USRE38392E1 - Method for identifying metastatic sequences - Google Patents

Method for identifying metastatic sequences Download PDF

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USRE38392E1
USRE38392E1 US09/985,799 US98579901A USRE38392E US RE38392 E1 USRE38392 E1 US RE38392E1 US 98579901 A US98579901 A US 98579901A US RE38392 E USRE38392 E US RE38392E
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cells
sequence
seq
cell
metastasis
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Timothy C. Thompson
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Baylor College of Medicine
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Priority claimed from US09/977,371 external-priority patent/USRE38490E1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification

Definitions

  • the present invention relates to methods for the identification and isolation of metastatic sequences, to diagnostic probes and kits which contain metastatic sequences and to therapeutic treatments for neoplastic disorders based on metastatic sequences.
  • Cancer cells are defined by two heritable properties, uncontrolled growth and uncontrolled invasion of normal tissue.
  • a cancerous cell can divide in defiance of the normal growth constraints in a cell leading to a localized growth or tumor.
  • some cancer cells also gain the ability to migrate away from their initial site and invade other healthy tissues in a patient. It is the combination of these two features that make a cancer cell especially dangerous.
  • a tumor or neoplasm is counted as a cancer if it is malignant, that is, if its cells have the ability to invade surrounding tissue.
  • True malignancy begins when the cells cross the basal lamina and begin to invade the underlying connective tissue. Malignancy occurs when the cells gain the ability to detach from the main tumor mass, enter the bloodstream or lymphatic vessels, and form secondary tumors or metastases at other sites in the body. The more widely a tumor metastasizes, the harder it is to eradicate and treat.
  • Malignant cancer usually begins as a benign localized cell population with abnormal growth characteristic called a dysplasia.
  • the abnormal cells acquire abnormal growth characteristic resulting in a neoplasia characterized as a cell population of localized growth and swelling. If untreated, the neoplasia in situ may progress into a malignant neoplasia.
  • Several years, or tens of years may elapse from the first sign of dysplasia to the onset of full blown malignant cancer. This characteristic process is observed in a number of cancers.
  • Prostate cancer provides one of the more clear examples of the progression of normal tissue to benign neoplasm to malignant neoplasm.
  • the walnut-sized prostate is an encapsulated organ of the mammalian male urogenital system. Located at the base of the bladder, the prostate is partitioned into zones referred to as the central, peripheral and transitional zones, all of which surround the urethra. Histologically, the prostate is a highly microvascularized gland comprising fairly large glandular spaces lined with epithelium which, along with the seminal vesicles, supply the majority of fluid to the male ejaculate. As an endocrine-dependent organ, the prostate responds to both the major hormone, testosterone, and the major female hormones, estrogen and progesterone. Testicular androgen is considered important for prostate growth and development because, in both humans and other animals, castration leads to prostate atrophy and, in most cases, an absence of any incidence of prostatic carcinoma.
  • BPH benign prostatic hyperplasia
  • prostatic carcinoma a type of neoplasia.
  • BPH benign prostatic hyperplasia
  • prostatic carcinoma a type of neoplasia.
  • BPH is very common in men over the age of 50. It is characterized by the presence of a number of large distinct nodules in the periurethral area of the prostate. Although benign and not malignant, these nodules can produce obstruction of the urethra causing nocturia, hesitancy to void, and difficulty in starting and stopping a urine stream upon voiding the bladder. Left untreated, a percentage of these prostate hyperplasia and neoplasias may develop into malignant prostate carcinoma.
  • Metastasis defined as tumor implants which are discontinuous with the primary tumor, can occur through direct seeding, lymphatic spread and hematogenous spread. All three routes have been found to occur with prostatic carcinoma. Local invasions typically involve the seminal vesicles, the base of the urinary bladder, and the urethra. Direct seeding occurs when a malignant neoplasm penetrates a natural open field such as the peritoneal, pleural or pericardial cavities. Cells seed along the surfaces of various organs and tissues within the cavity or can simply fill the cavity spaces.
