immobilized metal affinity chromatography for polyhistidine tags. Other techniques common in the art include ammonium sulfate fractionation, immunoprecipitation, fast protein liquid chromatography (FPLC), high performance liquid chromatography (HPLC), and preparative gel electrophoresis.

Polypeptides

Another object of the invention is to provide polypeptides encoded by the nucleic acid molecules of the instant invention. In a preferred embodiment, the polypeptide is a colon specific polypeptide (CSP). In an even more preferred embodiment, the polypeptide is derived from a polypeptide comprising the amino acid sequence of SEQ ID NO: 148 through 250. A polypeptide as defined herein may be produced recombinantly, as discussed supra, may be isolated from a cell that naturally expresses the protein, or may be chemically synthesized following the teachings of the specification and using methods well-known to those having ordinary skill in the art.

In another aspect, the polypeptide may comprise a fragment of a polypeptide, wherein the fragment is as defined herein. In a preferred embodiment, the polypeptide fragment is a fragment of a CSP. In a more preferred embodiment, the fragment is derived from a polypeptide comprising the amino acid sequence of SEQ ID NO: 148 through 250. A polypeptide that comprises only a fragment of an entire CSP may or may not be a polypeptide that is also a CSP. For instance, a full-length polypeptide may be colon-specific, while a fragment thereof may be found in other tissues as well as in colon. A polypeptide that is not a CSP, whether it is a fragment, analog, mutein, homologous protein or derivative, is nevertheless useful, especially for immunizing animals to prepare anti-CSP antibodies. However, in a preferred embodiment, the part or fragment is a CSP. Methods of determining whether a polypeptide is a CSP are described infra.

Fragments of at least 6 contiguous amino acids are useful in mapping B cell and T cell epitopes of the reference protein. See, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1984) and U.S. Pat. Nos. 4,708,871 and 5,595,915, the disclosures of which are incorporated herein by reference in their entireties. Because the fragment need not itself be immunogenic, part of an immunodominant epitope, nor even recognized by native antibody, to be useful in such epitope mapping, all fragments of at least 6 amino acids of the proteins of the present invention have utility in such a study.

Fragments of at least 8 contiguous amino acids, often at least 15 contiguous amino acids, are useful as immunogens for raising antibodies that recognize the proteins of the present invention. See, e.g., Lerner, Nature 299: 592-596 (1982); Shinnick et al., Annu. Rev. Microbiol. 37: 425-46 (1983); Sutcliffe et al., Science 219: 660-6 (1983), the disclosures of which are incorporated herein by reference in their entireties. As further described in the above-cited references, virtually all 8-mers, conjugated to a carrier, such as a protein, prove immunogenic, meaning that they are capable of eliciting antibody for the conjugated peptide; accordingly, all fragments of at least 8 amino acids of the proteins of the present invention have utility as immunogens.

Fragments of at least 8, 9, 10 or 12 contiguous amino acids are also useful as competitive inhibitors of binding of the entire protein, or a portion thereof, to antibodies (as in epitope mapping), and to natural binding partners, such as subunits in a multimeric complex or to receptors or ligands of the subject protein; this competitive inhibition permits identification and separation of molecules that bind specifically to the protein of interest, U.S. Pat. Nos. 5,539,084 and 5,783,674, incorporated herein by reference in their entireties.

The protein, or protein fragment, of the present invention is thus at least 6 amino acids in length, typically at least 8, 9, 10 or 12 amino acids in length, and often at least 15 amino acids in length. Often, the protein of the present invention, or fragment thereof, is at least 20 amino acids in length, even 25 amino acids, 30 amino acids, 35 amino acids, or 50 amino acids or more in length. Of course, larger fragments having at least 75 amino acids, 100 amino acids, or even 150 amino acids are also useful, and at times preferred.

One having ordinary skill in the art can produce fragments of a polypeptide by truncating the nucleic acid molecule, e.g., a CSNA, encoding the polypeptide and then expressing it recombinantly. Alternatively, one can produce a fragment by chemically synthesizing a portion of the full-length polypeptide. One may also produce a fragment by enzymatically cleaving either a recombinant polypeptide or an isolated naturally-occurring polypeptide. Methods of producing polypeptide fragments are well-known in the art. See, e.g., Sambrook (1989), supra; Sambrook (2001), supra; Ausubel (1992), supra; and Ausubel (1999), supra. In one embodiment, a polypeptide comprising only a fragment of polypeptide of the invention, preferably a CSP, may be produced by chemical or enzymatic cleavage of a polypeptide. In a preferred embodiment, a polypeptide fragment is produced by expressing a nucleic acid molecule encoding a fragment of the polypeptide, preferably a CSP, in a host cell.

By “polypeptides” as used herein it is also meant to be inclusive of mutants, fusion proteins, homologous proteins and allelic variants of the polypeptides specifically exemplified.

A mutant protein, or mutein, may have the same or different properties compared to a naturally-occurring polypeptide and comprises at least one amino acid insertion, duplication, deletion, rearrangement or substitution compared to the amino acid sequence of a native protein. Small deletions and insertions can often be found that do not alter the function of the protein. In one embodiment, the mutein may or may not be colon-specific. In a preferred embodiment, the mutein is colon-specific. In a preferred embodiment, the mutein is a polypeptide that comprises at least one amino acid insertion, duplication, deletion, rearrangement or substitution compared to the amino acid sequence of SEQ ID NO: 148 through 250. In a more preferred embodiment, the mutein is one that exhibits at least 50% sequence identity, more preferably at least 60% sequence identity, even more preferably at least 70%, yet more preferably at least 80% sequence identity to a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250. In yet a more preferred embodiment, the mutein exhibits at least 85%, more preferably 90%, even more preferably 95% or 96%, and yet more preferably at least 97%, 98%, 99% or 99.5% sequence identity to a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250.

A mutein may be produced by isolation from a naturally-occurring mutant cell, tissue or organism. A mutein may be produced by isolation from a cell, tissue or organism that has been experimentally mutagenized. Alternatively, a mutein may be produced by chemical manipulation of a polypeptide, such as by altering the amino acid residue to another amino acid residue using synthetic or semi-synthetic chemical techniques. In a preferred embodiment, a mutein may be produced from a host cell comprising an altered nucleic acid molecule compared to the naturally-occurring nucleic acid molecule. For instance, one may produce a mutein of a polypeptide by introducing one or more mutations into a nucleic acid sequence of the invention and then expressing it recombinantly. These mutations may be targeted, in which particular encoded amino acids are altered, or may be untargeted, in which random encoded amino acids within the polypeptide are altered. Muteins with random amino acid alterations can be screened for a particular biological activity or property, particularly whether the polypeptide is colon-specific, as described below. Multiple random mutations can be introduced into the gene by methods well-known to the art, e.g., by error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis and site-specific mutagenesis. Methods of producing muteins with targeted or random amino acid alterations are well-known in the art. See, e.g., Sambrook (1989), supra; Sambrook (2001), supra; Ausubel (1992), supra; and Ausubel (1999), U.S. Pat. No. 5,223,408, and the references discussed supra, each herein incorporated by reference.

By “polypeptide” as used herein it is also meant to be inclusive of polypeptides homologous to those polypeptides exemplified herein. In a preferred embodiment, the polypeptide is homologous to a CSP. In an even more preferred embodiment, the polypeptide is homologous to a CSP selected from the group having an amino acid sequence of SEQ ID NO: 148 through 250. In a preferred embodiment, the homologous polypeptide is one that exhibits significant sequence identity to a CSP. In a more preferred embodiment, the polypeptide is one that exhibits significant sequence identity to an comprising an amino acid sequence of SEQ ID NO: 148 through 250. In an even more preferred embodiment, the homologous polypeptide is one that exhibits at least 50% sequence identity, more preferably at least 60% sequence identity, even more preferably at least 70%, yet more preferably at least 80% sequence identity to a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250. In a yet more preferred embodiment, the homologous polypeptide is one that exhibits at least 85%, more preferably 90%, even more preferably 95% or 96%, and yet more preferably at least 97% or 98% sequence identity to a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250. In another preferred embodiment, the homologous polypeptide is one that exhibits at least 99%, more preferably 99.5%, even more preferably 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250. In a preferred embodiment, the amino acid substitutions are conservative amino acid substitutions as discussed above.

In another embodiment, the homologous polypeptide is one that is encoded by a nucleic acid molecule that selectively hybridizes to a CSNA. In a preferred embodiment, the homologous polypeptide is encoded by a nucleic acid molecule that hybridizes to a CSNA under low stringency, moderate stringency or high stringency conditions, as defined herein. In a more preferred embodiment, the CSNA is selected from the group consisting of SEQ ID NO: 1 through 147. In another preferred embodiment, the homologous polypeptide is encoded by a nucleic acid molecule that hybridizes to a nucleic acid molecule that encodes a CSP under low stringency, moderate stringency or high stringency conditions, as defined herein. In a more preferred embodiment, the CSP is selected from the group consisting of SEQ ID NO: 148 through 250.

The homologous polypeptide may be a naturally-occurring one that is derived from another species, especially one derived from another primate, such as chimpanzee, gorilla, rhesus macaque, baboon or gorilla, wherein the homologous polypeptide comprises an amino acid sequence that exhibits significant sequence identity to that of SEQ ID NO: 148 through 250. The homologous polypeptide may also be a naturally-occurring polypeptide from a human, when the CSP is a member of a family of polypeptides. The homologous polypeptide may also be a naturally-occurring polypeptide derived from a non-primate, mammalian species, including without limitation, domesticated species, e.g., dog, cat, mouse, rat, rabbit, guinea pig, hamster, cow, horse, goat or pig. The homologous polypeptide may also be a naturally-occurring polypeptide derived from a non-mammalian species, such as birds or reptiles. The naturally-occurring homologous protein may be isolated directly from humans or other species. Alternatively, the nucleic acid molecule encoding the naturally-occurring homologous polypeptide may be isolated and used to express the homologous polypeptide recombinantly. In another embodiment, the homologous polypeptide may be one that is experimentally produced by random mutation of a nucleic acid molecule and subsequent expression of the nucleic acid molecule. In another embodiment, the homologous polypeptide may be one that is experimentally produced by directed mutation of one or more codons to alter the encoded amino acid of a CSP. Further, the homologous protein may or may not encode polypeptide that is a CSP. However, in a preferred embodiment, the homologous polypeptide encodes a polypeptide that is a CSP.

Relatedness of proteins can also be characterized using a second functional test, the ability of a first protein competitively to inhibit the binding of a second protein to an antibody. It is, therefore, another aspect of the present invention to provide isolated proteins not only identical in sequence to those described with particularity herein, but also to provide isolated proteins (“cross-reactive proteins”) that competitively inhibit the binding of antibodies to all or to a portion of various of the isolated polypeptides of the present invention. Such competitive inhibition can readily be determined using immunoassays well-known in the art.

As discussed above, single nucleotide polymorphisms (SNPs) occur frequently in eukaryotic genomes, and the sequence determined from one individual of a species may differ from other allelic forms present within the population. Thus, by “polypeptide” as used herein it is also meant to be inclusive of polypeptides encoded by an allelic variant of a nucleic acid molecule encoding a CSP. In a preferred embodiment, the polypeptide is encoded by an allelic variant of a gene that encodes a polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 148 through 250. In a yet more preferred embodiment, the polypeptide is encoded by an allelic variant of a gene that has the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 through 147.

In another embodiment, the invention provides polypeptides which comprise derivatives of a polypeptide encoded by a nucleic acid molecule according to the instant invention. In a preferred embodiment, the polypeptide is a CSP. In a preferred embodiment, the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 148 through 250, or is a mutein, allelic variant, homologous protein or fragment thereof. In a preferred embodiment, the derivative has been acetylated, carboxylated, phosphorylated, glycosylated or ubiquitinated. In another preferred embodiment, the derivative has been labeled with, e.g., radioactive isotopes such as ¹²⁵I, ³²P, ³⁵S, and ³H. In another preferred embodiment, the derivative has been labeled with fluorophores, chemiluminescent agents, enzymes, and antiligands that can serve as specific binding pair members for a labeled ligand.

Polypeptide modifications are well-known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as, for instance Creighton, Protein Structure and Molecular Properties, 2nd ed., W. H. Freeman and Company (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, in Johnson (ed.), Posttranslational Covalent Modification of Proteins, pgs. 1-12, Academic Press (1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Ann. N.Y. Acad. Sci. 663: 48-62 (1992).

It will be appreciated, as is well-known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be branched as a result of ubiquitination, and they may be circular, with or without branching, generally as a result of posttranslation events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translation natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli, prior to proteolytic processing, almost invariably will be N-formylmethionine.

Useful post-synthetic (and post-translational) modifications include conjugation to detectable labels, such as fluorophores. A wide variety of amine-reactive and thiol-reactive fluorophore derivatives have been synthesized that react under nondenaturing conditions with N-terminal amino groups and epsilon amino groups of lysine residues, on the one hand, and with free thiol groups of cysteine residues, on the other.

Kits are available commercially that permit conjugation of proteins to a variety of amine-reactive or thiol-reactive fluorophores: Molecular Probes, Inc. (Eugene, Oreg., USA), e.g., offers kits for conjugating proteins to Alexa Fluor 350, Alexa Fluor 430, Fluorescein-EX, Alexa Fluor 488, Oregon Green 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, and Texas Red-X.

A wide variety of other amine-reactive and thiol-reactive fluorophores are available commercially (Molecular Probes, Inc., Eugene, Oreg., USA), including Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, Oreg., USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg., USA).

The polypeptides of the present invention can also be conjugated to fluorophores, other proteins, and other macromolecules, using bifunctional linking reagents. Common homobifunctional reagents include, e.g., APG, AEDP, BASED, BMB, BMDB, BMH, BMOE, BM[PEO]3, BM[PEO]4, BS3, BSOCOES, DFDNB, DMA, DMP, DMS, DPDPB, DSG, DSP (Lomant's Reagent), DSS, DST, DTBP, DTME, DTSSP, EGS, HBVS, Sulfo-BSOCOES, Sulfo-DST, Sulfo-EGS (all available from Pierce, Rockford, Ill., USA); common heterobifunctional cross-linkers include ABH, AMAS, ANB-NOS, APDP, ASBA, BMPA, BMPH, BMPS, EDC, EMCA, EMCH, EMCS, KMUA, KMUH, GMBS, LC-SMCC, LC-SPDP, MBS, M2C2H, MPBH, MSA, NHS-ASA, PDPH, PMPI, SADP, SAED, SAND, SANPAH, SASD, SATP, SBAP, SFAD, SIA, SIAB, SMCC, SMPB, SMPH, SMPT, SPDP, Sulfo-EMCS, Sulfo-GMBS, Sulfo-HSAB, Sulfo-KMUS, Sulfo-LC-SPDP, Sulfo-MBS, Sulfo-NHS-LC-ASA, Sulfo-SADP, Sulfo-SANPAH, Sulfo-SIAB, Sulfo-SMCC, Sulfo-SMPB, Sulfo-LC-SMPT, SVSB, TFCS (all available Pierce, Rockford, Ill., USA).

The polypeptides, fragments, and fusion proteins of the present invention can be conjugated, using such cross-linking reagents, to fluorophores that are not amine- or thiol-reactive. Other labels that usefully can be conjugated to the polypeptides, fragments, and fusion proteins of the present invention include radioactive labels, echosonographic contrast reagents, and MRI contrast agents.

The polypeptides, fragments, and fusion proteins of the present invention can also usefully be conjugated using cross-linking agents to carrier proteins, such as KLH, bovine thyroglobulin, and even bovine serum albumin (BSA), to increase immunogenicity for raising anti-CSP antibodies.

The polypeptides, fragments, and fusion proteins of the present invention can also usefully be conjugated to polyethylene glycol (PEG); PEGylation increases the serum half-life of proteins administered intravenously for replacement therapy. Delgado et al., Crit. Rev. Ther. Drug Carrier Syst. 9(3-4): 249-304 (1992); Scott et al., Curr. Pharm. Des. 4(6): 423-38 (1998); DeSantis et al., Curr. Opin. Biotechnol. 10(4): 324-30 (1999), incorporated herein by reference in their entireties. PEG monomers can be attached to the protein directly or through a linker, with PEGylation using PEG monomers activated with tresyl chloride (2,2,2-trifluoroethanesulphonyl chloride) permitting direct attachment under mild conditions.

In yet another embodiment, the invention provides analogs of a polypeptide encoded by a nucleic acid molecule according to the instant invention. In a preferred embodiment, the polypeptide is a CSP. In a more preferred embodiment, the analog is derived from a polypeptide having part or all of the amino acid sequence of SEQ ID NO: 148 through 250. In a preferred embodiment, the analog is one that comprises one or more substitutions of non-natural amino acids or non-native inter-residue bonds compared to the naturally-occurring polypeptide. In general, the non-peptide analog is structurally similar to a CSP, but one or more peptide linkages is replaced by a linkage selected from the group consisting of —CH ₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH-(cis and trans), —COCH₂—, —CH(OH)CH₂— and —CH₂SO—. In another embodiment, the non-peptide analog comprises substitution of one or more amino acids of a CSP with a D-amino acid of the same type or other non-natural amino acid in order to generate more stable peptides. D-amino acids can readily be incorporated during chemical peptide synthesis: peptides assembled from D-amino acids are more resistant to proteolytic attack; incorporation of D-amino acids can also be used to confer specific three-dimensional conformations on the peptide. Other amino acid analogues commonly added during chemical synthesis include ornithine, norleucine, phosphorylated amino acids (typically phosphoserine, phosphothreonine, phosphotyrosine), L-malonyltyro sine, a non-hydrolyzable analog of phosphotyrosine (see, e.g., Kole et al., Biochem. Biophys. Res. Com. 209: 817-821 (1995)), and various halogenated phenylalanine derivatives.

Non-natural amino acids can be incorporated during solid phase chemical synthesis or by recombinant techniques, although the former is typically more common. Solid phase chemical synthesis of peptides is well established in the art. Procedures are described, inter alia, in Chan et al. (eds.), Fmoc Solid Phase Peptide Synthesis: A Practical Approach (Practical Approach Series), Oxford Univ. Press (March 2000); Jones, Amino Acid and Peptide Synthesis (Oxford Chemistry Primers, No 7), Oxford Univ. Press (1992); and Bodanszky, Principles of Peptide Synthesis (Springer Laboratory), Springer Verlag (1993); the disclosures of which are incorporated herein by reference in their entireties.

Amino acid analogues having detectable labels are also usefully incorporated during synthesis to provide derivatives and analogs. Biotin, for example can be added using biotinoyl-(9-fluorenylmethoxycarbonyl)-L-lysine (FMOC biocytin) (Molecular Probes, Eugene, Oreg., USA). Biotin can also be added enzymatically by incorporation into a fusion protein of a E. coli BirA substrate peptide. The FMOC and tBOC derivatives of dabcyl-L-lysine (Molecular Probes, Inc., Eugene, Oreg., USA) can be used to incorporate the dabcyl chromophore at selected sites in the peptide sequence during synthesis. The aminonaphthalene derivative EDANS, the most common fluorophore for pairing with the dabcyl quencher in fluorescence resonance energy transfer (FRET) systems, can be introduced during automated synthesis of peptides by using EDANS-FMOC-L-glutamic acid or the corresponding tBOC derivative (both from Molecular Probes, Inc., Eugene, Oreg., USA). Tetramethylrhodamine fluorophores can be incorporated during automated FMOC synthesis of peptides using (FMOC)-TMR-L-lysine (Molecular Probes, Inc. Eugene, Oreg., USA).

Other useful amino acid analogues that can be incorporated during chemical synthesis include aspartic acid, glutamic acid, lysine, and tyrosine analogues having allyl side-chain protection (Applied Biosystems, Inc., Foster City, Calif., USA); the allyl side chain permits synthesis of cyclic, branched-chain, sulfonated, glycosylated, and phosphorylated peptides.

A large number of other FMOC-protected non-natural amino acid analogues capable of incorporation during chemical synthesis are available commercially, including, e.g., Fmoc-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, Fmoc-3-endo-aminobicyclo[2.2.1]heptane-2-endo-carboxylic acid, Fmoc-3-exo-aminobicyclo[2.2.1]heptane-2-exo-carboxylic acid, Fmoc-3-endo-amino-bicyclo[2.2.1]hept-5-ene-2-endo-carboxylic acid, Fmoc-3-exo-amino-bicyclo[2.2.1]hept-5-ene-2-exo-carboxylic acid, Fmoc-cis-2-amino-1-cyclohexanecarboxylic acid, Fmoc-trans-2-amino-1-cyclohexanecarboxylic acid, Fmoc-1-amino-1-cyclopentanecarboxylic acid, Fmoc-cis-2-amino-1-cyclopentanecarboxylic acid, Fmoc-1-amino-1-cyclopropanecarboxylic acid, Fmoc-D-2-amino-4-(ethylthio)butyric acid, Fmoc-L-2-amino-4-(ethylthio)butyric acid, Fmoc-L-buthionine, Fmoc-S-methyl-L-Cysteine, Fmoc-2-aminobenzoic acid (anthranillic acid), Fmoc-3-aminobenzoic acid, Fmoc-4-aminobenzoic acid, Fmoc-2-aminobenzophenone-2′-carboxylic acid, Fmoc-N-(4-aminobenzoyl)-β-alanine, Fmoc-2-amino-4,5-dimethoxybenzoic acid, Fmoc-4-aminohippuric acid, Fmoc-2-amino-3-hydroxybenzoic acid, Fmoc-2-amino-5-hydroxybenzoic acid, Fmoc-3-amino-4-hydroxybenzoic acid, Fmoc-4-amino-3-hydroxybenzoic acid, Fmoc-4-amino-2-hydroxybenzoic acid, Fmoc-5-amino-2-hydroxybenzoic acid, Fmoc-2-amino-3-methoxybenzoic acid, Fmoc-4-amino-3-methoxybenzoic acid, Fmoc-2-amino-3-methylbenzoic acid, Fmoc-2-amino-5-methylbenzoic acid, Fmoc-2-amino-6-methylbenzoic acid, Fmoc-3-amino-2-methylbenzoic acid, Fmoc-3-amino-4-methylbenzoic acid, Fmoc-4-amino-3-methylbenzoic acid, Fmoc-3-amino-2-naphtoic acid, Fmoc-D,L-3-amino-3-phenylpropionic acid, Fmoc-L-Methyldopa, Fmoc-2-amino-4,6-dimethyl-3-pyridinecarboxylic acid, Fmoc-D,L-amino-2-thiophenacetic acid, Fmoc-4-(carboxymethyl)piperazine, Fmoc-4-carboxypiperazine, Fmoc-4-(carboxymethyl)homopiperazine, Fmoc-4-phenyl-4-piperidinecarboxylic acid, Fmoc-L-1,2,3,4-tetrahydronorharman-3-carboxylic acid, Fmoc-L-thiazolidine-4-carboxylic acid, all available from The Peptide Laboratory (Richmond, Calif., USA).

Non-natural residues can also be added biosynthetically by engineering a suppressor tRNA, typically one that recognizes the UAG stop codon, by chemical aminoacylation with the desired unnatural amino acid. Conventional site-directed mutagenesis is used to introduce the chosen stop codon UAG at the site of interest in the protein gene. When the acylated suppressor tRNA and the mutant gene are combined in an in vitro transcription/translation system, the unnatural amino acid is incorporated in response to the UAG codon to give a protein containing that amino acid at the specified position. Liu et al., Proc. Natl. Acad. Sci. USA 96(9): 4780-5 (1999); Wang et al., Science 292(5516): 498-500 (2001).

Fusion Proteins

The present invention further provides fusions of each of the polypeptides and fragments of the present invention to heterologous polypeptides. In a preferred embodiment, the polypeptide is a CSP. In a more preferred embodiment, the polypeptide that is fused to the heterologous polypeptide comprises part or all of the amino acid sequence of SEQ ID NO: 148 through 250, or is a mutein, homologous polypeptide, analog or derivative thereof. In an even more preferred embodiment, the nucleic acid molecule encoding the fusion protein comprises all or part of the nucleic acid sequence of SEQ ID NO: 1 through 147, or comprises all or part of a nucleic acid sequence that selectively hybridizes or is homologous to a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 1 through 147.

The fusion proteins of the present invention will include at least one fragment of the protein of the present invention, which fragment is at least 6, typically at least 8, often at least 15, and usefully at least 16, 17, 18, 19, or 20 amino acids long. The fragment of the protein of the present to be included in the fusion can usefully be at least 25 amino acids long, at least 50 amino acids long, and can be at least 75, 100, or even 150 amino acids long. Fusions that include the entirety of the proteins of the present invention have particular utility.

The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length, and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as the IgG Fc region, and even entire proteins (such as GFP chromophore-containing proteins) are particular useful.

As described above in the description of vectors and expression vectors of the present invention, which discussion is incorporated here by reference in its entirety, heterologous polypeptides to be included in the fusion proteins of the present invention can usefully include those designed to facilitate purification and/or visualization of recombinantly-expressed proteins. See, e.g., Ausubel, Chapter 16, (1992), supra. Although purification tags can also be incorporated into fusions that are chemically synthesized, chemical synthesis typically provides sufficient purity that further purification by HPLC suffices; however, visualization tags as above described retain their utility even when the protein is produced by chemical synthesis, and when so included render the fusion proteins of the present invention useful as directly detectable markers of the presence of a polypeptide of the invention.

As also discussed above, heterologous polypeptides to be included in the fusion proteins of the present invention can usefully include those that facilitate secretion of recombinantly expressed proteins—into the periplasmic space or extracellular milieu for prokaryotic hosts, into the culture medium for eukaryotic cells—through incorporation of secretion signals and/or leader sequences. For example, a His ⁶tagged protein can be purified on a Ni affinity column and a GST fusion protein can be purified on a glutathione affinity column. Similarly, a fusion protein comprising the Fc domain of IgG can be purified on a Protein A or Protein G column and a fusion protein comprising an epitope tag such as myc can be purified using an immunoaffinity column containing an anti-c-myc antibody. It is preferable that the epitope tag be separated from the protein encoded by the essential gene by an enzymatic cleavage site that can be cleaved after purification. See also the discussion of nucleic acid molecules encoding fusion proteins that may be expressed on the surface of a cell.

Other useful protein fusions of the present invention include those that permit use of the protein of the present invention as bait in a yeast two-hybrid system. See Bartel et al. (eds.), The Yeast Two-Hybrid System, Oxford University Press (1997); Zhu et al., Yeast Hybrid Technologies, Eaton Publishing (2000); Fields et al., Trends Genet. 10(8): 286-92 (1994); Mendelsohn et al., Curr. Opin. Biotechnol. 5(5): 482-6 (1994); Luban et al., Curr. Opin. Biotechnol. 6(1): 59-64 (1995); Allen et al., Trends Biochem. Sci. 20(12): 511-6 (1995); Drees, Curr. Opin. Chem. Biol. 3(1): 64-70 (1999); Topcu et al., Pharm. Res. 17(9): 1049-55 (2000); Fashena et al., Gene 250(1-2): 1-14 (2000);; Colas et al, (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature 380, 548-550; Norman, T. et al., (1999) Genetic selection of peptide inhibitors of biological pathways. Science 285, 591-595, Fabbrizio et al., (1999) Inhibition of mammalian cell proliferation by genetically selected peptide aptamers that functionally antagonize E2F activity. Oncogene 18, 4357-4363; Xu et al., (1997) Cells that register logical relationships among proteins. Proc Natl Acad Sci USA. 94, 12473-12478; Yang, et al., (1995) Protein-peptide interactions analyzed with the yeast two-hybrid system. Nuc. Acids Res. 23, 1152-1156; Kolonin et al., (1998) Targeting cyclin-dependent kinases in Drosophila with peptide aptamers. Proc Natl Acad Sci USA 95, 14266-14271; Cohen et al., (1998) An artificial cell-cycle inhibitor isolated from a combinatorial library. Proc Natl Acad Sci U SA 95, 14272-14277; Uetz, P.; Giot, L.; al, e.; Fields, S.; Rothberg, J. M. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623-627; Ito, et al., (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA 98, 4569-4574, the disclosures of which are incorporated herein by reference in their entireties. Typically, such fusion is to either E. coli LexA or yeast GAL4 DNA binding domains. Related bait plasmids are available that express the bait fused to a nuclear localization signal.

Other useful fusion proteins include those that permit display of the encoded protein on the surface of a phage or cell, fusions to intrinsically fluorescent proteins, such as green fluorescent protein (GFP), and fusions to the IgG Fc region, as described above, which discussion is incorporated here by reference in its entirety.

The polypeptides and fragments of the present invention can also usefully be fused to protein toxins, such as Pseudomonas exotoxin A, diphtheria toxin, shiga toxin A, anthrax toxin lethal factor, ricin, in order to effect ablation of cells that bind or take up the proteins of the present invention.

Fusion partners include, inter alia, myc, hemagglutinin (HA), GST, immunoglobulins, β-galactosidase, biotin trpE, protein A, β-lactamase, -amylase, maltose binding protein, alcohol dehydrogenase, polyhistidine (for example, six histidine at the amino and/or carboxyl terminus of the polypeptide), lacZ, green fluorescent protein (GFP), yeast_mating factor, GAL4 transcription activation or DNA binding domain, luciferase, and serum proteins such as ovalbumin, albumin and the constant domain of IgG. See, e.g., Ausubel (1992), supra and Ausubel (1999), supra. Fusion proteins may also contain sites for specific enzymatic cleavage, such as a site that is recognized by enzymes such as Factor XIII, trypsin, pepsin, or any other enzyme known in the art. Fusion proteins will typically be made by either recombinant nucleic acid methods, as described above, chemically synthesized using techniques well-known in the art (e.g., a Merrifield synthesis), or produced by chemical cross-linking.

Another advantage of fusion proteins is that the epitope tag can be used to bind the fusion protein to a plate or column through an affinity linkage for screening binding proteins or other molecules that bind to the CSP.

As further described below, the isolated polypeptides, muteins, fusion proteins, homologous proteins or allelic variants of the present invention can readily be used as specific immunogens to raise antibodies that specifically recognize CSPs, their allelic variants and homologues. The antibodies, in turn, can be used, inter alia, specifically to assay for the polypeptides of the present invention, particularly CSPs, e.g. by ELISA for detection of protein fluid samples, such as serum, by immunohistochemistry or laser scanning cytometry, for detection of protein in tissue samples, or by flow cytometry, for detection of intracellular protein in cell suspensions, for specific antibody-mediated isolation and/or purification of CSPs, as for example by immunoprecipitation, and for use as specific agonists or antagonists of CSPs.

One may determine whether polypeptides including muteins, fusion proteins, homologous proteins or allelic variants are functional by methods known in the art. For instance, residues that are tolerant of change while retaining function can be identified by altering the protein at known residues using methods known in the art, such as alanine scanning mutagenesis, Cunningham et al., Science 244(4908): 1081-5 (1989); transposon linker scanning mutagenesis, Chen et al., Gene 263(1-2): 39-48 (2001); combinations of homolog- and alanine-scanning mutagenesis, Jin et al., J. Mol. Biol. 226(3): 851-65 (1992); combinatorial alanine scanning, Weiss et al., Proc. Natl. Acad. Sci USA 97(16): 8950-4 (2000), followed by functional assay. Transposon linker scanning kits are available commercially (New England Biolabs, Beverly, Mass., USA, catalog. no. E7-102S; EZ::TN™ In-Frame Linker Insertion Kit, catalogue no. EZI04KN, Epicentre Technologies Corporation, Madison, Wis., USA).

Purification of the polypeptides including fragments, homologous polypeptides, muteins, analogs, derivatives and fusion proteins is well-known and within the skill of one having ordinary skill in the art. See, e.g., Scopes, Protein Purification, 2d ed. (1987). Purification of recombinantly expressed polypeptides is described above. Purification of chemically-synthesized peptides can readily be effected, e.g., by HPLC.

Accordingly, it is an aspect of the present invention to provide the isolated proteins of the present invention in pure or substantially pure form in the presence of absence of a stabilizing agent. Stabilizing agents include both proteinaceous or non-proteinaceous material and are well-known in the art. Stabilizing agents, such as albumin and polyethylene glycol (PEG) are known and are commercially available.

Although high levels of purity are preferred when the isolated proteins of the present invention are used as therapeutic agents, such as in vaccines and as replacement therapy, the isolated proteins of the present invention are also useful at lower purity. For example, partially purified proteins of the present invention can be used as immunogens to raise antibodies in laboratory animals.

In preferred embodiments, the purified and substantially purified proteins of the present invention are in compositions that lack detectable ampholytes, acrylamide monomers, bis-acrylamide monomers, and polyacrylamide.

The polypeptides, fragments, analogs, derivatives and fusions of the present invention can usefully be attached to a substrate. The substrate can be porous or solid, planar or non-planar; the bond can be covalent or noncovalent.

For example, the polypeptides, fragments, analogs, derivatives and fusions of the present invention can usefully be bound to a porous substrate, commonly a membrane, typically comprising nitrocellulose, polyvinylidene fluoride (PVDF), or cationically derivatized, hydrophilic PVDF; so bound, the proteins, fragments, and fusions of the present invention can be used to detect and quantify antibodies, e.g. in serum, that bind specifically to the immobilized protein of the present invention.

As another example, the polypeptides, fragments, analogs, derivatives and fusions of the present invention can usefully be bound to a substantially nonporous substrate, such as plastic, to detect and quantify antibodies, e.g. in serum, that bind specifically to the immobilized protein of the present invention. Such plastics include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, or mixtures thereof; when the assay is performed in a standard microtiter dish, the plastic is typically polystyrene.

The polypeptides, fragments, analogs, derivatives and fusions of the present invention can also be attached to a substrate suitable for use as a surface enhanced laser desorption ionization source; so attached, the protein, fragment, or fusion of the present invention is useful for binding and then detecting secondary proteins that bind with sufficient affinity or avidity to the surface-bound protein to indicate biologic interaction there between. The proteins, fragments, and fusions of the present invention can also be attached to a substrate suitable for use in surface plasmon resonance detection; so attached, the protein, fragment, or fusion of the present invention is useful for binding and then detecting secondary proteins that bind with sufficient affinity or avidity to the surface-bound protein to indicate biological interaction there between.

Antibodies

In another aspect, the invention provides antibodies, including fragments and derivatives thereof, that bind specifically to polypeptides encoded by the nucleic acid molecules of the invention, as well as antibodies that bind to fragments, muteins, derivatives and analogs of the polypeptides. In a preferred embodiment, the antibodies are specific for a polypeptide that is a CSP, or a fragment, mutein, derivative, analog or fusion protein thereof. In a more preferred embodiment, the antibodies are specific for a polypeptide that comprises SEQ ID NO: 148 through 250, or a fragment, mutein, derivative, analog or fusion protein thereof.

The antibodies of the present invention can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of such proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, as, e.g., by solubilization in SDS. New epitopes may be also due to a difference in post translational modifications (PTMs) in disease versus normal tissue. For example, a particular site on a CSP may be glycosylated in cancerous cells, but not glycosylated in normal cells or visa versa. In addition, alternative splice forms of a CSP may be indicative of cancer. Differential degradation of the C or N-terminus of a CSP may also be a marker or target for anticancer therapy. For example, a CSP may be N-terminal degraded in cancer cells exposing new epitopes to which antibodies may selectively bind for diagnostic or therapeutic uses.

As is well-known in the art, the degree to which an antibody can discriminate as among molecular species in a mixture will depend, in part, upon the conformational relatedness of the species in the mixture; typically, the antibodies of the present invention will discriminate over adventitious binding to non-CSP polypeptides by at least 2-fold, more typically by at least 5-fold, typically by more than 10-fold, 25-fold, 50-fold, 75-fold, and often by more than 100-fold, and on occasion by more than 500-fold or 1000-fold. When used to detect the proteins or protein fragments of the present invention, the antibody of the present invention is sufficiently specific when it can be used to determine the presence of the protein of the present invention in samples derived from human colon.

Typically, the affinity or avidity of an antibody (or antibody multimer, as in the case of an IgM pentamer) of the present invention for a protein or protein fragment of the present invention will be at least about 1×10 ⁻⁶molar (M), typically at least about 5×10⁻⁷M, 1×10⁻⁷M, with affinities and avidities of at least 1×10⁻⁸M, 5×10⁻⁹M, 1×10⁻¹⁰M and up to 1×10⁻¹³M proving especially useful.

The antibodies of the present invention can be naturally-occurring forms, such as IgG, IgM, IgD, IgE, IgY, and IgA, from any avian, reptilian, or mammalian species.

Human antibodies can, but will infrequently, be drawn directly from human donors or human cells. In this case, antibodies to the proteins of the present invention will typically have resulted from fortuitous immunization, such as autoimmune immunization, with the protein or protein fragments of the present invention. Such antibodies will typically, but will not invariably, be polyclonal. In addition, individual polyclonal antibodies may be isolated and cloned to generate monoclonals.

Human antibodies are more frequently obtained using transgenic animals that express human immunoglobulin genes, which transgenic animals can be affirmatively immunized with the protein immunogen of the present invention. Human Ig-transgenic mice capable of producing human antibodies and methods of producing human antibodies therefrom upon specific immunization are described, inter alia, in U.S. Pat. Nos. 6,162,963; 6,150,584; 6,114,598; 6,075,181; 5,939,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,770,429; 5,661,016; 5,633,425; 5,625,126; 5,569,825; 5,545,807; 5,545,806, and 5,591,669, the disclosures of which are incorporated herein by reference in their entireties. Such antibodies are typically monoclonal, and are typically produced using techniques developed for production of murine antibodies.

Human antibodies are particularly useful, and often preferred, when the antibodies of the present invention are to be administered to human beings as in vivo diagnostic or therapeutic agents, since recipient immune response to the administered antibody will often be substantially less than that occasioned by administration of an antibody derived from another species, such as mouse.

IgG, IgM, IgD, IgE, IgY, and IgA antibodies of the present invention can also be obtained from other species, including mammals such as rodents (typically mouse, but also rat, guinea pig, and hamster) lagomorphs, typically rabbits, and also larger mammals, such as sheep, goats, cows, and horses, and other egg laying birds or reptiles such as chickens or alligators. For example, avian antibodies may be generated using techniques described in WO 00/29444, published May 25, 2000, the contents of which are hereby incorporated in their entirety. In such cases, as with the transgenic human-antibody-producing non-human mammals, fortuitous immunization is not required, and the non-human mammal is typically affirmatively immunized, according to standard immunization protocols, with the protein or protein fragment of the present invention.

As discussed above, virtually all fragments of 8 or more contiguous amino acids of the proteins of the present invention can be used effectively as immunogens when conjugated to a carrier, typically a protein such as bovine thyroglobulin, keyhole limpet hemocyanin, or bovine serum albumin, conveniently using a bifunctional linker such as those described elsewhere above, which discussion is incorporated by reference here.

Immunogenicity can also be conferred by fusion of the polypeptide and fragments of the present invention to other moieties. For example, peptides of the present invention can be produced by solid phase synthesis on a branched polylysine core matrix; these multiple antigenic peptides (MAPs) provide high purity, increased avidity, accurate chemical definition and improved safety in vaccine development. Tam et al., Proc. Natl. Acad. Sci. USA 85: 5409-5413 (1988); Posnett et al., J. Biol. Chem. 263: 1719-1725 (1988).

Protocols for immunizing non-human mammals or avian species are well-established in the art. See Harlow et al. (eds.), Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1998); Coligan et al. (eds.), Current Protocols in Immunology, John Wiley & Sons, Inc. (2001); Zola, Monoclonal Antibodies: Preparation and Use of Monoclonal Antibodies and Engineered Antibody Derivatives (Basics: From Background to Bench), Springer Verlag (2000); Gross M, Speck J. Dtsch. Tierarztl. Wochenschr. 103: 417-422 (1996), the disclosures of which are incorporated herein by reference. Immunization protocols often include multiple immunizations, either with or without adjuvants such as Freund's complete adjuvant and Freund's incomplete adjuvant, and may include naked DNA immunization (Moss, Semin. Immunol. 2: 317-327 (1990).

Antibodies from non-human mammals and avian species can be polyclonal or monoclonal, with polyclonal antibodies having certain advantages in immunohistochemical detection of the proteins of the present invention and monoclonal antibodies having advantages in identifying and distinguishing particular epitopes of the proteins of the present invention. Antibodies from avian species may have particular advantage in detection of the proteins of the present invention, in human serum or tissues (Vikinge et al., Biosens. Bioelectron. 13: 1257-1262 (1998).

Following immunization, the antibodies of the present invention can be produced using any art-accepted technique. Such techniques are well-known in the art, Coligan, supra; Zola, supra; Howard et al. (eds.), Basic Methods in Antibody Production and Characterization, CRC Press (2000); Harlow, supra; Davis (ed.), Monoclonal Antibody Protocols, Vol. 45, Humana Press (1995); Delves (ed.), Antibody Production: Essential Techniques, John Wiley & Son Ltd (1997); Kenney, Antibody Solution: An Antibody Methods Manual, Chapman & Hall (1997), incorporated herein by reference in their entireties, and thus need not be detailed here.

Briefly, however, such techniques include, inter alia, production of monoclonal antibodies by hybridomas and expression of antibodies or fragments or derivatives thereof from host cells engineered to express immunoglobulin genes or fragments thereof. These two methods of production are not mutually exclusive: genes encoding antibodies specific for the proteins or protein fragments of the present invention can be cloned from hybridomas and thereafter expressed in other host cells. Nor need the two necessarily be performed together: e.g., genes encoding antibodies specific for the proteins and protein fragments of the present invention can be cloned directly from B cells known to be specific for the desired protein, as further described in U.S. Pat. No. 5,627,052, the disclosure of which is incorporated herein by reference in its entirety, or from antibody-displaying phage.

Recombinant expression in host cells is particularly useful when fragments or derivatives of the antibodies of the present invention are desired.

Host cells for recombinant production of either whole antibodies, antibody fragments, or antibody derivatives can be prokaryotic or eukaryotic.

Prokaryotic hosts are particularly useful for producing phage displayed antibodies of the present invention.

The technology of phage-displayed antibodies, in which antibody variable region fragments are fused, for example, to the gene III protein (pIII) or gene VIII protein (PVIII) for display on the surface of filamentous phage, such as M13, is by now well-established. See, e.g., Sidhu, Curr. Opin. Biotechnol. 11(6): 610-6 (2000); Griffiths et al., Curr. Opin. Biotechnol. 9(1): 102-8 (1998); Hoogenboom et al., Immunotechnology, 4(1): 1-20 (1998); Rader et al., Current Opinion in Biotechnology 8: 503-508 (1997); Aujame et al., Human Antibodies 8: 155-168 (1997); Hoogenboom, Trends in Biotechnol. 15: 62-70 (1997); de Kruif et al., 17: 453-455 (1996); Barbas et al., Trends in Biotechnol. 14: 230-234 (1996); Winter et al., Ann. Rev. Immunol. 433-455 (1994). Techniques and protocols required to generate, propagate, screen (pan), and use the antibody fragments from such libraries have recently been compiled. See, e.g., Barbas (2001), supra; Kay, supra; Abelson, supra, the disclosures of which are incorporated herein by reference in their entireties.

Typically, phage-displayed antibody fragments are scFv fragments or Fab fragments; when desired, full length antibodies can be produced by cloning the variable regions from the displaying phage into a complete antibody and expressing the full length antibody in a further prokaryotic or a eukaryotic host cell.

Eukaryotic cells are also useful for expression of the antibodies, antibody fragments, and antibody derivatives of the present invention.

For example, antibody fragments of the present invention can be produced in Pichia pastoris and in Saccharomyces cerevisiae. See, e.g., Takahashi et al., Biosci. Biotechnol. Biochem. 64(10): 2138-44 (2000); Freyre et al., J. Biotechnol. 76(2-3):1 57-63 (2000); Fischer et al., Biotechnol. Appl. Biochem. 30 (Pt 2): 117-20 (1999); Pennell et al., Res. Immunol. 149(6): 599-603 (1998); Eldin et al., J. Immunol. Methods. 201(1): 67-75 (1997);, Frenken et al., Res. Immunol. 149(6): 589-99 (1998); Shusta et al., Nature Biotechnol. 16(8): 773-7 (1998), the disclosures of which are incorporated herein by reference in their entireties.

Antibodies, including antibody fragments and derivatives, of the present invention can also be produced in insect cells. See, e.g., Li et al., Protein Expr. Purif 21(1): 121-8 (2001); Ailor et al., Biotechnol. Bioeng. 58(2-3): 196-203 (1998); Hsu et al., Biotechnol. Prog. 13(1): 96-104 (1997); Edelman et al., Immunology 91(1): 13-9 (1997); and Nesbit et al., J. Immunol. Methods 151(1-2): 201-8 (1992), the disclosures of which are incorporated herein by reference in their entireties.

Antibodies and fragments and derivatives thereof of the present invention can also be produced in plant cells, particularly maize or tobacco, Giddings et al., Nature Biotechnol. 18(11): 1151-5 (2000); Gavilondo et al., Biotechniques 29(1): 128-38 (2000); Fischer et al., J. Biol. Regul. Homeost. Agents 14(2): 83-92 (2000); Fischer et al., Biotechnol. Appl. Biochem. 30 (Pt 2): 113-6 (1999); Fischer et al., Biol. Chem. 380(7-8): 825-39 (1999); Russell, Curr. Top. Microbiol. Immunol. 240: 119-38 (1999); and Ma et al., Plant Physiol. 109(2): 341-6 (1995), the disclosures of which are incorporated herein by reference in their entireties.

Antibodies, including antibody fragments and derivatives, of the present invention can also be produced in transgenic, non-human, mammalian milk. See, e.g. Pollock et al., J. Immunol Methods. 231: 147-57 (1999); Young et al., Res. Immunol. 149: 609-10 (1998); Limonta et al., Immunotechnology 1: 107-13 (1995), the disclosures of which are incorporated herein by reference in their entireties.

Mammalian cells useful for recombinant expression of antibodies, antibody fragments, and antibody derivatives of the present invention include CHO cells, COS cells, 293 cells, and myeloma cells.

Verma et al., J. Immunol. Methods 216(1-2):165-81 (1998), herein incorporated by reference, review and compare bacterial, yeast, insect and mammalian expression systems for expression of antibodies.

Antibodies of the present invention can also be prepared by cell free translation, as further described in Merk et al., J. Biochem. (Tokyo) 125(2): 328-33 (1999) and Ryabova et al., Nature Biotechnol. 15(1): 79-84 (1997), and in the milk of transgenic animals, as further described in Pollock et al., J. Immunol. Methods 231(1-2): 147-57 (1999), the disclosures of which are incorporated herein by reference in their entireties.

The invention further provides antibody fragments that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention.

Among such useful fragments are Fab, Fab′, Fv, F(ab)′ ₂, and single chain Fv (scFv) fragments. Other useful fragments are described in Hudson, Curr. Opin. Biotechnol. 9(4): 395-402 (1998).

It is also an aspect of the present invention to provide antibody derivatives that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention.

Among such useful derivatives are chimeric, primatized, and humanized antibodies; such derivatives are less immunogenic in human beings, and thus more suitable for in vivo administration, than are unmodified antibodies from non-human mammalian species. Another useful derivative is PEGylation to increase the serum half life of the antibodies.

Chimeric antibodies typically include heavy and/or light chain variable regions (including both CDR and framework residues) of immunoglobulins of one species, typically mouse, fused to constant regions of another species, typically human. See, e.g., U.S. Pat. No. 5,807,715; Morrison et al., Proc. Natl. Acad. Sci USA. 81(21): 6851-5 (1984); Sharon et al., Nature 309(5966): 364-7 (1984); Takeda et al., Nature 314(6010): 452-4 (1985), the disclosures of which are incorporated herein by reference in their entireties. Primatized and humanized antibodies typically include heavy and/or light chain CDRs from a murine antibody grafted into a non-human primate or human antibody V region framework, usually further comprising a human constant region, Riechmann et al., Nature 332(6162): 323-7 (1988); Co et al., Nature 351(6326): 501-2 (1991); U.S. Pat. Nos. 6,054,297; 5,821,337; 5,770,196; 5,766,886; 5,821,123; 5,869,619; 6,180,377; 6,013,256; 5,693,761; and 6,180,370, the disclosures of which are incorporated herein by reference in their entireties.

Other useful antibody derivatives of the invention include heteromeric antibody complexes and antibody fusions, such as diabodies (bispecific antibodies), single-chain diabodies, and intrabodies.

It is contemplated that the nucleic acids encoding the antibodies of the present invention can be operably joined to other nucleic acids forming a recombinant vector for cloning or for expression of the antibodies of the invention. The present invention includes any recombinant vector containing the coding sequences, or part thereof, whether for eukaryotic transduction, transfection or gene therapy. Such vectors may be prepared using conventional molecular biology techniques, known to those with skill in the art, and would comprise DNA encoding sequences for the immunoglobulin V-regions including framework and CDRs or parts thereof, and a suitable promoter either with or without a signal sequence for intracellular transport. Such vectors may be transduced or transfected into eukaryotic cells or used for gene therapy (Marasco et al., Proc. Natl. Acad. Sci. (USA) 90: 7889-7893 (1993); Duan et al., Proc. Natl. Acad. Sci. (USA) 91: 5075-5079 (1994), by conventional techniques, known to those with skill in the art.

