Patent application title: NOVEL COMPOSITIONS AND METHODS IN CANCER WITH ALTERED EXPRESSION OF KCNJ9
Inventors:
David W. Morris (Davis, CA, US)
Eric K. Engelhard (Davis, CA, US)
Assignees:
Sagres Discovery
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2010-12-16
Patent application number: 20100317003
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Patent application title: NOVEL COMPOSITIONS AND METHODS IN CANCER WITH ALTERED EXPRESSION OF KCNJ9
Inventors:
David W. Morris
Eric K. Engelhard
Agents:
McDermott Will & Emery LLP
Assignees:
Origin: WASHINGTON, DC US
IPC8 Class: AC12Q168FI
USPC Class:
Publication date: 12/16/2010
Patent application number: 20100317003
Abstract:
The present invention relates to novel sequences for use in diagnosis and
treatment of carcinomas, especially breast cancers. In addition, the
present invention describes the use of novel compositions for use in
screening methods.Claims:
1-10. (canceled)
11. A method of diagnosing carcinoma comprising:a) determining the expression of one or more genes comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1, 2, and 3 in a first tissue type of a first individual; andb) comparing said expression of said gene(s) from a second normal tissue type from said first individual or a second unaffected individual;wherein a difference in said expression indicates that the first individual has carcinoma.
12-19. (canceled)
Description:
[0001]The present application is a continuing application of U.S.
Application entitled Novel Compositions and Methods for Cancer, U.S. Ser.
No. 10/034,650, filed Dec. 20, 2001, and U.S. Ser. Nos. 09/747,377, filed
Dec. 22, 2000, and 09/798,586, filed Mar. 2, 2001 all of which are
expressly incorporated herein by reference.
FIELD OF THE INVENTION
[0002]The present invention relates to novel sequences for use in diagnosis and treatment of cancer, especially carcinomas including breast cancer, as well as the use of the novel compositions in screening methods.
BACKGROUND OF THE INVENTION
[0003]Oncogenes are genes that can cause cancer. Carcinogenesis can occur by a wide variety of mechanisms, including infection of cells by viruses containing oncogenes, activation of protooncogenes in the host genome, and mutations of protooncogenes and tumor suppressor genes.
[0004]There are a number of viruses known to be involved in human cancer as well as in animal cancer. Of particular interest here are viruses that do not contain oncogenes themselves; these are slow-transforming retroviruses. They induce tumors by integrating into the host genome and affecting neighboring protooncogenes in a variety of ways, including promoter insertion, enhancer insertion, and/or truncation of a protooncogene or tumor suppressor gene. The analysis of sequences at or near the insertion sites led to the identification of a number of new protooncogenes.
[0005]With respect to lymphoma and leukemia, murine leukemia retrovirus (MuLV), such as SL3-3 or Akv, is a potent inducer of tumors when inoculated into susceptible newborn mice, or when carried in the germline. A number of sequences have been identified as relevant in the induction of lymphoma and leukemia by analyzing the insertion sites; see Sorensen et al., J. of Virology 74:2161 (2000); Hansen et al., Genome Res. 10(2):237-43 (2000); Sorensen et al., J. Virology 70:4063 (1996); Sorensen et al., J. Virology 67:7118 (1993); Joosten et al., Virology 268:308 (2000); and Li et al., Nature Genetics 23:348 (1999); all of which are expressly incorporated by reference herein.
[0006]In addition, breast cancer is one of the most significant diseases that affects women. At the current rate, American women have a 1 in 8 risk of developing breast cancer by age 95 (American Cancer Society, 1992). Treatment of breast cancer at later stages is often futile and disfiguring, making early detection a high priority in medical management of the disease.
[0007]KCNJ9 (Kir 3.3, GIRK3) is a member of the G-protein-activated inwardly rectifying potassium (GIRK) channel family. Vaughn J. et al., Biochem Biophys Res Commun (2000) August 2; 274(2):302-9. In particular, the KCNJ9 gene encodes a G-protein-coupled inwardly rectifying potassium channel. Wolford J. K. et al., Mol Genet Metab (2001) May; 73(1):97-103.
[0008]The gene spans approximately 7.6 kb and contains one noncoding and two coding exons separated by approximately 2.2 and approximately 2.6 kb introns, respectively. Fourteen single nucleotide polymorphisms (SNPs), including one that predicts a Val366Ala substitution, and an 8 base-pair (bp) insertion/deletion have been identified, and earlier expression studies have revealed the presence of the transcript in various human tissues including pancreas, and two major insulin-responsive tissues: fat and skeletal muscle.
[0009]The comparative gene content and order are identical between mouse and human, indicating a high degree of conservation between the two species in the KCNJ9 region. Doudney K. et al., Genomics (2001) March 1; 72(2):180-92. The genomic organization of the KCNJ9 locus on chromosome 1q21-23 makes it a candidate gene for Type II diabetes mellitus in the Pima Indian population. KCNJ9, however, has not before been associated with or implicated in cancer. All references cited herein are expressly incorporated in their entirety.
[0010]As demonstrated below, mutations that interrupt the KCNJ9 coding sequence result in cancer. Moreover, altered expression of KCNJ9 correlate with cancer, in particular with breast cancer.
[0011]Accordingly, it is an object of the invention to provide sequences involved in cancer and in particular in oncogenesis and breast cancer.
SUMMARY OF THE INVENTION
[0012]In accordance with the objects outlined above, the present invention provides methods for screening for compositions which modulate carcinomas, especially breast cancer. Also provided herein are methods of inhibiting proliferation of a cell, preferably a breast cancer cell. Methods of treatment of carcinomas, including diagnosis, are also provided herein.
[0013]In one aspect, a method of screening drug candidates comprises providing a cell that expresses a carcinoma associated (CA) gene or fragments thereof, such as KCNJ9. Preferred embodiments of CA genes are genes which are differentially expressed in cancer cells, preferably lymphatic, breast, prostate or epithelial cells, compared to other cells. Preferred embodiments of CA genes used in the methods herein include, but are not limited to the nucleic acids selected from Table 1. The method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the CA gene.
[0014]In one embodiment, the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate.
[0015]Also provided herein is a method of screening for a bioactive agent capable of binding to a CA protein (CAP), the method comprising combining the CAP and a candidate bioactive agent, and determining the binding of the candidate agent to the CAP.
[0016]Further provided herein is a method for screening for a bioactive agent capable of modulating the activity of a CAP. In one embodiment, the method comprises combining the CAP and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of the CAP.
[0017]Also provided is a method of evaluating the effect of a candidate carcinoma drug comprising administering the drug to a patient and removing a cell sample from the patient. The expression profile of the cell is then determined. This method may further comprise comparing the expression profile of the patient to an expression profile of a healthy individual.
[0018]In a further aspect, a method for inhibiting the activity of an CA protein is provided. In one embodiment, the method comprises administering to a patient an inhibitor of a CA protein preferably selected from the group consisting of the sequences outlined in Table 1 or their complements.
[0019]A method of neutralizing the effect of a CA protein, preferably a protein encoded by a nucleic acid selected from the group of sequences outlined in Table 1, is also provided. Preferably, the method comprises contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.
[0020]Moreover, provided herein is a biochip comprising a nucleic acid segment which encodes a CA protein, preferably selected from the sequences outlined in Table 1.
[0021]Also provided herein is a method for diagnosing or determining the propensity to carcinomas, especially breast cancer by sequencing at least one carcinoma or breast cancer gene of an individual. In yet another aspect of the invention, a method is provided for determining carcinoma including breast cancer gene copy number in an individual.
[0022]Novel sequences are also provided herein. Preferred compositions include the sequences set forth in Table 1. Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0023]FIG. 1 depicts mRNA expression of KCNJ9 in breast cancer tissue compared with expression in normal tissue. Samples 1-50 are breast cancer samples. Samples 51 and 52 are normal tissue. Bars represent the mean of expression level. Error bars represent standard deviation.
DETAILED DESCRIPTION OF THE INVENTION
[0024]The present invention is directed to a number of sequences associated with carcinomas, especially lymphoma, breast cancer or prostate cancer. The relatively tight linkage between clonally-integrated proviruses and protooncogenes forms "provirus tagging", in which slow-transforming retroviruses that act by an insertion mutation mechanism are used to isolate protooncogenes. In some models, uninfected animals have low cancer rates, and infected animals have high cancer rates. It is known that many of the retroviruses involved do not carry transduced host protooncogenes or pathogenic trans-acting viral genes, and thus the cancer incidence must therefor be a direct consequence of proviral integration effects into host protooncogenes. Since proviral integration is random, rare integrants will "activate" host protooncogenes that provide a selective growth advantage, and these rare events result in new proviruses at clonal stoichiometries in tumors.
[0025]The use of oncogenic retroviruses, whose sequences insert into the genome of the host organism resulting in carcinoma, allows the identification of host sequences involved in carcinoma. These sequences may then be used in a number of different ways, including diagnosis, prognosis, screening for modulators (including both agonists and antagonists), antibody generation (for immunotherapy and imaging), etc. However, as will be appreciated by those in the art, oncogenes that are identified in one type of cancer such as breast cancer have a strong likelihood of being involved in other types of cancers as well. Thus, while the sequences outlined herein are initially identified as correlated with breast cancer, they can also be found in other types of cancers as well, outlined below.
[0026]Accordingly, the present invention provides nucleic acid and protein sequences that are associated with carcinoma, herein termed "carcinoma associated" or "CA" sequences. In a preferred embodiment, the present invention provides nucleic acid and protein sequences that are associated with carcinomas which originate in mammary tissue, which are known as breast cancer sequences or "BA".
[0027]Suitable cancers which can be diagnosed or screened for using the methods of the present invention include cancers classified by site or by histological type. Cancers classified by site include cancer of the oral cavity and pharynx (lip, tongue, salivary gland, floor of mouth, gum and other mouth, nasopharynx, tonsil, oropharynx, hypopharynx, other oral/pharynx); cancers of the digestive system (esophagus; stomach; small intestine; colon and rectum; anus, anal canal, and anorectum; liver; intrahepatic bile duct; gallbladder; other biliary; pancreas; retroperitoneum; peritoneum, omentum, and mesentery; other digestive); cancers of the respiratory system (nasal cavity, middle ear, and sinuses; larynx; lung and bronchus; pleura; trachea, mediastinum, and other respiratory); cancers of the mesothelioma; bones and joints; and soft tissue, including heart; skin cancers, including melanomas and other non-epithelial skin cancers; Kaposi's sarcoma and breast cancer; cancer of the female genital system (cervix uteri; corpus uteri; uterus, nos; ovary; vagina; vulva; and other female genital); cancers of the male genital system (prostate gland; testis; penis; and other male genital); cancers of the urinary system (urinary bladder; kidney and renal pelvis; ureter; and other urinary); cancers of the eye and orbit; cancers of the brain and nervous system (brain; and other nervous system); cancers of the endocrine system (thyroid gland and other endocrine, including thymus); cancers of the lymphomas (hodgkin's disease and non-hodgkin's lymphoma), multiple myeloma, and leukemias (lymphocytic leukemia; myeloid leukemia; monocytic leukemia; and other leukemias).
[0028]Other cancers, classified by histological type, that may be associated with the sequences of the invention include, but are not limited to, Neoplasm, malignant; Carcinoma, NOS; Carcinoma, undifferentiated, NOS; Giant and spindle cell carcinoma; Small cell carcinoma, NOS; Papillary carcinoma, NOS; Squamous cell carcinoma, NOS; Lymphoepithelial carcinoma; Basal cell carcinoma, NOS; Pilomatrix carcinoma; Transitional cell carcinoma, NOS; Papillary transitional cell carcinoma; Adenocarcinoma, NOS; Gastrinoma, malignant; Cholangiocarcinoma; Hepatocellular carcinoma, NOS; Combined hepatocellular carcinoma and cholangiocarcinoma; Trabecular adenocarcinoma; Adenoid cystic carcinoma; Adenocarcinoma in adenomatous polyp; Adenocarcinoma, familial polyposis coli; Solid carcinoma, NOS; Carcinoid tumor, malignant; Branchiolo-alveolar adenocarcinoma; Papillary adenocarcinoma, NOS; Chromophobe carcinoma; Acidophil carcinoma; Oxyphilic adenocarcinoma; Basophil carcinoma; Clear cell adenocarcinoma, NOS; Granular cell carcinoma; Follicular adenocarcinoma, NOS; Papillary and follicular adenocarcinoma; Nonencapsulating sclerosing carcinoma; Adrenal cortical carcinoma; Endometroid carcinoma; Skin appendage carcinoma; Apocrine adenocarcinoma; Sebaceous adenocarcinoma; Ceruminous adenocarcinoma; Mucoepidermoid carcinoma; Cystadenocarcinoma, NOS; Papillary cystadenocarcinoma, NOS; Papillary serous cystadenocarcinoma; Mucinous cystadenocarcinoma, NOS; Mucinous adenocarcinoma; Signet ring cell carcinoma; Infiltrating duct carcinoma; Medullary carcinoma, NOS; Lobular carcinoma; Inflammatory carcinoma; Paget's disease, mammary; Acinar cell carcinoma; Adenosquamous carcinoma; Adenocarcinoma w/squamous metaplasia; Thymoma, malignant; Ovarian stromal tumor, malignant; Thecoma, malignant; Granulosa cell tumor, malignant; Androblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; Lipid cell tumor, malignant; Paraganglioma, malignant; Extra-mammary paraganglioma, malignant; Pheochromocytoma; Glomangiosarcoma; Malignant melanoma, NOS; Amelanotic melanoma; Superficial spreading melanoma; Malig melanoma in giant pigmented nevus; Epithelioid cell melanoma; Blue nevus, malignant; Sarcoma, NOS; Fibrosarcoma, NOS; Fibrous histiocytoma, malignant; Myxosarcoma; Liposarcoma, NOS; Leiomyosarcoma, NOS; Rhabdomyosarcoma, NOS; Embryonal rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma, NOS; Mixed tumor, malignant, NOS; Mullerian mixed tumor; Nephroblastoma; Hepatoblastoma; Carcinosarcoma, NOS; Mesenchymoma, malignant; Brenner tumor, malignant; Phyllodes tumor, malignant; Synovial sarcoma, NOS; Mesothelioma, malignant; Dysgerminoma; Embryonal carcinoma, NOS; Teratoma, malignant, NOS; Struma ovarii, malignant; Choriocarcinoma; Mesonephroma, malignant; Hemangiosarcoma; Hemangioendothelioma, malignant; Kaposi's sarcoma; Hemangiopericytoma, malignant; Lymphangiosarcoma; Osteosarcoma, NOS; Juxtacortical osteosarcoma; Chondrosarcoma, NOS; Chondroblastoma, malignant; Mesenchymal chondrosarcoma; Giant cell tumor of bone; Ewing's sarcoma; Odontogenic tumor, malignant; Ameloblastic odontosarcoma; Ameloblastoma, malignant; Ameloblastic fibrosarcoma; Pinealoma, malignant; Chordoma; Glioma, malignant; Ependymoma, NOS; Astrocytoma, NOS; Protoplasmic astrocytoma; Fibrillary astrocytoma; Astroblastoma; Glioblastoma, NOS; Oligodendroglioma, NOS; Oligodendroblastoma; Primitive neuroectodermal; Cerebellar sarcoma, NOS; Ganglioneuroblastoma; Neuroblastoma, NOS; Retinoblastoma, NOS; Olfactory neurogenic tumor; Meningioma, malignant; Neurofibrosarcoma; Neurilemmoma, malignant; Granular cell tumor, malignant; Malignant lymphoma, NOS; Hodgkin's disease, NOS; Hodgkin's; paragranuloma, NOS; Malignant lymphoma, small lymphocytic; Malignant lymphoma, large cell, diffuse; Malignant lymphoma, follicular, NOS; Mycosis fungoides; Other specified non-Hodgkin's lymphomas; Malignant histiocytosis; Multiple myeloma; Mast cell sarcoma; Immunoproliferative small intestinal disease; Leukemia, NOS; Lymphoid leukemia, NOS; Plasma cell leukemia; Erythroleukemia; Lymphosarcoma cell leukemia; Myeloid leukemia, NOS; Basophilic leukemia; Eosinophilic leukemia; Monocytic leukemia, NOS; Mast cell leukemia; Megakaryoblastic leukemia; Myeloid sarcoma; and Hairy cell leukemia.
[0029]In addition, the genes may be involved in other diseases, such as but not limited to diseases associated with aging or neurodegenerative diseases.
[0030]Association in this context means that the nucleotide or protein sequences are either differentially expressed, activated, inactivated or altered in carcinomas as compared to normal tissue. As outlined below, CA sequences include those that are up-regulated (i.e. expressed at a higher level), as well as those that are down-regulated (i.e. expressed at a lower level), in carcinomas. CA sequences also include sequences which have been altered (i.e., truncated sequences or sequences with substitutions, deletions or insertions, including point mutations) and show either the same expression profile or an altered profile. In a preferred embodiment, the CA sequences are from humans; however, as will be appreciated by those in the art, CA sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other CA sequences are provided, from vertebrates, including mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc). In some cases, prokaryotic CA sequences may be useful. CA sequences from other organisms may be obtained using the techniques outlined below.
[0031]CA sequences can include both nucleic acid and amino acid sequences. In a preferred embodiment, the CA sequences are recombinant nucleic acids. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by polymerases and endonucleases, in a form not normally found in nature. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
[0032]Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid as depicted above. A recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample. A substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred. The definition includes the production of an CA protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
[0033]In a preferred embodiment, the CA sequences are nucleic acids. As will be appreciated by those in the art and is more fully outlined below, CA sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids, as well as screening applications; for example, biochips comprising nucleic acid probes to the CA sequences can be generated. In the broadest sense, then, by "nucleic acid" or "oligonucleotide" or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below (for example in antisense applications or when a candidate agent is a nucleic acid), nucleic acid analogs may be used that have alternate backbones, comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example to increase the stability and half-life of such molecules in physiological environments for use in anti-sense applications or as probes on a biochip.
[0034]As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
[0035]The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand "Watson" also defines the sequence of the other strand "Crick"; thus the sequences described herein also includes the complement of the sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. As used herein, the term "nucleoside" includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, "nucleoside" includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
[0036]An CA sequence can be initially identified by substantial nucleic acid and/or amino acid sequence homology to the CA sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
[0037]The CA sequences of the invention were initially identified as described herein; basically, infection of mice with murine leukemia viruses (MLV) resulted in lymphoma. The sequences were subsequently validated by determining expression levels of the gene product, i.e. mRNA, in breast cancer samples.
[0038]The CA sequences outlined herein comprise the insertion sites for the virus. In general, the retrovirus can cause carcinomas in three basic ways: first of all, by inserting upstream of a normally silent host gene and activating it (e.g. promoter insertion); secondly, by truncating a host gene that leads to oncogenesis; or by enhancing the transcription of a neighboring gene. For example, retrovirus enhancers, including SL3-3, are known to act on genes up to approximately 200 kilobases of the insertion site.
[0039]In a preferred embodiment, CA sequences are those that are up-regulated in carcinomas; that is, the expression of these genes is higher in carcinoma tissue as compared to normal tissue of the same differentiation stage. "Up-regulation" as used herein means at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably, at least about 200%, with from 300 to at least 1000% being especially preferred.
[0040]In a preferred embodiment, CA sequences are those that are down-regulated in carcinomas; that is, the expression of these genes is lower in carcinoma tissue as compared to normal I tissue of the same differentiation stage. "Down-regulation" as used herein means at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably, at least about 200%, with from 300 to at least 1000% being especially preferred.
[0041]In a preferred embodiment, CA sequences are those that are altered but show either the same expression profile or an altered profile as compared to normal lymphoid tissue of the same differentiation stage. "Altered CA sequences" as used herein refers to sequences which are truncated, contain insertions or contain point mutations.
[0042]CA proteins of the present invention may be classified as secreted proteins, transmembrane proteins or intracellular proteins.
[0043]In a preferred embodiment the CA protein is an intracellular protein. Intracellular proteins may be found in the cytoplasm and/or in the nucleus. Intracellular proteins are involved in all aspects of cellular function and replication (including, for example, signaling pathways); aberrant expression of such proteins results in unregulated or disregulated cellular processes. For example, many intracellular proteins have enzymatic activity such as protein kinase activity, protein phosphatase activity, protease activity, nucleotide cyclase activity, polymerase activity and the like. Intracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are involved in maintaining the structural integrity of organelles.
[0044]An increasingly appreciated concept in characterizing intracellular proteins is the presence in the proteins of one or more motifs for which defined functions have been attributed. In addition to the highly conserved sequences found in the enzymatic domain of proteins, highly conserved sequences have been identified in proteins that are involved in protein-protein interaction. For example, Src-homology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner. PTB domains, which are distinct from SH2 domains, also bind tyrosine phosphorylated targets. SH3 domains bind to proline-rich targets. In addition, PH domains, tetratricopeptide repeats and WD domains to name only a few, have been shown to mediate protein-protein interactions. Some of these may also be involved in binding to phospholipids or other second messengers. As will be appreciated by one of ordinary skill in the art, these motifs can be identified on the basis of primary sequence; thus, an analysis of the sequence of proteins may provide insight into both the enzymatic potential of the molecule and/or molecules with which the protein may associate.
[0045]In a preferred embodiment, the CA sequences are transmembrane proteins. Transmembrane proteins are molecules that span the phospholipid bilayer of a cell. They may have an intracellular domain, an extracellular domain, or both. The intracellular domains of such proteins may have a number of functions including those already described for intracellular proteins. For example, the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins. Frequently the intracellular domain of transmembrane proteins serves both roles. For example certain receptor tyrosine kinases have both protein kinase activity and SH2 domains. In addition, autophosphorylation of tyrosines on the receptor molecule itself, creates binding sites for additional SH2 domain containing proteins.
[0046]Transmembrane proteins may contain from one to many transmembrane domains. For example, receptor tyrosine kinases, certain cytokine receptors, receptor guanylyl cyclases and receptor serine/threonine protein kinases contain a single transmembrane domain. However, various other proteins including channels and adenylyl cyclases contain numerous transmembrane domains. Many important cell surface receptors are classified as "seven transmembrane domain" proteins, as they contain 7 membrane spanning regions. Important transmembrane protein receptors include, but are not limited to insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporters, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors, e.g. IL-1 receptor, IL-2 receptor, etc.
[0047]Characteristics of transmembrane domains include approximately 20 consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted.
[0048]The extracellular domains of transmembrane proteins are diverse; however, conserved motifs are found repeatedly among various extracellular domains. Conserved structure and/or functions have been ascribed to different extracellular motifs. For example, cytokine receptors are characterized by a cluster of cysteines and a WSXWS (W=tryptophan, S=serine, X=any amino acid) motif. Immunoglobulin-like domains are highly conserved. Mucin-like domains may be involved in cell adhesion and leucine-rich repeats participate in protein-protein interactions.
[0049]Many extracellular domains are involved in binding to other molecules. In one aspect, extracellular domains are receptors. Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like. For example, growth factors such as EGF, FGF and PDGF are circulating growth factors that bind to their cognate receptors to initiate a variety of cellular responses. Other factors include cytokines, mitogenic factors, neurotrophic factors and the like. Extracellular domains also bind to cell-associated molecules. In this respect, they mediate cell-cell interactions. Cell-associated ligands can be tethered to the cell for example via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be transmembrane proteins. Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
[0050]CA proteins that are transmembrane are particularly preferred in the present invention as they are good targets for immunotherapeutics, as are described herein. In addition, as outlined below, transmembrane proteins can be also useful in imaging modalities.
[0051]It will also be appreciated by those in the art that a transmembrane protein can be made soluble by removing transmembrane sequences, for example through recombinant methods. Furthermore, transmembrane proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.
[0052]In a preferred embodiment, the CA proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins have a signal peptide or signal sequence that targets the molecule to the secretory pathway. Secreted proteins are involved in numerous physiological events; by virtue of their circulating nature, they serve to transmit signals to various other cell types. The secreted protein may function in an autocrine manner (acting on the cell that secreted the factor), a paracrine manner (acting on cells in close proximity to the cell that secreted the factor) or an endocrine manner (acting on cells at a distance). Thus secreted molecules find use in modulating or altering numerous aspects of physiology. CA proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, for example for blood tests.
[0053]An CA sequence is initially identified by substantial nucleic acid and/or amino acid sequence homology to the CA sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
[0054]As used herein, a nucleic acid is a "CA nucleic acid" if the overall homology of the nucleic acid sequence to one of the nucleic acids of Table 1 is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%. In some embodiments the homology will be as high as about 93 to 95 or 98%. In a preferred embodiment, the sequences which are used to determine sequence identity or similarity are selected from those of the nucleic acids of Table 1. In another embodiment, the sequences are naturally occurring allelic variants of the sequences of the nucleic acids of Table 1. In another embodiment, the sequences are sequence variants as further described herein.
[0055]Homology in this context means sequence similarity or identity, with identity being preferred. A preferred comparison for homology purposes is to compare the sequence containing sequencing errors to the correct sequence. This homology will be determined using standard techniques known in the art, including, but not limited to, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387-395 (1984), preferably using the default settings, or by inspection.
[0056]One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
[0057]Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzymology, 266: 460-480 (1996); http://blast.wustl]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
[0058]Thus, "percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of the nucleic acids of Table 1. A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
[0059]The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer nucleotides than those of the nucleic acids of Table 1, it is understood that the percentage of homology will be determined based on the number of homologous nucleosides in relation to the total number of nucleosides. Thus, for example, homology of sequences shorter than those of the sequences identified herein and as discussed below, will be determined using the number of nucleosides in the shorter sequence.
[0060]In one embodiment, the nucleic acid homology is determined through hybridization studies. Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acids identified in the figures, or their complements, are considered CA sequences. High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
[0061]In another embodiment, less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and Ausubel, supra, and Tijssen, supra.
[0062]In addition, the CA nucleic acid sequences of the invention are fragments of larger genes, i.e. they are nucleic acid segments. Alternatively, the CA nucleic acid sequences can serve as indicators of oncogene position, for example, the CA sequence may be an enhancer that activates a protooncogene. "Genes" in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, additional sequences of the CA genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences; see Maniatis et al., and Ausubel, et al., supra, hereby expressly incorporated by reference. In general, this is done using PCR, for example, kinetic PCR.
[0063]Once the CA nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire CA nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant CA nucleic acid can be further used as a probe to identify and isolate other CA nucleic acids, for example additional coding regions. It can also be used as a "precursor" nucleic acid to make modified or variant CA nucleic acids and proteins.
[0064]The CA nucleic acids of the present invention are used in several ways. In a first embodiment, nucleic acid probes to the CA nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, for example for gene therapy and/or antisense applications. Alternatively, the CA nucleic acids that include coding regions of CA proteins can be put into expression vectors for the expression of CA proteins, again either for screening purposes or for administration to a patient.
[0065]In a preferred embodiment, nucleic acid probes to CA nucleic acids (both the nucleic acid sequences outlined in the figures and/or the complements thereof) are made. The nucleic acid probes attached to the biochip are designed to be substantially complementary to the CA nucleic acids, i.e. the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by "substantially complementary" herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, as outlined herein.
[0066]A nucleic acid probe is generally single stranded but can be partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. In general, the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.
[0067]In a preferred embodiment, more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular target. The probes can be overlapping (i.e. have some sequence in common), or separate.
[0068]As will be appreciated by those in the art, nucleic acids can be attached or immobilized to a solid support in a wide variety of ways. By "immobilized" and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below. The binding can be covalent or non-covalent. By "non-covalent binding" and grammatical equivalents herein is meant one or more of either electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin. By "covalent binding" and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
[0069]In general, the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
[0070]The biochip comprises a suitable solid substrate. By "substrate" or "solid support" or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon®, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, etc. In general, the substrates allow optical detection and do not appreciably fluoresce.
[0071]In a preferred embodiment, the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two. Thus, for example, the biochip is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred. Using these functional groups, the probes can be attached using functional groups on the probes. For example, nucleic acids containing amino groups can be attached to surfaces comprising amino groups, for example using linkers as are known in the art; for example, homo- or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference). In addition, in some cases, additional linkers, such as alkyl groups (including substituted and heteroalkyl groups) may be used.
[0072]In this embodiment, the oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
[0073]In an additional embodiment, the immobilization to the solid support may be very strong, yet non-covalent. For example, biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
[0074]Alternatively, the oligonucleotides may be synthesized on the surface, as is known in the art. For example, photoactivation techniques utilizing photopolymerization compounds and techniques are used. In a preferred embodiment, the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Pat. Nos. 5,700,637 and 5,445,934; and references cited within, all of which are expressly incorporated by reference; these methods of attachment form the basis of the Affymetrix GeneChip technology.
[0075]In addition to the solid-phase technology represented by biochip arrays, gene expression can also be quantified using liquid-phase arrays. One such system is kinetic polymerase chain reaction (PCR). Kinetic PCR allows for the simultaneous amplification and quantification of specific nucleic acid sequences. The specificity is derived from synthetic oligonucleotide primers designed to preferentially adhere to single-stranded nucleic acid sequences bracketing the target site. This pair of oligonucleotide primers form specific, non-covalently bound complexes on each strand of the target sequence. These complexes facilitate in vitro transcription of double-stranded DNA in opposite orientations. Temperature cycling of the reaction mixture creates a continuous cycle of primer binding, transcription, and re-melting of the nucleic acid to individual strands. The result is an exponential increase of the target dsDNA product. This product can be quantified in real time either through the use of an intercalating dye or a sequence specific probe. SYBR® Greene I, is an example of an intercalating dye, that preferentially binds to dsDNA resulting in a concomitant increase in the fluorescent signal. Sequence specific probes, such as used with TaqMan® technology, consist of a fluorochrome and a quenching molecule covalently bound to opposite ends of an oligonucleotide. The probe is designed to selectively bind the target DNA sequence between the two primers. When the DNA strands are synthesized during the PCR reaction, the fluorochrome is cleaved from the probe by the exonuclease activity of the polymerase resulting in signal dequenching. The probe signaling method can be more specific than the intercalating dye method, but in each case, signal strength is proportional to the dsDNA product produced. Each type of quantification method can be used in multi-well liquid phase arrays with each well representing primers and/or probes specific to nucleic acid sequences of interest. When used with messenger RNA preparations of tissues or cell lines, and an array of probe/primer reactions can simultaneously quantify the expression of multiple gene products of interest. See Germer, S., et al., Genome Res. 10:258-266 (2000); Heid, C. A., et al., Genome Res. 6, 986-994 (1996).
[0076]In a preferred embodiment, CA nucleic acids encoding CA proteins are used to make a variety of expression vectors to express CA proteins which can then be used in screening assays, as described below. The expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the CA protein. The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
[0077]Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the CA protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the CA protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
[0078]In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences. In a preferred embodiment, the regulatory sequences include a promoter and transcriptional start and stop sequences.
[0079]Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
[0080]In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
[0081]In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used.
[0082]The CA proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding an CA protein, under the appropriate conditions to induce or cause expression of the CA protein. The conditions appropriate for CA protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
[0083]Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect, plant and animal cells, including mammalian cells. Of particular interest are Drosophila melanogaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, THP1 cell line (a macrophage cell line) and human cells and cell lines.
[0084]In a preferred embodiment, the CA proteins are expressed in mammalian cells. Mammalian expression systems are also known in the art, and include retroviral systems. A preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and PCT/US97/01048, both of which are hereby expressly incorporated by reference. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. Examples of transcription terminator and polyadenlytion signals include those derived form SV40.
[0085]The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used. Techniques include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
[0086]In a preferred embodiment, CA proteins are expressed in bacterial systems. Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. The expression vector may also include a signal peptide sequence that provides for secretion of the CA protein in bacteria. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include Vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others. The bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
[0087]In one embodiment, CA proteins are produced in insect cells. Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
[0088]In a preferred embodiment, CA protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
[0089]The CA protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, for the creation of monoclonal antibodies. If the desired epitope is small, the CA protein may be fused to a carrier protein to form an immunogen. Alternatively, the CA protein may be made as a fusion protein to increase expression, or for other reasons. For example, when the CA protein is an CA peptide, the nucleic acid encoding the peptide may be linked to other nucleic acid for expression purposes.
[0090]In one embodiment, the CA nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the CA nucleic acids, proteins and antibodies at any position. For example, the label should be capable of producing, either directly or indirectly, a detectable signal. The detectable moiety may be a radioisotope, such as 3H, 14C, 32P, 35S, or 125I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).
[0091]Accordingly, the present invention also provides CA protein sequences. An CA protein of the present invention may be identified in several ways. "Protein" in this sense includes proteins, polypeptides, and peptides. As will be appreciated by those in the art, the nucleic acid sequences of the invention can be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the CA protein has homology to some protein in the database being used. Generally, the nucleic acid sequences are input into a program that will search all three frames for homology. This is done in a preferred embodiment using the following NCBI Advanced BLAST parameters. The program is blastx or blastn. The database is nr. The input data is as "Sequence in FASTA format". The organism list is "none". The "expect" is 10; the filter is default. The "descriptions" is 500, the "alignments" is 500, and the "alignment view" is pairwise. The "query Genetic Codes" is standard (1). The matrix is BLOSUM62; gap existence cost is 11, per residue gap cost is 1; and the lambda ratio is 0.85 default. This results in the generation of a putative protein sequence.
[0092]Also included within one embodiment of CA proteins are amino acid variants of the naturally occurring sequences, as determined herein. Preferably, the variants are preferably greater than about 75% homologous to the wild-type sequence, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%. In some embodiments the homology will be as high as about 93 to 95 or 98%. As for nucleic acids, homology in this context means sequence similarity or identity, with identity being preferred. This homology will be determined using standard techniques known in the art as are outlined above for the nucleic acid homologies.
[0093]CA proteins of the present invention may be shorter or longer than the wild type amino acid sequences. Thus, in a preferred embodiment, included within the definition of CA proteins are portions or fragments of the wild type sequences herein. In addition, as outlined above, the CA nucleic acids of the invention may be used to obtain additional coding regions, and thus additional protein sequence, using techniques known in the art.
[0094]In a preferred embodiment, the CA proteins are derivative or variant CA proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative CA peptide will contain at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. The amino acid substitution, insertion or deletion may occur at any residue within the CA peptide.
[0095]Also included in an embodiment of CA proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the CA protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant CA protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the CA protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
[0096]While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed CA variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and LAR mutagenesis. Screening of the mutants is done using assays of CA protein activities.
[0097]Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
[0098]Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the CA protein are desired, substitutions are generally made in accordance with the following chart:
TABLE-US-00001 CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu
[0099]Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
[0100]The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the CA proteins as needed. Alternatively, the variant may be designed such that the biological activity of the CA protein is altered. For example, glycosylation sites may be altered or removed, dominant negative mutations created, etc.
[0101]Covalent modifications of CA polypeptides are included within the scope of this invention, for example for use in screening. One type of covalent modification includes reacting targeted amino acid residues of an CA polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of an CA polypeptide. Derivatization with bifunctional agents is useful, for instance, for crosslinking CA polypeptides to a water-insoluble support matrix or surface for use in the method for purifying anti-CA antibodies or screening assays, as is more fully described below. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0102]Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl, threonyl or tyrosyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains [T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
[0103]Another type of covalent modification of the CA polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide. "Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence CA polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence CA polypeptide.
[0104]Addition of glycosylation sites to CA polypeptides may be accomplished by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence CA polypeptide (for O-linked glycosylation sites). The CA amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the CA polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
[0105]Another means of increasing the number of carbohydrate moieties on the CA polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 Sep. 1987, and in Aplin and Wriston, L A Crit. Rev. Biochem., pp. 259-306 (1981).
[0106]Removal of carbohydrate moieties present on the CA polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
[0107]Another type of covalent modification of CA comprises linking the CA polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
[0108]CA polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising an CA polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion of an CA polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl-terminus of the CA polypeptide, although internal fusions may also be tolerated in some instances. The presence of such epitope-tagged forms of an CA polypeptide can be detected using an antibody against the tag polypeptide. Also, provision of the epitope tag enables the CA polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. In an alternative embodiment, the chimeric molecule may comprise a fusion of an CA polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
[0109]Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
[0110]Also included with the definition of CA protein in one embodiment are other CA proteins of the CA family, and CA proteins from other organisms, which are cloned and expressed as outlined below. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related CA proteins from humans or other organisms. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the CA nucleic acid sequence. As is generally known in the art, preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditions for the PCR reaction are well known in the art.
[0111]In addition, as is outlined herein, CA proteins can be made that are longer than those encoded by the nucleic acids of the figures, for example, by the elucidation of additional sequences, the addition of epitope or purification tags, the addition of other fusion sequences, etc.
[0112]CA proteins may also be identified as being encoded by CA nucleic acids. Thus, CA proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein.
[0113]In a preferred embodiment, the invention provides CA antibodies. In a preferred embodiment, when the CA protein is to be used to generate antibodies, for example for immunotherapy, the CA protein should share at least one epitope or determinant with the full length protein. By "epitope" or "determinant" herein is meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller CA protein will be able to bind to the full length protein. In a preferred embodiment, the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
[0114]In one embodiment, the term "antibody" includes antibody fragments, as are known in the art, including Fab, Fab2, single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
[0115]Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
[0116]The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a polypeptide encoded by a nucleic acid of Table 1, or fragment thereof or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
[0117]In one embodiment, the antibodies are bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for a protein encoded by a nucleic acid of Table 1, or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific.
[0118]In a preferred embodiment, the antibodies to CA are capable of reducing or eliminating the biological function of CA, as is described below. That is, the addition of anti-CA antibodies (either polyclonal or preferably monoclonal) to CA (or cells containing CA) may reduce or eliminate the CA activity. Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.
[0119]In a preferred embodiment the antibodies to the CA proteins are humanized antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework residues (FR) regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
[0120]Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
[0121]Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies [Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[0122]By immunotherapy is meant treatment of a carcinoma with an antibody raised against an CA protein. As used herein, immunotherapy can be passive or active. Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient). Active immunization is the induction of antibody and/or T-cell responses in a recipient (patient). Induction of an immune response is the result of providing the recipient with an antigen to which antibodies are raised. As appreciated by one of ordinary skill in the art, the antigen may be provided by injecting a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen and under conditions for expression of the antigen.
[0123]In a preferred embodiment, oncogenes which encode secreted growth factors may be inhibited by raising antibodies against CA proteins that are secreted proteins as described above. Without being bound by theory, antibodies used for treatment, bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted CA protein.
[0124]In another preferred embodiment, the CA protein to which antibodies are raised is a transmembrane protein. Without being bound by theory, antibodies used for treatment, bind the extracellular domain of the CA protein and prevent it from binding to other proteins, such as circulating ligands or cell-associated molecules. The antibody may cause down-regulation of the transmembrane CA protein. As will be appreciated by one of ordinary skill in the art, the antibody may be a competitive, non-competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the CA protein. The antibody is also an antagonist of the CA protein. Further, the antibody prevents activation of the transmembrane CA protein. In one aspect, when the antibody prevents the binding of other molecules to the CA protein, the antibody prevents growth of the cell. The antibody may also sensitize the cell to cytotoxic agents, including, but not limited to TNF-α, TNF-β, IL-1, INF-γ and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like. In some instances the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity. Thus, carcinomas may be treated by administering to a patient antibodies directed against the transmembrane CA protein.