  • Hematogenous spread is typical of sarcomas and carcinomas. Hematogenous spread of prostatic carcinoma occurs primarily to the bones, but can include massive visceral invasion as well. It has been estimated that about 60% of newly diagnosed prostate cancer patients will have metastases at the time of initial diagnosis.
  • Surgery or radiotherapy is the treatment of choice for early prostatic neoplasia. Surgery involves complete removal of the entire (radical prostatectomy), and often removal of the surrounding lymp nodes, lymphadenectomy. Radiotherapy, occasionally used as adjuvant therapy, may be either external or interstitial using 125 I. Encodrine therapy is the treatment of choice for more advanced forms. The aim of this therapy is to deprive the prostate cells, and presumably the transformed prostate cells as well, of testosterone. This is accomplished by orchiectomy (castration) or administration of estrogens or synthetic hormones which are agonists of luteinizing hormone-releasing hormone.
  • the present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new methods for the identification of sequences related to metastasis.
  • One embodiment of the invention is directed to methods for the identification of a metastatic sequence.
  • One or more oncogenic sequences are transfected into a cell to form a transfected cell.
  • the transfected cell is introduced into a primary site of a host animal to establish a colony which is incubated in the animal for a period of time sufficient to develop both a primary tumor and a metastatic tumor.
  • Expressed sequences are harvested from the primary tumor and the metastasis.
  • Harvested sequences are compared to each other and to non-metastatic cells to identify sequences related to metastasis.
  • Dominant metastatic genes are genes whose expression leads to metastasis. Such genes are typically expressed at high levels in metastatic cells and not significantly expressed in normal or nonmetastatic cells.
  • Recessive metastatic genes genes whose expression prevents metastatis, may be selectively expressed in normal and nonmetastatic cells and absent in metastatic cells. Dominant and recessive metastatic genes may act directly or act pleiotropically by enhancing or inhibiting the expression or function of other dominant and recessive metastatic genes.
  • Another embodiment of the invention is directed to methods for identifying metastatic sequences.
  • a mammalian cell is treated with a metastatic agent and the treated cell is implanted into a primary site of a host mammal.
  • the host animal is maintained for a period of time sufficient for the cells to proliferate and to develop a metastatsis metastasis at a secondary cite site.
  • Expressed squences from cells of the primary cite and cells of the secondary site are reverse transcribed into cDNA by differential display polymerase chain reaction to identify differentially expressed sequences.
  • Sequences may be in the form of DNA, RNA or PNA.
  • the nucleic acid may be single-stranded or double-stranded. Single stranded nucleic acid may be in the form of a sense strand or an antisense strand.
  • the sequence may be part of a homologous recombination vector designed to recombine with another metastatic sequence.
  • Another embodiment of the invention is directed to a method for treating a neoplastic disorder comprising administering a pharmaceutically effective amount of a metastatic nucleic acid to a patient.
  • the nucleic acid may be single-stranded in the sense or the antisense direction.
  • the nucleic acid may be packaged in a viral vector such as, for example, a retroviral, a vaccinia or an adenoviral vector.
  • Administration may be performed by injection, pulmonary absorption, topical application or delayed release of the nucleic acid along with a pharmaceutically acceptable carrier such as water, alcohols, salts, oils, fatty acids, saccharides, polysaccharides and combinations thereof.
  • Another embodiment of the invention is directed to a kit for detecting of the presence or absence of a metastatic sequence.
  • FIG. 1 Schematic showing two paths in the multistep progression to cancer.
  • FIG. 2 A-B Staining of primary tumor (A) and metastatic deposite (B) from the lung of the same animal
  • FIG. 3 A-D Staining of normal human prostate (A), moderately differentiated human prostate tumor (B and C), and poorly differentiated prostate tumor (D).
  • FIG. 4 Schematic of method for isolating a metastatic gene from a gene ablated mouse strain.