The antibodies of the present invention, including fragments and derivatives thereof, can usefully be labeled. It is, therefore, another aspect of the present invention to provide labeled antibodies that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention.

The choice of label depends, in part, upon the desired use.

For example, when the antibodies of the present invention are used for immunohistochemical staining of tissue samples, the label is preferably an enzyme that catalyzes production and local deposition of a detectable product.

Enzymes typically conjugated to antibodies to permit their immunohistochemical visualization are well-known, and include alkaline phosphatase, β-galactosidase, glucose oxidase, horseradish peroxidase (HRP), and urease. Typical substrates for production and deposition of visually detectable products include o-nitrophenyl-beta-D-galactopyranoside (ONPG); o-phenylenediamine dihydrochloride (OPD); p-nitrophenyl phosphate (PNPP); p-nitrophenyl-beta-D-galactopryanoside (PNPG); 3′,3′-diaminobenzidine (DAB); 3-amino-9-ethylcarbazole (AEC); 4-chloro-1-naphthol (CN); 5-bromo-4-chloro-3-indolyl-phosphate (BCIP); ABTS®; BluoGal; iodonitrotetrazolium (INT); nitroblue tetrazolium chloride (NBT); phenazine methosulfate (PMS); phenolphthalein monophosphate (PMP); tetramethyl benzidine (TMB); tetranitroblue tetrazolium (TNBT); X-Gal; X-Gluc; and X-Glucoside.

Other substrates can be used to produce products for local deposition that are luminescent. For example, in the presence of hydrogen peroxide (H ₂O₂), horseradish peroxidase (HRP) can catalyze the oxidation of cyclic diacylhydrazides, such as luminol. Immediately following the oxidation, the luminol is in an excited state (intermediate reaction product), which decays to the ground state by emitting light. Strong enhancement of the light emission is produced by enhancers, such as phenolic compounds. Advantages include high sensitivity, high resolution, and rapid detection without radioactivity and requiring only small amounts of antibody. See, e.g., Thorpe et al., Methods Enzymol. 133: 331-53 (1986); Kricka et al., J. Immunoassay 17(1): 67-83 (1996); and Lundqvist et al., J. Biolumin. Chemilumin. 10(6): 353-9 (1995), the disclosures of which are incorporated herein by reference in their entireties. Kits for such enhanced chemiluminescent detection (ECL) are available commercially.

The antibodies can also be labeled using colloidal gold.

As another example, when the antibodies of the present invention are used, e.g., for flow cytometric detection, for scanning laser cytometric detection, or for fluorescent immunoassay, they can usefully be labeled with fluorophores.

There are a wide variety of fluorophore labels that can usefully be attached to the antibodies of the present invention.

For flow cytometric applications, both for extracellular detection and for intracellular detection, common useful fluorophores can be fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Texas Red, Cy3, Cy5, fluorescence resonance energy tandem fluorophores such as PerCP-Cy5.5, PE-CyS, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7.

Other fluorophores include, inter alia, Alexa Fluor® 350, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, Oreg., USA), BODIPY dyes, such as BODIPY 493/503, BODIPY FL, BODIPY R6G, BODIPY 530/550, BODIPY TMR, BODIPY 558/568, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, Oreg., USA), and Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, all of which are also useful for fluorescently labeling the antibodies of the present invention.

For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the present invention can usefully be labeled with biotin.

When the antibodies of the present invention are used, e.g., for Western blotting applications, they can usefully be labeled with radioisotopes, such as ³³P, ³²P, ³⁵S, 3H, and ¹²⁵I.

As another example, when the antibodies of the present invention are used for radioimmunotherapy, the label can usefully be ²²⁸Th, ²²⁷Ac, ²²⁵Ac, ²²³Ra, ²¹³Bi, ²¹²Pb, ²¹²Bi, ²¹¹At, ²⁰³Pb, ¹⁹⁴Os, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁵³Sm, ¹⁴⁹Tb, ¹³¹I, ¹²⁵I, ¹¹¹In, ¹⁰⁵Tc, ^99mTc, ⁹⁷Ru, ⁹⁰Y, ⁹⁰Sr, ⁸⁸Y, ⁷²Se, ⁶⁷Cu, or ⁴⁷Sc.

As another example, when the antibodies of the present invention are to be used for in vivo diagnostic use, they can be rendered detectable by conjugation to MRI contrast agents, such as gadolinium diethylenetriaminepentaacetic acid (DTPA), Lauffer et al., Radiology 207(2): 529-38 (1998), or by radioisotopic labeling.

As would be understood, use of the labels described above is not restricted to the application for which they are mentioned.

The antibodies of the present invention, including fragments and derivatives thereof, can also be conjugated to toxins, in order to target the toxin's ablative action to cells that display and/or express the proteins of the present invention. Commonly, the antibody in such immunotoxins is conjugated to Pseudomonas exotoxin A, diphtheria toxin, shiga toxin A, anthrax toxin lethal factor, or ricin. See Hall (ed.), Immunotoxin Methods and Protocols (Methods in Molecular Biology, vol. 166), Humana Press (2000); and Frankel et al. (eds.), Clinical Applications of Immunotoxins, Springer-Verlag (1998), the disclosures of which are incorporated herein by reference in their entireties.

The antibodies of the present invention can usefully be attached to a substrate, and it is, therefore, another aspect of the invention to provide antibodies that bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, attached to a substrate.

Substrates can be porous or nonporous, planar or nonplanar.

For example, the antibodies of the present invention can usefully be conjugated to filtration media, such as NHS-activated Sepharose or CNBr-activated Sepharose for purposes of immunoaffinity chromatography.

For example, the antibodies of the present invention can usefully be attached to paramagnetic microspheres, typically by biotin-streptavidin interaction, which microspheres can then be used for isolation of cells that express or display the proteins of the present invention. As another example, the antibodies of the present invention can usefully be attached to the surface of a microtiter plate for ELISA.

As noted above, the antibodies of the present invention can be produced in prokaryotic and eukaryotic cells. It is, therefore, another aspect of the present invention to provide cells that express the antibodies of the present invention, including hybridoma cells, B cells, plasma cells, and host cells recombinantly modified to express the antibodies of the present invention.

In yet a further aspect, the present invention provides aptamers evolved to bind specifically to one or more of the proteins and protein fragments of the present invention, to one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention, or the binding of which can be competitively inhibited by one or more of the proteins and protein fragments of the present invention or one or more of the proteins and protein fragments encoded by the isolated nucleic acids of the present invention.

In sum, one of skill in the art, provided with the teachings of this invention, has available a variety of methods which may be used to alter the biological properties of the antibodies of this invention including methods which would increase or decrease the stability or half-life, immunogenicity, toxicity, affinity or yield of a given antibody molecule, or to alter it in any other way that may render it more suitable for a particular application.

Transgenic Animals and Cells

In another aspect, the invention provides transgenic cells and non-human organisms comprising nucleic acid molecules of the invention. In a preferred embodiment, the transgenic cells and non-human organisms comprise a nucleic acid molecule encoding a CSP. In a preferred embodiment, the CSP comprises an amino acid sequence selected from SEQ ID NO: 148 through 250, or a fragment, mutein, homologous protein or allelic variant thereof. In another preferred embodiment, the transgenic cells and non-human organism comprise a CSNA of the invention, preferably a CSNA comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 through 147, or a part, substantially similar nucleic acid molecule, allelic variant or hybridizing nucleic acid molecule thereof.

In another embodiment, the transgenic cells and non-human organisms have a targeted disruption or replacement of the endogenous orthologue of the human CSG. The transgenic cells can be embryonic stem cells or somatic cells. The transgenic non-human organisms can be chimeric, nonchimeric heterozygotes, and nonchimeric homozygotes. Methods of producing transgenic animals are well-known in the art. See, e.g., Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, 2d ed., Cold Spring Harbor Press (1999); Jackson et al., Mouse Genetics and Transgenics: A Practical Approach, Oxford University Press (2000); and Pinkert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press (1999).

Any technique known in the art may be used to introduce a nucleic acid molecule of the invention into an animal to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection. (see, e.g., Paterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology 11: 1263-1270 (1993); Wright et al., Biotechnology 9: 830-834 (1991); and U.S. Pat. No. 4,873,191 (1989 retrovirus-mediated gene transfer into germ lines, blastocysts or embryos (see, e.g., Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)); gene targeting in embryonic stem cells (see, e.g., Thompson et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (see, e.g., Lo, 1983, Mol. Cell. Biol. 3: 1803-1814 (1983)); introduction using a gene gun (see, e.g., Ulmer et al., Science 259: 1745-49 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (see, e.g., Lavitrano et al., Cell 57: 717-723 (1989)).

Other techniques include, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (see, e.g., Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997)). The present invention provides for transgenic animals that carry the transgene (i.e., a nucleic acid molecule of the invention) in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric animals.

The transgene may be integrated as a single transgene or as multiple copies, such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, e.g., the teaching of Lasko et al. et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (RT-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.

Methods for creating a transgenic animal with a disruption of a targeted gene are also well-known in the art. In general, a vector is designed to comprise some nucleotide sequences homologous to the endogenous targeted gene. The vector is introduced into a cell so that it may integrate, via homologous recombination with chromosomal sequences, into the endogenous gene, thereby disrupting the function of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type. See, e.g., Gu et al., Science 265: 103-106 (1994). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. See, e.g., Smithies et al., Nature 317: 230-234 (1985); Thomas et al., Cell 51: 503-512 (1987); Thompson et al., Cell 5: 313-321 (1989).

In one embodiment, a mutant, non-functional nucleic acid molecule of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous nucleic acid sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene. See, e.g., Thomas, supra and Thompson, supra. However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.

In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from an animal or patient or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.

The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. See, e.g., U.S. Pat. Nos. 5,399,349 and 5,460,959, each of which is incorporated by reference herein in its entirety.

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well-known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.

Computer Readable Means

A further aspect of the invention relates to a computer readable means for storing the nucleic acid and amino acid sequences of the instant invention. In a preferred embodiment, the invention provides a computer readable means for storing SEQ ID NO: 1 through 147 and SEQ ID NO: 148 through 250 as described herein, as the complete set of sequences or in any combination. The records of the computer readable means can be accessed for reading and display and for interface with a computer system for the application of programs allowing for the location of data upon a query for data meeting certain criteria, the comparison of sequences, the alignment or ordering of sequences meeting a set of criteria, and the like.

The nucleic acid and amino acid sequences of the invention are particularly useful as components in databases useful for search analyses as well as in sequence analysis algorithms. As used herein, the terms “nucleic acid sequences of the invention” and “amino acid sequences of the invention” mean any detectable chemical or physical characteristic of a polynucleotide or polypeptide of the invention that is or may be reduced to or stored in a computer readable form. These include, without limitation, chromatographic scan data or peak data, photographic data or scan data therefrom, and mass spectrographic data.

This invention provides computer readable media having stored thereon sequences of the invention. A computer readable medium may comprise one or more of the following: a nucleic acid sequence comprising a sequence of a nucleic acid sequence of the invention; an amino acid sequence comprising an amino acid sequence of the invention; a set of nucleic acid sequences wherein at least one of said sequences comprises the sequence of a nucleic acid sequence of the invention; a set of amino acid sequences wherein at least one of said sequences comprises the sequence of an amino acid sequence of the invention; a data set representing a nucleic acid sequence comprising the sequence of one or more nucleic acid sequences of the invention; a data set representing a nucleic acid sequence encoding an amino acid sequence comprising the sequence of an amino acid sequence of the invention; a set of nucleic acid sequences wherein at least one of said sequences comprises the sequence of a nucleic acid sequence of the invention; a set of amino acid sequences wherein at least one of said sequences comprises the sequence of an amino acid sequence of the invention; a data set representing a nucleic acid sequence comprising the sequence of a nucleic acid sequence of the invention; a data set representing a nucleic acid sequence encoding an amino acid sequence comprising the sequence of an amino acid sequence of the invention. The computer readable medium can be any composition of matter used to store information or data, including, for example, commercially available floppy disks, tapes, hard drives, compact disks, and video disks.

Also provided by the invention are methods for the analysis of character sequences, particularly genetic sequences. Preferred methods of sequence analysis include, for example, methods of sequence homology analysis, such as identity and similarity analysis, RNA structure analysis, sequence assembly, cladistic analysis, sequence motif analysis, open reading frame determination, nucleic acid base calling, and sequencing chromatogram peak analysis.

A computer-based method is provided for performing nucleic acid sequence identity or similarity identification. This method comprises the steps of providing a nucleic acid sequence comprising the sequence of a nucleic acid of the invention in a computer readable medium; and comparing said nucleic acid sequence to at least one nucleic acid or amino acid sequence to identify sequence identity or similarity.

A computer-based method is also provided for performing amino acid homology identification, said method comprising the steps of: providing an amino acid sequence comprising the sequence of an amino acid of the invention in a computer readable medium; and comparing said an amino acid sequence to at least one nucleic acid or an amino acid sequence to identify homology.

A computer-based method is still further provided for assembly of overlapping nucleic acid sequences into a single nucleic acid sequence, said method comprising the steps of: providing a first nucleic acid sequence comprising the sequence of a nucleic acid of the invention in a computer readable medium; and screening for at least one overlapping region between said first nucleic acid sequence and a second nucleic acid sequence.

Diagnostic Methods for Colon Cancer

The present invention also relates to quantitative and qualitative diagnostic assays and methods for detecting, diagnosing, monitoring, staging and predicting cancers by comparing expression of a CSNA or a CSP in a human patient that has or may have colon cancer, or who is at risk of developing colon cancer, with the expression of a CSNA or a CSP in a normal human control. For purposes of the present invention, “expression of a CSNA” or “CSNA expression” means the quantity of CSG mRNA that can be measured by any method known in the art or the level of transcription that can be measured by any method known in the art in a cell, tissue, organ or whole patient. Similarly, the term “expression of a CSP” or “CSP expression” means the amount of CSP that can be measured by any method known in the art or the level of translation of a CSG CSNA that can be measured by any method known in the art.

The present invention provides methods for diagnosing colon cancer in a patient, in particular squamous cell carcinoma, by analyzing for changes in levels of CSNA or CSP in cells, tissues, organs or bodily fluids compared with levels of CSNA or CSP in cells, tissues, organs or bodily fluids of preferably the same type from a normal human control, wherein an increase, or decrease in certain cases, in levels of a CSNA or CSP in the patient versus the normal human control is associated with the presence of colon cancer or with a predilection to the disease. In another preferred embodiment, the present invention provides methods for diagnosing colon cancer in a patient by analyzing changes in the structure of the mRNA of a CSG compared to the mRNA from a normal control. These changes include, without limitation, aberrant splicing, alterations in polyadenylation and/or alterations in 5′ nucleotide capping. In yet another preferred embodiment, the present invention provides methods for diagnosing colon cancer in a patient by analyzing changes in a CSP compared to a CSP from a normal control. These changes include, e.g., alterations in glycosylation and/or phosphorylation of the CSP or subcellular CSP localization.

In a preferred embodiment, the expression of a CSNA is measured by determining the amount of an mRNA that encodes an amino acid sequence selected from SEQ ID NO: 148 through 250, a homolog, an allelic variant, or a fragment thereof. In a more preferred embodiment, the CSNA expression that is measured is the level of expression of a CSNA mRNA selected from SEQ ID NO: 1 through 147, or a hybridizing nucleic acid, homologous nucleic acid or allelic variant thereof, or a part of any of these nucleic acids. CSNA expression may be measured by any method known in the art, such as those described supra, including measuring mRNA expression by Northern blot, quantitative or qualitative reverse transcriptase PCR (RT-PCR), microarray, dot or slot blots or in situ hybridization. See, e.g., Ausubel (1992), supra; Ausubel (1999), Supra; Sambrook (1989), supra; and Sambrook (2001), supra. CSNA transcription may be measured by any method known in the art including using a reporter gene hooked up to the promoter of a CSG of interest or doing nuclear run-off assays. Alterations in mRNA structure, e.g., aberrant splicing variants, may be determined by any method known in the art, including, RT-PCR followed by sequencing or restriction analysis. As necessary, CSNA expression may be compared to a known control, such as normal colon nucleic acid, to detect a change in expression.

In another preferred embodiment, the expression of a CSP is measured by determining the level of a CSP having an amino acid sequence selected from the group consisting of SEQ ID NO: 148 through 250, a homolog, an allelic variant, or a fragment thereof. Such levels are preferably determined in at least one of cells, tissues, organs and/or bodily fluids, including determination of normal and abnormal levels. Thus, for instance, a diagnostic assay in accordance with the invention for diagnosing over- or underexpression of CSNA or CSP compared to normal control bodily fluids, cells, or tissue samples may be used to diagnose the presence of colon cancer. The expression level of a CSP may be determined by any method known in the art, such as those described supra. In a preferred embodiment, the CSP expression level may be determined by radioimmunoassays, competitive-binding assays, ELISA, Western blot, FACS, immunohistochemistry, immunoprecipitation, proteomic approaches: two-dimensional gel electrophoresis (2D electrophoresis) and non-gel-based approaches such as mass spectrometry or protein interaction profiling. See, e.g, Harlow (1999), supra; Ausubel (1992), supra; and Ausubel (1999), supra. Alterations in the CSP structure may be determined by any method known in the art, including, e.g., using antibodies that specifically recognize phosphoserine, phosphothreonine or phosphotyrosine residues, two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and/or chemical analysis of amino acid residues of the protein. Id.

In a preferred embodiment, a radioimmunoassay (RIA) or an ELISA is used. An antibody specific to a CSP is prepared if one is not already available. In a preferred embodiment, the antibody is a monoclonal antibody. The anti-CSP antibody is bound to a solid support and any free protein binding sites on the solid support are blocked with a protein such as bovine serum albumin. A sample of interest is incubated with the antibody on the solid support under conditions in which the CSP will bind to the anti-CSP antibody. The sample is removed, the solid support is washed to remove unbound material, and an anti-CSP antibody that is linked to a detectable reagent (a radioactive substance for RIA and an enzyme for ELISA) is added to the solid support and incubated under conditions in which binding of the CSP to the labeled antibody will occur. After binding, the unbound labeled antibody is removed by washing. For an ELISA, one or more substrates are added to produce a colored reaction product that is based upon the amount of a CSP in the sample. For an RIA, the solid support is counted for radioactive decay signals by any method known in the art. Quantitative results for both RIA and ELISA typically are obtained by reference to a standard curve.

Other methods to measure CSP levels are known in the art. For instance, a competition assay may be employed wherein an anti-CSP antibody is attached to a solid support and an allocated amount of a labeled CSP and a sample of interest are incubated with the solid support. The amount of labeled CSP detected which is attached to the solid support can be correlated to the quantity of a CSP in the sample.

Of the proteomic approaches, 2D PAGE is a well-known technique. Isolation of individual proteins from a sample such as serum is accomplished using sequential separation of proteins by isoelectric point and molecular weight. Typically, polypeptides are first separated by isoelectric point (the first dimension) and then separated by size using an electric current (the second dimension). In general, the second dimension is perpendicular to the first dimension. Because no two proteins with different sequences are identical on the basis of both size and charge, the result of 2D PAGE is a roughly square gel in which each protein occupies a unique spot. Analysis of the spots with chemical or antibody probes, or subsequent protein microsequencing can reveal the relative abundance of a given protein and the identity of the proteins in the sample.

Expression levels of a CSNA can be determined by any method known in the art, including PCR and other nucleic acid methods, such as ligase chain reaction (LCR) and nucleic acid sequence based amplification (NASBA), can be used to detect malignant cells for diagnosis and monitoring of various malignancies. For example, reverse-transcriptase PCR (RT-PCR) is a powerful technique which can be used to detect the presence of a specific mRNA population in a complex mixture of thousands of other mRNA species. In RT-PCR, an mRNA species is first reverse transcribed to complementary DNA (cDNA) with use of the enzyme reverse transcriptase; the cDNA is then amplified as in a standard PCR reaction.

Hybridization to specific DNA molecules (e.g., oligonucleotides) arrayed on a solid support can be used to both detect the expression of and quantitate the level of expression of one or more CSNAs of interest. In this approach, all or a portion of one or more CSNAs is fixed to a substrate. A sample of interest, which may comprise RNA, e.g., total RNA or polyA-selected mRNA, or a complementary DNA (cDNA) copy of the RNA is incubated with the solid support under conditions in which hybridization will occur between the DNA on the solid support and the nucleic acid molecules in the sample of interest. Hybridization between the substrate-bound DNA and the nucleic acid molecules in the sample can be detected and quantitated by several means, including, without limitation, radioactive labeling or fluorescent labeling of the nucleic acid molecule or a secondary molecule designed to detect the hybrid.

The above tests can be carried out on samples derived from a variety of cells, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a patient. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, urine, saliva or any other bodily secretion or derivative thereof. By blood it is meant to include whole blood, plasma, serum or any derivative of blood. In a preferred embodiment, the specimen tested for expression of CSNA or CSP includes, without limitation, colon tissue, fluid obtained by bronchial alveolar lavage (BAL), sputum, colon cells grown in cell culture, blood, serum, lymph node tissue and lymphatic fluid. In another preferred embodiment, especially when metastasis of a primary colon cancer is known or suspected, specimens include, without limitation, tissues from brain, bone, bone marrow, liver, adrenal glands and colon. In general, the tissues may be sampled by biopsy, including, without limitation, needle biopsy, e.g., transthoracic needle aspiration, cervical mediatinoscopy, endoscopic lymph node biopsy, video-assisted thoracoscopy, exploratory thoracotomy, bone marrow biopsy and bone marrow aspiration. See Scott, supra and Franklin, pp. 529-570, in Kane, supra. For early and inexpensive detection, assaying for changes in CSNAs or CSPs in cells in sputum samples may be particularly useful. Methods of obtaining and analyzing sputum samples is disclosed in Franklin, supra.

All the methods of the present invention may optionally include determining the expression levels of one or more other cancer markers in addition to determining the expression level of a CSNA or CSP. In many cases, the use of another cancer marker will decrease the likelihood of false positives or false negatives. In one embodiment, the one or more other cancer markers include other CSNA or CSPs as disclosed herein. Other cancer markers useful in the present invention will depend on the cancer being tested and are known to those of skill in the art. In a preferred embodiment, at least one other cancer marker in addition to a particular CSNA or CSP is measured. In a more preferred embodiment, at least two other additional cancer markers are used. In an even more preferred embodiment, at least three, more preferably at least five, even more preferably at least ten additional cancer markers are used.

Diagnosing

In one aspect, the invention provides a method for determining the expression levels and/or structural alterations of one or more CSNAs and/or CSPs in a sample from a patient suspected of having colon cancer. In general, the method comprises the steps of obtaining the sample from the patient, determining the expression level or structural alterations of a CSNA and/or CSP and then ascertaining whether the patient has colon cancer from the expression level of the CSNA or CSP. In general, if high expression relative to a control of a CSNA or CSP is indicative of colon cancer, a diagnostic assay is considered positive if the level of expression of the CSNA or CSP is at least two times higher, and more preferably are at least five times higher, even more preferably at least ten times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control. In contrast, if low expression relative to a control of a CSNA or CSP is indicative of colon cancer, a diagnostic assay is considered positive if the level of expression of the CSNA or CSP is at least two times lower, more preferably are at least five times lower, even more preferably at least ten times lower than in preferably the same cells, tissues or bodily fluid of a normal human control. The normal human control may be from a different patient or from uninvolved tissue of the same patient.

The present invention also provides a method of determining whether colon cancer has metastasized in a patient. One may identify whether the colon cancer has metastasized by measuring the expression levels and/or structural alterations of one or more CSNAs and/or CSPs in a variety of tissues. The presence of a CSNA or CSP in a certain tissue at levels higher than that of corresponding noncancerous tissue (e.g., the same tissue from another individual) is indicative of metastasis if high level expression of a CSNA or CSP is associated with colon cancer. Similarly, the presence of a CSNA or CSP in a tissue at levels lower than that of corresponding noncancerous tissue is indicative of metastasis if low level expression of a CSNA or CSP is associated with colon cancer. Further, the presence of a structurally altered CSNA or CSP that is associated with colon cancer is also indicative of metastasis.

In general, if high expression relative to a control of a CSNA or CSP is indicative of metastasis, an assay for metastasis is considered positive if the level of expression of the CSNA or CSP is at least two times higher, and more preferably are at least five times higher, even more preferably at least ten times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control. In contrast, if low expression relative to a control of a CSNA or CSP is indicative of metastasis, an assay for metastasis is considered positive if the level of expression of the CSNA or CSP is at least two times lower, more preferably are at least five times lower, even more preferably at least ten times lower than in preferably the same cells, tissues or bodily fluid of a normal human control.

The CSNA or CSP of this invention maybe used as element in an array or a multi-analyte test to recognize expression patterns associated with colon cancers or other colon related disorders. In addition, the sequences of either the nucleic acids or proteins may be used as elements in a computer program for pattern recognition of colon disorders.

Staging

The invention also provides a method of staging colon cancer in a human patient. The method comprises identifying a human patient having colon cancer and analyzing cells, tissues or bodily fluids from such human patient for expression levels and/or structural alterations of one or more CSNAs or CSPs. First, one or more tumors from a variety of patients are staged according to procedures well-known in the art, and the expression level of one or more CSNAs or CSPs is determined for each stage to obtain a standard expression level for each CSNA and CSP. Then, the CSNA or CSP expression levels are determined in a biological sample from a patient whose stage of cancer is not known. The CSNA or CSP expression levels from the patient are then compared to the standard expression level. By comparing the expression level of the CSNAs and CSPs from the patient to the standard expression levels, one may determine the stage of the tumor. The same procedure may be followed using structural alterations of a CSNA or CSP to determine the stage of a colon cancer.

Monitoring

Further provided is a method of monitoring colon cancer in a human patient. One may monitor a human patient to determine whether there has been metastasis and, if there has been, when metastasis began to occur. One may also monitor a human patient to determine whether a preneoplastic lesion has become cancerous. One may also monitor a human patient to determine whether a therapy, e.g., chemotherapy, radiotherapy or surgery, has decreased or eliminated the colon cancer. The method comprises identifying a human patient that one wants to monitor for colon cancer, periodically analyzing cells, tissues or bodily fluids from such human patient for expression levels of one or more CSNAs or CSPs, and comparing the CSNA or CSP levels over time to those CSNA or CSP expression levels obtained previously. Patients may also be monitored by measuring one or more structural alterations in a CSNA or CSP that are associated with colon cancer.

If increased expression of a CSNA or CSP is associated with metastasis, treatment failure, or conversion of a preneoplastic lesion to a cancerous lesion, then detecting an increase in the expression level of a CSNA or CSP indicates that the tumor is metastasizing, that treatment has failed or that the lesion is cancerous, respectively. One having ordinary skill in the art would recognize that if this were the case, then a decreased expression level would be indicative of no metastasis, effective therapy or failure to progress to a neoplastic lesion. If decreased expression of a CSNA or CSP is associated with metastasis, treatment failure, or conversion of a preneoplastic lesion to a cancerous lesion, then detecting an decrease in the expression level of a CSNA or CSP indicates that the tumor is metastasizing, that treatment has failed or that the lesion is cancerous, respectively. In a preferred embodiment, the levels of CSNAs or CSPs are determined from the same cell type, tissue or bodily fluid as prior patient samples. Monitoring a patient for onset of colon cancer metastasis is periodic and preferably is done on a quarterly basis, but may be done more or less frequently.

The methods described herein can further be utilized as prognostic assays to identify subjects having or at risk of developing a disease or disorder associated with increased or decreased expression levels of a CSNA and/or CSP. The present invention provides a method in which a test sample is obtained from a human patient and one or more CSNAs and/or CSPs are detected. The presence of higher (or lower) CSNA or CSP levels as compared to normal human controls is diagnostic for the human patient being at risk for developing cancer, particularly colon cancer. The effectiveness of therapeutic agents to decrease (or increase) expression or activity of one or more CSNAs and/or CSPs of the invention can also be monitored by analyzing levels of expression of the CSNAs and/or CSPs in a human patient in clinical trials or in in vitro screening assays such as in human cells. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the human patient or cells, as the case may be, to the agent being tested.

Detection of Genetic Lesions or Mutations

The methods of the present invention can also be used to detect genetic lesions or mutations in a CSG, thereby determining if a human with the genetic lesion is susceptible to developing colon cancer or to determine what genetic lesions are responsible, or are partly responsible, for a person's existing colon cancer. Genetic lesions can be detected, for example, by ascertaining the existence of a deletion, insertion and/or substitution of one or more nucleotides from the CSGs of this invention, a chromosomal rearrangement of CSG, an aberrant modification of CSG (such as of the methylation pattern of the genomic DNA), or allelic loss of a CSG. Methods to detect such lesions in the CSG of this invention are known to those having ordinary skill in the art following the teachings of the specification.

Methods of Detecting Noncancerous Colon Diseases

The invention also provides a method for determining the expression levels and/or structural alterations of one or more CSNAs and/or CSPs in a sample from a patient suspected of having or known to have a noncancerous colon disease. In general, the method comprises the steps of obtaining a sample from the patient, determining the expression level or structural alterations of a CSNA and/or CSP, comparing the expression level or structural alteration of the CSNA or CSP to a normal colon control, and then ascertaining whether the patient has a noncancerous colon disease. In general, if high expression relative to a control of a CSNA or CSP is indicative of a particular noncancerous colon disease, a diagnostic assay is considered positive if the level of expression of the CSNA or CSP is at least two times higher, and more preferably are at least five times higher, even more preferably at least ten times higher, than in preferably the same cells, tissues or bodily fluid of a normal human control. In contrast, if low expression relative to a control of a CSNA or CSP is indicative of a noncancerous colon disease, a diagnostic assay is considered positive if the level of expression of the CSNA or CSP is at least two times lower, more preferably are at least five times lower, even more preferably at least ten times lower than in preferably the same cells, tissues or bodily fluid of a normal human control. The normal human control may be from a different patient or from uninvolved tissue of the same patient.

One having ordinary skill in the art may determine whether a CSNA and/or CSP is associated with a particular noncancerous colon disease by obtaining colon tissue from a patient having a noncancerous colon disease of interest and determining which CSNAs and/or CSPs are expressed in the tissue at either a higher or a lower level than in normal colon tissue. In another embodiment, one may determine whether a CSNA or CSP exhibits structural alterations in a particular noncancerous colon disease state by obtaining colon tissue from a patient having a noncancerous colon disease of interest and determining the structural alterations in one or more CSNAs and/or CSPs relative to normal colon tissue.

Methods for Identifying Colon Tissue

In another aspect, the invention provides methods for identifying colon tissue. These methods are particularly useful in, e.g., forensic science, colon cell differentiation and development, and in tissue engineering.

In one embodiment, the invention provides a method for determining whether a sample is colon tissue or has colon tissue-like characteristics. The method comprises the steps of providing a sample suspected of comprising colon tissue or having colon tissue-like characteristics, determining whether the sample expresses one or more CSNAs and/or CSPs, and, if the sample expresses one or more CSNAs and/or CSPs, concluding that the sample comprises colon tissue. In a preferred embodiment, the CSNA encodes a polypeptide having an amino acid sequence selected from SEQ ID NO: 148 through 250, or a homolog, allelic variant or fragment thereof. In a more preferred embodiment, the CSNA has a nucleotide sequence selected from SEQ ID NO: 1 through 147, or a hybridizing nucleic acid, an allelic variant or a part thereof. Determining whether a sample expresses a CSNA can be accomplished by any method known in the art. Preferred methods include hybridization to microarrays, Northern blot hybridization, and quantitative or qualitative RT-PCR. In another preferred embodiment, the method can be practiced by determining whether a CSP is expressed. Determining whether a sample expresses a CSP can be accomplished by any method known in the art. Preferred methods include Western blot, ELISA, RIA and 2D PAGE. In one embodiment, the CSP has an amino acid sequence selected from SEQ ID NO: 148 through 250, or a homolog, allelic variant or fragment thereof. In another preferred embodiment, the expression of at least two CSNAs and/or CSPs is determined. In a more preferred embodiment, the expression of at least three, more preferably four and even more preferably five CSNAs and/or CSPs are determined.

In one embodiment, the method can be used to determine whether an unknown tissue is colon tissue. This is particularly useful in forensic science, in which small, damaged pieces of tissues that are not identifiable by microscopic or other means are recovered from a crime or accident scene. In another embodiment, the method can be used to determine whether a tissue is differentiating or developing into colon tissue. This is important in monitoring the effects of the addition of various agents to cell or tissue culture, e.g., in producing new colon tissue by tissue engineering. These agents include, e.g., growth and differentiation factors, extracellular matrix proteins and culture medium. Other factors that may be measured for effects on tissue development and differentiation include gene transfer into the cells or tissues, alterations in pH, aqueous:air interface and various other culture conditions.

Methods for Producing and Modifying Colon Tissue

In another aspect, the invention provides methods for producing engineered colon tissue or cells. In one embodiment, the method comprises the steps of providing cells, introducing a CSNA or a CSG into the cells, and growing the cells under conditions in which they exhibit one or more properties of colon tissue cells. In a preferred embodiment, the cells are pluripotent. As is well-known in the art, normal colon tissue comprises a large number of different cell types. Thus, in one embodiment, the engineered colon tissue or cells comprises one of these cell types. In another embodiment, the engineered colon tissue or cells comprises more than one colon cell type. Further, the culture conditions of the cells or tissue may require manipulation in order to achieve full differentiation and development of the colon cell tissue. Methods for manipulating culture conditions are well-known in the art.

Nucleic acid molecules encoding one or more CSPs are introduced into cells, preferably pluripotent cells. In a preferred embodiment, the nucleic acid molecules encode CSPs having amino acid sequences selected from SEQ ID NO: 148 through 250, or homologous proteins, analogs, allelic variants or fragments thereof. In a more preferred embodiment, the nucleic acid molecules have a nucleotide sequence selected from SEQ ID NO: 1 through 147, or hybridizing nucleic acids, allelic variants or parts thereof. In another highly preferred embodiment, a CSG is introduced into the cells. Expression vectors and methods of introducing nucleic acid molecules into cells are well-known in the art and are described in detail, supra.

Artificial colon tissue may be used to treat patients who have lost some or all of their colon function.

Pharmaceutical Compositions

In another aspect, the invention provides pharmaceutical compositions comprising the nucleic acid molecules, polypeptides, antibodies, antibody derivatives, antibody fragments, agonists, antagonists, and inhibitors of the present invention. In a preferred embodiment, the pharmaceutical composition comprises a CSNA or part thereof. In a more preferred embodiment, the CSNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 through 147, a nucleic acid that hybridizes thereto, an allelic variant thereof, or a nucleic acid that has substantial sequence identity thereto. In another preferred embodiment, the pharmaceutical composition comprises a CSP or fragment thereof. In a more preferred embodiment, the CSP having an amino acid sequence that is selected from the group consisting of SEQ ID NO: 148 through 250, a polypeptide that is homologous thereto, a fusion protein comprising all or a portion of the polypeptide, or an analog or derivative thereof. In another preferred embodiment, the pharmaceutical composition comprises an anti-CSP antibody, preferably an antibody that specifically binds to a CSP having an amino acid that is selected from the group consisting of SEQ ID NO: 148 through 250, or an antibody that binds to a polypeptide that is homologous thereto, a fusion protein comprising all or a portion of the polypeptide, or an analog or derivative thereof.

Such a composition typically contains from about 0.1 to 90% by weight of a therapeutic agent of the invention formulated in and/or with a pharmaceutically acceptable carrier or excipient.

Pharmaceutical formulation is a well-established art, and is further described in Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20^thed., Lippincott, Williams & Wilkins (2000); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7^thed., Lippincott Williams & Wilkins (1999); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3^rded. (2000), the disclosures of which are incorporated herein by reference in their entireties, and thus need not be described in detail herein.

Briefly, formulation of the pharmaceutical compositions of the present invention will depend upon the route chosen for administration. The pharmaceutical compositions utilized in this invention can be administered by various routes including both enteral and parenteral routes, including oral, intravenous, intramuscular, subcutaneous, inhalation, topical, sublingual, rectal, intra-arterial, intramedullary, intrathecal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal, intrapulmonary, and intrauterine.

Oral dosage forms can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Solid formulations of the compositions for oral administration can contain suitable carriers or excipients, such as carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or microcrystalline cellulose; gums including arabic and tragacanth; proteins such as gelatin and collagen; inorganics, such as kaolin, calcium carbonate, dicalcium phosphate, sodium chloride; and other agents such as acacia and alginic acid.

Agents that facilitate disintegration and/or solubilization can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate, microcrystalline cellulose, corn starch, sodium starch glycolate, and alginic acid.

Tablet binders that can be used include acacia, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone (Povidone™), hydroxypropyl methylcellulose, sucrose, starch and ethylcellulose.

Lubricants that can be used include magnesium stearates, stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica.

Fillers, agents that facilitate disintegration and/or solubilization, tablet binders and lubricants, including the aforementioned, can be used singly or in combination.

Solid oral dosage forms need not be uniform throughout. For example, dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which can also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.

Oral dosage forms of the present invention include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Additionally, dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Liquid formulations of the pharmaceutical compositions for oral (enteral) administration are prepared in water or other aqueous vehicles and can contain various suspending agents such as methylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquid formulations can also include solutions, emulsions, syrups and elixirs containing, together with the active compound(s), wetting agents, sweeteners, and coloring and flavoring agents.

The pharmaceutical compositions of the present invention can also be formulated for parenteral administration. Formulations for parenteral administration can be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions.

For intravenous injection, water soluble versions of the compounds of the present invention are formulated in, or if provided as a lyophilate, mixed with, a physiologically acceptable fluid vehicle, such as 5% dextrose (“D5”), physiologically buffered saline, 0.9% saline, Hanks' solution, or Ringer's solution. Intravenous formulations may include carriers, excipients or stabilizers including, without limitation, calcium, human serum albumin, citrate, acetate, calcium chloride, carbonate, and other salts.

Intramuscular preparations, e.g. a sterile formulation of a suitable soluble salt form of the compounds of the present invention, can be dissolved and administered in a pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or 5% glucose solution. Alternatively, a suitable insoluble form of the compound can be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, such as an ester of a long chain fatty acid (e.g., ethyl oleate), fatty oils such as sesame oil, triglycerides, or liposomes.

Parenteral formulations of the compositions can contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, liquid polyethylene glycol, and the like).

Aqueous injection suspensions can also contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Non-lipid polycationic amino polymers can also be used for delivery. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical compositions of the present invention can also be formulated to permit injectable, long-term, deposition. Injectable depot forms may be made by forming microencapsulated matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in microemulsions that are compatible with body tissues.

The pharmaceutical compositions of the present invention can be administered topically.

For topical use the compounds of the present invention can also be prepared in suitable forms to be applied to the skin, or mucus membranes of the nose and throat, and can take the form of lotions, creams, ointments, liquid sprays or inhalants, drops, tinctures, lozenges, or throat paints. Such topical formulations further can include chemical compounds such as dimethylsulfoxide (DMSO) to facilitate surface penetration of the active ingredient. In other transdermal formulations, typically in patch-delivered formulations, the pharmaceutically active compound is formulated with one or more skin penetrants, such as 2-N-methyl-pyrrolidone (NMP) or Azone. A topical semi-solid ointment formulation typically contains a concentration of the active ingredient from about 1 to 20%, e.g., 5 to 10%, in a carrier such as a pharmaceutical cream base.

For application to the eyes or ears, the compounds of the present invention can be presented in liquid or semi-liquid form formulated in hydrophobic or hydrophilic bases as ointments, creams, lotions, paints or powders.

For rectal administration the compounds of the present invention can be administered in the form of suppositories admixed with conventional carriers such as cocoa butter, wax or other glyceride.

Inhalation formulations can also readily be formulated. For inhalation, various powder and liquid formulations can be prepared. For aerosol preparations, a sterile formulation of the compound or salt form of the compound may be used in inhalers, such as metered dose inhalers, and nebulizers. Aerosolized forms may be especially useful for treating respiratory disorders.

Alternatively, the compounds of the present invention can be in powder form for reconstitution in the appropriate pharmaceutically acceptable carrier at the time of delivery.

The pharmaceutically active compound in the pharmaceutical compositions of the present invention can be provided as the salt of a variety of acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.

After pharmaceutical compositions have been prepared, they are packaged in an appropriate container and labeled for treatment of an indicated condition.

The active compound will be present in an amount effective to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.

A “therapeutically effective dose” refers to that amount of active ingredient, for example CSP polypeptide, fusion protein, or fragments thereof, antibodies specific for CSP, agonists, antagonists or inhibitors of CSP, which ameliorates the signs or symptoms of the disease or prevents progression thereof; as would be understood in the medical arts, cure, although desired, is not required.

The therapeutically effective dose of the pharmaceutical agents of the present invention can be estimated initially by in vitro tests, such as cell culture assays, followed by assay in model animals, usually mice, rats, rabbits, dogs, or pigs. The animal model can also be used to determine an initial preferred concentration range and route of administration.

For example, the ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population) can be determined in one or more cell culture of animal model systems. The dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies are used in formulating an initial dosage range for human use, and preferably provide a range of circulating concentrations that includes the ED50 with little or no toxicity. After administration, or between successive administrations, the circulating concentration of active agent varies within this range depending upon pharmacokinetic factors well-known in the art, such as the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors specific to the subject requiring treatment. Factors that can be taken into account by the practitioner include the severity of the disease state, general health of the subject, age, weight, gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Where the therapeutic agent is a protein or antibody of the present invention, the therapeutic protein or antibody agent typically is administered at a daily dosage of 0.01 mg to 30 mg/kg of body weight of the patient (e.g., 1 mg/kg to 5 mg/kg). The pharmaceutical formulation can be administered in multiple doses per day, if desired, to achieve the total desired daily dose.

Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Conventional methods, known to those of ordinary skill in the art of medicine, can be used to administer the pharmaceutical formulation(s) of the present invention to the patient. The pharmaceutical compositions of the present invention can be administered alone, or in combination with other therapeutic agents or interventions.

Therapeutic Methods

The present invention further provides methods of treating subjects having defects in a gene of the invention, e.g., in expression, activity, distribution, localization, and/or solubility, which can manifest as a disorder of colon function. As used herein, “treating” includes all medically-acceptable types of therapeutic intervention, including palliation and prophylaxis (prevention) of disease. The term “treating” encompasses any improvement of a disease, including minor improvements. These methods are discussed below.

Gene Therapy and Vaccines

The isolated nucleic acids of the present invention can also be used to drive in vivo expression of the polypeptides of the present invention. In vivo expression can be driven from a vector, typically a viral vector, often a vector based upon a replication incompetent retrovirus, an adenovirus, or an adeno-associated virus (AAV), for purpose of gene therapy. In vivo expression can also be driven from signals endogenous to the nucleic acid or from a vector, often a plasmid vector, such as pVAX1 (Invitrogen, Carlsbad, Calif., USA), for purpose of “naked” nucleic acid vaccination, as further described in U.S. Pat. Nos. 5,589,466; 5,679,647; 5,804,566; 5,830,877; 5,843,913; 5,880,104; 5,958,891; 5,985,847; 6,017,897; 6,110,898; and 6,204,250, the disclosures of which are incorporated herein by reference in their entireties. For cancer therapy, it is preferred that the vector also be tumor-selective. See, e.g., Doronin et al., J. Virol 75: 3314-24 (2001).

In another embodiment of the therapeutic methods of the present invention, a therapeutically effective amount of a pharmaceutical composition comprising a nucleic acid of the present invention is administered. The nucleic acid can be delivered in a vector that drives expression of a CSP, fusion protein, or fragment thereof, or without such vector. Nucleic acid compositions that can drive expression of a CSP are administered, for example, to complement a deficiency in the native CSP, or as DNA vaccines. Expression vectors derived from virus, replication deficient retroviruses, adenovirus, adeno-associated (AAV) virus, herpes virus, or vaccinia virus can be used as can plasmids. See, e.g., Cid-Arregui, supra. In a preferred embodiment, the nucleic acid molecule encodes a CSP having the amino acid sequence of SEQ ID NO: 148 through 250, or a fragment, fusion protein, allelic variant or homolog thereof.

In still other therapeutic methods of the present invention, pharmaceutical compositions comprising host cells that express a CSP, fusions, or fragments thereof can be administered. In such cases, the cells are typically autologous, so as to circumvent xenogeneic or allotypic rejection, and are administered to complement defects in CSP production or activity. In a preferred embodiment, the nucleic acid molecules in the cells encode a CSP having the amino acid sequence of SEQ ID NO: 148 through 250, or a fragment, fusion protein, allelic variant or homolog thereof.

Antisense Administration

Antisense nucleic acid compositions, or vectors that drive expression of a CSG antisense nucleic acid, are administered to downregulate transcription and/or translation of a CSG in circumstances in which excessive production, or production of aberrant protein, is the pathophysiologic basis of disease.

Antisense compositions useful in therapy can have a sequence that is complementary to coding or to noncoding regions of a CSG. For example, oligonucleotides derived from the transcription initiation site, e.g., between positions −10 and +10 from the start site, are preferred.

Catalytic antisense compositions, such as ribozymes, that are capable of sequence-specific hybridization to CSG transcripts, are also useful in therapy. See, e.g., Phylactou, Adv. Drug Deliv. Rev. 44(2-3): 97-108 (2000); Phylactou et al., Hum. Mol. Genet. 7(10): 1649-53 (1998); Rossi, Ciba Found. Symp. 209: 195-204 (1997); and Sigurdsson et al., Trends Biotechnol. 13(8): 286-9 (1995), the disclosures of which are incorporated herein by reference in their entireties.

Other nucleic acids useful in the therapeutic methods of the present invention are those that are capable of triplex helix formation in or near the CSG genomic locus. Such triplexing oligonucleotides are able to inhibit transcription. See, e.g., Intody et al., Nucleic Acids Res. 28(21): 4283-90 (2000); McGuffie et al., Cancer Res. 60(14): 3790-9 (2000), the disclosures of which are incorporated herein by reference. Pharmaceutical compositions comprising such triplex forming oligos (TFOs) are administered in circumstances in which excessive production, or production of aberrant protein, is a pathophysiologic basis of disease.

In a preferred embodiment, the antisense molecule is derived from a nucleic acid molecule encoding a CSP, preferably a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250, or a fragment, allelic variant or homolog thereof. In a more preferred embodiment, the antisense molecule is derived from a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 through 147, or a part, allelic variant, substantially similar or hybridizing nucleic acid thereof.

Polypeptide Administration

In one embodiment of the therapeutic methods of the present invention, a therapeutically effective amount of a pharmaceutical composition comprising a CSP, a fusion protein, fragment, analog or derivative thereof is administered to a subject with a clinically-significant CSP defect.

Protein compositions are administered, for example, to complement a deficiency in native CSP. In other embodiments, protein compositions are administered as a vaccine to elicit a humoral and/or cellular immune response to CSP. The immune response can be used to modulate activity of CSP or, depending on the immunogen, to immunize against aberrant or aberrantly expressed forms, such as mutant or inappropriately expressed isoforms. In yet other embodiments, protein fusions having a toxic moiety are administered to ablate cells that aberrantly accumulate CSP.

In a preferred embodiment, the polypeptide is a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250, or a fusion protein, allelic variant, homolog, analog or derivative thereof. In a more preferred embodiment, the polypeptide is encoded by a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 through 147, or a part, allelic variant, substantially similar or hybridizing nucleic acid thereof.

Antibody, Agonist and Antagonist Administration

In another embodiment of the therapeutic methods of the present invention, a therapeutically effective amount of a pharmaceutical composition comprising an antibody (including fragment or derivative thereof) of the present invention is administered. As is well-known, antibody compositions are administered, for example, to antagonize activity of CSP, or to target therapeutic agents to sites of CSP presence and/or accumulation. In a preferred embodiment, the antibody specifically binds to a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250, or a fusion protein, allelic variant, homolog, analog or derivative thereof. In a more preferred embodiment, the antibody specifically binds to a CSP encoded by a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 through 147, or a part, allelic variant, substantially similar or hybridizing nucleic acid thereof.

The present invention also provides methods for identifying modulators which bind to a CSP or have a modulatory effect on the expression or activity of a CSP. Modulators which decrease the expression or activity of CSP (antagonists) are believed to be useful in treating colon cancer. Such screening assays are known to those of skill in the art and include, without limitation, cell-based assays and cell-free assays. Small molecules predicted via computer imaging to specifically bind to regions of a CSP can also be designed, synthesized and tested for use in the imaging and treatment of colon cancer. Further, libraries of molecules can be screened for potential anticancer agents by assessing the ability of the molecule to bind to the CSPs identified herein. Molecules identified in the library as being capable of binding to a CSP are key candidates for further evaluation for use in the treatment of colon cancer. In a preferred embodiment, these molecules will downregulate expression and/or activity of a CSP in cells.