[0125]In another preferred embodiment, the antibody is conjugated to a therapeutic moiety. In one aspect the therapeutic moiety is a small molecule that modulates the activity of the CA protein. In another aspect the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the CA protein. The therapeutic moiety may inhibit enzymatic activity such as protease or protein kinase activity associated with carcinoma.
[0126]In a preferred embodiment, the therapeutic moiety may also be a cytotoxic agent. In this method, targeting the cytotoxic agent to tumor tissue or cells, results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with carcinomas, including lymphoma or breast cancer. Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like. Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against CA proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody. Targeting the therapeutic moiety to transmembrane CA proteins not only serves to increase the local concentration of therapeutic moiety in the carcinoma of interest, i.e., lymphoma or breast cancer, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.
[0127]In another preferred embodiment, the CA protein against which the antibodies are raised is an intracellular protein. In this case, the antibody may be conjugated to a protein which facilitates entry into the cell. In one case, the antibody enters the cell by endocytosis. In another embodiment, a nucleic acid encoding the antibody is administered to the individual or cell. Moreover, wherein the CA protein can be targeted within a cell, i.e., the nucleus, an antibody thereto contains a signal for that target localization, i.e., a nuclear localization signal.
[0128]The CA antibodies of the invention specifically bind to CA proteins. By "specifically bind" herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10-4-10-6 M.sup.-1, with a preferred range being 10-7-10-9 M.sup.-1.
[0129]In a preferred embodiment, the CA protein is purified or isolated after expression. CA proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, and chromatofocusing. For example, the CA protein may be purified using a standard anti-CA antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the CA protein. In some instances no purification will be necessary.
[0130]Once expressed and purified if necessary, the CA proteins and nucleic acids are useful in a number of applications.
[0131]In one aspect, the expression levels of genes are determined for different cellular states in the carcinoma phenotype; that is, the expression levels of genes in normal tissue and in carcinoma tissue (and in some cases, for varying severities of lymphoma or breast cancer that relate to prognosis, as outlined below) are evaluated to provide expression profiles. An expression profile of a particular cell state or point of development is essentially a "fingerprint" of the state; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. By comparing expression profiles of cells in different states, information regarding which genes are important (including both up- and down-regulation of genes) in each of these states is obtained. Then, diagnosis may be done or confirmed: does tissue from a particular patient have the gene expression profile of normal or carcinoma tissue.
[0132]"Differential expression," or grammatical equivalents as used herein, refers to both qualitative as well as quantitative differences in the genes temporal and/or cellular expression patterns within and among the cells. Thus, a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus carcinoma tissue. That is, genes may be turned on or turned off in a particular state, relative to another state. As is apparent to the skilled artisan, any comparison of two or more states can be made. Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both. Alternatively, the determination is quantitative in that expression is increased or decreased; that is, the expression of the gene is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript. The degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of Affymetrix GeneChip® expression arrays, Lockhart, Nature Biotechnology, 14:1675-1680 (1996), hereby expressly incorporated by reference. Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, Northern analysis and RNase protection. As outlined above, preferably the change in expression (i.e. upregulation or downregulation) is at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably, at least about 200%, with from 300 to at least 1000% being especially preferred.
[0133]As will be appreciated by those in the art, this may be done by evaluation at either the gene transcript, or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, for example through the use of antibodies to the CA protein and standard immunoassays (ELISAs, etc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc. Thus, the proteins corresponding to CA genes, i.e. those identified as being important in a particular carcinoma phenotype, i.e., breast cancer or lymphoma, can be evaluated in a diagnostic test specific for that carcinoma.
[0134]In a preferred embodiment, gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well. Similarly, these assays may be done on an individual basis as well.
[0135]In this embodiment, the CA nucleic acid probes may be attached to biochips as outlined herein for the detection and quantification of CA sequences in a particular cell. The assays are done as is known in the art. As will be appreciated by those in the art, any number of different CA sequences may be used as probes, with single sequence assays being used in some cases, and a plurality of the sequences described herein being used in other embodiments. In addition, while solid-phase assays are described, any number of solution based assays may be done as well.
[0136]In a preferred embodiment, both solid and solution based assays may be used to detect CA sequences that are up-regulated or down-regulated in carcinomas as compared to normal tissue. In instances where the CA sequence has been altered but shows the same expression profile or an altered expression profile, the protein will be detected as outlined herein.
[0137]In a preferred embodiment nucleic acids encoding the CA protein are detected. Although DNA or RNA encoding the CA protein may be detected, of particular interest are methods wherein the mRNA encoding a CA protein is detected. The presence of mRNA in a sample is an indication that the CA gene, such as KCNJ9 has been transcribed to form the mRNA, and suggests that the protein is expressed. Probes to detect the mRNA can be any nucleotide/deoxynucleotide probe that is complementary to and base pairs with the mRNA and includes but is not limited to oligonucleotides, cDNA or RNA. Probes also should contain a detectable label, as defined herein. In one method the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample. Following washing to remove the non-specifically bound probe, the label is detected. In another method detection of the mRNA is performed in situ. In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the non-specifically bound probe, the label is detected. For example a digoxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding a CA protein is detected by binding the digoxygenin with an anti-digoxygenin secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
[0138]In a preferred embodiment, any of the three classes of proteins as described herein (secreted, transmembrane or intracellular proteins) are used in diagnostic assays. The CA proteins, antibodies, nucleic acids, modified proteins and cells containing CA sequences are used in diagnostic assays. This can be done on an individual gene or corresponding polypeptide level, or as sets of assays.
[0139]As described and defined herein, CA proteins find use as markers of carcinomas, including breast cancer or lymphomas such as, but not limited to, Hodgkin's and non-Hodgkin lymphoma. Detection of these proteins in putative carcinoma tissue or patients allows for a determination or diagnosis of the type of carcinoma. Numerous methods known to those of ordinary skill in the art find use in detecting carcinomas. In one embodiment, antibodies are used to detect CA proteins. A preferred method separates proteins from a sample or patient by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be any other type of gel including isoelectric focusing gels and the like). Following separation of proteins, the CA protein is detected by immunoblotting with antibodies raised against the CA protein. Methods of immunoblotting are well known to those of ordinary skill in the art.
[0140]In another preferred method, antibodies to the CA protein find use in in situ imaging techniques. In this method cells are contacted with from one to many antibodies to the CA protein(s). Following washing to remove non-specific antibody binding, the presence of the antibody or antibodies is detected. In one embodiment the antibody is detected by incubating with a secondary antibody that contains a detectable label. In another method the primary antibody to the CA protein(s) contains a detectable label. In another preferred embodiment each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a plurality of CA proteins. As will be appreciated by one of ordinary skill in the art, numerous other histological imaging techniques are useful in the invention.
[0141]In a preferred embodiment the label is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths. In addition, a fluorescence activated cell sorter (FACS) can be used in the method.
[0142]In another preferred embodiment, antibodies find use in diagnosing carcinomas from blood samples. As previously described, certain CA proteins are secreted/circulating molecules. Blood samples, therefore, are useful as samples to be probed or tested for the presence of secreted CA proteins. Antibodies can be used to detect the CA proteins by any of the previously described immunoassay techniques including ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology and the like, as will be appreciated by one of ordinary skill in the art.
[0143]In a preferred embodiment, in situ hybridization of labeled CA nucleic acid probes to tissue arrays is done. For example, arrays of tissue samples, including CA tissue and/or normal tissue, are made. In situ hybridization as is known in the art can then be done.
[0144]It is understood that when comparing the expression fingerprints between an individual and a standard, the skilled artisan can make a diagnosis as well as a prognosis. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis.
[0145]In a preferred embodiment, the CA proteins, antibodies, nucleic acids, modified proteins and cells containing CA sequences are used in prognosis assays. As above, gene expression profiles can be generated that correlate to carcinoma, especially breast cancer or lymphoma, severity, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being preferred. As above, the CA probes are attached to biochips for the detection and quantification of CA sequences in a tissue or patient. The assays proceed as outlined for diagnosis.
[0146]In a preferred embodiment, any of the CA sequences as described herein are used in drug screening assays. The CA proteins, antibodies, nucleic acids, modified proteins and cells containing CA sequences are used in drug screening assays or by evaluating the effect of drug candidates on a "gene expression profile" or expression profile of polypeptides. In one embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, Zlokarnik, et al., Science 279, 84-8 (1998), Heid, et al., Genome Res., 6:986-994 (1996).
[0147]In a preferred embodiment, the CA proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified CA proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the carcinoma phenotype. As above, this can be done by screening for modulators of gene expression or for modulators of protein activity. Similarly, this may be done on an individual gene or protein level or by evaluating the effect of drug candidates on a "gene expression profile". In a preferred embodiment, the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, see Zlokarnik, supra.
[0148]Having identified the CA genes herein, a variety of assays to evaluate the effects of agents on gene expression may be executed. In a preferred embodiment, assays may be run on an individual gene or protein level. That is, having identified a particular gene as aberrantly regulated in carcinoma, candidate bioactive agents may be screened to modulate the genes response. "Modulation" thus includes both an increase and a decrease in gene expression or activity. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tumor tissue, with changes of at least 10%, preferably 50%, more preferably 100-300%, and in some embodiments 300-1000% or greater. Thus, if a gene exhibits a 4 fold increase in tumor compared to normal tissue, a decrease of about four fold is desired; a 10 fold decrease in tumor compared to normal tissue gives a 10 fold increase in expression for a candidate agent is desired, etc. Alternatively, where the CA sequence has been altered but shows the same expression profile or an altered expression profile, the protein will be detected as outlined herein.
[0149]As will be appreciated by those in the art, this may be done by evaluation at either the gene or the protein level; that is, the amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the level of the gene product itself can be monitored, for example through the use of antibodies to the CA protein and standard immunoassays. Alternatively, binding and bioactivity assays with the protein may be done as outlined below.
[0150]In a preferred embodiment, gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well.
[0151]In this embodiment, the CA nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of CA sequences in a particular cell. The assays are further described below.
[0152]Generally, in a preferred embodiment, a candidate bioactive agent is added to the cells prior to analysis. Moreover, screens are provided to identify a candidate bioactive agent which modulates a particular type of carcinoma, modulates CA proteins, binds to a CA protein, or interferes between the binding of a CA protein and an antibody.
[0153]The term "candidate bioactive agent" or "drug candidate" or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic or inorganic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactive agents that are capable of directly or indirectly altering either the carcinoma phenotype, binding to and/or modulating the bioactivity of an CA protein, or the expression of a CA sequence, including both nucleic acid sequences and protein sequences. In a particularly preferred embodiment, the candidate agent suppresses a CA phenotype, for example to a normal tissue fingerprint. Similarly, the candidate agent preferably suppresses a severe CA phenotype. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
[0154]In one aspect, a candidate agent will neutralize the effect of an CA protein. By "neutralize" is meant that activity of a protein is either inhibited or counter acted against so as to have substantially no effect on a cell.
[0155]Candidate agents encompass numerous chemical classes, though typically they are organic or inorganic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
[0156]Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
[0157]In a preferred embodiment, the candidate bioactive agents are proteins. By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
[0158]In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, may be used. In this way libraries of procaryotic and eucaryotic proteins may be made for screening in the methods of the invention. Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
[0159]In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
[0160]In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
[0161]In a preferred embodiment, the candidate bioactive agents are nucleic acids, as defined above.
[0162]As described above generally for proteins, nucleic acid candidate bioactive agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
[0163]In a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
[0164]In assays for altering the expression profile of one or more CA genes, after the candidate agent has been added and the cells allowed to incubate for some period of time, the sample containing the target sequences to be analyzed is added to the biochip. If required, the target sequence is prepared using known techniques. For example, the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR occurring as needed, as will be appreciated by those in the art. For example, an in vitro transcription with labels covalently attached to the nucleosides is done. Generally, the nucleic acids are labeled with a label as defined herein, with biotin-FITC or PE, cy3 and cy5 being particularly preferred.
[0165]In a preferred embodiment, the target sequence is labeled with, for example, a fluorescent, chemiluminescent, chemical, or radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe. The label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected. Alternatively, the label can be a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme. The label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin. For the example of biotin, the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. As known in the art, unbound labeled streptavidin is removed prior to analysis.
[0166]As will be appreciated by those in the art, these assays can be direct hybridization assays or can comprise "sandwich assays", which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference. In this embodiment, in general, the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
[0167]A variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above. The assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
[0168]These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding.
[0169]The reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target. In addition, either solid phase or solution based (i.e., kinetic PCR) assays may be used.
[0170]Once the assay is run, the data is analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.
[0171]In a preferred embodiment, as for the diagnosis and prognosis applications, having identified the differentially expressed gene(s) or mutated gene(s) important in any one state, screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.
[0172]In addition, screens can be done for novel genes that are induced in response to a candidate agent. After identifying a candidate agent based upon its ability to suppress a CA expression pattern leading to a normal expression pattern, or modulate a single CA gene expression profile so as to mimic the expression of the gene from normal tissue, a screen as described above can be performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent treated CA tissue reveals genes that are not expressed in normal tissue or CA tissue, but are expressed in agent treated tissue. These agent specific sequences can be identified and used by any of the methods described herein for CA genes or proteins. In particular these sequences and the proteins they encode find use in marking or identifying agent treated cells. In addition, antibodies can be raised against the agent induced proteins and used to target novel therapeutics to the treated CA tissue sample.
[0173]Thus, in one embodiment, a candidate agent is administered to a population of CA cells, that thus has an associated CA expression profile. By "administration" or "contacting" herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface. In some embodiments, nucleic acid encoding a proteinaceous candidate agent (i.e. a peptide) may be put into a viral construct such as a retroviral construct and added to the cell, such that expression of the peptide agent is accomplished; see PCT US97/01019, hereby expressly incorporated by reference.
[0174]Once the candidate agent has been administered to the cells, the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein.
[0175]Thus, for example, CA tissue may be screened for agents that reduce or suppress the CA phenotype. A change in at least one gene of the expression profile indicates that the agent has an effect on CA activity. By defining such a signature for the CA phenotype, screens for new drugs that alter the phenotype can be devised. With this approach, the drug target need not be known and need not be represented in the original expression screening platform, nor does the level of transcript for the target protein need to change.
[0176]In a preferred embodiment, as outlined above, screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of either the expression of the gene or the gene product itself can be done. The gene products of differentially expressed genes are sometimes referred to herein as "CA proteins" or an "CAP". The CAP may be a fragment, or alternatively, be the full length protein to the fragment encoded by the nucleic acids of Table 1. Preferably, the CAP is a fragment. In another embodiment, the sequences are sequence variants as further described herein.
[0177]Preferably, the CAP is a fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment. Preferably, the fragment includes a non-transmembrane region. In a preferred embodiment, the fragment has an N-terminal Cys to aid in solubility. In one embodiment, the a c-terminus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, i.e., to cysteine.
[0178]In one embodiment the CA proteins are conjugated to an immunogenic agent as discussed herein. In one embodiment the CA protein is conjugated to BSA.
[0179]In a preferred embodiment, screening is done to alter the biological function of the expression product of the CA gene, such as KCNJ9. Again, having identified the importance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can be run as is more fully outlined below.
[0180]In a preferred embodiment, screens are designed to first find candidate agents that can bind to CA proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate the CAP activity and the carcinoma phenotype. Thus, as will be appreciated by those in the art, there are a number of different assays which may be run; binding assays and activity assays.
[0181]In a preferred embodiment, binding assays are done. In general, purified or isolated gene product is used; that is, the gene products of one or more CA nucleic acids are made. In general, this is done as is known in the art. For example, antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present. Alternatively, cells comprising the CA proteins can be used in the assays.
[0182]Thus, in a preferred embodiment, the methods comprise combining a CA protein and a candidate bioactive agent, and determining the binding of the candidate agent to the CA protein. Preferred embodiments utilize the human or mouse CA protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease. In some embodiments, as outlined herein, variant or derivative CA proteins may be used.
[0183]Generally, in a preferred embodiment of the methods herein, the CA protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.). The insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape. Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, Teflon®, etc. Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable. Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
[0184]In a preferred embodiment, the CA protein is bound to the support, and a candidate bioactive agent is added to the assay. Alternatively, the candidate agent is bound to the support and the CA protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
[0185]The determination of the binding of the candidate bioactive agent to the CA protein may be done in a number of ways. In a preferred embodiment, the candidate bioactive agent is labeled, and binding determined directly. For example, this may be done by attaching all or a portion of the CA protein to a solid support, adding a labeled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as is known in the art.
[0186]By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. For the specific binding members, the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal.
[0187]In some embodiments, only one of the components is labeled. For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using 125I, or with fluorophores. Alternatively, more than one component may be labeled with different labels; using 125I for the proteins, for example, and a fluorophore for the candidate agents.
[0188]In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to the target molecule (i.e. CA protein), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent.
[0189]In one embodiment, the candidate bioactive agent is labeled. Either the candidate bioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimal activity, typically between 4 and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
[0190]In a preferred embodiment, the competitor is added first, followed by the candidate bioactive agent. Displacement of the competitor is an indication that the candidate bioactive agent is binding to the CA protein and thus is capable of binding to, and potentially modulating, the activity of the CA protein. In this embodiment, either component can be labeled. Thus, for example, if the competitor is labeled, the presence of label in the wash solution indicates displacement by the agent. Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
[0191]In an alternative embodiment, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive agent is bound to the CA protein with a higher affinity. Thus, if the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the CA protein.