  • FIG. 5 A-B Schematic showing method to establish a tumor and metastatic transplant from fetal tissue(A) and from cell lines and tumors (b).
  • FIG. 6 Isolation and characterization of nmb gene expression by DD-PCR and RNA blot in primary and metastatic cells.
  • FIG. 7 Differential expression of multiple genes is determined by DD-PCR and RNA blot of primary and metastatic cells.
  • FIG. 8 Caveolin identified as a differentially expressed gene by DD-PCR.
  • FIG. 9 Differential expression of genes isolated by DD-PCR confirmed by RNA blots.
  • FIG. 10 RNA blot analysis of total tumor mRNA using clone 29 GADPH probes.
  • FIG. 11 RNA blot of three independent MPR metastatic tumors and 5 MPR non-metastatic tumors.
  • FIG. A-RR 12 Nucleotide sequences of metastatic nucleic acids.
  • FIG. 13 A-D Characterization of metastatic sequences isolated.
  • FIG. 14 Immunohistological staining of primary and metastatic human prostate tumors using anti-caveolin antibodies.
  • the present invention is directed to methods for identifying metastatic sequences, to the metastatic sequences identified, to methods for the detection, diagnosis and treatment of disorders related to metastasis, and to diagnostic kits which comprise these sequences.
  • Malignant cancer involves a multistage progression from, for example, normal tissue through hyper plasia, early adenoma, early carcinoma and finally to a metastatic tumor (FIG. 1 ).
  • Cells of a typical tumor loosen their adhesion to their original cellular neighbors and cross the basal lamina and endothelial lining to enter the body's circulation. Once in circulation, the metastatic cell exits from the circulation to disseminate throughout the body and proliferate in a new environment.
  • the ability of a cell to metastasize requires additional mutationic or epigenetic changes.
  • An understanding of the molecular mechanisms of metastasis allow for the design of treatments to inhibit metastasis.
  • Knowledge of stage specific gene expression for neoplastic disorders allows for early detection and typing of tumors. With early detection and typing, proper treatment may be administered to a patient with the neoplastic disorder earlier, which will lead to a higher probability of a complete cure.
  • stage specific tumors For human prostate tumors, the study of stage specific tumors is difficult, if not impossible, as cell lines are extremely difficult to grow and it is rare that tissue becomes available from the primary tumor as well as metastatic disease from the same patient. This problem is exacerbated because of the infrequent biopsy of metastatic deposits in conjuntion with isolation of material from the primary tumor. Furthermore, the growth of cell lines from malignant prostates has proved to be problematic over the last few decades. This is evidenced by the lack of cell lines from prostate cancer obtained under any conditions.
  • One embodiment of the invention is directed to a method for identifying a metastatic sequence.
  • a mammalian cell is transformed into a pre-neoplastic or neoplastic state or phenotype by transfection with one or more oncogenic sequences.
  • the mammalian cell may be treated with an agent or subjected to a condition that potentiates the metastatic character of the cell or predisposes the cell to metastasis.
  • the transfected or treated cell is implanted into a host animal at a primary site and grown for a period of time sufficient to develop a metastasis at a secondary site. Expressed sequences from cells of the primary site and cells at the secondary site are amplified by differential display polymerase chain reactions. PCR products from these reactions are compared and the metastatic sequence identified by alteration in the levels or patterns of the resulting products.
  • Mammalian cells from a wide variety of tissue types and species are suitable for transection or treatment including surgically obtained or primary or immortalized cells and cell lines.
  • Cells may be from humans or primates, mice, rats, sheep, cows, rabbits, horses, pigs or guinea pigs or from transgenic or xenogenic host mammals.
  • Cells may be obtained from adult, juvenile or fetal tissue, and used directly from the mammal, from cryogenically preserved samples, or after culturing in vitro or in vivo for a period of time. In vitro culturing typically involves tissue culture conditions (e.g.
  • tissue type from which the cells are derived or obtained may be any tissue which is susceptible to transfection or other treatment including, for example, urogenital tissues, epithelial cells, hepatic cells, fibroblasts lymphatic tissues, hematopoietic cells, cells of the immune system, cells of the gastrointestinal system and cells of the nervous system.