In another embodiment of the therapeutic methods of the present invention, a pharmaceutical composition comprising a non-antibody antagonist of CSP is administered. Antagonists of CSP can be produced using methods generally known in the art. In particular, purified CSP can be used to screen libraries of pharmaceutical agents, often combinatorial libraries of small molecules, to identify those that specifically bind and antagonize at least one activity of a CSP.

In other embodiments a pharmaceutical composition comprising an agonist of a CSP is administered. Agonists can be identified using methods analogous to those used to identify antagonists.

In a preferred embodiment, the antagonist or agonist specifically binds to and antagonizes or agonizes, respectively, a CSP comprising an amino acid sequence of SEQ ID NO: 148 through 250, or a fusion protein, allelic variant, homolog, analog or derivative thereof. In a more preferred embodiment, the antagonist or agonist specifically binds to and antagonizes or agonizes, respectively, a CSP encoded by a nucleic acid molecule having a nucleotide sequence of SEQ ID NO: 1 through 147, or a part, allelic variant, substantially similar or hybridizing nucleic acid thereof.

Targeting Colon Tissue

The invention also provides a method in which a polypeptide of the invention, or an antibody thereto, is linked to a therapeutic agent such that it can be delivered to the colon or to specific cells in the colon. In a preferred embodiment, an anti-CSP antibody is linked to a therapeutic agent and is administered to a patient in need of such therapeutic agent. The therapeutic agent may be a toxin, if colon tissue needs to be selectively destroyed. This would be useful for targeting and killing colon cancer cells. In another embodiment, the therapeutic agent may be a growth or differentiation factor, which would be useful for promoting colon cell function.

In another embodiment, an anti-CSP antibody may be linked to an imaging agent that can be detected using, e.g., magnetic resonance imaging, CT or PET. This would be useful for determining and monitoring colon function, identifying colon cancer tumors, and identifying noncancerous colon diseases.

EXAMPLES

Example 1

Gene Expression Analysis

CSGs were identified by a systematic analysis of gene expression data in the LIFESEQ® Gold database available from Incyte Genomics Inc (Palo Alto, Calif.) using the data mining software package CLASP™ (Candidate Lead Automatic Search Program). CLASP™ is a set of algorithms that interrogate Incyte's database to identify genes that are both specific to particular tissue types as well as differentially expressed in tissues from patients with cancer. LifeSeq® Gold contains information about which genes are expressed in various tissues in the body and about the dynamics of expression in both normal and diseased states. CLASP™ first sorts the LifeSeq® Gold database into defined tissue types, such as breast, ovary and prostate. CLASP™ categorizes each tissue sample by disease state. Disease states include “healthy,” “cancer,” “associated with cancer,” “other disease” and “other.” Categorizing the disease states improves our ability to identify tissue and cancer-specific molecular targets. CLASP™ then performs a simultaneous parallel search for genes that are expressed both (1) selectively in the defined tissue type compared to other tissue types and (2) differentially in the “cancer” disease state compared to the other disease states affecting the same, or different, tissues. This sorting is accomplished by using mathematical and statistical filters that specify the minimum change in expression levels and the minimum frequency that the differential expression pattern must be observed across the tissue samples for the gene to be considered statistically significant. The CLASPTM algorithm quantifies the relative abundance of a particular gene in each tissue type and in each disease state. [0445]
To find the CSGs of this invention, the following specific CLASPTM profiles were utilized: tissue-specific expression (CLASP 1), detectable expression only in cancer tissue (CLASP 2), highest differential expression for a given cancer (CLASP 4); differential expression in cancer tissue (CLASP 5), and. cDNA libraries were divided into 60 unique tissue types (early versions of LifeSeq® had 48 tissue types). Genes or ESTs were grouped into “gene bins,” where each bin is a cluster of sequences grouped together where they share a common contig. The expression level for each gene bin was calculated for each tissue type. Differential expression significance was calculated with rigorous statistical significant testing taking into account variations in sample size and relative gene abundance in different libraries and within each library (for the equations used to determine statistically significant expression see Audic and Clayerie “The significance of digital gene expression profiles,” Genome Res 7(10): 986-995 (1997), including Equation 1 on page 987 and Equation 2 on page 988, the contents of which are incorporated by reference). Differentially expressed tissue-specific genes were selected based on the percentage abundance level in the targeted tissue versus all the other tissues (tissue-specificity). The expression levels for each gene in libraries of normal tissues or non-tumor tissues from cancer patients were compared with the expression levels in tissue libraries associated with tumor or disease (cancer-specificity). The results were analyzed for statistical significance. [0446]
The selection of the target genes meeting the rigorous CLASP™ profile criteria were as follows: [0447]
(a) CLASP 1: tissue-specific expression: To qualify as a CLASP 1 candidate, a gene must exhibit statistically significant expression in the tissue of interest compared to all other tissues. Only if the gene exhibits such differential expression with a 90% of confidence level is it selected as a CLASP 1 candidate. [0448]
(b) CLASP 2: detectable expression only in cancer tissue: To qualify as a CLASP 2 candidate, a gene must exhibit detectable expression in tumor tissues and undetectable expression in libraries from normal individuals and libraries from normal tissue obtained from diseased patients. In addition, such a gene must also exhibit further specificity for the tumor tissues of interest. [0449]
(c) CLASP 5: differential expression in cancer tissue: To qualify as a CLASP 5 candidate, a gene must be differentially expressed in tumor libraries in the tissue of interest compared to normal libraries for all tissues. Only if the gene exhibits such differential expression with a 90% of confidence level is it selected as a CLASP 5 candidate. [0450]

The CLASP™ scores for SEQ ID NO: 1-147 are listed below:



The CLASP ™ scores for SEQ ID NO: 1-147 are listed below:

SEQ ID NO: 1	DEX0253_1 CLASP2
SEQ ID NO: 2	DEX0253_2 CLASP2
SEQ ID NO: 3	DEX0253_3 CLASP2
SEQ ID NO: 4	DEX0253_4 CLASP2 CLASP1
SEQ ID NO: 5	DEX0253_5 CLASP2 CLASP1
SEQ ID NO: 6	DEX0253_6 CLASP5 CLASP1
SEQ ID NO: 7	DEX0253_7 CLASP5 CLASP1
SEQ ID NO: 8	DEX0253_8 CLASP2
SEQ ID NO: 9	DEX0253_9 CLASP2
SEQ ID NO: 10	DEX0253_10 CLASP2
SEQ ID NO: 11	DEX0253_11 CLASP2 CLASP1
SEQ ID NO: 12	DEX0253_12 CLASP2
SEQ ID NO: 13	DEX0253_13 CLASP2
SEQ ID NO: 14	DEX0253_14 CLASP5 CLASP1
SEQ ID NO: 15	DEX0253_15 CLASP2
SEQ ID NO: 16	DEX0253_16 CLASP2
SEQ ID NO: 17	DEX0253_17 CLASP2
SEQ ID NO: 18	DEX0253_18 CLASP2
SEQ ID NO: 19	DEX0253_19 CLASP2
SEQ ID NO: 20	DEX0253_20 CLASP2
SEQ ID NO: 21	DEX0253_21 CLASP2
SEQ ID NO: 22	DEX0253_22 CLASP2
SEQ ID NO: 23	DEX0253_23 CLASP2
SEQ ID NO: 24	DEX0253_24 CLASP2 CLASP1
SEQ ID NO: 25	DEX0253_25 CLASP2 CLASP1
SEQ ID NO: 26	DEX0253_26 CLASP2 CLASP1
SEQ ID NO: 27	DEX0253_27 CLASP2 CLASP1
SEQ ID NO: 28	DEX0253_28 CLASP2
SEQ ID NO: 29	DEX0253_29 CLASP2
SEQ ID NO: 30	DEX0253_30 CLASP2
SEQ ID NO: 31	DEX0253_31 CLASP2
SEQ ID NO: 32	DEX0253_32 CLASP2
SEQ ID NO: 33	DEX0253_33 CLASP2
SEQ ID NO: 34	DEX0253_34 CLASP2
SEQ ID NO: 35	DEX0253_35 CLASP2
SEQ ID NO: 36	DEX0253_36 CLASP2
SEQ ID NO: 37	DEX0253_37 CLASP2
SEQ ID NO: 38	DEX0253_38 CLASP2
SEQ ID NO: 39	DEX0253_39 CLASP2
SEQ ID NO: 40	DEX0253_40 CLASP2
SEQ ID NO: 41	DEX0253_41 CLASP2
SEQ ID NO: 42	DEX0253_42 CLASP2
SEQ ID NO: 43	DEX0253_43 CLASP2
SEQ ID NO: 44	DEX0253_44 CLASP2
SEQ ID NO: 45	DEX0253_45 CLASP2
SEQ ID NO: 46	DEX0253_46 CLASP2
SEQ ID NO: 47	DEX0253_47 CLASP2
SEQ ID NO: 48	DEX0253_48 CLASP2 CLASP1
SEQ ID NO: 50	DEX0253_50 CLASP2 CLASP1
SEQ ID NO: 51	DEX0253_51 CLASP2 CLASP1
SEQ ID NO: 52	DEX0253_52 CLASP2 CLASP1
SEQ ID NO: 53	DEX0253_53 CLASP2
SEQ ID NO: 54	DEX0253_54 CLASP2 CLASP1
SEQ ID NO: 55	DEX0253_55 CLASP2 CLASP1
SEQ ID NO: 56	DEX0253_56 CLASP2
SEQ ID NO: 57	DEX0253_57 CLASP2
SEQ ID NO: 58	DEX0253_58 CLASP2
SEQ ID NO: 59	DEX0253_59 CLASP2
SEQ ID NO: 60	DEX0253_60 CLASP2
SEQ ID NO: 61	DEX0253_61 CLASP2
SEQ ID NO: 62	DEX0253_62 CLASP5 CLASP1
SEQ ID NO: 63	DEX0253_63 CLASP5 CLASP1
SEQ ID NO: 64	DEX0253_64 CLASP5 CLASP1
SEQ ID NO: 65	DEX0253_65 CLASP5 CLASP1
SEQ ID NO: 66	DEX0253_66 CLASP2
SEQ ID NO: 68	DEX0253_68 CLASP2
SEQ ID NO: 69	DEX0253_69 CLASP2 CLASP1
SEQ ID NO: 70	DEX0253_70 CLASP5 CLASP1
SEQ ID NO: 71	DEX0253_71 CLASP5 CLASP1
SEQ ID NO: 72	DEX0253_72 CLASP2
SEQ ID NO: 73	DEX0253_73 CLASP2
SEQ ID NO: 74	DEX0253_74 CLASP2
SEQ ID NO: 75	DEX0253_75 CLASP2
SEQ ID NO: 76	DEX0253_76 CLASP2 CLASP1 CLASP4
SEQ ID NO: 77	DEX0253_77 CLASP2
SEQ ID NO: 78	DEX0253_78 CLASP5 CLASP1
SEQ ID NO: 79	DEX0253_79 CLASP5 CLASP1
SEQ ID NO: 80	DEX0253_80 CLASP5 CLASP1
SEQ ID NO: 81	DEX0253_81 CLASP5 CLASP1
SEQ ID NO: 82	DEX0253_82 CLASP2
SEQ ID NO: 83	DEX0253_83 CLASP2
SEQ ID NO: 84	DEX0253_84 CLASP1
SEQ ID NO: 85	DEX0253_85 CLASP1
SEQ ID NO: 86	DEX0253_86 CLASP2
SEQ ID NO: 87	DEX0253_87 CLASP2
SEQ ID NO: 88	DEX0253_88 CLASP2
SEQ ID NO: 89	DEX0253_89 CLASP2
SEQ ID NO: 90	DEX0253_90 CLASP2
SEQ ID NO: 91	DEX0253_91 CLASP2
SEQ ID NO: 92	DEX0253_92 CLASP2
SEQ ID NO: 93	DEX0253_93 CLASP2
SEQ ID NO: 94	DEX0253_94 CLASP2
SEQ ID NO: 95	DEX0253_95 CLASP2 CLASP1
SEQ ID NO: 96	DEX0253_96 CLASP2 CLASP1
SEQ ID NO: 97	DEX0253_97 CLASP2
SEQ ID NO: 98	DEX0253_98 CLASP2
SEQ ID NO: 99	DEX0253_99 CLASP2
SEQ ID NO: 100	DEX0253_100 CLASP2
SEQ ID NO: 101	DEX0253_101 CLASP2
SEQ ID NO: 102	DEX0253_102 CLASP2
SEQ ID NO: 103	DEX0253_103 CLASP2
SEQ ID NO: 104	DEX0253_104 CLASP5 CLASP1
SEQ ID NO: 105	DEX0253_105 CLASP2
SEQ ID NO: 106	DEX0253_106 CLASP2
SEQ ID NO: 107	DEX0253_107 CLASP2
SEQ ID NO: 108	DEX0253_108 CLASP2
SEQ ID NO: 109	DEX0253_109 CLASP2 CLASP1
SEQ ID NO: 110	DEX0253_110 CLASP2
SEQ ID NO: 111	DEX0253_111 CLASP2
SEQ ID NO: 112	DEX0253_112 CLASP2
SEQ ID NO: 113	DEX0253_113 CLASP2
SEQ ID NO: 114	DEX0253_114 CLASP2
SEQ ID NO: 115	DEX0253_115 CLASP5 CLASP1
SEQ ID NO: 116	DEX0253_116 CLASP2
SEQ ID NO: 117	DEX0253_117 CLASP2
SEQ ID NO: 118	DEX0253_118 CLASP2
SEQ ID NO: 119	DEX0253_119 CLASP2
SEQ ID NO: 120	DEX0253_120 CLASP2
SEQ ID NO: 121	DEX0253_121 CLASP2
SEQ ID NO: 123	DEX0253_123 CLASP2
SEQ ID NO: 124	DEX0253_124 CLASP2
SEQ ID NO: 125	DEX0253_125 CLASP5 CLASP1
SEQ ID NO: 126	DEX0253_126 CLASP5 CLASP1
SEQ ID NO: 127	DEX0253_127 CLASP2
SEQ ID NO: 128	DEX0253_128 CLASP2
SEQ ID NO: 129	DEX0253_129 CLASP2
SEQ ID NO: 130	DEX0253_130 CLASP2
SEQ ID NO: 131	DEX0253_131 CLASP2
SEQ ID NO: 132	DEX0253_132 CLASP2
SEQ ID NO: 133	DEX0253_133 CLASP5 CLASP1
SEQ ID NO: 134	DEX0253_134 CLASP2
SEQ ID NO: 135	DEX0253_135 CLASP2
SEQ ID NO: 136	DEX0253_136 CLASP2
SEQ ID NO: 139	DEX0253_139 CLASP2
SEQ ID NO: 140	DEX0253_140 CLASP5
SEQ ID NO: 141	DEX0253_141 CLASP2
SEQ ID NO: 142	DEX0253_142 CLASP2
SEQ ID NO: 143	DEX0253_143 CLASP2
SEQ ID NO: 144	DEX0253_144 CLASP2
SEQ ID NO: 145	DEX0253_145 CLASP2
SEQ ID NO: 146	DEX0253_146 CLASP2
SEQ ID NO: 147	DEX0253_147 CLASP2

Example 2

Relative Quantitation of Gene Expression

Real-Time quantitative PCR with fluorescent Taqman probes is a quantitation detection system utilizing the 5′-3′ nuclease activity of Taq DNA polymerase. The method uses an internal fluorescent oligonucleotide probe (Taqman) labeled with a 5′ reporter dye and a downstream, 3′ quencher dye. During PCR, the 5′-3′ nuclease activity of Taq DNA polymerase releases the reporter, whose fluorescence can then be detected by the laser detector of the Model 7700 Sequence Detection System (PE Applied Biosystems, Foster City, Calif., USA). Amplification of an endogenous control is used to standardize the amount of sample RNA added to the reaction and normalize for Reverse Transcriptase (RT) efficiency. Either cyclophilin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ATPase, or 18S ribosomal RNA (rRNA) is used as this endogenous control. To calculate relative quantitation between all the samples studied, the target RNA levels for one sample were used as the basis for comparative results (calibrator). Quantitation relative to the “calibrator” can be obtained using the standard curve method or the comparative method (User Bulletin #2: ABI PRISM 7700 Sequence Detection System). [0452]
The tissue distribution and the level of the target gene are evaluated for every sample in normal and cancer tissues. Total RNA is extracted from normal tissues, cancer tissues, and from cancers and the corresponding matched adjacent tissues. Subsequently, first strand cDNA is prepared with reverse transcriptase and the polymerase chain reaction is done using primers and Taqman probes specific to each target gene. The results are analyzed using the ABI PRISM 7700 Sequence Detector. The absolute numbers are relative levels of expression of the target gene in a particular tissue compared to the calibrator tissue. [0453]
One of ordinary skill can design appropriate primers. The relative levels of expression of the CSNA versus normal tissues and other cancer tissues can then be determined. All the values are compared to normal thymus (calibrator). These RNA samples are commercially available pools, originated by pooling samples of a particular tissue from different individuals. [0454]
The relative levels of expression of the CSNA in pairs of matching samples and 1 cancer and 1 normal/normal adjacent of tissue may also be determined. All the values are compared to normal thymus (calibrator). A matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. [0455]
In the analysis of matching samples, the CSNAs that show a high degree of tissue specificity for the tissue of interest. These results confirm the tissue specificity results obtained with normal pooled samples. [0456]
Further, the level of mRNA expression in cancer samples and the isogenic normal adjacent tissue from the same individual are compared. This comparison provides an indication of specificity for the cancer stage (e.g. higher levels of mRNA expression in the cancer sample compared to the normal adjacent). [0457]
Altogether, the high level of tissue specificity, plus the mRNA overexpression in matching samples tested are indicative of SEQ ID NO: 1 through 147 being a diagnostic marker for cancer. [0458]

DEX0095_71

cln150 -sqcln033

Sequence Sequence ID#

Dex0095_71 (cln150-sqcln033) DEX0253_104 (SEQ ID No.: 104)
Semi quantitative PCR was done using the following primers: [0459]

Primer DexSeqID From To Primer Length

Sqcln033F DEX0253_104 36 56 21

Sqcln033R DEX0253_104 256 236 21
The relative levels of expression of sqcln0033 in 12 normal samples from 12 different tissues were determined. These RNA samples are individual samples or are commercially available pools, originated by pooling samples of a particular tissue from different individuals. Using Polymerase Chain Reaction (PCR) technology expression levels were analyzed from four 10× serial cDNA dilutions in duplicate. Relative expression levels of 0, 1, 10, 100 and 1000 are used to evaluate gene expression. A positive reaction in the most dilute sample indicates the highest relative expression value. [0460]

Tissue Normal

Breast 0

Colon 1000

Endometrium 0

Kidney 0

Liver 0

Lung 0

Ovary 0

Prostate 0

Small Intestine 1000

Stomach 0

Testis 10

Uterus 0
Relative levels of expression in the table above show that none of the normal tissues examined here except colon and small intestine exhibit appreciable levels of sqcln033 gene expression. [0461]
The relative levels of expression of sqcln033 in 12 cancer samples from 12 different tissues were determined. Using Polymerase Chain Reaction (PCR) technology expression levels were analyzed from four 10× serial cDNA dilutions in duplicate. Relative expression levels of 0, 1, 10, 100 and 1000 are used to evaluate gene expression. A positive reaction in the most dilute sample indicates the highest relative expression value. [0462]

Tissue Cancer

Bladder 1

Breast 0

Colon 1000

Kidney 1

Liver 0

Lung 0

Ovary 0

pancreas 0

prostate 1

Stomach 0

Testes 1

Uterus 0
Relative levels of expression in Table 2 show that sqcln033 is expressed in very low levels in bladder, kidney, prostate and testicular cancer. High levels of expression were observed for colon cancer. [0463]
The relative levels of expression of sqcln033 in 6 matching pair colon samples were determined. Each matching pair is formed by mRNA from the cancer sample for a particular tissue and mRNA from the normal adjacent sample for that same tissue from the same individual. [0464]
Using Polymerase Chain Reaction (PCR) technology expression levels were analyzed from four 10× serial cDNA dilutions in duplicate. Relative expression levels of 0, 1, 10, 100 and 1000 are used to evaluate gene expression. A positive reaction in the most dilute sample indicates the highest relative expression value. [0465]

Sample ID Tissue Cancer NAT

9609B019 Colon 10 100

9709C074RA Colon 1 10

9705F002D Colon 10 100

9608B012 Colon 0 100

4004709A1 Colon 10 10

9707C004GB Colon 100 10
Relative levels of expression in the table above show that sqcln033 is expressed in low levels in four and in moderate levels in one of the six colon cancer samples. Moderate levels of expression was observed in matching normal adjacent tissue (NAT). [0466]
From the semi-quantitative PCR experiments exhibited above especially from the results in the tables above high degree of specificity of sqCln033 for colon tissue is observed. [0467]
Experiments are underway to design and test primers and probe for quantitative PCR experiments. [0468]

Example 3

Protein Expression

The CSNA is amplified by polymerase chain reaction (PCR) and the amplified DNA fragment encoding the CSNA is subcloned in pET-21d for expression in [0469] E. coli. In addition to the CSNA coding sequence, codons for two amino acids, Met-Ala, flanking the NH₂-terminus of the coding sequence of CSNA, and six histidines, flanking the COOH-terminus of the coding sequence of CSNA, are incorporated to serve as initiating Met/restriction site and purification tag, respectively.
An over-expressed protein band of the appropriate molecular weight may be observed on a Coomassie blue stained polyacrylamide gel. This protein band is confirmed by Western blot analysis using monoclonal antibody against 6× Histidine tag. [0470]
Large-scale purification of CSP was achieved using cell paste generated from 6-liter bacterial cultures, and purified using immobilized metal affinity chromatography (IMAC). Soluble fractions that had been separated from total cell lysate were incubated with a nickle chelating resin. The column was packed and washed with five column volumes of wash buffer. CSP was eluted stepwise with various concentration imidazole buffers. [0471]

Example 4

Protein Fusions

Briefly, the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5′ and 3′ ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector. For example, if pC4 (Accession No. 209646) is used, the human Fc portion can be ligated into the BamHI cloning site. Note that the 3′ BamHI site should be destroyed. Next, the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and a polynucleotide of the present invention, isolated by the PCR protocol described in Example 2, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced. If the naturally occurring signal sequence is used to produce the secreted protein, pC4 does not need a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector can be modified to include a heterologous signal sequence. See, e.g., WO 96/34891. [0472]

Example 5

Production of an Antibody from a Polypeptide

In general, such procedures involve immunizing an animal (preferably a mouse) with polypeptide or, more preferably, with a secreted polypeptide-expressing cell. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100, μg/ml of streptomycin. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP20), available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al., [0473] Gastroenterology 80: 225-232 (1981).

The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the polypeptide. Alternatively, additional antibodies capable of binding to the polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, protein specific antibodies are used to immunize an animal, preferably a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the protein-specific antibody can be blocked by the polypeptide. Such antibodies comprise anti-idiotypic antibodies to the protein specific antibody and can be used to immunize an animal to induce formation of further protein-specific antibodies. Using the Jameson-Wolf methods the following epitopes were predicted. (Jameson and Wolf, CABIOS, 4(1), 181-186, 1988, the contents of which are incorporated by reference).



	DEX0253_151	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	68-108	1.02 41
	DEX0253_154	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	13-31	1.00 19
	DEX0253_155	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	8-22	1.25 15
	67-98	1.06 32
	DEX0253_156	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	14-33	1.13 20
	DEX0253_161	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	32-46	0.98 15
	DEX0253_170	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	6-48	1.02 43
	DEX0253_172	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	19-28	1.00 10
	DEX0253_174	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	13-29	1.01 17
	DEX0253_178	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	2-17	0.99 16
	DEX0253_179	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	7-20	0.96 14
	DEX0253_184	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	18-33	1.00 16
	DEX0253_187	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	42-54	1.09 13
	DEX0253_188	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	14-41	1.03 28
	81-97	0.96 17
	DEX0253_192	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	25-75	0.97 51
	DEX0253_197	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	31-58	1.06 28
	DEX0253_198	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	73-82	1.22 10
	154-165	1.21 12
	213-224	1.06 12
	38-58	1.05 21
	314-323	1.03 10
	250-267	0.99 18
	275-295	0.98 21
	137-149	0.96 13
	DEX0253_203	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	3-28	0.99 26
	DEX0253_204	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	29-39	1.10 11
	52-65	0.97 14
	DEX0253_208	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	3-14	1.04 12
	DEX0253_211	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	33-54	1.22 22
	59-76	1.08 18
	DEX0253_212	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	22-31	1.00 10
	DEX025 3_214	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	7-22	1.24 16
	DEX0253_217	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	34-46	1.06 13
	DEX0253_218	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	23-32	1.16 10
	DEX0253_219	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	19-30	1.24 12
	DEX0253_221	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	58-69	1.08 12
	42-51	1.04 10
	DEX0253_222	Antigenicity Index(Jameson-Wolf)
	positions	AI avg length
	58-69	1.08 12
	42-51	1.04 10
	DEX0253_226	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	8-18	1.01 11
	DEX0253_229	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	8-36	1.02 29
	DEX0253_237	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	14-31	0.91 18
	DEX0253_245	Antigenicity Index (Jameson-Wolf)
	positions	AI avg length
	3-27	1.12 25
	DEX0253_246	Antigenicity Index (Jameson-Wolf if)
	positions	AI avg length
	23-32	0.93 10

Examples of post-translational modifications (PTMs) of the BSPs of this invention are listed below. In addition, antibodies that specifically bind such post-translational modifications may be useful as a diagnostic or as therapeutic. Using the ProSite database (Bairoch et al., Nucleic Acids Res. 25(1):217-221 (1997), the contents of which are incorporated by reference), the following PTMs were predicted for the LSPs of the invention (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_prosite.html most recently accessed Oct. 23, 2001). For full definitions of the PTMs see http://www.expasy.org/cgi-bin/prosite-list.pl most recently accessed Oct. 23, 2001.



DEX0253_148	Tyr_Phospho_Site 3-9;
DEX0253_150	Ck2_Phospho_Site 19-22; 23-26;
DEX0253_151	Ck2_Phospho_Site 83-86; 128-131; Myristyl 28-33;
	49-54; Pkc_Phospho_Site 77-79; 83-85; 105-107;
	139-141;
DEX0253_152	Leucine_Zipper 6-27; Pkc_Phospho_Site 16-18;
DEX0253_153	Pkc_Phospho_Site 16-18; 24-26;
DEX0253_154	Ck2_Phospho_Site 2-5; 23-26;
DEX0253_155	Amidation 12-15; Myristyl 12-17; 31-36; 34-39;
	Pkc_Phospho_Site 75-77;
DEX0253_156	Ck2_Phospho_Site 8-11; Pkc_Phospho_Site 16-18;
DEX0253_158	Pkc_Phospho_Site 2-4;
DEX0253_159	Myristyl 26-31; Pkc_Phospho_Site 9-11; 14-16;
DEX0253_160	Myristyl 13-18; Pkc_Phospho_Site 31-33;
DEX0253_161	Pkc_Phospho_Site 8-10; 17-19;
DEX0253_162	Ck2_Phospho_Site 28-31; Myristyl 48-53;
	Pkc_Phospho_Site 28-30;
DEX0253_164	Asn_Glycosylation 21-24; Myristyl 17-22;
DEX0253_165	Pkc_Phospho_Site 44-46;
DEX0253_166	Asn_Glycosylation 7-10; Camp_Phospho_Site 3-6;
	Ck2_Phospho_Site 19-22; Myristyl 32-37;
	Pkc_Phospho_Site 6-8; 45-47; Tyr_Phospho_Site
	28-34;
DEX0253_167	Asn_Glycosylation 14-17; Myristyl 23-28;
	Pkc_Phospho_Site 9-11;
DEX0253_168	Asn_Glycosylation 38-41; Ck2_Phospho_Site 40-43;
	Myristyl 49-54; Pkc_Phospho_Site 16-18; 35-37;
DEX0253_170	Camp_Phospho_Site 43-46; Myristyl 36-41;
DEX0253_171	Asn_Glycosylation 37-40; Ck2_Phospho_Site
	79-82; 86-89; Myristyl 22-27; 34-39; 47-52;
	Pkc_Phospho_Site 7-9; 15-17;
DEX0253_172	Asn_Glycosylation 7-10; 11-14; 21-24;
	Pkc_Phospho_Site 52-54; 55-57;
DEX0253_173	Ck2_Phospho_Site 27-30;
DEX0253_174	Myristyl 56-61;
DEX0253_175	Myristyl 24-29;
DEX0253_176	Pkc_Phospho_Site 8-10;
DEX0253_177	Pkc_Phospho_Site 9-11;
DEX0253_178	Ck2_Phospho_Site 52-55; Myristyl 24-29;
	Pkc_Phospho_Site 45-47;
DEX0253_179	Asn_Glycosylation 63-66; Ck2_Phospho_Site 25-28;
	Pkc_Phospho_Site 27-29;
DEX0253_180	Ck2_Phospho_Site 26-29;
DEX0253_181	Amidation 51-54 Pkc_Phospho_Site 6-8; 51-53;
DEX0253_183	Ck2_Phospho_Site 17-20; Pkc_Phospho_Site
	36-38; 39-41;
DEX0253_184	Pkc_Phospho_Site 31-33;
DEX0253_186	Pkc_Phospho_Site 18-20;
DEX0253_187	Ck2_Phospho_Site 42-45; 77-80;
DEX0253_188	Ck2_Phospho_Site 21-24; 86-89; Myristyl 25-30;
	Pkc_Phospho_Site 73-75;
DEX0253_189	Amidation 25-28; Camp_Phospho_Site 10-13;
	Ck2_Phospho_Site 37-40;
DEX0253_191	Ck2_Phospho_Site 11-14; Myristyl 16-21;
DEX0253_192	Ck2_Phospho_Site 48-51 ; 6 1-64; Myristyl 57-62;
	Pkc_Phospho_Site 22-24; 58-60;
DEX0253_193	Ck2_Phospho_Site 43-46; Pkc_Phospho_Site 18-20;
	34-36;
DEX0253_196	Asn_Glycosylation 3-6; Ck2_Phospho_Site 8-11;
DEX0253_197	Myristyl 4-9;
DEX0253_198	Asn_Glycosylation 46-49; 54-57; 99-102; 123-126;
	135-138; 482-185; 195-198; 223-226; 229-232;
	272-275; 305-308 Camp_Phospho_Site
	259-262 Ck2_Phospho_Site 75-78; 82-85; 114-117;
	197-200; 208-211 ; 247-250; 308-311; 382-385;
	395-398; Myristyl 173-178; 316-321; 320-325;
	334-339; 339-344; Pkc_Phospho_Site 63-65; 82-84;
	101-103; 114-116; 157∫159; 187-189; 197-199;
	212-214; 225-227; 293-295; 395-397;
	Receptor_Cytokines_1 126-138;
	Receptor_Cytokines_2 304-310;
DEX0253_199	Camp_Phospho_Site 6-9;
DEX0253_202	Amidation 38-41; Asn_Glycosylation 2-5; 45-48;
DEX0253_203	Camp_Phospho_Site 24-27; Pkc_Phospho_Site 20-22;
DEX0253_204	Asn_Glycosylation 65-68; Pkc_Phospho_Site 54-56;
DEX0253_205	Ck2_Phospho_Site 6-9; Myristyl 46-51; 47-52; 59-64;
	63-68; 78-83 Pkc_Phospho_Site 6-8; 19-21;
DEX0253_207	Ck2_Phospho_Site 57-60; 61-64; Myristyl 69-74;
	Pkc_Phospho_Site 34-36;
DEX0253_208	Camp_Phospho_Site 5-8; Ck2_Phospho_Site 37-40;
	Myristyl 26-31;
DEX0253_209	Pkc_Phospho_Site 52-54;
DEX0253_210	Camp_Phospho_Site 34-37 Ck2_Phospho_Site 9-12;
	Pkc_Phospho_Site 9-11; 33-35;
DEX0253_211	Amidation 67-70; Camp_Phospho_Site 69-72;
	Ck2_Phospho_Site 5-8; 10-13 ; 23-26;
	Myristyl 35-40; 54-59; Pkc_Phospho_Site 67-69;
DEX0253_212	Prokar_Lipoprotein 6-16;
DEX0253_213	Asn_Glycosylation 44-47; Camp_Phospho_Site 37-40;
	Ck2_Phospho_Site 2-5; Pkc_Phospho_Site 8-10;
DEX0253_215	Asn_Glycosylation 3-6; Ck2_Phospho_Site 28-31;
DEX0253_216	Ck2_Phospho_Site 9-12; Pkc_Phospho_Site 32-34;
DEX0253_217	Camp_Phospho_Site 28-31; Myristyl 12-17;
	Rieske_2 35-40;
DEX0253_218	Ck2_Phospho_Site 21-24; Myristyl 29-34;
DEX0253_221	Camp_Phospho_Site 51-54; Ck2_Phospho_Site
	29-32; 98-101; Myristyl 21-26; 70-75; 75-80;
	Pkc_Phospho_Site 50-52; 57-59; 104-106;
	Prokar_Lipoprotein 66-76; 68-78;
DEX0253_222	Camp_Phospho_Site 51-54; Ck2_Phospho_Site
	29-32; 98-101; Myristyl 21-26; 70-75; 75-80;
	Pkc_Phospho_Site 50-52; 57-59; 104-
	106; Prokar_Lipoprotein 66-76; 68-78;
DEX0253_223	Ck2_Phospho_Site 75-78;
DEX0253_224	Ck2_Phospho_Site 96-99; Myristyl 72-77; 76-81;
	105-110; Pkc_Phospho_Site 63-65; 87-89;
DEX0253_225	Asn_Glycosylation 3-6; 8-11 ; 9-12;
	Pkc_Phospho_Site 12-14;
DEX0253_226	Camp_Phospho_Site 14-17;
DEX0253_228	Ck2_Phospho_Site 14-17;
DEX0253_229	Asn_Glycosylation 41-44; Ck2_Phospho_Site
	20-23; 39-42; Myristyl 25-30; 30-35; 37-42;
	45-50; Pkc_Phospho_Site 9-11;
	20-22;
DEX0253_230	Ck2_Phospho_Site 15-18;
DEX0253_231	Camp_Phospho_Site 25-28;
DEX0253_232	Ck2_Phospho_Site 7-10; Rgd 8-10;
DEX0253_235	Ck2_Phospho_Site 21-24; Myristyl 4-9;
	Pkc_Phospho_Site 21-23;
DEX0253_236	Ck2_Phospho_Site 12-15 ; 26-29; Myristyl
	10-15; 24-29; 30-35;
DEX0253_237	Ck2_Phospho_Site 41-44;
DEX0253_238	Ck2_Phospho_Site 2-5; Pkc_Phospho_Site 46-48;
DEX0253_239	Pkc_Phospho_Site 9-11; 17-19;
DEX0253_240	Camp_Phospho_Site 2-5; Ck2_Phospho_Site 5-8;
DEX0253_241	Pkc_Phospho_Site 20-22;
DEX0253_242	Ck2_Phospho_Site 30-33; Myristyl 4-9; 27-32; 36-41;
DEX0253_243	Asn_Glycosylation 40-43; Pkc_Phospho_Site 12-14;
	48-50;
DEX0253_245	Asn_Glycosylation 7-10; Glycosaminoglycan 9-12;
	Myristyl 10-15; 19-24; Pkc_Phospho_Site 23-25;
DEX0253_246	Ck2_Phospho_Site 3-6; 35-38;
DEX0253_247	Ck2_Phospho_Site 12-15; 27-30;
DEX0253_248	Glycosaminoglycan 30-33; Myristyl 18-23;
	Pkc_Phospho_Site 22-24;
DEX0253_250	Asn_Glycosylation 21-24; Ck2_Phospho_Site
	14-17; 28-31; Myristyl 26-31;

Example 6

Method of Determining Alterations in a Gene Corresponding to a Polynucleotide

RNA is isolated from individual patients or from a family of individuals that have a phenotype of interest. cDNA is then generated from these RNA samples using protocols known in the art. See, Sambrook (2001), supra. The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NO: 1 through 147. Suggested PCR conditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 seconds at 52-58° C.; and 60-120 seconds at 70° C., using buffer solutions described in Sidransky et al., [0476] Science 252(5006): 706-9 (1991). See also Sidransky et al., Science 278(5340): 1054-9 (1997).
PCR products are then sequenced using primers labeled at their 5′ end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons is also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations are then cloned and sequenced to validate the results of the direct sequencing. PCR products is cloned into T-tailed vectors as described in Holton et al., [0477] Nucleic Acids Res., 19: 1156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations not present in unaffected individuals.
Genomic rearrangements may also be determined. Genomic clones are nick-translated with digoxigenin deoxyuridine 5′ triphosphate (Boehringer Manheim), and FISH is performed as described in Johnson et al., [0478] Methods Cell Biol. 35: 73-99 (1991). Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the corresponding genomic locus.
Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C-and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, Vt.) in combination with a cooled charge-coupled device camera (Photometrics, Tucson, Ariz.) and variable excitation wavelength filters. Id. Image collection, analysis and chromosomal fractional length measurements are performed using the ISee Graphical Program System. (Inovision Corporation, Durham, N.C.) Chromosome alterations of the genomic region hybridized by the probe are identified as insertions, deletions, and translocations. These alterations are used as a diagnostic marker for an associated disease. [0479]

Example 7

Method of Detecting Abnormal Levels of a Polypeptide in a Biological Sample

Antibody-sandwich ELISAs are used to detect polypeptides in a sample, preferably a biological sample. Wells of a microtiter plate are coated with specific antibodies, at a final concentration of 0.2 to 10 μg/ml. The antibodies are either monoclonal or polyclonal and are produced by the method described above. The wells are blocked so that non-specific binding of the polypeptide to the well is reduced. The coated wells are then incubated for >2 hours at RT with a sample containing the polypeptide. Preferably, serial dilutions of the sample should be used to validate results. The plates are then washed three times with deionized or distilled water to remove unbound polypeptide. Next, 50 μl of specific antibody-alkaline phosphatase conjugate, at a concentration of 25-400 ng, is added and incubated for 2 hours at room temperature. The plates are again washed three times with deionized or distilled water to remove unbound conjugate. 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution are added to each well and incubated 1 hour at room temperature. [0480]
The reaction is measured by a microtiter plate reader. A standard curve is prepared, using serial dilutions of a control sample, and polypeptide concentrations are plotted on the X-axis (log scale) and fluorescence or absorbance on the Y-axis (linear scale). The concentration of the polypeptide in the sample is calculated using the standard curve. [0481]

Example 8

Formulating a Polypeptide

The secreted polypeptide composition will be formulated and dosed in a fashion consistent with good medical practice, taking into account the clinical condition of the individual patient (especially the side effects of treatment with the secreted polypeptide alone), the site of delivery, the method of administration, the scheduling of administration, and other factors known to practitioners. The “effective amount” for purposes herein is thus determined by such considerations. [0482]
As a general proposition, the total pharmaceutically effective amount of secreted polypeptide administered parenterally per dose will be in the range of about 1, μg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone. If given continuously, the secreted polypeptide is typically administered at a dose rate of about 1 μg/kg/hour to about 50 mg/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed. The length of treatment needed to observe changes and the interval following treatment for responses to occur appears to vary depending on the desired effect. [0483]
Pharmaceutical compositions containing the secreted protein of the invention are administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, gels, drops or transdermal patch), bucally, or as an oral or nasal spray. “Pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. [0484]
The secreted polypeptide is also suitably administered by sustained-release systems. Suitable examples of sustained-release compositions include semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., [0485] Biopolymers 22: 547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981), and R. Langer, Chem. Tech. 12: 98-105 (1982)), ethylene vinyl acetate (R. Langer et al.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustained-release compositions also include liposomally entrapped polypeptides. Liposomes containing the secreted polypeptide are prepared by methods known per se: D E Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal secreted polypeptide therapy.
For parenteral administration, in one embodiment, the secreted polypeptide is formulated generally by mixing it at the desired degree of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a pharmaceutically acceptable carrier, I.e., one that is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. [0486]
For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to polypeptides. Generally, the formulations are prepared by contacting the polypeptide uniformly and intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. [0487]
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG. [0488]
The secreted polypeptide is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of polypeptide salts. [0489]
Any polypeptide to be used for therapeutic administration can be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). Therapeutic polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. [0490]
Polypeptides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous polypeptide solution, and the resulting mixture is lyophilized. The infusion solution is prepared by reconstituting the lyophilized polypeptide using bacteriostatic Water-for-Injection. [0491]
The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the polypeptides of the present invention may be employed in conjunction with other therapeutic compounds. [0492]

Example 9

Method of Treating Decreased Levels of the Polypeptide

It will be appreciated that conditions caused by a decrease in the standard or normal expression level of a secreted protein in an individual can be treated by administering the polypeptide of the present invention, preferably in the secreted form. Thus, the invention also provides a method of treatment of an individual in need of an increased level of the polypeptide comprising administering to such an individual a pharmaceutical composition comprising an amount of the polypeptide to increase the activity level of the polypeptide in such an individual. [0493]
For example, a patient with decreased levels of a polypeptide receives a daily dose 0.1-100 μg/kg of the polypeptide for six consecutive days. Preferably, the polypeptide is in the secreted form. The exact details of the dosing scheme, based on administration and formulation, are provided above. [0494]

Example 10

Method of Treating Increased Levels of the Polypeptide

Antisense technology is used to inhibit production of a polypeptide of the present invention. This technology is one example of a method of decreasing levels of a polypeptide, preferably a secreted form, due to a variety of etiologies, such as cancer. [0495]
For example, a patient diagnosed with abnormally increased levels of a polypeptide is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the treatment was well tolerated. The formulation of the antisense polynucleotide is provided above. [0496]

Example 11

Method of Treatment Using Gene Therapy

One method of gene therapy transplants fibroblasts, which are capable of expressing a polypeptide, onto a patient. Generally, fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37° C. for approximately one week. [0497]
At this time, fresh media is added and subsequently changed every several days. After an additional two weeks in culture, a monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into larger flasks. pMV-7 (Kirschmeier, P. T. et al., DNA, 7: 219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and HindIII and subsequently treated with calf intestinal phosphatase. The linear vector is fractionated on agarose gel and purified, using glass beads. [0498]
The cDNA encoding a polypeptide of the present invention can be amplified using PCR primers which correspond to the 5′ and 3′ end sequences respectively as set forth in Example 1. Preferably, the 5′ primer contains an EcoRI site and the 3′ primer includes a HindIII site. Equal quantities of the Moloney murine sarcoma virus linear backbone and the amplified EcoRI and HindIII fragment are added together, in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions appropriate for ligation of the two fragments. The ligation mixture is then used to transform bacteria HB 101, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector has the gene of interest properly inserted. [0499]
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is then added to the media and the packaging cells transduced with the vector. The packaging cells now produce infectious viral particles containing the gene (the packaging cells are now referred to as producer cells). [0500]
Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells. The spent media, containing the infectious viral particles, is filtered through a millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells. Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. [0501]
If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether protein is produced. [0502]
The engineered fibroblasts are then transplanted onto the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads. [0503]

Example 12

Method of Treatment Using Gene Therapy—in vivo

Another aspect of the present invention is using in vivo gene therapy methods to treat disorders, diseases and conditions. The gene therapy method relates to the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences into an animal to increase or decrease the expression of the polypeptide. [0504]
The polynucleotide of the present invention may be operatively linked to a promoter or any other genetic elements necessary for the expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are known in the art, see, for example, WO 90/11092, WO 98/11779; U.S. Pat. Nos. 5,693,622; 5,705,151; 5,580,859; Tabata H. et al. (1997) Cardiovasc. Res. 35 (3): 470-479, Chao J et al. (1997) Pharmacol. Res. 35 (6): 517-522, Wolff J. A. (1997) Neuromuscul. Disord. 7 (5): 314-318, Schwartz B. et al. (1996) Gene Ther. 3 (5): 405-411, Tsurumi Y. et al. (1996) Circulation 94 (12): 3281-3290 (incorporated herein by reference). [0505]
The polynucleotide constructs may be delivered by any method that delivers injectable materials to the cells of an animal, such as, injection into the interstitial space of tissues (heart, muscle, skin, lung, liver, intestine and the like). The polynucleotide constructs can be delivered in a pharmaceutically acceptable liquid or aqueous carrier. [0506]
The term “naked” polynucleotide, DNA or RNA, refers to sequences that are free from any delivery vehicle that acts to assist, promote, or facilitate entry into the cell, including viral sequences, viral particles, liposome formulations, lipofectin or precipitating agents and the like. However, the polynucleotides of the present invention may also be delivered in liposome formulations (such as those taught in Felgner P. L. et al. (1995) Ann. NY Acad. Sci. 772: 126-139 and Abdallah B. et al. (1995) Biol. Cell 85 (1): 1-7) which can be prepared by methods well known to those skilled in the art. [0507]
The polynucleotide vector constructs used in the gene therapy method are preferably constructs that will not integrate into the host genome nor will they contain sequences that allow for replication. Any strong promoter known to those skilled in the art can be used for driving the expression of DNA. Unlike other gene therapies techniques, one major advantage of introducing naked nucleic acid sequences into target cells is the transitory nature of the polynucleotide synthesis in the cells. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide production of the desired polypeptide for periods of up to six months. [0508]
The polynucleotide construct can be delivered to the interstitial space of tissues within the an animal, including of muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue. Interstitial space of the tissues comprises the intercellular fluid, mucopolysaccharide matrix among the reticular fibers of organ tissues, elastic fibers in the walls of vessels or chambers, collagen fibers of fibrous tissues, or that same matrix within connective tissue ensheathing muscle cells or in the lacunae of bone. It is similarly the space occupied by the plasma of the circulation and the lymph fluid of the lymphatic channels. Delivery to the interstitial space of muscle tissue is preferred for the reasons discussed below. They may be conveniently delivered by injection into the tissues comprising these cells. They are preferably delivered to and expressed in persistent, non-dividing cells which are differentiated, although delivery and expression may be achieved in non-differentiated or less completely differentiated cells, such as, for example, stem cells of blood or skin fibroblasts. In vivo muscle cells are particularly competent in their ability to take up and express polynucleotides. [0509]
For the naked polynucleotide injection, an effective dosage amount of DNA or RNA will be in the range of from about 0.05 μg/kg body weight to about 50 mg/kg body weight. Preferably the dosage will be from about 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill will appreciate, this dosage will vary according to the tissue site of injection. The appropriate and effective dosage of nucleic acid sequence can readily be determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, such as, inhalation of an aerosol formulation particularly for delivery to lungs or bronchial tissues, throat or mucous membranes of the nose. In addition, naked polynucleotide constructs can be delivered to arteries during angioplasty by the catheter used in the procedure. [0510]
The dose response effects of injected polynucleotide in muscle in vivo is determined as follows. Suitable template DNA for production of mRNA coding for polypeptide of the present invention is prepared in accordance with a standard recombinant DNA methodology. The template DNA, which may be either circular or linear, is either used as naked DNA or complexed with liposomes. The quadriceps muscles of mice are then injected with various amounts of the template DNA. [0511]
Five to six week old female and male Balb/C mice are anesthetized by intraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incision is made on the anterior thigh, and the quadriceps muscle is directly visualized. The template DNA is injected in 0.1 ml of carrier in a 1 cc syringe through a 27 gauge needle over one minute, approximately 0.5 cm from the distal insertion site of the muscle into the knee and about 0.2 cm deep. A suture is placed over the injection site for future localization, and the skin is closed with stainless steel clips. [0512]
After an appropriate incubation time (e.g., 7 days) muscle extracts are prepared by excising the entire quadriceps. Every fifth 15 um cross-section of the individual quadriceps muscles is histochemically stained for protein expression. A time course for protein expression may be done in a similar fashion except that quadriceps from different mice are harvested at different times. Persistence of DNA in muscle following injection may be determined by Southern blot analysis after preparing total cellular DNA and HIRT supernatants from injected and control mice. [0513]
The results of the above experimentation in mice can be use to extrapolate proper dosages and other treatment parameters in humans and other animals using naked DNA. [0514]

Example 13

Transgenic Animals

The polypeptides of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol. [0515]
Any technique known in the art may be used to introduce the transgene (i.e., polynucleotides of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., [0516] Biotechnology (NY) 9: 830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol. 3: 1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259: 1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115: 171-229 (1989), which is incorporated by reference herein in its entirety.
Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., [0517] Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810813 (1997)).
The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, I. e., mosaic animals or chimeric. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., [0518] Science 265: 103-106 (1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product. [0519]
Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest. [0520]
Transgenic animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders. [0521]