[0192]In a preferred embodiment, the methods comprise differential screening to identity bioactive agents that are capable of modulating the activity of the CA proteins. In this embodiment, the methods comprise combining a CA protein and a competitor in a first sample. A second sample comprises a candidate bioactive agent, a CA protein and a competitor. The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the CA protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the CA protein.
[0193]Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native CA protein, but cannot bind to modified CA proteins. The structure of the CA protein may be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug candidates that affect CA bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
[0194]Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
[0195]A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
[0196]Screening for agents that modulate the activity of CA proteins may also be done. In a preferred embodiment, methods for screening for a bioactive agent capable of modulating the activity of CA proteins comprise the steps of adding a candidate bioactive agent to a sample of CA proteins, as above, and determining an alteration in the biological activity of CA proteins. "Modulating the activity of an CA protein" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the candidate agent should both bind to CA proteins (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of CA proteins.
[0197]Thus, in this embodiment, the methods comprise combining a CA sample and a candidate bioactive agent, and evaluating the effect on CA activity. By "CA activity" or grammatical equivalents herein is meant one of the CA protein's biological activities, including, but not limited to, its role in tumorigenesis, including cell division, preferably in lymphatic tissue, cell proliferation, tumor growth and transformation of cells. In one embodiment, CA activity includes activation of or by a protein encoded by a nucleic acid of Table 1. An inhibitor of CA activity is the inhibition of any one or more CA activities.
[0198]In a preferred embodiment, the activity of the CA protein is increased; in another preferred embodiment, the activity of the CA protein is decreased. Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.
[0199]In a preferred embodiment, the invention provides methods for screening for bioactive agents capable of modulating the activity of a CA protein. The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising CA proteins. Preferred cell types include almost any cell. The cells contain a recombinant nucleic acid that encodes a CA protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells.
[0200]In one aspect, the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts). In another example, the determinations are determined at different stages of the cell cycle process.
[0201]In this way, bioactive agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the CA protein.
[0202]In one embodiment, a method of inhibiting carcinoma cancer cell division, is provided. The method comprises administration of a carcinoma cancer inhibitor.
[0203]In a preferred embodiment, a method of inhibiting lymphoma carcinoma cell division is provided comprising administration of a lymphoma carcinoma inhibitor.
[0204]In a preferred embodiment, a method of inhibiting breast cancer carcinoma cell division is provided comprising administration of a breast cancer carcinoma inhibitor.
[0205]In another embodiment, a method of inhibiting tumor growth is provided. The method comprises administration of a carcinoma cancer inhibitor. In a particularly preferred embodiment, a method of inhibiting tumor growth in lymphatic tissue is provided comprising administration of a lymphoma inhibitor.
[0206]In another embodiment, a method of inhibiting tumor growth is provided. The method comprises administration of a carcinoma cancer inhibitor. In a particularly preferred embodiment, a method of inhibiting tumor growth in mammary tissue is provided comprising administration of a breast cancer inhibitor.
[0207]In a further embodiment, methods of treating cells or individuals with cancer are provided. The method comprises administration of a carcinoma cancer inhibitor. In one embodiment the carcinoma is a breast cancer carcinoma. In an alternative embodiment, the carcinoma is a lymphoma carcinoma. In one embodiment, a carcinoma cancer inhibitor is an antibody as discussed above. In another embodiment, the carcinoma cancer inhibitor is an antisense molecule. Antisense molecules as used herein include antisense or sense oligonucleotides comprising a singe-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for carcinoma cancer molecules. Antisense or sense oligonucleotides, according to the present invention, comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988) and van der Krol et al., BioTechniques 6:958, (1988).
[0208]Antisense molecules may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell. Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
[0209]The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways. The concentration of therapeutically active compound in the formulation may vary from about 0.1-100% wgt/vol. The agents may be administered alone or in combination with other treatments, i.e., radiation.
[0210]The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
[0211]Without being bound by theory, it appears that the various CA sequences are important in carcinomas. Accordingly, disorders based on mutant or variant CA genes may be determined. In one embodiment, the invention provides methods for identifying cells containing variant CA genes comprising determining all or part of the sequence of at least one endogenous CA genes in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the CA genotype of an individual comprising determining all or part of the sequence of at least one CA gene, such as KCNJ9 of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue. The method may include comparing the sequence of the sequenced CA gene to a known CA gene, such as KCNJ9, i.e., a wild-type gene. As will be appreciated by those in the art, alterations in the sequence of some oncogenes can be an indication of either the presence of the disease, or propensity to develop the disease, or prognosis evaluations.
[0212]The sequence of all or part of the CA gene, such as KCNJ9, can then be compared to the sequence of a known CA gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc. In a preferred embodiment, the presence of a difference in the sequence between the CA gene, such as KCNJ9 of the patient and the known CA gene is indicative of a disease state or a propensity for a disease state, as outlined herein.
[0213]In a preferred embodiment, the CA genes are used as probes to determine the number of copies of the CA gene, such as KCNJ9 in the genome. For example, some cancers exhibit chromosomal deletions or insertions, resulting in an alteration in the copy number of a gene.
[0214]In another preferred embodiment CA genes are used as probes to determine the chromosomal location of the CA genes. Information such as chromosomal location finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in CA gene, such as KCNJ9, loci.
[0215]Thus, in one embodiment, method's of modulating CA in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-CA antibody that reduces or eliminates the biological activity of an endogenous CA protein. Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a CA protein. As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment, for example when the CA sequence is down-regulated in carcinoma, the activity of the CA gene is increased by increasing the amount of CA in the cell, for example by overexpressing the endogenous CA or by administering a gene encoding the CA sequence, using known gene-therapy techniques, for example. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety. Alternatively, for example when the CA sequence is up-regulated in carcinoma, the activity of the endogenous CA gene is decreased, for example by the administration of a CA antisense nucleic acid.
[0216]In one embodiment, the CA proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to CA proteins, which are useful as described herein. Similarly, the CA proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify CA antibodies. In a preferred embodiment, the antibodies are generated to epitopes unique to a CA protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications. For example, the CA antibodies may be coupled to standard affinity chromatography columns and used to purify CA proteins. The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the CA protein.
[0217]In one embodiment, a therapeutically effective dose of a CA or modulator thereof is administered to a patient. By "therapeutically effective dose" herein is meant a dose that produces the effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for CA degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
[0218]A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
[0219]The administration of the CA proteins and modulators of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly. In some instances, for example, in the treatment of wounds and inflammation, the CA proteins and modulators may be directly applied as a solution or spray.
[0220]The pharmaceutical compositions of the present invention comprise a CA protein in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically acceptable base addition salts" include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
[0221]The pharmaceutical compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.
[0222]In a preferred embodiment, CA proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above. Similarly, CA genes (including both the full-length sequence, partial sequences, or regulatory sequences of the CA coding regions) can be administered in gene therapy applications, as is known in the art. These CA genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
[0223]In a preferred embodiment, CA genes, such as KCNJ9, are administered as DNA vaccines, either single genes or combinations of CA genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16:1304-1305 (1998).
[0224]In one embodiment, CA genes of the present invention are used as DNA vaccines. Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a CA gene or portion of a CA gene under the control of a promoter for expression in a patient with carcinoma. The CA gene used for DNA vaccines can encode full-length CA proteins, but more preferably encodes portions of the CA proteins including peptides derived from the CA protein. In a preferred embodiment a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a CA gene. Similarly, it is possible to immunize a patient with a plurality of CA genes or portions thereof as defined herein. Without being bound by theory, expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced which recognize and destroy or eliminate cells expressing CA proteins.
[0225]In a preferred embodiment, the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine. Such adjuvant molecules include cytokines that increase the immunogenic response to the CA polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.
[0226]In another preferred embodiment CA genes find use in generating animal models of carcinomas, particularly breast cancer or lymphoma carcinomas. As is appreciated by one of ordinary skill in the art, when the CA gene identified is repressed or diminished in CA tissue, gene therapy technology wherein antisense RNA directed to the CA gene will also diminish or repress expression of the gene. An animal generated as such serves as an animal model of CA that finds use in screening bioactive drug candidates. Similarly, gene knockout technology, for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence of the CA protein. When desired, tissue-specific expression or knockout of the CA protein may be necessary.
[0227]It is also possible that the CA protein is overexpressed in carcinoma. As such, transgenic animals can be generated that overexpress the CA protein. Depending on the desired expression level, promoters of various strengths can be employed to express the transgene. Also, the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of CA and are additionally useful in screening for bioactive molecules to treat carcinoma.
[0228]The CA nucleic acid sequences of the invention are depicted in Table 1. The sequences in each Table include genomic sequence, mRNA and coding sequences for both mouse and human. N/A indicates a gene that has been identified, but for which there has not been a name ascribed. The different sequences are assigned the following SEQ ID Nos: [0229]Table 1 (mouse gene: mCG2257; human gene KCNJ9) [0230]Mouse genomic sequence (SEQ ID NO: 1) [0231]Mouse mRNA sequence (SEQ ID NO: 2) [0232]Mouse coding sequence (SEQ ID NO: 3) [0233]Human genomic sequence (SEQ ID NO: 4) [0234]Human mRNA sequence (SEQ ID NO: 5) [0235]Human coding sequence (SEQ ID NO: 6)
TABLE-US-00002 [0235]TABLE 1 MOUSE SEQUENCE - GENOMIC NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN- NNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN- NNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN- NNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCC- TCATGAATGCTGAGACTAAAGGT GTGCATCACCCCTGCCCAATTTCAAAATAGTGACCCAAGGGAAGACCAGATTACAAGGTGCTGCACTACAAAGT- GAGAAAATGTTAACGGTTACCCT TTAAAAACTTTGCTTAGAGGGAAAAAAAAAACCCCACAATCATAACCAAAGCAATGGACCAGGAACTATTTTCC- TGCCTGTTTTGTCTTTTCAAATT TCTGTCATCTTCTGCTCCTAGAGAGGAACGGCTACAGTAAGATGGTCTGAAGACCTGGTAGTTTTTTTTTTTTT- TTTTTTTTTAAGATTTATTTATT TATTATATGTAAGTACATGTAAGTAAGTACATTGTAGCTGTCCTCAGATACTCCAGAAGAGGGCATCAGATTTC- GTTACGGATGGTTGTGAGCCACC ATGTGGTTGCTGGGATTTGAACTCGGGACCTTTGGAAAAGCAGTCGGTGCTCTTAACCACTGAGCCATCTCGCC- AGCCCAGACCTGGTAGTTTAAGC CTGCAATCTCAGCTGTTTGGGGAGGGGAAGCAGGAGGGTTGCAAGCTCAAAGCCTGAGCTACAGAATGAGTTCA- AAGCCAGTGTGAATAACTTAGCA GGGCTCACAGTCTTGACATTCAGAGATGGGGAAGATTATGGGGCTGAGCTCAGACCACAATATAAAATGAAGAA- GGAACACAGAGGAGAGAAGCCAA GAACTGTCGGGGTTTATGAAATCATTACAAGACACAAGAATTTATTATTTTTCCAGAATTGTTACCCAAGCATT- TGGCATCCATCGCCACCTACATG TCAGTGTCCACCTGGACAGAAATCTCAAACTTAGTCCAGCGTAGAACATCTTACCCACAGGAGCGCTCCTCATG- GGACTATGTCACCATCATCCAAC TAGAAACACAGCAGTCATCTCAGCCTCCTTAGTCTTCCTTACAGCAGCAACTCCATCCTCTAACCAAAGCATCT- CCCACTGAGCACGCCCTCCTGCC CCCCTCTCTCTCTCTCCCTTTATCGCTGCTGCAGTCTACAGCAGATGCACCTCTCAGCAGGGATCCTGGAGCAG- CCATCTAGTGCCTTATCCCCTCC AGTCTTTCTACACTCCAATAATGCTTCAGGTCACTAACTCCTTTATGTAAAAACAATTAAGGCTCAGCAAGATG- GCTCAGAGGGGTAAAGGCAACTT GCTGCCAAACTTGATGACCTGGGGTCAATCTCCAGTACTGATGTGGTAGGAGAGACTCAACTACCAAGAGTTAT- CCTCTGACCTCTACATGTGTGTT GTGGTACACCCACAAACACAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGACAGACAGACAGACAGACAGACA- GACAGACAGAGACACACACAGAG AGAAATGTAACGTTTAGAGAAGAATCCATCCATATTCTTTAGCACAGAACAGAAGGCACATTAATTATAACCTG- GGCATCCTGCCCTGTCTTCCTCA CATCCAACTCTATAGCTGCTTCCTCCTCTAACACCCAAGGTTGTTAAGTCTTGTGTCCCCTTCTGTATCTTGCT- CCTTGTTCTTTGGTCACACAGTG ACCAAAGTCACTGAGTGTTGTGCAAACCTCTTCTTCTTGACTCCTGTATCTCTCTGGAGCTCTACTTAGGCTCC- AGTACCTGCAAGGGATTAATGCC CTCACATGACAGGCCCCAGACAGAACCCATCCTCTTTCCCTCTCACCAAGGTTGGGAATGCTCACAGCTCCCTG- ATTTCTGTGTAACTCCTGTCAAG CAGACTGAAACACCGACATTACATCTTGCTCTTTATGCTTGCCTATGTCCCATTCTGTGTCATGACAATTCAGC- CACCAAGTTCTGTTAACTCTCCC TTGGTTATATTTCTCTAGGATACACATTTTCATTTCTATGGCCAGAATCATAAAATTACCACTAGCCCAGGACC- TGACCCATCCCTCACCCCTCTTT CCAGTATCAAAGGGAGACAAACTGTTTTTATTAAAGATGTACTGTATTTAAAAAAACCTAGAATCAAAACTTTG- AACAAAGTGGGGTGTGATGGTAT ACACCTTTAATCCCAGCACTTGGGAGGCAGAGGCAGGTGGATTTCTGAGTTCAAGGCCAGCCTGGTCTACAAAG- TGAGTTCCAGGACAGCCAGGTCT ACACAGAGAAATCCTGTCTCGAAAAAACCAACCAACCAAATAAATAAATAAATAAATAAATAAATAAATAAATA- AATACAAGGTCTCTGGATAAACT CCTTCCAAATAGAAATGAGAAGCCATCTGGTGAAGCTCAGTGTGAGGGTGAGGTGGCACGAGATGGAACTGGGC- AATTGGAAAGAGTTAGGTATTGT AGAGCCACAGGAGAGCAGGACTGTGGTGACTTCTGTGGCCCTGTGATGTTCTCACTCAAGAGTGACTTACATCA- GGATTCCATTCTTAAATAAGCAC ACTTTATTAGCAACTATAACTCTGTATACATTGTGTTTGCTTTTAATATTTAACTTTTTGTTTTCCAAAAAGAG- TTCCTGAAACATACAACAAGCAG AAATTGTCATTGCTGAAGGATGCTTAGCATGCTCATGTTTCTGAGTGTTTACTAGGCGTGATAAATTTGACTTT- TCTTGTTTTCTTTCAGTTCACTC TGTCTGATGCTCCTGCCCCGGTCTCCTAAATGCAGGGATTATAGGTGTGCACCGCCACACCTAACTGTGTACAG- TAGATCGTAAGATGGGAAATCCC AGAGTCAGGGACCTTAGGTGGCTGACCTATACACAGTGACATGCCCAGGAAGTGTTAAATCTGGCATTTGAATC- CACCTGTTTGACCCCAGAGTTTG TCAAAGGGTAATAGTACAGCGCTCTTGCATGACTTAAAGAGATGCTCATTTTCCCAAGAGAACCAAGAGGTTCT- AGTGGCCAAATGTCAGTATGAAT AAATCTGCTGAGATGCGCTGTGCAGCGTCCGTCGACCTTACAGGAGGACAGAGCAATCCTTTTCCTTTTTGATT- CATCGCTCCTTTCAGACTTGATC CTCTCACCACAGATCTCTTTCCTTCCACTTCCTCATTCAAAATGGGGTCAGTTCCCCCTCAGAACAAAAGAGGA- ACATGAGGCGAAGACCCTTTGCA GAGGGAAAATCCACAGCTGGGCGTAGGCCGAGGGAGCTTTCGCTGGGAGAAGCAGGTGAGTTCGGATGAAGGGA- AGCAACTGAGAGAGGCAAGGCAG ATCCTCAGACGGGGCGGGTTGGGGGGGGGGCGACTCGGAGAGGGAGTTTTCGGGGAGTCATCAGAGCTGGCCAG- GAAGAACTAGGCATGAACATGAG TCCCAGGGACTCCGAGGGACACATTTCTGCTTAGGTCCCACAGTATTAACACGGTCCACTAAAAGCAGATACGC- TCAGCAGGATGAGCGGCCACAGA GGAGAGCCTATCAGTACTCGGTTTAGTCATTACCTTTTAATACACATGATTTATATAAGCCTGTATGTGTATAA- GACTTAAGTTATAAATGGCTAAT TACATTACAGAAGGACTACAGAAGGCAGAGAGAGGGAGGGAGGGGAGGGCAAGGGTGGGGAGGGGAGGGAAAGG- AAGATGCTCTTTACCTATAAGGT TTATCTAGTATCTTTCTAATTGGTCCTTTTAGTGGCAATTCTGTTAACATTCAAATACACCATGGAGAGGGAAG- AACAGAAAACCCCCAGATGCCTG GAACTGGGGAAGCTGTCTTAACCCTGACCTCTCTTGGGATGCTCTTCTCATCTATAAACTAATGATTACTTTAG- ATCACTTCTGAATGACCATGGTT AAGTCCTGGTCTAACTCTATCCAGCCCCGTAGACCTGGTAGACAAGATGGACCTGTGCGTAACTCTTCTAGGGC- TGATTCCACATGGAATTTACCTA CTTTTATTTAGAGATGAGGTCTCACTGTGTCCCTCTGGATGAGCTGGAACTCACCACACACACCAGGGTGGCCT- CAGACTCAGAGATTTACTTGCCA GTGCTTCTCAAATGTTGGGGTAAAAAGCGTAAGCCACCACCCACAGACCCCATGAATTCATATCAATTGTTATT- TGAACTAACTTGACCTTCCTACT CCCCTCAGCTCACATCCTCAACCGTCCCTGCCTTCCCCTCCAGACTTCCTCCCCCATTTCCACGCTTTTGCTCA- AGAAGTCTCATGATCTCGTTCAA GGAAGCTCTCCCAGGTTGGCTGACCTCATAGCTGGCAACAAAGGCAACTACTGCTAGGGGTGAACACAAGGCTA- CAGTGCACTCATCCTGCACCCAA ACTCAGAATTGCACCAAAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT- GTCTGTGTCTGTGTCTGTGTCTG TGTCTGTGTGTCTGTGTGTCTCTGTGTGTGTCTGTGTCTGTGTTTGCTTTCACTGTGTATGCATATGTGGAGAC- CAGAGACGAATGAGGAGATCATA GACATACACTGTCACACCCAGCCTTTCTGTGGATGCTGGGGATCCAAACTCAGGTCCCCAGATCCATGCAGCAA- GTCCTTTGCCCACTAGGCTGTCT CCCGAGCTCTGCACCTAGGCTCTTTATAGGACCAGCAGTGTGGCCTCACTGTCCTCTATTTCCAATCTGTGTTT- ATTACAACTCCGCTGACATATTG GGGTTGATTTCTTGGAGGGATGCTTTTATTCTCTTGGTGAAATATTTTTCTGTGCACTGATGGCTTGTGAAATT- TTCTTCTCTGTTGCCTCAGTTCA AGCCAGACGAACAAGGAGCTGAGATTAAGCTTAGTAAGTAAAGCCCAGGACCTGGAGGATCTGGAAACTGGGTG- AAAGAGTTGTCCTCTGTTGGCTA GGTTAGGTTCAGGGCAGCCAGGATGGAGTCAGAGGGGTGGCTGACAACACCCAGGGGCCACTGTCAGCTCTGTG- ACTTTCCCTCCAGAAAAAGGGGC CAGTTCTGTGCAAACATGTTCTTGTCCAGGAGTTTGGTTTCTTCTCTCTGAGCACCTGGCACAGTGGCACCAAT- GTGAGCAGTCACTTGGCAGGGCA GAGAAAAGCAAGCTAGCAGTCCCCAGGCTCAGGTGACAGAGCCAGGCCCAGGAGACAGGGATATTGACTGGGGC- TTTAACAGCACTATTGATGCCAA TCTCGGGCAAAAACCTGATATTTCCACTTGGAATAACAAGAAACAGCCAAGAGGATTGGAGAGAGGTCAGTGGA- CAAGGAGAGCCCTCTGCAGGTCG TGCTGGGTGATTCCAGAACAGAAGAGGGCAGCCCCTGCTGGACAGGGTCTCCTGAGATGATGGTGATGGTGACG- GTGATGGTAATAGTGATGGTGAC AGGGACAATGACAGTAGTAGTGGCTAGGAGAAGGAAAAAGAAGAAAGAGAAAAACACAATGTCAGGCTTTAAAT- AAATAATCCTCATGAAGTAGATA CTATTTATTGTGGTTTTGATATGAAACACCCCTCCCCCAAGGGCTCGGGTATTGGAGATTTGAGCCCCAGCGTG- TGGTGCTATTGAGAGGTGACTTT GTTGATGGCGACTTTGTTTGATGAGGAGATAAGAGGTGGACTGTAAGGAGGTGAAAGCTGTCTGAGGAAGTAGG- TCACCAGTGGTGTGCTCTCAAAG GTGGGTCTCAACCTTCCCAATGCTGTGACGCTTCATTTAACACAGTTCCTTATGTTGTGGTGGCCCCCCCAATC- ATAAAATTAGTTTTGTTGCTGCT TCATAACTGTAATTTTACTACTGTTATAAATCATAATGTAAATATTTTTGGAGCTTAGAGGCTTGCCAAAGGGG- TCACGACCCACAGGTTGAGAACC ACTGCTCTAGAAGGAAGTCACCTCCTCTTCTCTCCTTGTCATTCTCTTTTCTTCCTCTCCCTTACACTCCCCTT- CTTCTCTTCTCTCTCCCTACTCC TCACCTCTCCACTCTCCATGAAATCTGATTTCCCCTGCTATCTACCACAATGCCGGATCTCATATTCCCAAGAA- GAATGGAGACAAACAACCCTGGA CTGGATTCCTCCTCTTTTAAGTGTGACTTGGGTGTTCTGTTACAGCAATGAAAAGCTAGCAATATAAGATGGCT- AGTCTCATCTCTTAGATTTAAAA AACTAACATTTTCCAAACATAGTGGCTCATGTCTGTAGACACAGAGCCAGGGAAGCAGAGGCAGAAGGATCCAC- TGCAGGTCCAAGGCTGGCCTGGA CTATGTAACAAGAGAGAGAGAGAGAGAGAGAGAGAGAGTAGAGAGAGAGAGAGGAGAGATTGAAGGAAAGAAAG- ATTGAAGAAAAGAAAGATGAAAG AAAGAAAGAAAGAAAGAAAGATTGAAGGAAAGAAAGAGAGATTTAAAGAAAGAAAAAAGAAAGATGAAAGAAAG- AAAAGGGAAGAAAGATTGAAGGA AAGAAAAGAACGGAAGGAAGGAAAGAAGGAAGGAAGGAAGGAGTGCAGGGGGAGGAGGGAAGAAAAGAGTGGAG- GGGGAGGAGGGAGAAAAGAAAGA AAAGGAGACTATATGAAGCTATTTGCTCAAAGCCATGCATCTTCTATCAGAGAGTAGAATTTGAACTCAAGTCA- TTGCCTCTGAAGCTTGTATTACC CCACACACCTGTCATAGCTCGTGAGCACATTTCAGAAACTTCTAGTCTTCTATTGTGCTGTTTCTTCCTGTTCT- TTCTAGTTATGTATTCTTGCAGT GTTAAGGCTTAGGGGATTGGATATAAAATATCTTGTGCATAACAATATTGGCAATAGTAATAACACCAGCTTAA- ATTTATTTTTTATAGCTTTAGTA ATTTAATTTATGTATATGAGTTCATGGTAGCTGTCTTCAGACACACCAGAAGAGGGCATCAGATCCTATTACAG- ATGGTTGTGAGCCACCATGTGGT TGCTGGGAATTGAACTCAGGACTACTGGAAGAACAGCCAGTGCTCTTAACTGCTGCAAAATGGTACAGTTACTC- TGGAAGACAGTTTGGCAGTCACC TGAAAAACTAAACATACTCTTTCCATATGATATTGCAACCATACTCCTTGGTATTTACCGCACCCCCAAAAGCT- GAAAACTTGTCTAAATAAAAACC CTGCACACAGATGTTTGTAGCAACTTTATTTGGAATCGGCAAAAACTGGAAATGAAATGACTTTCAGTGGCTCA- ATGGACAAATGAATTGTGGTACT TTCCTGGCCGTGGACCATCATTCAGTACCAAAATGAGATGAGCTGTGGAGCTAAAAAAGACATGAAGCAACCTT- AAATGCACAAGTGGAAGAAGCCA ATCCAAGGAGCTGCATACTGTATAATTCCAACCCCATGGCATCCTGGAAAAGGCAGAACCATGGAAACAGGTTT- TTAAAAAATCAGAGATTGCCAAA GGCTAAGGGGAGAGTGGATGGCTGGGGGCAGCAGAGAGGAAAGCACCCCACAACCATCATGGCGGATACACATC- CTCGTGGCCGTTCTGGGTTTACA GCAAGAGAAACCACACCAAGAGAAAGTCCTAATGTGAACTAGAAACCAGTGATCATGCTGTGCCAAGTTAGATT- TGTAAGTCGTAAACAAGCTACTA TTCTCACTGGAGATGTCTAGAGTAGAGGAGACTGTGTATGCCAGGCAGAAGGCATGTGGAAACTCTTAGTGCCT-
TCTCTCAGTTTATATGTGTTTGT GTGTATGTATACATCTTTGTGTGTATGTGTGTGCATGTACACGTGCGTACACACAGAAGTCTGAAGTCGATGTT- TTCCTATATCACTCTCCACCTTA GTTTTTCAGACAGGGTCCCTCATGAAACCTGGAATTCACCAGTTTGTTGGGGCTAACTGGCCAGTGAGCTCTGG- GCATCCTCATGTCTCTGCCTTCT CAGCTGGGATTCCACGTGTTTGCCACCACATCCTGCATTTACACGGGTGCTGAGAACCCAAGCTCAGGTCCTCA- TCAGTAGGGCAAGCACTTAACTG ACTGGGCCATCTTCCCAGGCTCTTCTCTTGCTGTACAATTAAAAGTATTCTTTGAAAAAGTCTAATATGCATGC- CTATATTTCCAGCACCGAGTAAG TGGAGCTAACCTGGGCTAGACAGTAAGACCCGGTCTTGGGGGTGGGGAACACCTAACAAAAAAATAAAAACAAA- ACAAAACAAAACAAAAACCAAAA ACATTAAATGAAGAGCCAGGGCAGTGACAAGACACGTGACTCCTCAATCTCTGTCCAACTCTGGAATTCAATAG- GCTACTTTTTCTGTTTTCCTCAT CCATAAATAGAAAAAGGGATAACTGTCTCACAGGATTGTCACAGAAATTAAATGAGATGCTGCTGGATGGATTA- GCAGTAGGAGCATGTAGCAGCAG ACCTGTGCAACTCTGTGTCTTTCCACTGATGGCATCATAGGCTACTGCTGGGCAAGGACCTATTCATTTCATAA- TCGCCTCTACCTAGCCCAGTATG TGGTGTTTGAGCCCCCTGAGTCTGCTGGGTTGATGGTAAGAACTAGCCTAGACTTCTCTCTCTCTCTGTTGGAC- ATTTGAGGGTTTTCTCAACTTTT TGCTATGAGCAAAGTACATCTCAAAACCCTTTTATTTACATCACCTAATTTGATCTGCATCCCAGGTGAAGCCA- GCAGAAGAGGGCTGTTTGCCCAC GCCCACACTCTGAGACAGACAGAATCACTATGGCTCAGAGAAGTGAAGGGACCTCTTCGGGTCACAGGTATATC- AGTGATGGTGATGACGATGGCGG AGCCTCTGGCCCTGCTTCTCTAGCCCCTACCTCTGCAGACCTTTTTCTCTCTGCCTGCTGCCTTCTGCATCAGA- GGTCTCTTAAAAAATTGCAGCCT TGTCACGCTGGGCCTGGTCCTTCTGTCCGCTGTCTGGAGGGCAGCACCTTTGCCCAGTGGTCCCTGCTGGGGAT- TGTGAACTGCAAACTCCCAGATG GCCTCTGAAATCAAATATTTTATTTCCAATGCCTCTATTTTCCCAGAATGAGGAGCACACCAGTTCCCCCACAC- ACACACTTGCTTTCGTCCCTATA AAGAGGTGAGGAGATGACTCTCCGTGTCCAGGAGGAAGGACTTTGGCTAAAAATAGCTGTGGCGTGTGGATTAG- CCAGAGTGGTACCCAGGACTGGG AAAGGGAGGGGGACGCTGTGGAGCTGTAGCCAGACTGGTTGCCATAGAAACGAGAGAGGAGCAGGGGAACCTGG- GAAGTGGGGATGACACAGATACC AAGTCCTAGTCTGAGCTGCCGTTACATTCAGGAGAAACAGCAGTGTCGGCGGCTCCCAATCTCAGAGGGAACCT- AGGGTACTGGGGGAGATGGTGTC AGGGACATGGACGCCAACCCCCAAGGGTCTCTGCTGCTGGCTACTCTTCTCTCCAGGCTCTGTGAGTTGAGTTG- TGGGACTTGGGGTTTGGGCCCCT ATTTCTGAGCCAAGAGGGGTTTGGGTGGAGCTGCTCCCAGAGGGACTTCTCCCCGACAGACCCCTTTCCAAAAG- ATAAGCCCCCTGTACTGGCCAGC GCTCTCTAGAGGGAGGTGGAGTACTCCAAGATAATGTGGTGCTCGGATCTTACTGAAAGGGGTCACAGCATGCC- CAAGAACTGTGGTCGGAAGAACT GGAGTTATTTGGAGGGAAGAGGAAGAAATGAAGACGTTGCTCTTCAGGTGGTGGACACTGCACACCTTTCCTGT- CCCATGAAGAAGAGAGCTTTTCT CGAGATGGCAATGGCTAGGATGTCATCAGTAGGCTCCCTGGGCAGTCGTGTTCTGGGAATGATCAGACACTGGG- AATCCTTCCCCATTCCTGGCCGT AGATGGAGGTCAGATCACCTTAGACCCTACGAAGACTGTCTAGAAGCCCACCTGAAGTTAATACTAGGATGAAA- GAGACCTGGGGTCTCGAGGCACT GAAAACTTACAGATGAGGTGCAGAGGACATCCTGGGCTGCAGAGAGGGAAAAAACAAGCCTGCTTGCTGTTGGG- GGAGGGGAAGATCTTAATCTGCC ATTGCCGAAGTGTTCCCAGGTCATGTCTCCTGACTTCCATGGAAAATAAGTGTGTGGGGTTACAAACCATCTTT- TTGGGGTTTTTTCCTTGTGCCTT TCTTTAACATACACACACCCTCCAAAGGTCTGCTGGCTACAGAACACTTGGCTCCAAAGTTTAAAAATGGAATG- TCGGGTTTGTGGGTATATATTCA TGCAGTTTCTCCCTAGGATCTGGTCAAACATCCAAACCATCTGAGATCCTTATGTCACATTTCTGCCCCCACAG- GGCCACCTGCTCTCCCCACTTCC CCAGCCTTCCTGCCCCACCCCTCACCCTGAATGGGAGGAGATGGCAAATCCCAGGAAAGAGAAAGGAAGGTTGA- TGAGTCTTAATCCTTATTCTACA GACTTCTGTTCATACGGTCCATATCTCCTAGGGGACCCTGAAAGCCTAGGAACCGACTCTGGCCATCCATCTCT- CCGGGAAGATTATAACCCAGAGT GCTTCTCAGGGGGGAAGAATTTGAAGCAAAACCAGGTGGGTTTTGCTTGGAATCTGGGCTTTGTGTGGAATGTG- GGCTTTGGGACATATGGCAGGAG TGGGTGGGGTTGCTGGTAGGGTAGTAAATGCAAATCAGGAAATTGGTAGGGGGGGTCGATGTGGGTGTTTGGTG- TTTCGATTGGTCTGATTTCTTAT CTCTTAGAAGAATACGAATCTGAGAGATACTAGACTAGCGTAACTCTGGATGGCCTGGCGCCTCCTTCATCCTT- GCCGTGGGCAGTTGAGCTCACGC GTGGCCCCCAATCTCCTATTGCCCACCCTTTTCAGCGTGTCTCCTGTGGGAAAGAGCCCTGGCGGGAAATGGGC- TGGTATCAGAGCATCAGTGACCA CGGTGAAGCAGTTAGAATTGCCAGTGGGAAGTTCCCAATGCTGAGGACATCCAACCTTTGCACACTGGAGGTTT- TTGTGCACAGTCTGCATTGCTTT CTCCTTGGGAAGTCTGGGGTGGAGGGGAAATGTAGCAGGAGAAAGAGTGAGGCCAGGGAGAACACCGAGGGAAC- AGTCTTCAGGTGGGGCTTCTGGC AGGATGCTGAAGAGTGCTGGGGGAAGGGATAATTGCCAGGGAAAGGGCTGTGGAAGTCCTCATCGCAGGGAGGG- CTTTGCATGGAGAAGGAACTGCC AAGAAGTCTACCTCTTCAGTACCCTAAATGTCTGATCCGGGGTGCCTGTGAGTTGCTACATACACCAGCTTGAG- GTAGTGACGCTGAGATCTGTGAC ATCGAGATGGCTAATGCCTCTTTTCTTACTGAACTTCGACACCCAGTCTGTGCTCTTTATCCTGTGTAATCTGT- ACAACTCTCTCTCTCTCTCTCTC TCTCTCTCTCTCTCTCTCATAATTCTTTATTCTTTTTTAAAAAGATTTATTTACTTAATGTATATGATTACACT- GCCGCTGCCTTCAGACACATCAG AAGAGGGCATAAGATCCCATTACAGATTGTTGTAAGCCACCATGTGGTTCCTGGGAATTGAACTCAGAACCTCT- CTGGAAGTGCAGGCAGCGCTCTT AACCCCGCTGAGTCACCTCTCCAGCCATACAACTTTTTCTTAACCATTGTTTTATTTTATGTAATAGTTTGCCC- TCATGTACGTCTGTGCATTACCC TCGGAGGCCAGCAGAGTGCGAGTTACAGCCGGTTGTGAGCCGACTTGTGGGTGCTGGGAATCGAAATCAGATCC- GCTGGAAGAGCAACCAGTGAATC ATTTGAGCCATCTCCCCAGCACTTGTGCCCCAACTTTCTGAGATTTATGGGATGTTAGGGATTATCGTTCCCAA- TCCACCAGTGGGGAAAAACTAAG GCTAAAGAGACAGGAAGGGAGATTGTCTCACAGCATTGGCCCTGAGTTCGGGGCAGATCCATCAACTCGGCACA- CCTTTATTAAGACCCCGCAGGAT CCCCGCTGCGGCCGCCATGGCGCAGGAGAACGCCGCTTTCTCTCCCGGGTCGGAGGAGCCGCCACGCCGCCGCG- GTCGCCAGCGCTACGTGGAGAAG GACGGTCGCTGTAACGTGCAGCAGGGCAACGTCCGCGAGACCTACCGCTACCTGACCGACCTGTTCACCACGCT- GGTGGACCTGCAGTGGCGCCTCA GCCTGCTCTTCTTCGTGCTCGCCTACGCGCTCACTTGGCTCTTCTTCGGCGCCATCTGGTGGCTCATCGCCTAC- GGCCGCGGCGACCTGGAGCACCT GGAGGACACCGCGTGGACCCCGTGCGTCAACAACCTCAACGGCTTCGTGGCCGCCTTCCTCTTCTCCATCGAGA- CGGAGACCACCATCGGCTATGGG CACCGCGTCATCACCGACCAGTGTCCCGAGGGCATCGTGCTGCTGCTGCTGCAGGCTATCCTGGGCTCCATGGT- GAACGCTTTCATGGTGGGCTGCA TGTTCGTCAAGATCTCGCAGCCCAACAAGCGCGCCGCCACTCTCGTCTTCTCCTCGCACGCCGTGGTGTCTCTG- CGCGACGGGCGCCTCTGTCTCAT GTTTCGCGTGGGCGACCTGCGATCCTCACACATCGTCGAGGCCTCCATCCGAGCCAAGCTCATCCGCTCCCGTC- AGACGCTCGAGGGCGAGTTCATC CCTTTGCACCAGACCGACCTCAGCGTGGGCTTTGACACGGGGGACGACCGCCTCTTTCTCGTCTCACCTCTCGT- CATCAGCCACGAAATCGATGCCG CCAGCCCCTTCTGGGAGGCATCGCGCCGCGCCCTCGAGAGGGACGACTTCGAGATCGTAGTCATTCTCGAGGGC- ATGGTGGAGGCCACGGGTGCGGG CAGGCTGGAGGATGGGAGCAGGGATGCAGGACAAGGGCAAGAAAAGCAGCCAGGGGAGGCGCAGAAAGATGGAC- AGAGAATGGAGTGTAGGGTGACA GGCCTGAGGGGTAGCGGGGGCCGGGGAGAGGACGGGAGATGACAGGGATGGACAGGGTGACTTTGCAGAGTCAA- GAAAAGCTTGGAAGAGGTCTATG AAATGGCACTAGCTTGAGGCCCTGACCTGACAGCTATGTCACTTTGAACTACATTTTACATCTCTGAATTCATT- TAAGCCCAGCAAAGCTCCCCTGG AGGTTACTTTTGACTGTGCTCGGTTTTCAGAGAATGAGTAGCCCCAAAGAAAGGTCCCATAAATAGCCCGCTGT- CACAAGCCAATAAATAGCACAGC CTGGGTTGAACATAGGACATCTATCTTCAGTGTTTCCTGGTACAGTGTTGGGATGAAGGTTAAGTGCAGGGTTC- TTGAAGCCCAGAGGTCCATAGCT CTGGAATTTAACTGACCTAAGTAAAAGGGAGGTAGGTAGGAAAAAGACTAGTACTGGAGCAAAAACAGGTCCTT- GAAGAGGTCCTAGCCGTCAGGGA GCATAAGGAAGACGCAGGTGAACCAAGAGGCCACTAGGAGGAGCTGCGGAGCTGCTACGGACAGGCTAGCTCCC- TGCTGCTAGCCTTGAAACCTGGC TCCTGGGCCTAGACAAAAACATCATCTTCTCCATGGCCACCTCAGGTCTTCCCACTCCCCTCTCCTCCTTCACT- CCAACTAGGCTGGTTCTAGCCCA TGCCCATTCCACACTGCTCCCTCTGTCTCTGCGCTGTCCCTCTCTCTGACACAATCTCGGACAGGTTTCTATCA- GGGACTTTTTCATCTGCCTTCTC TTCCCCCTCTGCCACTGCCTCCACTTTGCACCTAACCCTACTCCCCCAAGCCCTACCTCTGCTTCTCAGGCCTT- CTCCCTGCAGAGGCCCCGGTGGC CTCTCTTTCCCTACGATCCCTGATACATCTTATTCCAGCTTTGCCAAAGAATACCAATGACCCCAAGATGTCTC- AGGGCCAGACTTCCGATGTCAGA GCCGGTCTCTGATTAGTGAATGCTTACTCCTCTGTTTTTGAGATGGATTCCGGTTTGGGAAGATTCTGAGGTAG- GAACAAAATGATCTGCCCCGAGG GGAGGGTGCACAAACCCAACAGAGAAGACAGGACACAGGCTCAGGGCAAGAACTGGGAAGGGGCAGTGTAAAGG- ACATGGGGATGGGAGCTTGCTTG ACTTTTCTAGAGATAAGGCTGGGAAGGATGGTAGTATTTTGGGATTCAAACTGCTTTTGAAAAGCAAGAATAAT- GAGCCAAAACCCAACATGATGAC ATTTAAGGGGAATAAATATAAAATTCTACATTTAGGCTTTAAAAAAATCACTTATGTAAGCACAGCATGGAAAG- GCTCCGGTGGAGAAAGAACTGGG GGTTTTAGTTGGCCACTGGCTTTGCTGCAGCAACGTGATGCAGCTTCCAAAGGCGTTTATGTAATGTAATCATG- GGCCCGCTTCACCAAAGCATCTG GGCGAGAAGCAAGAGATAGTAAGCCTTCTTTTATGCACAGATAAGGCCACAGTTGAAAAAGCACTTCAGATGAG- CCCTTACCTGGGCCTGGTGGCCA TTCTGATTTGCAATGAAGATTGTAAGCTTTGGGGGAGTCAGATGAAGTAAGAAATGGCCATGAGTGTTCAATCT- GAGGAAGAGAAGATGTAAGGGAA CCCCATATTTACACTCAAGGGGGTGTCAGGTGGTAAGGGAATGGAACCAGGGGCCACGGGTCCTAGGAGACAGA- TTTTAGTTTATGTAAGAGAAAAC CCAGAGCCAAAGAGATGTCTCAGCTTGCAACCACGCCTGACTACTGACCTGAGTTGAATTACCAGGTCTCACAT- TGGGGAGTCAACTGTCTCCCCAA GTTGTCCTCTGACCTCCACATACATACATATGCACGCATATAGACACATAAATGTAAAACACATTTGTAAAGAC- GATTGGCACGTTGCACAAAGGAC TGGACTTTTAATGAGATGGTGAGCTTTCAATCCTGGGGTGTAATCAGTTCAGCCCATTGTCTGGGAATGCTTGG- GGGTGGGTGGAGGCGGCTCTGTG GGAAACAGGAAGGTTAGGCTTAAGGTTAAGCTTCTCAATGGAGAGTAGGGGAAAACATAGGCTGGCAGATAGAG- AAGAGGGCTAACTAAAAAGAGAG GTGGGACTCTCAGAGAGAGAAGAGGGTTGTGGGATGACAGACAGGAGAAGGAATCCTCTGTCAGGGGCCCCTTT- GACTGATGCCGCTTCTCCTCCCC CCACCCCCCAGGAATGACGTGCCAAGCTCGAAGCTCGTACCTGGTGGATGAAGTGTTGTGGGGCCACCGGTTCA- CATCCGTGCTCACCCTGGAGGAT GGTTTCTATGAGGTGGACTACGCCAGCTTCCACGAAACCTTTGAGGTGCCCACACCCTCGTGCAGTGCTCGGGA- ACTGGCAGAAGCCGCGGCCCGCC TTGATGCCCATCTCTACTGGTCCATCCCCAGCAGGCTGGATGAGAAGGTGGAGGAAGAAGGGGCTGGGGAGGGG- GCAGGTGCGGGAGATGGAGCTGA CAAGGAGCACAATGGCTGCCTGCCACCCCCAGAGAGTGAGTCCAAGGTGTGACTGGTTTCCTCCCACCCCCTGT- GGCAGACCAGGGGGCCGGACTCA GGTACACAGAAGCTGCGAGTGGAGGTGGAAGAAGAGGAGGCAGGCAGTGTCCCGAGGAACAGCTAAAGTTGGGA- GAGGCCCGCTGAGTCCAGGATCG AGTAGGGAAGGCTGAGGTCCTGGTTTGAAGAGAGAGGGTTGCAGGGCGGGGTGAGAGAACATGTCAGTCTGTCT- GTGTTTGACCTTCACATCGGTTC