Example 14

Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or “knocking out” the gene and/or its promoter using targeted homologous recombination. (E.g., see Smithies et al., [0522] Nature 317: 230-234 (1985); Thomas & Capecchi, Cell 51: 503512 (1987); Thompson et al., Cell 5: 313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (e.g., see Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art.
In further embodiments of the invention, cells that are genetically engineered to express the polypeptides of the invention, or alternatively, that are genetically engineered not to express the polypeptides of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (I.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. [0523]
The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally. [0524]
Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporated by reference herein in its entirety). [0525]
When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system. [0526]
Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of polypeptides of the present invention, studying conditions and/or disorders associated with aberrant expression, and in screening for compounds effective in ameliorating such conditions and/or disorders. [0527]
All patents, patent publications, and other published references mentioned herein are hereby incorporated by reference in their entireties as if each had been individually and specifically incorporated by reference herein. While preferred illustrative embodiments of the present invention are described, one skilled in the art will appreciate that the present invention can be practiced by other than the described embodiments, which are presented for purposes of illustration only and not by way of limitation. The present invention is limited only by the claims that follow. [0528]
1 250 1 421 DNA Homo sapiens 1 cttaaaaata atttctagat tgttggcatt attaaaaccc taaatccttt taggaactat 60 tgcgaagaaa gaatatgata ttcgtaagag ctcagtgcta atattagcat tggttatggt 120 agtgaaagac cagataaatc ttttagttgg gaagtatgtc ttgaggtata cttccttata 180 atcattaagt aaataagtaa aactatatta catagataat gtgtaactct ctgtattaca 240 tagaatgtct gcagaatgta gataggaaaa ataaagtttg tcaataattt tcaacatctt 300 tattgagata cagttaatct gccatgacga tttgcctact ataaagtgta catttcagtg 360 tgtttagcta gtgtatttgc agagttgtgc agtcatcacc acagtaactt ccctaacact 420 c 421 2 426 DNA Homo sapiens 2 agaaacccat tcctaagtga actgccactg ctctagtcta acttaggttg gcagagagcc 60 agcactttct tcagcattca gggcagggag cactgaggat attggcattg cttattacta 120 agcacacaga tacaagtatg tgcttgatat gtaaccaaag taagttaaac tccttattta 180 atcttagcac ctgtctaaag gctgggtgac tgtatttata gatgaggaaa actgaaaatt 240 gggggccaag gggcagtgaa gtgaagtgac ttgttctatg atacacagct agtaggaata 300 ttagcactgg aatttgaatt tcatgccatc ccattccaac ctgggtgttt actacttccc 360 actatctccc aagcatgggt attttaggaa atatagaaca ttttctcagc aatacagact 420 tatttc 426 3 1016 DNA Homo sapiens misc_feature (469)..(574) n=a, c, g or t 3 agaaacccat tcctaagtga actgccactg ctctagtcta acttaggttg gcagagagcc 60 agcactttct tcagcattca gggcagggag cactgaggat attggcattg cttattacta 120 agcacacaga tacaagtatg tgcttgatat gtaaccaaag taagttaaac tccttattta 180 atcttagcac ctgtctaaag gctgggtgac tgtatttata gatgaggaaa actgaaaatt 240 gggggccaag gggcagtgaa gtgaagtgac ttgttctatg atacacagct agtaggaata 300 ttagcactgg aatttgaatt tcatgccatc ccattccaac cctgggtgtt tactacttcc 360 cactatctcc caagcatggg tattttagga aatatagaac attttctcag caatacagac 420 ttatttctct attctccttt ccacatactc tcttttccct taacaacann nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnntcacat catttattct taggccactt 600 tgatgctttt tcattgatgc tctttataga catagtgaag taaaagttta tctaggatat 660 atggtgggag gtgaggaaga cttaggtaga gaggttccaa accagttgtt actgcttagc 720 tcaatttcag acatacttcc tccagccctc tctaaactac ccaccagtct tcgcccctct 780 tttcttagtt ctgtggcact tgccctgggt gccctaactg tatggcatgc tgttctcatc 840 agtcgaggtg agactagcat cgaaaggcac atcaacaaga aggagagacg tcggctacag 900 gccaagggca gagtgagtag ggttgaaggc tcggggtggg taggtgggta actgaacttg 960 ctctccctgt aaacagaggc catgggcagg gctgactagg gcaagcatta taaaag 1016 4 1358 DNA Homo sapiens 4 ctcctggggc tcgttttctc caggaggctg cattctgatc cataaacctt ctcctcgggg 60 tttagggtcg agctgttcct gatgtttatc ggagactggg atcaaagcta tccaggtcat 120 aaatctctct ctgtggctgt tgggccccag ggcagctgaa gagggttgac agccctttgg 180 acctcaaagg aaaaaatgtg ctctactcca cccactccca gctctgccaa gaagctgtcc 240 tctgagaagc catggctggg ccgttccatt ctggggagct gctgaaaaga gctgggaggc 300 cgagaagaac ttgcgtgtgc tgggggagag gaagcctggc cttgagggag gggtgcaggt 360 gtggctcctg tgtgtgtggg ggctggggga ccttgtgtgc cttttccttg tggctgtgaa 420 atgctttatg agtacttcca taggaggatg gacagggagt cggggagata aactcagcca 480 caaggcccca gggcctcagg aaacttgcac ccaaccctct cattttacag aagaaaactg 540 tgcctggaag gttgaagggt ttgttcccag tcacacaacc agggatcctt aggacagcca 600 gaccaggaaa ccatttccaa actgccaagc catggcagag tatcaagacc tcaggaacca 660 tcgagacacc atggaagcat tgggaaaagc ctccttagct tttgaagctc ctcattgttc 720 ttgagtgtgc atggagccca tgactgcggg gttttgtaga cacctcaggg attacatgac 780 tggtacccct gacaaagtca aggctgctgg acaaaatgag tccgaggatt tcaggggcac 840 gctgggcgca ggagctggtg ggctgttggg agtgcccctt tactgggcag gcttccttcc 900 tcctggtgat ggggggttcc tcagcacaaa agtgaagggg tggaggggct ggaggagcag 960 gaatctctct tgttgatagg tatgaggcct tgaagtcctt ttctttgtcc caggattcat 1020 ggacgcttcg gggctgatct ttgagttttc aagcatgggg tgcagagacg tttaggtaaa 1080 ctcttaccgt cctctctctt cgtcagggct tcccaggaat caacaatgcc caagaaggaa 1140 gggattgtag aaatagctta accctttcat ttaccaacgt ggaaattgaa gcccagggaa 1200 gggaagggac cggtcgtgga agggagagcc atcagcagaa agagaccctg agatcttcgc 1260 ctgggattcc caggaagtcc agcccgagct gattcacaga acaaatgcat gcaaaccttg 1320 ctatcaataa attacacatg cacttacgta aaacacat 1358 5 2375 DNA Homo sapiens 5 cttttctctt gttgagtgca aatggagaac agctgctcac gctcgtcgtc tgacatcagc 60 tatttctcag gatgaccctg cgagacaggc cagggtcatt agacccaatt tggttctcag 120 caaatatgtg tttattcctg catgcgtggg ccacaggctg gtttcttggg tgcaatgaat 180 agctgcaggt ttattagggt gtctttttag atggatgtat gtttcccgat gtctatagaa 240 cactccggac cccggagagt gaagactctg cctgtcggac ttgctttgag aagatccttc 300 tccacctccc catggcagaa gttgcttcac agaggggaac agttttatgg atgtggctga 360 gaccttaaac ttgaggcaac ccatctgagg tggcatccag aggagactgg ctggcccctc 420 cttcaccttg gatgtagtgc tgtttctagg atctcttttc aatcagcaaa acaggggatg 480 ttccaagagg gtgtggattc cctgccatcc cacatggtca agtggagggg acgggaaaaa 540 gctatgaagg gtttgtgacc acacagactc tcctggcccc ctgtcctttt ggaaagaaga 600 cagggatgaa atataatcaa gcaattaacc acccccatca tcaccaagaa caacagtatc 660 aacaagaaga acagggacaa caaaacccac ggatgaaaca ttcctttctc agctcagatc 720 ttatctggtg cgttctctct ctgctctgtc ttggtgtgtg gtttagagaa acatggacaa 780 cgctgtttgg aagaacaggt gagcgagggt ggggaatttc agaggcctgg gcccaccgcc 840 tccacccctt ccccagttta acctttgaca ggatcttcac ctctctctga tcagcattgc 900 ttcttgttca aaggcctcag ccacccagct gtgtcccttt ccccagaaag caagggcaga 960 tggcagtggg tctgttgatg agagaacttt aagggcccaa tcagtccctg ggcaccccct 1020 cctgggctcg ttttctccag gaggctgcat tctgatccat aaaccttctc ctcggggttt 1080 agggtcgagc tgttcctgat gtttatcgga gactgggatc aaagctatcc aggtcataaa 1140 tctctctctg tggctgttgg gccccagggc agctgaagag ggttgacagc cctttggacc 1200 tcaaaggaaa aaatgtgctc tactccaccc actcccagct ctgccaagaa gctgtcctct 1260 gagaagccat ggctgggccg ttccattctg gggagctgct gaaaagagct gggaggccga 1320 gaagaacttg cgtgtgctgg gggagaggaa gcctggcctt gagggagggg tgcaggtgtg 1380 gctcctgtgt gtgtgggggc tgggggacct tgtgtgcctt ttccttgtgg ctgtgaaatg 1440 ctttatgagt acttccatag gaggatggac agggagtcgg ggagataaac tcagccacaa 1500 ggccccaggg cctcaggaaa cttgcaccca accctctcat tttacagaag aaaactgtgc 1560 ctggaaggtt gaagggtttg ttcccagtca cacaaccagg gatccttagg acagccagac 1620 caggaaacca tttccaaact gccaagccat ggcagagtat caagacctca ggaaccatcg 1680 agacaccatg gaagcattgg gaaaagcctc cttagctttt gaagctcctc attgttcttg 1740 agtgtgcatg gagcccatga ctgcggggtt ttgtagacac ctcagggatt acatgactgg 1800 tacccctgac aaagtcaagg ctgctggaca aaatgagtcc gaggatttca ggggcacgct 1860 gggcgcagga gctggtgggc tgttgggagt gcccctttac tgggcaggct tccttcctcc 1920 tggtgatggg gggttcctca gcacaaaagt gaaggggtgg aggggctgga ggagcaggaa 1980 tctctcttgt tgataggtat gaggccttga agtccttttc tttgtcccag gattcatgga 2040 cgcttcgggg ctgatctttg agttttcaag catggggtgc agagacgttt aggtaaactc 2100 ttaccgtcct ctctcttcgt cagggcttcc caggaatcaa caatgcccaa gaaggaaggg 2160 attgtagaaa tagcttaacc ctttcattta ccaacgtgga aattgaagcc cagggaaggg 2220 aagggaccgg tcgtggaagg gagagccatc agcagaaaga gaccctgaga tcttcgcctg 2280 ggattcccag gaagtccagc ccgagctgat tcacagaaca aatgcatgca aaccttgcta 2340 tcaataaatt acacatgcac ttacgtaaaa cacat 2375 6 410 DNA Homo sapiens misc_feature (34)..(34) n=a, c, g or t 6 cagagtcaag gccccaaggc cgtgggtctt tganggaggg gtttttgaga catgtncagg 60 gacaaaccta gcaacaagag aactcttaat cccatacgtg atattgcnaa ttagcttttc 120 ctttcacaaa tattgtccac cctaagtatg tttactataa tgttagctgt taaagacccc 180 tcctaccccc aaaccattta cccttcaata aaaatggtgc caagttgcaa gggttagaca 240 ggtatgtatt gaaatttaga aagtttgaat aatttcttta acacaaaagc atttttttct 300 tatttctcat acttttgaat ctatttaaat acaacttcag tgctgattaa tctactaaat 360 gtgaaagttt aagatttata gctgggtgca gtggctacac ctgtaatcct 410 7 416 DNA Homo sapiens misc_feature (114)..(114) n=a, c, g or t 7 ctcgagcaga gtcaaggccc caaggccgtg ggtctttgaa ggaggggttt ttgagacatg 60 tacagggaca aacctagcaa caagagaact cttaatccca tacgtgatat tgcnaattag 120 cttttccttt cacaaatatt gtccacccta agtatgttta ctataatgtt agctgttaaa 180 gacccctcct acccccaaac catttaccct tcaataaaaa tggtgccaag ttgcaagggt 240 tagacaggta tgtattgaaa tttagaaagt ttgaataatt tctttaacac aaaagcattt 300 ttttcttatt tctcatactt ttgaatctat ttaaatacaa cttcagtgct gattaatcta 360 ctaaatgtga aagtttaaga tttatagctg ggtgcagtgg ctacacctgt aatcct 416 8 786 DNA Homo sapiens 8 atgttcctag tagaacacaa agtttgctca ggtaacacac aagtaagcat taaatgcctt 60 cctgttgtat ctgagaagtt tgttatgaaa tattttggta accgctgcat agtcagtgta 120 ggaggagcag atgaatttta gctgtggtta tgtgtgctgt aaaagactat acgtgcttgt 180 attagtcaga atgagtacac cactaatttt tgtatggtaa gagatttata ctaagctcat 240 catcagtttc tataattcag tgagataaaa ctgagtcaga ttgattttta ggtagcacat 300 gtagaaacag ctaattttat tcccctgatt tgatcctcat ctattgatta tataaactaa 360 agaagctaag aacaattaac ccttacgagg ttacacagtc aggagatgct gaactgagat 420 tcagtgtaga aagtctgtct tcagagccta tgcttttagt ctttatgcta agtttaactt 480 gtttaaatag caagattatg aagcactata cagtgacctc gtatagacaa aaatatagta 540 tattgattat tagagaaact acatattaga ctgttgtaca tacgtgggca agtatttgtt 600 aaatcatttc agttgcctaa atttaagcaa ctgtgctgtt taaaacatgc tcattcacat 660 tttttcttaa tctagaaagt cacttctgaa taattgcttg tttagatttt ctcatttggt 720 gtgggaaatt tatattaaaa ttttaactaa tattctaaca atacagagtc tgaacctaaa 780 gtccac 786 9 1509 DNA Homo sapiens 9 atcagaccta gtgcgtaggc ttctggatct cagaatcact tatacttaag tccaggctgt 60 tctcaaataa ggcaagaagc atctgctgtt aatagctgac agtaaattac acaaagtaaa 120 acatggaaaa ttaaagtcag aaaagctagg aagcttttct atcattttca attttctgca 180 aaaatacaga cataatcagg tttaggatct gcttgtgatg gataaattac atctgtaatt 240 ccttcttttc catattactg cattcagacg ataatttgct ttcagatatc ttgctcatct 300 aatcgttcat agactggaaa taagtagtaa catctcccaa tcctaggaag catttataac 360 tagtctttgc ctttttgggt gttgatagac tagtggtgat tataagcttt cgagcttctg 420 aaaagcacaa cgaagattaa aataatcata ggataataaa atactttaaa acccttctag 480 tctttaattt taaaatgttc cagtagaaca caaatttgct caggtaacac acaagtaagc 540 attaaatgcc ttcctgtgta tctgagaagt ttgttatgaa atattttgga aaccgctgca 600 tagtcagtgt aggaggagca gatgaatttt agctgtggtt atgtgtgctg taaaagacta 660 tacgtgcttg tattagtcag aatgagtaca ccactaattt ttgtatggta agagatttat 720 actaagctca tcatcagttt ctataattca gtgagataaa actgagtcag attgattttt 780 aggtagcaca tgtagaaaca gctaatttta ttcccctgat ttgatcctca tctattgatt 840 atataaacta aagaagctaa gaacaattaa cccttacgag gttacacagt caggagatgc 900 tgaactgaga ttcagtgtag aaagtctgtc ttcagagcct atgcttttag tctttatgct 960 aagtttaact tgtttaaata gcaagattat gaagcactat acagtgacct cgtatagaca 1020 aaaatatagt atattgatta ttagagaaac tacatattag actgttgtac atacgtgggc 1080 aagtatttgt taaatcattt cagttgccta aatttaagca actgtgctgt ttaaaacatg 1140 ctcattcaca ttttttctta atctagaaag tcacttctga ataattgctt gtttagattt 1200 tctcatttgg tgtgggaaat ttatattaaa attttaacta atattctaac aatacagagt 1260 ctgaacctaa agtccagaag aattttaagt catgccgcag acaggatgaa cagtatagca 1320 aatcagaata atagactgtg agggggggta ggggggaacc catgagaatt tcaggatgtc 1380 aagataaagc ttggaattga ggtaaaggca tcagataagg aagtgatcat ttcataactt 1440 gtttttgctt gaaatatatt atattttaca tcacaaaagt agtataactg ttattttgct 1500 aatgcacag 1509 10 283 DNA Homo sapiens 10 ctaagtaatc cttgtcaggg gaggtggttc ccaattcgtg actcttggac cttggggcat 60 cttatgattt attgttatca ctaacaatag ctcgctatgt gtcatgtctt ctgctacata 120 ttttatgttt tatttcagct tttaaaaaga ttttcatgat tcatgattgt tgtaaagcag 180 gactaggctg tatgtacata tttgaaatga aagtttcaca aaacatcatt tacctttact 240 atgtgtgaca cactttgcta tttttcattt aatctatttt att 283 11 736 DNA Homo sapiens 11 gtctttctga aaggaagcac tcggaatcct tccgaacttt ccaagtccat ccatgattca 60 gagatactgc cttctctctc tctgggattt tatgtgtttc tgatagtgaa ttgttgatgt 120 atttgctact ttgcttcttt tctctttcaa gacttgatca ttttatatgc tgtttggaga 180 aaaaaagaac ttttgttagc aaggaggttt cagaaatgat tttggatttt ctgtaagtgt 240 ttaatttagt tctaggggac agcatctctc atcccggagt aaatttctgc ctttgacctg 300 catggattat tttttcaggc tgcggaattt ctcggcacct acctgtagta tggggcactt 360 ggtttggttg cagagtaaga aggtggaaga atgagctgta cttggttaag cagttgaaac 420 cttttttgag caggatctgt aaaagcataa ttgaatttgt ttcacccccg tggattccag 480 tgggcccgac agcgcaacag gtttgcagat ttcttttgaa attccttttt cccccctccc 540 tctgcctcag caaaagaaaa gaatccatat aacaggttca tgttcaattg cttggctttt 600 cagcacttat tctgaagact ttataatatt tttaaacttg accttggaac acagagggct 660 ttgtgggtga ggtgtattta tatttactta agggtgcaca ttttaaaaat cttattctgt 720 gtttgtacaa agacgc 736 12 547 DNA Homo sapiens 12 ccggttagaa tagagcttcc acaagctcct actttgatat ctgccctcct agcactgggg 60 ccactgtttc ctgctttccc tctatgtgaa ctctccgtgt ttctaatatc atctggatta 120 atcacatcct ctctggccta ctcaaagata gtaactctaa caacttttcc ctctctttca 180 tgcaattcct actttgcctc tctctgctgg actttttctc atcgacatat aaacatgctg 240 ttatgtctcc caaccaaaaa aaatgcaaaa accctttcag ccctatgctc acccatcatc 300 cagctgtagt cctcttcctt ccttttactc tcctttatta tagctaaatt tcttgaaagg 360 atggaatgtc cacttcctct cctcccatcc tttcctgaac ctaccccaat ctgccttttg 420 tccccactgt gccagtgaga gggctcttga taagctctcc cttcattgac ttccagttgc 480 tcaatgaaat gggcagttct cagtcctcat cttacttgac tttccagcag catttagtac 540 taccaga 547 13 1559 DNA Homo sapiens misc_feature (1337)..(1337) n=a, c, g or t 13 gttctacgct taaaacaacc tcttccccct aactttaaaa tcagatacag taaaagcctc 60 ttgttgagga tgtggttatc ttggtagatg agagtgtgtc agaaacaggt agaaacttac 120 ctagcaaaag aactagtact gtatcttgac ttgttacatg gcaacaatca attagatgat 180 aatttctatt taaaagcatt ctatatgggg aaagacatgt tcattttgat aagtaaagac 240 aaaatctagg tttttagttg atgtgtgttg tacatgtggt ctttggaaag caaacctaac 300 tatgtattat tgacattaaa aatgatgact taatgctggg taaatcctgt actcagaaga 360 tactcactga tgatccattc ctggctataa cctatgaact aaacgaattt tttaatcttg 420 gtgcttatta ttagcttcag cttgcctctc taataatccc aacaccttgt gctctcatcc 480 tgctctcagc ttattacttt gccccgtttt tcactgagaa gacagaagca gttagaatag 540 agcttccaca agctcctact ttgatatctg ccctcctagc actggggcca ctgtttcctg 600 ctttccctct atgtgaactc tccgtgtttc taatatcatc tggattaatc acatcctctc 660 tggcctactc aaagatagta actctaacaa cttttccctc tctttcatgc aattcctact 720 ttgcctctct ctgctggact ttttctcatc gacatataaa catgctgtta tgtctcccaa 780 ccaaaaaaaa tgcaaaaacc ctttcagccc tatgctcacc catcatccag ctgtagtcct 840 cttccttcct tttactctcc tttattatag ctaaatttct tgaaaggatg gaatgtccac 900 ttcctctcct cccatccttt cctgaaccta ccccaatctg ccttttgtcc ccactgtgcc 960 agtgagaggg ctcttgataa gctctccctt cattgacttc cagttgctca atgaaatggg 1020 cagttctcag tcctcatctt acttgacttt ccagcagcat ttagtactac cagccagtcc 1080 tcatccttga aatactttct tttcccatat ctctaactgc ttaagtcaaa agggttccat 1140 gatccagtcc ttacataact taccttcttt ggctacgctc attatctggg atctcatcca 1200 gtcttggggc tttaaatact atatggggac aactacagcc gagaaccttt ccctgaactt 1260 tagactcttt tgtccagaag attatacaaa ttctctgttt ggttatagaa tttagaatgc 1320 cccaaatcaa gataatnctc cctcaattct gttcctccta taagcttccc caatcggtaa 1380 atgaaaactg tgtccttcta gttaatcata ccaaaatcct aaaaatcatc cttaactcct 1440 ctcatctctg atatccatat ccaacccatg agcaaatact gtcaatctgc cagaatccaa 1500 acatctctcc agccccattg ccaccaccct ggtccaagcc accaccaggc cttgcctag 1559 14 1455 DNA Homo sapiens 14 ggagtgtgaa ggtggtgagt catgggagtt ccaagggaat gggtgataaa gggaggtctc 60 aaatgaggca caagtggaga aggtagcttg ggaaaggaga aggatgcttc tccttataag 120 atgggaaagg cagaggaaga gggtcaagat acagtgatct aggggtgata tggaagtgag 180 ttgagagaac tcaactctgg gttctgaaac ccctaggttt ggggggcttt gagataggga 240 agaggtttaa agtcagttgt tctagcaaat atggtttgga atttatttgt gatgcttaaa 300 aatattgctg aagagaagtg aagtctatcc tagagttgga tggtgagatt atttagtgga 360 actaccagat ccatgttgtg attctttcca gtatcattca gcagcccttg ggcagttgcg 420 aggcaagtca tcagtggtgt atggagattt tcccaggtgg gtgtggttga aggcagggaa 480 gaacgagttc aggagcacat tacaagaaga aggtgactgt aaggtccagg ctgagcagga 540 aggtaaagca agaaggaaac atgaggttgt gaagagaagt ttagagggat gaggaggcag 600 gagagatgaa cagttgcagg atgtagctag agtggcgatg ttagatcttg gggccagaga 660 tctttacaat gattatgaag atcaaagggc attagaatca agctataaag agccactgtt 720 tgatgttggg atgtgaggat gctgcaggtg gatgtctgca cattgatggt gagaacatgg 780 tcatcctggc cctgctgggt ctttgctaaa gagactgtgc tctgttcttg gggccgtttt 840 catcatctga ttagagcagt ggtccccaca tggtgttctt tggaccatct gtataaaatg 900 ttcataggtc aaggataaaa tggaaaaaca gagaaaatgt cacagaaatg tgcccattgt 960 tgaaagacca ccagctgtcc tttttggagg attgttcttt attctaaaaa tgtatatatt 1020 ctattctatt aaaacatttt tgtattggca tttttttctc ttttatgaaa tgccatgggg 1080 tagaaatttg taatgtatcc aattctcctg tcttcatgta ttgccctgtg gtgggggagg 1140 ggatgtggct agtactggcc aagaggctgg gggcagaggt gcaatgttag acttctagcc 1200 tggagcattt aattcttagt acaagactct ctaacattct tctccctctg ttccctgctt 1260 ggtgatactc gaggtattgc aacccccatt aaccttagtc ttagggcaag tttgatggga 1320 aacagagcac cccacacctc cctgcagatg aagcatgagt gagaaaaaca acttctgatg 1380 tttgaagtta ccaagatttg ggagttgttt gttattgcag caaaacctca cctattctga 1440 ccaatcatgg tggaa 1455 15 904 DNA Homo sapiens misc_feature (281)..(281) n=a, c, g or t 15 tggaaaacca aaaaattgat atgctaaagt atactcttaa aggtcttaat actttaaaag 60 tatatagatc tcatgaacat aattcatttg aggaaaaaaa tacaaatcat ttcttgtccc 120 aggaaaacag taaatcttta atggaacttt ttagcaatta tgacaaaaag aatggaaaaa 180 tgtttaaaca tatataaaag gctagacgtt tatcgccaaa tagtatctaa aggtcataga 240 atagttagga attctgtcat tttgttttgt gtaataaata nccccttcct ttaccctttc 300 accctaataa tagatatcca ccattttgnt gtgattatcc aactatagag tacctttttc 360 aagaactcat tatataccaa agtaggagct tgctgacact gataatgctt tatttagttt 420 tgtagtgaca tacaattacc atttgcttag gaaaaaaaat aaagaacana nacaagtaaa 480 ttttttaaaa ctatggttgt gtatatataa gttgataaaa atcctttggg agaaaactnn 540 tgtcttgtgt gttaagagca ttaaatagtc atacccctta gcctagtgtg tcttctatcc 600 tgaaaaaaaa ttaacaaagc aaatactaac ttaagaaaaa aaactacagc actgaaaaga 660 tntgttgtaa tattgtttat gctaacataa annatgtaaa nnnttatata ttgtttatac 720 tgacttataa tttattacta tacatagtgt aaattatgat acattggctt tggtangcag 780 ttttntaacc gctaataata taaataccat actattaaca atctagaaaa atgattctgg 840 tataggttat gtgaaaaggc acaaaataaa attgtatata gtacactagc aatgaacagt 900 ctga 904 16 984 DNA Homo sapiens 16 acagatttac tctcctgaat tttccagaaa tgtagatact tttaaatcaa aggaaggctg 60 tattttgttt tgttcagaac ttttctattc cagaaaatca tgtcaattga cagcaaagcc 120 acttgtggtc attgagcctc ctgtgtaaag caccgacgtc attctgtagt tgtcatcact 180 gtattcaggg tgattctaca cgtaggagtg agcatttgac agcttccatg tcttctagtg 240 cggctgagaa tttacatatt aagatacaca ttatttatta tcaattactt tcctgtttca 300 atgtccattt agagcactaa aaatatcttt gtaggtagtt gatattactt atgaatttta 360 tttcaggaga gcaaaggaaa atacaagata gttgtatgaa aagggggcac cgggtgtgct 420 agagtggctc accaccgccc tacacagtgg gctaattggc tggagagtag agctgactct 480 gcacagttgc atgctgaccc tctgaagaat ttttttacaa aagcgtgacg tcgcgtgaag 540 accttgacag aattagcaaa gcggttgaga tgcatacttt ggagtcagac agactccagt 600 tcacatcttg gcttttatac ttacagctgt ataaccgtag acaatctatc taccctctgg 660 ccgactccat ttcctcaatt ataagatagg ataacttgtg aaatgctttc cacaagatta 720 ctattgcatt tattctcctc accactctta atgaagagag tcttgtaaca gataactcta 780 attgtcttca gagttcaggt ccccaagaaa gattatgcct tctaaaagct agtctgtttc 840 cttccagtgg gagccatttc attcatgctg ctctactctt tacttggact gctagcaaac 900 atggagctaa gtactcatgc ttaatttctg tggctttcct caaatagggt ttcaatacta 960 tagtttgccc tcactccatt ccct 984 17 429 DNA Homo sapiens 17 cgtgataaaa atagtttgct ctgagttttt gcctttctgg aatttaatag caagaaaaat 60 atgttcccta ccctctcagc ccccactcta cctccctgtg gcttgttaag ccttccttct 120 gcctcctgca tcaacttcct gatggagagt gtatgaatgc aaaagctcct cccttagcac 180 ttacctagtg cttcactctc tgggctcctg ccactgggtc ccagctaaga gagtttgatt 240 ttaaaatcca gagtttatgg ctttttaaaa ataacctctc acctatttat caaaagctcc 300 ttctaaataa tatttacaac aacaacaatg ataatggcta ctatctagta tttcccattt 360 tccagacact gtgctgggct ctttccaaac actgttttaa tctttaccaa cacccagtcc 420 gccgctcta 429 18 734 DNA Homo sapiens 18 cttttggacc ataagcctca ggaagctata aggattattt gcattcttac acctgggcac 60 tcttcctttt tgctgaatac cagtttttca atcttttcta tttttgaaat aggtaagaaa 120 agaaaataat tttctagaat ttgaagaaaa atcttaaaac atttgaaatt ctttgttatg 180 atgactaata taacgaatag cactcaggtt tatcaaatat taacattttt ccatatttgt 240 tatagaattt ttttccatat ttgctacaga aataatttct ttatatatat aatacatatt 300 tgaacactga ttttacttga tacattaata taatgctgat gtgctgagat gaataaatca 360 aagaacctct tggagctctt ggtgtgcaat aagcatagtt aacgaatata aaataagtga 420 tattttctag aaaataaata ctggtctaca atgccttatc tgtcatttca aagtctctaa 480 aaagatctga aaatccaatg ccttttaaaa ataaaattac ggtaatctca tttggccaca 540 aaacctgttc agaattgatg tgaggctatt aagatattta tttctcttat ttattagtga 600 atattcatct ttcactacag aaatactaac gagtttgatt acagggtgct ttagacttcc 660 ctcaaggtgt acatatttgc tacttttctc taaaatccca aacatcctgg attctgaaac 720 acatctaaac cccc 734 19 1184 DNA Homo sapiens 19 attctaactc tgtgacatgc agtctgtgac actgagagtt acttgcacct tcctctggac 60 tggagatcct ttctagtgca gacattttat aattctattc tgtatcgtgt tcatttaagt 120 agtctgcttt atcattacat taacatttat gaaagacttg ctggtatcat tggcttagcg 180 attatttttc catctagatg ctttttttaa agaaatgaag agaatatgta atgttttaaa 240 tgtacatttt agtttgattt aaattttaat caaggatttt tattttatac attacatact 300 gatcactgtt ttatgttaac tctggtccta ataaacagaa aataacaatt tggaatatct 360 acaacaatga gagctcgagg taaaatatag cataaataag acatatatgt gtatgaactg 420 agatatatag aaataattaa atgtaacaat cttttggacc ataagcctca ggaagctata 480 aggattattt gcattcttac acctgggcac tcttcctttt tgctgaatac cagtttttca 540 atcttttcta tttttgaaat aggtaagaaa agaaaataat tttctagaat ttgaagaaaa 600 atcttaaaac atttgaaatt ctttgttatg atgactaata taacgaatag cactcaggtt 660 tatcaaatat taacattttt ccatatttgt tatagaattt ttttccatat ttgctacaga 720 aataatttct ttatatatat aatacatatt tgaacactga ttttacttga tacattaata 780 taatgctgat gtgctgagat gaataaatca aagaacctct tggagctctt ggtgtgcaat 840 aagcatagtt aacgaatata aaataagtga tattttctag aaaataaata ctggtctaca 900 atgccttatc tgtcatttca aagtctctaa aaagatctga aaatccaatg ccttttaaaa 960 ataaaattac ggtaatctca tttggccaca aaacctgttc agaattgatg tgaggctatt 1020 aagatattta tttctcttat ttattagtga atattcatct ttcactacag aaatactaac 1080 gagtttgatt acagggtgct ttagacttcc ctcaaggtgt acatatttgc tacttttctc 1140 taaaatccca aacatcctgg attctgaaac acatctaaac cccc 1184 20 550 DNA Homo sapiens 20 ctttcccgct cccggcccca gtgccttgca tgcagcaagg tcttggcatg tgcaagcttc 60 cttaaggagc ctgcagcttt gctccaaagc acacactggc agaccttggc cagatgcctg 120 gcacaggggc tggggaggga aaggctgccc aacccccgtt ttccctttgc agatgagcat 180 tctccaaatc catgtttacc cagtcctcct taatgctgcc ttccaaactg tcagcgggtg 240 ctaaaaagca cacattagga tgaattagaa catgccaggc tgcaagggcg ggtgtcatcc 300 cagaactcac agagcacgtt gagggctcag ccgctcagcc acatctttag gtcccaccag 360 catctccccc caggcatgga cctccccaat ttaccctgtg aaggctgcat ggagaagatg 420 caggtcttag gaacagccag catcaccaga ggtgccactt agtgagtacc cagtgggctc 480 ccaacaccgt gctgagctcc cagtgggaga accggaaccg tctgcctgtt ctctgttgta 540 ttccagcatc 550 21 599 DNA Homo sapiens 21 tactatgtgc cagacacagg agttttcagg atgagtcaat aagataataa acacaaagtc 60 ccggccccag tgccttgcat gcagcaaggt cttggcatgt gcaagcttcc ttaaggagcc 120 tgcagctttg ctccaaagca cacactggca gaccttggcc agatgcctgg cacaggggct 180 ggggagggaa aggctgccca acccccgttt tccctttgca gatgagcatt ctccaaatcc 240 atgtttaccc agtcctcctt aatgctgcct tccaaactgt cagcgggtgc taaaaagcac 300 acattaggat gaattagaac atgccaggct gcaagggcgg gtgtcatccc agaactcaca 360 gagcacgttg agggctcagc cgctcagcca catctttagg tcccaccagc atctcccccc 420 aggcatggac ctccccaatt taccctgtga aggctgcatg gagaagatgc aggtcttagg 480 aacagccagc atcaccagag gtgccactta gtgagtaccc agtgggctcc caacaccgtg 540 ctgagctccc agtgggagaa ccggaaccgt ctgcctgttc tctgttgtat tccagcatc 599 22 618 DNA Homo sapiens 22 gaaaaactac tctttttggt gtaaagatat tttttatatt ttctttgctt gtaaagagtt 60 attatcaatt tgtaagtata aaaactgcaa gtatagttgg tagttgataa gaaaggtaga 120 taataaaact taaaagggat ggacacagat tgaaaaaggc cttgagtgcc aagacaagag 180 ctctgaactt taacaggcac tggaaaccgt cataggtctt aggtaggaat atgctgtgct 240 cccaccatct taattaggtc ttatggaggt ttgatagcaa gagggtagga atatcattta 300 gcaggctact gcaagtatcc aggtgaaatg tacagaggtt ttgaactagg ctgctgggga 360 gggtgcagag aagaaatatt ttggaaataa aatggacaga aagtgtataa atggataaag 420 agaggaatag aactgacacc aggcttcaag cctgatgcct gagaataaag gtgtaattat 480 gaagggaatc caggaagaca tggaaagagt ggttggagta aggttaaagt gatagtttta 540 gattgggtta ttttgacgtt gaagtgttga ccaacttctt aagtgaaaat gtgcaacagt 600 cattgaaaat atgagttt 618 23 711 DNA Homo sapiens 23 gaaaaataag tttttgttaa tggttgggat tttcttactg gcctcgtggc aagttttgtt 60 atctcttatt atatatattc taccttttta tgggaaaaac tactcttttt ggtgtaaaga 120 tattttttat attttctttg cttgtaaaga gttattatca atttgtaagt ataaaaactg 180 caagtatagt tggtagttga taagaaaggt agataataaa acttaaaagg gatggacaca 240 gattgaaaaa ggccttgagt gccaagacaa gagctctgaa ctttaacagg cactggaaac 300 cgtcataggt cttaggtagg aatatgctgt gctcccacca tcttaattag gtcttatgga 360 ggtttgatag caagagggta ggaatatcat ttagcaggct actgcaagta tccaggtgaa 420 atgtacagag gttttgaact aggctgctgg ggagggtgca gagaagaaat attttggaaa 480 taaaatggac agaaagtgta taaatggata aagagaggaa tagaactgac accaggcttc 540 aagcctgatg cctgagaata aaggtgtaat tatgaaggga atccaggaag acatggaaag 600 agtggttgga gtaaggttaa agtgatagtt ttagattggg ttattttgac gttgaagtgt 660 tgaccaactt cttaagtgaa aatgtgcaac agtcattgaa aatatgagtt t 711 24 547 DNA Homo sapiens 24 aacaaggtaa gcatagccgg ttttcatggg cttattttct catggaaatg attctgtgta 60 gaattgatta ttcatgaaga cacaatgtaa catcaagttt gggttaatgt tcctcagtgc 120 aacaacaaag acgtatttgt aatcactccc atgagtctac tttgcagcaa gaacatgcat 180 tttggaatta ttcccatcct gtgtgctgaa tactggatgt gactcttagt cagctctgtg 240 acccttgtca agtaacttaa gctctttgat catcagcttt gtcatctgta aaatgggcat 300 tctgcctact tcaaagagaa gttgaaggga ttaaacgaga taacctacaa agagcaccca 360 gcacaatggc ctaaaaaagg aaggcactga atcattctca ctcccctacc ttcagtctga 420 tcctgctctt attgtcaaaa ggataatttc aattttaata gatctgagat cctgtttttt 480 aataataatt ttatagaatt tttcatttta tggcaggcac agggctcatg cctgtaatcc 540 cagcact 547 25 549 DNA Homo sapiens 25 gcaaagacct catgaggggt caacgagggg aagccctcgt gggtcagagt acgccacggg 60 acagactatg ctggcagctt ctagatcgtt gaactctgtt cttgaagact gggcagaatc 120 taggaagaac ggaggcacct gagttcacca ggtgggacga acctggcctt agcacggaat 180 gtggcattta ggtgcttaag tttgttgttt tttttaaatt aaagtggttg acctggagag 240 ctggtgtgga aatgtagcag gaggtctatt tggaaagaag gatggagtag attatgaaag 300 ttcttaaata tcataatgag gcttgtggat tttattctgt ggtttggatg ctctcttctt 360 ccatcccttg gatgccaaca ggcatgcact gtttaatctt ggaattcaaa cggtggcctc 420 aaacagtgag gctgagtatg tggcctcatt agcttcagac ccagcagggc tgggctcaca 480 ggcgtgtcat ttatcaaggg cttgaatctc tgccagctaa tttatctaag acaactctat 540 gagatgggg 549 26 350 DNA Homo sapiens 26 ctttaagata gatgggtaca catattatga atatactttc cttttgccag accttgacat 60 tctgtagact tttaatggaa tattatttgc ctctttcatc ttaccttgac gtatgaggtg 120 gatggcttac gtgcagggta atgtatgaac cttcccaagc tctgtacaaa tataacttgt 180 cattcgtaga gacgtatgta tttatatgtg tgcatgcagt cttatttgta gattttcttc 240 ccatttgctt aatactgaac gctatggcct agatgtgaaa tttaccaggt actactcata 300 gcaggcagtg aaaccgtgga ctcagctgct ctttccttct ttcctcccca 350 27 627 DNA Homo sapiens 27 ccacgcgtcc ggtttcaaaa aagaagagta agtcaaaggt taaacttttg gggcggagga 60 aaaaggataa gaaagaggat acagagttta atcagagttg gcatcagata gagtaaccat 120 ggacatttgg aagctgtaac ctctctcata tttcgccaag gataactgct tcctgtatta 180 tcatgtaatg agttttatgc gtgatggaaa atgtaaaagt aatcttaacc caaacctgca 240 ttttaatgcc acatggaccg gctgtaattt atggcatctt taagatagat gggtacacat 300 attatgaata tactttcctt ttgccagacc ttgacattct gtagactttt aatggaatat 360 tatttgcctc tttcatctta ccttgacgta tgaggtggat ggcttacgtg cagggtaatg 420 tatgaacctt cccaagctct gtacaaatat aacttgtcat tcgtagagac gtatgtattt 480 atatgtgtgc atgcagtctt atttgtagat tttcttccca tttgcttaat actgaacgct 540 atggcctaga tgtgaaattt accaggtact actcatagca ggcagtgaaa ccgtggactc 600 agctgctctt tccttctttc ctcccca 627 28 548 DNA Homo sapiens misc_feature (133)..(348) n=a, c, g or t 28 gttgcatgtg ttggggatat ttctccatta gcaagaagtt tccaaacctt accagtgttt 60 tgatgaatct aggaacagat ctggcagtga gacctacatc cattttcccc acggacagca 120 tcttgctgga gtnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnntc ctcctgttaa 360 atgttgatag aaaaatggtt tgatggcagc atatatccag attgtagatt tcataatatt 420 aaaggggagt gggcaaataa taaaatgcaa gaaatgaaag catttgaaaa tttagaggac 480 agaaatgact tttaagtaag tgattttagg tgtactggaa tgagtaatct agaatatttg 540 atatgaga 548 29 988 DNA Homo sapiens 29 aaatccacaa ataataattt acatttgaga aaatccccca gtacttctat gaataagatc 60 aagggcaaaa gtgtgctctt ttacatgcca gaaacctcaa gaatttttcg taaggtacag 120 ttcaaggaaa accaagcagc tcttgactca acaaataaaa atgtaagtct gtctgaagaa 180 ttagtgaacc agggcaccca gtcagctttc tcctaaaata aatttggaga gctgaaagat 240 atggatgagg tcagatttct aaaaaatcag tatacacaca gtgttttaag aataaaaaac 300 agattgatta aagggaaaaa taatttgtaa ataacagaag ccataactta gagataaaaa 360 taactgtcct ctgattaaca gaacttttag aatgatgaga aaaattaata acacagttaa 420 agatatcaca gtgattttta aaaatatttc aaggttgaag aaaaaatatt cctatgagaa 480 tacaggctga aaaagatcaa agtaaaatga atcaggtcgg tatcagaaat ttcagtgata 540 tacaatgaag gaataaaatg gagcagcagc tatagttttg aaacaaaatg tattttccaa 600 ggttcttgta cccaaccaaa ttataactta tgtgttagga caatagagaa gtaattttag 660 ccaaagaaat aatctgaaat tatagcatct atgcacattt attgaaacaa gaaactcaga 720 aatcaaaata gccgagaaat taataaaata ttcaaaagga ggaaaataca ttttagaata 780 aagcataatg aggaataaaa tcactatgac tttttgaaag tataaaaatt gttatttttt 840 tctatgaata cttgctcaaa tttaaagtag tggatttaat gttgtagcgc taagtattca 900 gccaagaggt agaactaata aataaaaatg atagttcttt taaaaaaaca taaaaataat 960 tatctcatga gtagcctaag aaaaaagc 988 30 651 DNA Homo sapiens 30 acaccaaata aggtaatgga gataaacttt agaaatcatg tttttaaact gatgtttaaa 60 agggatggaa ctcacactat ttaaaaggtg aagactgcca cgtcagtgtg aaattgttta 120 aaaaagtcca acacatttgg ggctggacac accagtcaaa tggttgaaat tagaagatgg 180 ggaaaaaata tgtcaggtaa atactttatt tcattggatt tatgacttcc cctgtaagaa 240 gcattattat tttatataaa tacccaaaaa aaaaaaacaa caaaggcagc taaattctga 300 aattaattgc atatgcatca tgatttcaga tatattaaac tgtgaaaaaa gtgcgttaaa 360 atggtaaagc acaataatca aaataaagtt tgtatagcaa tattaatatc acataaaata 420 taaattagaa caaaaaagca cttataggga taaagagaaa caccagagaa aaacaaagaa 480 aaaatcctaa gaaaatataa ccttcacata cttatatggt ttaacagcaa agcccgtgaa 540 ctgtttaata taggaagcac aaacgtgact gaagttacaa gagactgaga caactttcaa 600 aactcatggg gggagaattt tatcacttca acagaaactt aacaatttaa c 651 31 553 DNA Homo sapiens 31 actggacttc ctctttcttc catcaaagac taagatgcct tttttccttg atgtacttta 60 ttttgtggag catattatct actttcctga aaaaatggtt tatgggagat aaatcataaa 120 aaggttttat tagattctac atctcatgat tgatccaaaa gacgttttaa aaacaaaaca 180 aaaaaaggcc ttgtaggtct taactcttac ttagcctcac atttatttga tagtttgagt 240 gagtatctta aaaattgaag atgattataa aaattttaat gtagacatta ttttttctca 300 gaattttgaa ggcactgctc tgtcttttgc agttggagag tctgatgcca ttctgattct 360 taaatctttt atacaaaaca tgtttttgct tttggcagga agctttacct tttctttctt 420 tcaagtgtcc tgaaacttca ctgagatgta tcatggtata ggtccacttt gatccactgt 480 cctggacact tgctaggcct tttcagtctc gaagctcatg actttcaggt aagagaaatt 540 tacgtctaag acc 553 32 2159 DNA Homo sapiens 32 ggccgcttaa ttaaagatct tttttttttt ttttttttag tgctgaataa tagtccattg 60 tctttatgta ccacagttta tccactcacc tactgaagga catcttagtt gcttcaatgt 120 tttggaggtt acagataatg ctactataaa catccatgtg caggtttttg tgtgaatgta 180 aagtttccaa ttcatttgag taaataccaa agcatgcaat tgctacatca tataaaagta 240 tgtttggtac tataagaaac tgccaaactg tcctcttaag tggctatgca tattttcact 300 tccaccagca ataaatggag ttcctgttgc tccacatgct cactagcatt tggtgttgtc 360 agtgttctgg attttggtca ttctaataag tacatagtca tatctcgttg ttttaattta 420 caattcccta atgacatatg atgttgaaca tcttctcata tgcttatttg ccatctgtat 480 atctactttg gtgaggtatc tgttcagatc ttttgccttt tttttttctt tgagacagag 540 tctcactctt gtcacccagg ctggagtgca gtggcacgat ctcagctcac tgcaacctct 600 gcctgctggg ttcaagcaat tcttctgcct cagcctccca agtagctggg attacaggca 660 cccaccacca cgcccaggta atttttatat ttttcataga gatggggttt cgccatattg 720 gccaggctgg tctcaaactc ctgacctcag gtgatccacc tgcctcagcc tccgaaagtg 780 ctgggattac aggcgtgaga caccacaccc ggctcttgtg cacgtaattc tattttattt 840 gagatggagt cttgctctgt tgcccaggct ggagtgcagt ggcatgatct cggctcaccg 900 caacctccgc ctctcaggct caagagattc ttgtgcctca gccttccagg tagctgggac 960 tgtgcaccac catgctgggc taatgtttgt atttttagta gagttggggt ttcacttagc 1020 caggctggtc ccgaacttct ggccycaaaa gatctgcccg cctcggcttc tcaaagtgcc 1080 ttggattccc aaagtgctgg gattacaggt gtgaaccatc atgactggca aagcatatgc 1140 ttttgaggcc cattgtcttt cctaatttgt tgaatacata ctacatgagt atcttcaaac 1200 actgagcaac tacgaaattt tttgtgaaat gccagtagaa atactaataa gtattatatt 1260 tccaggtaaa atgagacacg ggttttttaa agtcactgaa tgtgcatgga agtatttttg 1320 agactcacta aggaaataga ggcaccagca ctctctgtaa tttttagtaa aagactccta 1380 tctgagggaa tctgggattc cccccaaaag gatctcagtt tgatcaccct acagtgaagg 1440 tcaacaagtc ctacccaaga attcaaaaca cctgtcagtc tttagttccc tagtcttgaa 1500 gtttgagcag agtcacatat taccagagaa ttcgaggata gtatctccga gaagccggga 1560 aaaaactcag ttaagagaga agggatgctt taaaaaaaaa aaaagaggtc ttagacgtaa 1620 atttctctta cctgaaagtc atgagcttcg agactgaaaa ggcctagcaa gtgtccagga 1680 cagtggatca aagtggacct ataccatgat acatctcagt gaagtttcag gacacttgaa 1740 agaaagaaaa ggtaaagctt cctgccaaaa gcaaaaacat gttttgtata aaagatttaa 1800 gaatcagaat ggcatcagac tctccaactg caaaagacag agcagtgcct tcaaaattct 1860 gagaaaaaat aatgtctaca ttaaaatttt tataatcatc ttcaattttt aagatactca 1920 ctcaaactat caaataaatg tgaggctaag taagagttaa gacctacaag gccttttttt 1980 gttttgtttt taaaacgtct tttggatcaa tcatgagatg tagaatctaa taaaaccttt 2040 ttatgattta tctcccataa accatttttt caggaaagta gataatatgc tccacaaaat 2100 aaagtacatc aaggaaaaaa ggcatcttag tctttgatgg aagaaagagg aagtccagt 2159 33 450 DNA Homo sapiens 33 agaaaacaag atccagatac aaaaatcgat tgtattttaa ctatgctaat aattagcaga 60 tattgaaact ttttaaacat acaatttatt atagcatcag aaaaatggaa tgcttaagta 120 taaatctgac aaaaaatgtg agctacctgt acactggacc actaaacact agtgaaacaa 180 aattgaagag ctacttaatt ggaaatcagt ttccccccag atttatctat agagtcagtg 240 aaatcccaat caaaatctca gcaaggtctt taagaaattg acaatcttat tttaaaattt 300 aagtggagat gcgaaataac taaagcaatt ctctgacaaa aacaagaaaa aagctagaag 360 gctaacaacc acactgattg caagatttat cagaacaggt ataataatca ggccagtgtc 420 atatcggcat acacgataga ccaggagatc 450 34 584 DNA Homo sapiens 34 ctagacttat ggatttgagg gagctgtgtg aaactcatca tggcaaatat gcttatgtgt 60 atatatcctt tgccatacat gtgctgcaaa ctgtaatgaa atgttattta taagactggt 120 aaggcatgtg ttattagact ggacacacaa aagcccttga ttatctagga agcaatcctc 180 tagggtccag atgtagtttg gaatgtgggt gtttagtatc actgtacttc attactgatt 240 tttatttcta tgctgtttga ctgtattagc tctttgttat tattggggag gtagccagag 300 gtctccagat tcccataatg aatttacagg tgtgatctta tggacaagga ggagtcagct 360 gtattagttg ggggttcaat cttgcctgat aagcttttcc tagttggttt tacagatacg 420 agccctgatc tactccctgc tgccactgtc tgtttctatg atgcatgtca ccatgatatc 480 tgagtatgta tgaaaatata tttaggctaa ttttaactag aatatggaaa ggaaaaagtt 540 ctattgctct gcattgctct gttttcagca atcactgttt ttca 584 35 642 DNA Homo sapiens 35 gctagactta tggatttgag ggagctgtgt gaaactcatc atggcaaata tgcttatgtg 60 tatatatcct ttgccataca tgtgctgcaa actgtaatga aatgttattt ataagactgg 120 taaggcatgt gttattagac tggacacaca aaagcccttg attatctagg aagcaatcct 180 ctagggtcca gatgtagttt ggaatgtggg tgtttagtat cactgtactt cattactgat 240 ttttatttct atgctgtttg actgtattag ctctttgtta ttattgggga ggtagccaga 300 ggtctccaga ttcccataat gaatttacag gtgtgatctt atggacaagg aggagtcagc 360 tgtattagtt gggggttcaa tcttgcctga taagcttttc ctagttggtt ttacagatac 420 gagccctgat ctactccctg ctgccactgt ctgtttctat gatgcatgtc accatgatat 480 ctgagtatgt atgaaaatat atttaggcta attttaacta gaatatggaa aggaaaaagt 540 tctattgctc tgcatttgct ctgtttttca gcaatcactg tttttcaccc acatatagaa 600 agtttgaaag ctctctctga tgtctggcaa ccagatctcc ca 642 36 669 DNA Homo sapiens 36 ccaaaattta ctagaatgtc ctgaaccaca tctttcataa tgttgctgac tcaaagactc 60 ttgaaggctc ctgaccacat tattcgcaat tctaactctc ttgccacccc ttccccatga 120 cccatgtaca attacatgct ctagatcttc tcctcaaaga tgaacataag tctgaaatat 180 caacaccttg gcagccctat tatcaattgc tgatctgtag tccccatgta agtacgcctt 240 ttttagcaac cagtttttgt cccagccata ttaatacttg tggtcagtgg ttaacaatgt 300 tgaagcttaa attatatcca gatgaaattt taaaaaggaa tcacttgtgt tcctctgtgt 360 taacacagga atcccagcat gtgtttctct tccaggaaac cataattata tgtacaaata 420 tctacccgga caattagggg cataatcatg ctctaaatag aagtgttcaa acaagtcaac 480 accttctctc cagttattcc tctttcttct ttctcttaga tgtcatggtt tctgtgtctc 540 aagacattta tgatttgatt tttctaaccc tttctaggtt ctattagagt caattagaca 600 acatattcct tctttctaag aatctggaca aggaggtata cttttctaaa ttttaatcct 660 attaatgcc 669 37 1006 DNA Homo sapiens 37 tcttaaaatg agcaccctca ggactgttag gtaggagagg tgttagattt caagtagata 60 caaataggtc cagaaggtaa aatgaggacc caaggataga agagcgacag tgatttcagc 120 tgagcctcag ttccaagcac agaacttttc agaaacagaa tgggttgcat aatatgtccc 180 cttttaaaag acactttgca gacctggatg cctgtgtgtt ggcatggagc atagaggttt 240 cctgtcctgg gtaaacatgc tgtgctggac taggttctct ctgaaagtct ctccctgctt 300 caggagtcta gaattctaag tttcttctca ggagactcca aaatttacta gaatgtcctg 360 aaccacatct ttcataatgt tgctgactca aagactcttg aaggctcctg accacattat 420 tcgcaattct aactctcttg ccaccccttc cccatgaccc atgtacaatt acatgctcta 480 gatcttctcc tcaaagatga acataagtct gaaatatcaa caccttggca gccctattat 540 caattgctga tctgtagtcc ccatgtaagt acgccttttt tagcaaccag tttttgtccc 600 agccatatta atacttgtgg tcagtggtta acaatgttga agcttaaatt atatccagat 660 gaaattttaa aaaggaatca cttgtgttcc tctgtgttaa cacaggaatc ccagcatgtg 720 tttctcttcc aggaaaccat aattatatgt acaaatatct acccggacaa ttaggggcat 780 aatcatgctc taaatagaag tgttcaaaca agtcaacacc ttctctccag ttattcctct 840 ttcttctttc tcttagatgt catggtttct gtgtctcaag acatttatga tttgattttt 900 ctaacccttt ctaggttcta ttagagtcaa ttagacaaca tattccttct ttctaagaat 960 ctggacaagg aggtatactt ttctaaattt taatcctatt aatgcc 1006 38 589 DNA Homo sapiens 38 aggagctggg ttttgcttaa cagaaggagc actgacccat gttatagaca atcgcagaat 60 ttcatatccc catctataaa atgaaaacac aatacttctc accaacactt atacagcacc 120 tactatgtgc taggttagag atcataaact ggtgatatgt aagtggaata taaccctcag 180 acttggtctg tgtgttctac gcagttgatc tgcaccagcc tttgttaaaa ttggaaggaa 240 attgctaata tttaaaatca ggatatttcc cacgaaaatc tacatttcta gtatctcaga 300 aaaatcatta tttggcagca ctgggccaga atttctgcag ggcaattgtt gtcctgactt 360 gggtggctgg tggaaatggg cgtgtactcc taagtttgtc ccaattgcta ccgctctatt 420 acttcatcct ttaatgttca ctactcttgg ccctgtggga tttttgaggc tgagattcct 480 atattaggtt ctgaaggcaa aacacacaca gaaaagaatg atttcaggcc cttcctgagc 540 atactcatga tgtataactt ttatgacagt aatagtagta tctagcaat 589 39 528 DNA Homo sapiens 39 aagacctgtc tttattttta gaagtaagaa taaaagagat tgtggtggag tatcacaggc 60 agcgtgggag cactgaggga gcccctgacc caccctagga gtggatcagg atgacttctg 120 aaaggccaaa ctgattaata agggataaat aaagtcatgc aaatgaaaag gttgtatatg 180 tgttggggga aagcattcca gacagaagga ccagtgtgtg caaaggccct ggggtgagag 240 gtgcctaatc agtactgaat atacaaagag gtagagctgg gactaaacca ctgtgctcac 300 tttgcctgct tgaattccga ttccaaggag tggaatagac ttcaaatgtc ttcaagtcca 360 cttgtttctg ccaagttctc atttttgttc catgaaggca gagcaccttc tttatttcat 420 ccactgatga cttctcagcc tctagaattc tgccttatga tggatttctc agaaatatgt 480 ttgtgtaatg aagacaagga cagtggttag agtttacatt ctactggg 528 40 673 DNA Homo sapiens 40 caaaaaataa aaaccaaaac attagttggg cgtggtagtg tgtcccaggt actcaggaag 60 ctgaggtggg aggattgctt gagtcccgga gttggatgct gcagtgagct atgattgtgc 120 cactgcagcc tgggtgacag aacaagaccc tgtctttaaa aacaagaagt aagaataaaa 180 gagattgtgg tggagtatca caggcagcgt gggagcactg agggagcccc tgacccaccc 240 taggagtgga tcaggatgac ttctgaaagg ccaaactgat taataaggga taaataaagt 300 catgcaaatg aaaaggttgt atatgtgttg ggggaaagca ttccagacag aaggaccagt 360 gtgtgcaaag gccctggggt gagaggtgcc taatcagtac tgaatataca aagaggtaga 420 gctgggacta aaccactgtg ctcactttgc ctgcttgaat tccgattcca aggagtggaa 480 tagacttcaa atgtcttcaa gtccacttgt ttctgccaag ttctcatttt tgttccatga 540 aggcagagca ccttctttat ttcatccact gatgacttct cagcctctag aattctgcct 600 tatgatggat ttctcagaaa tatgtttgtg taatgaagac aaggacagtg gttagagttt 660 acattctact ggg 673 41 447 DNA Homo sapiens 41 ctcaagcagg gctagcacct ccaatctaga gcaccctgca cttccggctc caccggtctt 60 cttgtccctt cactgccttg cctaggggtg ccttctcctc ctctcttaag ctgagtacaa 120 gtgataatat agtgattaac acaatgctgt agtgttttcc tgttaaacag ggaatggttg 180 attttccagg agaatagaaa atgaaattgt cattggagga cctcctcagt tgaaatcatt 240 ctgtggctga tttcctccta ttttgttttt tgttggttgg ttggtttttg ctttttcagt 300 agctacccag gtatacaaat agcttctttg cagttctgat catctttagg ggccgcattg 360 ggcataattg gaataataat actagctaac ctgcttgcag ggcttgctct gtgctgtgca 420 ctttgtgagc actttaaata taggagc 447 42 562 DNA Homo sapiens 42 ctcaagcagg gctagcacct ccaatctaga gcaccctgca cttccggctc caccggtctt 60 cttgtccctt cactgccttg cctaggggtg ccttctcctc ctctcttaag ctgagtacaa 120 gtgataatat agtgattaac acaatgctgt agtgttttcc tgttaaacag ggaatggttg 180 attttccagg agaatagaaa atgaaattgt cattggagga cctcctcagt tgaaatcatt 240 ctgtggctga tttcctccta ttttgttttt tgttggttgg ttggtttttg ctttttcagt 300 agctacccag gtatacaaat agcttctttg cagttctgat catctttagg ggccgcattg 360 ggcataattg gaataataat actagctaac ctgcttgcag ggcttgctct gtgctgtgca 420 ctttgtgagc actttaaata taggagccaa acctctcttt ccaaaagcct gaagggcagg 480 tgtcctcgca gttcccattc catagatcac catccttcca tggaaagtac tctgtggact 540 gtaacttgcc atctagactt tt 562 43 848 DNA Homo sapiens 43 gggtctttct agctttcttg tcctttgtga agctggactg gtgatgtgca gttgaagaca 60 gcatcatcgg gggccttctg ctccatgtgt accctccagt atttgcaaaa gattgaacct 120 acaagatacg ttattagggc aagtatttac atggaaaggc tctgagttct ccaagacttt 180 ggtcattttt tacaagatga tgtactaccc tgatgatttg tggaatcttc ttaggaaccg 240 tgactgtgtt gcttttctga tcatgggtac agggccatct ttgttgaggc ttcccatgtg 300 tgtgggcaca gagcttctgt ggcattccag cagtagatta atggagctgt catcctctga 360 agcctcatgg gttgtgcatg caaacctggt cctgtgaact gcatgggagt ctcttaaaag 420 ggcagaggga ttccttcctt tgtgaaaggt ttagaatggc acatatttgt aatttccaga 480 ctcatctttt cccactctca cattcactct gtatttggcc gtactaaatt gttgacagtt 540 ctccaaatac aacagcattg ctattctgct gccttcgtac atgccgttta cattactgtc 600 acattgtcca ggaattcatc cctgccatga ctgcagtgcc ccctctggga gctccccgtg 660 ccctgtgcct gccgctgtca gagcttccag catgctgggc tgtggaggtg ttggtctgtt 720 tgcccaccca gcaagcctct aagctcctca aagacaccaa ctgtcacgca tatctggagc 780 agcacctggt accttacggg tccttaaatg ccggctgaat gaatgatgtc ttctgtctct 840 ttaaaccc 848 44 1111 DNA Homo sapiens 44 gggtctttct agctttcttg tcctttgtga agctggactg gtgatgtgca gttgaagaca 60 gcatcatcgg gggccttctg ctccatgtgt accctccagt atttgcaaaa gattgaacct 120 acaagatacg ttattagggc aagtatttac atggaaaggc tctgagttct ccaagacttt 180 ggtcattttt tacaagatga tgtactaccc tgatgatttg tggaatcttc ttaggaaccg 240 tgactgtgtt gcttttctga tcatgggtac agggccatct ttgttgaggc ttcccatgtg 300 tgtgggcaca gagcttctgt ggcattccag cagtagatta atggagctgt catcctctga 360 agcctcatgg gttgtgcatg caaacctggt cctgtgaact gcatgggagt ctcttaaaag 420 ggcagaggga ttccttcctt tgtgaaaggt ttagaatggc acatatttgt aatttccaga 480 ctcatctttt cccactctca cattcactct gtatttggcc gtactaaatt gttgacagtt 540 ctccaaatac aacagcattg ctattctgct gccttcgtac atgccgttta cattactgtc 600 acattgtcca ggaattcatc cctgccatga ctgcagtgcc ccctctggga gctccccgtg 660 ccctgtgcct gccgctgtca gagcttccag catgctgggc tgtggaggtg ttggtctgtt 720 tgcccaccca gcaagcctct aagctcctca aagacaccaa ctgtcacgca tatctggagc 780 agcacctggt accttacggg tccttaaatg ccggctgaat gaatgatgtc ttctgtctct 840 ttaaacccac cttctactat gctaccataa tggatatttc ttctaactgg caattttaaa 900 gatcctgctg tggcctttgg tcaggctttt gagcagggtt tggcaaatcc gtggcctatg 960 gaccaggtct ggcccgcggc ctgatggtca tccttgcgct ggccgtttca ggatgaattt 1020 acagttactg acaccaattc ctgtggaaaa taataaaaga ctcgcggctt tcacatcacg 1080 tagcttaaaa agggaacacg gggacaaact g 1111 45 626 DNA Homo sapiens 45 tgttctgaca tcaacaggaa aaatggtaca agaatatttt cagatcatgc caaaaagcag 60 cacttcgtta aaaggaagaa aaaatttcaa gtaaaacata aacaggtttt tagattgctc 120 gataattcaa ttagtgaatc aaacaatgat aaaagctata tatttcctgc tgatttgtca 180 ggaaatagtg acactgacaa agatagcatt acctaagaat ataaaagcaa agatagcgtt 240 gccacagact gcttaatgtg tgtcatctat caaaggggta tatgtgatga gaagaaaaac 300 ttgaaatgcc ctcaaatgtt tcagctatca gaaactgaaa aaactcttac tagtgtgttc 360 cgcataattg tgagcaatat tctaaagatc gacgtttctt cagttatgat tttcttgagg 420 ctacatcaga gaacttcctt aaacctgtcg gtaatacaaa atcagtgagt catggcaaag 480 gggagacatt atctatctgt tcttgactat ggaaaataat gttgcagaat ctttgtcctg 540 tgtgtgaaga agcgatgagt acaggaccag aactgtccgg aagacgtatt tcaggagacg 600 cacatggcag tcgggcgccg ctctag 626 46 185 DNA Homo sapiens 46 gaagaaactg tgaggtcaca atacttttga ttcattatgt gaatatacat acacactcac 60 atctctatta ctgtatccat ctctatatac ttgaactcca tatgctctat attaacttcg 120 ccaaatccaa cccaacaaac agggttcatc tctgattttt ccccccatat ttatgattct 180 cagac 185 47 268 DNA Homo sapiens 47 atggatttgc cacaagctgg ctttgaaagc agtggtagag tgtgaaagaa gttaccttaa 60 gacttcttgc cagttgcact gtaggtacga tgtactgttt gttgtgattt gactttcctc 120 caccaccccc ctgccccagg aagatgtgat cttgtgcatc ttgtgttcac gcagagtagg 180 gtagttggat ctttgtcaag tctcagtgat ccacatgcgt gcatctattt tgtcagtctg 240 cttgtctttg tatccatgtc atactgtc 268 48 108 DNA Homo sapiens 48 gtcgacgacg acagcaatgc cgatccgcgt cacgcccgca accggctgcg gctgcaggtg 60 atgcctgccc tgcgcgaggc cttcccgcag gcgccgctgg cgctggcc 108 49 83 DNA Homo sapiens 49 gatcgagatc ggcggcgtgc cgctggtgca tctgcccgcc gaggcggtgc gcgcgccctg 60 gccgctcgac gagcgcgagg tgc 83 50 475 DNA Homo sapiens 50 aaagaaacaa gcaacaaata ggaaaatcaa atttttagaa gtaggtgcat aataggggaa 60 tagcttaagg ggagaactat gatgttaatt ctttgaaagt gagtaatgta attagaacaa 120 taacactatg agtttttcta taaacaaaat atagcaagat taagttgata acatacattt 180 ctaaaatttt ggcttcctta gagaaagcca accaaatata aaattttaca gcagagtcaa 240 gttttttcag tttggcctat attttctttg gtaacactgt tctgaatgta tatgcagtgt 300 ttatttcaca acttccctct gaatgacctt tcaaaaatta atgattcttc acattcatga 360 ccagatgttt tctctgatgg aagcatctga tgtttgcagt catcaaataa gattcaaaat 420 gtctgtttca agcaaatcaa gtaaaacttc tccatcacat caaaagtaag gcttg 475 51 607 DNA Homo sapiens 51 aaagaaacaa gcaacaaata ggaaaatcaa atttttagaa gtaggtgcat aataggggaa 60 tagcttaagg ggagaactat gatgttaatt ctttgaaagt gagtaatgta attagaacaa 120 taacactatg agtttttcta taaacaaaat atagcaagat taagttgata acatacattt 180 ctaaaatttt ggcttcctta gagaaagcca accaaatata aaattttaca gcagagtcaa 240 gttttttcag tttggcctat attttctttg gtaacactgt tctgaatgta tatgcagtgt 300 ttatttcaca acttccctct gaatgacctt tcaaaaatta atgattcttc acattcatga 360 ccagatgttt tctctgatgg aagcatctga tgtttgcagt catcaaataa gattcaaaat 420 gtctgtttca agcaaatcaa gtaaaacttc tccatcacat caaaagtaag gctttatatg 480 gttcacaagt agctatatga aataaacaga atttaaacga tcttaataat ttttttcttt 540 aaacaaggtg acaaaataac aatgccaata tataaaaact cctcattaat gataagtgct 600 agatgga 607 52 590 DNA Homo sapiens 52 ctcctcatta atgataattg ctagatggac accatgtaaa gtatggaaaa tgcctgtctg 60 aacaaatgct tttgctaaat tctctgaatt tttttttgtt tttcctcacc agttagcttt 120 gatgttttga tcagagtttt tagaaaattt ctaggatctg ttgcctttgg actttagagc 180 ttcttggagc cacatgtcag tactaaaacg ttttcttaag ccctcgcttt ccatagcaaa 240 aacatgttat gtccattatc cacctaactc atacttaaaa acaacaccca agatgctcta 300 ttttgttttc aaagtcagag aagaaaatag aggggaagta tttttatgtt cttttccctg 360 aattggtcga agctagttag ttcaaaaaag atacaaaata tggaatacca cctattttat 420 ttcctggcaa ctgtttcatt caaatcatag agtaacatat gatttactac actcctttat 480 gaatattaat ctcgtatctt cacagaatga cttaatatca ttgatcagct agaacatcga 540 cctcacctgt ctgttgtttt taacgaaatg tttattccta gtcaaaccac 590 53 217 DNA Homo sapiens 53 agtctgctaa ctcattccag tggttttttc caactgcatc tcagttatct tacatagact 60 gcaagaagtg agaaagacaa gaggttatct agtccagcct tgctatttta tagtttaaat 120 ccctcaacca catccctgat gaacttttgc cagtgccggt aattaacaat atcacaaggc 180 tgttctgatt gtctgtattt ctcagtgttt gttagag 217 54 430 DNA Homo sapiens 54 aataaagata agaatgacaa cagatttctt tttgggaaca atgagagtgg gaagacaatg 60 agcaacatct ttaaagtact gaaaggtatc agcagaccca tgctacaaaa aatgtaaaag 120 aacatcatca ggcagaagga aaaaaatagt atcagattga agtctgttct acacaaagta 180 atgaatacca gaaatgataa ctacctgggt aaatatataa gattattttc ttcttattta 240 aagtaagagt gagattctta tcaacaatag cataaaggct gaaggggaga aatggaagtc 300 tattagtgta atcttataca tgatgtggta tgatgtcact tgaatgtaga attataaaga 360 taaacagcat aaactcttaa agcaaccacc aaaataacaa agagttataa ctaataattc 420 agcaaaggag 430 55 2956 DNA Homo sapiens 55 gttgttgttg ttttttttga gacagagtct tgctctgtcg tctaggctag agtgcagtgg 60 cgccacctcg gctcactgca acctccacct cctgggttca agtgattttc ctgcctcagc 120 ctcccgagta gctgggttta caggtgctcg ccaccacgcc cggctaattt ttgtttcttt 180 agtagggttt caccgtgttg gccaggctgg tctcgaactg ctgacctcgt gatctgccca 240 ccttggcctc ccaaagtggt gagattacag gcgtgagcca ctgcacctgg ctttttattt 300 ttttaacttt gtatacggta ttttcttttt ctgtatagaa gtcaaactat tttccttcat 360 ggattctggt ttttgtctct tcattccaag accatttaaa aaaatgtgtt cacattttcc 420 tctgatactt ttaaggtgtc tttctgaaga taaaacctga tgtgtctgca atgctagagt 480 gaggcttgag tatgggcaag cttcctgagt gcacgtgtga gctgaggaca gcatggcgtg 540 tgaggaagga tcagtccaca cagctcatgt aagctcacga gagaggctac tggcttcact 600 gcacgtgtct actgggtgtt ttgacaacgt ggagtgaata cttcatgtcc tcacaaattc 660 aaatgctgtt tttatcatgt ataaatatta tattggaaaa aaataaaatc ataatgaagt 720 tatttgctca cttatcttga agaaaaacac atacatgttg cacttctgaa tttaccttaa 780 cctgtttaat acctactgag aaagtctact attcagaatg cagaaaaagg tggaaggagt 840 ggttagggcc ctaaaagtca aactgggtcc ccgcagccca gagatcaaca ttatttaaaa 900 actcaccatg caaagctaat agagaacgaa ccatgtaacc ctttttgaac tattacattt 960 tcaactcaaa gcttggccct atcttccagt tacacgtcta taaatgtcaa ctacgaagcc 1020 tttcagaggc cctacacttt gcaaatgaag tcagtggaac cctcctgcac acagacagag 1080 cccaaaggac aggagtgcag ctggcagtgc agcccttggt ggggccaagg ggcaggtcac 1140 atggaagggt gcgggttcct cccatgtcca tacgctgacc cctcactcat gctcccagac 1200 ccctctggac accgtgctgc tggcagatgc tgtgctcctg ggaggtggga tgcaagctga 1260 accttgctca ctccctttgg gctaaatgac aggtgagcac tgggcacagc aaatgtgact 1320 ggccacagcc tcatctgcag gggcaacaag tttcccacac aagatcccgt taccatccca 1380 cacaccccgt ctccatctct ctggatcctt gttcagacac agtgttttta tcaacaccca 1440 cagaggaaaa tgggtaaatg cgaaaactcg tttttgcagc tttaaattac ctatgtcctc 1500 agaatgtagc agaattcaca gctggctggg aaaagctata atacatgcac tgcacacact 1560 aacgcgtttg aatataaata agcgtatctt taagttctgt aaagttcctt accgccaagt 1620 agaataaaga caccaacctc ttttgtcatg aggctcaaag tctcctctgg ataccgttct 1680 ataatctgaa gtaatctagg aaacttcaat ctggcttcat tggaatttaa ttttaaagct 1740 ttcaacattt tctccaccac aagtgctgga tacgcctgca gttctgcaga atcaataact 1800 atcaaggaca ccaaagaaga aagcaatggt caatgtatcc caatatccat aaactatgat 1860 gttaaatgct aacactttcc ctttttggct tgtattttgt agtgtcattg ttctcttctt 1920 aactaccact ttacaccaac aaacaccagg tacagttttg tatctatcct ggagccaaat 1980 ccttccatta gagtgcccat tctgcatgaa gcacagtttg aatcctgggc tgggaacata 2040 aggggcaatt ggtggttatt gaatttattc caggagcatg aagcaggcca cacgagccag 2100 taatattgaa gctgcaagca aaatatcaaa gtagaaatta aacaaatgga aacagaggac 2160 cacttgactc catttaaatg taggtcatgt tgcttagaga ggccattgtc tctctctttt 2220 ttttttttta agatggagtc tcgctctgtc acccaggctg gtgtgcagta gtggatatcg 2280 gctcactgca acctctgcct cctgggttca agcaattctc ctgccccagc ctcctgagta 2340 gctgggacta caggcatggg ccaccacgcc cagctaattt ttttgtattc ttagtagaga 2400 tggggtttca ccacgttggc caggctggtc ccgaactcct gacctcaagt gatccacctg 2460 ccttagcttc ccaaagtgct gggattacag gcgtgagcca cctcacctgg cctaatttca 2520 ttttatctcc tttgctgaat tattagttat aactctttgt tattttggtg gttgctttaa 2580 gagtttatgc tgtttatctt tataattcta cattcaagtg acatcatacc acatcatgta 2640 taagattaca ctaatagact tccatttctc cccttcagcc tttatgctat tgttgataag 2700 aatctcactc ttactttaaa taagaagaaa ataatcttat atatttaccc aggtagttat 2760 catttctggt attcattact ttgtgtagaa cagacttcaa tctgatacta tttttttcct 2820 tctgcctgat gatgttcttt tacatttttt gtagcatggg tctgctgata cctttcagta 2880 ctttaaagat gttgctcatt gtcttcccac tctcattgtt cccaaaaaga aatctgttgt 2940 cattcttatc tttatt 2956 56 517 DNA Homo sapiens 56 cctggctgga gcggacacgg tcaagaccgt cctccctacc ttctcccttc aacccaagct 60 caactcaacc aaaaatggcc cctctgtccc catgcctgat aggaaagtca ggggaaagtc 120 tgtccgatta ctgtcaaaga agacaggagg taagggtcag agtggaccac tgactgaata 180 tgagtcgcag aagtgttaga ggcagaagtc cagggccatt tccttaatat cgaagtgtct 240 ctgctggagg tctgggatgg atttttgccc tgcatttaga agttctgggg tcctgggaga 300 ggggagagaa gcccaatagc agaggagaca gagtgtgggc ggggcgagcc ggaggggtgc 360 atcctgggag agcaccaggg tgagggaggg gtgaagatga gccccgtcag ggaagcgctg 420 gcgagtgtgg gaagtcacct gcccctcggc ctgtgagctg ctctgcttgg agtgactaag 480 gctcgggagg tccaggctcg gccagaggca gctcata 517 57 1490 DNA Homo sapiens 57 ggggaaccag acgcccagtc acaggcgaga gccctgggat gcaccggcca gaggccatgc 60 tgctgctgct cacgcttgcc ctcctggggg gccccacctg ggcagggagt aagtcagtgg 120 ggtctgccct caatctcccc tgcctccctc caggagagcc agggactcac ccggcccttg 180 tcccagacta actctggtca cagaaccatc ctgtctgcct ggaggggcgg ggtcccctgt 240 tctggcagag gtcaccccca tatcaccgca tggggatttt cttccctttg ggtctctctt 300 ttcttcagag atgtatggcc ctggaggagg caagtatttc agcaccactg aagactacga 360 ccatgaaatc acagggctgc gggtgtctgt aggtcttctc ctggtgaaaa ggtgagtagg 420 gctatggtca tgggcccagc gccatgtccc ctcccatccc acagtttcag gaactcaggg 480 cagcgggtaa gcacccgtgg ccacttttgc cacacatgcc tggctactgt cgatgcttcc 540 tggctcccgc tgatgcttcc tggctggagc ggacacggtc agaccgtcct ccctaccttc 600 tcccttcaac ccaagctcaa ctcaaccaaa aatggcccct ctgtccccat gcctgatagg 660 aaagtcaggg gaaagtctgt ccgattactg tcaaagaaga caggaggtaa gggtcagagt 720 ggaccactga ctgaatatga gtcgcagaag tgttagaggc agaagtccag ggccatttcc 780 ttaatatcga agtgtctctg ctggaggtct gggatggatt tttgccctgc atttagaagt 840 tctggggtcc tgggagaggg gagagaagcc caatagcaga ggagacagag tgtgggcggg 900 gcgagccgga ggggtgcatc ctgggagagc accagggtga gggaggggtg aagatgagcc 960 ccgtcaggga agcgctggcg agtgtgggaa gtcacctgcc cctcggcctg tgagctgctc 1020 tgcttggagt gactaaggct cgggaggtcc aggctcggcc agaggcagct catatgtggg 1080 ccacagtgac ggcagctggt gccttctggg tcacggagac ctggcgctgc acgcagctct 1140 cctcaccagg atctcagtga ctcctcccaa aagtcacacc cactttgcag acggggaaac 1200 tgagtccgga gaggctgggt aacgagctca agatcacagg gcccaaaagt ggtagaatca 1260 gggttggtga ccagtgagtc tgtgtcagag acccaaagtc tgatggtgct ggactctctg 1320 catcccggga aggaggatgg gggcgctgag gacccgggat gtgctgggcc atcccagatc 1380 tggacgtcca aagctttgcc tctctcccag tgtccaggtg aaacttggag actcctggga 1440 cgtgaaactg ggagccttag gtgggaatac ccaggaagtc accctgcagc 1490 58 436 DNA Homo sapiens misc_feature (197)..(197) n=a, c, g or t 58 ctctgtctcc tcataggaat ttcttagttt cttggctttc gaatgtgact caacccctcc 60 cttggcctgt ctgtctgctg tgtcgctttt aggttctgct gccacggcta actatgtttc 120 cctgtgtttc cagataaact tgtgagggtc agaagctgac agaccaagct catttttcaa 180 gccaatctgt gtcatanaga gaccacgggt tttccttggg ttgggtcctt ctacctggtt 240 cagtcagctg tgaacaaaac ttgtggaatt tggtcatttt ccttaaaatg gagatacgag 300 agatcaccat ggctggcgtg aaactagttc tggatctgat tgtcttttca attgtttgtc 360 catcaggtga acccactctg aagggacttt tggtaacatt ttccccaaaa taaagatcat 420 taattaatta tnaaaa 436 59 458 DNA Homo sapiens 59 ctctgtctcc tcataggaat ttcttagttt cttggctttc gaatgtgact caacccctcc 60 cttggcctgt ctgtctgctg tgtcgctttt aggttctgct gccacggcta actatgtttc 120 cctgtgtttc cagataaact tgtgagggtc agaagctgac agaccaagct catttttcaa 180 gccaatctgt gtcatacaga gaccacgggt tttccttggg ttgggtcctt ctacctggtt 240 cagtcagctg tgaacaaaac ttgtggaatt tggtcatttt ccttaaaatg gagatacgag 300 agatcaccat ggctggcgtg aaactagttc tggatctgat tgtcttttca attgtttgtc 360 catcaggtga acccactctg aagggacttt tggtaacatt ttccccaaaa taaagatcat 420 taattaatta taaaaaaaaa aaaaaaaaat gagcggcc 458 60 359 DNA Homo sapiens misc_feature (336)..(336) n=a, c, g or t 60 cggacgcgtg ggaaacacaa actgcatcat ccaaaaatac acctttggtc cacggatgcc 60 actggaagac atctgaattt tagacctcca gagagaagat ctgggtggct agctccagag 120 tggaggcatg cttgcttttt ctttacactt gtgaagagga atggatccgg acatctgcaa 180 tctgggtaga ggacggcagg cagcaagctt agccactcgg ccaggcttct cagcccttac 240 tctagacatg tgatccttcc tccacgtgat atacttcaca actttcttac ggctactcaa 300 ggcatcccaa gttaaaagga aggtcagatg tgattnatca ctttattatg ataaaaaaa 359 61 932 DNA Homo sapiens misc_feature (161)..(180) n=a, c, g or t 61 tggccagaga catatgaaaa gatgccttag acatatagca tcttttctca tccacttact 60 aggagaaatg ctcactaaaa ttatcctgta atgccattta aaaaaatctc agattgttga 120 agtacaaaaa gttagataac atattatcaa ccaaaatgtg nnnnnnnnnn nnnnnnnnnn 180 ttgggccagc tgtgtttggg taaactagtt aaggtggtag ggttgtttgg tcaggaatta 240 aatcataaag aaaaacaaaa cctctgaaat gaaaactcat ggtgagggta aaacttcacc 300 ccttgtagtc acttatgttt aactggtcta ctggattttt ttaaaggtta agaaaacaca 360 aactgcatca tccaaaaata cacctttggt ccacggatgc cactggaaga catctgaatt 420 ttagacctcc agagagaaga tctgggtggc tagctccaga gtggaggcat gcttgctttt 480 tctttacact tgtgaagagg aatggatccg gacatctgca atctgggtag aggacggcag 540 gcagcaagct tagccactcg gccaggcttc tcagccctta ctctagacat gtgatccttc 600 ctccacgtga tatacttcac aactttctta cggctactca aggcatccca agttaaaagg 660 aaggtcagat gtgattctca ctttattatg ataaaaaaaa ttactattta aatactataa 720 ataaatatta taataaatac taagctagaa ccatcagaat acatcacttc tgtatccagt 780 tttcaaagta tctttggtgt ttgtcaggaa taaataaaag taatcatttt atttctatta 840 aattatatct ggcactagtg gctagtactt ttgtacttat tagtacaacc ttaaaaagtc 900 ttaaaaagat ttcttttggt ttcagaacat aa 932 62 554 DNA Homo sapiens 62 ctggcagatc cggacgggca ggactgggtg tgtcccatga gagcacctcc ttcctggcct 60 ttcctgtgga ctttgtccca caccacctgc ctgggttcct tcctttagtc acttccagct 120 ccaggcacag cagttggtga ctccttggtg ggagccgtgt cccacccggt cctgatactg 180 ccgtcttctc tttcacagtc ctccaggctt gggccagcct tgggggcagc agagcttctg 240 gggtgagtgt cgagatcctg tgtcctgaga gcggtagtca gggagagggc tggtcggggc 300 agggctgccc gggcaggaca caggatgcgg ccggccaggc tggggccaag gtgttcagac 360 ctggactttg ggctcgtgct ttcttcatgg ttgcgccttg ctcgctgtcc cttggagtct 420 tcatttggtt ttgctttttt tgtttgtttg ttttcaccta atttttgcca gacttaagct 480 agttttgctg ccttttgaaa ctagtggaag aatcatttta tttcctgggg ataatttggg 540 ggcttttgaa tcca 554 63 786 DNA Homo sapiens 63 ccagtggcct gtgtcctagc aaatgagagc caccctgaaa aataaaatcc tgtctcccca 60 acgccagccc tggcaaggca cccagaactc tccggaatgc ttgaaggcag ggcctggcct 120 ttccatgggg tccagggctg tggggtccct ggcggtactg tgggcctgca gagcggggca 180 tgtgggctga agaccgtctc cccaccatgg tgggaaggga caaagggtgg ccctggcaga 240 tccggacggg caggactggg tgtgtcccat gagagcacct ccttcctggc ctttcctgtg 300 gactttgtcc cacaccacct gcctgggttc cttcctttag tcacttccag ctccaggcac 360 agcagttggt gactccttgg tgggagccgt gtcccacccg gtcctgatac tgccgtcttc 420 tctttcacag tcctccaggc ttgggccagc cttgggggca gcagagcttc tggggtgagt 480 gtcgagatcc tgtgtcctga gagcggtagt cagggagagg gctggtcggg gcagggctgc 540 ccgggcagga cacaggatgc ggccggccag gctggggcca aggtgttcag acctggactt 600 tgggctcgtg ctttcttcat ggttgcgcct tgctcgctgt cccttggagt cttcatttgg 660 ttttgctttt tttgtttgtt tgttttcacc taatttttgc cagacttaag ctagttttgc 720 tgccttttga aactagtgga agaatcattt tatttcctgg ggataatttg ggggcttttg 780 aatcca 786 64 575 DNA Homo sapiens misc_feature (411)..(411) n=a, c, g or t 64 ggcacagcta gttggtgact ccttggtggg agccgtgtcc cacccggtcc tgatactgcc 60 gtcttctctt tcacagtcct ccaggcttgg gccagccttg ggggcagcag agctttctgg 120 gctgacatgg ggctcattgc tcctttctcc aagccctctg agggacatca aaagcgtggg 180 acgcatccac ttttccacca tcttggcttg ccccactgtt ccctccatcc tggagggcct 240 tccttaagca catgtgtggg ggtgggcagg cacactggct gatagctgtg gatgcggccg 300 tgacatcctt cacccctgcc cccatggcat gcatgatcca ttagggagga ccgtctgcac 360 aaaggtctct tgccctgtgc aagcttcctg caagactgga cttgcaaaag ntccagcctg 420 tatggctgga gttccccatg cctgccaatc tcctgtcgac tgcgagtcag ctccgatact 480 tcaccagatt cagccacctg ggggagctgg aagtgaatct cctcgtagct gagccttctg 540 atgagactgc agccccggct gacacctgga ttgca 575 65 834 DNA Homo sapiens 65 cagcagttgg tgactccttg gtgggagccg tgtcccaccc ggtcctgata ctgccgtctt 60 ctctttcaca gtcctccagg cttgggccag ccttgggggc agcagagctt ctgggctgac 120 atgggctcat tgctccttct ccaagccctc tgaggacatc aaaagcgtgg acgcatcact 180 ttccaccatc ttgctgccca ctgtccctcc atcctgaggc ctcctaagca catgtgtggg 240 gtggcaggca cactgctgat agctgtggat gcggccgtga catccttcac ccctgccccc 300 atggcatgca tgatccatta gggaggaccg tctgcacaaa ggtctcttgc cctgtgcagc 360 ttcctgcaga ctggacttgc aaagtccagc ctgtatggct ggagttccca tgcctgccaa 420 tctcctgtcg actgcgagtc agctccgata cttcaccaga ttcagccacc tgggggagct 480 ggaagtgaat ctcctcgtag ctgagccttc tgatgagact gcagccccgg ctgacacctg 540 gattgcagca ctcatgaaag accctgagca gcaggaccag tttggcagag cccgaattcc 600 tgacccacag gaactgggag ataaaactct gtggttttaa tcttctcatt ttagagtgct 660 cagtgtccat gtggtgtgaa cacgcttcat tcaacctggg cccttgggag agatgctgag 720 tggttcccgg gctgtcccca ctccacacca tggcagtgaa gagctgctga agtacatgct 780 tcatagtccc ttgcgtctcc tctatgagta cagttcctgt ttgtggagta gcaa 834 66 437 DNA Homo sapiens 66 cgagaaagaa aaggtatagc ttaaagtggc ttttgagcag gcatgagttt atggaaccaa 60 ggattcctgt gaagacattt tcttttgata aaagaatatt gataagaata ttataccaaa 120 ttgaacaaaa gtagccacag tatgaaggat tcagtacatg gccaaataac ttatttcaaa 180 atagtttaga gttatattcc ttgaagacgg aggttggatg gggattaaat tttgtaaaga 240 cgccaatggc tgttaaacaa aagagctgag atggatgtgc tcttgaatta aaaataaaaa 300 tattttaaat atactattac atcataaaca ttctatgtct ctacttttcc atctagaagc 360 aagaattctt tagtactttc cgagcatcta ctgtgtagac tatcttgtgt tatgaccaat 420 tgcttatatt tatttac 437 67 80 DNA Homo sapiens 67 acaaaaccat atgcttcaac acctcaggtt gaccatttgg ggggagtgtg tatgggtgtt 60 ttaagatggc ggggtatgcc 80 68 663 DNA Homo sapiens 68 gtgtagagca tggaagcagg gagaccagtt aggagtctat tgtaatagtc ctggtgagag 60 accacagcgg cttggactaa gatggcaact aagataatga tggttgcagg gcccctcttc 120 aatggaggca ttgccagcct tctggccatg aaggagaaag tgatttcaac taacccagga 180 aactcttacc tctaaatgga gatacttcct gataacagaa gaaactgggc atctaaccca 240 gaaataccag ctgagtagga gaagagaaaa ggcatcagcc agtcaaggtt tcagaaggct 300 gccaacagtc tttgtaagcc accttgggag tagatgagaa cggcaatcaa tcaacatggt 360 ttggtgaaca aaccatatat tacaaagtgc ttctgtgaag tctgcatcct cacaactaat 420 gagtgagaca tttctcattg tttctgctca cccaggaata ccatgctgtg ccagctcttg 480 ccatttatta accaactgat aatggtgcag tgctgtagtc atggaagcta tttcaaaagg 540 ttaaggaagt ctactggaat cctggttctt ctagttgcca ttcagactta tttttaaagt 600 ctcattgaaa tgtaatgcat gttatggaaa gtcaggatga aataaaattg agattttttt 660 ttt 663 69 695 DNA Homo sapiens misc_feature (309)..(482) n=a, c, g or t 69 gaaacacaga aagaggggag aaacaggaga ggggaaagag agaggagaga gaaaccaagg 60 aaatgtgaca tataataatt ttttaaagaa tattttttca tttttttatt gaggtataaa 120 atacatgtag taaggtatgt caataactca aatcttatgt gattttttta tgtacatgta 180 tacctgtgta cacctgtgta accactacct aagtcaagat agagaacatt ttaatcatct 240 taaaagattt cctgtgtctc ttcccaccaa tacctgctga tgagcccact ctccttacag 300 ctatcagcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnccttcatg ttaatgaaca tttgaattgt tttcatgttc ttgttatgaa tcaacatggt 540 tatgaatagt ttggttatga agagttttac acatgttttt agtctatttt gtttctctta 600 aatatatact tagtcacggg attactggtc atatagtata ggcaggcaga tgttcagctt 660 taattgacac aaccaactgt ttttgaaagg ggttg 695 70 739 DNA Homo sapiens 70 ggtttctctt catggacatt gtttgcatct acatgtgaca cttaggaatg atctgtttag 60 tctcaatcac tcactcctgg atctgcctgt ctctctctga gataacaaag gccttaatgt 120 ttagccacct gcatcagagt tggtgaggtg gtttgaaaca attcatccta atataaaaag 180 aacagctttt gtaagggggc actgagtgtc tcaaacagcc gcatgggcag gaagagtgct 240 cagtccagtt ttggttgaac ttgtcttgtt gccctaaggc ctcctatgaa agactgacag 300 gcttggactg aatcttgtga tctggacacc aagggtcacc tgtgggccca gagctagctc 360 tgaagaatgg ggtagtttct ttgagaacct ccacagcaaa agtttggtcc tctgttccca 420 atgcatgtcc cactttacca gctacatccc ccagtacctg cccatggctc atgactcatg 480 aaatataaaa ctcagtaggc aggcataact ggttcagacc tgccagggct atgtgggaac 540 tatcattggt acaaaaactc taagtgtgga gaagactgtg gtagacaaga ggggacatgt 600 ctgttctaaa cgcacatcag aaacttccaa tgactatggc caagtgagat aagggtgtac 660 agaacttctc aggacatgca gacctatgtg tcactcataa ctgaaattca aataaatatt 720 ttgtggattt cctgtggac 739 71 9883 DNA Homo sapiens misc_feature (7153)..(7153) n=a, c, g or t 71 ataagaataa aaattacccc aaatttccaa atcaagaagt aatcatggtt caggtttggg 60 cagatgttct ttctaggcat gaacacacgt tatctcattg tttacttaac accgggttat 120 aaacatttac ccatagcatt tgaaaggtag ctatagatag aaaagaatca gagaagttct 180 aaaacagctc ttgcgctttg tttcaaattc tctgcaggaa agatgaggtc ttcagccttt 240 tttttagctg gacggcaccg ttgcagcagt ggtgaacagg gcactggatt gagtcaggaa 300 acccagctgt gaccttgggc aagccacttg ccctctttga gcttcactcc tgctaaggca 360 aggggcgcta ttcgtaccct gtctgcccac ctcacaggct ctggtgaagt ccttgatttg 420 aacgccttta gctcccaagg ttgtggtttg gagatagggc aggtcacatg accatgaaga 480 ctgaaggaga aacgtggaag cacgtgtgcc tgttgcttct tttccaactt aaaatgcttg 540 gtgatctcct gaagactcca gcctcctctc tgggaagcca ggatccacag accctttacc 600 tgcgggtcat gggcagtccc agatggtccc cctccccaac agagggggtg cagtgagacc 660 tccggaagtt actgcctctg ttaccctcaa agggattttc agatcagaca gcccccctac 720 tccaagggac gtgtgtggag cttggtacct ttatttatct cctgctccaa cccctgtgga 780 ctgcctgcac ccagaatggg gcctctcctg ctggcaagtg gctgagaacc tccactccac 840 tcagcagggc tgttccccat ttaccgaaaa gctccgagag aaaataacta accccatggc 900 gccgctgtag ctactggcag agcctcctgg tccccacctc tagcgcctgt ggtttttgtt 960 tcatgcagag tgagcagtga atctgggatc ccatcagcag tcagtttggg tgcctgcgag 1020 gcacaatgat agatgttggt gaagggtatg tgtgaggata ttaattaata ttaacatgct 1080 agttatatta atattctcat tggaatttgt ggggctttgc aagttatatt tcaaatatat 1140 ggttttatgt aatcctttta actgccctga aaccttgaaa ttattgcacc tattttatag 1200 atggagagac tgaggctcag aggggtgaat tgcctaagat cctgggggag gaagcaccca 1260 ggttttctgg ttttgagtcc tgggcccttc ctgctgagta gctaccccca acacagacct 1320 gcccttggag agcttgcagc cacactggga aggccagtgt attggattgc tgcttagacc 1380 tggaaagcac gtgaataaag cttcaggtta aaaccatggg ggttccagga ggcagcagtc 1440 ggctctgcct gggggtgagc tgaggagccg gtgctctctg gaacaagggt agttgggctg 1500 aggctcagtg gacagtggag gttggcaggt gaagtgcagg aggtctttgc agggagtggg 1560 accaccttga gcacacacag aggaatgaga caggcaggtt actcaaggag cagaggtctc 1620 gtgaccactt cccagagcat gtggggtcct agcctcatct ccaggaggag aaagtgcatc 1680 tatacacaga tttgtcaatg gagtttaaat aggatgtggg aaaatctaga ttttccaaaa 1740 cagtacatat ttgctttgag aagaaaggta gatgcaggat gcataggtta gataatttta 1800 atagcagtaa cctcagagca tgtaagtatg atttgattta ctggagtgcc tggccgtctc 1860 agtcagtggg agcacggctt gggctgggag atgaggttga caagggttct ttctctcaaa 1920 tgcttccttt ggtttgctaa gaggtatctc ctactcggcc gggggcagaa gacttttcct 1980 tcttttccca gtttgcagta gttgggccag atttgtggaa gtgggagaaa ggcctgccct 2040 gcttctacat agagttggct gtcctgactt gatactcggt gtgccttcca gagacccgcc 2100 tccatctcct caactccctg gcttgatgct taggtggtga tggctgttgg gcacaggagt 2160 tacataacag atctgtgatg gacccaggag cagagccagc tgagtgaatg tcatggagtg 2220 ggagtggtct tgcatggctg tggtgtcccc tgcagcttgt gcagggtatg tggcaagagg 2280 tgctcaccac tcatctggaa tggctagact ggaagcactt ggccctcttg ggctctgcac 2340 ccccaccccc tcccacctgg cctgcctgct catcttcatg ggcgcctggg gagaccaatt 2400 atggctgctt gtcatagtgg ctcaggtcac cgttcacact tcctgggacc aggacatcag 2460 agccctgaga agggtcaagg ggccaagtgg gcctagcctt ttactgacag ctgggaaatg 2520 caagcgtgtg gaccagagca ccaagtgagt tggggccggt gtgggttcag caccgtgtcc 2580 ctacccagag ctccatttgt tgaaaacagc ctttctctac cgtttcttca cttggacaac 2640 tttaaactat gtattggctg gtcgcggtgg ctcacgcctg taatcccagc actttgggag 2700 gccgaggtgg gcaggtaact tgaggtcagg aggtcgagac cagcctggcc aacatggtga 2760 aacctcatct ctactaaaaa tacaaaaatt agcccagcgt ggtgacacgc acctgtaatc 2820 ccagctactc gggaggctga ggcagaagaa tcgcttgaac ttgggaggca aagattgcag 2880 tgagctgaga ttgcatcacc gcacttcagc ctgggagaca gagcgagact gcatctcaaa 2940 aaacaaacag aaacctacat attttctata tttcccccaa cattgaggct catttcttgg 3000 atgaacaatt taaatgtact gtgcctctct ggcaatattt tccaaaatta cagatgtttc 3060 tatactttca ccggcagctc tgcctcccag aatttattct acggatgggt taacacgtgt 3120 gcaaaatgat ttatttgcaa ggttcgtcat tgttgcctta tttttaatag caaaagattg 3180 gaggcagctt aaatgttcat tcgcaggggc caatgaacaa accatggccc gtctaaacat 3240 gggataccgc gtggccataa tacataagat ggacgctcaa cgcactgtgc cggattgagc 3300 agcaaggtgg attgccgagg gaagaagcag gtctgggcgg tgtgtctcgg agctgccatc 3360 agtgtaaaag ggaagagaat caaaagtgtc tttgcttgtc tatgcccagg gggtctctgg 3420 gcagacaccg caagtcggtg attgtgatgc ctctggaggg ggtgctggtc atgggagatt 3480 gcttgtttgc tggagatccc atgtaccttt tgattgctga agcaggtgaa tgtacgcctt 3540 tccaagaaat taaaatgggc caggtgcggt ggctcacgcc tgtaatccca gcagtttggg 3600 aggctgatgt tggaggatca cttgaggtga ggggttcgag accagcctgg ccaacatgat 3660 gaaaccccat ctccactaaa aatacacaaa ttagccagac atggtggtac atgcctgtaa 3720 tcccagctgc tctagaggct gaggcaggag aatcatgtga accctagagg ctgagtttac 3780 agtgagccaa gatcatgcca ttgtactcca gcctgagcta cagagcgaga ctctgtctca 3840 aataataaaa taaaataaat taaaaacata aggactgtaa ccttgcctcc tgcccagtgt 3900 aggaaggtca aggttctggc tacttctcaa gtacaggagc ctcactcagg ccccagacca 3960 ctaatcaaaa aatatgtgct tggttctcac aaaggggccg agtgtgaggg cttgggtgtt 4020 gcttggtaaa tacgaccccc ggtcccggcc ttggagagat ggagccctct ctgggcccct 4080 tggacacact gctgttggct gactttgtca ttttcaaccc ttgctccgat tggctcacgt 4140 catgatttct gaaacctttg ggggcttccc cactgacaga aagatacact ttaactcagc 4200 actgggcatc ccaggccctc tttactgggc ctcttcttga gccgcacttg gcctgtcacc 4260 ccttcctctg tctgccctct taactcccca cctccgtgcc tttgctcata tagttccctt 4320 tgcctgcctt tccgtccaga gcagtctcca cgtgcccagg tcctgtctga ctttcaaggg 4380 ccagcttagt ttccacttct gcactgcctt ctgacctccc tggcttctgt gtaaactgcc 4440 cagatcaagc cacacaatgg ttcctgcacc caaggaagct ccctggggcc ccctcctggc 4500 cactcgctct tcgccggtag tcaccactca caccttggca ctttcgcgtg gtgcctgccg 4560 ctgcctgttt gggcctccca cacacagagt gtacagaacg gactcctcgg tgtctggctg 4620 ccttcccgca gcactgtcag atcatccagg ttgcctgtag tggccctttg gtttttttct 4680 ctgctgcgta ggagttcacc aaatatacca ctatttattc attctcctgt ggacaggcat 4740 tgggttatgt ccagcctctt cggtgaattc attcttgtct ttgggggcgc gtgtgcgctc 4800 tttgctgggt atacacccag ggtgggttga tggcttacct gactcagaat gtgtttgcat 4860 gaatgaaatt caggttggta tgagaaatct agggtgtcct ggctggagcc aggcttcttg 4920 attacaggga cagagcaggt acagggatcc tggtttagac agcctgctcc catggggtgg 4980 tagcattgtt ggggtgcagg atgctgaatc tgcaggggac ctatccgctc agtgcccagt 5040 gggattttag ctggctggaa aggtggtcac atgtagaggg gctcaacaat ccagctaaag 5100 aggctgagcg ttggtccatt gttctcaatt tgagagaaaa ctgagatcat caaaattagg 5160 actggtatgt actaaaggaa agaacctaat tacaaggctg aattgagtaa gccctcgctg 5220 agggactttg gatttctttg ttgttcccct ttatttctgc acccccaccc aagtgacaga 5280 tatgtacatg attggatgat tttgctttcc tggttgagag attcctggga acttggccca 5340 ggagaagggg gagaaatgtg gagccgctag agtggcctcc gcttgtttgt gttgattgaa 5400 ggggagacgg aaggagagct gtggacccct gaccccttgt gagggcatgt gatccttttc 5460 aaaaggctca ccaggcagaa gtgcctggcc aggggccgct ctttccctct aatcccctct 5520 ggagaagggc caggctgtgg gttgctgacc tgctctgatg tggatcagcc tcccccaata 5580 atgcagctgc ccagaagctc agagagccca ggcaaccccc aaaggcagga gggccggctg 5640 tcattcccgt tgtcattccc aggcggctgg agtgggagca gagcggtcag ttcagatgaa 5700 cagtgctcga gtctgacccc aaccagcgag ttatggtaag atggaaggtt ctccatctat 5760 attaaataag agaacaaaag ccctcccagg ctgcatgaat attccaggga tatatatgtg 5820 aacgggttgc cagtttagct tggcctgtgg gtggcagccg cctgagtgag cacttcgtgg 5880 ctgcagctct aaagggtttg gatctgaaac taatgaatga aaatatgacc tcagaagatt 5940 taaagagagc aaatacccag caacagaacc tgggtcccag agactgttgg gagcatgaaa 6000 tcccaggctg gccgaaggag gaagtgggag agcaatggca gctgacatca catggtgcca 6060 gaccttctca gtgctttctg tgttcactca ttattccgtc cctctctctc agaggcaggt 6120 atggctgctt ccccatttta tagatgagga agctaaggca aggagaggtt gtgtaacttg 6180 ctcacagaca caaagctagc cagtggcaaa gctggaggtc aggtctaggt ggtcaggctc 6240 cagagttctg cggatttcac agcacggcag tggcagtcgg aagaaccatt tgtcaggtga 6300 ttgtgggcaa atgacgtcag cccttcaaac ctctgttttg catctgcaag ctgcttgctg 6360 ctgcaacaaa ttaccagaaa cttagtgact taaaacacaa attaggtcgg gtgcggtggc 6420 tcacatctgt aatcccagca ctttgggagg ctgaggtgag tggatcactt gaggtcagga 6480 gttcgagacc agcctggcca acatgatgaa accctgtctc taacaaaaat ataaaaaatt 6540 agccaggcat ttggccgggt gtggtggatc acgcctgtaa tcccagaact ttgggaggac 6600 aaggtgggcg gaacacaagg tcaggagttc aagaccagcc tgaccaatat ggtgaaagcc 6660 tgtctctact aagaatacaa aattagcagg acgtggtggc acgcgcctgt agtcccagtt 6720 actgggaggc ggaggttgca gtgagccaag atcacgccac tgcactccag cctgggtgac 6780 agagtgagac tccatctcaa aaaaaaaaaa aaaaagtaca aaagagcaaa acaaaacaaa 6840 agttatgaaa atgaaaacct gagccatcct ttatcttatt tccccaaatc cactaattat 6900 taacagaaag taaaagctat gaaaaatgaa tgaaagtgac tgcaatttcc ttgaagtgtg 6960 ttagaacctg cctttagtgt cagctatggg ttccctcatg aaggtcagct gagccatgac 7020 ccatgaacca tggaagcttg actctagatt gaccatcttg agatgccaaa gatgtccacg 7080 tcctaatccc atgtgggaga cagaataatg gccctgcaga ccttcccagc tggccatgac 7140 ccctcatttg acnagctctt cccttctctc tgaccagcac catgcttctc ctggtgacaa 7200 gccttctgct ctgtgagtta ccacacccag cattcctcct gatcccagag aaatcggatc 7260 tgcgaacagt ggcaccagcc tctagtctca atgtgaggtt tgactccagg acgatgaatt 7320 taagctggga ctgccaagaa aacacaacct tcagcaagtg tttcttaact gacaagaaga 7380 acagagtcgt ggaacccagg ctcagtaaca acgaatgttc gtgcacattt cgtgaaattt 7440 gtctgcatga aggagtcaca tttgaggttc acgtgaatac tagtcaaaga ggatttcaac 7500 agaaactgct ttatccaaat tcaggaaggg agggtaccgc tgctcagaat ttctcctgtt 7560 tcatctacaa tgcggattta atgaactgta cctgggcgag gggtccgacg gccccccgtg 7620 acgtccagta ttttttgtac atacgaaact caaagagaag gagggagatc cggtgtcctt 7680 attacataca agactcagga acccatgtgg gatgtcacct ggataacctg tcaggattaa 7740 cgtctcgcaa ttactttctg gttaacggaa ccagccgaga aattggcatc caattctttg 7800 attcactttt ggacacaaag aaaatagaac gattcaaccc tcccagcaat gtcaccgtac 7860 gttgcaacac gacgcactgc ctcgtacggt ggaaacagcc caggacctat cagaagctgt 7920 cgtacctgga ctttcagtac cagctggacg tccacagaaa gaatacccag cctggcacgg 7980 aaaacctact gattaatgtt tctggtgatt tggaaaatag atacaacttt ccaagctctg 8040 agcccagagc aaaacacagt gtgaagatca gagctgcaga cgtccgcatc ttgaattgga 8100 gctcctggag tgaagccatt gaatttggtt ctgacgacgg gaacctcggc tctgtgtaca 8160 tttatgtgct cctaatcgtg ggaacccttg tctgtggcat cgtcctcggc ttcctcttta 8220 aaaggttcct taggatacag cggctgttcc cgccagttcc acagatcaaa gacaaactga 8280 atgataacca tgaggtggaa gacgagatca tctgggagga attcacccca gaggaaggga 8340 aaggctaccg cgaagaggtc ttgaccgtga aggaaattac ctgagaccca gagggtgtag 8400 gaatggcatg gacatctccg cctccgcgac acgggggaac tgttttcttg atgatgctgt 8460 gaacctttat atcattttct atgtttttat ttaaaaacat gacatttggg gccaggcgcg 8520 gtggctcacg cctgtaatcc cagcactttg ggaggccaag gcaggcggat cacttgaggt 8580 caggagttcg agaccagcct gcccaacatg gtgaaacccc atctctacta aaaatacaaa 8640 aaaattagcc gggcgtggtg gtgggcgcct atagtcccag ctacttggga ggctgaggca 8700 ggagaattgc ttgaaccctg ggaagtggag gttgcagtca gccgagattt gtgccactgc 8760 actcccagcc tgggcgacag agccagactc catctggctc aaacaaacag acaaaacaaa 8820 acaaaataaa ataggcccag tatgatggct catgcctata atcccagcac tttgggaggc 8880 aaggcaggtg gatcacttga ggtccggagt tcgagacaag cctggtcaat acagtgaaac 8940 cctgtctcta ctaaaaatac aaaaattagc tgggcatggt ggtgcatgcc tgtaacccca 9000 gctactcggg aggctgaggc aggagactca cttgaacccg ggagatggag gttgcagtga 9060 gctgagattt gccactgcac tccagcctgg gcgacaccgt gagactccat ctaaaataga 9120 agaaaaggtt tctcttcatg gacattgttt gcatctacat gtgacactta ggaatgatct 9180 gtttagtctc aatcactcac tcctggatct gcctgtctct ctctgagata acaaaggcct 9240 taatgtttag ccacctgcat cagagttggt gaggtggttt gaaacaattc atcctaatat 9300 aaaaagaaca gcttttgtaa gggggcactg agtgtctcaa acagccgcat gggcaggaag 9360 agtgctcagt ccagttttgg ttgaatttgt cttgttgccc taaggcctcc tatgaaagac 9420 tgacaggctt ggactgaatc ttgtgatctg gacaccaagg gtcacctgtg ggcccagagc 9480 tagctctgaa gaatggggta gtttctttga gaacctccac agcaaaagtt tggtcctctg 9540 ttcccaatgc atgtcccact ttaccagcta catcccccag tacctgccca tggctcatga 9600 ctcatgaaat ataaaactca gtaggcaggc ataactggtt cagacctgcc agggctatgt 9660 gggaactatc attggtacaa aaactctaag tgtggagaag actgtggtag acaagagggg 9720 acatgtctgt tctaaacgca catcagaaac ttccaatgac tatggccaag tgagataagg 9780 gtgtacagaa cttctcagga catgcagacc tatgtgtcac tcataactga aattcaaata 9840 aatattttgt ggatttccaa aaaaaaaaaa aaaaaaggcg gcc 9883 72 93 DNA Homo sapiens 72 gttatattaa aacaatagaa acattaatct gtctgtcttt tctccattct atccattcgt 60 tctttaatgt ggtcactttt gaatgctgta tac 93 73 299 DNA Homo sapiens 73 ctcgagcgct cacatattac cacctctgta aatccttttc taacttattc agggtgaccg 60 aattctgtgt ttctgtgccc ccttaatact tgttatataa gtctccttcc ccaaccaccc 120 ccacacttac cacatcacgt tagcaagaat gagagcaatt tgagggcagt ggctttgtat 180 cttatttata gccctggcac caaaacagtt tgtaaaaagt taatctggtg cagggtggca 240 taacacataa gagtctgttt cttttgagat atttggcaga ggttgtggtg tgcggagat 299 74 94 DNA Homo sapiens misc_feature (85)..(85) n=a, c, g or t 74 gctgtgttta tgctgctggc tgtactggga ggaatatggt cctttgtctc tgacccagga 60 gtttcatgtc ttctgccaag atacnttaca tgga 94 75 433 DNA Homo sapiens 75 gctgtgttta tgctgctggc tgtactggga ggaatatggt cctttgtctc tgacccagga 60 gtttcatgtc ttctgccaag atacattaca tggatagata cattaggtag gtagatacat 120 tagatataga tagatacatt agatatagat agatacatta gatatagata gatacattag 180 atatagatac attagatata gatggataca cagatagata cacagataga tagatagata 240 gatagataga tagatagata gatagataga tagattcatt tatttattga gacagagtct 300 tgctctgtca ccgaagctgg agggtagtgg cttgttcttg gctcactgca acctccacct 360 cctgggttca ggtgattctc ctgcctcagc ctccacagca gctgggatta catgcccacc 420 tattttgtac ttc 433 76 334 DNA Homo sapiens 76 gctcgaggtt aatggaccat tcgggttata tggttcatat tttttgctca tttttatgtc 60 atggtgttta tcttttctgt gctgatttgt aaaagctatt ttaaaaccct tcatctgcca 120 tatatgttac atttctttcc tgctttctgc caccttccaa tttgttacca actttcttct 180 ccaaccttgg gccactggca tatacactca ttttaaatat cagaacttgt agtgctcttt 240 gaaatgcaga cagactatgg ttcattctgc aactgcatat tagttaacag gcaaaaatac 300 cttagtaaga gaaagtgtct tttccttcta atgt 334 77 547 DNA Homo sapiens 77 ggcttatatg tggagaactg acgtctgaac ccagatctga ttcccaagtg taatactttc 60 caataggcag ccttatatct ctgtacctca aaagagaagg ctatattatt taaaagatta 120 ggaattgtcc tatatggttt taaaatacac ttgctatagc acaataataa gtggtttagt 180 ggtgactgct actcctgtga gtttggttta aaaacagccc agtttgtacc ctgttggtca 240 tgataaaagc ataccaccct tactttgaga attttaacca tagagcacaa tatgtgtcaa 300 acaagctaaa aaagtattct tttcagttgc attttgatgg acattgaaat tgcttagact 360 ctttgaccaa aagtacaaac tgctgttaaa ctggtgacaa aatctgtttt catggacgct 420 aggctactta agctttattt tcctcctaag cattctctgc ctttgtaaag cactctagca 480 gcagtatttg cttagcttct aattttggtt ttgcttttgt gttttctctc tttctcttgg 540 ttgttcc 547 78 263 DNA Homo sapiens 78 tcgagggttg aaatgagtgt cattagccaa gtgacattta agtgccttgg tttgtctgct 60 tgcttttctg tggattgaaa aaaactgacc actgttaata tgattgtaca gtgacactgg 120 aaattatgag atgtgtgtct ggttagtcct gcttgtattt cagttgagat gcataccaag 180 tctgataatg cagagctttt ccatttcatg tgtctgttta ccattttcat gatcttaagc 240 aataaacatt tcttgacaac agc 263 79 765 DNA Homo sapiens 79 gcgggaagag cacgcagccc tgcgagtact atttccgcgt gtaccactcg ctgtgcccca 60 tcagctgggt gagtcggcag agggggcgcc gggccaggcg tgtgcagggc tcggccgagg 120 ctgagccggc gtcccgctcc ctgcctttct gcttcccagg tggagagctg gaacgagcag 180 atcaagaacg ggattttcgc cggcaaaatc tgactgcccc agcgcggctt cctctgaaga 240 tgcagtgatc ctgcatcttt ttgtctcgcg gagccccggg tctcggttat ccacccctac 300 ctcccagtgt ctaagccacg aataatgcca ccagccttcg agttccttgt ttcccttgct 360 ctggtctcca cgtgtatgat ggggttctca ggcccaggct tcgaccagag gaccctctgc 420 caccaccgtt tcttcctgtc cttgagctac cttggtgaac tcatgacccc aggcccctgc 480 tccaccagga tgtcccccag gtcctgccag ctgggaagtg ccagcatgaa cgcctccaac 540 ttcgtggaag ccagggtccc ctgcagctga gggacgccaa gcagacacac ctgccctccc 600 cagccagctc ctgtctgtat gggcgagatg actgagagcg cccacgtccc taaggctgtc 660 ctgaccctcc atgctgcgac aaggacaggg aatggtcggt cactatgggc ctggtgtctc 720 ccctccccca ccacccggtg ctgcccagct caagccagaa gtgac 765 80 162 DNA Homo sapiens 80 cgctgcctca agaccaggac ccgccgcggg aagagcacgc agccctgcga gtactatttc 60 cgcgtgtacc actcgctgtg ccccatcagc tgggtggaga gctggaacga gcagatcgaa 120 gaacgggatt ttctgcctgt gcaaacatct tgacttgccc ca 162 81 986 DNA Homo sapiens 81 agcgggcggt gcacgacggc tcccattggc tggggctcgg gcgtcctagc caatccggcc 60 gcggggtgcg tttctcctga cccgggtggg accgcacccc gcggactcag aagcgagcgg 120 caccccggga ccatcccaca gcagatccag tggccgccaa cgtcaggctg gagttgcctc 180 cttcgtggat gttggatgtg gaagcccagg agccccccaa ggggaaatgg tcgacgccgc 240 ccttcgaccc gcgcttcccc agccagaacc agatccgtaa ctgctaccag aacttcctgg 300 actaccaccg ctgcctcaag accaggaccc gccgcgggaa gagcacgcag ccctgcgagt 360 actatttcct gcgtgtacca ctcgctgtgc cccatcagct gggtggagag ctggaacgag 420 cagatcaaga acgggatttt cgccggcaaa atctgactgc cccagcgcgg cttcctctga 480 agatgcagtg atcctgcatc tttttgtctc gcggagcccc gggtctcggt tatccacccc 540 tacctcccag tgtctaagcc acgaataatg ccaccagcct tcgagttcct tgtttgccct 600 tgctcgtggt ctccacgtgt atgatggggt tctcaggccc aggcttcgac cagaggagcc 660 ctctggccac caccgtttct tcctgtgcct tgagctacct tggtgaactc atgaccccag 720 gccccctgct ccaccaggat gtcccccagg gtcctgccag ctgggaagtg ccagcatgaa 780 cgcctccaac ttcgtggaag ccaggtcccc tgcagctgag ggacgccaag cagacacacc 840 tgccctcccc agacagctcc tgtctgtatg ggcgagatga ctgagagcgc ccacgtccct 900 aaggctgtcc tgacctccat gctgcgacaa ggacagggaa tggtcggtca ctatgggcct 960 ggtgtctccc ctcccccatc aaccgg 986 82 369 DNA Homo sapiens 82 aacccaagat gactcgtctt ttggtgggag aattcactct gttcatgttt catttaacaa 60 ttgatctact gtacttaatt acctttggct tattttacat ttattggttt atcttgtgtt 120 tttcttccct ctgatctggt tatcgatttc ctttttcttc ccctgttgca ctttccattt 180 cattattggc agctgtccct tctctggggt tcctaatcaa acacatattc tttagcacat 240 gcctcgatgg ggattctttt cgcagcaccc tcatctggag ctcacagaac ctgtcactct 300 gtaggttctg gtcttttttc agcttaggaa catctatttg ttgcttgatt tgattattgt 360 tagtttgtt 369 83 923 DNA Homo sapiens misc_feature (354)..