ATGGGTGGATGGATGGACAGAAGGATGGGCTCATGGGGGTTGATCGGGAAGGTGGAGCAGATAGAGACAGCCAA- TGGATAATCGCTCAGGTGGTAAG TGGCTTGGCAGTCGATGATCGTCACCTGCAGCACACCTTTGTGAGAAATCCATGGGCATCCTTTTCTTCCAGAT- ATAGGTAGCCTCAAACCAGGGAG CGTGGCTTAGGGAGCAGGCTGTCAGGTGGACTACCACCCCCACTCACCTCCCCTCAACTGGCCTCCCTATGTGT- GACACGCCTGCCTAACTAGAGAA GAGAGCACTGGGTAGAGGTGGGCACAGGTGTGGGTGCCCTCCCCAGCATCACTGTCCCATGGCGAGAGGTCAGA- AAGGCAAACAAGCAATGGGGGTA GATGCTGAGCAGGGAGGGGCCCTGAAGCAGGACCTGGGGACAGCCAAGGACAACTATTTTGTGAGAGAGGAATG- AAACCTTGCAGGTCCTGCCACAG AAGCAAGAAGCAGAGGAAAGGCCATGGAGAGACTTAATAAAGGGTTTTACAAGGGTACCTGGATCCCAGGGGGA- AGTAGTTTATCCTTGGGGCACAG TGGCAGGGCTCATTCAGAACGGTGAGTAAGTGTCAGGTGTGATATTCAAAGACCTGGTTCTTAACACGAGAGCA- CAGCGAAGGTGGAGGTCAGAAAT AACTCCCAGCCACTGAAGGAAGTATGGCTTCAGTCTGGAGAGCTCAGAAAAGACTCGACCCTAGGAGCCCACAC- AAGCGGTTATAGCCACAAGTGAG AGGGCATTAGGGACAGGAAGCTAAGGATTGAGTAAGGCAGTGGGGAATGGTGGGAGCCAGCAGTTACAAAGCTT- TACTCACCTGGATGGGCTTGTTA AAACACAGATTACCAGCCCCACTCCCTGCATTCTGACTCAGTAGGTCCGGGACGGAAACCAAAAAAAAAAAAAA- AAAATCTGCACGTCTAACTAGTT CCCAGACCTAACAGGTTCCCAGATCGCGGTGACACTGTCTGTCTGGGGACTGCACTTGGGTGAAGCATCTAAGC- GGAAGAGAAGCTGGAGGAACTGA AAAGCACCCCAGGTTCCTCAAGGAACAGAGAAACAAGAAGGGAAATGTTGGGGAGAGGGGACCCAGGTCCAGAC- TCGAAGGGCTTAACTCTGGGTCC AAGAAACGTCATTGGTAACTGGCCAGTGGCACCCGAGAGGGCAACAGAGATAGGAGAAGGCCATTTAGGGACCC- CCAAGGAGGCAGTGGGGGGTCTG TAGCTGAATTGGCCTTACCACAAAAGACCAACTCTCTTAAGAGACTCACAAGGCAAGACTGACTAGGGGAGAAA- ATGGAGCCTGTACCTACAGGTGT CTGCTGTCTGCCACCTGTCCTCCCAGGACAGGGCACCCTGGAGACACATTCCACCTCCACTGCATCCTTGTCTT- GCCCCAGTCATCTTGGGATGGTT GAGGGGACAGCAACAGCATGGCAATGGACCTGAGGCTGGCCCCCCTGGAGCTAAGTGTAGCCCAAGTGAGCACG- TAACCTGATAGGACTGGCTCAGA CTCTGGCCCTGGCTATACCCATCCCTGCCCTCGAATAAAAGTCTGCTGCTCTGTCCCAGGCTAAGAAGCCAGCA- TCCAATGGGGCATCGAGGCCTCC CTCCCAGTGCCCAGCTCAGAGTGGGTCCACGCAGAGAGGACTCAAGCTGCCTGTTGCCTCTCCCCTTCCATCTA- GCAATGGCCACAGGTTTCGGGAC CAGCTGGGTCACTCTCAAAGATGAGGTCCACGCACATGAACCTGCTGGGATCCCACGAACACATATTGGACCTG- AGCACAGGGACTGAGCAGGGTTT GAATTCAGAGAAAATCGAGGAGTCTAGACAAGAGGGGTGGGGTGCTTGGTATCCGCACACGAAGCAATGGAATG- GAGACATTGAAGCTGTTCCTGGA GGTCACTCAGGGCACCGCTGTCCAGGGCACAGCCAGGAGACCTGTGTTCTAGCACCAATGCTGATTGTCACTAA- TTACCTCTATGACTCTCAGCAAG ACCTATTACTTCTCTGGGCCTCAGCTTCCTTATCTGTTAAAAAAAAATGATATGTTGGCAAACTCAATAATGCT- CAATAAACTTTCAACTACTGAAT GAAAAAAGGTAGACTGGATGCCGCTCAAAGTATTAGGACAGCTGAGGCTCTTAGGACCGGAGAACCCTTTAGGC- GGGGAGTTGCGGCTAGCCAGCAG GCAAGTCCTGGCATCAGATGTAAGCAGATGAGGCGGCTCTTGTGTACACAGAGGACACAGGCTCTCCCAACTGC- TGCTGTCCTTAAGTAGGCAGCCG TGTTCTGAAGCTCCTATTCGGCTGCTGTCAGAGAATAATTAAGGGCAGGAGGAAAAAGACTGAGGCCCCAGGGC- CTGTGGGAGGAGTCTGGTCCAAG ACTAGTTCAACCAGGAGAAATGGACCAGAGGAGGGTGTGCCCCAGTCTGGAGAGCTCAGAAAAGACTCGTCCCT- TGGAGCTCTGTGAAAGGGGCAAA GCTCAGCTGGAACTCACCCCTCCTCTTCCTAGGTCCCCCTTCCCAAATAGAAGCCCCATTAGGACTTGGCTCAG- CACAGACATTTTGGACAACAGAT GGGACCCCGGCATCCCCTCATGCAGTTGGTGGGTAACAAGGCCCACGAAGGGACAGATGGTGTTTATGGTGGGA- AGAGAGGCCCGGGTTGTCCAGCA ACCACCCTACTACCACCCCACCCCCACCCCCGATGCTGCCTTTTATAGCTTCACCGCAAGAGAAGACACAACAG- GCCTCGATTTTACAAAACCAGTT TATTCACATTTTAGAAAAACTAGTTTGAGGACAGGAACTGGCCTTCCTACAACATGAGTGTGGGACTAAGAACG- GCAGCCAGGAAACTTGAGGGAAG GTGGGGACAGGGGAGCCATGTCTCCCACTCTAGGTGATGGCTGGTCAAATAAATTAAAGGTGGGCTGGACAGAG- GGAGAGGGTATCCAGGCAACCAG AGGAGGGGTGGCACTGGCTGGAAGACAGTCAACACCTGCAAGAACTGGAAAGAGCATGTGGAGTCGGCTGAGGA- AGAGGCTCCCTTTGACCCTTACC CTGCTATACGATCCTGCAGGACTGTGAAGCTGGCTGCTTCTCCCCCTGATGGTGCCCAGGTACAGCTCAGCACA- GGAAGCCTGAGGAAAGGCAGTTC CTTTCCCTCACCTTGGGGTGCTACAGATCACCGCTTCCGCATCCTCTTCATAAAGCAGCAGGTGATGGTAGCCA- GGACGCTGGCGCCGGTAACTAGG GCGACTCCTGTACCCACCAACAGGGGCACAAATAGGGTATCCACAGCTGGCAGAAAAGAAGACAGGCTCTGCTC- AGAGAGTACCACGGTATCTGACA CTCTCCCCTGCAGATTTTCTAGACTCAGCCCTCCCCAAGGGAGAGCTGAGCGCCAGTCCTGCCTACCTACACTT- CACACACAAACACAACCATCCCC CATCCCCCATCCCCACCCCCTCCCCTCGGTCTCAGCACTCAGGCCGGCTTGGGGCCCTTCATGCAAAGGGATGT- GGAAAAAGGATTGCAAGGGAAGA CAGGAAGATGGAAAGGGGCAAACAGAGCAGGAACAGGTGGGTAGATGGTGGCTGTCACTCACCATGCGTGTAGG- GGTAGACTGTAACAGGCCCGGAG CGCGCACTGCCTGCCTGGTACCAGCTGTAGTCCGCATGCTGCACCCAGGCACTTGGGGCACAGTGGTATATGCC- TTCATCCTCAGGCCCCAAGCCAT GCAGTCTTAGCCGATGACTTCTGGGTCCCACCAGCTCCACACTGACAGGACCCCCTCCAGGCCGGACTCCCAGC- TCTGCCACACCGTCCTGACCCAC TCCACCCACAAGCTGAGCAGGGCCAGAGCTCAGCTCGCCCTCCTCTGGCCTCTCCACCCACCAGCTGGCTGCTA- GTCGCAGCCCTGGGGGGCCGCCC CGCACAGAGATGTTGCATAGCAGGGAGGCCGTCTCTCCCCGGTACACAGTGCCCCCTGCTAGCCACGCCACGGC- CTCTAGCACCACGCCTGCAGAGC AAAGAACACGGGGGTTACCAGGTGAAGGCCCAGGGGCTAAGAGGTTAGGAAATAAATTCTATAAGTTCTGAACC- CCGTCAAGGGCTCAACATCCTCT TACCTTCTTCTCTCACATGCACAGGGAGAGGCCGGGAACGAGCACTGGCCGCTTCACGAAGTCGGGTCCCAGAC- CCTCGAACATAGGCTTTGGCGAG GCAGCGGTAGGTACCTGCATCAGCGGGCCTGGCAGCCTCCAGCCGCAGTCGGTAGGTTCTGGATGCTACTTTCT- CCATGGCAATGTGCCGGTCCTCA TAGCCAGGGCCCAGGCTGCCTATACCTTCCGTGTCTAGCTGGGCCACCAGGCGGCCGGGTCCAGGAGCCCCTGC- AGGGGCCATCTCCCAGCCCACAG AGTACGCAGCATGACGGCCTGGTGGGGGCAGTGCACCGGACACATTGCACAGCAGTTCTAAGGGTTCGCCTGGG- CCAATCCGACGTTCACCAGGTCC CACGGTCACCGCCAGCTGGCTGGCTGAAACACAGCAGGAGATGGGAGGAGTCACTGAGATGCCTGGGCCCCCCA- CCTGTAATTCTTCTTTGCAGAAA TTTAGAGGCCTCTTATATCTCCCTCACCCCAGGACCCGAATTTCACCCTTCCCCCCATAGCCTTTGTATCTCCA- TGCTTGTGCGGCACTCCCGATGC CCAACTGAGAGACACCCCCCCCCCCCCAGTGGGCTATGCTGCACTCACATAGAGTCTGCACATCAACATGAGCC- AGGACTGCCCTCTTCTCTGCGAC CTGGACCCAGGAGCCGTCAGGATCCTGAATCCACTCAGCGGCCGTACAGTGGTAGGTGCCCGAGTCTCCAGCCT- GGGCACCCCCAACCACCATTCGG TACCGATCAGTCCCTTCCTTGCTCAGCCGAAGCTCCCCAGAAGCTAGCCTCTCAGCGTAGGGCGCTCCAGCCTC- CACCGCCATGTCGGAGCGCAGTC CCACTACTTCCTGTAGAGTGGCTCGCCCCACTGGCGCCTCCGGAATGGCTCTCCCAAAGGACACCGACAGGTGT- GTGTGTTTCTTTGTTTTGGTCTG AGCCAGGCAGCCCAGCGCAAGCTCCTGCCCCTCGTGCACTGTGAGGCGTGAGGGGGAGGTGGCAGCCTGGCGCC- CTCGGGGCCCTGGAGGGGCAGCA GATACCTGCAGCTCATCTGGAAGAACTGGAGAGAAAGGCTTTAGTGAGAGAGGGCTTGGAGCAGCATCCCTCCT- GTTTCCTGTGCGTATCCTGTTTC ACTACACACTCTCTAGGCTTCTAGAATGTAAGAACTGTGCTCTGTAGCTTTTCTTCTATACCGCAGAGATGCCA- AGCTTGGTCTGGGCACATCAAGA TATTCAATAACTACTTGCTGAACGTCACAGAGCAAGCCTACTGACCCCTACTCTGATGTCTAAGACTGATCCAT- TTTAAATACTCAAAAAAAGTAAT CCTGTCTTCCTTCTCTAAAGATAAAGAGGCTGGACCTGATGGTGCCGGCCTATAATCGCAACTACCCAGGAAGC- TAAGGCAGAAGAGTCAAGTTCAA GGGCCGTCTGGGCTACAGAACAAGTTCAGTGCTGGTCTGGTCAACTTGGAAAGTTTCTGATTCAGCCCATCCCC- CCCCCCAAAAAAAAAAAAAAAAA ACTGCTGGAAAGACTGGCTTGATGGCACTGGAGCTGACACAATACTGCCTGAGCTACACAGTGAATTGTGGGAG- TTTTGTCACCAGTTTCAGGCCAA CCTAGGCTAGTTGTAAGCTAGCCTGGGCTACAAGAGTGAGCCCTCAAAAAACAACACAGGGAATATAGCTTAGT- AGAGTGCTCCCTTGGTATGTTCC ATGCTGTAGGTTCAATTTCCAGTACTGAGAAGATGGGGGTGGGGGGAGAAGAGGAGAAGGAAGAAGATGACAGA- GAAGGAGGAAGACGAAGCAAAAA TAGATCTGAGCGTGCTGGCTTATACCTATAACCCCAGTGCTTGTGAGGCTCTCTCACCACCTAGCTCAGAGCCC- AGTACCTCTCAGCTCCACCTTGG CACTGTAGTTGCCCAGGTACTGCGTATCCGTGGAGGGGGTGTAGCACTCATAAAAGCCAGAGTCCTGGGCCTGC- AGGCGAGCAATCTTGAGCACCAC CGAATCTCCCTTCAGGCGCTGCACCTGCAGGTCACCAGATGCCACACGAGGCCCAAAGACAGCATAGGAGAACT- GGCTATCCTTGGTGCTGACAATG CCCAGGGACGTAGCTGGGGCCTCTGGTCTGTACATGAACCACTCGAAGTCTTGCTGGGCAGGGCCCTCATAGTC- ACTCACGTTGCAGGAGATAGAGA CAGCGGTGCCAGCCACCCGGTAAAGAGGTCCCCTGGGGACATGCACCTGCCGGGCGTAGCACCTGGTTCCTGTG- GGGTAAAAGCAGAAAGAACTGGA ATCTTTTTAGAGAGAACGAGTCCCCACTTGATGGCCAGTATCATCAGCACCATTCTTGACTGCTGCCTGTGAAG- GAAAGGGAAACCTAGGGATGGTT AGAACATAGCTGGGCAAAGACACAGATGGGAACACAAGATGGGACACGAGACACCAGCATCCCAGCACATCACG- TAGGTTCAGATCCACCTGAACAG AGGAATACTCTAGCTAACTAGAGGCAGAGCACCTACATGAACAGAGGAGTCCATCCGGAGTCTGGGGTATAGAC- AGCCCGGTAAGTAAAATGCTTTC CATGCAAGCATCAGGACCTGAGTTCAATTCCCGGCAACTATGGAAAAAGCTTGGCACGGTGGCTGGTGAGTAGC- CACATAAGCCTGACAACCTGGGT TTGAGCCCCACAAGGGAAGGAAGGAACTGAATCCTGAAAGTTGTCTCCTCTCTCTCTCTCTCTCTCTCTCTCTC- TCTCTCTCTCTCTCTCTCTCACA CACACACACGCACACGCACACGCACACGAATAAATGCAGTGGACGGTTCCTGAGCCATGACCCCTGAGGTTGAC- CTCTGACCTTTACACAGACACCT GCACCCATGCATACATGTGGACCCTCATACGCATGCAATTGGAAATAACAAATAAAAGAAGCATGCCTGATTCC- CAACTCTCCAGCTCGTGGCTGGA TCTTTATCGCTCCTACCCTGCCATGTGTGGCGTCTCTCCGGGCTCAGAAAGAACTTCTAGCTAAGGGATTCTGA- GCCTTTTGCTGGAGGGGAGCTGA CAACATCTTACAGAAAGGCTGCTTGGCTTAGCTCTGTCACCTGGGCTAGACAATGGAGCCAGTGGCCCAGGCTG- GCTGGGGTACTGCACTGGATGGG GCACCATGCTGGACAGGGCACGGACCTAGTCAGTCCTCACTACACAATACCCTCCCCACTACAGCTGTGCCATG- AGCTCACTGCTTCTCCCAGCCCA CAAGGCTACACAGGCAGCTGTGGCTTCTGGGGCAAGAACCAGGCTCTGCCCAGGCCTGGGGCAGAAGATCCCTT- CCCCGCCCCCAGAATCTGTCAAC CTTCTCGCTAACCCAGATGATGCACTATGCACAGCCCCCCAAACAAAAGCTTTCATTTACATATGATTTGCCTA- TCTGCGCAGCATTTGCATAGACC TCCTTTTAATAAGGAGACCCCAAACACCTGCTGCCCCTTCCCCCTTCTCTCTCTCTCCCTCCCTCCACCCATTT- TACCCCACCACTGTTCCAACAGA CAGCCGGTTCCAAAACCTTGGACTGTTCAGCTTCTTTCTCCTCTCCACTACTTCTAGGTAAGACCGCAGAACCC- TAGAATGCAAGACGGGCTGTCAA TCATTCCTCAGATGAGAGAAATGAAGAAACTCCCAGAGAGCACCTCTAGGGAGCATTGCCACTAGGTTCTAACC- ACAGATGTGAGTCTAGAGCTCTC TCCCAGCCAACACGGAAGGCCTGGCCGTGAGAACCATCTCTCTTCTAGATGAGAAAAGTAAAGTGAAATGTGAC- ATTGTGGCCAAGTCCCTGCCCTC TCTGGAGGGCCTCTACACACCCCTGAAGAGGGACAAAACCAAGAAGGCGGAGATGCTTCCAAGGAGAGCCCTGG- TTTACAAAACTGTAATTTCCCGG
ATTTCCAGGGGTAAGTCCACAGCTTGCTACTGGCCCTGGAGGAAACCACCCAAGAGCCTGAGAGCCTGCTCTAC- AGTTCTCGCTCCTTTCCTTCTTC TGGCTACGTCACGCAGAGAACACATGACCACCGCCTGAAGCAGAGGCTAGAGTCAGAAAGCCAACATGAGACCA- ACCCTTTCCTCTCCTGGATCTCA GTTTCTATCTCTCAGAACTAAGAGCTCCCAGTTCTGATGTTGAACCCGTGAGTATATGTGTGTGACTCAGGCAC- ATATCGCTCCAGGCACATTTCAT AATCAGGAGGATGGTTAAGGCAGCCAAGCTGACACCATCCTGGCTGCCCATGGGTAAACTCTGCTGAGAAGGTC- CCCTCCCACCTTCCTGGCTCCAC ACAAGCTGATGCTTGGAGATCCCCATGGGAAAGTTGTCCCCACAGCCTCAGGACATAGGCTGCTACAAGGTTCT- CAATGGCTGGGCTGCCTCCTTTC TAACAGCCAAAGGTCTGCTTTGAAGTCAGTTCTGAGTTCAAATCCACCCCCACCCTCACTGGATACACAGTGGT- ATTTCACGACATCTGTGAAGTGA ATGCCAGTGCTGGCTGCAGTGGGCTAAAATGACATTCAGCTCCTCTCCCGCCCCTGAATAACACTCACTCCTCC- CAACCACCCCTGGGCTCATCCCT GCTCGGGTTAAGCCCAAAGGAAAAGAAGCAATCGCTAGGCAACCAAGCCCCACAGCTCCTTCAACTCCCTACAT- CACTCTGCCTCCCGCCTTGTTCC CAAAGGAGTTTTCATCCTGGCCCCAGAAGCCCAAGGAACCATTCTGTACAACACTGCACAGTTCTGGTATAAAC- CAGAGAAGGAGGTTGGGGTGCCC CACTATAGTATCTTCTCCATATGCATATCACACACACACACACACACACACACACACACACACACACACACGTT- CCTTTCAAGGGCTTCAGTCTCCT GGCAACTGCTCCATGCCATATCTTTCCCAGACCACCTCCTACAGGGAGCCCTCCAAGTCAGACCCCAAACATGG- TAATGTTAGCAACCTCCACAGGC CTCAACACACACACACTCACACTCACACACACACACCAGACATGACGCAAGGTTGGCCCAGAAAACACACCATC- ATAAACACCCACCAGGACAGACA CTGGGTGCTTAGAGATCCCAGGTTCAGTTTCCATGGAGCCTAGTTTCTCCTGAGGCAGGGATGTTGGGACCAAC- TGAGTCTGACAACCAGGCAAATA TCTGGGAGCGTGGAAGGGCAAAGAGGGAACTGGCCCAGGGTGGAGACACGTGAGGGAAGAAGCCTCAGATGGTG- ACATGTTATATTGGGAGGTGGGG GTGTTGGGGAGACTTTTTTCAGAGATCGTGGTCAGAATCAGCCCCTGGGCCTCCAGCCAACTCTGGGCAATTAT- GAAGACCGCCAGGCACTGCCCAC GCAGAGCAAACACCCAAAACCAGGCCTTGAGCCGAGAGTGGGGCAGAAGGTTGTCACGGTATTTGGTAGCAACG- ACCCCAGACGCTGGGTGTAACCG ATGAGAAGTGGTGCCTGCCTCCGGAGGCCCGATGGTGTCTCAGGGGATACCTCAGTAGGTCGCCCATATGCCCC- AGCTAGGAACCTAGAGCGAGGAC ACCACCACCCTCCCCATAACTGATTGGGCAGACAGGCGCAAAAGGAAGCGAGACGCCGAGCCCAGAGACAGTGG- AGGCACGTCTGTTGGAGAAGTAG GGATGCAACCAGCTCTGAAATGCTAGGAAGGTGGGCTGGTGGGCTGCACTATGTTAGGCACCTACCCGGCCGGG- ACAGGGACGCGGCGACCACCACC TGGCTTACCAAGTATTAGCAGCAGCAGCAGGAGCGAACTCAGCGGCGTGGGGCTAGGGACGCCCATTCTGCGTA- GGCGGCTCTGGGGAGACTCCTGG GGGCGGCGTAGGCTCTGGGGGGCCAGGGCCGCGGGGGGCGCATGCCCAGGTGGGGGGCAGAAAGCGGAGCAGTG- AAGCGTGGGTGCGCAGAGCCCAG CCGAGCGGGAGCCGCCAACTCCCCGCCCTCCACCCTTCTTCCCCTCCTCCCTCCGCTCTTCCCGCCCTCCGCAG- CTCGGGAGACCAGTCCCAGCCGC GCCCCGCTGCCCGGCCCCGCCCCCGCCTCGCCCCGCCCCAGGCCGTCGCCTCGGCCAGACTTCGACCCTGATGG- TGGCTCCGCCTCTGGCCTCAGGC TGGGCGAACTGGCGGCACCTGGGCTCCTCTATCCCCATTTCCTCGCTCAGAGGGCACCCCGCCCTGCACCTGCC- AGCCTTCCAGGGAGAATGGGGTG CTTTCAGGGCCTCTGGGGATGCATGATGGGGTGACTGTGGTTACGCACTCAGAATCCAATTGGG MOUSE SEQUENCE - mRNA CTGAGCTGCCGTTACATTCAGGAGAAACAGCAGTGTCGGCGGCTCCCAATCTCAGAGGGAACCTAGGGTACTGG- GGGAGATGGTGTCAGGGACATGG ACGCCAACCCCCAAGGGTTTCTGCTGCTGGCTACTCTTCTCTCCAGGCTCTACTTCTGTTCATACGGTCCATAT- CTCCTAGGGGACCCTGAAAGCCT AGGAACCGACTCTGGCCATCCATCTCTCCGGGAAGATTATAACCCAGAGTGCTTCTCAGGGGGGAAGAATTTGA- AGCAAAACCAGACCCCGCAGGAT CCCCGCTGCGGCCGCCATGGCGCAGGAGAACGCCGCTTTCTCTCCCGGGTCGGAGGAGCCGCCACGCCGCCGCG- GTCGCCAGCGCTACGTGGAGAAG GACGGTCGCTGTAACGTGCAGCAGGGCAACGTCCGCGAGACCTACCGCTACCTGACCGACCTGTTCACCACGCT- GGTGGACCTGCAGTGGCGCCTCA GACTGCTCTTCTTCGTGCTCGCCTACGCGCTCACTTGGCTCTTCTTCGGTGTCATCTGGTGGCTCATCGCCTAC- GGTCGCGGCGACCTGGAGCACCT GGAGGACACCGCGTGGACCCCGTGCGTCAACAACCTCAACGGCTTCGTGGCCGCCTTCCTCTTCTCCATCGAGA- CGGAGACCACCATCGGCTATGGG CACCGCGTCATCACCGACCAGTGTCCCGAGGGCATCGTGCTGCTGCTGCTGCAGGCTATCCTGGGCTCCATGGT- GAACGCTTTCATGGTGGGCTGCA TGTTCGTCAAGATCTCGCAGCCCAACAAGCGCGCCGCCACTCTCGTCTTCTCCTCGCACGCCGTGGTGTCTCTG- CGCGACGGGCGCCTCTGTCTCAT GTTTCGCGTGGGCGACCTGCGATCCTCACACATCGTCGAGGCCTCCATCCGAGCCAAGCTCATCCGCTCCCGTC- AGACGCTCGAGGGCGAGTTCATC CCTTTGCACCAGACCGACCTCAGCGTGGGCTTTGACACGGGGGACGACCGCCTCTTTCTCGTCTCACCTCTCGT- CATCAGCCACGAAATCGATGCCG CCAGCCCCTTCTGGGAGGCATCGCGCCGCGCCCTCGAGAGGGACGACTTCGAGATCGTAGTCATTCTCGAGGGC- ATGGTGGAGGCCACGGGAATGAC GTGCCAAGCTCGAAGCTCGTACCTGGTGGATGAAGTGTTGTGGGGACACCGGTTCACATCCGTGCTCACCCTGG- AGGATGGTTTCTATGAGGTGGAC TACGCCAGCTTCCACGAAACCTTTGAGGTGCCCACACCCTCGTGCAGTGCTCGGGAACTGGCAGAAGCCGCGGC- CCGCCTTGATGCCCATCTCTACT GGTCCATCCCCAGCAGGCTGGATGAGAAGGTGGAGGAAGAAGGGGCTGGGGAGGGGGGCAGGTGCGGGAGATGG- AGCTGACAAGGAGCACAATGGCT GCCACCCCCAGAGAGTGAGTCCAAGGTGTGACTGGTTTCCTCCCACCCCCTGTGGCAGACCAGGGGGCCGGACT- CAGGTACACAGAAGCTGCGAGTG GAGGTGGAAGAAGAGGAGGCAGGCAGTGTCCCGAGGAACAGCTAAAGTTGGGAGAGGCCCGCTGAGTCCAGGAT- CGAGTAGGGAAGGCTGAGGTCCT GGTTTGAAGAGAGAGGGTTGCAGGGCGGGGTGAGAGAACATGTCAGTCTGTCTGTGTTTGACCTTCACATCGGT- TCATGGGTGGATGGATGGACAGA AGGATGGGCTCATGGGGGTTGATCGGGAAGGTGGAGCAGATAGAGACAGCCAATGGATAATCGCTCAGGTGGTA- AGTGGCTTGGCAGTCGATGATCG TCACCTGCAGCACACCTTTGTGAGAAATCCATGGGCATCCTTTTCTTCCAGATATAGGTAGCCTCAAACCAGGG- AGCGTGGCTTAGGGAGCAGGCTG TCAGGTGGACTACCACCCCCACTCACCTCCCCTCAACTGGCCTCCCTGATGTGTGACACGCCTGCCTAACTAGA- GAAGAGAGCACTGGGTAGAGGTG GACACAGGTGTGGCTGCCCTCCCCAGTATCACTGTCCCATGGCGAGAGGTCAGAAAGGCAAACAAACAATGGGG- GTAGATGCTGAGCAGGGAGGGGC CCTGAAGCAGGACCTGGGGACAGCCAAGGACAACTATTTTGTGAGAGAGGAATGAAACCTTGCAGGTCCTGCCA- CAGAAGCAAGAAGCAGAGGAAAG GCCATGGAGAGACTTAATAAAGGGTTTTACAAGGGA MOUSE SEQUENCE - CODING ATGGCGCAGGAGAACGCCGCTTTCTCTCCCGGGTCGGAGGAGCCGCCACGCCGCCGCGGTCGCCAGCGCTACGT- GGAGAAGGACGGTCGCTGTAACG TGCAGCAGGGCAACGTCCGCGAGACCTACCGCTACCTGACCGACCTGTTCACCACGCTGGTGGACCTGCAGTGG- CGCCTCAGACTGCTCTTCTTCGT GCTCGCCTACGCGCTCACTTGGCTCTTCTTCGGTGTCATCTGGTGGCTCATCGCCTACGGTCGCGGCGACCTGG- AGCACCTGGAGGACACCGCGTGG ACCCCGTGCGTCAACAACCTCAACGGCTTCGTGGCCGCCTTCCTCTTCTCCATCGAGACGGAGACCACCATCGG- CTATGGGCACCGCGTCATCACCG ACCAGTGTCCCGAGGGCATCGTGCTGCTGCTGCTGCAGGCTATCCTGGGCTCCATGGTGAACGCTTTCATGGTG- GGCTGCATGTTCGTCAAGATCTC GCAGCCCAACAAGCGCGCCGCCACTCTCGTCTTCTCCTCGCACGCCGTGGTGTCTCTGCGCGACGGGCGCCTCT- GTCTCATGTTTCGCGTGGGCGAC CTGCGATCCTCACACATCGTCGAGGCCTCCATCCGAGCCAAGCTCATCCGCTCCCGTCAGACGCTCGAGGGCGA- GTTCATCCCTTTGCACCAGACCG ACCTCAGCGTGGGCTTTGACACGGGGGACGACCGCCTCTTTCTCGTCTCACCTCTCGTCATCAGCCACGAAATC- GATGCCGCCAGCCCCTTCTGGGA GGCATCGCGCCGCGCCCTCGAGAGGGACGACTTCGAGATCGTAGTCATTCTCGAGGGCATGGTGGAGGCCACGG- GAATGACGTGCCAAGCTCGAAGC TCGTACCTGGTGGATGAAGTGTTGTGGGGACACCGGTTCACATCCGTGCTCACCCTGGAGGATGGTTTCTATGA- GGTGGACTACGCCAGCTTCCACG AAACCTTTGAGGTGCCCACACCCTCGTGCAGTGCTCGGGAACTGGCAGAAGCCGCGGCCCGCCTTGATGCCCAT- CTCTACTGGTCCATCCCCAGCAG GCTGGATGAGAAGGTGGAGGAAGAAGGGGCTGGGGAGGGGGGCAGGTGCGGGAGATGGAGCTGA HUMAN SEQUENCE - GENOMIC GGAGTAGGAGAAAGCTATGGCATTTTAGGAAAATTAATCGGGAGGTGACAAAATAGTTTGAACCAAGTGGATAT- AGTAGGCAAGTAGACGATAGAAA ATAATTGCAATAATATAAGCATGAAGAGATGACAGCCCAAATCAGCGTGGCAATGGTGAAAAGTGGAACACAGA- AAATGAATTGGAGTACAGAAAAA TCAAAAGAAAATGAAAAAAGTTTGAAGCCAACTTGACATGTTGAGCAAAAGAGGGAAGCTTCAGAGATCATACT- AGAGTCTCAAGTCAGGTGATCAG AACTGCGAGGTCATTCACGGGCATAGGGGAGCCTGGGGGGGATCACACCTGGTGAGGAGACTGAGGTGGGGGAA- GAGGAAGTGATGAGTTCAGAGCT GGAAGCTGTGGAGAGGGGTCAGAACCAGAGAGAGAAAGGAGGTCATTGCTGCCAGGGCAGTGTGAGTTGAAGCT- ATGAGAACAGGGTAGATCCCAAC AAAGACTGCACAGAGAAATGAGAGCCTGGCACAGAGAGTGAGGAACACCTATGTTTAGGGGATGGGAAGAAGAA- GGACCCCCAAAGAGTGAAAGAGA ATCCACCAGACAGGCAGGAAGGAGACAAAGAAAGTGAGATGTCATGGAGCTAAGGAAGGAGAGACTGTTAAGGA- GGAGGTTCTAACAGTGCCAACAA GTACAGAGAGAAGAGGCATTGGGTTTGGCAGTGACAAAGTCTCTAGTGACATTTGAGAGCAATTTCAGAAGAGT- GAGCAAGGTGGGAACCAGATTAC AAGTTACCACTAGAAAGTGAGAAACTGTCAGCAAGTATAGGTTACACTTTTGAGAACTCTACTCATAGAGAGGA- GAGAAATAGAAACCAGACAATGT ACTAGAAACAGGCCAGGCCAGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGTGGATCAC- CTGAGGTCAGGAGTTTGAGACCA GCCTGGCCAACATGGCAAAAACCCATCTCTACTAAAAATACAAAAATTAGCCGGGCCTGGTGGCACGCGCCTGT- AATCCCAGCTACTTGGGAGACTG AGGCATGTGAATCGCTTGAACCTGGGAGGCAGAGGTTGCAGTGAGCCGAGATCACGCCACTGCACTCCAGCCTG- GGGGACAGAGCTAGACTCTGTCT CCAAAAACAAAACAAAACAAAACACAAAATAAAAAGAAAAAGAAACAGTCTTCCAGTTTTTCTTCTTCACACTC- CGAATGCCCTCTCTTCCTAAGTC AATATGGATGAGGGGCTGTGGTGAGGTGGTCTGAGGGCCAGCCTGCAAGACTGGTATAAGACCTTTAAGTTTCA- AAAAATAGGACATCCAAAAGATC CTTAAGGGGGCCACAGTCTTGACATTCACAGACAGAGAGGACTTAGGCAGGGGTGTCCAATTTTTTGGTTTCCC- TGGGCCACATTGGAAGAAGAAGA ATTGTCTTGGGCCACACATAAAATACACTAACACTAACAGTAGCTGATGAGCTTTAAAAAAAAAAAATCACAAA- AAAAACCCTCATGATGTTTTAAG AAAGTTTACAAATTTGTGTTGGGCCTCATTCAAAGCTGTCCTGGGCTGCATGCAGCCCACAGGCGGTGGGTTGG- ACAAGCTTAGCTTGGAGGCTCTG GTGGAACTCCAAAATAAACATGAAGAACACCACAGAAGAGAAAGCAAAGGGACTGTAATGATTTATGGATCATT- AACAGACATTTATTGTGCACTTA TTATTTTTCCAAAAATGTTATCCATCCATTTAGCTTCAACTACCACCCATGTGTCAATATGTCCAGCCCACCCG- GATATCCATTTCAAACTCAACAT ATTTAAAGTCGAACATGTCACCTTGCTCACAAGAGTGCTCCTCTCCATTTATTCTCTACCATGGTAGATACACT- ATCATCACCCAACCAGAAACATG GCAGCCATCCTAGATTCTTCAATCTTCCTCACCTCATCTCCCTTATTGAATCAATGCATCTGTATTCTAAATAG- CCTCAATATTGTCCCCTTCCTCT CTATTCCACTATCATTGCTGTAGTCTAGGACACCATTACCTCTCACCAGGTAATAATAGTTTGGATCTTTGATC- CTGCTCAAATTTCATGCTGGATT TTAATGCCAATGCTGGACATGGGCCTGGTGGGAGGTGTTTTGATCATGGGGGCAGGTCCCTCAGCGGCTTGGTG-
CTATCTTCATGATAGTGAGTTCT TGTGAGATCTGGGTGTTTAAAAGTATATGGCAACATCCCCCATCATCAACTCTCTCTTGCTCCTGCTTTTGCCA- TGTGATGTGCCTGCTCCTGCTTT GCCTTCCACCACGAGTAAAAGCTTCCTGAGGTCTCCTGAGAAGCTGAGCAGATGTCAGCACCATGCTTCCTGTA- AATCCTGCAGAACTGTGTGCCAA TTAAACCTTTTTTCTTTATAAATTACCCACTCTCTGGGTTTTTTTTTTTTTAATTTTTAATCTTTTTTTTTTTT- TTGAGACAGGGTTTCACTCTGTC CCCTAAGCTGGAGTGCAGTGGTGTGATCACAGCTCACTTGTACCCCTGAACTCCTGTGCTGAAGTAGTCTTCCT- GCCTCAACCTCAAACGTAGCTGG AACTACAGGTGTTCACCATTACACCCAGCTATTTTTTTTTTTTTTTAACTTTCAGTAGAGACGAAGAATCGCTA- TGTAGATCAGGATGGTCTTGAAC TTGTGAGCTCAAGCAGTCCTCCCACCTCAGCCTCCCAAAATGCTGGATTACAGGCTTGAGCCACCATGGCCTAT- CTCAGGTATTTCATTATAGCAAT GCAAGAATGGCCTAATACACCAGGGCTACTGCAGCAGCCTTCTAACTACTCTCCCTGCCTCCAGTCTTCCTCCA- CTCTAATAATTCTTTGGATTATG AATTTCTTTATTTGAAAGTAATTAAGCACCAGTAAAGTACATCTCTCTGAAACACACATCTGACCGTACCACTT- CCAAGTTTTAAAACCTTCAGTAA CTGCCAACTATCTATAAGTAAAGTCCGAGTTCCTTTCCCTGGAAGAGAAGGCCTATTATAACCTGGACCTGGTG- CCATTCCAGCCTTATCTTCTTCC ACTGCCCCTATACACCCAAAGCTACAGCTACTTCTTTTAACACTCAAGGTTCAGCCTTATGTTCTCTTTCTGTG- TCTTGCCCCTTGAGCCTTTGTCA TTTACATAGCTCCAACGATTGTCCCTGAGTGATGCCCAAATCTCTATCTTCAATCCTACACTCTCTTTGGAGCT- CCATATTTCTAGTTGCTTGCAGG GGATTTCCATCTTGACTTGACAGGCCCCAAACTGAACTCAGTACCTTCCTCCCCAAAGGTGGAAGTGCTCGTGA- CTTCCTTAGTTCTGTGTTACTCC TGGTCAATTAGAATAAAAAACTACAAGTGACCTTTACTCTTCACCGTTGCCTTGGGCCCATTCCTGGACATGTC- AAATAAGCCAACAAATGCTGTCA AGTCTCCCTTTCTTTCATCTGTTTGCAATGTGCTTTTTCATTCCTATGACCACTATCAGAATCAGAAAGATCAG- GACCTGACCCATCCTTCACCTTT CTCTCCAGTCTCAAAGGAAGAGGCAACCTGTTTTCATCATGTCGGTCCCTGTGCCCTTGTTTTAAAACCCCAAA- CAGTTGCCTCGTGCTTACAGGTC ACAGTGAAGGAGGTCTTCACCACAGAAGACCTAGAAAAAAAAAAAAGAAGATAAAAAACGTGACAGGCCCTCAG- ACTGAACTCGGCATCTTTCTCTC TGAGGCTGGAAGTGCTCATGACTTCCTTAATTCTATGTTACTTCTGGTCAATCAGACTAAAAAACTACAAGTGA- TCTACAGAAGTGTCCTCTACTAA CAATCAGAGTGAGGATAGAGTCGGGTGGGACTGGGCAGTTAGAAAGACTTTATAAGTCCTTGAACAGCAGGGGT- GGGAGCTTGTGGAAAAGTACACA GGTAGCTTCAACAGCACTGTAATGTTCTGAATTTAAAAGAGTGACTTAAATTGAGTTTTTGTTCTTAAATTATG- CTTTATAACATATAGACATATGT CCACCATCTATATTCTTTTGTACATATCAAATGTCAGGTTTTCATTTTTAAATTTGTTTGCAAAAGAGAAGTCC- TAGGACAGTCTCTAGGAGCCCAG TAGGGAATCAGTAATAAGGGGCATAGGACACTAATATTTGTGAGTGTTTACTACATCAGATAGATCAGAAGATG- GGGAAACTGAAGTTCTGAGGAGT TAAGTGGTTTGCCTATGGTAACATAGCTGGAAAGTGTTTTGAGATTTGAATCCACATATATTTGACCCCAAAGC- TTGTCTCAGAATAATGCCAGAGG GAATTTGCACGTTTTAAACACAATCTTGCCAACCAGAGGCTATGACCCCTGAGTACATGTTGGTATGAAAAATT- CCCCAGAATTACAACATCCAATG TCCACCATGAAACATGACAGAGGAAACTTCTCTTTTTGAAGACCCCTCTCTCTTCTTTCAGTTTCCCAACTTGC- GTCTTCCTTATTCTCCTCCATTT CTCCTTTCAGACTCACTGCTTCCAGCTTTGGCCTCATCTCTACTTTTACTTCATTTGTAATGGGGCAGAGGCTA- CCTCAGAGCAGAGGAGGAGGAGA GTTGGGGCGTGTCACCTGTTTTAGAAAGAATCCACAAGTGGGCAGCAGTCTGAGGGGCTTGCGCTGGGCAAAGC- AGATGTGGACAGAGGGAATCAGG AAAGCTTTGGGTTGGGAGGCATGATAGAGACTCAGAATAGTCAGTATTTAACAAGTCAGGGGAAGTGGCTAGAA- AGAACAGAGACACTGGCATGGCT CACCACAGGATTCAGGATTCCAAGTGGCGTTTTGGTGCTCACATCCCACAGTGCGGAACAAATTCCATTAGTAG- TGGAGCATCTCATAGCTGAATGA CTCAGGCCGCAGAGGAGAAATCCAAGAGAAGGACTGAGCTACATTCCCCTAGTCACTAACGAATCATTATGTAA- GTAGATCACCCCCTTTAAATAAA TGCAATATACACAAACCCACATTTATAAGACATAATTTAGGGAATACTTAGTTACCTACTAAAGAATTCTTTCC- TTTAAAAAAGAAAAACATGGCCA GGCACGGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAAGCCAAGGTGGGTGGATCACCTAAGGTCAGGAGTT- CAAGACCAGCCTGGCCAACATGG TGAAACCCCATCTCTACTGAAAATACAAAAAAAAAAAAAAAATTAGCCAGGCATAGTGGTGTGTGCCTGTAATC- TCAGCTACTCAGGAGGGCGAGGC AAGAGAATCACTTGAATCCGGGCGGTGGGGGTTGCAGTGAGCGGAGATCGCGCCACTGCACTCCAGCCTGGGCA- ACAGAGCGGAACTCTGTCTCAAA AAGGAATAAAAAAAAAGGAAAAAAGAAAAAAACAAATTTCTCTAACTAGGGACTTCTAGTACCTTTCCAGTTGG- GTCCAATTGATAGAAATTCCATT AACATCCAATGCACTGTGATAGGAGGGAGGCAACTGGGAATAAAGAAACACGAGGAATCTCGAGTCGGGTGGCC- TGAGTCTTAGTCCTGACTATGTT CTTGGGACCTATTCCTACCTGTAAAGTAAGGGCTAATCCTGTACCACCTCTAACCGTCATATAACTTTTAAATC- TTAGCCTATCTCTACCCAGTCCT ATAAAGCAAGATAGAACTCTGTGTGAAGGCTTCTGATCCTCCTGCTCTGCTGAAAGTAGCCAGAAAGGCAGCAA- GCTCCTCAGCCTCAGGAACCCAG CCTGAGGCGAGGGGCTGGCTGAAATTGCCTCCGTCTGGCCTGGAGCTGTGCTCTGCTTCTCCCCATTTCACTCT- AATCTTCAGCTTCAGTCATTTGC CACATCTACTCCTTCAACCATATCTTTCCTCTGCTCTGAGTTTTCTAGAGCCCCATCCCCCTTGAATTTATACA- AATTTTTGCAATCAACCAGATTG GCCTCCCTGCTCCACTAAACTCATATCCTCAACTGTCTGCTGTCTTCCCCATCATGCTTCCTCTTCCACTTGCC- AGATTTTGCACAAGATGTCTCAT GATCTTGTCCAGGGAAGCCTCCCCAGTTTGGCTGATCTCAGGGCTGCCACAAAGGCACCTGCTGATGGGGCAAG- TTGAGGACTGAACTGCATTCAGC TTGCCAATTCCTGCACCCAGCTCAGAGCTGTGTCTGCTGGAGGAAGGGAACCTTTTATTTTCTCCCAAAAGTAT- CACCTGTTCCCTGTTCTCCAAGT GACAGGCCACAGTAGGCTCTTTTTAAGCTCTTTTCCTATTTTGCACCACGGTTCCCTTTTTTTTTTTTTTTTTT- TTTTTTTTTTTTTTATGAGACAA GGTCTCACTCTGTTGTCCAGGCTGGAGTGCAGTGGCGCAATCACGGCTCACTGCAGCCTTGAGCTCCCAGGCTC- AGGTGATCCTCCCACCTCAACCT CCAAGGTGGCTGGGACCACATGCACATACCACTACACCCATCTAATTTTGTATTTTTTGTAGAGACAGGGTTTC- GCCATGTTGCCCAGGCTGGTCTC CATCTCCTGGGTTCAAGCGATCCGTGCACCTCAGCCTCCCAAAGTGCTGGGATTATAGGTTCGAGCCACCGTGC- CAAGCCAAAAGCTAGAATCTTGT CTATGCTTTTGTGTCCTGGTGCCTGGGAAAACTTTTTTTCTCCTGCCTCAGTTCAGCTCAGTGATAAATAAGGA- ACTGAGGTTAGATAACAGGTAAA GTCTAGGACCTGCAGGATGAGTGAATCAGGTGAGGGAGTGGTAGTCTTCTTCCTTGTCAGCCAGGCTAGGTTCA- GGGGCACCTGGACTGAGGCGAGG GGCTGGCTGAAATTGCCTTGTGGAGGGCCCTGCCAGTGATGCCCCCTCCAGCAAATAGGGCCAGCTCTATGCAA- ATGTGTTCTTGCCCAGGAGTTTG GTTTCTTCTCTCTGAGCTCCTGGCACAGTGGAACCAATGTGAGCAGCTGCTTGGCAGGACAGAGAAGGGCAGGC- TAGCAGTCCCAAAGCTCGGGTGA CAGGACCAGGCCCAGGAGACGGGGATGTTGACTGGGGCTTTAACAGCACTCTTGATGCCAATCTCGGGCTGAAA- ACTCGATATTTCCACTTGGAACA ACAAGAATCACCAGCAAGAGAGCTGAGGAGAGGGCAGTATACCGGGGGCGCCCCCTGCAGGCCTCACAGGGTGG- TGCCAGAACAGAGGAAGGTGGCA CAGGCAGGGTGGGGCTTTCAGGACATCCCTGAGATGATGGTGATGACGGTGACAATGATGATGACCATGAAGAA- GACAATGAGGAGGAGGAAGAGGA AGACAGTAGCTAGCATTTACTGAGTACTAACAATGTGTCAGGCATTGCCTTATGTAGTCTTCATGACAACCCTC- TAAGAGATGAATAATATGGTTTT CTTTTTTTTAGATGAAGAATCTGAGGTTTAACGGGTTAAATAATTGCTCAGGTTCACCCAGCTAGTAGTGGACA- GAGGTGGGATTTGAACCCAAGTC ATTGCCTCCTGAGCTTATATTATCCAGTACCGAATTTCCCACCTTGCCAGGTCATTCCAGGAGCTTCTAGCCCT- CCGTGTCCATCTCTATGTCTTCC TGCTCCTCTAGCTCATATTTTCTTGATCCAAATTTAAAGGATCTGGATAAGAATAGATCCATATCTGGGATATA- ATAATACTGATAACAACAGCAAC AACACTTTGCGTTTGTAAACCACTTTCTTCTCTTCATTATTTCCCTGGGGAAAAATAAACAATAAGATATTTCT- GTTTCTCCAAATTTTGTTCTGAT TTTTATCAGTGTTCCTGAAGCTATTTCAATATAGTCATGATCAATTTCTAAGAATATTTTTAGGTTCTGCTTTT- TTATGTAACAGTGTGTTATATAC ACATTCACATATTTAAACACAGCAATTATTATGGCTTTACAGTAACCCATGATATTAATATTCCACAGATATTA- CATTACTGAGGCACACTAGGCTA AGGCTGACAACACCAAATGCTGGCAGGAATGTGGAGCAACAGGAACAGGAATTCGTGGCTGATGGGAATGCAAA- ATGGTACAGCTACTTTGGAAGAA AGTGTGGCAGTTTCCTAAAAAACTAAACATACTCTTACCATACGATCCAGGAATCATGCTCCTTGGTATCTACC- CAAAGGAGATGAAAACTTACGTC CACATGAAAATCTGCCGATGGATGTTTATAGCAGCTGTATTCATCATGGCCAAATCTTGAAAGCAACCGAGATG- TCCTTCAGTAGGTAAATAGATAA ATAAACCATGGCCATCCTGAAATGGAATATTATTCAGTGCCAAAAAGAAATGAACTACAAAGCCATGAAAAGAC- ATGGAGGAACCTTAAATTTACAT TACTAAGTGAAAGAAGCCAATTTGAAATGGCTACATATTGTACAATTCCAACGACATGACATTCTGGAAAAGGA- AAATTATGGAAACAGGAAAAAGA GCAGTTGTTGCCAGGGGTTAGGGAAGGGGGATTGACTAGGCAGAGCATAGAGGACTTTTACAGCAATGAGACTA- TAATGGTGGATACACATCATTAT ATATTTGTCCAAACCCACAGAGTGTACAACACCAAAAGTGAACCCTAATGTCAGCTATGGACTTTGGGCGATTA- TGATGTCAATGTAGCTTCATCAC TTGTAACAAATGTGCCATTCTGGTGGATGTGTGGGGACAGGGGGCATACGGAAAATCTCTATAACTTTCCTCTC- AGTTTTGCTCTGAATCTAAAACT ACCCTTTAAGAAGTCTTCTTTTAAAACAATTTACAAAGCATGAGGTGATACAGATGTGGGAGTTTGGCTCCTGT- CTCTGCCCAACTCTGTGACATTC GATAAATTACTTAACATGTCTCTGTTTCAGTTTCCTCATCTATAACTGGGAAAAATAACACCTGTCTTATAGAG- TTGCCATGGGGATGACATGAGGC ATGTGTCTCGTTCATATCCCATGCTCAGTGAATTAGTAGCAGCAGCCACTGTGTGTTTGTGTGTCTTTATCCCT- CCTGGGTTAATGAGCTCCTTGTG GGCAGGGACTCACCCATTCTGTAACCACCCCATCTAACACACTGCCTGGCACTTGGGCTCCGCAGAAGTTTGCC- GAGTGAATACTTAGTAAGCCCTA ACCTAGGCTTTTCTCTCTGGTGGACATTTGGGTTGTTTCTAGGGTTTTTGCTATGAATAAAACACATTTCAAAG- CCCTTTGTGGTTTTTTTGGTTTT TGTTTGTTTGTTTTTTCTTCGTTTGATCTGCTGACTCTGTGAAGCAGGCAGAAAGGGGATATTTGCTCTTGTCC- ACACCCTGGTACAGATGGAATAA CTGTGGCTCAGGGAAGTGAAGTGACTCCTATGGGACACAGTGCAAATCAGTGGCAATAATTAGAACCCCTGACC- CTGCCTCCCTTCCTTTAGTAGAT CTATTTTCCTTCTAGCTACCGCCTTCTGGATCCATGGCCTCTCCAAAACTAGACCATGATGGTCAGCCTGACCT- GAGAGCAGCACCTGCACGCAGAG ACCCATGTTGAAGGTGGTGAGCTGCCAGCTACCAGATGGCCCTCTGAAACCCCAGGGAACCTAGCACCTTATTC- TCAAATACATGAGGGCTTGTATT TTCCCCCAGGAAGGAGCTTCTTAGGAAAGAGCCAGCGTGCCAGCTTTGTTTTTCTTTCTTCTTCTTTTTTTTTT- TTTCCTATGAGGGGGTGAGGAGC CAAGCTCTGAGTTGTCCAGGAGGAGGGACTTTGGCTAAAAATAGCTATGGCGTGTGGTTTGGATCAACCCCTAG- TGGTACCCAGGACTGGGGAGGGG AGGGGGATGCTCTGGAGCTGTCGCCAGACTGGTTGCCGTGGAAACAAGAGAGGAGCAGGGGAGCCTGGGAAGTA- GGGATGACACAGATAGCAAGTCC TAGTCAGAGCTGCCGCTACATTTAGGAGAAACAGCGGTGTCTGCGGCTCCCACCCTTCGGGGGGCCCGTGGGGG- GGGCGGTGTCAGGGGCATGGACG CCACCCCCCAGGGGTCTCTGCTGCCGGCTACTCTCCTCTCCACGTGCTGTGAGTTGAGTTGCGGGGGACTTGGG- GTTTGGGCCCCTATTTCCAAGGC AAGTGGGGGTTTGGGAGGAGCTGGTTCTTGGGGGAGTTTTCACCAGGTCTCTCCTTCCAAAAAATGAGCCCCCT- TACTCCCCAGCTCTCTAGAGGGA