(565) n=a, c, g or t 83 aacccaagat gactcgtctt ttggtgggag aattcactct gttcatgttt catttaacaa 60 ttgatctact gtacttaatt acctttggct tattttacat ttattggttt atcttgtgtt 120 tttcttccct ctgatctggt tatcgatttc ctttttcttc ccctgttgca ctttccattt 180 cattattggc agctgtccct tctctggggt tcctaatcaa acacatattc tttagcacat 240 gcctcgatgg ggattctttt cgcagcaccc tcatctggag ctcacagaac ctgtcactct 300 gtaggttctg gtcttttttc agcttaggaa catctatttg ttgcttgatt tgannnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540 nnnnnnnnnn nnnnnnnnnn nnnnnccctg gataggaagg gataggaaga gactacttgg 600 tgccatgggg taggggtgag ggtataagta gatcagagtg ggaagacctc agccttgggt 660 ggcttgtctc tgcttcttgc caggtgggag ggcctgtcca cacctggatc cccgtaccac 720 agtgccagcc atgcccttcc ctgggctacc attgtccctt tcctcaccca gttggtagag 780 gagtcaggag gtgggaggcc gtgggctttg gttttataat gtaaccactg tgggggtggg 840 ggaggatggt gaaccatgta tttcagtgaa atatttaata tatttaaata tcaataaaat 900 caaactcttt gtaaaaaaag ccg 923 84 338 DNA Homo sapiens misc_feature (12)..(12) n=a, c, g or t 84 ataatttttt tntttttaaa ggaaatgaac gtggaggact ggggtgaagg gccagcctgg 60 gtagtttaat ctttttggga agacatgact ttaaggagat tccctgcttt gtgacaggtt 120 gctccatgct gtcttgggga caagggcctg tactgccttc aaatctgggc tcaccccaca 180 ttttggtgag gggaagatag ggtgggggga taaggaggag aaaagactct agcttttttt 240 ttctatgcat gatatactgt gtgggtttat caagagtgta gacacagttg ctgttctcaa 300 ataataggcc aaataaaatg cgattctttt tttctttg 338 85 436 DNA Homo sapiens 85 ataatttttt tctttttaaa ggaaatgaac gtggaggact ggggtgaagg gccagcctgg 60 gtagtttaat ctttttggga agacatgact ttaaggagat tccctgcttt gtgacaggtt 120 gctccatgct gtcttgggga caagggcctg tactgccttc aaatctgggc tcaccccaca 180 ttttggtgag gggaagatag ggtgggggga taaggaggag aaaagactct agcttttttt 240 ttctatgcat gatatactgt gtgggtttat caagagtgta gacacagttg ctgttctcaa 300 ataataggcc aaataaaatg cgattctttt tttctttgaa acacacagaa cagcccagct 360 ataaaacagg caactgagga agaaccaaac cgcataccgg caagactcta gcatgtcaag 420 gtcaaagact ctccag 436 86 462 DNA Homo sapiens 86 agggaacgtt ggatgtagtc acactgctgt tggtgttact tagaccttca tttttccacc 60 agactgtagt gttcaaaatt ctttttagta agagaaccct ttttttctga actttttaca 120 accatctcca aattataaaa cataagactt ttttttagta aaaatatatt tttttacaag 180 cacagtggct tgcaccatgg aggggagagg aggtgttttg tccttggagc tgctggcctg 240 agagaacctt gtcatcgtgg gagctgggcc attcctacac agtggtctgg caatgacccg 300 gtggtggtgg aggcctgtga gtgggcactg gtaatgggaa cagctgtaaa accctggagg 360 ccagccccag gagagtgacc ttacccagga aagttctggg aaacaaacca cagggaggct 420 ttacaggaat ttttggttgt gcccacaggc aaggcacatg ag 462 87 1435 DNA Homo sapiens misc_feature (1012)..(1119) n=a, c, g or t 87 ttagaggtag aagaactgac tataagcaga agtgtttgag gaggctgcat ggagaacaag 60 gggcatcatc ttggcccttg gcaggttggc aggatttgac ttggtgaaga gaacgagaaa 120 ggggacttta actgggagga ctactctggc tttgatttct ccatcatgcg gagattggtc 180 tttggaagtt gtagcttcca gagaccttcg atgtttgcta acatgtccaa gctctacatt 240 tattgattgt tggttctgtt catggctatg ttcaaattct tgtacctttt tgtcctccac 300 agtttcttgt ctcatccctg tcttccacct ctgctccccg ctcttgtctg gtctaattaa 360 cttcctctgt tggagcagct tcccctcttg ggtaaactca gacatgaccg cagcaaagca 420 gcgtggaatc ttctgtttgg tcagtgttcc ccccagcttc cccgcagata cagctgcatt 480 ggagcccctg aagacaaacc agagaagtgc tgcatcctgg ggggcaggag gctttgcttt 540 gcccagggct gggctcctga atgaattttg gtgcagcctt aacggccgag ttgtgctgtt 600 gaaggtgcac tgctctgtgt ccaggcactt catggagggg agaggaggtg ttttgtcctt 660 ggagctgctg gcctgagaga accttgtcat cgtgggagct gggccattcc tacacagtgg 720 tctggcaatg acccggtggt ggtggaggcc tgtgagtggg cactggtaat gggaacagct 780 gtaaaaccct ggaggccagc cccaggagag tgaccttacc aggaaagttc tgggaaacaa 840 accacaggga ggctttacag gaatttttgg ttgtgcccac aggcaaggca catgaggaaa 900 agaaatgtaa ttatagtttg taagtcgatg aaaagaggca atgagtgaca tgaaatagct 960 gctctaagtt tcttcttcct gtcggacagg aagaaatggg gttttatgca tnnnnnnnnn 1020 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1080 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc ttctgaggga agactgagat 1140 taagcagata actgtataaa tgcataatta cacagcatgg tgagtgctct gaaggataag 1200 tgtggggagc ctcatttaga ttggaggatt gtgaaagtca agagacagga gagtcaaggt 1260 gaggcaaggt gagtaagagc tatccaggca aagactgctt ggtaggggag tgtcccagca 1320 acgggaaaca acctggaaaa aatatgacac ctcaggggaa ctaaaagcag ttgtatgtgg 1380 ctgatgcaca gacagggaag ggcaggaagt gtgctgaaag aaggcaggag gagaa 1435 88 459 DNA Homo sapiens misc_feature (437)..(437) n=a, c, g or t 88 gtctggtttg agtctaggat gaaggtacct tcctccagga aggccctggt gttccttctg 60 ccagactcct gagggtctcg ccagttcaag cccacttgaa gcccagctcg tttggggtta 120 cttgaaccat ctgggggatt ccaactagta tctttagctc ctgacatgag ctgttctact 180 gtgggctcag cccttgtctg agactgtatc cctatagggt cccggtcttc tgttgacccc 240 tcaccttctg tgggcctggg catggacctc tgatccttcc atctgaagaa gtgtcaaaat 300 aaaagtccat gcttccggga atcaggaagt cgcctcaagg caaaagtagc tgagtgtttc 360 tatatctgtt ttgttttcct ttctaacttc tctttttggt gggtaattct tcaccatctt 420 gttgattctt taagtcntag cataacacac attttaaaa 459 89 1263 DNA Homo sapiens 89 gtctggtttg agtctaggat gaaggtacct tcctccagga aggccctggt gttccttctg 60 ccagactcct gagggtctcg ccagttcaag cccacttgaa gcccagctcg tttggggtta 120 cttgaaccat ctgggggatt ccaactagta tctttagctc ctgacatgag ctgttctact 180 gtgggctcag cccttgtctg agactgtatc cctatagggt cccggtcttc tgttgacccc 240 tcaccttctg tgggcctggg gcatggacct ctgatccttc catctgaaga agctgtcaaa 300 ataaaagtcc atgcttccgg gaatcaggaa gtcgcctcaa ggcaaaagta gctgagtgtt 360 tctatatctg ttttgttttc ctttctatct tctctttttg gtgggtaatt cttcaccatc 420 ttgttgattc tttaagtctt agcataacac acattttaaa aatccagttg ttttagttgc 480 tttctgtctc catagaaggt caccatggtt ctcagccctg tcggacctgg agcctggtac 540 catgaccagg gacagggagt cctcatgccg ttttaagcag tggtgatcta agttttattt 600 cttaggtgag tcaaggtcgg aaaagcttga gacccctgct ctaggggctg tacctgtccc 660 tttctccctt ttctcctgtc tggactaggg ttcgaagggg ctggtgggcc atgtggagac 720 caagtagctg acaatcccca ggacctgtgg gctcagacac agggccctgc acctctcagc 780 ccttccggtc tcagctcagc acctcccttg cctggcccct ctttcctgca tgagctccct 840 gcctctgcca ggaggaacct ctgtcctgtt tctagatgcg ccatatcctc tcccacctcc 900 tgctctttcc tccagttgtg tgcctcgtaa cctcttcctc cctccaaggc taaatcaaac 960 cctacctcct tatacaggag gaagtaattt ctgggttgat gtatgcatcc ggcagattca 1020 tgctgagcca acaggttagg ggctggagaa acagtgatga gcttaaccag gccctgccag 1080 cctgcccacc ccgagtctgg tgagggtagc aaaaaacata aagtggaatt gataaataat 1140 ataatctatc catatccata tttttatttt ttattatttt gggacgaagt cttgctctgt 1200 cactccagcc tgagctacag agtgagaccg tgtctcaaaa aaaaataaga aaaaaaaaaa 1260 aaa 1263 90 554 DNA Homo sapiens 90 gctcgagctg ttttcttcag gtgagtagaa caatggcatt ttaaatctaa gaggcaccta 60 gtaaatacat ttatttcaat tcctttccta cataggggaa gaaacagagg ctgcaaaaga 120 tttagttagt tcaagaaaaa acagtataat ttggagtttt tgactttgtg agttttgtta 180 cggcgctgac attcattctt ttgtgcgttc agtgtattca aatcttcaaa tctagagcac 240 attgtatgct gggcagaagg cacagtactt gaggattcag tggacagtga tacagaaaag 300 gctgctgtcc ttgggcactg atgagcctcg ggctactaca agtaagcagg cagtggcagt 360 aggtggaatg agggctgcag gtcctggcat catggatacc aatttgggct tagaatggaa 420 gcggaggctt ccttgaagaa cagcggtcta agctgagact tgtaggaata gtggtaatta 480 acaaacagac aggaagaaga gctttccagg aagacagcaa aacataggca aaggtctgga 540 gaggagagag agca 554 91 435 DNA Homo sapiens misc_feature (406)..(406) 91 tattagtcca taaaggctat ttctagtatt aaacaatgct taagaatagc ttggatccat 60 gaaaactttt gagaaggagg acaaagcaga cggaacctaa tctctgaaca atttcaatta 120 catcttttac aagtggctgt tggctagtca ttaaaaatga gccattcaca cttgtggaca 180 ccttttttgc catgcagact tgacttgcaa agcctttatt atccctggtt aagaacagca 240 cagctaataa aaacgaatca tatggcttta aactacttgc atccaacagg gacatcctaa 300 aaatggtccg gatagtgact tcatgaccat ttaggctgca agtgccatag ttactaatga 360 gaacagatat ttccaaatgg cggcaataga ttatggaaaa tggagnaagg nnagagagta 420 ntttactttc agcta 435 92 580 DNA Homo sapiens misc_feature (556)..(557) n=a, c, g or t 92 aaaaaaactg tttagaaaac cttcatattt actctcccgt tcaaactatt ggccctgatt 60 tttacagata atcaaaagtc aggctgccaa acttattttc tttgaatttg gaatatcttt 120 taaaatttgc ctttttcttt cttattatta gtccataaag gctatttcta gtattaaaca 180 atgcttaaga atagcttgga tccatgaaaa cttttgagaa ggaggacaaa gcagacggaa 240 cctaatctct gaacaatttc aattacatct tttacaagtg gctgttggct agtcattaaa 300 aatgagccat tcacacttgt ggacaccttt tttgccatgc agacttgact tgcaaagcct 360 ttattatccc tggttaagaa cagcacagct aataaaaacg aatcatatgg ctttaaacta 420 cttgcatcca acagggacat cctaaaaatg gtccggatag tgacttcatg accatttagg 480 ctgcaagtgc catagttact aatgagaaca gatatttcca aatggcggca atagattatg 540 gaaaatggag naaggnnaga gagtanttta ctttcagcta 580 93 724 DNA Homo sapiens misc_feature (297)..(602) n=a, c, g or t 93 tactgatgtg cttttgattt gtctggaggg tgactactac ctctttgagg tgcctcctgg 60 gaccctcaaa atattaactt ttatactctg tgtagcctgt actttaagcc agaacattca 120 aagtacactg aagaaatgtg ttgaaaatct atgcaaccat tttcgcatta tgtactagca 180 aataaacaat ctttaatttc tggaattttc cattttcctc agtgatattg ttgattgatt 240 tgtagttttc tttctttgct aggtttcagt atcagggctg taccaatttt tttcttnnnn 300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600 nntgtgccat ctttatgaag tgaattatga agctttccaa tcttttttat tttgtagaac 660 agtttaaata cacaacaata tactaagttc ttagattgaa gctgttttta aatcacaaag 720 acag 724 94 586 DNA Homo sapiens 94 ctaagacagt ggccaatctg actgtgaaaa taagggcagg ctacactgga gagcagggat 60 agggacaccc ggggggcaga gatgtgggtc accttagggg aggacacact caggaggccg 120 gcccatgatg gcacatgaag gctgggagca cggtgctcaa ggatcagctc atcagggaac 180 ttgaccaaat ttagagcaag gccctttgat agtgtataga gatgtttgtt ctaagcagca 240 atagaaagct tctggaatct gttccattaa gaggtgatag aaacaaaata tgagtcgttt 300 tggagttgtt ttcagcagag tcacaatgat agcaccatta tagatatttt acagacataa 360 tcctgatctt ttgggtggat gaccagaatg tctagttggt tcactgagcc ctggttttga 420 cccaatatgg taattcgtga actcttagga ggccagaaat atcctaatcc tgtgcaaggc 480 agggaccctt ggactgtaac tgtcttgtct gcttttggtc gtgaaggaga ctcagaggcc 540 caaacaagaa tttaggaaaa agagcaatag gattgtgttt aaaaaa 586 95 491 DNA Homo sapiens misc_feature (480)..(480) n=a, c, g or t 95 aaataattta acctaggaaa agaaaaagaa aattgaaaat tggagctaaa ataatttgat 60 ttttccctca acagggttat tggctgtctt ttaagtgact aaaagagcgt atctttatgt 120 gaattttagg catggtcata tgattaatac aaggataaag caaccaaatg ctctcagtat 180 ttattcccgt gctatttgtc tgttttttag ttcatggagt attgtattgt acttggtaat 240 ttgatgcttt tgagatgtcc tttagacaga tttttaacta caggacttcc tctgtagaat 300 cgacaatgtg tttcactctc tgtggcattg acaatgtttt tgaatgccta attgttcagt 360 agaactccgt ggttattatt acaactttgt acattattat aaatatttta tattagttgt 420 atattccact gcagatagca accagaaaac taaaatacag aaatattaca tattagaggn 480 gattataatg g 491 96 634 DNA Homo sapiens 96 aaataattta acctaggaaa agaaaaagaa aattgaaaat tggagctaaa ataatttgat 60 ttttccctca acagggttat tggctgtctt ttaagtgact aaaagagcgt atctttatgt 120 gaattttagg catggtcata tgattaatac aaggataaag caaccaaatg ctctcagtat 180 ttattcccgt gctatttgtc tgttttttag ttcatggagt attgtattgt acttggtaat 240 ttgatgcttt tgagatgtcc tttagacaga tttttaacta caggacttcc tctgtagaat 300 cgacaatgtg tttcactctc tgtggcattg acaatgtttt tgaatgccta attgttcagt 360 agaactccgt ggttattatt acaactttgt acattattat aaatatttta tattagttgt 420 atattccact gcagatagca accagaaaac taaatacaga aatattacat atagagagaa 480 tataatgtac aaaaaaaatc ttgggagatg agtgctttgg gtttaattct atttttactg 540 aaaccagaga ataataggat tcaaatctac ctaatttttc tatttttctg attttccatt 600 ctgtatgctc ttctttgaat tttttccttg gtca 634 97 397 DNA Homo sapiens misc_feature (326)..(326) n=a, c, g or t 97 aataattagc caagttgtgg tgctttgagt tttttgagtc tgtggtttaa tatctgtcaa 60 caattttgga aaattatcag ccattttatt tgaagtcttt cttctgtcac atatttcttt 120 tccttataca attagaattg catttatatt agggagtttg atattatccc acagatcctg 180 gatgatatat ttcattttct tccttttctt tttcctagtg tttcagtttg gacgagtttt 240 atcgacatat ctttaaggtc actaatgatt ttctcagctg tgtcaagtct cctgataagg 300 ccaataaaga gactatatct attatngtgt ntttaanttc tagcatttcc attttattct 360 tagagtttaa nctctctaat gaaattaccc atcttat 397 98 342 DNA Homo sapiens 98 ataaagatgg ggtgagggaa gaaaagatga caaaaggaga ggaccaggca tgagaagagg 60 aagaggagaa tgcggaggag gctgcttgcc tgctgtggga tggatggcag gggcacttcc 120 ccagactcac ttttctcaga tgtaaaactg accagccttg tgccacagat gtgaagatag 180 ccccatagaa cttaaagagc agaccataac ttcccatgaa tgagagctac taacatttac 240 atctgaaaaa caatttggat acttacccaa gtctccaaca aacaaagtca cactgaagct 300 ggagagcaca ctcataacac ccggaaaaac attttttttt aa 342 99 873 DNA Homo sapiens misc_feature (338)..(528) n=a, c, g or t 99 ataaagatgg ggtgagggaa gaaaagatga caaaaggaga ggaccaggca tgagaagagg 60 aagaggagaa tgcggaggag gctgcttgcc tgctgtggga tggatggcag gggcacttcc 120 ccagactcac ttttctcaga tgtaaaactg accagccttg tgccacagat gtgaagatag 180 ccccatagaa cttaaagagc agaccataac ttcccatgaa tgagagctac taacatttac 240 atctgaaaaa caatttggat acttacccaa gtctccaaca aacaaagtca cactgaagct 300 ggagagcaca ctcataacac ccggaaaaac attttttnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 420 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 480 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnntg aggagggcaa 540 aaatcacctg gtgaccattg gacaggcccc agagacaaat cttcttacct gggcaattca 600 gaagggagcc aagaccacct ggtgaccatc aaacaggcca tctggaggca aaactcctta 660 tctggggaat ttagaagtaa tcaaacttcc ctagtatctg aagacggcat ctgatcatga 720 tacaggaact agaaagaaat catttaggca gttagtgagg gtgagggaag agagaggccc 780 tctcatattg tttatttagg ccattagtga gggtgaggga agagagagac cctctcatat 840 tgtttcatat tgttttatac tcagtacctg att 873 100 297 DNA Homo sapiens misc_feature (48)..(48) n=a, c, g or t 100 ggaaaaggcc cccttaacct tcctcctcag gcccactcag caaatgtngc cactttgtgg 60 ccactttgtg taaggcattc cagagatctg gtgaggcacc tatctacaaa tatttataca 120 cacacattca tatatggttt cagtcacaaa atggggtcat tctctcccct gacctatcat 180 ttagggcatt ggaacatggc tgcatgtggc tctgtttgtg agggtccagg ggatggacag 240 ggaggctctg cattattttg cttttaccaa cattgcagca tgaacgtttt tttaact 297 101 258 DNA Homo sapiens 101 aatataaata cgcctttaat agtaacacct aattacctaa caccatcaaa aatggggtgc 60 tccatgaaga agcacataat tcaaattatt gaagtttatc ccttctaatg accacataga 120 tttctcttgc cccattaaaa aattagataa tcagtatttc taggatagtt gttttcttcc 180 aaccaattaa ggcataatct atgtagcaga acattcagag gatgatgcct ggtcaacatt 240 tgaataaaca atcactgt 258 102 712 DNA Homo sapiens misc_feature (603)..(603) n=a, c, g or t 102 aatataaata cgcctttaat agtaacacct aattacctaa caccatcaaa aatggggtgc 60 tccatgaaga agcacataat tcaaattatt gaagtttatc ccttctaatg accacataga 120 tttctcttgc cccattaaaa aattagataa tcagtatttc taggatagtt gttttcttcc 180 aaccaattaa ggcataatct atgtagcaga acattcagag gatgatgcct ggtcaacatt 240 tgaataaaca atcactgtga tgttacctct atttaagatg actccaataa aacttctatg 300 gtttgcatta ttagttgatc agactttaag cattatcttt tgatagggtc aaggaacctg 360 tcttaactcc ccatctctga ccaaaatata cttgttttct ataagctata aagccagata 420 gccaatttta tgagaattgt ccctatacta tatccatgtg agcgatgagt gcctggcatg 480 aagatgcata aaggaggcag taatatacaa caactgaagc ataacctctg gagccagtct 540 tcttcagaca aatcccaatt ccattactca ctggccacct aaacaagcta cttaattcat 600 ctncctcagt tttcttcaac tgtttaatgg gtatgatcaa caaaccaact tcagtgggtt 660 atcataaata ttaataaatg agagaatgca tgtgaaacaa agctataagc aa 712 103 173 DNA Homo sapiens misc_feature (96)..(96) n=a, c, g or t 103 gaatgtggct ggtgagtagg cacttggtgt ggcagtgtgg ctagtgggta agaacatggc 60 tggtgattag gcatgtggtg tggcagtgtg gctggngggg acgagcatgg ctggtgggta 120 agaacgtggc tgggagtagn agcatggccg gtggttggga atgtggctag tga 173 104 688 DNA Homo sapiens 104 tctgaatgtt ttggtgaata aatctgttct tcagcaaccc tacctgcttc tccaaactgc 60 ctaaagagat ccagtactga tgacgctgtt cttccatctt tactccctgg aaactaacca 120 cgttgtcttc tttccttcac caccacccag gagctcagag atctaagctg ctttccatct 180 tttctcccag ccccaggaca ctgactctgt acaggatggg gccgtcctct tgcctccttc 240 tcatcctaat cccccttctc cagctgatca acctggggag tactcagtgt tccttagact 300 ccgttatgga taagaagatc aaggatgttc tcaacagtct agagtacagt ccctctccta 360 taagcaagaa gctctcgtgt gctagtgtca aaagccaagg cagaccgtcc tcctgccctg 420 ctgggatggc tgtcactggc tgtgcttgtg gctatggctg tggttcgtgg gatgttcagc 480 tggaaaccac ctgccactgc cagtgcagtg tggtggactg gaccactgcc cgctgctgcc 540 acctgacctg acagggagga ggctgagaac tcagttttgt gaccatgaca gtaatgaaac 600 cagggtccca accaagaaat ctaactcaaa cgtcccactt catttgttcc attcctgatt 660 cttgggtaat aaagacaaac tttgtaaa 688 105 977 DNA Homo sapiens 105 ggcttggaga gggtcacaga ggctagtagc tgtgtggact tgcaggcagc cccaaatgct 60 cacctatgtg cagagtcagc atgtcctgcc tcccctggta atgtggtcgc ctgcatctct 120 gtggccagcg ctctcgttca tcattcagtc tgatggcttg agtgcctcta tgtttgctac 180 atgctgagac cgtattctag tgccgtattc tggaggtact gggtgtacct acagatttaa 240 gaatgcaaat ctggaggtac acccagtgga ttcaaagtag tctcatagaa caaagagact 300 tatatagtga cctttgctgc atccactagt atacaccatc tgaggtctct tgaactgaaa 360 atgaatgtgg aagcaaggga acagtgtgat gttcagctct cagatctcac atggcatctg 420 atttggcttg aggtgcctcc cctcctctct gtcccctggc tgtgggctca tggattggca 480 gagcccagtt atggcttccg ttttacttgc tataatatcc agaggcaatg tactagtcta 540 cctagaaaat tgtgctcacg gcatcccttt gtcacattaa taagcattat ggacactacg 600 acattttatt aagtattttg ttctggtatc tacttgatta tagtaaatta tcaaaatcct 660 tatttagctc atggactctc attaaagcat gttctggaaa ccttggccat aggttaggag 720 cctgtaaagt ttgattcatt gcaagatata agtgattagc agttggtagt agtgacattg 780 atgggcccca ttaaaaggtc tattggatgt ggtggtggca tagcgatagg ttggagttgg 840 aggtcagcat ggatgtctct gatttagaac caagcttacc tttgcataac ctatagtgac 900 actctcttca tctccccacg ccttagccat gtctccctga ggttcatact gtttggaatt 960 tcacaggctc atttatc 977 106 500 DNA Homo sapiens misc_feature (357)..(357) n=a, c, g or t 106 cagagcaggc attgacctag atgtcttccc ctgccttcat tgggagggtg ctgagccacg 60 ggttccacct ctgccaaagg cacacctagg agactcctca tgtccagctg agaagagggg 120 gacacctcct gtctgagact gcagctcaca ctgctgcatg cttcctggac accatctctc 180 tgaccttggt cgcatctgcc tagcctgcag ctacgttctc tgacctccag ctcttcctct 240 ttctcccctc ggtaatacca aagtctcaag aacacagccc tcacttctag acagaaaggc 300 ctcaccagga cccacctgtg tggcccaggt gtgacctcat gtacaaacac atctccnaaa 360 atcaccntct cgtcatcatg gaccctagta ntatccatga gttaacnctn atttctgtgt 420 taatcggggn tgcagcacat tttggtgcag attcattgtg gctttggggt gccatttggg 480 actctccccc atgcacaatg 500 107 476 DNA Homo sapiens misc_feature (466)..(466) n=a, c, g or t 107 gccatctttc cactcattcc ttctcaaaag gaatgtagta ccatatagta gttaagaata 60 tagacactgg agccgatctt cttgagttcc aatagtggct cttctacttt ttaaatctca 120 ttttccttca tctttaaatt gaagatagta acaatctcat ggggttgtga taactaaggg 180 ggtaatgcat gtaaagtgct tagaaaatgc ctggacatag gaagctctaa gtttgctgct 240 actactgtta ttatggttac tattattaat cattgcaagg aaaatgtatc aacagatgaa 300 tttggttcaa tactgccttc tagttttgtg accttagaat ttataggaac aaaaaagatt 360 tgaagggagg ttgggctgga tcatagagag ccttgattcc atgttttagg atgtatacac 420 agtgagaagt ccttcaggtt ttggtcctgg gaagagttgt gaatcngaaa gttaac 476 108 834 DNA Homo sapiens misc_feature (824)..(824) n=a, c, g or t 108 ataagtatgc atgcttcata tacttcattt attctttctt ccttgaagcc tctcctcttt 60 attaggcact attcatttgt ctacttggta cctgtatttt tttaatgtca ctattttgac 120 agtaccaata aaggtaaagc cactcaatta cgcagggctc tctctttatg ctttgggtag 180 gtgcacctgt gcaactgagg ggacggtcag tgttatcaag gttacctgtt attacaagta 240 gaagaaccca caaagatcag gagagagctc attttcctcc attagtagga ggtaggacta 300 tacattcaca aacacgaacc ttaaaatagc tcacaaaata gtgtcataca tgtacccagc 360 catctttcca ctcattcctt ctcaaaagga atgtagtacc atatagtagt taagaatata 420 gacactggag ccgatcttct tgagttccaa tagtggctct tctacttttt aaatctcatt 480 ttccttcatc tttaaattga agatagtaac aatctcatgg ggttgtgata actaaggggg 540 taatgcatgt aaagtgctta gaaaatgcct ggacatagga agctctaagt ttgctgctac 600 tactgttatt atggttacta ttattaatca ttgcaaggaa aatgtatcaa cagatgaatt 660 tggttcaata ctgccttcta gttttgtgac cttagaattt ataggaacaa aaaagatttg 720 aagggaggtt gggctggatc atagagagcc ttgattccat gttttaggat gtatacacag 780 tgagaagtcc ttcaggtttt ggtcctggga agagttgtga atcngaaagt taac 834 109 498 DNA Homo sapiens 109 tttaaattgg gagttaagga tgagcacttt tactgtatta aaaaatactc accagttaaa 60 aaaaaatact cttttcccct tcctcggaca cctaaatcta agagaacaac tcctatataa 120 aaatgatata aaaatcatac attttggaag tatgtttcta actgttctga gaggctgcat 180 ggtaaagctg aagtgaaaga tgtattttaa atctgtatat atgagcaagt atatattgat 240 gattgaagct aggtgctgcc taaatacatg gcccagactt tgaggaatta tagtgtaatg 300 gctgggaata caggtttgga gtcacaccgt agagctgaaa gcttggcttt tatttagctg 360 tgggtccttg ggcaggatac gtaatctgtc tgtgcctgaa atacccacca cacccatcct 420 gtaatggggg gataataagc ctgcctatct catggggcta ttaagaattt tcagttaact 480 tttacttatg aagtgcta 498 110 259 DNA Homo sapiens 110 tttaatgtgg tttagtttta gtcacttaga tttgcttttt atggagtgac tggagtttgg 60 ggaggggagc agggaggttt ttcttttttt ctttataaca ctggctaaat attttaatta 120 ctgctataga aggaagaagc taaaagtatt gcattcacaa atattgcata gattatacaa 180 acacagaaat atatgcatat gcatgtttaa aatatatgcc acatatcaac accatgtatc 240 caacttgaat aaggtcatt 259 111 414 DNA Homo sapiens 111 atgaaaggga tgaggggaac tcaaagttac aatgtcctac ttggagcagt aagttcagta 60 gacatatcac ttgcctcatt aacatcaagc atcccaaaac ccagtctggg tcagttttgc 120 ccagagtggg gtttgtagaa cacgggttct cctgggatcc tatacctagc ccagaatcag 180 ttgcaaaagc caggccatag caaattgtcc tgccagccag atagcagaga atctgacggc 240 agcaggcaga aggagccgct ccattgcagt aagccaagat cgcgccactt gcctcattac 300 atcaagcatc ccaaaaccca gtctgggtca gttttgccca gagtgaggtt tgtagaacac 360 gggttctcct gggatctata cctagcccag aatcagttcc aaaagtccaa aaga 414 112 589 DNA Homo sapiens 112 ctgggcaaca ttggggagac tctgtctcta aagaaaaaaa ggagagctgg tggtgaaagt 60 gtgaaggacc caggaagtac agacactggt ggtcaaagaa caagggtagg agtgtcatca 120 aatgatagtg ttggcagcat gggagctgtg ggtagagagt gagataccta aatttatgat 180 ttctgggtgg cagtaacttc tagggtgtgg ctgtgggagt gggcctctga atggggtgga 240 ggagaaaatc attaaagatt agaaaatctt gggatttaga ggataggttg tgggatgggt 300 gatacacgtt agtgttgcat ttgcccaggg taacgccaag agttggcaga gaaaataata 360 ctgacctaga ctttaataaa ggatttggga atgacagaga agcaacagta aaaataaggg 420 ataattagat gtttgggtgt ttcgcctggc tgtgtctgtc ctgtgtctgg ccaattatta 480 caatgtattt acactgtaaa tacatgtaat tcatataata gttttataag tagcaaaatg 540 tagtttaata aaaaaccatc ttagtcttct tacagaatat ttagttacc 589 113 471 DNA Homo sapiens 113 cccaggctgg gggtcaggtg aggagggagc tgggatccag caagcctagt gaaacccagg 60 ggacagtgga ctcggtcaca tccaggatgg tgatcaacag ctgcatcatc ccgcttcctt 120 ctcaagcgac aattccagag ccttggccac acggtgcttg tatctttcgt attcagaccc 180 cctggggttc cagcccctta ctgccttcac tttcctctca ccccttgact catctttcct 240 gctacttgtc acttgagata cctaagatga tgtgtgttat ggagaggtta gagcaccagc 300 ttcagaacca ccctgtgact ttggcctagt cacctgacat ttctagactt tggtgtcttc 360 attcataaag gcagtgtgga ctgcttgctg atgttatcgt gaacctgaat tccttcttag 420 agtttctaag tgctttctgg ggattaacct tttaaatcct tgcagtagcc c 471 114 1032 DNA Homo sapiens 114 aatgagggag ctcttgagct cccttgatga gcaccacaca gggccctctg ggaagcagta 60 agaacccatc ccagggctca ataagaacct aacccagcct gggatggccc ttccctttct 120 gccaaggtcc ttcccatgcc aaacctcagg cccttatctt ggtatctgtc accacccacc 180 acccccccga cacacacaca gtcatgcaag ttgtaagaca gtgacagaag atttgaagaa 240 gaccaccaga gcaggggata gcagaacatg cagacttagg gggaagccag gcgttcatac 300 caaagaatta gacctgttgg gtacccaggc tgggggtcag gtgaggaggg agctgggatc 360 cagcaagcct agtgaaaccc aggggacagt ggactcggtc acatccagga tggtgatcaa 420 cagctgcatc atcccgcttc cttctcaagc gacaattcca gagccttggc cacacggtgc 480 ttgtatcttt cgtattcaga ccccctgggg ttccagcccc ttactgcctt cactttcctc 540 tcaccccttg actcatcttt cctgctactt gtcacttgag atacctaaga tgatgtgtgt 600 tatggagagg ttagagcacc agcttcagaa ccaccctgtg actttggcct agtcacctga 660 catttctaga ctttggtgtc ttcattcata aaggcagtgt ggactgcttg ctgatgttat 720 cgtgaacctg aattccttct tagagtttct aagtgctttc tggggattaa ccttttaaat 780 ccttgcagta gcccaataag gtaggtattg ttgttatccc cattttacag gtaaggaaac 840 tgaggcacag agagtaattt gcacaaggct tatggctttt tagtggagga gccaagagtc 900 aaattaagag tggttgagtc aggcatggtg gcccctgcct atagtcccag ctacttgaaa 960 gagtgaggtg ggaggatcgc ttgagcccag gagttcaatg ctacagagca agacctcaac 1020 tctttaaaaa aa 1032 115 440 DNA Homo sapiens misc_feature (428)..(428) n=a, c, g or t 115 ggactacatc catgttccac cacaccaggc tccaattaca ttttgacttc tccacttgga 60 tgtttaaaat gcttctcaaa tttaacatat cctaaagata attttgtgtc tccccacaaa 120 acttgctctt tttgcattca ttgctgtctt agttaatggc accaccatcc atactgttac 180 tttagccaga aacctttgaa acatcccaat tggtctttct gattttctct gtttcacaac 240 ttattctcca cagacaggat actccaaaca gtacccaaag ccattgtctc ttatactttt 300 caatctataa aatatacata cataagagta tataaaatat attataaagt aaatatccat 360 gtatccaaac acacaggttt agaactggga acacaatatg caaaagaata atattgggac 420 ccccctancc tcatgtcata 440 116 249 DNA Homo sapiens 116 aaaaaaagtt ctgacaattt gtttgctttt acattttcaa atttgtgaaa tgtagagata 60 attttgtttt caaatctttg taattccctg aagcaaatac tttcaagcca gttgcaaaat 120 gctgctttag aaataattca tataaacatg cttctctatt taatcacaag gggagatgtg 180 gagaatggat gttttatttt ttcagtagtt tttgctctat aaaaatatta aattgctatt 240 atgattact 249 117 1017 DNA Homo sapiens 117 gccctttttt ggtgtgcccg ctgaatgagc actccaggct gtggagttcg ggacatgcct 60 tggtttgtgg ggaccatgct gcctgcctgt cgagaccaag catcgatact gtgtgtctac 120 ctgatgaaag tgtccagtat gtgtctgcat gacttgggga cactaagaaa accaaaggga 180 ttagcaacaa agagagcttg tcacctttgt gcggaaccag ctggcatctc acagggacaa 240 cctacaacct gagctgctgc gtcctcacta aatctgggcc cctagggacc ccgttttact 300 cctgctctcc tggagcttat tacgggcctg gctaccaaag ggaaagaggg gaaaatagac 360 caggagcctt atgctagaac catttatttt gtttcacgtg atgcagacag agataaaact 420 gcaaatttaa tgaaacttta acaatcagta caatgtttct ccttaagaac tttgtaaata 480 gcatttatct ttcaagagtt ctttctctct ttttgtgatt attttataaa cttaaaggaa 540 aaagagaaaa agtcagtggt tccagcattt gctttagtct gtgacttaaa tggattataa 600 ctcttgaccg ctgacattta ccaagataaa tcagtggtca tagatgtgga gcttgatgtc 660 tcttcggctc tgggaccaat ccccttggac aaaagttttc ctgtgttctt agtattctga 720 actggctaca gcaactttta ggaaaataaa ggttacaaaa aaagttctga caatttgttt 780 gcttttacat tttcaaattt gtgaaatgta gagataattt tgttttcaaa tctttgtaat 840 tccctgaagc aaatactttc aagccagttg caaaatgctg ctttagaaat aattcatata 900 aacatgcttc tctatttaat cacaagggga gatgtggaga atggatgttt tattttttca 960 gtagtttttg ctctataaaa atattaaatt gctattatga ttactaaaga taaaaaa 1017 118 332 DNA Homo sapiens 118 ctgcctccac gtggattacc acatttctca cctcatccta caaggcagtt cctgtttcta 60 ttcccccttc acacaaaata acttcgtatg ttgttagtaa gcaggagaac cagcctttga 120 actcaggact gtttaaagac caaggtcctg gccactgaaa taaaacatct gcaactggca 180 gattaatgaa aggctctaga aggaaacaaa aaacccaaga gactgctggc agtgatagct 240 gagttttagg gggaaaagtt gttttagttt tccctgtata ctttcttgtg tagttttaaa 300 aatctacagt atttacactt tcaaaacaaa at 332 119 344 DNA Homo sapiens 119 gcgcagggga aattataggt ggctgtggtt gtaattacaa agttctgtca cgtcttcatt 60 gttaggagga aaagaattca ataatcctat cagttctgct gtaaaacaaa tgagctatga 120 aattctggtg aacactgatt ttatgtctcc attcttgagg acactgttag tttgttttca 180 tctgtatgcc ttgattagag caaataacct taaatatcct taaggaaact tagatataca 240 tcatttccag tttttatcaa atgtgaattt tttttgtcat actgcccacc taacatggga 300 tgttttctca gaatattgtt cacttatgtg tttgagtttc ttaa 344 120 718 DNA Homo sapiens 120 aaaaaatcat aatagtttat gatcttgaag ggtttaaaag tatttgatga agatgtcttt 60 tgaatttatt tgtaggtctt cttgtgtatt taaaagctaa gttatcttgt aatcattttt 120 ttctatacct ttgtcagtaa cctcttagtg atgaaataaa aaagattagg taatcatcca 180 gcaatgggga agaagttaag gaacaaagag ctcagattaa actagttttt agaatctaag 240 catttctgca tgaatttgaa tcatggaaaa caaaatgtag cactccaaca tttgatgcaa 300 aactaaaagt ggaatactgc tttgatattt gaatgaattg aaaaataatt aacatccttg 360 gaactgtatg taaagaagga cttcacaagt attatagata cccccaacct cagccctttt 420 cccatgtatc tctttgatca catccctacc tcatagatca cccatgtgct gaagactttc 480 agttctgtat cttcattcta gatctcctga actcaagatc agaatatctt tctgacttct 540 gactgtgtat ttctggatgt tatacaagaa cctcagctca aactcagtat tccctaaacc 600 attgtttttg aaactttatg ttggatgtga aatctgtatt gtagaataac attaaaaaaa 660 gaaagaatag tatgcaaaat atcagagtgc attgtatgta gcaagagtag gtattttc 718 121 2617 DNA Homo sapiens 121 atgtggaatc aacctacctg tccaggaaca gatgaagaga taagaaaatg cagtgtgtat 60 acacagtgga atgctcttca ccataaaaaa ttcacggaat catgtcattg cagcaacatg 120 gtggacaatg taagaaaagc tccccggaga agctgtacag aagctgcctc ctcagcagtc 180 agggccaggt accggagctg tttttaccca aggacagggc cggccccaag tcatcccaga 240 gctgccatgg caccccctca gtcgggtcct gaggaatcct acacaagcta cttatatcag 300 tgatcactag gataatccat agaacttttg ggaaagaagt ttaagacctt tctcccacca 360 tttcagcagg ataaattcca actggattag aaaatgaaat gttaataatg caaataagta 420 catatttata tctgtatata aaatacagtt gatatttgcc tggtgtttag gtgtctaaag 480 gactttctaa gcataaaggc aaaaaaaagt cataaaaatg ctatagcagt ttgagactct 540 atgcaggaaa gggcatcatc acgtgcatgg atgaatctgt atctaatttt aaacaatttc 600 caatggtgcc tgtttccttt tctttgaaaa tctctggaga aatagttcct cttgctgtgt 660 ctttctttag gcaagaattt ttactaattg atgtgtagtc tgaatcctgg ctaagtataa 720 accttttatt ttttatacct gttcttagtg aaaatgaaac tgtgactttt tttttaattc 780 cttttgttgg tcaaaaacta caattaactc ttctgagttt cttctctggc tgaacaaaca 840 atggtcccat tggcctttca gggaactcca ggccgtctca aaaaccttca tgtttcattt 900 cttttcagag ctcccaaaaa gaatagcttg ctcttgacgt tgtacatgtt agtggaatga 960 tcaggactac tttgcaaaga tgaaaaattt gtgtttctag tgatttgaaa atagaaatct 1020 gatgtaacta ttagatattg ggaaagaagg tgacgaaggt aggtatcacc gaaagcactt 1080 aacaattctg aataattctg tacttgattg catttatgtg tatcatagga acagttgggt 1140 ttccttgagt gttaaattat ttattcactt attccacttc aagccagcta aatgattgtt 1200 tccctgatgg caaaagtctc agattgattg cacagtttat ttggttggat tgtttatgct 1260 ctttttatta tttattctta tttcaccaat gaaaatatca ctaagttctt tggtttgttg 1320 acctgattgt acctactttg acaaatcact gcctttctgg acccagtttt ctcattaagt 1380 ggcagtgata acctgtcata cttacagata taaaaacatg aaagttaaag tattgggtaa 1440 tactttcctc ctatcttttt tttattttga aaaagataaa aaattggcat aatgtattag 1500 ttaagatgga ataatcatat gttgatatcc agccatttct tctctcaaat gataggaaga 1560 tttttatgtg aaactacttg tgagagatct taacaatttg tagttagaga aagcactatt 1620 atatcatttg gaaatgcaag aaacaagtta cctttggggc aacagaggcc cttgtcattt 1680 tctcaaaaga aggaagcatc agcattttga tgatgatgtt gagattgtag aaatgatgaa 1740 ggtgaaaaag ttattctagc ttatgtttag caaaatgaaa tgaacccaaa taataaaaca 1800 gttacaacat tgaatctctt tgggagaaaa aaaaaagata gaatgctaat gtccttcaga 1860 acttcttaaa ccagaacctt aaaaaaaaga gaagctttta aaaaatcata atagtttatg 1920 atcttgaagg gtttaaaagt atttgatgaa gatgtctttt gaatttattt gtaggtcttc 1980 ttgtgtattt aaaagctaag ttatcttgta atcatttttt tctatacctt tgtcagtaac 2040 ctcttagtga tgaaataaaa aagattaggt aatcatccag caatggggaa gaagttaagg 2100 aacaaagagc tcagattaaa ctagttttta gaatctaagc atttctgcat gaatttgaat 2160 catggaaaac aaaatgtagc actccaacat ttgatgcaaa actaaaagtg gaatactgct 2220 ttgatatttg aatgaattga aaaataatta acatccttgg aactgtatgt aaagaaggac 2280 ttcacaagta ttatagatac ccccaacctc agcccttttc ccatgtatct ctttgatcac 2340 atccctacct catagatcac ccatgtgctg aagactttca gttctgtatc ttcattctag 2400 atctcctgaa ctcaagatca gaatatcttt ctgacttctg actgtgtatt tctggatgtt 2460 atacaagaac ctcagctcaa actcagtatt ccctaaacca ttgtttttga aactttatgt 2520 tggatgtgaa atctgtattg tagaataaca ttaaaaaaag aaagaatagt atgcaaaata 2580 tcagagtgca ttgtatgtag caagagtagg tattttc 2617 122 373 DNA Homo sapiens misc_feature (74)..(294) n=a, c, g or t 122 gtattataat aatggcctta atgaataaca ttctctatat tcacacttat ttgcaatata 60 atactgccat tctnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnncactaa 300 tctcaaagag ggcatgatct tcaagaatta ataaccctct caagtctcta caatctaatg 360 caattacctt ggg 373 123 308 DNA Homo sapiens 123 gctgaaagcc cagagcagag ctgttctcat ggggaaggac cctgtcttcc ccatcatcct 60 aggcgttcat tgaggatgag gactgtcttc ctccatcaga ccgagagttc ccaagggcaa 120 gggctgtctc tccctggtca gacagggagc tccccgaggg cagaggtcct gtctcctcca 180 tcagactggt agcccccaca accacaaagc tatgtctact ttcatcagaa ggagctccct 240 aagtggggaa gggttctccc tattttcccc ttccaggtgg gaaattcctg gccagggtcc 300 cctgtctc 308 124 774 DNA Homo sapiens 124 gccaacacca aagggggcac gggagaagga caggaggggt ggtttccctc agcaagctct 60 cagtcccact gacactggcc caagagggct gagtgtactg ggcactcacg cagggagatt 120 gttcccgaag gccctcggga aagttggtga atgcaaacag caggcagcca gagagcctgc 180 tgcagaggag accagagacg atgccccagg agggcacaga agtgtgcaaa agactcagca 240 gtgggaagga gcctggtccg tgagtgtgag gagataaccc gggccctagg cccttcctgc 300 cccaactttc caccacctgg cccagcccct tgcagcggtg aggcttagca tctctctgct 360 gggtttgtga gagcccagac tgccccagtg agggtacagg agtactctcc ccaggcagga 420 agggtgggcg gcctccctcc aggtacccaa gaggaaatgt tagcagctga aagccccaga 480 gcagagctgt tctcatgggg aaggaccctg tcttccccat catcctaggc gttcattgag 540 gatgaggact gtcttcctcc atcagaccga gagttcccaa gggcaagggc tgtctctccc 600 tggtcagaca gggagctccc cgagggcaga ggtcctgtct cctccatcag actggtagcc 660 cccacaacca caaagctatg tctactttca tcagaaggag ctccctaagt ggggaagggt 720 tctccctatt ttccccttcc aggtgggaaa ttcctggcca gggtcccctg tctc 774 125 271 DNA Homo sapiens 125 aagtcgtacg catggttaaa aaaaaaagaa aagaaaatcc aaaatagtac tgaaggtatg 60 cagtacacag gaagcctccg cccacctcca cctcccagct tccccctttg gaggtatctg 120 ctgtagtggg ctcctcaaga tacttctagc catgctctgt ttgtgcatgc ttatccctgc 180 acagacagca gaagctgtct tggccaacaa gaccaggaag cattggtatt tgcaggttaa 240 ttgaaaaatt catttaaggt ggagaaccat a 271 126 1950 DNA Homo sapiens 126 atgatgccac aggatgagcg cacttcaaag ctggaaggaa gcctggtgag ggagcagggc 60 agaatcttct cctggactgt gagggtacat acggtggatg tgtatggctt cattgaagat 120 gccagtcctt gcattggcat ctgcagattt gaagaagtag gcccctcttc tagtcttcat 180 ggactggatt tggcaagaaa agtccttcat cagtcagcca ttcagaaact ctgggaagcc 240 tatctggtaa cgtccatggg caggcaaaat ttgccattca gctacaagaa gtgcagttgg 300 cagacagcct tcaacttcag catcttcaga gtctgccttg actttcaagc tgaggccatg 360 gacttctcag gagctcctag ccaatggctg agaacaacgt gtctaacaca tgttctcttt 420 ctctttgatg gccaaggcat ggctggccaa tgggatgctt ctctctccaa aggagcaggg 480 agagctggag ataccctcct tgcaaacagc agcttgagga tccagcgcct ggtgcacagc 540 ccacagcgac cccaagaagc tgctccaacc cctgggacta tggagctcta cagctgtaga 600 gaccaccagg aagtggactg caggcccctg gcctctccat tcagattctg caaagagatc 660 ctgatgggtt gggccaatgg gtcaggcatc cagtcagctc tggctaaggg agctgcctgg 720 tgccaggacg agcgtaacac ggacccacag tgtccccaga agggggcagg cgttctgaga 780 gccacaaagt cctggctgcc agtgctccct ggtctgatcc taaacccgtc ctcctggggt 840 gacagcttcg ccgtgagcgc tgcctgggct cggaagggca tcgaggagtg gatcgggaga 900 cagcgctgcc cgggcggtgt ctcgggaccc cgacagctgc ggttggcggg caccataggc 960 cgaagcaccc gggaaagaga ccctgagacg ctgctgaatg agcaaagcaa ctgcaaaaca 1020 ttcataggcc atggtcctgt ttcttacagt gtgaaaaagt ctattcaggc ctgtgtcact 1080 gtgtatctgc agatggttgg atcagagcac cttcttgtga tgtcacaaat cggggccttt 1140 ctagccttct taaccttgga ggttctgctc agcagctgct actggcgtct cgtcctcttg 1200 gctctgggtc tggggcactg gaaggtaaac tccctgctga gttggaggca gcagcattga 1260 gtgggtggct gttttccagc caggatttac ccagggcttt atggcttgca aagccttcct 1320 cacagggctt tgtcaggcat ttaatattca caaaaatgtg gccaggatca aaattattat 1380 tatggggaaa ctgaggccag actgtaaagt ccacaggtca ggttctttgt ggctcactct 1440 tgtatccctg ggccttttgc actgattggc acatggcaga tcctcaagaa cattttccag 1500 gtggatgagg ttcagagggg ccatgcagct tggccagagg gcacacagcc agagaggcag 1560 ggattctgtt ctgttctgtc caagtcccca cctcttttat ggagccaggc tgttctgtgt 1620 ctttgaagag agcctctgcc cttcagaaag ggtcctcacc tttttccttt ctgtaaatta 1680 agtcgtacgc atggttaaaa aaaaaagaaa agaaaatcca aaatagtact gaaggtatgc 1740 agtacacagg aagcctccgc ccacctccac ctcccagctt ccccctttgg aggtatctgc 1800 tgtagtgggc tcctcaagat acttctagcc atgctctgtt tgtgcatgct tatccctgca 1860 cagacagcag aagctgtctt ggccaacaag accaggaagc attggtattt gcaggttaat 1920 tgaaaaattc atttaaggtg gagaaccata 1950 127 209 DNA Homo sapiens 127 gttgggtgtg gtggggtgtt ttgttgttaa tgttgttttt gccagtctgt gttgataaga 60 tttattattg agaatagtgc ttgttctctg agtactcctg acttagaaaa ggagcatagc 120 cctactaaag gggacttcaa agtagaaatc gtcaataacc ttttacttgc tacagttagt 180 ggcctcaaca tgatgttttt aaagatctt 209 128 496 DNA Homo sapiens misc_feature (101)..(403) n=a, c, g or t 128 gcctccggtg gatggaatga agcaaggatg ggggctgcct gcagagctgt gtcactcact 60 tgtattcagc tttcctgcct ctggctctct gtcttttacc nnnnnnnnnn nnnnnnnnnn 120 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnttttatc acttttaaaa 420 ctaagaaaac aatgatcacc atacatgctc tgcttccaaa ctatactttc acatccaaag 480 taaccccaga ttcata 496 129 252 DNA Homo sapiens 129 catttctaac atttattgtc ctccagtaca aagaagtaac ccattgtcat gtctactcta 60 tgataggcta gaactatagg gttgctctat attgatcagg tttttaaaga taaaaatgaa 120 aaaaaaatcc tatccagaca aaataaatca gtgttttata tttttggagc atcagaactt 180 actttaagac ctcactggta attctttagc ctctcacatg tgataaagac attgtgctta 240 cattttttta aa 252 130 149 DNA Homo sapiens 130 atcagaatcc tgggaagggt ttgttaaaac actactaggc agggtgaggt aacctaagag 60 cttttggagg cccaggtgag agggatcact tgcggccagc agagttcaag agcagcccag 120 gcaacacagg gagacctctt ctctacaaa 149 131 390 DNA Homo sapiens misc_feature (217)..(273) n=a, c, g or t 131 agcaagtacg cagcattggg aatgaaccaa actcgtagga ggcacagccc actcagtgtg 60 cgggcccggg cgagctgcag gcctgaaacc cacccaccct cttagatgtg tctgtgggcc 120 atagaaatta ctagggttgt cttgggtgtg gcctcaacct gttcaacaac aggtgtgctg 180 tttccattct ggaaaccagt cctctgtctt ccagaannnn nnnnnnnnnn nnnnnnnnnn 240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnntactagg cagggtgagg taacctaaga 300 gcttttggag gcccaggtga gagggatcac ttgaggccag cagagttcaa gagcagccca 360 ggcaacacag ggagacctct tctctacaaa 390 132 1079 DNA Homo sapiens misc_feature (874)..(874) n=a, c, g or t 132 gggatgaaaa cttcctttaa aagaatcctg ttgtatttta atattgttcc ggggttcttt 60 gcatatgtat atgctctata tgaacaatac tgaaatgaac atccatatct atgacctctc 120 tctgcactcc aggctcagat atgcaactcc ctatttgaca ggtctgcttg aaaacttgct 180 gggcatccca gaggtaacat ggatctaatg gaaggtttga ttttgtcctc caagccagtt 240 cttcccttga ctttctacat ttcaccaaat gataccccaa ccactcactt attctagccc 300 aagatctagg agttattctt aggttttcct ttaccccctc cacatggatc catcagcagg 360 tcttgttctt ttttcttccc aaatatatct caagtccatg ctcttctgtc tgtccctact 420 gccactatcc aagctctgag gccatccatt acatggacaa ctataaacta catgtcctaa 480 tgacatatta gcagtagagt tgctaggtca aaagatttgt gtgttttatt ttgatagact 540 ttgctacatt attctcaaag aggctttctc agtgttatct gcttattata tgagaatttc 600 tgtttctgta ctctgtcacc accactgaat atcagggtca ctcttagccc atagcctcgt 660 gagaattaga agtcacttcc tctgggtgag gcagctagct ccacagcaca gacttaacaa 720 gtggaacttt agcatgtatt taattcccac tcattctctt acctatgtgt ccttctgcag 780 tcaacactct acacaactgt acatgaccac aatgctgtgc ataaataatt ttttagactc 840 tttgtaaatc tatatgtaaa aaatggcatc ttantttgna taagnanggn ggangncant 900 taaaattcct tttccttgga ntgncnaatt nanagacttt cctnattttn agggttccta 960 acaaattgga aaatncnggg gttaaccnaa ggncnatcat atatttnacc atnaaaaatt 1020 ttttcctggn accttangtt tgttaaaagn acttttttat ngaaaccttt aaattttta 1079 133 303 DNA Homo sapiens misc_feature (295)..(295) n=a, c, g or t 133 ttaagtattc aatttctgtt ttaaatgcca agaggtagaa attaaaggta ggcatggtgg 60 tcacagtcca ctaaaaaact agtattccaa cttctattcc ctggcacact actaaatagg 120 caaccaggga tttaaaaaat ggtttctggt gtccaggtaa gtttgcataa aaccaaaata 180 aaactgttta atactgggcc cactacatta atctatggtg ctaacacgtg ctgtgaaccg 240 tggggtcagg ggctggggga taaagttgca accatttttt ggggggttgg gggangagga 300 ggg 303 134 546 DNA Homo sapiens 134 ccggcaaatt taaccaaaaa aaaaaagtaa tatgaccata attaatatca gtcaaaatat 60 tctttaaagg aaaaaaatac taataagaga actctataaa aataaagaat ataataaaaa 120 gagatcacat ttgcaaattt acattgttta atatcatagc ctcaaaataa attgcatata 180 aattttaaaa cctatggaga aattgacaaa tccaccaaca ctgtgggaaa tttttaatac 240 atatctctta gctattaatg cataaagtag gtaaggaaaa ccaataggat gcaaataatt 300 tgaacaataa aatcaacaac tttgatttag ttgatataca tatacagaca cttgcattta 360 gtaattggaa aatatacatt attttccaac acacacaaaa aaacacttgc aaaaatgggc 420 tgtgtcttaa atttttcaaa gaactgatat catacagaac acatgttatg accataatgt 480 agttacatta gaaaatgtgg cagggattct gattctcctt tctgtgctag ggcatacagt 540 taaatc 546 135 590 DNA Homo sapiens 135 aaaaaagtaa tatgaccata attaatatca gtcaaaatat tctttaaagg aaaaaaatac 60 taataagaga actctataaa aataaagaat ataataaaaa gagatcacat ttgcaaattt 120 acattgttta atatcatagc ctcaaaataa attgcatata aattttaaaa cctatggaga 180 aattgacaaa tccaccaaca ctgtgggaaa tttttaatac atatctctta gctattaatg 240 cataaagtag gtaaggaaaa ccaataggat gcaaataatt tgaacaataa aatcaacaac 300 tttgatttag ttgatataca tatacagaca cttgcattta gtaattggaa aatatacatt 360 attttccaac acacacaaaa aaacacttgc aaaaatgggc tgtgtcttaa atttttcaaa 420 gaactgatat catacagaac acatgttatg accataatgt agttacatta gaaaatgtgg 480 cagggattct gattctcctt tctgtgctag ggcatacagt taaatcacat tttcaccttc 540 cttgtattta tgagacttag ctctgtcctt atgaatgtgg gcagaagtga 590 136 165 DNA Homo sapiens 136 gctcgaggcc tggcatctga gttcttctgt tcaggagaaa cactttcagc aggccattga 60 gagggtcatc ggaggtgagc ctgggagccc ttagggaggg aggggtgttt gcagctctgg 120 gcctggcagg ctcaccccct ggccccagtt tcaattctgc atgca 165 137 172 DNA Homo sapiens 137 tagttacagt ccttaaatat atgtcttggg tgccctgtgg ctgtgatttt ttaagggaaa 60 ttaacttatt ttaaataaaa taaacttaat ttaaaataaa attttgttat ctaaagccaa 120 atagaaaaaa ttccacattt tttcttacag tgctcattca tcagaacctt tt 172 138 809 DNA Homo sapiens 138 agtacgtaca gtatcaaaca gtctccctcc ttttctctgt gatttggtct ttctccttag 60 agaatgtcct ccctccaact ccaaaagaca tgcctctgtg gtatagttac agtccttaaa 120 tatatgtctt gggtgccctg tggctgtgat tttttaaggg aaattaactt attttaaata 180 aaataaactt aatttaaaat aaaattttgt tatctaaagc caaatagaaa aaattccaca 240 ttttttctta cagtgctcat tcatcagaac cttttttttt tcttcttatt ttttcttttt 300 ttggggagaa tgggtcctcc ctttggtgcg catcaggggg aataagaggt acaaacaggc 360 ggtgattata cgctcacttg ggagtttgga aactccgggg gcatcattgg gattcccatt 420 ttgtcctcaa gcctccggag tagctaggac atacgggttt tgcaccacaa ggccgggata 480 aatttcaaaa tttttctcac gagacaaagt ttgggattct tggccccagg attgggacgg 540 ggtatatcac aaaagaaact atttcagggg cgcttagaga ggctcaagtg acacctactt 600 atcaggggtt tccagtggag agaactgtac cctaccctta ctacctttta agtggtgcct 660 ctccctccac ctttaacctt tacacattac ggaactggcg ctatcatttt aaagtcaact 720 aacctggact ttggacttct ttaacacttc agctccggga tccaaactaa aatcttaggc 780 aaggcctaat ggacggtaga agtctacgc 809 139 294 DNA Homo sapiens 139 gtctttttca ttcatagtaa ccctgcaaaa caaacatata gaacagagac attatggaga 60 cttgaggatt gattttatgt attgattatg tatgtaagtc ccgataacat ctctggttca 120 ggaaattgca agaaaaagat tgggaatcag aacagcagaa aggtattttt ggaagggtaa 180 tttactgatt tttcgtttta aattgttgac attgccttcg ccggtggaaa tgaattactt 240 atgtgaatct ggcaggaaca caatttttaa aattagaaaa ttagtcctcc ttat 294 140 1056 DNA Homo sapiens 140 acctaaacac attttaatta tattttgctc atttttggag aacccattcc ctttgacatc 60 tattatgaac attctaaaac ttaaatttgt gaaaacaaaa ctctgggaga tagattgtaa 120 ttttattcca tgaggaaggt gttaaaccag ctttgcagtt tgaattttat tcttaaaggc 180 tctgcagttc ttacctggat gtcgaaatga tttttaattt caactgctgt agacctcatc 240 ctgtgggaac tagaaataat gtccaactgc cgtccagtct ggcgacattc cagccgttcc 300 cccaccccac gataacggcc tgactcttcc tcaattcatg acagcccatt ctacacataa 360 cctttctcct ctggcaccgg tcctcccagc agagagggat cctgcccttc ccttcccact 420 ctccagcata cagaccagca ggaagccaca agagggaaaa acaaaagcct tctgtataag 480 gcctatgaaa ggaccatggg ccagcctcag aatctgctgc ccctacaaac cagtattcct 540 caaatgatag ttccacattt acttaataag gaggactaat tttctaattt taaaaattgt 600 gttcctgcca gattcacata agtaattcat ttccaccggc gaaggcaatg tcaacaattt 660 aaaacgaaaa atcagtaaat tacccttcca aaaatacctt tctgctgttc tgattcccaa 720 tctttttctt gcaatttcct gaaccagaga tgttatcggg acttacatac ataatcaata 780 cataaaatca atcctcaagt ctccataatg tctctgttct atatgtttgt tttgcagggt 840 tactatgaat gaaaaagaca atttcatgaa tgcagaaaat ctggggatcg tgtttgggcc 900 cactctgatg aggccccctg aggacagcac cctgaccacc ctgcatgata tgcggtacca 960 aaagctgatt gtgcagattt taatagaaaa cgaagacgtt ttattctaat ccatcaggga 1020 aatgagctga atggccccag caccatccaa gttgac 1056 141 968 DNA Homo sapiens misc_feature (319)..(319) n=a, c, g or t 141 acgagatgtc ccagtaacct aaaattatcc agtcggtctt cttactttac aactaagaaa 60 aataaggctt agaaagaggg attgccagaa actttggcag ctggattgcc tgtgcttgtt 120 cctctaagcc atacctaaat tctgcagtaa atacttaact ttttaatagg gaaattgctt 180 caagataact tgaccagtga tacggtaaaa taattagact attggactaa tggtttaaca 240 caagtggctt taaaaagtct gcttaaaaaa caatttttat ttagaaaaaa tagaaaaata 300 aaaacatctt caaaatttng gagcctgaag gggctgtttg tttcatatat ggataatctt 360 tgaaaaggca agtcctgtat gtatttttca tttgttgaaa gaagattggt tatcagtagg 420 cttgcaaaca taatttgctt ttaagttctt tcaaggtttt atgcaataaa acctattgat 480 ttggaacttt aaaaaanaaa acaacaaaaa aatactttca gggttttgta atttcaagtg 540 gttttttaag gggagcaata gtttgccatt taccaaaggc ttctccagat aatttcttaa 600 atgtttctac ttaaaaataa aagctattaa taataagctg tcatgggatc catttgaaga 660 cagggaaaat agaaaatttt tattgtaaag ggaagaactt atccttttaa ttttatggac 720 taacagagtc tgcaggtctt aactcatttc agcctgtcaa atgtgcaatt aaaaatgaat 780 tttctaattg tattcaaatg aggctctata gtgaatacag aatcactctt ctaagttttt 840 tcccagttaa tttgtttaaa agtgttgtac tctcttgcaa gaacgtttaa aagttaagtc 900 ttgtaactgt taacatctaa tgtattaata taagccattt gttttttacc atttttttaa 960 ggccgtat 968 142 1466 DNA Homo sapiens 142 gaaaatttga gtatcttttt gaaattttaa attgaaattt ggatagagat ggttatggag 60 agaaatcaaa caactggaat agctgtttga tatcacttaa aagtgataaa attttaagtt 120 gaatctggtc agtttgcaat ggcctatttg taagaaatat caagacttct tgagaaaaat 180 gaaaagtgaa tacataaatg cttaaaatct ggtacttctg agttaaggtt ttgctctttg 240 agcttaatcc aatttgggat gatttttcat cctagggctt tttgttttcc ttttttattt 300 ttattttttc tttttttagg ggaaggggac ttgctttctt ttccaaaaag gtgaatcctt 360 cttgtaggac ataggtaaaa aaaacaaagc tgaaatatat gttttgaata tagatagcta 420 attccctggg atataatatc ctttcaattt tttttttttt ttgggcccag tctgcctttg 480 gatgtttcaa aagtctgaac gagatgtccc agtaacctaa aattatccag tcggtcttct 540 tactttacaa ctaagaaaaa taaggcttag aaagagggat tgccagaaac tttggcagct 600 ggattgcctg tgcttgttcc tctaagccat acctaaattc tgcagtaaat acttaacttt 660 ttaataggga aattgcttca agataacttg accagtgata cggtaaaata attagactat 720 tggactaatg gtttaacaca agtggcttta aaaagtctgc ttaaaaaaca atttttattt 780 agaaaaaata gaaaaataaa aacatcttca aaatttagga gcctgaaggg gctgtttgtt 840 tcatatatgg ataatctttg aaaaggcaag tcctgtatgt atttttcatt tgttgaaaga 900 agattggtta tcagtaggct tgcaaacata atttgctttt aagttctttc aaggttttat 960 gcaataaaac ctattgattt ggaactttaa aaaaaaaaac aacaaaaaaa tactttcagg 1020 gttttgtaat ttcaagtggt tttttaaggg gagcaatagt ttgccattta ccaaaggctt 1080 ctccagataa tttcttaaat gtttctactt aaaaataaaa gctattaata ataagctgtc 1140 atgggatcca tttgaagaca gggaaaatag aaaattttta ttgtaaaggg aagaacttat 1200 ccttttaatt ttatggacta acagagtctg caggtcttaa ctcatttcag cctgtcaaat 1260 gtgcaattaa aaatgaattt tctaattgta ttcaaatgag gctctatagt gaatacagaa 1320 tcactcttct aagttttttc ccagttaatt tgtttaaaag tgttgtactc tcttgcaaga 1380 acgtttaaaa gttaagtctt gtaactgtta acatctaatg tattaatata agccatttgt 1440 tttttaccat ttttttaagg ccgtat 1466 143 306 DNA Homo sapiens misc_feature (289)..(289) n=a, c, g or t 143 gacacagcct atctcaaaga gagatgagaa gagccaggcc ccctctcttc ttcctccatg 60 ctgttagctc accagggcag atcttgacct caaagaatgc cgtcttccct tctggagctg 120 gtcctgtgat gtgaacctgg ctatcttcaa ttcacaggat agggagtaag acatttcatt 180 ttggccttag gtccaagcca tcttcttcaa tgtagctact actagagagc ccacaatgaa 240 gccaataatt ggctccccat ttggcaattt gtgtcctttt cagaaagang aagggttagt 300 aatcac 306 144 494 DNA Homo sapiens 144 gacacagcct atctcaaaga gagatgagaa gagccaggcc ccctctcttc ttcctccatg 60 ctgttagctc accagggcag atcttgacct caaagaatgc cgtcttccct tctggagctg 120 gtcctgtgat gtgaacctgg ctatcttcaa ttcacaggat agggagtaag acatttcatt 180 ttggccttag gtccaagcca tcttcttcaa tgtagctact actagagagc ccacaatgaa 240 gccaataatt ggctccccat ttggcaattt gtgtcccttt tcagaaagag gaagggttag 300 taatcagcac ttttaagtac cagcatgcag cattaacaag ttctcaaggc ctgcaagcca 360 tagggtttct gtcttccctg tattggcctt gtaatctctg accatgatta gggtaagagt 420 taagagactc ccaggacagg aaacggaaaa catcagattg tgtatggaat gaaccctctt 480 ggctggatgt ggtg 494 145 174 DNA Homo sapiens 145 gtggaacaac tctatgccat aaaatttctt atttcacagt taaatgaaca tatttgtgtt 60 atgtcacttt cttttagctt gcattccttt tataggaagg ccattttagg agtcctgggg 120 cattttgact caacttctta aatcatttat tctattcaca aaaggtttat tgaa 174 146 445 DNA Homo sapiens misc_feature (371)..(371) n=a, c, g or t 146 tgatttttaa caattgtgtg tgtgcaccca gctaaccatc tctacaatcg atctagaaca 60 ttttcatcac ttcagtgctt ctcgtatatt ccttcccagc taacccatga tccccaaccc 120 tggccatagg aacccgctga tccatcttct atcactttag attgaatttg tctttcctac 180 tgttttatat aaagaaatta cctcctttaa gtcctatcaa attcctgatc acccttaaaa 240 aacaattttt aggtattacc ataaaacctt ccatgacatt ctctgcttta tcttctctgt 300 gctactttgt ccattcattg ttgcattgta atgtatttct gtacatgtta tatcactaaa 360 ctgtctcctc nttgaaggga gggacatgtg ntcatcatct atttcnaagg cttatacaga 420 aactganaca tagtagatgc ttact 445 147 734 DNA Homo sapiens 147 tgatttttaa caattgtgtg tgtgcaccca gctaaccatc tctacaatcg atctagaaca 60 ttttcatcac ttcagtgctt ctcgtatatt ccttcccagc taacccatga tccccaaccc 120 tggccatagg aacccgctga tccatcttct atcactttag attgaatttg tctttcctac 180 tgttttatat aaagaaatta cctcctttaa gtcctatcaa attcctgatc acccttaaaa 240 aacaattttt aggtattacc ataaaacctt ccatgacatt ctctgcttta tcttctctgt 300 gctactttgt ccattcattg ttgcattgta atgtatttct gtacatgtta tatcactaaa 360 ctgtctcctc cttgaaggga gggacatgtg ttcactcatc tattttcaag gcttattaca 420 gaaactgaaa catagtagat gcttacttgg gaatattata tctcaaaata gaaaaacacc 480 cagcaaatcg catcttatat tagtctttag aattagtatc aaagcctaat tattatgaca 540 cttgaaacat taaataactt agaaaacaaa gacttaaaag ttttatgata aagccagaaa 600 ctttttatac tgaccatttt taaatactga catttcagat taattggggg cagatgatat 660 atgaaattat agtttatact gtgacttctt aatacttcag ttgtgttaga taaactgata 720 gttcgtcaca tttt 734 148 29 PRT Homo sapiens 148 Met Leu Lys Ile Ile Asp Lys Leu Tyr Phe Ser Tyr Leu His Ser Ala 1 5 10 15 Asp Ile Leu Cys Asn Thr Glu Ser Tyr Thr Leu Ser Met 20 25 149 87 PRT Homo sapiens 149 Met Gly Trp His Glu Ile Gln Ile Pro Val Leu Ile Phe Leu Leu Ala 1 5 10 15 Val Tyr His Arg Thr Ser His Phe Thr Ser Leu Pro Leu Gly Pro Gln 20 25 30 Phe Ser Val Phe Leu Ile Tyr Lys Tyr Ser His Pro Ala Phe Arg Gln 35 40 45 Val Leu Arg Leu Asn Lys Glu Phe Asn Leu Leu Trp Leu His Ile Lys 50 55 60 His Ile Leu Val Ser Val Cys Leu Val Ile Ser Asn Ala Asn Ile Leu 65 70 75 80 Ser Ala Pro Cys Pro Glu Cys 85 150 45 PRT Homo sapiens 150 Ser Ser Val Ala Leu Ala Leu Gly Ala Leu Thr Val Trp His Ala Val 1 5 10 15 Leu Ile Ser Arg Gly Glu Thr Ser Ile Glu Arg His Ile Asn Lys Lys 20 25 30 Glu Arg Arg Arg Leu Gln Ala Lys Gly Arg Val Ser Arg 35 40 45 151 152 PRT Homo sapiens 151 Met Val Pro Glu Val Leu Ile Leu Cys His Gly Leu Ala Val Trp Lys 1 5 10 15 Trp Phe Pro Gly Leu Ala Val Leu Arg Ile Pro Gly Cys Val Thr Gly 20 25 30 Asn Lys Pro Phe Asn Leu Pro Gly Thr Val Phe Phe Cys Lys Met Arg 35 40 45 Gly Leu Gly Ala Ser Phe Leu Arg Pro Trp Gly Leu Val Ala Glu Phe 50 55 60 Ile Ser Pro Thr Pro Cys Pro Ser Ser Tyr Gly Ser Thr His Lys Ala 65 70 75 80 Phe His Ser His Lys Glu Lys Ala His Lys Val Pro Gln Pro Pro His 85 90 95 Thr Gln Glu Pro His Leu His Pro Ser Leu Lys Ala Arg Leu Pro Leu 100 105 110 Pro Gln His Thr Gln Val Leu Leu Gly Leu Pro Ala Leu Phe Ser Ser 115 120 125 Ser Pro Glu Trp Asn Gly Pro Ala Met Ala Ser Gln Arg Thr Ala Ser 130 135 140 Trp Gln Ser Trp Glu Trp Val Glu 145 150 152 29 PRT Homo sapiens MISC_FEATURE (14)..(14) X=any amino acid 152 Met Gly Leu Arg Val Leu Leu Leu Leu Gly Leu Ser Leu Xaa Met Ser 1 5 10 15 Gln Lys Pro Leu Xaa Gln Arg Pro Thr Ala Leu Gly Pro 20 25 153 46 PRT Homo sapiens 153 Met Phe Leu Val Glu His Lys Val Cys Ser Gly Asn Thr Gln Val Ser 1 5 10 15 Ile Lys Cys Leu Pro Val Val Ser Glu Lys Phe Val Met Lys Tyr Phe 20 25 30 Gly Asn Arg Cys Ile Val Ser Val Gly Gly Ala Asp Glu Phe 35 40 45 154 34 PRT Homo sapiens 154 Met Thr His Ser Glu Leu Leu Leu Val Ile Thr Ile Asn His Lys Met 1 5 10 15 Pro Gln Gly Pro Arg Val Thr Asn Trp Glu Pro Pro Pro Leu Thr Arg 20 25 30 Ile Thr 155 99 PRT Homo sapiens 155 Met Asp Ser Phe Leu Leu Leu Arg Gln Arg Glu Gly Gly Lys Arg Asn 1 5 10 15 Phe Lys Arg Asn Leu Gln Thr Cys Cys Ala Val Gly Pro Thr Gly Ile 20 25 30 His Gly Gly Glu Thr Asn Ser Ile Met Leu Leu Gln Ile Leu Leu Lys 35 40 45 Lys Gly Phe Asn Cys Leu Thr Lys Tyr Ser Ser Phe Phe His Leu Leu 50 55 60 Thr Leu Gln Pro Asn Gln Val Pro His Thr Thr Gly Arg Cys Arg Glu 65 70 75 80 Ile Pro Gln Pro Glu Lys Ile Ile His Ala Gly Gln Arg Gln Lys Phe 85 90 95 Thr Pro Gly 156 55 PRT Homo sapiens 156 Met Gln Phe Leu Leu Cys Leu Ser Leu Leu Asp Phe Phe Ser Ser Thr 1 5 10 15 Tyr Lys His Ala Val Met Ser Pro Asn Gln Lys Lys Cys Lys Asn Pro 20 25 30 Phe Ser Pro Met Leu Thr His His Pro Ala Val Val Leu Phe Leu Pro 35 40 45 Phe Thr Leu Leu Tyr Tyr Ser 50 55 157 59 PRT Homo sapiens 157 Met Leu Gln Val Asp Val Cys Thr Leu Met Val Arg Thr Trp Ser Ser 1 5 10 15 Trp Pro Cys Trp Val Phe Ala Lys Glu Thr Val Leu Cys Ser Trp Gly 20 25 30 Arg Phe His His Leu Ile Arg Ala Val Val Pro Thr Trp Cys Ser Leu 35 40 45 Asp His Leu Tyr Lys Met Phe Ile Gly Gln Gly 50 55 158 62 PRT Homo sapiens MISC_FEATURE (41)..(41) X=any amino acid 158 Met Thr Lys Arg Met Glu Lys Cys Leu Asn Ile Tyr Lys Arg Leu Asp 1 5 10 15 Val Tyr Arg Gln Ile Val Ser Lys Gly His Arg Ile Val Arg Asn Ser 20 25 30 Val Ile Leu Phe Cys Val Ile Asn Xaa Pro Phe Leu Tyr Pro Phe Thr 35 40 45 Leu Ile Ile Asp Ile His His Phe Xaa Val Ile Ile Gln Leu 50 55 60 159 47 PRT Homo sapiens 159 His Leu Asn Arg Phe Ala Asn Ser Val Lys Val Phe Thr Arg Arg His 1 5 10 15 Ala Phe Val Lys Lys Phe Phe Arg Gly Ser Ala Cys Asn Cys Ala Glu 20 25 30 Ser Ala Leu Leu Ser Ser Gln Leu Ala His Cys Val Gly Arg Trp 35 40 45 160 43 PRT Homo sapiens 160 Met Gln Glu Ala Glu Gly Arg Leu Asn Lys Pro Gln Gly Gly Arg Val 1 5 10 15 Gly Ala Glu Arg Val Gly Asn Ile Phe Phe Leu Leu Leu Asn Ser Arg 20 25 30 Lys Ala Lys Thr Gln Ser Lys Leu Phe Leu Ser 35 40 161 62 PRT Homo sapiens 161 Met Phe Gly Ile Leu Glu Lys Ser Ser Lys Tyr Val His Leu Glu Gly 1 5 10 15 Ser Leu Lys His Pro Val Ile Lys Leu Val Ser Ile Ser Val Val Lys 20 25 30 Asp Glu Tyr Ser Leu Ile Asn Lys Arg Asn Lys Tyr Leu Asn Ser Leu 35 40 45 Thr Ser Ile Leu Asn Arg Phe Cys Gly Gln Met Arg Leu Pro 50 55 60 162 78 PRT Homo sapiens 162 Met Thr Pro Ala Leu Ala Ala Trp His Val Leu Ile His Pro Asn Val 1 5 10 15 Cys Phe Leu Ala Pro Ala Asp Ser Leu Glu Gly Ser Ile Lys Glu Asp 20 25 30 Trp Val Asn Met Asp Leu Glu Asn Ala His Leu Gln Arg Glu Asn Gly 35 40 45 Gly Trp Ala Ala Phe Pro Ser Pro Ala Pro Val Pro Gly Ile Trp Pro 50 55 60 Arg Ser Ala Ser Val Cys Phe Gly Ala Lys Leu Gln Ala Pro 65 70 75 163 51 PRT Homo sapiens 163 Met Ser Ser Trp Ile Pro Phe Ile Ile Thr Pro Leu Phe Ser Gly Ile 1 5 10 15 Arg Leu Glu Ala Trp Cys Gln Phe Tyr Ser Ser Leu Tyr Pro Phe Ile 20 25 30 His Phe Leu Ser Ile Leu Phe Pro Lys Tyr Phe Phe Ser Ala Pro Ser 35 40 45 Pro Ala Ala 50 164 27 PRT Homo sapiens 164 Met Gly Ile Ile Pro Lys Cys Met Phe Leu Leu Gln Ser Arg Leu Met 1 5 10 15 Gly Val Ile Thr Asn Thr Ser Leu Leu Leu His 20 25 165 52 PRT Homo sapiens 165 Met Lys Val Leu Lys Tyr His Asn Glu Ala Cys Gly Phe Tyr Ser Val 1 5 10 15 Val Trp Met Leu Ser Ser Ser Ile Pro Trp Met Pro Thr Gly Met His 20 25 30 Cys Leu Ile Leu Glu Phe Lys Arg Trp Pro Gln Thr Val Arg Leu Ser 35 40 45 Met Trp Pro His 50 166 47 PRT Homo sapiens 166 Met Gly Arg Lys Ser Thr Asn Lys Thr Ala Cys Thr His Ile Asn Thr 1 5 10 15 Tyr Val Ser Thr Asn Asp Lys Leu Tyr Leu Tyr Arg Ala Trp Glu Gly 20 25 30 Ser Tyr Ile Thr Leu His Val Ser His Pro Pro His Thr Ser Arg 35 40 45 167 42 PRT Homo sapiens 167 Met Cys Trp Gly Tyr Phe Ser Ile Ser Lys Lys Phe Pro Asn Leu Thr 1 5 10 15 Ser Val Leu Met Asn Leu Gly Thr Asp Leu Ala Val Arg Pro Thr Ser 20 25 30 Ile Phe Pro Thr Asp Ser Ile Leu Leu Glu 35 40 168 55 PRT Homo sapiens 168 Met Asn Lys Ile Lys Gly Lys Ser Val Leu Phe Tyr Met Pro Glu Thr 1 5 10 15 Ser Arg Ile Phe Arg Lys Val Gln Phe Lys Glu Asn Gln Ala Ala Leu 20 25 30 Asp Ser Thr Asn Lys Asn Val Ser Leu Ser Glu Glu Leu Val Asn Gln 35 40 45 Gly Thr Gln Ser Ala Phe Ser 50 55 169 24 PRT Homo sapiens 169 Met Met His Met Gln Leu Ile Ser Glu Phe Ser Cys Leu Cys Cys Phe 1 5 10 15 Phe Phe Leu Gly Ile Tyr Ile Lys 20 170 68 PRT Homo sapiens 170 Met Ile His Leu Ser Glu Val Ser Gly His Leu Lys Glu Arg Lys Gly 1 5 10 15 Lys Ala Ser Cys Gln Lys Gln Lys His Val Leu Tyr Lys Arg Phe Lys 20 25 30 Asn Gln Asn Gly Ile Arg Leu Ser Asn Cys Lys Arg Gln Ser Ser Ala 35 40 45 Phe Lys Ile Leu Arg Lys Asn Asn Val Tyr Ile Lys Ile Phe Ile Ile 50 55 60 Ile Phe Asn Phe 65 171 100 PRT Homo sapiens 171 Ser Phe Ala Phe Phe Phe Ser Leu Arg Gln Ser Leu Thr Leu Ser Pro 1 5 10 15 Arg Leu Glu Cys Ser Gly Thr Ile Ser Ala His Cys Asn Leu Cys Leu 20 25 30 Leu Gly Ser Ser Asn Ser Ser Ala Ser Ala Ser Gln Val Ala Gly Ile 35 40 45 Thr Gly Thr His His His Ala Gln Val Ile Phe Ile Phe Phe Ile Glu 50 55 60 Met Gly Phe Arg His Ile Gly Gln Ala Gly Leu Lys Leu Leu Thr Ser 65 70 75 80 Gly Asp Pro Pro Ala Ser Ala Ser Glu Ser Ala Gly Ile Thr Gly Val 85 90 95 Arg His His Thr 100 172 58 PRT Homo sapiens 172 Met Glu Cys Leu Ser Ile Asn Leu Thr Lys Asn Val Ser Tyr Leu Tyr 1 5 10 15 Thr Gly Pro Leu Asn Thr Ser Glu Thr Lys Leu Lys Ser Tyr Leu Ile 20 25 30 Gly Asn Gln Phe Pro Pro Arg Phe Ile Tyr Arg Val Ser Glu Ile Pro 35 40 45 Ile Lys Ile Ser Ala Arg Ser Leu Arg Asn 50 55 173 47 PRT Homo sapiens 173 Met Asp Lys Glu Glu Ser Ala Val Leu Val Gly Gly Ser Ile Leu Pro 1 5 10 15 Asp Lys Leu Phe Leu Val Gly Phe Thr Asp Thr Ser Pro Asp Leu Leu 20 25 30 Pro Ala Ala Thr Val Cys Phe Tyr Asp Ala Cys His His Asp Ile 35 40 45 174 106 PRT Homo sapiens 174 Met Thr His Val Gln Leu His Ala Leu Asp Leu Leu Leu Lys Asp Glu 1 5 10 15 His Lys Ser Glu Ile Ser Thr Pro Trp Gln Pro Tyr Tyr Gln Leu Leu 20 25 30 Ile Cys Ser Pro His Val Ser Thr Pro Phe Leu Ala Thr Ser Phe Cys 35 40 45 Pro Ser His Ile Asn Thr Cys Gly Gln Trp Leu Thr Met Leu Lys Leu 50 55 60 Lys Leu Tyr Pro Asp Glu Ile Leu Lys Arg Asn His Leu Cys Ser Ser 65 70 75 80 Val Leu Thr Gln Glu Ser Gln His Val Phe Leu Phe Gln Glu Thr Ile 85 90 95 Ile Ile Cys Thr Asn Ile Tyr Pro Asp Asn 100 105 175 35 PRT Homo sapiens 175 Met Ser Met Leu Arg Lys Gly Leu Lys Ser Phe Phe Ser Val Cys Val 1 5 10 15 Leu Pro Ser Glu Pro Asn Ile Gly Ile Ser Ala Ser Lys Ile Pro Gln 20 25 30 Gly Gln Glu 35 176 54 PRT Homo sapiens 176 Met Ser Ser Ser Pro Leu Val Ser Ala Lys Phe Ser Phe Leu Phe His 1 5 10 15 Glu Gly Arg Ala Pro Ser Leu Phe His Pro Leu Met Thr Ser Gln Pro 20 25 30 Leu Glu Phe Cys Leu Met Met Asp Phe Ser Glu Ile Cys Leu Cys Asn 35 40 45 Glu Asp Lys Asp Ser Gly 50 177 20 PRT Homo sapiens 177 Met Arg Pro Leu Lys Met Ile Arg Thr Ala Lys Lys Leu Phe Val Tyr 1 5 10 15 Leu Gly Ser Tyr 20 178 66 PRT Homo sapiens 178 Met Met Tyr Tyr Pro Asp Asp Leu Trp Asn Leu Leu Arg Asn Arg Asp 1 5 10 15 Cys Val Ala Phe Leu Ile Met Gly Thr Gly Pro Ser Leu Leu Arg Leu 20 25 30 Pro Met Cys Val Gly Thr Glu Leu Leu Trp His Ser Ser Ser Arg Leu 35 40 45 Met Glu Leu Ser Ser Ser Glu Ala Ser Trp Val Val His Ala Asn Leu 50 55 60 Val Leu 65 179 70 PRT Homo sapiens 179 Met Cys Val Ile Tyr Gln Arg Gly Ile Cys Asp Glu Lys Lys Asn Leu 1 5 10 15 Lys Cys Pro Gln Met Phe Gln Leu Ser Glu Thr Glu Lys Thr Leu Thr 20 25 30 Ser Val Phe Arg Ile Ile Val Ser Asn Ile Leu Lys Ile Asp Val Ser 35 40 45 Ser Val Met Ile Phe Leu Arg Leu His Gln Arg Thr Ser Leu Asn Leu 50 55 60 Ser Val Ile Gln Asn Gln 65 70 180 30 PRT Homo sapiens 180 Met Asn Pro Val Cys Trp Val Gly Phe Gly Glu Val Asn Ile Glu His 1 5 10 15 Met Glu Phe Lys Tyr Ile Glu Met Asp Thr Val Ile Glu Met 20 25 30 181 55 PRT Homo sapiens 181 Met His Ala Cys Gly Ser Leu Arg Leu Asp Lys Asp Pro Thr Thr Leu 1 5 10 15 Leu Cys Val Asn Thr Arg Cys Thr Arg Ser His Leu Pro Gly Ala Gly 20 25 30 Gly Trp Trp Arg Lys Val Lys Ser Gln Gln Thr Val His Arg Thr Tyr 35 40 45 Ser Ala Thr Gly Lys Lys Ser 50 55 182 16 PRT Homo sapiens 182 Met Pro Ala Leu Arg Glu Ala Phe Pro Gln Ala Pro Leu Ala Leu Ala 1 5 10 15 183 48 PRT Homo sapiens 183 Met Thr Phe Gln Lys Leu Met Ile Leu His Ile His Asp Gln Met Phe 1 5 10 15 Ser Leu Met Glu Ala Ser Asp Val Cys Ser His Gln Ile Arg Phe Lys 20 25 30 Met Ser Val Ser Ser Lys Ser Ser Lys Thr Ser Pro Ser His Gln Lys 35 40 45 184 55 PRT Homo sapiens 184 Met Ser Val Leu Lys Arg Phe Leu Lys Pro Ser Leu Ser Ile Ala Lys 1 5 10 15 Thr Cys Tyr Val His Tyr Pro Pro Asn Ser Tyr Leu Lys Thr Thr Pro 20 25 30 Lys Met Leu Tyr Phe Val Phe Lys Val Arg Glu Glu Asn Arg Gly Glu 35 40 45 Val Phe Leu Cys Ser Phe Pro 50 55 185 14 PRT Homo sapiens 185 Met Trp Leu Arg Asp Leu Asn Tyr Lys Ile Ala Arg Leu Asp 1 5 10 186 42 PRT Homo sapiens 186 Met Met Phe Phe Tyr Ile Phe Cys Ser Met Gly Leu Leu Ile Pro Phe 1 5 10 15 Ser Thr Leu Lys Met Leu Leu Ile Val Phe Pro Leu Ser Leu Phe Pro 20 25 30 Lys Arg Asn Leu Leu Ser Phe Leu Ser Leu 35 40 187 100 PRT Homo sapiens 187 Leu Phe Phe Phe Leu Arg Trp Ser Leu Ala Leu Val Thr Gln Ala Gly 1 5 10 15 Val Gln Val Val Asp Ile Gly Ser Leu Gln Pro Leu Pro Pro Gly Phe 20 25 30 Lys Gln Phe Ser Cys Pro Ser Leu Leu Ser Ser Trp Asp Tyr Arg His 35 40 45 Gly Pro Pro Arg Pro Ala Asn Phe Phe Val Phe Leu Val Glu Met Gly 50 55 60 Phe His His Val Gly Gln Ala Gly Pro Glu Leu Leu Thr Ser Ser Asp 65 70 75 80 Pro Pro Ala Leu Ala Ser Gln Ser Ala Gly Ile Thr Gly Val Ser His 85 90 95 Leu Thr Trp Pro 100 188 106 PRT Homo sapiens 188 Met Ser Cys Leu Trp Pro Ser Leu Asp Leu Pro Ser Leu Ser His Ser 1 5 10 15 Lys Gln Ser Ser Ser Gln Ala Glu Gly Gln Val Thr Ser His Thr Arg 20 25 30 Gln Arg Phe Pro Asp Gly Ala His Leu His Pro Ser Leu Thr Leu Val 35 40 45 Leu Ser Gln Asp Ala Pro Leu Arg Leu Ala Pro Pro Thr Leu Cys Leu 50 55 60 Leu Cys Tyr Trp Ala Ser Leu Pro Ser Pro Arg Thr Pro Glu Leu Leu 65 70 75 80 Asn Ala Gly Gln Lys Ser Ile Pro Asp Leu Gln Gln Arg His Phe Asp 85 90 95 Ile Lys Glu Met Ala Leu Asp Phe Cys Leu 100 105 189 46 PRT Homo sapiens 189 Met Val Ile Ser Arg Ile Ser Ile Leu Arg Lys Met Thr Lys Phe His 1 5 10 15 Lys Phe Cys Ser Gln Leu Thr Glu Pro Gly Arg Arg Thr Gln Pro Lys 20 25 30 Glu Asn Pro Trp Ser Leu Tyr Asp Thr Asp Trp Leu Glu Lys 35 40 45 190 46 PRT Homo sapiens 190 Met Ser Arg Val Arg Ala Glu Lys Pro Gly Arg Val Ala Lys Leu Ala 1 5 10 15 Ala Cys Arg Pro Leu Pro Arg Leu Gln Met Ser Gly Ser Ile Pro Leu 20 25 30 His Lys Cys Lys Glu Lys Ala Ser Met Pro Pro Leu Trp Ser 35 40 45 191 50 PRT Homo sapiens 191 Met Arg Pro Ala Arg Leu Gly Pro Arg Cys Ser Asp Leu Asp Phe Gly 1 5 10 15 Leu Val Leu Ser Ser Trp Leu Arg Leu Ala Arg Cys Pro Leu Glu Ser 20 25 30 Ser Phe Gly Phe Ala Phe Phe Val Cys Leu Phe Ser Pro Asn Phe Cys 35 40 45 Gln Thr 50 192 76 PRT Homo sapiens 192 Met Glu Gly Thr Val Gly Gln Ala Lys Met Val Glu Lys Trp Met Arg 1 5 10 15 Pro Thr Leu Leu Met Ser Leu Arg Gly Leu Gly Glu Arg Ser Asn Glu 20 25 30 Pro His Val Ser Pro Glu Ser Ser Ala Ala Pro Lys Ala Gly Pro Ser 35 40 45 Leu Glu Asp Cys Glu Arg Glu Asp Gly Ser Ile Arg Thr Gly Trp Asp 50 55 60 Thr Ala Pro Thr Lys Glu Ser Pro Thr Ser Cys Ala 65 70 75 193 54 PRT Homo sapiens 193 Arg Thr Val Cys Thr Lys Val Ser Cys Pro Val Gln Leu Pro Ala Asp 1 5 10 15 Trp Thr Cys Lys Val Gln Pro Val Trp Leu Glu Phe Pro Cys Leu Pro 20 25 30 Ile Ser Cys Arg Leu Arg Val Ser Ser Asp Thr Ser Pro Asp Ser Ala 35 40 45 Thr Trp Gly Ser Trp Lys 50 194 27 PRT Homo sapiens 194 Met Glu Pro Arg Ile Pro Val Lys Thr Phe Ser Phe Asp Lys Arg Ile 1 5 10 15 Leu Ile Arg Ile Leu Tyr Gln Ile Glu Gln Lys 20 25 195 17 PRT Homo sapiens 195 Met Leu Gln His Leu Arg Leu Thr Ile Trp Gly Glu Cys Val Trp Val 1 5 10 15 Phe 196 51 PRT Homo sapiens 196 Met Arg Asn Val Ser Leu Ile Ser Cys Glu Asp Ala Asp Phe Thr Glu 1 5 10 15 Ala Leu Cys Asn Ile Trp Phe Val His Gln Thr Met Leu Ile Asp Cys 20 25 30 Arg Ser His Leu Leu Pro Arg Trp Leu Thr Lys Thr Val Gly Ser Leu 35 40 45 Leu Lys Pro 50 197 62 PRT Homo sapiens 197 Met Ser His Gly Gln Val Leu Gly Asp Val Ala Gly Lys Val Gly His 1 5 10 15 Ala Leu Gly Thr Glu Asp Gln Thr Phe Ala Val Glu Val Leu Lys Glu 20 25 30 Thr Thr Pro Phe Phe Arg Ala Ser Ser Gly Pro Thr Gly Asp Pro Trp 35 40 45 Cys Pro Asp His Lys Ile Gln Ser Lys Pro Val Ser Leu Ser 50 55 60 198 400 PRT Homo sapiens 198 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Glu Lys Ser Asp Leu Arg Thr Val Ala Pro 20 25 30 Ala Ser Ser Leu Asn Val Arg Phe Asp Ser Arg Thr Met Asn Leu Ser 35 40 45 Trp Asp Cys Gln Glu Asn Thr Thr Phe Ser Lys Cys Phe Leu Thr Asp 50 55 60 Lys Lys Asn Arg Val Val Glu Pro Arg Leu Ser Asn Asn Glu Cys Ser 65 70 75 80 Cys Thr Phe Arg Glu Ile Cys Leu His Glu Gly Val Thr Phe Glu Val 85 90 95 His Val Asn Thr Ser Gln Arg Gly Phe Gln Gln Lys Leu Leu Tyr Pro 100 105 110 Asn Ser Gly Arg Glu Gly Thr Ala Ala Gln Asn Phe Ser Cys Phe Ile 115 120 125 Tyr Asn Ala Asp Leu Met Asn Cys Thr Trp Ala Arg Gly Pro Thr Ala 130 135 140 Pro Arg Asp Val Gln Tyr Phe Leu Tyr Ile Arg Asn Ser Lys Arg Arg 145 150 155 160 Arg Glu Ile Arg Cys Pro Tyr Tyr Ile Gln Asp Ser Gly Thr His Val 165 170 175 Gly Cys His Leu Asp Asn Leu Ser Gly Leu Thr Ser Arg Asn Tyr Phe 180 185 190 Leu Val Asn Gly Thr Ser Arg Glu Ile Gly Ile Gln Phe Phe Asp Ser 195 200 205 Leu Leu Asp Thr Lys Lys Ile Glu Arg Phe Asn Pro Pro Ser Asn Val 210 215 220 Thr Val Arg Cys Asn Thr Thr His Cys Leu Val Arg Trp Lys Gln Pro 225 230 235 240 Arg Thr Tyr Gln Lys Leu Ser Tyr Leu Asp Phe Gln Tyr Gln Leu Asp 245 250 255 Val His Arg Lys Asn Thr Gln Pro Gly Thr Glu Asn Leu Leu Ile Asn 260 265 270 Val Ser Gly Asp Leu Glu Asn Arg Tyr Asn Phe Pro Ser Ser Glu Pro 275 280 285 Arg Ala Lys His Ser Val Lys Ile Arg Ala Ala Asp Val Arg Ile Leu 290 295 300 Asn Trp Ser Ser Trp Ser Glu Ala Ile Glu Phe Gly Ser Asp Asp Gly 305 310 315 320 Asn Leu Gly Ser Val Tyr Ile Tyr Val Leu Leu Ile Val Gly Thr Leu 325 330 335 Val Cys Gly Ile Val Leu Gly Phe Leu Phe Lys Arg Phe Leu Arg Ile 340 345 350 Gln Arg Leu Phe Pro Pro Val Pro Gln Ile Lys Asp Lys Leu Asn Asp 355 360 365 Asn His Glu Val Glu Asp Glu Ile Ile Trp Glu Glu Phe Thr Pro Glu 370 375 380 Glu Gly Lys Gly Tyr Arg Glu Glu Val Leu Thr Val Lys Glu Ile Thr 385 390 395 400 199 10 PRT Homo sapiens 199 Met Asp Arg Met Glu Lys Arg Gln Thr Asp 1 5 10 200 20 PRT Homo sapiens 200 Met Cys Tyr Ala Thr Leu His Gln Ile Asn Phe Leu Gln Thr Val Leu 1 5 10 15 Val Pro Gly Leu 20 201 31 PRT Homo sapiens 201 Met Cys Leu Cys Cys Trp Leu Tyr Trp Glu Glu Tyr Gly Pro Leu Ser 1 5 10 15 Leu Thr Gln Glu Phe His Val Phe Cys Gln Asp Thr Leu His Gly 20 25 30 202 54 PRT Homo sapiens 202 Met Asn His Ser Leu Ser Ala Phe Gln Arg Ala Leu Gln Val Leu Ile 1 5 10 15 Phe Lys Met Ser Val Tyr Ala Ser Gly Pro Arg Leu Glu Lys Lys Val 20 25 30 Gly Asn Lys Leu Glu Gly Gly Arg Lys Gln Glu Arg Asn Val Thr Tyr 35 40 45 Met Ala Asp Glu Gly Phe 50 203 35 PRT Homo sapiens 203 Met Ile Lys Ala Tyr His Pro Tyr Phe Glu Asn Phe Asn His Arg Ala 1 5 10 15 Gln Tyr Val Ser Asn Lys Leu Lys Lys Tyr Ser Phe Gln Leu His Phe 20 25 30 Asp Gly His 35 204 76 PRT Homo sapiens 204 Met Lys Met Val Asn Arg His Met Lys Trp Lys Ser Ser Ala Leu Ser 1 5 10 15 Asp Leu Val Cys Ile Ser Thr Glu Ile Gln Ala Gly Leu Thr Arg His 20 25 30 Thr Ser His Asn Phe Gln Cys His Cys Thr Ile Ile Leu Thr Val Val 35 40 45 Ser Phe Phe Gln Ser Thr Glu Lys Gln Ala Asp Lys Pro Arg His Leu 50 55 60 Asn Val Thr Trp Leu Met Thr Leu Ile Ser Thr Leu 65 70 75 205 94 PRT Homo sapiens 205 Met Glu Gly Gln Asp Ser Leu Arg Asp Val Gly Ala Leu Ser His Leu 1 5 10 15 Ala His Thr Asp Arg Ser Trp Leu Gly Arg Ala Gly Val Ser Ala Trp 20 25 30 Arg Pro Ser Ala Ala Gly Asp Pro Gly Phe His Glu Val Gly Gly Val 35 40 45 His Ala Gly Thr Ser Gln Leu Ala Gly Pro Gly Gly His Pro Gly Gly 50 55 60 Ala Gly Ala Trp Gly His Glu Phe Thr Lys Val Ala Gln Gly Gln Glu 65 70 75 80 Glu Thr Val Val Ala Glu Gly Pro Leu Val Glu Ala Trp Ala 85 90 206 53 PRT Homo sapiens 206 Met Pro Gln Asp Gln Asp Pro Pro Arg Glu Glu His Ala Ala Leu Arg 1 5 10 15 Val Leu Phe Pro Arg Val Pro Leu Ala Val Pro His Gln Leu Gly Gly 20 25 30 Glu Leu Glu Arg Ala Asp Arg Arg Thr Gly Phe Ser Ala Cys Ala Asn 35 40 45 Ile Leu Thr Cys Pro 50 207 75 PRT Homo sapiens 207 Trp Ser Thr Pro Pro Phe Asp Pro Arg Phe Pro Ser Gln Asn Gln Ile 1 5 10 15 Arg Asn Cys Tyr Gln Asn Phe Leu Asp Tyr His Arg Cys Leu Lys Thr 20 25 30 Arg Thr Arg Arg Gly Lys Ser Thr Gln Pro Cys Glu Tyr Tyr Ser Cys 35 40 45 Val Tyr His Ser Leu Cys Pro Ile Ser Trp Val Glu Ser Trp Asn Glu 50 55 60 Gln Ile Lys Asn Gly Ile Phe Ala Gly Lys Ile 65 70 75 208 44 PRT Homo sapiens 208 Met Arg Val Leu Arg Lys Glu Ser Pro Ser Arg His Val Leu Lys Asn 1 5 10 15 Met Cys Leu Ile Arg Asn Pro Arg Glu Gly Thr Ala Ala Asn Asn Glu 20 25 30 Met Glu Ser Ala Thr Gly Glu Glu Lys Gly Asn Arg 35 40 209 83 PRT Homo sapiens MISC_FEATURE (80)..(80) X=any amino acid 209 Met His Arg Lys Lys Lys Leu Glu Ser Phe Leu Leu Leu Ile Pro Pro 1 5 10 15 Pro Tyr Leu Pro Leu Thr Lys Met Trp Gly Glu Pro Arg Phe Glu Gly 20 25 30 Ser Thr Gly Pro Cys Pro Gln Asp Ser Met Glu Gln Pro Val Thr Lys 35 40 45 Gln Gly Ile Ser Leu Lys Ser Cys Leu Pro Lys Lys Ile Lys Leu Pro 50 55 60 Arg Leu Ala Leu His Pro Ser Pro Pro Arg Ser Phe Pro Leu Lys Xaa 65 70 75 80 Lys Lys Leu 210 40 PRT Homo sapiens 210 Met Thr Arg Phe Ser Gln Ala Ser Ser Ser Lys Asp Lys Thr Pro Pro 1 5 10 15 Leu Pro Ser Met Val Gln Ala Thr Val Leu Val Lys Lys Tyr Ile Phe 20 25 30 Thr Lys Lys Lys Ser Tyr Val Leu 35 40 211 87 PRT Homo sapiens 211 Met Pro Arg Pro Thr Glu Gly Glu Gly Ser Thr Glu Asp Arg Asp Pro 1 5 10 15 Ile Gly Ile Gln Ser Gln Thr Arg Ala Glu Pro Thr Val Glu Gln Leu 20 25 30 Met Ser Gly Ala Lys Asp Thr Ser Trp Asn Pro Pro Asp Gly Ser Ser 35 40 45 Asn Pro Lys Arg Ala Gly Leu Gln Val Gly Leu Asn Trp Arg Asp Pro 50 55 60 Gln Glu Ser Gly Arg Arg Asn Thr Arg Ala Phe Leu Glu Glu Gly Thr 65 70 75 80 Phe Ile Leu Asp Ser Asn Gln 85 212 38 PRT Homo sapiens 212 Met Met Pro Gly Pro Ala Ala Leu Ile Pro Pro Thr Ala Thr Ala Cys 1 5 10 15 Leu Leu Val Val Ala Arg Gly Ser Ser Val Pro Lys Asp Ser Ser Leu 20 25 30 Phe Cys Ile Thr Val His 35 213 88 PRT Homo sapiens 213 Met Ser Leu Leu Asp Ala Ser Ser Leu Lys Pro Tyr Asp Ser Phe Leu 1 5 10 15 Leu Ala Val Leu Phe Leu Thr Arg Asp Asn Lys Gly Phe Ala Ser Gln 20 25 30 Val Cys Met Ala Lys Lys Val Ser Thr Ser Val Asn Gly Ser Phe Leu 35 40 45 Met Thr Ser Gln Gln Pro Leu Val Lys Asp Val Ile Glu Ile Val Gln 50 55 60 Arg Leu Gly Ser Val Cys Phe Val Leu Leu Leu Lys Ser Phe His Gly 65 70 75 80 Ser Lys Leu Phe Leu Ser Ile Val 85 214 42 PRT Homo sapiens 214 Met Val Ile Arg Glu Leu Leu Gly Gly Gln Lys Tyr Pro Asn Pro Val 1 5 10 15 Gln Gly Arg Asp Pro Trp Thr Val Thr Val Leu Ser Ala Phe Gly Arg 20 25 30 Glu Gly Asp Ser Glu Ala Gln Thr Arg Ile 35 40 215 49 PRT Homo sapiens MISC_FEATURE (46)..(46) X=any amino acid 215 Met Pro Asn Cys Ser Val Glu Leu Arg Gly Tyr Tyr Tyr Asn Phe Val 1 5 10 15 His Tyr Tyr Lys Tyr Phe Ile Leu Val Val Tyr Ser Thr Ala Asp Ser 20 25 30 Asn Gln Lys Thr Lys Ile Gln Lys Tyr Tyr Ile Leu Glu Xaa Ile Ile 35 40 45 Met 216 37 PRT Homo sapiens MISC_FEATURE (6)..(6) X=any amino acid 216 Met Glu Met Leu Glu Xaa Lys Xaa Thr Ile Ile Asp Ile Val Ser Leu 1 5 10 15 Leu Ala Leu Ser Gly Asp Leu Thr Gln Leu Arg Lys Ser Leu Val Thr 20 25 30 Leu Lys Ile Cys Arg 35 217 72 PRT Homo sapiens 217 Met Gly Ser Tyr Gly Leu Leu Phe Lys Phe Tyr Gly Ala Ile Phe Thr 1 5 10 15 Ser Val Ala Gln Gly Trp Ser Val Leu His Leu Arg Lys Val Ser Leu 20 25 30 Gly Lys Cys Pro Cys His Pro Ser His Ser Arg Gln Ala Ala Ser Ser 35 40 45 Ala Phe Ser Ser Ser Ser Ser His Ala Trp Ser Ser Pro Phe Val Ile 50 55 60 Phe Ser Ser Leu Thr Pro Ser Leu 65 70 218 49 PRT Homo sapiens 218 Met Gly Ser Phe Ser Pro Leu Thr Tyr His Leu Gly His Trp Asn Met 1 5 10 15 Ala Ala Cys Gly Ser Val Cys Glu Gly Pro Gly Asp Gly Gln Gly Gly 20 25 30 Ser Ala Leu Phe Cys Phe Tyr Gln His Cys Ser Met Asn Val Phe Leu 35 40 45 Thr 219 34 PRT Homo sapiens 219 Met Leu Thr Arg His His Pro Leu Asn Val Leu Leu His Arg Leu Cys 1 5 10 15 Leu Asn Trp Leu Glu Glu Asn Asn Tyr Pro Arg Asn Thr Asp Tyr Leu 20 25 30 Ile Phe 220 34 PRT Homo sapiens MISC_FEATURE (17)..(17) X=any amino acid 220 Met Leu Leu Leu Pro Ala Thr Phe Leu Pro Thr Ser His Ala Arg Pro 1 5 10 15 Xaa Gln Pro His Cys His Thr Thr Cys Leu Ile Thr Ser His Val Leu 20 25 30 Thr His 221 111 PRT Homo sapiens 221 Met Gly Pro Ser Ser Cys Leu Leu Leu Ile Leu Ile Pro Leu Leu Gln 1 5 10 15 Leu Ile Asn Leu Gly Ser Thr Gln Cys Ser Leu Asp Ser Val Met Asp 20 25 30 Lys Lys Ile Lys Asp Val Leu Asn Ser Leu Glu Tyr Ser Pro Ser Pro 35 40 45 Ile Ser Lys Lys Leu Ser Cys Ala Ser Val Lys Ser Gln Gly Arg Pro 50 55 60 Ser Ser Cys Pro Ala Gly Met Ala Val Thr Gly Cys Ala Cys Gly Tyr 65 70 75 80 Gly Cys Gly Ser Trp Asp Val Gln Leu Glu Thr Thr Cys His Cys Gln 85 90 95 Cys Ser Val Val Asp Trp Thr Thr Ala Arg Cys Cys His Leu Thr 100 105 110 222 111 PRT Homo sapiens 222 Met Gly Pro Ser Ser Cys Leu Leu Leu Ile Leu Ile Pro Leu Leu Gln 1 5 10 15 Leu Ile Asn Leu Gly Ser Thr Gln Cys Ser Leu Asp Ser Val Met Asp 20 25 30 Lys Lys Ile Lys Asp Val Leu Asn Ser Leu Glu Tyr Ser Pro Ser Pro 35 40 45 Ile Ser Lys Lys Leu Ser Cys Ala Ser Val Lys Ser Gln Gly Arg Pro 50 55 60 Ser Ser Cys Pro Ala Gly Met Ala Val Thr Gly Cys Ala Cys Gly Tyr 65 70 75 80 Gly Cys Gly Ser Trp Asp Val Gln Leu Glu Thr Thr Cys His Cys Gln 85 90 95 Cys Ser Val Val Asp Trp Thr Thr Ala Arg Cys Cys His Leu Thr 100 105 110 223 83 PRT Homo sapiens 223 Met Asn Val Glu Ala Arg Glu Gln Cys Asp Val Gln Leu Ser Asp Leu 1 5 10 15 Thr Trp His Leu Ile Trp Leu Glu Val Pro Pro Leu Leu Ser Val Pro 20 25 30 Trp Leu Trp Ala His Gly Leu Ala Glu Pro Ser Tyr Gly Phe Arg Phe 35 40 45 Thr Cys Tyr Asn Ile Gln Arg Gln Cys Thr Ser Leu Pro Arg Lys Leu 50 55 60 Cys Ser Arg His Pro Phe Val Thr Leu Ile Ser Ile Met Asp Thr Thr 65 70 75 80 Thr Phe Tyr 224 132 PRT Homo sapiens MISC_FEATURE (3)..(3) X=any amino acid 224 Met Asp Xaa Thr Arg Val His Asp Asp Glu Xaa Val Ile Xaa Gly Asp 1 5 10 15 Val Phe Val His Glu Val Thr Pro Gly Pro His Arg Trp Val Leu Val 20 25 30 Arg Pro Phe Cys Leu Glu Val Arg Ala Val Phe Leu Arg Leu Trp Tyr 35 40 45 Tyr Arg Gly Glu Lys Glu Glu Glu Leu Glu Val Arg Glu Arg Ser Cys 50 55 60 Arg Leu Gly Arg Cys Asp Gln Gly Gln Arg Asp Gly Val Gln Glu Ala 65 70 75 80 Cys Ser Ser Val Ser Cys Ser Leu Arg Gln Glu Val Ser Pro Ser Ser 85 90 95 Gln Leu Asp Met Arg Ser Leu Leu Gly Val Pro Leu Ala Glu Val Glu 100 105 110 Pro Val Ala Gln His Pro Pro Asn Glu Gly Arg Gly Arg His Leu Gly 115 120 125 Gln Cys Leu Leu 130 225 38 PRT Homo sapiens 225 Met Ile Asn Asn Ser Asn His Asn Asn Ser Ser Ser Ser Lys Leu Arg 1 5 10 15 Ala Ser Tyr Val Gln Ala Phe Ser Lys His Phe Thr Cys Ile Thr Pro 20 25 30 Leu Val Ile Thr Thr Pro 35 226 58 PRT Homo sapiens 226 Met Ser Thr Phe Thr Val Leu Lys Asn Thr His Gln Leu Lys Lys Asn 1 5 10 15 Thr Leu Phe Pro Phe Leu Gly His Leu Asn Leu Arg Glu Gln Leu Leu 20 25 30 Tyr Lys Asn Asp Ile Lys Ile Ile His Phe Gly Ser Met Phe Leu Thr 35 40 45 Val Leu Arg Gly Cys Met Val Lys Leu Lys 50 55 227 26 PRT Homo sapiens 227 Met His Met His Ile Phe Leu Cys Leu Tyr Asn Leu Cys Asn Ile Cys 1 5 10 15 Glu Cys Asn Thr Phe Ser Phe Phe Leu Leu 20 25 228 47 PRT Homo sapiens 228 Met Leu Asp Val Met Arg Gln Val Ala Arg Ser Trp Leu Thr Ala Met 1 5 10 15 Glu Arg Leu Leu Leu Pro Ala Ala Val Arg Phe Ser Ala Ile Trp Leu 20 25 30 Ala Gly Gln Phe Ala Met Ala Trp Leu Leu Gln Leu Ile Leu Gly 35 40 45 229 53 PRT Homo sapiens 229 Met Gly Asn Ile Gly Glu Thr Leu Ser Leu Lys Lys Lys Arg Arg Ala 1 5 10 15 Gly Gly Glu Ser Val Lys Asp Pro Gly Ser Thr Asp Thr Gly Gly Gln 20 25 30 Arg Thr Arg Val Gly Val Ser Ser Asn Asp Ser Val Gly Ser Met Gly 35 40 45 Ala Val Gly Arg Glu 50 230 80 PRT Homo sapiens 230 Met Val Ile Asn Ser Cys Ile Ile Pro Leu Pro Ser Gln Ala Thr Ile 1 5 10 15 Pro Glu Pro Trp Pro His Gly Ala Cys Ile Phe Arg Ile Gln Thr Pro 20 25 30 Trp Gly Ser Ser Pro Leu Leu Pro Ser Leu Ser Ser His Pro Leu Thr 35 40 45 His Leu Ser Cys Tyr Leu Ser Leu Glu Ile Pro Lys Met Met Cys Val 50 55 60 Met Glu Arg Leu Glu His Gln Leu Gln Asn His Pro Val Thr Leu Ala 65 70 75 80 231 40 PRT Homo sapiens 231 Met Phe Gln Arg Phe Leu Ala Lys Val Thr Val Trp Met Val Val Pro 1 5 10 15 Leu Thr Lys Thr Ala Met Asn Ala Lys Arg Ala Ser Phe Val Gly Arg 20 25 30 His Lys Ile Ile Phe Arg Ile Cys 35 40 232 24 PRT Homo sapiens 232 Met Leu Leu Tyr Leu Ile Thr Arg Gly Asp Val Glu Asn Gly Cys Phe 1 5 10 15 Ile Phe Ser Val Val Phe Ala Leu 20 233 26 PRT Homo sapiens 233 Met Pro Pro Arg Gly Leu Pro His Phe Ser Pro His Pro Thr Arg Gln 1 5 10 15 Phe Leu Phe Leu Phe Pro Leu His Thr Lys 20 25 234 37 PRT Homo sapiens 234 Met Ser Tyr Glu Ile Leu Val Asn Thr Asp Phe Met Ser Pro Phe Leu 1 5 10 15 Arg Thr Leu Leu Val Cys Phe His Leu Tyr Ala Leu Ile Arg Ala Asn 20 25 30 Asn Leu Lys Tyr Pro 35 235 40 PRT Homo sapiens 235 Met Gly Lys Gly Leu Arg Leu Gly Val Ser Ile Ile Leu Val Lys Ser 1 5 10 15 Phe Phe Thr Tyr Ser Ser Lys Asp Val Asn Tyr Phe Ser Ile His Ser 20 25 30 Asn Ile Lys Ala Val Phe His Phe 35 40 236 40 PRT Homo sapiens 236 Met Glu Glu Thr Gly Pro Leu Pro Ser Gly Ser Ser Leu Ser Asp Gln 1 5 10 15 Gly Glu Thr Ala Leu Ala Leu Gly Asn Ser Arg Ser Asp Gly Gly Arg 20 25 30 Gln Ser Ser Ser Ser Met Asn Ala 35 40 237 50 PRT Homo sapiens 237 Met His Lys Gln Ser Met Ala Arg Ser Ile Leu Arg Ser Pro Leu Gln 1 5 10 15 Gln Ile Pro Pro Lys Gly Glu Ala Gly Arg Trp Arg Trp Ala Glu Ala 20 25 30 Ser Cys Val Leu His Thr Phe Ser Thr Ile Leu Asp Phe Leu Phe Phe 35 40 45 Phe Phe 50 238 49 PRT Homo sapiens 238 Ser Ser Trp Gly Asp Ser Phe Ala Val Ser Ala Ala Trp Ala Arg Lys 1 5 10 15 Gly Ile Glu Glu Trp Ile Gly Arg Gln Arg Cys Pro Gly Gly Val Ser 20 25 30 Gly Pro Arg Gln Leu Arg Leu Ala Gly Thr Ile Gly Arg Ser Thr Arg 35 40 45 Glu 239 54 PRT Homo sapiens 239 Met Leu Arg Pro Leu Thr Val Ala Ser Lys Arg Leu Leu Thr Ile Ser 1 5 10 15 Thr Leu Lys Ser Pro Leu Val Gly Leu Cys Ser Phe Ser Lys Ser Gly 20 25 30 Val Leu Arg Glu Gln Ala Leu Phe Ser Ile Ile Asn Leu Ile Asn Thr 35 40 45 Asp Trp Gln Lys Gln His 50 240 23 PRT Homo sapiens 240 Met Lys Lys Lys Ser Tyr Pro Asp Lys Ile Asn Gln Cys Phe Ile Phe 1 5 10 15 Leu Glu His Gln Asn Leu Leu 20 241 59 PRT Homo sapiens MISC_FEATURE (6)..(7) X=any amino acid 241 Met Val Lys Tyr Met Xaa Xaa Leu Xaa Leu Thr Pro Xaa Phe Ser Asn 1 5 10 15 Leu Leu Gly Thr Leu Lys Xaa Arg Lys Val Xaa Xaa Xaa Xaa Xaa Pro 20 25 30 Arg Lys Arg Asn Phe Xaa Xaa Xaa Pro Pro Xaa Leu Xaa Lys Xaa Arg 35 40 45 Cys His Phe Leu His Ile Asp Leu Gln Arg Val 50 55 242 55 PRT Homo sapiens MISC_FEATURE (53)..(53) X=any amino acid 242 Met Val Ser Gly Val Gln Val Ser Leu His Lys Thr Lys Ile Lys Leu 1 5 10 15 Phe Asn Thr Gly Pro Thr Thr Leu Ile Tyr Gly Ala Asn Thr Cys Cys 20 25 30 Glu Pro Trp Gly Gln Gly Leu Gly Asp Lys Val Ala Thr Ile Phe Trp 35 40 45 Gly Val Gly Gly Xaa Gly Gly 50 55 243 75 PRT Homo sapiens 243 Met Val Ile Thr Cys Val Leu Tyr Asp Ile Ser Ser Leu Lys Asn Leu 1 5 10 15 Arg His Ser Pro Phe Leu Gln Val Phe Phe Cys Val Cys Trp Lys Ile 20 25 30 Met Tyr Ile Phe Gln Leu Leu Asn Ala Ser Val Cys Ile Cys Ile Ser 35 40 45 Thr Lys Ser Lys Leu Leu Ile Leu Leu Phe Lys Leu Phe Ala Ser Tyr 50 55 60 Trp Phe Ser Leu Pro Thr Leu Cys Ile Asn Ser 65 70 75 244 17 PRT Homo sapiens 244 Met Ser Trp Val Pro Cys Gly Cys Asp Phe Leu Arg Glu Ile Asn Leu 1 5 10 15 Phe 245 30 PRT Homo sapiens 245 Met Tyr Val Ser Pro Asp Asn Ile Ser Gly Ser Gly Asn Cys Lys Lys 1 5 10 15 Lys Ile Gly Asn Gln Asn Ser Arg Lys Val Phe Leu Glu Gly 20 25 30 246 57 PRT Homo sapiens 246 Arg Val Thr Met Asn Glu Lys Asp Asn Phe Met Asn Ala Glu Asn Leu 1 5 10 15 Gly Ile Val Phe Gly Pro Thr Leu Met Arg Pro Pro Glu Asp Ser Thr 20 25 30 Leu Thr Thr Leu His Asp Met Arg Tyr Gln Lys Leu Ile Val Gln Ile 35 40 45 Leu Ile Glu Asn Glu Asp Val Leu Phe 50 55 247 70 PRT Homo sapiens MISC_FEATURE (38)..(38) X=any amino acid 247 Met Phe Ala Ser Leu Leu Ile Thr Asn Leu Leu Ser Thr Asn Glu Lys 1 5 10 15 Tyr Ile Gln Asp Leu Pro Phe Gln Arg Leu Ser Ile Tyr Glu Thr Asn 20 25 30 Ser Pro Phe Arg Leu Xaa Asn Phe Glu Asp Val Phe Ile Phe Leu Phe 35 40 45 Phe Leu Asn Lys Asn Cys Phe Leu Ser Arg Leu Phe Lys Ala Thr Cys 50 55 60 Val Lys Pro Leu Val Gln 65 70 248 36 PRT Homo sapiens 248 Met Arg Arg Ala Arg Pro Pro Leu Phe Phe Leu His Ala Val Ser Ser 1 5 10 15 Pro Gly Gln Ile Leu Thr Ser Lys Asn Ala Val Phe Pro Ser Gly Ala 20 25 30 Gly Pro Val Met 35 249 26 PRT Homo sapiens 249 Met Ser Leu Ser Phe Ser Leu His Ser Phe Tyr Arg Lys Ala Ile Leu 1 5 10 15 Gly Val Leu Gly His Phe Asp Ser Thr Ser 20 25 250 43 PRT Homo sapiens MISC_FEATURE (6)..(6) X=any amino acid 250 Met Ser Leu Pro Ser Xaa Arg Arg Gln Phe Ser Asp Ile Thr Cys Thr 1 5 10 15 Glu Ile His Tyr Asn Ala Thr Met Asn Gly Gln Ser Ser Thr Glu Lys 20 25 30 Ile Lys Gln Arg Met Ser Trp Lys Val Leu Trp 35 40