GGAAGAGGGGCCCAGGAAAAGTGGTATTGCAATCTTCTGCAAAGGGGTCATAGCATGCACAAGAAATGAGGAGT- AGGTTGGAGGAACTGAAATTCTT GGAGGGAAGATGGAGAAATCAAGTCCTTGATCTTGAGATAGAGGTAACAATTTCACACTTTTCCTTCCCCTGAG- AAAAGTGCAGTCCCCCACTCAGG AAGACAGGATGTGGGACACATTCAAAATAAGGTTTACCTAGATCCCTGGGGCAATGGAGAGTGAGAGAGTTCTG- GGGGTGATCCGACATCGGGGTTC CTTCCCCATCCCTGGGCAGAGAGATCTGTCTAGGCAAGCCGACTGGGGGTCAGATTACCTAAGACCCTGAGAGA- ACATCTGGAAGCCCACCTGGGAC TAAAGCTAGGATAATGGGAGCAGGGTCGTTTTCTGCATGACCTGGGGTCTCTGAGCCAGTCAATGCTTACTCTT- CCTGAGGACATCTGAGCTTCAGG AAAGGAAAAGGAAGCCCATTGTTGGGGGCAGGGGAAACCCTAATCTTCCATTGCCATGGGGCTCTTGGACCCTG- TGTCCCCTGACTCCATGGACAAT AAATGCAGGGGGTGCCCCTAAGCTCAAAGCCATTTCATTTTGATTTCTCTTCCTACCTTCTCTACCCCAAGACA- CACAAACACACACACACACACCC TCTCCAGAGTGCTGACTGCAGAGGACCTCACCCCAGAACATAAGATGCTGGAGTGCTAGGTTTAGAGTCACATA- CCCAGGCAGTTTCTCCCCAGGAC CTGGTCAACCATCCAGGCCATCTGTGGTTCCTATGGCACACTCCTCCATCCCCCACCCACTAGCCAGCCCACGT- TTCCGTGGAGTGGGAGGAGAGGA TCATTCCCAGGAAAGAGAAGGGAAGGTGGAAGAGTCCCAAATCCTATTCTAAACCTTTCCCTGTATGGTCCATA- TCTCCTAGAGGACCCTGGGTGCT TTGGGGAAGGGCTCTGGACCTCTCTCAGAGCAGATTGCAGCTCAGAGAGCTCCTCAGAGGCAAGCATGTGAAGA- AAAATCAGGTGGGCTTCGCTTGG AATGTGGGCTTTGGGGCATATGGCAGGTGGGGGCGGGGCTGGTGTTAGGATAGTCCATGGGAAGTAAGAGGCTG- GGGGAAAATATAACTAGAGGGAG TGGGGAAATAAATGTGGGTGCTTAGTGCTTCACCTGATCTGATTCCATGTCTCTCATGAAGAATAGGATCCCAG- AGGGATACGAGCCTAACTCTTTA TAACTCTGGGCTTCCTTTCCCAGGCTTCTGTGTTGGGATCTTCCAGTTCCCCTCCCCATTTGCAGGCTGTCTCC- ACTAGGAGAAAAAACCCAAGGGA AATGAGGCTGGCCCAAGAGCAGCAGTGATCGTGGGTAGGTCTCAGGGAGGATTTCTAGTGGGAATTTCCTAATG- TTCCACCCTTGTGCACTGGAGGG TTTCCACTGACTTTCCACAGCTTTCATTTCTTTCTCGTTTGTAAGCATGTTGAGGGGAGGGAATGGAGCGGAGT- GAGTGAGGTCCAAGGAGGGAAGA ATGAGAAAGACTGTGTATCAGTCTTGGGGTGAACTTCAAAACAGCCTGCGAGGAGAGCCATTGGTGGCTGCACT- GGCTACAGCTGGGGAAGGGATGG TGGAAGTCCTTAGGGCAGGGAGGGCTCCATTACCCGCCTGCCCCCCTCCCCAAAAAGCCCCCAGTCTATTGATT- TCAGGAAATCACTAGGGGGATCT GGGCCTGGGTCTTTGGCCCCGGGGCTGCCCCTGAGGTGCTGCACACCCCAGCTGGAGGTGATGGCACCAAAATA- TCTGGTACCTCCTTCCCCTGAAA ATCATCGTGGAACTTGCACAGTTCTATCCAGTTCAGGTACATCATTCCATTTGACCCTCACAACTTTCTGAGCC- TGGGGGGCAGTTAGGGCTGAATG TGTTATTCCCAGAAATAGAGGCCAGGCAACACGAAGGGACTCGCCCAGGGCCCCCCAGGGCTCGGTGCTGGCCC- TGATGCCCCGTGCCTCCCCATCT CCCGAGGGGCCACTCATTCGGCAAACCTTTATTAAGCCCCTCCAGGACCCCCGACGCCGCCTAGGCGCCCAGCG- ACGCGCGGCAGGTGGCAGCAGCT CGGGCCCCCGCCGCACTCCAGGCGCCCGCAGCGCTCGCCCTGACGCGGCCGCCATGGCGCAGGAGAACGCGGCC- TTCTCGCCCGGGCAGGAGGAGCC GCCGCGGCGCCGCGGCCGCCAGCGCTACGTGGAGAAGGATGGCCGGTGCAACGTGCAGCAGGGCAACGTGCGCG- AGACATACCGCTACCTGACGGAC CTGTTCACCACGCTGGTGGACCTGCAGTGGCGCCTCAGCCTGTTGTTCTTCGTCCTGGCCTACGCGCTCACCTG- GCTCTTCTTCGGCGCCATCTGGT GGCTGATCGCCTACGGCCGCGGCGACCTGGAGCACCTGGAGGACACCGCGTGGACGCCGTGCGTCAACAACCTC- AACGGCTTCGTGGCCGCCTTCCT CTTCTCCATCGAGACCGAGACCACCATCGGCTACGGGCACCGCGTCATCACCGACCAGTGCCCCGAGGGCATCG- TGCTGCTGCTGCTGCAGGCCATC CTGGGCTCCATGGTGAACGCCTTCATGGTGGGCTGCATGTTCGTCAAGATCTCGCAGCCCAACAAGCGCGCAGC- CACGCTCGTCTTCTCCTCGCACG CCGTGGTGTCGCTGCGCGACGGGCGCCTCTGCCTCATGTTCCGCGTGGGCGACTTGCGCTCCTCACACATAGTG- GAGGCCTCCATCCGCGCCAAGCT CATCCGCTCGCGCCAGACGCTGGAGGGCGAGTTCATCCCGCTGCACCAGACCGACCTCAGCGTGGGCTTCGACA- CGGGAGACGACCGCCTCTTCCTC GTCTCGCCGCTGGTTATCAGCCACGAGATCGACGCCGCCAGCCCCTTCTGGGAGGCGTCGCGCCGTGCCCTCGA- GAGGGACGACTTCGAGATCGTCG TTATCCTCGAGGGCATGGTGGAAGCCACGGGTGCGAGCAGGCCTGGGGAGGGGAGCGGGGTTGGCAGAGGGTGG- GCGGGACCGAGGAAGGCAGGGGC GAGACTAGGGGCCAGGGGAGCTGGGGAGGATGGATGGAGGGGCTGGTGGAGGATGAGACAGTGAGGTGAGACAG- GGGTCGGAGGCGGGAGTGGAACC GAGCAACGCCGCAGAAGGCCAAGAGAAAGCTTGGAGGAATTCTCCGAAATGGCACTGGCGTGGGGCCCTGGGCC- CAGAGGAATGTGTCACTTGGAAT AGGGACAGTAATAATAGCTAGTGCTCGCCCAGTATTCACCCTGTGTCATGCGCAGTTCCAAAGCACTTTCTACC- TCTGAGTCGATTTAATCCTAACA AGAACCCTCTGAAGGTAACTTCTTGTTATTGTGCTCACTTTTTAGAGATGAGATTGCTCCAATGAGAAATTAAG- GAAGTTGTCCACTTTCCTAAGCC AATAAGTGGCCATGCCTGGATTGGACACAGGCAATGTGGCTTCAATGTTTAGTGGTCCCGAGTTGGAAGGAGGG- GTTAGGTTCAGGGGTTTTCTCAC TGCAGTCAGGTTCAGGCCCCTGGAATTTGACGGTGAAGGTTTTCCATTGCCTGAGTTATTTCTAGGCCGGATCT- TGAGGGGAGTTTAATACCTAGTC TCACTTGTACCTCGGTTTCCCAATTCATCCATTTCCACTGACAAGGGATATAGATGATGTTACCTTTTCTAGCT- CTTTTCCAAAAGGAACTGGCAAC TCATCTGTGATGTCAATAAGTCCAACCCAGACCTACACAGTGAAGGCTTTGGGAGCAGGTGAAAAAAGACCAGT- GTTACAGGAGTCGCAAAGGAGGT CACTTAGGACTTGAGATCTAGAGGATAGATGAGGATGAGGAAACTGCGGGTGGAGGACCAAAGGCCCACTAGGG- GGCGCCGCAGTCCCTCCTCTGAC GCCAGAGCTGCTGATGCTCCCTGCCGGCTTCGCTGACAAGCTGGTGCCTTCAGATCCTTTCCCTGGCCCCTTTA- GGCTGAGACTCCGCTTCACACCC CAACCCCAGCTCCGCATCACTGTTCCCATTCCTGCTTCACCCCGACTCTTTCCTCTTCCCCCACTCACCCCGTT- CCCTTTCCTCTCTCTCCAGCTGT CACTCCTTTTCTGCCAGTATCTCAGGCAGGCCCCTCACCCTCCAGGGAAGTTGCTGCCCGGCCCTCTTTTCTCT- TTGTACCCCCAGCCCTGCCCTCT CCTCCTCGAAGCCCTTCTCTCCCCAGTGTCCCTTATGCCTCTTTCTCTTCTCTCCCACTGGATACTTTCTATTC- CAACTTCACCGAGGAATACCAAT GTCTCAGCGCCAGGCTTTCCGAGTTGACAGCCACTCTCCGGTTAGCTAATGTTCACTCTTCTGTTTCCCCTTGT- TCCGAGATGGATATGGGTTGGGG GCAAGACCCTGTGGCAGAAAGGAGAATGACCTGCCCTGAGGGGTGCACCAGCCCAACAGGAAGATAGGACACAA- GCCCCGGGCAGGGAGGACCAGGA CAGAGGAGATGAGGATAGGAATCTGTCTGTTTTTCTAGAGAGATAAAGCTGGAAAGGATGGTAATATTTTGGGT- GAGACAGTCAGGATTCAAAACGC TTTTGAAAAGCAAGAATAATGAGCCAAAACCCAGCAAGATGACATTTAAAATGAATAAATATAAAATTCTACAT- TTAGGCTTTAAAAAAATCACTTA TGTAAGCACAGCATGGAAGAGCACTGGTGAAAAAAGAACTGGGAGTTTTAGTTGGCTACAGTCTTGATGTCGTA- GCAATGTGATGCAGCCTCCAAAA TGATTATGTAATGTTATCCTGGGCCCTATTAGTGAAAGCATCATGGCCAGAAGAGAGAGATGGTGCGCGCTCTC- TTATGCACGGAGCAGGCCACAGT TGGAAAATTTACTATACTCAAAATGCTTAAAGGGCCCTCCTTGGCCATTCTGGCTTGTAATCAAAAAAGTAGAG- TTCTGGAAAACCAGGTCAAATGA GGAATCGTGGAGGAAGCCAGGGATGTTAAGTCAAGAGAGAAAACATGAGGGAATCTGAGACTCCTGTTTTCAGA- TACTCAGAGGACTGTGAAGTGGG AGGGGAATGAAGCCAAGAGTTGGAAATCCCAGGGTACAGGTTTTAGCTCTGTATAAAGAACAACCCAACTATTA- GAGCTATCATACAAAGGAGTGGG CCCTTTATGAAGTGGTGAGCTATCAATCCTGGGAGGTAATCAAGTATAAGCTAGATGCCCATTGTTAGAAATGC- TCCTTTGGGGAGCCCTGTATGGA GTGAGAAGTTGGACTAGAGGATCCCTAAGGTTAGTTTCAAGGTTAAGCTTTTTTTGGTTGGCATCACCAAATGA- CAGGAGGGGAAAAAAGAGCTGGA CATTAAGAGGAGTTGGGGCAAATGGAGAAGACACGAGGGAGCTGGGTAAGAACAGGAGCTAGGGAGGGGGGGAA- ATGGACTGGACCAAAGGGAGGTG GGAGCCCTTAGGAAGGAATAGAAGGGAGGGTGCTGGGAGTAGGGTTGTGGAATGAGAAGAGGAGAGGGAAGCCT- GGAGCTGAGATTCCCCCTGACCG GTGCCCCTCCTCCCAGGAATGACATGCCAAGCTCGGAGCTCCTACCTGGTAGACGAGGTGCTGTGGGGCCACCG- CTTCACGTCAGTGCTGACTCTGG AGGACGGCTTCTACGAAGTGGACTATGCCAGCTTTCACGAGACTTTTGAGGTGCCCACACCTTCGTGCAGTGCT- CGAGAGCTGGCAGAGGCTGCCGC CCGCCTTGATGCCCATCTCTACTGGTCCATCCCCAGCCGGCTGGATGAGAAGGTGGAGGAGGAGGGGGCGGGGG- AGGGGGCGGGTGGGGAAGCTGGG GCTGACAAGGAGCAGAATGGCTGCCTGCCACCCCCAGAGAGTGAGTCCAAGGTGTGACCAGCTTCCTCCAGACC- CCTGTGGCAGACCGGGGGCCAGA CACAGATACATGGGGAACTGCATATCGGAGGTGGTGGAGGAGGAGGAGGAGGAGGAAGGCAAAGCCCCTGGAAA- TGTGCTAAAGTTGGAAAGTCCCC GTCCCCCAGAACCTCAAGTCTAGAAACCAGTATGGAAGGGAGGGGTCCTGATTTCAGGGAAATGGAGGGTGGGG- CCGGGTGAAAATGCCAGTCTGTG TTTGACCTTCACATTTGTTCATGAGTGGATGGATGGACAGAATGATGGACTTTTGGGGGTTGGATGGGAAGATG- GTAGCAGATAAAGACAGCTGACA GATACATAGATGGACCAGTAGACAACTGGTCCACTCAGGGCTGCCACTAACCTGTAGAACACCCCTGTGCAAAT- TTTAAAAAGGAACCCTTTTCCTC CAGACAGATACAGCCCCAAACCAGGGTGCATGGCTTGGGGAGCAGAGTATAGGATGGATTGCAGTCCCCAGTCA- CCTCTTCTGCCAGCCTCCCCACA TATGGCACAACTGTCTAATGACACGGTAGGCCAAGCTGAAGTGAAGGAGAAAGGAGCCGGACCAAGATGGGCAC- ATGAGGAGGGTGCCCTCCTAGCT CCACCCTCACCAGGATGAAGGCGTGCAAGGGGCTCAGCAAGGTGTGAATGACCTTAGTCCGCAAGTTCAGGGAA- GCAGGCAGAGCGGGGAGGTGCCT GAGCTGGGGCCTGGAGAGGGGCCTGGGAAAGGAAAACCAGGGATAGCTATTTTCTTACAGTGGAGTGAGATCTT- ACAGGTATCAGGCACAGGCAGGA AGAGAGAGAGAGAGGTTCTGGGGAGGAAGGGCCAGGAGAGAGATCTAGAAAGTGGGTTCACTAGAGCTGGGAAA- CAGGGAGCCCCTAGGAAAGCAGT GTGTCCTTGGGGCACAGTCATTCACATCACTGATTGGGTGCCATGTGGAGTGGACATTCAAAAACCTGGTTCCT- GTCCTCAAAATAAGGGGCACCTG GGAAAACAGAGGAATCTACCTGTGGTGACTGAACGAGGGATAATTCAAACTGACAACCTGTGCAGTCCCGTGGA- GGGTAGGGGAGTGTGGGTGATCA GAAGGCTGGGGCCAGTGTAAGGCATAGGGAATATGTAAGTCAGGAGTTAGAAATCTCCAGTGTGCGTTGGAATC- ACCTGGAGGGCTTGGTAAAACAC AGATTTTTGGGCTCCACTCCAAGGGTTTCTGACCCAAGAGGTGGGGACCAAAACCATGCATTCCTAAGAAGTCC- CCAGGTCATGCTGCTGTTGCTGG ACTGAGGACCACACTTTGAGAACCTGTGCTCTAAGTGAATACTTGGAAGTCGTTTCAGGACATGGGGCATAGAA- ACTGAGGAGTAGCTGAGAGGAAG ATGAAGAGAAGCTGAGAAGAAGCTGAGGATCCTCACAGGAGCAGACAGAGAAATGTGAAGGGTGGGGTTTTATG- TGTGGGAAAGGGACCCGAAGCCC AGGCTGAAGAGTTTAACTTTGGGCCCAGAAACTCAACCATCAATGGAAACAGGGCAGTGACAAGTGGAGGGGGT- GTCTGGAAGCTGAGCAGGCCCGA CAGAGAGATGAAGCCATCAGAAGGACTTGAGGGGGCTCCTGGGGAGGTCGGGGGGAGGTGGAGCAGGAAGAGTT- TTAGGGGCAAAGGACAGAACCCC TTGTAGGACTGGAGGCAAGATTGAATGTGGGAGAAAATCGGAGAGAAGCGATAGGAGTTAGAACATCTGGATGT- GTCTGCAGCCTGCTGTCAGCCCA ATTGGGCCAGGGGGTCCCAAAGACGCATATTCTCACCCCACCTCCACCTGCTTCCTGATCACATCCCAGTCACC- AGCGGCAGCTTCCTGGATAGTGA GGGAGAACAACTGCAAGTTGAGAGAGGCAGAGGGGTGGAAGGGACCTGAAGCTGGCCTGGAGAAAAGCATAGGC- CCAGGAGAGCCTGCCCTGGGACA GCGCCTGTCTCCCACACAGCAGCACTGGCCCAGCAAGGACCTCCTCCCTTGGCCCTGGCCACATCCCACTCCTG- CCCTTTCATAAGCCCCCTGGGGA AAGCACTCCAGTCTTCTCTGTTCCAGGCTGGGCAGATAGGGTCCTATGGGGCACAGCCAGGGTCCTATGGGCAT- AGCCAGGGCCCTATGGGTCCTCT
GGAAGCAAGAAAGGGGGCCATGGAAGCAGCCCAGACAGCTGGGGTTCACTCAGAGAGGACCCAAGTCCCAGTCC- CTTCCTTTCAGTCAAAACACGGA TATCTTTGCCTCAGGTCACAGGGCCACTGGGGCCCTGTCATCAAAGATGAGATTCCTGAAGCCTGGCATTGACT- GGTCCCCTAAGAACAGATGTTGG GATGGAGAATGGGGATTCATTTGGGTTTCAGTAAAACAGGGGGGTCTGGACAAGAGCGGGTGGGCTACTTGGTA- TCCACACACACGCACTCACACAG GAGCCAACCCATTGCAGCTGAACAAGCAGAGAAACTCAGTCTGGAAAGGCCCCTCCTGCCTGCTGAAGTCACTG- AGACCCTGCCACACCTCTCCTCG CCACTGTCACCACTCAGGGCACCACTGTACAGTGCAACAAGTCAGGAGACCTAGGTCCTACTCCTGACACTTGC- TAATTAGCTCTATGACTCTGGGC AAATCGCATATCTGGGCCTCAGTTTCCTCATCTGTAAAAATGACAGCAAACTCGTAATGCTCAATAAATGTTTA- AATAACAACTGAAGGAGGCCTGC CAGATGCCTCTTAAGGTGCCGTGCAGGTAAGAATTTTAGGATCAGAGAATCCTTAGGCAAGAAAATTCATGAAA- CTCCTGGGGCACTGGAGGAGGGG TGAAGCTGAAGGGTGGGAGGGAGGAGACCCCAGGGTAGGTACAGGCAGGTGAAGCGGGTATATGCAGGTGTAGT- GGGTATATGCGGGTAGAGGGTAT ATGCAGGTACAGCGAGTACATGTGGGTGCAATGGCTCTGTGGACACACAGGCCCTCCCCTGACTGCCTGTTGTC- CCAGCCTGAGTATCAGTTGTGTT CTGAGGCTTCTATTCTGCTGCTATGGGTCAGAAGGAACAACAATTTCAGCCCCAGGGCCTAGTGGGAGGAGTCA- GGTCCAAGACTAGCCTGACCAGG AGAATGAGACGTGGGAAGAGTTGGGGAAAGTCTGGGAAGCTCAGAAAAGGCACTGCCCCTGGAGGCCCATGCCC- TTTAACATGGGAGAAGCTGGTGC GGGGGTGACCACAGGCAGCTGGAACCTACCCTCCTTTTCTATGCTTCCCTCCCCAAGTAGGAGTCCAATCAGGA- GTTGTCTCAGCCCCGACAGTTCA GGCTGCAGATGGAACCCAGGTGTCCCCTCCTGGGGTGGGTGGCATGGCCCATGGAGGCCAGATGGTGTTTGTGG- TGGGAAGAGAGGCCTGGGTCATC CAGAATAGGTTGTCAATCCCCAACCACCTCCCTACTATGCACCCTGAGCGTTTTACAGTCTCATGGTAGGGAAG- ACACAGCCAAGCCTGCTTTTTAT AAAACAAGTTTATTCACATTTTAGAAAAACTAATTCCAGGACAGGAAATGGCCTCCCTATAGGATCCCTAAGAG- ATCAAGAACAGAAGGCCAGAGGG AGGGGCTTGGGAGGGAAGGAGTGGGGAAGGGGAGGCACGTCTCCCATTCTGGGTAGTGGGAGGTCAAATAAATT- AAAGGAAGAGTGGACAGAGGGAG AGGGTGTCCAGGCAACCAGAGGAGGGCTTGGAGCTGGGCCGGAAGACAGTCGACACCTGCAAGACCTGAAAAGG- GTGCCCGGTGTGGGCTAAGGACA GAGAGCCCTGAGTGGGGCTCCCTCGCGGCCTCCACCCCTTAACAGGGCCCTGTGGATCTGAGCTGCCTACTCCT- CCTCCAGGTGGGGCCTGGGAGGG AGCAGCTTGGTTCAGGACTTGGGGGTGGGAAGCCCAATGAAAACAAGGTTGGGGGGTTCTTTTCCCTCACCTGG- GGAGTAAGGGATCACCGTTTTCG AAGCCTCTTCATGAAGCAGCAAGTGATGGTACCAAGGACAGTGGCACCAGTGACTAGGGCCACCCCTGTACCCA- CCAGCAGAGGCACAAATAGGGTG TCCAGGGCTGGGGGAGAGAGGATGACTGTTCAGAGAGGATGCCATCATCCTCCACCCATACACTTGCCTCTGCG- CTTTCCCCATCAAGTTCTCTGAA CCCACCTTCTCCATTCACAGACACCCCCATCCCTGCCCACAGCCTGCCCCCTCAGCATGCAAGTCAGCATCAAC- CACAGAGGACCCCGTGCAGGTGG GCACTGCAGGGCTGGAAGTTGGATTTTTTGAGACTTCATGTGACATAATGTGGAGGAGAGAGATAGTAGCAGGA- GGGTCAGAAGATGGGAAGGGAAG GCCAGTGGCAGAGGCCAGGAGGAAGGCAGAGTGAGGAGGGTGGAGGGGGTGTCACTCACCATGCATGTAGGGGT- AGACTGTAACAGGCCCTGAGCGG GCACTGCCCGCCTGGTACCAGCTGTAGTCGGCATGCTGCACCCAGGCGCTGGGGGCACAGTGGTACACGCCTTC- ATCCTCGGGCCCCAAGCTGTGTA GTCTCAGCCGATGGCTTCGGGGCCCCACCAGCTCTACGCTGACAGGGCCTCCTCCAGGCCGGACTCCCAGCTCT- GCCACACCATCCTGGCCTACGCC ACCCACCAGCTGGGCAGGGACAGAGCTGAGCTCTCCGTCCTCTGGTCGCTCCACCCACCAGCTGGCGGCCAGCC- GCAGTCCTGGGGGGCCACCCCGC ACAGAGATGTTGCACAGCAGGGAGGCAGTCTCCCCGCGGTACACTGTGCCTCCTGCTAGCCATGCCACAGCCTC- CAGCACCACACCTGCAGAACAAA GGACATGGGGTCAGAGGGTGCAGGGCCAGGGAGCATGGGGTTAGGGCTGCCGCCAAGCACCGCCCCAGGAAACT- CAGGGTATTCCCACAATCTTGGT AGAAGAGGAGCGTGAGGCTGTGGCCTGCAAACAGCTGACGGAGAGGGAGGGGTCATGGAAACAGAAGGAAAAGG- GGTTGACAATCCTCGAACCCCGT CCAGGGCCCAGCCCCCTCTCACCTTCCTCCCGCACATGTACAGGGAGAGGCCGGGAACGGGCACTGGCTGCTTC- ACGAAGCCGGGTCCCAGACCCTC GAACATAGGCTTTGGCGAGGCAGCGGTAGGTGCCCGCATCACCAGGCCTGGCAGCCTCTAGCCGTAGCCGGTAT- GTTCTGGATGCCACCTTCTCCAT GGCAATGTGTCGGCCCTCATAGCCAGGGCCCAGGCTGCCCACACCCTCTGTGTCCAGCTGGGCTACCAGGCGGC- CGGGCCCCAGGTGCCCCCGCAGG TGCCATCTCCCAACCTACAGAGTATGCAGCATGACGGCCTGCTGGGGGAAGTGCCCCTGACACATTGCACAGCA- GTTCCAAGGGCTCCCCTGGGCCG ATCCGACGTTCACCAGGCCCCACTGTCACTGCCAGCTGGCTGGCTGAAACACAGGTAGGGGAAGAGGTGTCATG- GAGGCAGGAGGGGACACAGAGGC ACCCGATTCCCCAACTTCCTGTTTCCTACTTGACAGCAGCAACTTCAAAACCTCCTGTCTCCCCCTCACTAGGT- ATGACCATCTTTCTATTTAGGGG CTTGAATCTCACCCCTCAGCATGGGCCTCCTATCTCTATACCCAATTTCTGAGCAGAGAAAACCCATCAAGGGC- CGGGGGAGAGAAATGCTAGCAAG GCTGCTCACTCTGTGGAAGATGAGTTCCTTGGAGTCAGATGATGGCTATCTGGTACCCCCTGTGGCCACAGTGC- CCACCAGGATACTGTCCCTCCCA GCTCCCACAGTGGGATGTATAAGTGGCACTTACACAGCGTCTGCACATCCACGTGGGCCAGGACGGCCCTTTTC- TCTGCAATCTGGGCCCAGCTGCC ATCAGGATCCTGAATCCACTCAGCGGCAGTGCAGTGGTAGGTGCCTGCGTCCCCTGCCTGGGCACCCCCTACTA- CCATGCGGTACCGATCGGTCCCT TCCTTGCCCAGACGAAGCTCCCCTGCAGCCAATCGCTCAGCATAGGGAGCTCCAGCCTCCACGGCCAAGTCTGA- CCGGATTCCCACCACTTCCTGCA GAGTTGACCGCCCAACTGGTGCCTCGGGCACAGATCGCCCAAAGGACACTGCCAGGTGTGTGTGCTTCTGTGTG- CTTGTCCTCGCCAGGCAGCCCAG TGCCAGCTCCTGCCCCTCATGCACCGTCATGCGTGGGGGTGAGGTTGGGGCCTGGCGGCCTCGGGGCCCTGGGG- GGGCAGCAGACACCTGGAGGACA TCTGGAAGAACTGGAGAGAACAGCTGGAGTGAGGGAGGGCTGGGAGCTGGCAGCCCTTGTTACTGTTTCCTGTG- TATAGCCTATCTCCCTAAATAAA CTGTGAGCTCCCAGAGGGCAAAGATCGCATGTTGTATTATTTCTTCTGTAACTCAGTGGTGCCAAGGGCAGTAC- TGGGCACAGCACAGGCGCTCAAT AAATACTTGTAGAATTTCATAGAACCAGCCCATCGCCTACTCACCCTTATGTTTGAGACTGACCTCTGTTTGAA- ATACTGAGAAAAGCGGCTCTTTC TTCTCAGAAGACAAAGAAACTTAAGAGAGTGAGAATGTCACATGGTCTAACTCCTTCCCTAACTCTACTCTCTT- TCCCAGATCTGGGTCCTGTACTG TCCAGGAGTAGAGGCTATTCAACCCAACAGTCTTCTTCGTTCTTGGGAATGGAAAGTGGACTGGACAACTTAAG- GACATTTCTTCTCCCAGGAGGGG TCTTAATATGATAAGATGAGCACTGGCCTGGGTGAGGAACTCTGGGTTTGAGTCCCACATCAGCCACTGAGTTA- TTGGGTGACTTTGTGCAAATCAC TTAACCTCTTTGGGCCTCAAGTTCCTTGGCTACAAAACCTAAGGGGCAACTAGATAGGTCACTTGTGGCCTTGA- CTTTCTGCCTTGAGAGGGTGTGT GGCTCCACCCCGTCCCAGGGCCCAGTACCTCTCAGCTCCACCTTGCCGCTGTAGCTGCCCAGGTAGCGGGTATC- AGTGGAGGGGGTGTGGCACTCAT AAATGCCGGCATCCTGGGCCTGCAGGCGGGCAATCTTGAGCACCACGGCATCACCTTGTAGGCGCTGCACCTGC- ACCTCACCCGCCACCACTCGGGA CTTGAAGACAGCATAGGAGAACTGGGTATCCTTGGTACTGACAATGCCCAGTGCAGTATCTGGGGCCTCGGGCC- TATACAGGAACCACTCGAAGTTC TGCTGGGCAGGGCCCTCATAGCCGGTCACATTGCAGGAGATGGAGACAGCTGTGCCAGCCACGCGGTACAAGGG- CCCCTCGGGGACCAGCACCTCCC GGGCCCAGCATCCCATTCCTGTAGGGAAAGGCAGAAGGAGTTGGAGATGCCTGGTTCCTCATTCCATGCCCTCT- GCCGCCACAAGCACCATTCTTGA TCTCTGCCTACAAAAGGAAAGGAGACCTGGGAAAGCTTGTCCACAGCTTGGACCCTGTTCTGAGAATAGGAAAG- GGATGCTGTGATATAAGACACCT GGATCTCAAGGAGGTGGCATGGGCCCAGGATTGCCTTGGCATCCAGATGCATCCCATTTCTGGCGGACTAGAAG- CAGAGCACCTGAAGGCAGAAAGG AGTACATCTGATTCCTGACCTAACCAGGCCTTGGTTCCAACTGAACCTTGATCTGTCCCTGCCACTCACCCACC- TCCATGTCTGCCATTCCTTCCTC AGCACCTGGCAAGGGGAGCCTTCTGGCTAGGGGACTCTGAGACTACATGTCCCTCTCCTTTGCTTGAGGGGAGC- TGGCAGTCTTGCTCAGAAGTGCT AGTTGGCTCAGCTGTGTCACCTGGGCGAGACAATGGAGCCAGTGACCCTAGCTGGAAAGGGCACAGGCCCAGTC- AGTTCTCACCACACAATGCCCTC CCCTCTCCAGCTGCGCCATGAGCTCACTGCTTCTCTCACCCCACAGGGCTGCCCAGGCAGCTGGGGCTTCTGGG- GCAAGATCCAGGCTCTGCCCTGG CCATTGGGGGCAGAAGATCCCCTCCTCCAGTGCCTGCCAACCTTCCGGGCTAGCCCAGCAGATACAGAAGGTGC- CTGCCCCAGTTCCTTAACAAAAG CCTTCATTTGCACATGGTATGCATTCATTTACATATATGGCTCTCTTTCTGTAGGGAGGCACTAAATCCCCAGC- TGCCCCTTCTCATCTCTCTCCCT TCAGAAAGGCCAAACCTCTCTTCTTCACCCTACTCCACCCCTATGCCCAACCCTACCCCAGCAGATACTCCTGG- CAGACTTAGAGGGCTTAGCTCCT CCCTTCTTTCCTTCCATAGCTCCCACTAGATAAGATCACAGAACCTCAATGTAAAGAGGGCTAGGCCACCCCTC- CCCACCTCTCCCAATTTTACAGA TGAGAAAGGTAAGGCAGGAAAAGTATAATATGTTAGCCAAGATCATGCTGTCCCTAGATGGCTTCCACACACTC- CTCCAGAGGGGCAAAACCAGAGA GGAAGATGGGGAAACTCCAAGGCCAGGCCTGAAGGGACTGACCTCACCAACCAGAGTGTCACTTTTAGGCCTCC- CAGGGGGATACCATGGACTTTCT GCAGGAGCTAGAGGAAAATGCCCAGGAGTCTGTGGTCAAACTCTACCCTCCAGCTTCTCTAGAACGGCTCCTCT- GAACTTCCCCACCCCTGCTTCTG GGCTCCTAGCCCCTTCCTTCATCCTCTGGCTGGGTCACAGGGAGAACTCATGGTCTGTTGTTAAGGGCACAGCT- GCCAGTCAGGAAGTGGGATTCCA GCACCATCCCCATGCCCAGCTGTGTGGCCTGGGATCCAGTCTCTTTCTGTCCTAGGCCTCAGTTTCCACACTGG- AGGAGAACTAAGAGCTCCAGCTC TGACCATGTGTGAGTGCGTATGTGACTCAGGAGAGCCCTGCCCCAGGCCAGGCAAGTTTCATAATCAGAGTGAC- GGTGGAGACAGCCAAGCTGACAC CTTCCCTGACTGCCTCAGGGCAGACTGCTCAGAAGGCCCCCTCCCATTTTCCTGGCTCCACAACTGCTGATGCT- TGGAGATGCCCATGGGAAAGTCA CCTCCACAGCCTTAGGAAATCAGTTGCCACACAGCTCTCTCTCCCCTCCTCTGTATCAGTCGCAGCAAGGAAAG- GGACAGCAAAGAGGCCTGCTTTG GAATCAGATCTGTGTTCAAATCCTAGCCCCAACACTCACTAAATGTGCTCTCTGGGGCAAGTTACTTCATTTTC- CTCATTTGTGAAATGAATGTAAG TGCCCACAGGCAGTGGGTGCTCAGACCTCTGCGTGCTCCTTTTTCAAACACAGGCCAGCACTTCCCCACCTCCC- TGGGCTCCTCCCTGCTCCATGCT GCCCACTGGGGAAAACACACCAAGTGCTAGGCAACCCAGGCCCCACAGCGCCTTCCTCTCTGTACATCCTCCTG- CCACCTGCCCAGGGACCAGGGAG AGGACTCATCCTAACCCTGCAGGGCCCAGGGACCTGCAGCAGGGGAAGGCTTTGCTTGGTGCCACTGTGGAGCT- CTGGTCTAGAAACAGGCAGCTGG GGCTACCTTCAGCCTCTGCCTTGACGACAGCAGCTCTGAAGTCACCATCCCCACCCCCACGCTTCACTCTCATT- TCAAGGGCTTCAGCCTCATCAAC ATCTGTACTGGCAGTTTCACTGTCTCCATGCCATACTCTTCCCCAGACCACCTCCTACAGGGAGCCCTCCAGTT- CAGGCCAAAAACAATTCCACTGT CATTATCCCCATGCATCCATGCAAGATTGGCCCAGAACACCCCACCATGAACACCCACCACAGCAGGCACAAGG- TGCTTGGAGATCCCAGGATCAGT CTCCATGGAACCTGGTTTCTCCTGAGGCAAGGAAGCTGGAACTAAGCGGTGTGAAAACTGATGGGTGGCTGCAG- AGCCAAGTGCCATTTGGGAGACA GGAAGAAGGGCAAAGAGGGACCCAACCCAGGGTGGAGATGGGGGTGAGAGAGGGAACTGCCCCCAGTTGATGAA- GTGCGTGGAGCGCAACTGGGAGA GACTTACTTCAAAGATCGTGGGCAGAACTGGCCTCTGGGCCTCCAGCCAACTCTGGGGCAATTATGAAGCTGGG- CAGGCACTGCCCTCGTAGGGCGG GCACCCAAGGCCAGGCCTGGAGCTGAGTGTGGGGCAGAAAGGAGTCGCAGCATTTGGTGCAGCGACCCCAGTAC-
GTGGGTATGCTAGCTGAGATGTG TGGCCTGCCCCGGGAGGCCGAGCAGTGCCTGGGGCAGCACCTTAGTGGGTCCTCTCTACGCCCCAGTCCCTGGC- TTAGAGCTGGGGAGCCTGCACTC TTCCCAAGACTGGCTCGGCGGACAGCCACAAAGCGCAGCTGGACGCCGACCCCGGGGAGGCTGGAGGTACCCCT- GACGGAGGAGGATGTGAGGAGCC CCGAAATGCTAGGGGGGTGCTGGATGGCAGGCACCTGCCCGGCAGGGCCGGGAACCGGAACGGGGGCCTGGCTT- ACCTAGCATTAGCAGCAGCAGCA GCGGCAGCGAAGGCGGCAGCAGCGTGGGCCTGAGGGCGCCCATCCTGCGCGGCCAGCTCTGGGGAGGCTCCGGG- GGATGGCGCGGGTTCTGGGGGGC CGGAAGGGTGGGGGGCGCATGCCCAGGTTGAGGGCAGGAAGCGGGGCAGCGAGGCGTGGGTGCGCCGAGCGAGC- TGAACTGGAGCTGCCGAATCCCC TCCCTCCGCCCCTCCCGCTGCTTTCCCTCCAGCCCTCGGCAGTTCTGAAACCATTCTCGCCCCGGCCCGCCCCG- GCACCGCCCCTTCCACCGCCCCG TCTAGGCCCGCCAGGACTACAGTCGGACTCCAATCCTGGCTCCTCCCCGGGCCCCGGCCCCGCCCCAGTCCCAA- GCCGCACCCCTTCCCCGTCCCCG CAGGGCTAACGTCAGCCTCCAATCCTGGCTCCGCCCTGGACCCCGGCCTCGCCCCGCCCCTGGCCCTGGCTCCG- CCCGAGGCCCCCGCAGGAGTGAG CTAACTGCACCTCTGCGCATCGAAATTCCCACCCACCCTCGCACAGAGCGCATTCCACCCCGCACCTGCCAGCC- TTTCCTGGAGAGTTGGGTGCAGG GTCCCTGGGATTGGCGAGGTGACTGTGACCACGCATTTAGAATTCAGTTATTTGCTCTGAGCCATAGTCCTCGC- TGCAAACCCTGCTGAAGTAGGGG TTGGCGGAAGCCAGGAGTTCCTGAATGCGAAGGGTTTGAGCTGAAGGGCGCTTCCAGGATCCAGAAGGTCACTG- GAGACCTGTTTTTCACCCCCTCA GAGGGCAAAACCAAAAGAAAAATGGATTAGGAGAGGGGG HUMAN SEQUENCE - mRNA ACATTTAGGAGAAACAGCGGTGTCTGCGGCTCCCACCCTTCGGGGGGCCCGTGGGGGGGGCGGTGTCAGGGGCA- TGGACGCCACCCCCCAGGGGTCT CTGCTGCCGGCTACTCTCCTCTCCACGTGCTCCCCTCCAGGACCCCCGACGCCGCCTAGGCGCCCAGCGACGCG- CGGCAGGTGGCAGCAGCTCGGGC CCCCGCCGCACTCCAGGCGCCCGCAGCGCTCGCCCTGACGCGGCCGCCATGGCGCAGGAGAACGCGGCCTTCTC- GCCCGGGCAGGAGGAGCCGCCGC GGCGCCGCGGCCGCCAGCGCTACGTGGAGAAGGATGGCCGGTGCAACGTGCAGCAGGGCAACGTGCGCGAGACA- TACCGCTACCTGACGGACCTGTT CACCACGCTGGTGGACCTGCAGTGGCGCCTCAGCCTGTTGTTCTTCGTCCTGGCCTACGCGCTCACCTGGCTCT- TCTTCGGCGCCATCTGGTGGCTG ATCGCCTACGGCCGCGGCGACCTGGAGCACCTGGAGGACACCGCGTGGACGCCGTGCGTCAACAACCTCAACGG- CTTCGTGGCCGCCTTCCTCTTCT CCATCGAGACCGAGACCACCATCGGCTACGGGCACCGCGTCATCACCGACCAGTGCCCCGAGGGCATCGTGCTG- CTGCTGCTGCAGGCCATCCTGGG CTCCATGGTGAACGCCTTCATGGTGGGCTGCATGTTCGTCAAGATCTCGCAGCCCAACAAGCGCGCAGCCACGC- TCGTCTTCTCCTCGCACGCCGTG GTGTCGCTGCGCGACGGGCGCCTCTGCCTCATGTTCCGCGTGGGCGACTTGCGCTCCTCACACATAGTGGAGGC- CTCCATCCGCGCCAAGCTCATCC GCTCGCGCCAGACGCTGGAGGGCGAGTTCATCCCGCTGCACCAGACCGACCTCAGCGTGGGCTTCGACACGGGA- GACGACCGCCTCTTCCTCGTCTC GCCGCTGGTTATCAGCCACGAGATCGACGCCGCCAGCCCCTTCTGGGAGGCGTCGCGCCGTGCCCTCGAGAGGG- ACGACTTCGAGATGGTCGTTATC CTCGAGGGCATGGTGGAAGCCACGGGAATGACATGCCAAGCTCGGAGCTCCTACCTGGTAGACGAGGTGCTGTG- GGGCCACCGCTTCACGTCAGTGC TGACTCTGGAGGACGGCTTCTACGAAGTGGACTATGCCAGCTTTCACGAGACTTTTGAGGTGCCCACACCTTCG- TGCAGTGCTCGAGAGCTGGCAGA GGCTGCCGCCCGCCTTGATGCCCATCTCTACTGGTCCATCCCCAGCCGGCTGGATGAGAAGGTGGAGGAGGAGG- GGGCGGGGGAGGGGGCGGGTGGG GAAGCTGGGGCTGACAAGGAGCAGAATGGCTGCCTGCCACCCCCAGAGAGTGAGTCCAAGGTGTGACCAGCTTC- CTCCAGACCCCTGTGGCAGACCG GGGGCCAGACACAGATACATGGGGAACTGCATATCGGAGGTGGTGGAGGAGGAGGAGGAGGAGGAAGGCAAAGC- CCCTGGAAATGTGCTAAAGTTGG AAAGTCCCCGTCCCCCAGAACCTCAAGTCTAGAAACCAGTATGGAAGGGAGGGGTCCTGATTTCAGGGAAATGG- AGGGTGGGGCCGGGTGAAAATGC CAGTCTGTGTTTGACCTTCACATTTGTTCATGAGTGGATGGATGGACAGAATGATGGACTTTTGGGGGTTGGAT- GGGAAGATGGTAGCAGATAAAGA CAGCTGACAGATACATAGATGGACCAGTAGACAACTGGTCCACTCAGGGCTGCCACTAACCTGTAGAACACCCC- TGTGCAAATTTTAAAAAGGAACC CTTTTCCTCCAGACAGATACAGCCCCAAACCAGGGTGCATGGCTTGGGGAGCAGAGTATAGGATGGATTGCAGT- CCCCAGTCACCTCTTCTGCCAGC CTCCCCACATATGGCACAACTGTCTAATGACACGGTAGGCCAAGCTGAAGTGAAGGAGAAAGGAGCCGGACCAA- GATGGGCACATGAGGAGGGTGCC CTCCTAGCTCCACCCTCACCAGGATGAAGGCGTGCAAGGGGCTCAGCAAGGTGTGAATGACCTTAGTCCGCAAG- TTCAGGGAAGCAGGCAGAGCGGG GAGGTGCCTGAGCTGGGGCCTGGAGAGGGGCCTGGGAAAGGAAAACCAGGGATAGCTATTTTCTTACAGTGGAG- TGAGATCTTACAGGTATCAGGCA CAGGCAGGAAGAGAGAGAGAGAGGTTCTGGGGAGGAAGGGCCAGGAGAGAGATCTAGAAAGTGGGTTCACTAGA- GCTGGGAAACAGGGAGCCCCTAG GAAAGCAGTGTGTCCTTGGGGCACAGTCATTCACATCACTGATTGGGTGCCATGTGGAGTGGACATTCAAAAAC- CTGGTTCCTGTCCTCAAAATAAG GGGCACCTGGGAAAACAGAGGAATCTACCTGTGGTGACTGAACGAGGGATAATTCAAACTGACAACCTGTGCAG- TCCCGTGGAGGGTAGGGGAGTGT GGGTGATCAGAAGGCTGGGGCCAGTGTAAGGCATAGGGAATATGTAAGTCAGGAGTTAGAAATCTCCAGTGTGC- GTTGGAATCACCTGGAGGGCTTG GTAAAACACAGATTTTTGGGCTCCACTCCAAGGGTTTCTGACCCAAGAGGTGGGGACCAAAACCATGCATTCCT- AAGAAGTCCCCAGGTCATGCTGC TGTTGCTGGACTGAGGACCACACTTTGAGAACCTGTGCTCTAAGTGAATACTTGGAAGTCGTTTCAGGACATGG- GGCATAGAAACTGAGGAGTAGCT GAGAGGAAGATGAAGAGAAGCTGAGAAGAAGCTGAGGATCCTCACAGGAGCAGACAGAGAAATGTGAAGGGTGG- GGTTTTATGTGTGGGAAAGGGAC CCGAAGCCCAGGCTGAAGAGTTTAACTTTGGGCCCAGAAACTCAACCATCAATGGAAACAGGGCAGTGACAAGT- GGAGGGGGTGTCTGGAAGCTGAG CAGGCCCGACAGAGAGATGAAG HUMAN SEQUENCE - CODING ATGGCGCAGGAGAACGCGGCCTTCTCGCCCGGGCAGGAGGAGCCGCCGCGGCGCCGCGGCCGCCAGCGCTACGT- GGAGAAGGATGGCCGGTGCAACG TGCAGCAGGGCAACGTGCGCGAGACATACCGCTACCTGACGGACCTGTTCACCACGCTGGTGGACCTGCAGTGG- CGCCTCAGCCTGTTGTTCTTCGT CCTGGCCTACGCGCTCACCTGGCTCTTCTTCGGCGCCATCTGGTGGCTGATCGCCTACGGCCGCGGCGACCTGG- AGCACCTGGAGGACACCGCGTGG ACGCCGTGCGTCAACAACCTCAACGGCTTCGTGGCCGCCTTCCTCTTCTCCATCGAGACCGAGACCACCATCGG- CTACGGGCACCGCGTCATCACCG ACCAGTGCCCCGAGGGCATCGTGCTGCTGCTGCTGCAGGCCATCCTGGGCTCCATGGTGAACGCCTTCATGGTG- GGCTGCATGTTCGTCAAGATCTC GCAGCCCAACAAGCGCGCAGCCACGCTCGTCTTCTCCTCGCACGCCGTGGTGTCGCTGCGCGACGGGCGCCTCT- GCCTCATGTTCCGCGTGGGCGAC TTGCGCTCCTCACACATAGTGGAGGCCTCCATCCGCGCCAAGCTCATCCGCTCGCGCCAGACGCTGGAGGGCGA- GTTCATCCCGCTGCACCAGACCG ACCTCAGCGTGGGCTTCGACACGGGAGACGACCGCCTCTTCCTCGTCTCGCCGCTGGTTATCAGCCACGAGATC- GACGCCGCCAGCCCCTTCTGGGA GGCGTCGCGCCGTGCCCTCGAGAGGGACGACTTCGAGATCGTCGTTATCCTCGAGGGCATGGTGGAAGCCACGG- GAATGACATGCCAAGCTCGGAGC TCCTACCTGGTAGACGAGGTGCTGTGGGGCCACCGCTTCACGTCAGTGCTGACTCTGGAGGACGGCTTCTACGA- AGTGGACTATGCCAGCTTTCACG AGACTTTTGAGGTGCCCACACCTTCGTGCAGTGCTCGAGAGCTGGCAGAGGCTGCCGCCCGCCTTGATGCCCAT- CTCTACTGGTCCATCCCCAGCCG GCTGGATGAGAAGGTGGAGGAGGAGGGGGCGGGGGAGGGGGCGGGTGGGGAAGCTGGGGCTGACAAGGAGCAGA- ATGGCTGCCTGCCACCCCCAGAG AGTGAGTCCAAGGTGTGA MOUSE NOMENCLATURE ICS GNM Kcnj9 Celera mCG4483 HUMAN NOMENCLATURE HGNC KCNJ9 Celera hCG39735
EXAMPLES
Example 1 mRNA Expression Analysis of KCNJ9 in Breast Cancer Samples
[0236]mRNA was prepared from breast cancer samples as by standard procedures as are known in the art. Gene expression was measures by quantitative PCR on the ABI 7900HT Sequence Detection System using the 5' nuclease (TaqMan) chemistry. This chemistry differs from standard PCR by the addition of a dual-labeled (reporter and quencher) fluorescent probe which anneals between the two PCR primers. The fluorescence of the reporter dye is quenched by the quencher being in close proximity. During thermal cycling, the 5' nuclease activity of Taq DNA polymerase cleaves the annealed probe and liberates the reporter and quencher dyes. An increase in fluorescence is seen, and the cycle number in which the fluorescence increases above background is related to the starting template concentration in a log-linear fashion.
[0237]For data analysis, expression level of the target gene was normalized with the expression level of a house keeping gene. The mean level of expression of the housekeeping gene was subtracted from the mean expression level of the target gene. Standard deviation was then determined. In addition, the expression level of the target gene in cancer tissue is compared with the expression level of the target gene in normal tissue.
[0238]As shown in FIG. 1, KCNJ9 was up-regulated in approximately 12% of breast cancer samples examined.
Sequence CWU
1
7126642DNAMus Musculusmisc_feature(1)...(26642)n = A,T,C or G 1nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 120nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 180nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 240nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360nnncctcatg
aatgctgaga ctaaaggtgt gcatcacccc tgcccaattt caaaatagtg 420acccaaggga
agaccagatt acaaggtgct gcactacaaa gtgagaaaat gttaacggtt 480accctttaaa
aactttgctt agagggaaaa aaaaaacccc acaatcataa ccaaagcaat 540ggaccaggaa
ctattttcct gcctgttttg tcttttcaaa tttctgtcat cttctgctcc 600tagagaggaa
cggctacagt aagatggtct gaagacctgg tagttttttt tttttttttt 660tttttaagat
ttatttattt attatatgta agtacatgta agtaagtaca ttgtagctgt 720cctcagatac
tccagaagag ggcatcagat ttcgttacgg atggttgtga gccaccatgt 780ggttgctggg
atttgaactc gggacctttg gaaaagcagt cggtgctctt aaccactgag 840ccatctcgcc
agcccagacc tggtagttta agcctgcaat ctcagctgtt tggggagggg 900aagcaggagg
gttgcaagct caaagcctga gctacagaat gagttcaaag ccagtgtgaa 960taacttagca
gggctcacag tcttgacatt cagagatggg gaagattatg gggctgagct 1020cagaccacaa
tataaaatga agaaggaaca cagaggagag aagccaagaa ctgtcggggt 1080ttatgaaatc
attacaagac acaagaattt attatttttc cagaattgtt acccaagcat 1140ttggcatcca
tcgccaccta catgtcagtg tccacctgga cagaaatctc aaacttagtc 1200cagcgtagaa
catcttaccc acaggagcgc tcctcatggg actatgtcac catcatccaa 1260ctagaaacac
agcagtcatc tcagcctcct tagtcttcct tacagcagca actccatcct 1320ctaaccaaag
catctcccac tgagcacgcc ctcctgcccc cctctctctc tctcccttta 1380tcgctgctgc
agtctacagc agatgcacct ctcagcaggg atcctggagc agccatctag 1440tgccttatcc
cctccagtct ttctacactc caataatgct tcaggtcact aactccttta 1500tgtaaaaaca
attaaggctc agcaagatgg ctcagagggg taaaggcaac ttgctgccaa 1560acttgatgac
ctggggtcaa tctccagtac tgatgtggta ggagagactc aactaccaag 1620agttatcctc
tgacctctac atgtgtgttg tggtacaccc acaaacacag agagagagag 1680agagagagag
agagagagag acagacagac agacagacag acagacagac agagacacac 1740acagagagaa
atgtaacgtt tagagaagaa tccatccata ttctttagca cagaacagaa 1800ggcacattaa
ttataacctg ggcatcctgc cctgtcttcc tcacatccaa ctctatagct 1860gcttcctcct
ctaacaccca aggttgttaa gtcttgtgtc cccttctgta tcttgctcct 1920tgttctttgg
tcacacagtg accaaagtca ctgagtgttg tgcaaacctc ttcttcttga 1980ctcctgtatc
tctctggagc tctacttagg ctccagtacc tgcaagggat taatgccctc 2040acatgacagg
ccccagacag aacccatcct ctttccctct caccaaggtt gggaatgctc 2100acagctccct
gatttctgtg taactcctgt caagcagact gaaacaccga cattacatct 2160tgctctttat
gcttgcctat gtcccattct gtgtcatgac aattcagcca ccaagttctg 2220ttaactctcc
cttggttata tttctctagg atacacattt tcatttctat ggccagaatc 2280ataaaattac
cactagccca ggacctgacc catccctcac ccctctttcc agtatcaaag 2340ggagacaaac
tgtttttatt aaagatgtac tgtatttaaa aaaacctaga atcaaaactt 2400tgaacaaagt
ggggtgtgat ggtatacacc tttaatccca gcacttggga ggcagaggca 2460ggtggatttc
tgagttcaag gccagcctgg tctacaaagt gagttccagg acagccaggt 2520ctacacagag
aaatcctgtc tcgaaaaaac caaccaacca aataaataaa taaataaata 2580aataaataaa
taaataaata caaggtctct ggataaactc cttccaaata gaaatgagaa 2640gccatctggt
gaagctcagt gtgagggtga ggtggcacga gatggaactg ggcaattgga 2700aagagttagg
tattgtagag ccacaggaga gcaggactgt ggtgacttct gtggccctgt 2760gatgttctca
ctcaagagtg acttacatca ggattccatt cttaaataag cacactttat 2820tagcaactat
aactctgtat acattgtgtt tgcttttaat atttaacttt ttgttttcca 2880aaaagagttc
ctgaaacata caacaagcag aaattgtcat tgctgaagga tgcttagcat 2940gctcatgttt
ctgagtgttt actaggcgtg ataaatttga cttttcttgt tttctttcag 3000ttcactctgt
ctgatgctcc tgccccggtc tcctaaatgc agggattata ggtgtgcacc 3060gccacaccta
actgtgtaca gtagatcgta agatgggaaa tcccagagtc agggacctta 3120ggtggctgac
ctatacacag tgacatgccc aggaagtgtt aaatctggca tttgaatcca 3180cctgtttgac
cccagagttt gtcaaagggt aatagtacag cgctcttgca tgacttaaag 3240agatgctcat
tttcccaaga gaaccaagag gttctagtgg ccaaatgtca gtatgaataa 3300atctgctgag
atgcgctgtg cagcgtccgt cgaccttaca ggaggacaga gcaatccttt 3360tcctttttga
ttcatcgctc ctttcagact tgatcctctc accacagatc tctttccttc 3420cacttcctca
ttcaaaatgg ggtcagttcc ccctcagaac aaaagaggaa catgaggcga 3480agaccctttg
cagagggaaa atccacagct gggcgtaggc cgagggagct ttcgctggga 3540gaagcaggtg
agttcggatg aagggaagca actgagagag gcaaggcaga tcctcagacg 3600gggcgggttg
gggggggggc gactcggaga gggagttttc ggggagtcat cagagctggc 3660caggaagaac
taggcatgaa catgagtccc agggactccg agggacacat ttctgcttag 3720gtcccacagt
attaacacgg tccactaaaa gcagatacgc tcagcaggat gagcggccac 3780agaggagagc
ctatcagtac tcggtttagt cattaccttt taatacacat gatttatata 3840agcctgtatg
tgtataagac ttaagttata aatggctaat tacattacag aaggactaca 3900gaaggcagag
agagggaggg aggggagggc aagggtgggg aggggaggga aaggaagatg 3960ctctttacct
ataaggttta tctagtatct ttctaattgg tccttttagt ggcaattctg 4020ttaacattca
aatacaccat ggagagggaa gaacagaaaa cccccagatg cctggaactg 4080gggaagctgt
cttaaccctg acctctcttg ggatgctctt ctcatctata aactaatgat 4140tactttagat
cacttctgaa tgaccatggt taagtcctgg tctaactcta tccagccccg 4200tagacctggt
agacaagatg gacctgtgcg taactcttct agggctgatt ccacatggaa 4260tttacctact
tttatttaga gatgaggtct cactgtgtcc ctctggatga gctggaactc 4320accacacaca
ccagggtggc ctcagactca gagatttact tgccagtgct tctcaaatgt 4380tggggtaaaa
agcgtaagcc accacccaca gaccccatga attcatatca attgttattt 4440gaactaactt
gaccttccta ctcccctcag ctcacatcct caaccgtccc tgccttcccc 4500tccagacttc
ctcccccatt tccacgcttt tgctcaagaa gtctcatgat ctcgttcaag 4560gaagctctcc
caggttggct gacctcatag ctggcaacaa aggcaactac tgctaggggt 4620gaacacaagg
ctacagtgca ctcatcctgc acccaaactc agaattgcac caaagtgtgt 4680gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtctgtgtct 4740gtgtctgtgt
ctgtgtctgt gtgtctgtgt gtctctgtgt gtgtctgtgt ctgtgtttgc 4800tttcactgtg
tatgcatatg tggagaccag agacgaatga ggagatcata gacatacact 4860gtcacaccca
gcctttctgt ggatgctggg gatccaaact caggtcccca gatccatgca 4920gcaagtcctt
tgcccactag gctgtctccc gagctctgca cctaggctct ttataggacc 4980agcagtgtgg
cctcactgtc ctctatttcc aatctgtgtt tattacaact ccgctgacat 5040attggggttg
atttcttgga gggatgcttt tattctcttg gtgaaatatt tttctgtgca 5100ctgatggctt
gtgaaatttt cttctctgtt gcctcagttc aagccagacg aacaaggagc 5160tgagattaag
cttagtaagt aaagcccagg acctggagga tctggaaact gggtgaaaga 5220gttgtcctct
gttggctagg ttaggttcag ggcagccagg atggagtcag aggggtggct 5280gacaacaccc
aggggccact gtcagctctg tgactttccc tccagaaaaa ggggccagtt 5340ctgtgcaaac
atgttcttgt ccaggagttt ggtttcttct ctctgagcac ctggcacagt 5400ggcaccaatg
tgagcagtca cttggcaggg cagagaaaag caagctagca gtccccaggc 5460tcaggtgaca
gagccaggcc caggagacag ggatattgac tggggcttta acagcactat 5520tgatgccaat
ctcgggcaaa aacctgatat ttccacttgg aataacaaga aacagccaag 5580aggattggag
agaggtcagt ggacaaggag agccctctgc aggtcgtgct gggtgattcc 5640agaacagaag
agggcagccc ctgctggaca gggtctcctg agatgatggt gatggtgacg 5700gtgatggtaa
tagtgatggt gacagggaca atgacagtag tagtggctag gagaaggaaa 5760aagaagaaag
agaaaaacac aatgtcaggc tttaaataaa taatcctcat gaagtagata 5820ctatttattg
tggttttgat atgaaacacc cctcccccaa gggctcgggt attggagatt 5880tgagccccag
cgtgtggtgc tattgagagg tgactttgtt gatggcgact ttgtttgatg 5940aggagataag
aggtggactg taaggaggtg aaagctgtct gaggaagtag gtcaccagtg 6000gtgtgctctc
aaaggtgggt ctcaaccttc ccaatgctgt gacgcttcat ttaacacagt 6060tccttatgtt
gtggtggccc ccccaatcat aaaattagtt ttgttgctgc ttcataactg 6120taattttact
actgttataa atcataatgt aaatattttt ggagcttaga ggcttgccaa 6180aggggtcacg
acccacaggt tgagaaccac tgctctagaa ggaagtcacc tcctcttctc 6240tccttgtcat
tctcttttct tcctctccct tacactcccc ttcttctctt ctctctccct 6300actcctcacc
tctccactct ccatgaaatc tgatttcccc tgctatctac cacaatgccg 6360gatctcatat
tcccaagaag aatggagaca aacaaccctg gactggattc ctcctctttt 6420aagtgtgact
tgggtgttct gttacagcaa tgaaaagcta gcaatataag atggctagtc 6480tcatctctta
gatttaaaaa actaacattt tccaaacata gtggctcatg tctgtagaca 6540cagagccagg
gaagcagagg cagaaggatc cactgcaggt ccaaggctgg cctggactat 6600gtaacaagag
agagagagag agagagagag agagtagaga gagagagagg agagattgaa 6660ggaaagaaag
attgaagaaa agaaagatga aagaaagaaa gaaagaaaga aagattgaag 6720gaaagaaaga
gagatttaaa gaaagaaaaa agaaagatga aagaaagaaa agggaagaaa 6780gattgaagga
aagaaaagaa cggaaggaag gaaagaagga aggaaggaag gagtgcaggg 6840ggaggaggga
agaaaagagt ggagggggag gagggagaaa agaaagaaaa ggagactata 6900tgaagctatt
tgctcaaagc catgcatctt ctatcagaga gtagaatttg aactcaagtc 6960attgcctctg
aagcttgtat taccccacac acctgtcata gctcgtgagc acatttcaga 7020aacttctagt
cttctattgt gctgtttctt cctgttcttt ctagttatgt attcttgcag 7080tgttaaggct
taggggattg gatataaaat atcttgtgca taacaatatt ggcaatagta 7140ataacaccag
cttaaattta ttttttatag ctttagtaat ttaatttatg tatatgagtt 7200catggtagct
gtcttcagac acaccagaag agggcatcag atcctattac agatggttgt 7260gagccaccat
gtggttgctg ggaattgaac tcaggactac tggaagaaca gccagtgctc 7320ttaactgctg
caaaatggta cagttactct ggaagacagt ttggcagtca cctgaaaaac 7380taaacatact
ctttccatat gatattgcaa ccatactcct tggtatttac cgcaccccca 7440aaagctgaaa
acttgtctaa ataaaaaccc tgcacacaga tgtttgtagc aactttattt 7500ggaatcggca
aaaactggaa atgaaatgac tttcagtggc tcaatggaca aatgaattgt 7560ggtactttcc
tggccgtgga ccatcattca gtaccaaaat gagatgagct gtggagctaa 7620aaaagacatg
aagcaacctt aaatgcacaa gtggaagaag ccaatccaag gagctgcata 7680ctgtataatt
ccaaccccat ggcatcctgg aaaaggcaga accatggaaa caggttttta 7740aaaaatcaga
gattgccaaa ggctaagggg agagtggatg gctgggggca gcagagagga 7800aagcacccca
caaccatcat ggcggataca catcctcgtg gccgttctgg gtttacagca 7860agagaaacca
caccaagaga aagtcctaat gtgaactaga aaccagtgat catgctgtgc 7920caagttagat
ttgtaagtcg taaacaagct actattctca ctggagatgt ctagagtaga 7980ggagactgtg
tatgccaggc agaaggcatg tggaaactct tagtgccttc tctcagttta 8040tatgtgtttg
tgtgtatgta tacatctttg tgtgtatgtg tgtgcatgta cacgtgcgta 8100cacacagaag
tctgaagtcg atgttttcct atatcactct ccaccttagt ttttcagaca 8160gggtccctca
tgaaacctgg aattcaccag tttgttgggg ctaactggcc agtgagctct 8220gggcatcctc
atgtctctgc cttctcagct gggattccac gtgtttgcca ccacatcctg 8280catttacacg
ggtgctgaga acccaagctc aggtcctcat cagtagggca agcacttaac 8340tgactgggcc
atcttcccag gctcttctct tgctgtacaa ttaaaagtat tctttgaaaa 8400agtctaatat
gcatgcctat atttccagca ccgagtaagt ggagctaacc tgggctagac 8460agtaagaccc
ggtcttgggg gtggggaaca cctaacaaaa aaataaaaac aaaacaaaac 8520aaaacaaaaa
ccaaaaacat taaatgaaga gccagggcag tgacaagaca cgtgactcct 8580caatctctgt
ccaactctgg aattcaatag gctacttttt ctgttttcct catccataaa 8640tagaaaaagg
gataactgtc tcacaggatt gtcacagaaa ttaaatgaga tgctgctgga 8700tggattagca
gtaggagcat gtagcagcag acctgtgcaa ctctgtgtct ttccactgat 8760ggcatcatag
gctactgctg ggcaaggacc tattcatttc ataatcgcct ctacctagcc 8820cagtatgtgg
tgtttgagcc ccctgagtct gctgggttga tggtaagaac tagcctagac 8880ttctctctct
ctctgttgga catttgaggg ttttctcaac tttttgctat gagcaaagta 8940catctcaaaa
cccttttatt tacatcacct aatttgatct gcatcccagg tgaagccagc 9000agaagagggc
tgtttgccca cgcccacact ctgagacaga cagaatcact atggctcaga 9060gaagtgaagg
gacctcttcg ggtcacaggt atatcagtga tggtgatgac gatggcggag 9120cctctggccc
tgcttctcta gcccctacct ctgcagacct ttttctctct gcctgctgcc 9180ttctgcatca
gaggtctctt aaaaaattgc agccttgtca cgctgggcct ggtccttctg 9240tccgctgtct
ggagggcagc acctttgccc agtggtccct gctggggatt gtgaactgca 9300aactcccaga
tggcctctga aatcaaatat tttatttcca atgcctctat tttcccagaa 9360tgaggagcac
accagttccc ccacacacac acttgctttc gtccctataa agaggtgagg 9420agatgactct
ccgtgtccag gaggaaggac tttggctaaa aatagctgtg gcgtgtggat 9480tagccagagt
ggtacccagg actgggaaag ggagggggac gctgtggagc tgtagccaga 9540ctggttgcca
tagaaacgag agaggagcag gggaacctgg gaagtgggga tgacacagat 9600accaagtcct
agtctgagct gccgttacat tcaggagaaa cagcagtgtc ggcggctccc 9660aatctcagag
ggaacctagg gtactggggg agatggtgtc agggacatgg acgccaaccc 9720ccaagggtct
ctgctgctgg ctactcttct ctccaggctc tgtgagttga gttgtgggac 9780ttggggtttg
ggcccctatt tctgagccaa gaggggtttg ggtggagctg ctcccagagg 9840gacttctccc
cgacagaccc ctttccaaaa gataagcccc ctgtactggc cagcgctctc 9900tagagggagg
tggagtactc caagataatg tggtgctcgg atcttactga aaggggtcac 9960agcatgccca
agaactgtgg tcggaagaac tggagttatt tggagggaag aggaagaaat 10020gaagacgttg
ctcttcaggt ggtggacact gcacaccttt cctgtcccat gaagaagaga 10080gcttttctcg
agatggcaat ggctaggatg tcatcagtag gctccctggg cagtcgtgtt 10140ctgggaatga
tcagacactg ggaatccttc cccattcctg gccgtagatg gaggtcagat 10200caccttagac
cctacgaaga ctgtctagaa gcccacctga agttaatact aggatgaaag 10260agacctgggg
tctcgaggca ctgaaaactt acagatgagg tgcagaggac atcctgggct 10320gcagagaggg
aaaaaacaag cctgcttgct gttgggggag gggaagatct taatctgcca 10380ttgccgaagt
gttcccaggt catgtctcct gacttccatg gaaaataagt gtgtggggtt 10440acaaaccatc
tttttggggt tttttccttg tgcctttctt taacatacac acaccctcca 10500aaggtctgct
ggctacagaa cacttggctc caaagtttaa aaatggaatg tcgggtttgt 10560gggtatatat
tcatgcagtt tctccctagg atctggtcaa acatccaaac catctgagat 10620ccttatgtca
catttctgcc cccacagggc cacctgctct ccccacttcc ccagccttcc 10680tgccccaccc
ctcaccctga atgggaggag atggcaaatc ccaggaaaga gaaaggaagg 10740ttgatgagtc
ttaatcctta ttctacagac ttctgttcat acggtccata tctcctaggg 10800gaccctgaaa
gcctaggaac cgactctggc catccatctc tccgggaaga ttataaccca 10860gagtgcttct
caggggggaa gaatttgaag caaaaccagg tgggttttgc ttggaatctg 10920ggctttgtgt
ggaatgtggg ctttgggaca tatggcagga gtgggtgggg ttgctggtag 10980ggtagtaaat
gcaaatcagg aaattggtag ggggggtcga tgtgggtgtt tggtgtttcg 11040attggtctga
tttcttatct cttagaagaa tacgaatctg agagatacta gactagcgta 11100actctggatg
gcctggcgcc tccttcatcc ttgccgtggg cagttgagct cacgcgtggc 11160ccccaatctc
ctattgccca cccttttcag cgtgtctcct gtgggaaaga gccctggcgg 11220gaaatgggct
ggtatcagag catcagtgac cacggtgaag cagttagaat tgccagtggg 11280aagttcccaa
tgctgaggac atccaacctt tgcacactgg aggtttttgt gcacagtctg 11340cattgctttc
tccttgggaa gtctggggtg gaggggaaat gtagcaggag aaagagtgag 11400gccagggaga
acaccgaggg aacagtcttc aggtggggct tctggcagga tgctgaagag 11460tgctggggga
agggataatt gccagggaaa gggctgtgga agtcctcatc gcagggaggg 11520ctttgcatgg
agaaggaact gccaagaagt ctacctcttc agtaccctaa atgtctgatc 11580cggggtgcct
gtgagttgct acatacacca gcttgaggta gtgacgctga gatctgtgac 11640atcgagatgg
ctaatgcctc ttttcttact gaacttcgac acccagtctg tgctctttat 11700cctgtgtaat
ctgtacaact ctctctctct ctctctctct ctctctctct ctctcataat 11760tctttattct
tttttaaaaa gatttattta cttaatgtat atgattacac tgccgctgcc 11820ttcagacaca
tcagaagagg gcataagatc ccattacaga ttgttgtaag ccaccatgtg 11880gttcctggga
attgaactca gaacctctct ggaagtgcag gcagcgctct taaccccgct 11940gagtcacctc
tccagccata caactttttc ttaaccattg ttttatttta tgtaatagtt 12000tgccctcatg
tacgtctgtg cattaccctc ggaggccagc agagtgcgag ttacagccgg 12060ttgtgagccg
acttgtgggt gctgggaatc gaaatcagat ccgctggaag agcaaccagt 12120gaatcatttg
agccatctcc ccagcacttg tgccccaact ttctgagatt tatgggatgt 12180tagggattat
cgttcccaat ccaccagtgg ggaaaaacta aggctaaaga gacaggaagg 12240gagattgtct
cacagcattg gccctgagtt cggggcagat ccatcaactc ggcacacctt 12300tattaagacc
ccgcaggatc cccgctgcgg ccgccatggc gcaggagaac gccgctttct 12360ctcccgggtc
ggaggagccg ccacgccgcc gcggtcgcca gcgctacgtg gagaaggacg 12420gtcgctgtaa
cgtgcagcag ggcaacgtcc gcgagaccta ccgctacctg accgacctgt 12480tcaccacgct
ggtggacctg cagtggcgcc tcagcctgct cttcttcgtg ctcgcctacg 12540cgctcacttg
gctcttcttc ggcgccatct ggtggctcat cgcctacggc cgcggcgacc 12600tggagcacct
ggaggacacc gcgtggaccc cgtgcgtcaa caacctcaac ggcttcgtgg 12660ccgccttcct
cttctccatc gagacggaga ccaccatcgg ctatgggcac cgcgtcatca 12720ccgaccagtg
tcccgagggc atcgtgctgc tgctgctgca ggctatcctg ggctccatgg 12780tgaacgcttt
catggtgggc tgcatgttcg tcaagatctc gcagcccaac aagcgcgccg 12840ccactctcgt
cttctcctcg cacgccgtgg tgtctctgcg cgacgggcgc ctctgtctca 12900tgtttcgcgt
gggcgacctg cgatcctcac acatcgtcga ggcctccatc cgagccaagc 12960tcatccgctc
ccgtcagacg ctcgagggcg agttcatccc tttgcaccag accgacctca 13020gcgtgggctt
tgacacgggg gacgaccgcc tctttctcgt ctcacctctc gtcatcagcc 13080acgaaatcga
tgccgccagc cccttctggg aggcatcgcg ccgcgccctc gagagggacg 13140acttcgagat
cgtagtcatt ctcgagggca tggtggaggc cacgggtgcg ggcaggctgg 13200aggatgggag
cagggatgca ggacaagggc aagaaaagca gccaggggag gcgcagaaag 13260atggacagag
aatggagtgt agggtgacag gcctgagggg tagcgggggc cggggagagg 13320acgggagatg
acagggatgg acagggtgac tttgcagagt caagaaaagc ttggaagagg 13380tctatgaaat
ggcactagct tgaggccctg acctgacagc tatgtcactt tgaactacat 13440tttacatctc
tgaattcatt taagcccagc aaagctcccc tggaggttac ttttgactgt 13500gctcggtttt
cagagaatga gtagccccaa agaaaggtcc cataaatagc ccgctgtcac 13560aagccaataa
atagcacagc ctgggttgaa cataggacat ctatcttcag tgtttcctgg 13620tacagtgttg
ggatgaaggt taagtgcagg gttcttgaag cccagaggtc catagctctg 13680gaatttaact
gacctaagta aaagggaggt aggtaggaaa aagactagta ctggagcaaa 13740aacaggtcct
tgaagaggtc ctagccgtca gggagcataa ggaagacgca ggtgaaccaa 13800gaggccacta
ggaggagctg cggagctgct acggacaggc tagctccctg ctgctagcct 13860tgaaacctgg
ctcctgggcc tagacaaaaa catcatcttc tccatggcca cctcaggtct 13920tcccactccc
ctctcctcct tcactccaac taggctggtt ctagcccatg cccattccac 13980actgctccct
ctgtctctgc gctgtccctc tctctgacac aatctcggac aggtttctat 14040cagggacttt
ttcatctgcc ttctcttccc cctctgccac tgcctccact ttgcacctaa 14100ccctactccc
ccaagcccta cctctgcttc tcaggccttc tccctgcaga ggccccggtg 14160gcctctcttt
ccctacgatc cctgatacat cttattccag ctttgccaaa gaataccaat 14220gaccccaaga
tgtctcaggg ccagacttcc gatgtcagag ccggtctctg attagtgaat 14280gcttactcct
ctgtttttga gatggattcc ggtttgggaa gattctgagg taggaacaaa 14340atgatctgcc
ccgaggggag ggtgcacaaa cccaacagag aagacaggac acaggctcag 14400ggcaagaact
gggaaggggc agtgtaaagg acatggggat gggagcttgc ttgacttttc 14460tagagataag
gctgggaagg atggtagtat tttgggattc aaactgcttt tgaaaagcaa 14520gaataatgag
ccaaaaccca acatgatgac atttaagggg aataaatata aaattctaca 14580tttaggcttt
aaaaaaatca cttatgtaag cacagcatgg aaaggctccg gtggagaaag 14640aactgggggt
tttagttggc cactggcttt gctgcagcaa cgtgatgcag cttccaaagg 14700cgtttatgta
atgtaatcat gggcccgctt caccaaagca tctgggcgag aagcaagaga 14760tagtaagcct
tcttttatgc acagataagg ccacagttga aaaagcactt cagatgagcc 14820cttacctggg
cctggtggcc attctgattt gcaatgaaga ttgtaagctt tgggggagtc 14880agatgaagta
agaaatggcc atgagtgttc aatctgagga agagaagatg taagggaacc 14940ccatatttac
actcaagggg gtgtcaggtg gtaagggaat ggaaccaggg gccacgggtc 15000ctaggagaca
gattttagtt tatgtaagag aaaacccaga gccaaagaga tgtctcagct 15060tgcaaccacg
cctgactact gacctgagtt gaattaccag gtctcacatt ggggagtcaa 15120ctgtctcccc
aagttgtcct ctgacctcca catacataca tatgcacgca tatagacaca 15180taaatgtaaa
acacatttgt aaagacgatt ggcacgttgc acaaaggact ggacttttaa 15240tgagatggtg
agctttcaat cctggggtgt aatcagttca gcccattgtc tgggaatgct 15300tgggggtggg
tggaggcggc tctgtgggaa acaggaaggt taggcttaag gttaagcttc 15360tcaatggaga
gtaggggaaa acataggctg gcagatagag aagagggcta actaaaaaga 15420gaggtgggac
tctcagagag agaagagggt tgtgggatga cagacaggag aaggaatcct 15480ctgtcagggg
cccctttgac tgatgccgct tctcctcccc ccacccccca ggaatgacgt 15540gccaagctcg
aagctcgtac ctggtggatg aagtgttgtg gggccaccgg ttcacatccg 15600tgctcaccct
ggaggatggt ttctatgagg tggactacgc cagcttccac gaaacctttg 15660aggtgcccac
accctcgtgc agtgctcggg aactggcaga agccgcggcc cgccttgatg 15720cccatctcta
ctggtccatc cccagcaggc tggatgagaa ggtggaggaa gaaggggctg 15780gggagggggc
aggtgcggga gatggagctg acaaggagca caatggctgc ctgccacccc 15840cagagagtga
gtccaaggtg tgactggttt cctcccaccc cctgtggcag accagggggc 15900cggactcagg
tacacagaag ctgcgagtgg aggtggaaga agaggaggca ggcagtgtcc 15960cgaggaacag
ctaaagttgg gagaggcccg ctgagtccag gatcgagtag ggaaggctga 16020ggtcctggtt
tgaagagaga gggttgcagg gcggggtgag agaacatgtc agtctgtctg 16080tgtttgacct
tcacatcggt tcatgggtgg atggatggac agaaggatgg gctcatgggg 16140gttgatcggg
aaggtggagc agatagagac agccaatgga taatcgctca ggtggtaagt 16200ggcttggcag
tcgatgatcg tcacctgcag cacacctttg tgagaaatcc atgggcatcc 16260ttttcttcca
gatataggta gcctcaaacc agggagcgtg gcttagggag caggctgtca 16320ggtggactac
cacccccact cacctcccct caactggcct ccctatgtgt gacacgcctg 16380cctaactaga
gaagagagca ctgggtagag gtgggcacag gtgtgggtgc cctccccagc 16440atcactgtcc
catggcgaga ggtcagaaag gcaaacaagc aatgggggta gatgctgagc 16500agggaggggc
cctgaagcag gacctgggga cagccaagga caactatttt gtgagagagg 16560aatgaaacct
tgcaggtcct gccacagaag caagaagcag aggaaaggcc atggagagac 16620ttaataaagg
gttttacaag ggtacctgga tcccaggggg aagtagttta tccttggggc 16680acagtggcag
ggctcattca gaacggtgag taagtgtcag gtgtgatatt caaagacctg 16740gttcttaaca
cgagagcaca gcgaaggtgg aggtcagaaa taactcccag ccactgaagg 16800aagtatggct
tcagtctgga gagctcagaa aagactcgac cctaggagcc cacacaagcg 16860gttatagcca
caagtgagag ggcattaggg acaggaagct aaggattgag taaggcagtg 16920gggaatggtg
ggagccagca gttacaaagc tttactcacc tggatgggct tgttaaaaca 16980cagattacca
gccccactcc ctgcattctg actcagtagg tccgggacgg aaaccaaaaa 17040aaaaaaaaaa
aaatctgcac gtctaactag ttcccagacc taacaggttc ccagatcgcg 17100gtgacactgt
ctgtctgggg actgcacttg ggtgaagcat ctaagcggaa gagaagctgg 17160aggaactgaa
aagcacccca ggttcctcaa ggaacagaga aacaagaagg gaaatgttgg 17220ggagagggga
cccaggtcca gactcgaagg gcttaactct gggtccaaga aacgtcattg 17280gtaactggcc
agtggcaccc gagagggcaa cagagatagg agaaggccat ttagggaccc 17340ccaaggaggc
agtggggggt ctgtagctga attggcctta ccacaaaaga ccaactctct 17400taagagactc
acaaggcaag actgactagg ggagaaaatg gagcctgtac ctacaggtgt 17460ctgctgtctg
ccacctgtcc tcccaggaca gggcaccctg gagacacatt ccacctccac 17520tgcatccttg
tcttgcccca gtcatcttgg gatggttgag gggacagcaa cagcatggca 17580atggacctga
ggctggcccc cctggagcta agtgtagccc aagtgagcac gtaacctgat 17640aggactggct
cagactctgg ccctggctat acccatccct gccctcgaat aaaagtctgc 17700tgctctgtcc
caggctaaga agccagcatc caatggggca tcgaggcctc cctcccagtg 17760cccagctcag
agtgggtcca cgcagagagg actcaagctg cctgttgcct ctccccttcc 17820atctagcaat
ggccacaggt ttcgggacca gctgggtcac tctcaaagat gaggtccacg 17880cacatgaacc
tgctgggatc ccacgaacac atattggacc tgagcacagg gactgagcag 17940ggtttgaatt
cagagaaaat cgaggagtct agacaagagg ggtggggtgc ttggtatccg 18000cacacgaagc
aatggaatgg agacattgaa gctgttcctg gaggtcactc agggcaccgc 18060tgtccagggc
acagccagga gacctgtgtt ctagcaccaa tgctgattgt cactaattac 18120ctctatgact
ctcagcaaga cctattactt ctctgggcct cagcttcctt atctgttaaa 18180aaaaaatgat
atgttggcaa actcaataat gctcaataaa ctttcaacta ctgaatgaaa 18240aaaggtagac
tggatgccgc tcaaagtatt aggacagctg aggctcttag gaccggagaa 18300ccctttaggc
ggggagttgc ggctagccag caggcaagtc ctggcatcag atgtaagcag 18360atgaggcggc
tcttgtgtac acagaggaca caggctctcc caactgctgc tgtccttaag 18420taggcagccg
tgttctgaag ctcctattcg gctgctgtca gagaataatt aagggcagga 18480ggaaaaagac
tgaggcccca gggcctgtgg gaggagtctg gtccaagact agttcaacca 18540ggagaaatgg
accagaggag ggtgtgcccc agtctggaga gctcagaaaa gactcgtccc 18600ttggagctct
gtgaaagggg caaagctcag ctggaactca cccctcctct tcctaggtcc 18660cccttcccaa
atagaagccc cattaggact tggctcagca cagacatttt ggacaacaga 18720tgggaccccg
gcatcccctc atgcagttgg tgggtaacaa ggcccacgaa gggacagatg 18780gtgtttatgg
tgggaagaga ggcccgggtt gtccagcaac caccctacta ccaccccacc 18840cccacccccg
atgctgcctt ttatagcttc accgcaagag aagacacaac aggcctcgat 18900tttacaaaac
cagtttattc acattttaga aaaactagtt tgaggacagg aactggcctt 18960cctacaacat
gagtgtggga ctaagaacgg cagccaggaa acttgaggga aggtggggac 19020aggggagcca
tgtctcccac tctaggtgat ggctggtcaa ataaattaaa ggtgggctgg 19080acagagggag
agggtatcca ggcaaccaga ggaggggtgg cactggctgg aagacagtca 19140acacctgcaa
gaactggaaa gagcatgtgg agtcggctga ggaagaggct ccctttgacc 19200cttaccctgc
tatacgatcc tgcaggactg tgaagctggc tgcttctccc cctgatggtg 19260cccaggtaca
gctcagcaca ggaagcctga ggaaaggcag ttcctttccc tcaccttggg 19320gtgctacaga
tcaccgcttc cgcatcctct tcataaagca gcaggtgatg gtagccagga 19380cgctggcgcc
ggtaactagg gcgactcctg tacccaccaa caggggcaca aatagggtat 19440ccacagctgg
cagaaaagaa gacaggctct gctcagagag taccacggta tctgacactc 19500tcccctgcag
attttctaga ctcagccctc cccaagggag agctgagcgc cagtcctgcc 19560tacctacact
tcacacacaa acacaaccat cccccatccc ccatccccac cccctcccct 19620cggtctcagc
actcaggccg gcttggggcc cttcatgcaa agggatgtgg aaaaaggatt 19680gcaagggaag
acaggaagat ggaaaggggc aaacagagca ggaacaggtg ggtagatggt 19740ggctgtcact
caccatgcgt gtaggggtag actgtaacag gcccggagcg cgcactgcct 19800gcctggtacc
agctgtagtc cgcatgctgc acccaggcac ttggggcaca gtggtatatg 19860ccttcatcct
caggccccaa gccatgcagt cttagccgat gacttctggg tcccaccagc 19920tccacactga
caggaccccc tccaggccgg actcccagct ctgccacacc gtcctgaccc 19980actccaccca
caagctgagc agggccagag ctcagctcgc cctcctctgg cctctccacc 20040caccagctgg
ctgctagtcg cagccctggg gggccgcccc gcacagagat gttgcatagc 20100agggaggccg
tctctccccg gtacacagtg ccccctgcta gccacgccac ggcctctagc 20160accacgcctg
cagagcaaag aacacggggg ttaccaggtg aaggcccagg ggctaagagg 20220ttaggaaata
aattctataa gttctgaacc ccgtcaaggg ctcaacatcc tcttaccttc 20280ttctctcaca
tgcacaggga gaggccggga acgagcactg gccgcttcac gaagtcgggt 20340cccagaccct
cgaacatagg ctttggcgag gcagcggtag gtacctgcat cagcgggcct 20400ggcagcctcc
agccgcagtc ggtaggttct ggatgctact ttctccatgg caatgtgccg 20460gtcctcatag