Claims

We claim:

1. An isolated nucleic acid molecule comprising

(a) a nucleic acid molecule comprising a nucleic acid sequence that encodes an amino acid sequence of SEQ ID NO: 148 through 250;

(b) a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 1 through 147;

(c) a nucleic acid molecule that selectively hybridizes to the nucleic acid molecule of (a) or (b); or

(d) a nucleic acid molecule having at least 60% sequence identity to the nucleic acid molecule of (a) or (b).

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a cDNA.

3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is genomic DNA.

4. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is a mammalian nucleic acid molecule.

5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule is a human nucleic acid molecule.

6. A method for determining the presence of a colon specific nucleic acid (CSNA) in a sample, comprising the steps of:

(a) contacting the sample with the nucleic acid molecule according to claim 1 under conditions in which the nucleic acid molecule will selectively hybridize to a colon specific nucleic acid; and

(b) detecting hybridization of the nucleic acid molecule to a CSNA in the sample, wherein the detection of the hybridization indicates the presence of a CSNA in the sample.

7. A vector comprising the nucleic acid molecule of claim 1.

8. A host cell comprising the vector according to claim 7.

9. A method for producing a polypeptide encoded by the nucleic acid molecule according to claim 1, comprising the steps of (a) providing a host cell comprising the nucleic acid molecule operably linked to one or more expression control sequences, and (b) incubating the host cell under conditions in which the polypeptide is produced.

10. A polypeptide encoded by the nucleic acid molecule according to claim 1.

11. An isolated polypeptide selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence with at least 60% sequence identity to of SEQ ID NO: 148 through 250; or

(b) a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule comprising a nucleic acid sequence of SEQ ID NO: 1 through 147.

12. An antibody or fragment thereof that specifically binds to the polypeptide according to claim 11.

13. A method for determining the presence of a colon specific protein in a sample, comprising the steps of:

(a) contacting the sample with the antibody according to claim 12 under conditions in which the antibody will selectively bind to the colon specific protein; and

(b) detecting binding of the antibody to a colon specific protein in the sample, wherein the detection of binding indicates the presence of a colon specific protein in the sample.

14. A method for diagnosing and monitoring the presence and metastases of colon cancer in a patient, comprising the steps of:

(a) determining an amount of the nucleic acid molecule of claim 1 or a polypeptide of claim 6 in a sample of a patient; and

(b) comparing the amount of the determined nucleic acid molecule or the polypeptide in the sample of the patient to the amount of the colon specific marker in a normal control; wherein a difference in the amount of the nucleic acid molecule or the polypeptide in the sample compared to the amount of the nucleic acid molecule or the polypeptide in the normal control is associated with the presence of colon cancer.

15. A kit for detecting a risk of cancer or presence of cancer in a patient, said kit comprising a means for determining the presence the nucleic acid molecule of claim 1 or a polypeptide of claim 6 in a sample of a patient.

16. A method of treating a patient with colon cancer, comprising the step of administering a composition according to claim 12 to a patient in need thereof, wherein said administration induces an immune response against the colon cancer cell expressing the nucleic acid molecule or polypeptide.

17. A vaccine comprising the polypeptide or the nucleic acid encoding the polypeptide of claim 11.