ccagggccca ggctgcctat accttccgtg tctagctggg ccaccaggcg 20520gccgggtcca
ggagcccctg caggggccat ctcccagccc acagagtacg cagcatgacg 20580gcctggtggg
ggcagtgcac cggacacatt gcacagcagt tctaagggtt cgcctgggcc 20640aatccgacgt
tcaccaggtc ccacggtcac cgccagctgg ctggctgaaa cacagcagga 20700gatgggagga
gtcactgaga tgcctgggcc ccccacctgt aattcttctt tgcagaaatt 20760tagaggcctc
ttatatctcc ctcaccccag gacccgaatt tcacccttcc ccccatagcc 20820tttgtatctc
catgcttgtg cggcactccc gatgcccaac tgagagacac cccccccccc 20880ccagtgggct
atgctgcact cacatagagt ctgcacatca acatgagcca ggactgccct 20940cttctctgcg
acctggaccc aggagccgtc aggatcctga atccactcag cggccgtaca 21000gtggtaggtg
cccgagtctc cagcctgggc acccccaacc accattcggt accgatcagt 21060cccttccttg
ctcagccgaa gctccccaga agctagcctc tcagcgtagg gcgctccagc 21120ctccaccgcc
atgtcggagc gcagtcccac tacttcctgt agagtggctc gccccactgg 21180cgcctccgga
atggctctcc caaaggacac cgacaggtgt gtgtgtttct ttgttttggt 21240ctgagccagg
cagcccagcg caagctcctg cccctcgtgc actgtgaggc gtgaggggga 21300ggtggcagcc
tggcgccctc ggggccctgg aggggcagca gatacctgca gctcatctgg 21360aagaactgga
gagaaaggct ttagtgagag agggcttgga gcagcatccc tcctgtttcc 21420tgtgcgtatc
ctgtttcact acacactctc taggcttcta gaatgtaaga actgtgctct 21480gtagcttttc
ttctataccg cagagatgcc aagcttggtc tgggcacatc aagatattca 21540ataactactt
gctgaacgtc acagagcaag cctactgacc cctactctga tgtctaagac 21600tgatccattt
taaatactca aaaaaagtaa tcctgtcttc cttctctaaa gataaagagg 21660ctggacctga
tggtgccggc ctataatcgc aactacccag gaagctaagg cagaagagtc 21720aagttcaagg
gccgtctggg ctacagaaca agttcagtgc tggtctggtc aacttggaaa 21780gtttctgatt
cagcccatcc ccccccccaa aaaaaaaaaa aaaaaactgc tggaaagact 21840ggcttgatgg
cactggagct gacacaatac tgcctgagct acacagtgaa ttgtgggagt 21900tttgtcacca
gtttcaggcc aacctaggct agttgtaagc tagcctgggc tacaagagtg 21960agccctcaaa
aaacaacaca gggaatatag cttagtagag tgctcccttg gtatgttcca 22020tgctgtaggt
tcaatttcca gtactgagaa gatgggggtg gggggagaag aggagaagga 22080agaagatgac
agagaaggag gaagacgaag caaaaataga tctgagcgtg ctggcttata 22140cctataaccc
cagtgcttgt gaggctctct caccacctag ctcagagccc agtacctctc 22200agctccacct
tggcactgta gttgcccagg tactgcgtat ccgtggaggg ggtgtagcac 22260tcataaaagc
cagagtcctg ggcctgcagg cgagcaatct tgagcaccac cgaatctccc 22320ttcaggcgct
gcacctgcag gtcaccagat gccacacgag gcccaaagac agcataggag 22380aactggctat
ccttggtgct gacaatgccc agggacgtag ctggggcctc tggtctgtac 22440atgaaccact
cgaagtcttg ctgggcaggg ccctcatagt cactcacgtt gcaggagata 22500gagacagcgg
tgccagccac ccggtaaaga ggtcccctgg ggacatgcac ctgccgggcg 22560tagcacctgg
ttcctgtggg gtaaaagcag aaagaactgg aatcttttta gagagaacga 22620gtccccactt
gatggccagt atcatcagca ccattcttga ctgctgcctg tgaaggaaag 22680ggaaacctag
ggatggttag aacatagctg ggcaaagaca cagatgggaa cacaagatgg 22740gacacgagac
accagcatcc cagcacatca cgtaggttca gatccacctg aacagaggaa 22800tactctagct
aactagaggc agagcaccta catgaacaga ggagtccatc cggagtctgg 22860ggtatagaca
gcccggtaag taaaatgctt tccatgcaag catcaggacc tgagttcaat 22920tcccggcaac
tatggaaaaa gcttggcacg gtggctggtg agtagccaca taagcctgac 22980aacctgggtt
tgagccccac aagggaagga aggaactgaa tcctgaaagt tgtctcctct 23040ctctctctct
ctctctctct ctctctctct ctctctctct ctcacacaca cacacgcaca 23100cgcacacgca
cacgaataaa tgcagtggac ggttcctgag ccatgacccc tgaggttgac 23160ctctgacctt
tacacagaca cctgcaccca tgcatacatg tggaccctca tacgcatgca 23220attggaaata
acaaataaaa gaagcatgcc tgattcccaa ctctccagct cgtggctgga 23280tctttatcgc
tcctaccctg ccatgtgtgg cgtctctccg ggctcagaaa gaacttctag 23340ctaagggatt
ctgagccttt tgctggaggg gagctgacaa catcttacag aaaggctgct 23400tggcttagct
ctgtcacctg ggctagacaa tggagccagt ggcccaggct ggctggggta 23460ctgcactgga
tggggcacca tgctggacag ggcacggacc tagtcagtcc tcactacaca 23520ataccctccc
cactacagct gtgccatgag ctcactgctt ctcccagccc acaaggctac 23580acaggcagct
gtggcttctg gggcaagaac caggctctgc ccaggcctgg ggcagaagat 23640cccttccccg
cccccagaat ctgtcaacct tctcgctaac ccagatgatg cactatgcac 23700agccccccaa
acaaaagctt tcatttacat atgatttgcc tatctgcgca gcatttgcat 23760agacctcctt
ttaataagga gaccccaaac acctgctgcc ccttccccct tctctctctc 23820tccctccctc
cacccatttt accccaccac tgttccaaca gacagccggt tccaaaacct 23880tggactgttc
agcttctttc tcctctccac tacttctagg taagaccgca gaaccctaga 23940atgcaagacg
ggctgtcaat cattcctcag atgagagaaa tgaagaaact cccagagagc 24000acctctaggg
agcattgcca ctaggttcta accacagatg tgagtctaga gctctctccc 24060agccaacacg
gaaggcctgg ccgtgagaac catctctctt ctagatgaga aaagtaaagt 24120gaaatgtgac
attgtggcca agtccctgcc ctctctggag ggcctctaca cacccctgaa 24180gagggacaaa
accaagaagg cggagatgct tccaaggaga gccctggttt acaaaactgt 24240aatttcccgg
atttccaggg gtaagtccac agcttgctac tggccctgga ggaaaccacc 24300caagagcctg
agagcctgct ctacagttct cgctcctttc cttcttctgg ctacgtcacg 24360cagagaacac
atgaccaccg cctgaagcag aggctagagt cagaaagcca acatgagacc 24420aaccctttcc
tctcctggat ctcagtttct atctctcaga actaagagct cccagttctg 24480atgttgaacc
cgtgagtata tgtgtgtgac tcaggcacat atcgctccag gcacatttca 24540taatcaggag
gatggttaag gcagccaagc tgacaccatc ctggctgccc atgggtaaac 24600tctgctgaga
aggtcccctc ccaccttcct ggctccacac aagctgatgc ttggagatcc 24660ccatgggaaa
gttgtcccca cagcctcagg acataggctg ctacaaggtt ctcaatggct 24720gggctgcctc
ctttctaaca gccaaaggtc tgctttgaag tcagttctga gttcaaatcc 24780acccccaccc
tcactggata cacagtggta tttcacgaca tctgtgaagt gaatgccagt 24840gctggctgca
gtgggctaaa atgacattca gctcctctcc cgcccctgaa taacactcac 24900tcctcccaac
cacccctggg ctcatccctg ctcgggttaa gcccaaagga aaagaagcaa 24960tcgctaggca
accaagcccc acagctcctt caactcccta catcactctg cctcccgcct 25020tgttcccaaa
ggagttttca tcctggcccc agaagcccaa ggaaccattc tgtacaacac 25080tgcacagttc
tggtataaac cagagaagga ggttggggtg ccccactata gtatcttctc 25140catatgcata
tcacacacac acacacacac acacacacac acacacacac acacgttcct 25200ttcaagggct
tcagtctcct ggcaactgct ccatgccata tctttcccag accacctcct 25260acagggagcc
ctccaagtca gaccccaaac atggtaatgt tagcaacctc cacaggcctc 25320aacacacaca
cactcacact cacacacaca caccagacat gacgcaaggt tggcccagaa 25380aacacaccat
cataaacacc caccaggaca gacactgggt gcttagagat cccaggttca 25440gtttccatgg
agcctagttt ctcctgaggc agggatgttg ggaccaactg agtctgacaa 25500ccaggcaaat
atctgggagc gtggaagggc aaagagggaa ctggcccagg gtggagacac 25560gtgagggaag
aagcctcaga tggtgacatg ttatattggg aggtgggggt gttggggaga 25620cttttttcag
agatcgtggt cagaatcagc ccctgggcct ccagccaact ctgggcaatt 25680atgaagaccg
ccaggcactg cccacgcaga gcaaacaccc aaaaccaggc cttgagccga 25740gagtggggca
gaaggttgtc acggtatttg gtagcaacga ccccagacgc tgggtgtaac 25800cgatgagaag
tggtgcctgc ctccggaggc ccgatggtgt ctcaggggat acctcagtag 25860gtcgcccata
tgccccagct aggaacctag agcgaggaca ccaccaccct ccccataact 25920gattgggcag
acaggcgcaa aaggaagcga gacgccgagc ccagagacag tggaggcacg 25980tctgttggag
aagtagggat gcaaccagct ctgaaatgct aggaaggtgg gctggtgggc 26040tgcactatgt
taggcaccta cccggccggg acagggacgc ggcgaccacc acctggctta 26100ccaagtatta
gcagcagcag caggagcgaa ctcagcggcg tggggctagg gacgcccatt 26160ctgcgtaggc
ggctctgggg agactcctgg gggcggcgta ggctctgggg ggccagggcc 26220gcggggggcg
catgcccagg tggggggcag aaagcggagc agtgaagcgt gggtgcgcag 26280agcccagccg
agcgggagcc gccaactccc cgccctccac ccttcttccc ctcctccctc 26340cgctcttccc
gccctccgca gctcgggaga ccagtcccag ccgcgccccg ctgcccggcc 26400ccgcccccgc
ctcgccccgc cccaggccgt cgcctcggcc agacttcgac cctgatggtg 26460gctccgcctc
tggcctcagg ctgggcgaac tggcggcacc tgggctcctc tatccccatt 26520tcctcgctca
gagggcaccc cgccctgcac ctgccagcct tccagggaga atggggtgct 26580ttcagggcct
ctggggatgc atgatggggt gactgtggtt acgcactcag aatccaattg 26640gg
2664222267DNAMus
Musculus 2ctgagctgcc gttacattca ggagaaacag cagtgtcggc ggctcccaat
ctcagaggga 60acctagggta ctgggggaga tggtgtcagg gacatggacg ccaaccccca
agggtttctg 120ctgctggcta ctcttctctc caggctctac ttctgttcat acggtccata
tctcctaggg 180gaccctgaaa gcctaggaac cgactctggc catccatctc tccgggaaga
ttataaccca 240gagtgcttct caggggggaa gaatttgaag caaaaccaga ccccgcagga
tccccgctgc 300ggccgccatg gcgcaggaga acgccgcttt ctctcccggg tcggaggagc
cgccacgccg 360ccgcggtcgc cagcgctacg tggagaagga cggtcgctgt aacgtgcagc
agggcaacgt 420ccgcgagacc taccgctacc tgaccgacct gttcaccacg ctggtggacc
tgcagtggcg 480cctcagactg ctcttcttcg tgctcgccta cgcgctcact tggctcttct
tcggtgtcat 540ctggtggctc atcgcctacg gtcgcggcga cctggagcac ctggaggaca
ccgcgtggac 600cccgtgcgtc aacaacctca acggcttcgt ggccgccttc ctcttctcca
tcgagacgga 660gaccaccatc ggctatgggc accgcgtcat caccgaccag tgtcccgagg
gcatcgtgct 720gctgctgctg caggctatcc tgggctccat ggtgaacgct ttcatggtgg
gctgcatgtt 780cgtcaagatc tcgcagccca acaagcgcgc cgccactctc gtcttctcct
cgcacgccgt 840ggtgtctctg cgcgacgggc gcctctgtct catgtttcgc gtgggcgacc
tgcgatcctc 900acacatcgtc gaggcctcca tccgagccaa gctcatccgc tcccgtcaga
cgctcgaggg 960cgagttcatc cctttgcacc agaccgacct cagcgtgggc tttgacacgg
gggacgaccg 1020cctctttctc gtctcacctc tcgtcatcag ccacgaaatc gatgccgcca
gccccttctg 1080ggaggcatcg cgccgcgccc tcgagaggga cgacttcgag atcgtagtca
ttctcgaggg 1140catggtggag gccacgggaa tgacgtgcca agctcgaagc tcgtacctgg
tggatgaagt 1200gttgtgggga caccggttca catccgtgct caccctggag gatggtttct
atgaggtgga 1260ctacgccagc ttccacgaaa cctttgaggt gcccacaccc tcgtgcagtg
ctcgggaact 1320ggcagaagcc gcggcccgcc ttgatgccca tctctactgg tccatcccca
gcaggctgga 1380tgagaaggtg gaggaagaag gggctgggga ggggggcagg tgcgggagat
ggagctgaca 1440aggagcacaa tggctgccac ccccagagag tgagtccaag gtgtgactgg
tttcctccca 1500ccccctgtgg cagaccaggg ggccggactc aggtacacag aagctgcgag
tggaggtgga 1560agaagaggag gcaggcagtg tcccgaggaa cagctaaagt tgggagaggc
ccgctgagtc 1620caggatcgag tagggaaggc tgaggtcctg gtttgaagag agagggttgc
agggcggggt 1680gagagaacat gtcagtctgt ctgtgtttga ccttcacatc ggttcatggg
tggatggatg 1740gacagaagga tgggctcatg ggggttgatc gggaaggtgg agcagataga
gacagccaat 1800ggataatcgc tcaggtggta agtggcttgg cagtcgatga tcgtcacctg
cagcacacct 1860ttgtgagaaa tccatgggca tccttttctt ccagatatag gtagcctcaa
accagggagc 1920gtggcttagg gagcaggctg tcaggtggac taccaccccc actcacctcc
cctcaactgg 1980cctccctgat gtgtgacacg cctgcctaac tagagaagag agcactgggt
agaggtggac 2040acaggtgtgg ctgccctccc cagtatcact gtcccatggc gagaggtcag
aaaggcaaac 2100aaacaatggg ggtagatgct gagcagggag gggccctgaa gcaggacctg
gggacagcca 2160aggacaacta ttttgtgaga gaggaatgaa accttgcagg tcctgccaca
gaagcaagaa 2220gcagaggaaa ggccatggag agacttaata aagggtttta caaggga
226731131DNAMus Musculus 3atggcgcagg agaacgccgc tttctctccc
gggtcggagg agccgccacg ccgccgcggt 60cgccagcgct acgtggagaa ggacggtcgc
tgtaacgtgc agcagggcaa cgtccgcgag 120acctaccgct acctgaccga cctgttcacc
acgctggtgg acctgcagtg gcgcctcaga 180ctgctcttct tcgtgctcgc ctacgcgctc
acttggctct tcttcggtgt catctggtgg 240ctcatcgcct acggtcgcgg cgacctggag
cacctggagg acaccgcgtg gaccccgtgc 300gtcaacaacc tcaacggctt cgtggccgcc
ttcctcttct ccatcgagac ggagaccacc 360atcggctatg ggcaccgcgt catcaccgac
cagtgtcccg agggcatcgt gctgctgctg 420ctgcaggcta tcctgggctc catggtgaac
gctttcatgg tgggctgcat gttcgtcaag 480atctcgcagc ccaacaagcg cgccgccact
ctcgtcttct cctcgcacgc cgtggtgtct 540ctgcgcgacg ggcgcctctg tctcatgttt
cgcgtgggcg acctgcgatc ctcacacatc 600gtcgaggcct ccatccgagc caagctcatc
cgctcccgtc agacgctcga gggcgagttc 660atccctttgc accagaccga cctcagcgtg
ggctttgaca cgggggacga ccgcctcttt 720ctcgtctcac ctctcgtcat cagccacgaa
atcgatgccg ccagcccctt ctgggaggca 780tcgcgccgcg ccctcgagag ggacgacttc
gagatcgtag tcattctcga gggcatggtg 840gaggccacgg gaatgacgtg ccaagctcga
agctcgtacc tggtggatga agtgttgtgg 900ggacaccggt tcacatccgt gctcaccctg
gaggatggtt tctatgaggt ggactacgcc 960agcttccacg aaacctttga ggtgcccaca
ccctcgtgca gtgctcggga actggcagaa 1020gccgcggccc gccttgatgc ccatctctac
tggtccatcc ccagcaggct ggatgagaag 1080gtggaggaag aaggggctgg ggaggggggc
aggtgcggga gatggagctg a 1131427684DNAHomo Sapiens 4ggagtaggag
aaagctatgg cattttagga aaattaatcg ggaggtgaca aaatagtttg 60aaccaagtgg
atatagtagg caagtagacg atagaaaata attgcaataa tataagcatg 120aagagatgac
agcccaaatc agcgtggcaa tggtgaaaag tggaacacag aaaatgaatt 180ggagtacaga
aaaatcaaaa gaaaatgaaa aaagtttgaa gccaacttga catgttgagc 240aaaagaggga
agcttcagag atcatactag agtctcaagt caggtgatca gaactgcgag 300gtcattcacg
ggcatagggg agcctggggg ggatcacacc tggtgaggag actgaggtgg 360gggaagagga
agtgatgagt tcagagctgg aagctgtgga gaggggtcag aaccagagag 420agaaaggagg
tcattgctgc cagggcagtg tgagttgaag ctatgagaac agggtagatc 480ccaacaaaga
ctgcacagag aaatgagagc ctggcacaga gagtgaggaa cacctatgtt 540taggggatgg
gaagaagaag gacccccaaa gagtgaaaga gaatccacca gacaggcagg 600aaggagacaa
agaaagtgag atgtcatgga gctaaggaag gagagactgt taaggaggag 660gttctaacag
tgccaacaag tacagagaga agaggcattg ggtttggcag tgacaaagtc 720tctagtgaca
tttgagagca atttcagaag agtgagcaag gtgggaacca gattacaagt 780taccactaga
aagtgagaaa ctgtcagcaa gtataggtta cacttttgag aactctactc 840atagagagga
gagaaataga aaccagacaa tgtactagaa acaggccagg ccaggtggct 900catgcctgta
atcccagcac tttgggaggc caaggtgggt ggatcacctg aggtcaggag 960tttgagacca
gcctggccaa catggcaaaa acccatctct actaaaaata caaaaattag 1020ccgggcctgg
tggcacgcgc ctgtaatccc agctacttgg gagactgagg catgtgaatc 1080gcttgaacct
gggaggcaga ggttgcagtg agccgagatc acgccactgc actccagcct 1140gggggacaga
gctagactct gtctccaaaa acaaaacaaa acaaaacaca aaataaaaag 1200aaaaagaaac
agtcttccag tttttcttct tcacactccg aatgccctct cttcctaagt 1260caatatggat
gaggggctgt ggtgaggtgg tctgagggcc agcctgcaag actggtataa 1320gacctttaag
tttcaaaaaa taggacatcc aaaagatcct taagggggcc acagtcttga 1380cattcacaga
cagagaggac ttaggcaggg gtgtccaatt ttttggtttc cctgggccac 1440attggaagaa
gaagaattgt cttgggccac acataaaata cactaacact aacagtagct 1500gatgagcttt
aaaaaaaaaa aatcacaaaa aaaaccctca tgatgtttta agaaagttta 1560caaatttgtg
ttgggcctca ttcaaagctg tcctgggctg catgcagccc acaggcggtg 1620ggttggacaa
gcttagcttg gaggctctgg tggaactcca aaataaacat gaagaacacc 1680acagaagaga
aagcaaaggg actgtaatga tttatggatc attaacagac atttattgtg 1740cacttattat
ttttccaaaa atgttatcca tccatttagc ttcaactacc acccatgtgt 1800caatatgtcc
agcccacccg gatatccatt tcaaactcaa catatttaaa gtcgaacatg 1860tcaccttgct
cacaagagtg ctcctctcca tttattctct accatggtag atacactatc 1920atcacccaac
cagaaacatg gcagccatcc tagattcttc aatcttcctc acctcatctc 1980ccttattgaa
tcaatgcatc tgtattctaa atagcctcaa tattgtcccc ttcctctcta 2040ttccactatc
attgctgtag tctaggacac cattacctct caccaggtaa taatagtttg 2100gatctttgat
cctgctcaaa tttcatgctg gattttaatg ccaatgctgg acatgggcct 2160ggtgggaggt
gttttgatca tgggggcagg tccctcagcg gcttggtgct atcttcatga 2220tagtgagttc
ttgtgagatc tgggtgttta aaagtatatg gcaacatccc ccatcatcaa 2280ctctctcttg
ctcctgcttt tgccatgtga tgtgcctgct cctgctttgc cttccaccac 2340gagtaaaagc
ttcctgaggt ctcctgagaa gctgagcaga tgtcagcacc atgcttcctg 2400taaatcctgc
agaactgtgt gccaattaaa ccttttttct ttataaatta cccactctct 2460gggttttttt
ttttttaatt tttaatcttt tttttttttt tgagacaggg tttcactctg 2520tcccctaagc
tggagtgcag tggtgtgatc acagctcact tgtacccctg aactcctgtg 2580ctgaagtagt
cttcctgcct caacctcaaa cgtagctgga actacaggtg ttcaccatta 2640cacccagcta
tttttttttt tttttaactt tcagtagaga cgaagaatcg ctatgtagat 2700caggatggtc
ttgaacttgt gagctcaagc agtcctccca cctcagcctc ccaaaatgct 2760ggattacagg
cttgagccac catggcctat ctcaggtatt tcattatagc aatgcaagaa 2820tggcctaata
caccagggct actgcagcag ccttctaact actctccctg cctccagtct 2880tcctccactc
taataattct ttggattatg aatttcttta tttgaaagta attaagcacc 2940agtaaagtac
atctctctga aacacacatc tgaccgtacc acttccaagt tttaaaacct 3000tcagtaactg
ccaactatct ataagtaaag tccgagttcc tttccctgga agagaaggcc 3060tattataacc
tggacctggt gccattccag ccttatcttc ttccactgcc cctatacacc 3120caaagctaca
gctacttctt ttaacactca aggttcagcc ttatgttctc tttctgtgtc 3180ttgccccttg
agcctttgtc atttacatag ctccaacgat tgtccctgag tgatgcccaa 3240atctctatct
tcaatcctac actctctttg gagctccata tttctagttg cttgcagggg 3300atttccatct
tgacttgaca ggccccaaac tgaactcagt accttcctcc ccaaaggtgg 3360aagtgctcgt
gacttcctta gttctgtgtt actcctggtc aattagaata aaaaactaca 3420agtgaccttt
actcttcacc gttgccttgg gcccattcct ggacatgtca aataagccaa 3480caaatgctgt
caagtctccc tttctttcat ctgtttgcaa tgtgcttttt cattcctatg 3540accactatca
gaatcagaaa gatcaggacc tgacccatcc ttcacctttc tctccagtct 3600caaaggaaga
ggcaacctgt tttcatcatg tcggtccctg tgcccttgtt ttaaaacccc 3660aaacagttgc
ctcgtgctta caggtcacag tgaaggaggt cttcaccaca gaagacctag 3720aaaaaaaaaa
aagaagataa aaaacgtgac aggccctcag actgaactcg gcatctttct 3780ctctgaggct
ggaagtgctc atgacttcct taattctatg ttacttctgg tcaatcagac 3840taaaaaacta
caagtgatct acagaagtgt cctctactaa caatcagagt gaggatagag 3900tcgggtggga
ctgggcagtt agaaagactt tataagtcct tgaacagcag gggtgggagc 3960ttgtggaaaa
gtacacaggt agcttcaaca gcactgtaat gttctgaatt taaaagagtg 4020acttaaattg
agtttttgtt cttaaattat gctttataac atatagacat atgtccacca 4080tctatattct
tttgtacata tcaaatgtca ggttttcatt tttaaatttg tttgcaaaag 4140agaagtccta
ggacagtctc taggagccca gtagggaatc agtaataagg ggcataggac 4200actaatattt
gtgagtgttt actacatcag atagatcaga agatggggaa actgaagttc 4260tgaggagtta
agtggtttgc ctatggtaac atagctggaa agtgttttga gatttgaatc 4320cacatatatt
tgaccccaaa gcttgtctca gaataatgcc agagggaatt tgcacgtttt 4380aaacacaatc
ttgccaacca gaggctatga cccctgagta catgttggta tgaaaaattc 4440cccagaatta
caacatccaa tgtccaccat gaaacatgac agaggaaact tctctttttg 4500aagacccctc
tctcttcttt cagtttccca acttgcgtct tccttattct cctccatttc 4560tcctttcaga
ctcactgctt ccagctttgg cctcatctct acttttactt catttgtaat 4620ggggcagagg
ctacctcaga gcagaggagg aggagagttg gggcgtgtca cctgttttag 4680aaagaatcca
caagtgggca gcagtctgag gggcttgcgc tgggcaaagc agatgtggac 4740agagggaatc
aggaaagctt tgggttggga ggcatgatag agactcagaa tagtcagtat 4800ttaacaagtc
aggggaagtg gctagaaaga acagagacac tggcatggct caccacagga 4860ttcaggattc
caagtggcgt tttggtgctc acatcccaca gtgcggaaca aattccatta 4920gtagtggagc
atctcatagc tgaatgactc aggccgcaga ggagaaatcc aagagaagga 4980ctgagctaca
ttcccctagt cactaacgaa tcattatgta agtagatcac cccctttaaa 5040taaatgcaat
atacacaaac ccacatttat aagacataat ttagggaata cttagttacc 5100tactaaagaa
ttctttcctt taaaaaagaa aaacatggcc aggcacggtg gctcatgcct 5160gtaatcccag
cactttggga agccaaggtg ggtggatcac ctaaggtcag gagttcaaga 5220ccagcctggc
caacatggtg aaaccccatc tctactgaaa atacaaaaaa aaaaaaaaaa 5280ttagccaggc
atagtggtgt gtgcctgtaa tctcagctac tcaggagggc gaggcaagag 5340aatcacttga
atccgggcgg tgggggttgc agtgagcgga gatcgcgcca ctgcactcca 5400gcctgggcaa
cagagcggaa ctctgtctca aaaaggaata aaaaaaaagg aaaaaagaaa 5460aaaacaaatt
tctctaacta gggacttcta gtacctttcc agttgggtcc aattgataga 5520aattccatta
acatccaatg cactgtgata ggagggaggc aactgggaat aaagaaacac 5580gaggaatctc
gagtcgggtg gcctgagtct tagtcctgac tatgttcttg ggacctattc 5640ctacctgtaa
agtaagggct aatcctgtac cacctctaac cgtcatataa cttttaaatc 5700ttagcctatc
tctacccagt cctataaagc aagatagaac tctgtgtgaa ggcttctgat 5760cctcctgctc
tgctgaaagt agccagaaag gcagcaagct cctcagcctc aggaacccag 5820cctgaggcga
ggggctggct gaaattgcct ccgtctggcc tggagctgtg ctctgcttct 5880ccccatttca
ctctaatctt cagcttcagt catttgccac atctactcct tcaaccatat 5940ctttcctctg
ctctgagttt tctagagccc catccccctt gaatttatac aaatttttgc 6000aatcaaccag
attggcctcc ctgctccact aaactcatat cctcaactgt ctgctgtctt 6060ccccatcatg
cttcctcttc cacttgccag attttgcaca agatgtctca tgatcttgtc 6120cagggaagcc
tccccagttt ggctgatctc agggctgcca caaaggcacc tgctgatggg 6180gcaagttgag
gactgaactg cattcagctt gccaattcct gcacccagct cagagctgtg 6240tctgctggag
gaagggaacc ttttattttc tcccaaaagt atcacctgtt ccctgttctc 6300caagtgacag
gccacagtag gctcttttta agctcttttc ctattttgca ccacggttcc 6360cttttttttt
tttttttttt tttttttttt tttatgagac aaggtctcac tctgttgtcc 6420aggctggagt
gcagtggcgc aatcacggct cactgcagcc ttgagctccc aggctcaggt 6480gatcctccca
cctcaacctc caaggtggct gggaccacat gcacatacca ctacacccat 6540ctaattttgt
attttttgta gagacagggt ttcgccatgt tgcccaggct ggtctccatc 6600tcctgggttc
aagcgatccg tgcacctcag cctcccaaag tgctgggatt ataggttcga 6660gccaccgtgc
caagccaaaa gctagaatct tgtctatgct tttgtgtcct ggtgcctggg 6720aaaacttttt
ttctcctgcc tcagttcagc tcagtgataa ataaggaact gaggttagat 6780aacaggtaaa
gtctaggacc tgcaggatga gtgaatcagg tgagggagtg gtagtcttct 6840tccttgtcag
ccaggctagg ttcaggggca cctggactga ggcgaggggc tggctgaaat 6900tgccttgtgg
agggccctgc cagtgatgcc ccctccagca aatagggcca gctctatgca 6960aatgtgttct
tgcccaggag tttggtttct tctctctgag ctcctggcac agtggaacca 7020atgtgagcag
ctgcttggca ggacagagaa gggcaggcta gcagtcccaa agctcgggtg 7080acaggaccag
gcccaggaga cggggatgtt gactggggct ttaacagcac tcttgatgcc 7140aatctcgggc
tgaaaactcg atatttccac ttggaacaac aagaatcacc agcaagagag 7200ctgaggagag
ggcagtatac cgggggcgcc ccctgcaggc ctcacagggt ggtgccagaa 7260cagaggaagg
tggcacaggc agggtggggc tttcaggaca tccctgagat gatggtgatg 7320acggtgacaa
tgatgatgac catgaagaag acaatgagga ggaggaagag gaagacagta 7380gctagcattt
actgagtact aacaatgtgt caggcattgc cttatgtagt cttcatgaca 7440accctctaag
agatgaataa tatggttttc ttttttttag atgaagaatc tgaggtttaa 7500cgggttaaat
aattgctcag gttcacccag ctagtagtgg acagaggtgg gatttgaacc 7560caagtcattg
cctcctgagc ttatattatc cagtaccgaa tttcccacct tgccaggtca 7620ttccaggagc
ttctagccct ccgtgtccat ctctatgtct tcctgctcct ctagctcata 7680ttttcttgat
ccaaatttaa aggatctgga taagaataga tccatatctg ggatataata 7740atactgataa
caacagcaac aacactttgc gtttgtaaac cactttcttc tcttcattat 7800ttccctgggg
aaaaataaac aataagatat ttctgtttct ccaaattttg ttctgatttt 7860tatcagtgtt
cctgaagcta tttcaatata gtcatgatca atttctaaga atatttttag 7920gttctgcttt
tttatgtaac agtgtgttat atacacattc acatatttaa acacagcaat 7980tattatggct
ttacagtaac ccatgatatt aatattccac agatattaca ttactgaggc 8040acactaggct
aaggctgaca acaccaaatg ctggcaggaa tgtggagcaa caggaacagg 8100aattcgtggc
tgatgggaat gcaaaatggt acagctactt tggaagaaag tgtggcagtt 8160tcctaaaaaa
ctaaacatac tcttaccata cgatccagga atcatgctcc ttggtatcta 8220cccaaaggag
atgaaaactt acgtccacat gaaaatctgc cgatggatgt ttatagcagc 8280tgtattcatc
atggccaaat cttgaaagca accgagatgt ccttcagtag gtaaatagat 8340aaataaacca
tggccatcct gaaatggaat attattcagt gccaaaaaga aatgaactac 8400aaagccatga
aaagacatgg aggaacctta aatttacatt actaagtgaa agaagccaat 8460ttgaaatggc
tacatattgt acaattccaa cgacatgaca ttctggaaaa ggaaaattat 8520ggaaacagga
aaaagagcag ttgttgccag gggttaggga agggggattg actaggcaga 8580gcatagagga
cttttacagc aatgagacta taatggtgga tacacatcat tatatatttg 8640tccaaaccca
cagagtgtac aacaccaaaa gtgaacccta atgtcagcta tggactttgg 8700gcgattatga
tgtcaatgta gcttcatcac ttgtaacaaa tgtgccattc tggtggatgt 8760gtggggacag
ggggcatacg gaaaatctct ataactttcc tctcagtttt gctctgaatc 8820taaaactacc
ctttaagaag tcttctttta aaacaattta caaagcatga ggtgatacag 8880atgtgggagt
ttggctcctg tctctgccca actctgtgac attcgataaa ttacttaaca 8940tgtctctgtt
tcagtttcct catctataac tgggaaaaat aacacctgtc ttatagagtt 9000gccatgggga
tgacatgagg catgtgtctc gttcatatcc catgctcagt gaattagtag 9060cagcagccac
tgtgtgtttg tgtgtcttta tccctcctgg gttaatgagc tccttgtggg 9120cagggactca
cccattctgt aaccacccca tctaacacac tgcctggcac ttgggctccg 9180cagaagtttg
ccgagtgaat acttagtaag ccctaaccta ggcttttctc tctggtggac 9240atttgggttg
tttctagggt ttttgctatg aataaaacac atttcaaagc cctttgtggt 9300ttttttggtt
tttgtttgtt tgttttttct tcgtttgatc tgctgactct gtgaagcagg 9360cagaaagggg
atatttgctc ttgtccacac cctggtacag atggaataac tgtggctcag 9420ggaagtgaag
tgactcctat gggacacagt gcaaatcagt ggcaataatt agaacccctg 9480accctgcctc
ccttccttta gtagatctat tttccttcta gctaccgcct tctggatcca 9540tggcctctcc
aaaactagac catgatggtc agcctgacct gagagcagca cctgcacgca 9600gagacccatg
ttgaaggtgg tgagctgcca gctaccagat ggccctctga aaccccaggg 9660aacctagcac
cttattctca aatacatgag ggcttgtatt ttcccccagg aaggagcttc 9720ttaggaaaga
gccagcgtgc cagctttgtt tttctttctt cttctttttt tttttttcct 9780atgagggggt
gaggagccaa gctctgagtt gtccaggagg agggactttg gctaaaaata 9840gctatggcgt
gtggtttgga tcaaccccta gtggtaccca ggactgggga ggggaggggg 9900atgctctgga
gctgtcgcca gactggttgc cgtggaaaca agagaggagc aggggagcct 9960gggaagtagg
gatgacacag atagcaagtc ctagtcagag ctgccgctac atttaggaga 10020aacagcggtg
tctgcggctc ccacccttcg gggggcccgt ggggggggcg gtgtcagggg 10080catggacgcc
accccccagg ggtctctgct gccggctact ctcctctcca cgtgctgtga 10140gttgagttgc
gggggacttg gggtttgggc ccctatttcc aaggcaagtg ggggtttggg 10200aggagctggt
tcttggggga gttttcacca ggtctctcct tccaaaaaat gagccccctt 10260actccccagc
tctctagagg gaggaagagg ggcccaggaa aagtggtatt gcaatcttct 10320gcaaaggggt
catagcatgc acaagaaatg aggagtaggt tggaggaact gaaattcttg 10380gagggaagat
ggagaaatca agtccttgat cttgagatag aggtaacaat ttcacacttt 10440tccttcccct
gagaaaagtg cagtccccca ctcaggaaga caggatgtgg gacacattca 10500aaataaggtt
tacctagatc cctggggcaa tggagagtga gagagttctg ggggtgatcc 10560gacatcgggg
ttccttcccc atccctgggc agagagatct gtctaggcaa gccgactggg 10620ggtcagatta
cctaagaccc tgagagaaca tctggaagcc cacctgggac taaagctagg 10680ataatgggag
cagggtcgtt ttctgcatga cctggggtct ctgagccagt caatgcttac 10740tcttcctgag
gacatctgag cttcaggaaa ggaaaaggaa gcccattgtt gggggcaggg 10800gaaaccctaa
tcttccattg ccatggggct cttggaccct gtgtcccctg actccatgga 10860caataaatgc
agggggtgcc cctaagctca aagccatttc attttgattt ctcttcctac 10920cttctctacc
ccaagacaca caaacacaca cacacacacc ctctccagag tgctgactgc 10980agaggacctc
accccagaac ataagatgct ggagtgctag gtttagagtc acatacccag 11040gcagtttctc
cccaggacct ggtcaaccat ccaggccatc tgtggttcct atggcacact 11100cctccatccc
ccacccacta gccagcccac gtttccgtgg agtgggagga gaggatcatt 11160cccaggaaag
agaagggaag gtggaagagt cccaaatcct attctaaacc tttccctgta 11220tggtccatat
ctcctagagg accctgggtg ctttggggaa gggctctgga cctctctcag 11280agcagattgc
agctcagaga gctcctcaga ggcaagcatg tgaagaaaaa tcaggtgggc 11340ttcgcttgga
atgtgggctt tggggcatat ggcaggtggg ggcggggctg gtgttaggat 11400agtccatggg
aagtaagagg ctgggggaaa atataactag agggagtggg gaaataaatg 11460tgggtgctta
gtgcttcacc tgatctgatt ccatgtctct catgaagaat aggatcccag 11520agggatacga
gcctaactct ttataactct gggcttcctt tcccaggctt ctgtgttggg 11580atcttccagt
tcccctcccc atttgcaggc tgtctccact aggagaaaaa acccaaggga 11640aatgaggctg
gcccaagagc agcagtgatc gtgggtaggt ctcagggagg atttctagtg 11700ggaatttcct
aatgttccac ccttgtgcac tggagggttt ccactgactt tccacagctt 11760tcatttcttt
ctcgtttgta agcatgttga ggggagggaa tggagcggag tgagtgaggt 11820ccaaggaggg
aagaatgaga aagactgtgt atcagtcttg gggtgaactt caaaacagcc 11880tgcgaggaga
gccattggtg gctgcactgg ctacagctgg ggaagggatg gtggaagtcc 11940ttagggcagg
gagggctcca ttacccgcct gcccccctcc ccaaaaagcc cccagtctat 12000tgatttcagg
aaatcactag ggggatctgg gcctgggtct ttggccccgg ggctgcccct 12060gaggtgctgc
acaccccagc tggaggtgat ggcaccaaaa tatctggtac ctccttcccc 12120tgaaaatcat
cgtggaactt gcacagttct atccagttca ggtacatcat tccatttgac 12180cctcacaact
ttctgagcct ggggggcagt tagggctgaa tgtgttattc ccagaaatag 12240aggccaggca
acacgaaggg actcgcccag ggccccccag ggctcggtgc tggccctgat 12300gccccgtgcc
tccccatctc ccgaggggcc actcattcgg caaaccttta ttaagcccct 12360ccaggacccc
cgacgccgcc taggcgccca gcgacgcgcg gcaggtggca gcagctcggg 12420cccccgccgc
actccaggcg cccgcagcgc tcgccctgac gcggccgcca tggcgcagga 12480gaacgcggcc
ttctcgcccg ggcaggagga gccgccgcgg cgccgcggcc gccagcgcta 12540cgtggagaag
gatggccggt gcaacgtgca gcagggcaac gtgcgcgaga cataccgcta 12600cctgacggac
ctgttcacca cgctggtgga cctgcagtgg cgcctcagcc tgttgttctt 12660cgtcctggcc
tacgcgctca cctggctctt cttcggcgcc atctggtggc tgatcgccta 12720cggccgcggc
gacctggagc acctggagga caccgcgtgg acgccgtgcg tcaacaacct 12780caacggcttc
gtggccgcct tcctcttctc catcgagacc gagaccacca tcggctacgg 12840gcaccgcgtc
atcaccgacc agtgccccga gggcatcgtg ctgctgctgc tgcaggccat 12900cctgggctcc
atggtgaacg ccttcatggt gggctgcatg ttcgtcaaga tctcgcagcc 12960caacaagcgc
gcagccacgc tcgtcttctc ctcgcacgcc gtggtgtcgc tgcgcgacgg 13020gcgcctctgc
ctcatgttcc gcgtgggcga cttgcgctcc tcacacatag tggaggcctc 13080catccgcgcc
aagctcatcc gctcgcgcca gacgctggag ggcgagttca tcccgctgca 13140ccagaccgac
ctcagcgtgg gcttcgacac gggagacgac cgcctcttcc tcgtctcgcc 13200gctggttatc
agccacgaga tcgacgccgc cagccccttc tgggaggcgt cgcgccgtgc 13260cctcgagagg
gacgacttcg agatcgtcgt tatcctcgag ggcatggtgg aagccacggg 13320tgcgagcagg
cctggggagg ggagcggggt tggcagaggg tgggcgggac cgaggaaggc 13380aggggcgaga
ctaggggcca ggggagctgg ggaggatgga tggaggggct ggtggaggat 13440gagacagtga
ggtgagacag gggtcggagg cgggagtgga accgagcaac gccgcagaag 13500gccaagagaa
agcttggagg aattctccga aatggcactg gcgtggggcc ctgggcccag 13560aggaatgtgt
cacttggaat agggacagta ataatagcta gtgctcgccc agtattcacc 13620ctgtgtcatg
cgcagttcca aagcactttc tacctctgag tcgatttaat cctaacaaga 13680accctctgaa
ggtaacttct tgttattgtg ctcacttttt agagatgaga ttgctccaat 13740gagaaattaa
ggaagttgtc cactttccta agccaataag tggccatgcc tggattggac 13800acaggcaatg
tggcttcaat gtttagtggt cccgagttgg aaggaggggt taggttcagg 13860ggttttctca
ctgcagtcag gttcaggccc ctggaatttg acggtgaagg ttttccattg 13920cctgagttat
ttctaggccg gatcttgagg ggagtttaat acctagtctc acttgtacct 13980cggtttccca
attcatccat ttccactgac aagggatata gatgatgtta ccttttctag 14040ctcttttcca
aaaggaactg gcaactcatc tgtgatgtca ataagtccaa cccagaccta 14100cacagtgaag
gctttgggag caggtgaaaa aagaccagtg ttacaggagt cgcaaaggag 14160gtcacttagg
acttgagatc tagaggatag atgaggatga ggaaactgcg ggtggaggac 14220caaaggccca
ctagggggcg ccgcagtccc tcctctgacg ccagagctgc tgatgctccc 14280tgccggcttc
gctgacaagc tggtgccttc agatcctttc cctggcccct ttaggctgag 14340actccgcttc
acaccccaac cccagctccg catcactgtt cccattcctg cttcaccccg 14400actctttcct
cttcccccac tcaccccgtt ccctttcctc tctctccagc tgtcactcct 14460tttctgccag
tatctcaggc aggcccctca ccctccaggg aagttgctgc ccggccctct 14520tttctctttg
tacccccagc cctgccctct cctcctcgaa gcccttctct ccccagtgtc 14580ccttatgcct
ctttctcttc tctcccactg gatactttct attccaactt caccgaggaa 14640taccaatgtc
tcagcgccag gctttccgag ttgacagcca ctctccggtt agctaatgtt 14700cactcttctg
tttccccttg ttccgagatg gatatgggtt gggggcaaga ccctgtggca 14760gaaaggagaa
tgacctgccc tgaggggtgc accagcccaa caggaagata ggacacaagc 14820cccgggcagg
gaggaccagg acagaggaga tgaggatagg aatctgtctg tttttctaga 14880gagataaagc
tggaaaggat ggtaatattt tgggtgagac agtcaggatt caaaacgctt 14940ttgaaaagca
agaataatga gccaaaaccc agcaagatga catttaaaat gaataaatat 15000aaaattctac
atttaggctt taaaaaaatc acttatgtaa gcacagcatg gaagagcact 15060ggtgaaaaaa
gaactgggag ttttagttgg ctacagtctt gatgtcgtag caatgtgatg 15120cagcctccaa
aatgattatg taatgttatc ctgggcccta ttagtgaaag catcatggcc 15180agaagagaga
gatggtgcgc gctctcttat gcacggagca ggccacagtt ggaaaattta 15240ctatactcaa
aatgcttaaa gggccctcct tggccattct ggcttgtaat caaaaaagta 15300gagttctgga
aaaccaggtc aaatgaggaa tcgtggagga agccagggat gttaagtcaa 15360gagagaaaac
atgagggaat ctgagactcc tgttttcaga tactcagagg actgtgaagt 15420gggaggggaa
tgaagccaag agttggaaat cccagggtac aggttttagc tctgtataaa 15480gaacaaccca
actattagag ctatcataca aaggagtggg ccctttatga agtggtgagc 15540tatcaatcct
gggaggtaat caagtataag ctagatgccc attgttagaa atgctccttt 15600ggggagccct
gtatggagtg agaagttgga ctagaggatc cctaaggtta gtttcaaggt 15660taagcttttt
ttggttggca tcaccaaatg acaggagggg aaaaaagagc tggacattaa 15720gaggagttgg
ggcaaatgga gaagacacga gggagctggg taagaacagg agctagggag 15780ggggggaaat
ggactggacc aaagggaggt gggagccctt aggaaggaat agaagggagg 15840gtgctgggag
tagggttgtg gaatgagaag aggagaggga agcctggagc tgagattccc 15900cctgaccggt
gcccctcctc ccaggaatga catgccaagc tcggagctcc tacctggtag 15960acgaggtgct
gtggggccac cgcttcacgt cagtgctgac tctggaggac ggcttctacg 16020aagtggacta
tgccagcttt cacgagactt ttgaggtgcc cacaccttcg tgcagtgctc 16080gagagctggc
agaggctgcc gcccgccttg atgcccatct ctactggtcc atccccagcc 16140ggctggatga
gaaggtggag gaggaggggg cgggggaggg ggcgggtggg gaagctgggg 16200ctgacaagga
gcagaatggc tgcctgccac ccccagagag tgagtccaag gtgtgaccag 16260cttcctccag
acccctgtgg cagaccgggg gccagacaca gatacatggg gaactgcata 16320tcggaggtgg
tggaggagga ggaggaggag gaaggcaaag cccctggaaa tgtgctaaag 16380ttggaaagtc
cccgtccccc agaacctcaa gtctagaaac cagtatggaa gggaggggtc 16440ctgatttcag
ggaaatggag ggtggggccg ggtgaaaatg ccagtctgtg tttgaccttc 16500acatttgttc
atgagtggat ggatggacag aatgatggac ttttgggggt tggatgggaa 16560gatggtagca
gataaagaca gctgacagat acatagatgg accagtagac aactggtcca 16620ctcagggctg
ccactaacct gtagaacacc cctgtgcaaa ttttaaaaag gaaccctttt 16680cctccagaca
gatacagccc caaaccaggg tgcatggctt ggggagcaga gtataggatg 16740gattgcagtc
cccagtcacc tcttctgcca gcctccccac atatggcaca actgtctaat 16800gacacggtag
gccaagctga agtgaaggag aaaggagccg gaccaagatg ggcacatgag 16860gagggtgccc
tcctagctcc accctcacca ggatgaaggc gtgcaagggg ctcagcaagg 16920tgtgaatgac
cttagtccgc aagttcaggg aagcaggcag agcggggagg tgcctgagct 16980ggggcctgga
gaggggcctg ggaaaggaaa accagggata gctattttct tacagtggag 17040tgagatctta
caggtatcag gcacaggcag gaagagagag agagaggttc tggggaggaa 17100gggccaggag
agagatctag aaagtgggtt cactagagct gggaaacagg gagcccctag 17160gaaagcagtg
tgtccttggg gcacagtcat tcacatcact gattgggtgc catgtggagt 17220ggacattcaa
aaacctggtt cctgtcctca aaataagggg cacctgggaa aacagaggaa 17280tctacctgtg
gtgactgaac gagggataat tcaaactgac aacctgtgca gtcccgtgga 17340gggtagggga
gtgtgggtga tcagaaggct ggggccagtg taaggcatag ggaatatgta 17400agtcaggagt
tagaaatctc cagtgtgcgt tggaatcacc tggagggctt ggtaaaacac 17460agatttttgg
gctccactcc aagggtttct gacccaagag gtggggacca aaaccatgca 17520ttcctaagaa
gtccccaggt catgctgctg ttgctggact gaggaccaca ctttgagaac 17580ctgtgctcta
agtgaatact tggaagtcgt ttcaggacat ggggcataga aactgaggag 17640tagctgagag
gaagatgaag agaagctgag aagaagctga ggatcctcac aggagcagac 17700agagaaatgt
gaagggtggg gttttatgtg tgggaaaggg acccgaagcc caggctgaag 17760agtttaactt
tgggcccaga aactcaacca tcaatggaaa cagggcagtg acaagtggag 17820ggggtgtctg
gaagctgagc aggcccgaca gagagatgaa gccatcagaa ggacttgagg 17880gggctcctgg
ggaggtcggg gggaggtgga gcaggaagag ttttaggggc aaaggacaga 17940accccttgta
ggactggagg caagattgaa tgtgggagaa aatcggagag aagcgatagg 18000agttagaaca
tctggatgtg tctgcagcct gctgtcagcc caattgggcc agggggtccc 18060aaagacgcat
attctcaccc cacctccacc tgcttcctga tcacatccca gtcaccagcg 18120gcagcttcct
ggatagtgag ggagaacaac tgcaagttga gagaggcaga ggggtggaag 18180ggacctgaag
ctggcctgga gaaaagcata ggcccaggag agcctgccct gggacagcgc 18240ctgtctccca
cacagcagca ctggcccagc aaggacctcc tcccttggcc ctggccacat 18300cccactcctg
ccctttcata agccccctgg ggaaagcact ccagtcttct ctgttccagg 18360ctgggcagat
agggtcctat ggggcacagc cagggtccta tgggcatagc cagggcccta 18420tgggtcctct
ggaagcaaga aagggggcca tggaagcagc ccagacagct ggggttcact 18480cagagaggac
ccaagtccca gtcccttcct ttcagtcaaa acacggatat ctttgcctca 18540ggtcacaggg
ccactggggc cctgtcatca aagatgagat tcctgaagcc tggcattgac 18600tggtccccta
agaacagatg ttgggatgga gaatggggat tcatttgggt ttcagtaaaa 18660caggggggtc
tggacaagag cgggtgggct acttggtatc cacacacacg cactcacaca 18720ggagccaacc
cattgcagct gaacaagcag agaaactcag tctggaaagg cccctcctgc 18780ctgctgaagt
cactgagacc ctgccacacc tctcctcgcc actgtcacca ctcagggcac 18840cactgtacag
tgcaacaagt caggagacct aggtcctact cctgacactt gctaattagc 18900tctatgactc
tgggcaaatc gcatatctgg gcctcagttt cctcatctgt aaaaatgaca 18960gcaaactcgt
aatgctcaat aaatgtttaa ataacaactg aaggaggcct gccagatgcc 19020tcttaaggtg
ccgtgcaggt aagaatttta ggatcagaga atccttaggc aagaaaattc 19080atgaaactcc
tggggcactg gaggaggggt gaagctgaag ggtgggaggg aggagacccc 19140agggtaggta
caggcaggtg aagcgggtat atgcaggtgt agtgggtata tgcgggtaga 19200gggtatatgc
aggtacagcg agtacatgtg ggtgcaatgg ctctgtggac acacaggccc 19260tcccctgact
gcctgttgtc ccagcctgag tatcagttgt gttctgaggc ttctattctg 19320ctgctatggg
tcagaaggaa caacaatttc agccccaggg cctagtggga ggagtcaggt 19380ccaagactag
cctgaccagg agaatgagac gtgggaagag ttggggaaag tctgggaagc 19440tcagaaaagg
cactgcccct ggaggcccat gccctttaac atgggagaag ctggtgcggg 19500ggtgaccaca
ggcagctgga acctaccctc cttttctatg cttccctccc caagtaggag 19560tccaatcagg
agttgtctca gccccgacag ttcaggctgc agatggaacc caggtgtccc 19620ctcctggggt
gggtggcatg gcccatggag gccagatggt gtttgtggtg ggaagagagg 19680cctgggtcat
ccagaatagg ttgtcaatcc ccaaccacct ccctactatg caccctgagc 19740gttttacagt
ctcatggtag ggaagacaca gccaagcctg ctttttataa aacaagttta 19800ttcacatttt
agaaaaacta attccaggac aggaaatggc ctccctatag gatccctaag 19860agatcaagaa
cagaaggcca gagggagggg cttgggaggg aaggagtggg gaaggggagg 19920cacgtctccc
attctgggta gtgggaggtc aaataaatta aaggaagagt ggacagaggg 19980agagggtgtc
caggcaacca gaggagggct tggagctggg ccggaagaca gtcgacacct 20040gcaagacctg
aaaagggtgc ccggtgtggg ctaaggacag agagccctga gtggggctcc 20100ctcgcggcct
ccacccctta acagggccct gtggatctga gctgcctact cctcctccag 20160gtggggcctg
ggagggagca gcttggttca ggacttgggg gtgggaagcc caatgaaaac 20220aaggttgggg
ggttcttttc cctcacctgg ggagtaaggg atcaccgttt tcgaagcctc 20280ttcatgaagc
agcaagtgat ggtaccaagg acagtggcac cagtgactag ggccacccct 20340gtacccacca
gcagaggcac aaatagggtg tccagggctg ggggagagag gatgactgtt 20400cagagaggat
gccatcatcc tccacccata cacttgcctc tgcgctttcc ccatcaagtt 20460ctctgaaccc
accttctcca ttcacagaca cccccatccc tgcccacagc ctgccccctc 20520agcatgcaag
tcagcatcaa ccacagagga ccccgtgcag gtgggcactg cagggctgga 20580agttggattt
tttgagactt catgtgacat aatgtggagg agagagatag tagcaggagg 20640gtcagaagat
gggaagggaa ggccagtggc agaggccagg aggaaggcag agtgaggagg 20700gtggaggggg
tgtcactcac catgcatgta ggggtagact gtaacaggcc ctgagcgggc 20760actgcccgcc
tggtaccagc tgtagtcggc atgctgcacc caggcgctgg gggcacagtg 20820gtacacgcct
tcatcctcgg gccccaagct gtgtagtctc agccgatggc ttcggggccc 20880caccagctct
acgctgacag ggcctcctcc aggccggact cccagctctg ccacaccatc 20940ctggcctacg
ccacccacca gctgggcagg gacagagctg agctctccgt cctctggtcg 21000ctccacccac
cagctggcgg ccagccgcag tcctgggggg ccaccccgca cagagatgtt 21060gcacagcagg
gaggcagtct ccccgcggta cactgtgcct cctgctagcc atgccacagc 21120ctccagcacc
acacctgcag aacaaaggac atggggtcag agggtgcagg gccagggagc 21180atggggttag
ggctgccgcc aagcaccgcc ccaggaaact cagggtattc ccacaatctt 21240ggtagaagag
gagcgtgagg ctgtggcctg caaacagctg acggagaggg aggggtcatg 21300gaaacagaag
gaaaaggggt tgacaatcct cgaaccccgt ccagggccca gccccctctc 21360accttcctcc
cgcacatgta cagggagagg ccgggaacgg gcactggctg cttcacgaag 21420ccgggtccca
gaccctcgaa cataggcttt ggcgaggcag cggtaggtgc ccgcatcacc 21480aggcctggca
gcctctagcc gtagccggta tgttctggat gccaccttct ccatggcaat 21540gtgtcggccc
tcatagccag ggcccaggct gcccacaccc tctgtgtcca gctgggctac 21600caggcggccg
ggccccaggt gcccccgcag gtgccatctc ccaacctaca gagtatgcag 21660catgacggcc
tgctggggga agtgcccctg acacattgca cagcagttcc aagggctccc 21720ctgggccgat
ccgacgttca ccaggcccca ctgtcactgc cagctggctg gctgaaacac 21780aggtagggga
agaggtgtca tggaggcagg aggggacaca gaggcacccg attccccaac 21840ttcctgtttc
ctacttgaca gcagcaactt caaaacctcc tgtctccccc tcactaggta 21900tgaccatctt
tctatttagg ggcttgaatc tcacccctca gcatgggcct cctatctcta 21960tacccaattt
ctgagcagag aaaacccatc aagggccggg ggagagaaat gctagcaagg 22020ctgctcactc
tgtggaagat gagttccttg gagtcagatg atggctatct ggtaccccct 22080gtggccacag
tgcccaccag gatactgtcc ctcccagctc ccacagtggg atgtataagt 22140ggcacttaca
cagcgtctgc acatccacgt gggccaggac ggcccttttc tctgcaatct 22200gggcccagct
gccatcagga tcctgaatcc actcagcggc agtgcagtgg taggtgcctg 22260cgtcccctgc
ctgggcaccc cctactacca tgcggtaccg atcggtccct tccttgccca 22320gacgaagctc
ccctgcagcc aatcgctcag catagggagc tccagcctcc acggccaagt 22380ctgaccggat
tcccaccact tcctgcagag ttgaccgccc aactggtgcc tcgggcacag 22440atcgcccaaa
ggacactgcc aggtgtgtgt gcttctgtgt gcttgtcctc gccaggcagc 22500ccagtgccag
ctcctgcccc tcatgcaccg tcatgcgtgg gggtgaggtt ggggcctggc 22560ggcctcgggg
ccctgggggg gcagcagaca cctggaggac atctggaaga actggagaga 22620acagctggag
tgagggaggg ctgggagctg gcagcccttg ttactgtttc ctgtgtatag 22680cctatctccc
taaataaact gtgagctccc agagggcaaa gatcgcatgt tgtattattt 22740cttctgtaac
tcagtggtgc caagggcagt actgggcaca gcacaggcgc tcaataaata 22800cttgtagaat
ttcatagaac cagcccatcg cctactcacc cttatgtttg agactgacct 22860ctgtttgaaa
tactgagaaa agcggctctt tcttctcaga agacaaagaa acttaagaga 22920gtgagaatgt
cacatggtct aactccttcc ctaactctac tctctttccc agatctgggt 22980cctgtactgt
ccaggagtag aggctattca acccaacagt cttcttcgtt cttgggaatg 23040gaaagtggac
tggacaactt aaggacattt cttctcccag gaggggtctt aatatgataa 23100gatgagcact
ggcctgggtg aggaactctg ggtttgagtc ccacatcagc cactgagtta 23160ttgggtgact
ttgtgcaaat cacttaacct ctttgggcct caagttcctt ggctacaaaa 23220cctaaggggc
aactagatag gtcacttgtg gccttgactt tctgccttga gagggtgtgt 23280ggctccaccc
cgtcccaggg cccagtacct ctcagctcca ccttgccgct gtagctgccc 23340aggtagcggg
tatcagtgga gggggtgtgg cactcataaa tgccggcatc ctgggcctgc 23400aggcgggcaa
tcttgagcac cacggcatca ccttgtaggc gctgcacctg cacctcaccc 23460gccaccactc
gggacttgaa gacagcatag gagaactggg tatccttggt actgacaatg 23520cccagtgcag
tatctggggc ctcgggccta tacaggaacc actcgaagtt ctgctgggca 23580gggccctcat
agccggtcac attgcaggag atggagacag ctgtgccagc cacgcggtac 23640aagggcccct
cggggaccag cacctcccgg gcccagcatc ccattcctgt agggaaaggc 23700agaaggagtt
ggagatgcct ggttcctcat tccatgccct ctgccgccac aagcaccatt 23760cttgatctct
gcctacaaaa ggaaaggaga cctgggaaag cttgtccaca gcttggaccc 23820tgttctgaga
ataggaaagg gatgctgtga tataagacac ctggatctca aggaggtggc 23880atgggcccag
gattgccttg gcatccagat gcatcccatt tctggcggac tagaagcaga 23940gcacctgaag
gcagaaagga gtacatctga ttcctgacct aaccaggcct tggttccaac 24000tgaaccttga
tctgtccctg ccactcaccc acctccatgt ctgccattcc ttcctcagca 24060cctggcaagg
ggagccttct ggctagggga ctctgagact acatgtccct ctcctttgct 24120tgaggggagc
tggcagtctt gctcagaagt gctagttggc tcagctgtgt cacctgggcg 24180agacaatgga
gccagtgacc ctagctggaa agggcacagg cccagtcagt tctcaccaca 24240caatgccctc
ccctctccag ctgcgccatg agctcactgc ttctctcacc ccacagggct 24300gcccaggcag
ctggggcttc tggggcaaga tccaggctct gccctggcca ttgggggcag 24360aagatcccct
cctccagtgc ctgccaacct tccgggctag cccagcagat acagaaggtg 24420cctgccccag
ttccttaaca aaagccttca tttgcacatg gtatgcattc atttacatat 24480atggctctct
ttctgtaggg aggcactaaa tccccagctg ccccttctca tctctctccc 24540ttcagaaagg
ccaaacctct cttcttcacc ctactccacc cctatgccca accctacccc 24600agcagatact
cctggcagac ttagagggct tagctcctcc cttctttcct tccatagctc 24660ccactagata
agatcacaga acctcaatgt aaagagggct aggccacccc tccccacctc 24720tcccaatttt
acagatgaga aaggtaaggc aggaaaagta taatatgtta gccaagatca 24780tgctgtccct
agatggcttc cacacactcc tccagagggg caaaaccaga gaggaagatg 24840gggaaactcc
aaggccaggc ctgaagggac tgacctcacc aaccagagtg tcacttttag 24900gcctcccagg
gggataccat ggactttctg caggagctag aggaaaatgc ccaggagtct 24960gtggtcaaac
tctaccctcc agcttctcta gaacggctcc tctgaacttc cccacccctg 25020cttctgggct
cctagcccct tccttcatcc tctggctggg tcacagggag aactcatggt 25080ctgttgttaa
gggcacagct gccagtcagg aagtgggatt ccagcaccat ccccatgccc 25140agctgtgtgg
cctgggatcc agtctctttc tgtcctaggc ctcagtttcc acactggagg 25200agaactaaga
gctccagctc tgaccatgtg tgagtgcgta tgtgactcag gagagccctg 25260ccccaggcca
ggcaagtttc ataatcagag tgacggtgga gacagccaag ctgacacctt 25320ccctgactgc
ctcagggcag actgctcaga aggccccctc ccattttcct ggctccacaa 25380ctgctgatgc
ttggagatgc ccatgggaaa gtcacctcca cagccttagg aaatcagttg 25440ccacacagct
ctctctcccc tcctctgtat cagtcgcagc aaggaaaggg acagcaaaga 25500ggcctgcttt
ggaatcagat ctgtgttcaa atcctagccc caacactcac taaatgtgct 25560ctctggggca
agttacttca ttttcctcat ttgtgaaatg aatgtaagtg cccacaggca 25620gtgggtgctc
agacctctgc gtgctccttt ttcaaacaca ggccagcact tccccacctc 25680cctgggctcc
tccctgctcc atgctgccca ctggggaaaa cacaccaagt gctaggcaac 25740ccaggcccca
cagcgccttc ctctctgtac atcctcctgc cacctgccca gggaccaggg 25800agaggactca
tcctaaccct gcagggccca gggacctgca gcaggggaag gctttgcttg 25860gtgccactgt
ggagctctgg tctagaaaca ggcagctggg gctaccttca gcctctgcct 25920tgacgacagc
agctctgaag tcaccatccc cacccccacg cttcactctc atttcaaggg 25980cttcagcctc
atcaacatct gtactggcag tttcactgtc tccatgccat actcttcccc 26040agaccacctc
ctacagggag ccctccagtt caggccaaaa acaattccac tgtcattatc 26100cccatgcatc
catgcaagat tggcccagaa caccccacca tgaacaccca ccacagcagg 26160cacaaggtgc
ttggagatcc caggatcagt ctccatggaa cctggtttct cctgaggcaa 26220ggaagctgga
actaagcggt gtgaaaactg atgggtggct gcagagccaa gtgccatttg 26280ggagacagga
agaagggcaa agagggaccc aacccagggt ggagatgggg gtgagagagg 26340gaactgcccc
cagttgatga agtgcgtgga gcgcaactgg gagagactta cttcaaagat 26400cgtgggcaga
actggcctct gggcctccag ccaactctgg ggcaattatg aagctgggca 26460ggcactgccc
tcgtagggcg ggcacccaag gccaggcctg gagctgagtg tggggcagaa 26520aggagtcgca
gcatttggtg cagcgacccc agtacgtggg tatgctagct gagatgtgtg 26580gcctgccccg
ggaggccgag cagtgcctgg ggcagcacct tagtgggtcc tctctacgcc 26640ccagtccctg
gcttagagct ggggagcctg cactcttccc aagactggct cggcggacag 26700ccacaaagcg
cagctggacg ccgaccccgg ggaggctgga ggtacccctg acggaggagg 26760atgtgaggag
ccccgaaatg ctaggggggt gctggatggc aggcacctgc ccggcagggc 26820cgggaaccgg
aacgggggcc tggcttacct agcattagca gcagcagcag cggcagcgaa 26880ggcggcagca
gcgtgggcct gagggcgccc atcctgcgcg gccagctctg gggaggctcc 26940gggggatggc
gcgggttctg gggggccgga agggtggggg gcgcatgccc aggttgaggg 27000caggaagcgg
ggcagcgagg cgtgggtgcg ccgagcgagc tgaactggag ctgccgaatc 27060ccctccctcc
gcccctcccg ctgctttccc tccagccctc ggcagttctg aaaccattct 27120cgccccggcc
cgccccggca ccgccccttc caccgccccg tctaggcccg ccaggactac 27180agtcggactc
caatcctggc tcctccccgg gccccggccc cgccccagtc ccaagccgca 27240ccccttcccc
gtccccgcag ggctaacgtc agcctccaat cctggctccg ccctggaccc 27300cggcctcgcc
ccgcccctgg ccctggctcc gcccgaggcc cccgcaggag tgagctaact 27360gcacctctgc
gcatcgaaat tcccacccac cctcgcacag agcgcattcc accccgcacc 27420tgccagcctt
tcctggagag ttgggtgcag ggtccctggg attggcgagg tgactgtgac 27480cacgcattta
gaattcagtt atttgctctg agccatagtc ctcgctgcaa accctgctga 27540agtaggggtt
ggcggaagcc aggagttcct gaatgcgaag ggtttgagct gaagggcgct 27600tccaggatcc
agaaggtcac tggagacctg tttttcaccc cctcagaggg caaaaccaaa 27660agaaaaatgg
attaggagag gggg 2768453029DNAHomo
Sapiens 5acatttagga gaaacagcgg tgtctgcggc tcccaccctt cggggggccc
gtgggggggg 60cggtgtcagg ggcatggacg ccacccccca ggggtctctg ctgccggcta
ctctcctctc 120cacgtgctcc cctccaggac ccccgacgcc gcctaggcgc ccagcgacgc
gcggcaggtg 180gcagcagctc gggcccccgc cgcactccag gcgcccgcag cgctcgccct
gacgcggccg 240ccatggcgca ggagaacgcg gccttctcgc ccgggcagga ggagccgccg
cggcgccgcg 300gccgccagcg ctacgtggag aaggatggcc ggtgcaacgt gcagcagggc
aacgtgcgcg 360agacataccg ctacctgacg gacctgttca ccacgctggt ggacctgcag
tggcgcctca 420gcctgttgtt cttcgtcctg gcctacgcgc tcacctggct cttcttcggc
gccatctggt 480ggctgatcgc ctacggccgc ggcgacctgg agcacctgga ggacaccgcg
tggacgccgt 540gcgtcaacaa cctcaacggc ttcgtggccg ccttcctctt ctccatcgag
accgagacca 600ccatcggcta cgggcaccgc gtcatcaccg accagtgccc cgagggcatc
gtgctgctgc 660tgctgcaggc catcctgggc tccatggtga acgccttcat ggtgggctgc
atgttcgtca 720agatctcgca gcccaacaag cgcgcagcca cgctcgtctt ctcctcgcac
gccgtggtgt 780cgctgcgcga cgggcgcctc tgcctcatgt tccgcgtggg cgacttgcgc
tcctcacaca 840tagtggaggc ctccatccgc gccaagctca tccgctcgcg ccagacgctg
gagggcgagt 900tcatcccgct gcaccagacc gacctcagcg tgggcttcga cacgggagac
gaccgcctct 960tcctcgtctc gccgctggtt atcagccacg agatcgacgc cgccagcccc
ttctgggagg 1020cgtcgcgccg tgccctcgag agggacgact tcgagatcgt cgttatcctc
gagggcatgg 1080tggaagccac gggaatgaca tgccaagctc ggagctccta cctggtagac
gaggtgctgt 1140ggggccaccg cttcacgtca gtgctgactc tggaggacgg cttctacgaa
gtggactatg 1200ccagctttca cgagactttt gaggtgccca caccttcgtg cagtgctcga
gagctggcag 1260aggctgccgc ccgccttgat gcccatctct actggtccat ccccagccgg
ctggatgaga 1320aggtggagga ggagggggcg ggggaggggg cgggtgggga agctggggct
gacaaggagc 1380agaatggctg cctgccaccc ccagagagtg agtccaaggt gtgaccagct
tcctccagac 1440ccctgtggca gaccgggggc cagacacaga tacatgggga actgcatatc
ggaggtggtg 1500gaggaggagg aggaggagga aggcaaagcc cctggaaatg tgctaaagtt
ggaaagtccc 1560cgtcccccag aacctcaagt ctagaaacca gtatggaagg gaggggtcct
gatttcaggg 1620aaatggaggg tggggccggg tgaaaatgcc agtctgtgtt tgaccttcac
atttgttcat 1680gagtggatgg atggacagaa tgatggactt ttgggggttg gatgggaaga
tggtagcaga 1740taaagacagc tgacagatac atagatggac cagtagacaa ctggtccact
cagggctgcc 1800actaacctgt agaacacccc tgtgcaaatt ttaaaaagga acccttttcc
tccagacaga 1860tacagcccca aaccagggtg catggcttgg ggagcagagt ataggatgga
ttgcagtccc 1920cagtcacctc ttctgccagc ctccccacat atggcacaac tgtctaatga
cacggtaggc 1980caagctgaag tgaaggagaa aggagccgga ccaagatggg cacatgagga
gggtgccctc 2040ctagctccac cctcaccagg atgaaggcgt gcaaggggct cagcaaggtg
tgaatgacct 2100tagtccgcaa gttcagggaa gcaggcagag cggggaggtg cctgagctgg
ggcctggaga 2160ggggcctggg aaaggaaaac cagggatagc tattttctta cagtggagtg
agatcttaca 2220ggtatcaggc acaggcagga agagagagag agaggttctg gggaggaagg
gccaggagag 2280agatctagaa agtgggttca ctagagctgg gaaacaggga gcccctagga
aagcagtgtg 2340tccttggggc acagtcattc acatcactga ttgggtgcca tgtggagtgg
acattcaaaa 2400acctggttcc tgtcctcaaa ataaggggca cctgggaaaa cagaggaatc
tacctgtggt 2460gactgaacga gggataattc aaactgacaa cctgtgcagt cccgtggagg
gtaggggagt 2520gtgggtgatc agaaggctgg ggccagtgta aggcataggg aatatgtaag
tcaggagtta 2580gaaatctcca gtgtgcgttg gaatcacctg gagggcttgg taaaacacag
atttttgggc 2640tccactccaa gggtttctga cccaagaggt ggggaccaaa accatgcatt
cctaagaagt 2700ccccaggtca tgctgctgtt gctggactga ggaccacact ttgagaacct
gtgctctaag 2760tgaatacttg gaagtcgttt caggacatgg ggcatagaaa ctgaggagta
gctgagagga 2820agatgaagag aagctgagaa gaagctgagg atcctcacag gagcagacag
agaaatgtga 2880agggtggggt tttatgtgtg ggaaagggac ccgaagccca ggctgaagag
tttaactttg 2940ggcccagaaa ctcaaccatc aatggaaaca gggcagtgac aagtggaggg
ggtgtctgga 3000agctgagcag gcccgacaga gagatgaag
302961182DNAHomo Sapiens 6atggcgcagg agaacgcggc cttctcgccc
gggcaggagg agccgccgcg gcgccgcggc 60cgccagcgct acgtggagaa ggatggccgg
tgcaacgtgc agcagggcaa cgtgcgcgag 120acataccgct acctgacgga cctgttcacc
acgctggtgg acctgcagtg gcgcctcagc 180ctgttgttct tcgtcctggc ctacgcgctc
acctggctct tcttcggcgc catctggtgg 240ctgatcgcct acggccgcgg cgacctggag
cacctggagg acaccgcgtg gacgccgtgc 300gtcaacaacc tcaacggctt cgtggccgcc
ttcctcttct ccatcgagac cgagaccacc 360atcggctacg ggcaccgcgt catcaccgac
cagtgccccg agggcatcgt gctgctgctg 420ctgcaggcca tcctgggctc catggtgaac
gccttcatgg tgggctgcat gttcgtcaag 480atctcgcagc ccaacaagcg cgcagccacg
ctcgtcttct cctcgcacgc cgtggtgtcg 540ctgcgcgacg ggcgcctctg cctcatgttc
cgcgtgggcg acttgcgctc ctcacacata 600gtggaggcct ccatccgcgc caagctcatc
cgctcgcgcc agacgctgga gggcgagttc 660atcccgctgc accagaccga cctcagcgtg
ggcttcgaca cgggagacga ccgcctcttc 720ctcgtctcgc cgctggttat cagccacgag
atcgacgccg ccagcccctt ctgggaggcg 780tcgcgccgtg ccctcgagag ggacgacttc
gagatcgtcg ttatcctcga gggcatggtg 840gaagccacgg gaatgacatg ccaagctcgg
agctcctacc tggtagacga ggtgctgtgg 900ggccaccgct tcacgtcagt gctgactctg
gaggacggct tctacgaagt ggactatgcc 960agctttcacg agacttttga ggtgcccaca
ccttcgtgca gtgctcgaga gctggcagag 1020gctgccgccc gccttgatgc ccatctctac
tggtccatcc ccagccggct ggatgagaag 1080gtggaggagg agggggcggg ggagggggcg
ggtggggaag ctggggctga caaggagcag 1140aatggctgcc tgccaccccc agagagtgag
tccaaggtgt ga 118275PRTUnknownCytokine receptor
extracellular motif found in many species 7Trp Ser Xaa Trp Ser 1
5
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