  • Cell types useful for the identification of metastatic sequences related to prostate cancer include cells and cell lines of the fetal prostate lineage from normal or transgenic animals, and cells from normal or reconstituted prostate tissue.
  • One method of generating reconstituted prostate cells is to isolate fetal prostate tissue and microdissect the fetal prostate epithelium away from fetal mesenchyme.
  • Fetal prostate epithelium may be genetically manipulated before reassociation with fetal mesenchyme (FIG. 5 A). Genetic manipulation involves treatment or transfection with a metastatic agent or a nucleic acid sequence that affects neoplastic or metastatic potential of the cell.
  • Reassociation of fetal epithelium and mesenchyme is performed by implanting epithelial tissue within a pocket of mesenchymal tissue. After manipulation, cells are reimplanted into a mammalian host in a similar manner as other cells, such as reimplantation into or under the renal capsule.
  • Mammalian cells may be transfected by a variety of techniques, all of which are well-known to those of ordinary skill.
  • Direct methods involve the introduction of genetic material into the nucleus of a cell by injection. These techniques include high velocity projectile injection, microinjection, and electroporation.
  • Indirect methods involving the active or passive uptake of the genetic information by the cell, include transduction with recombinant vectors, and chemical or physical treatments such as calcium phosphate uptake, lipofection or dextran sulfate transfection.
  • Chemical techniques rely on chemical carriers to introduce nucleic acids into a cell. These methods, for example, utilize unilamellar phospholipid vesicles (e.g. liposomes) loaded with DNA (or RNA).
  • DOTMA N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTMA spontaneously associates with nucleic acids and forms unilamellar vesicles upon sonication. Genetic material is incorporated into these vesicles and subsequently transfected into the cell.
  • Calcium phosphate co-precipitation involves mixing of purified nucleic acid with buffers containing phosphate and calcium chloride which results in the formation of a fine precipitate. Presentation of this precipitate to cells results in incorporation of the nucleic acid into cellular genome.
  • Other chemicals such as DEAE dextran or polybrene, when present in media with nucleic acids, can also cause the transection of mammalian cells.
  • Electroporation is based on the principle that membranes, when subjected to an electric field, undergo a reversible breakdown resulting in pores large enough to permit the passage of nucleic acids.
  • micro-projectile mediated gene transfer micro-projectiles of subcellular dimensions are coated with nucleic acid and propelled at high velocity into a cell using a particle gun. The nucleic acid is introduced into the nucleus directly when the particles impinge upon the nucleus.
  • microinjection nucleic acid is injected directly into the nucleus of a cell with a needle. Lasers have also been used to introduce minute holes in cellular membrane to allow introduction of nucleic acids. All these methods may be used for transfection and the selection of the method will depend on the cell type, the desired transfection efficiency and the equipment available.
  • the efficiency of transfection may be monitored and enhanced by the co-transfection of a selectable marker. If a marker is co-transfected with a genetic construct, positively transformed cells may be separated from nontransformed cells by chemical selection. The efficiency of transfection will be increased in most cases because the chemicals will selectively kill non-transfected cells. The number of transfected cells may also be monitored by analyzing the degree of chemical resistance of the transfected cells.
  • Markers commonly used for selection purposes include, for example, nucleic acids encoding dihydrofolate reductase, metallothionein, CAD, adenosine deaminase, adenylate deaminase, UMP synthetase, IMP 5′-dehydrogenase, xanthine-guanine phosphoribosyltransferase, mutant thymidine kinase, mutant HGPRTase, thymi dylate synthetase, P-glycoprotein 170, ribonucleotide reductase, glutamine synthetase, asparagine synthetase, arginosuccinate synthetase, ornithine decarboxylase, HMG-CoA reductase, N-acetylglucosaminyl transferase, theronyl-tRNA synthetase
  • Markers may be used individually or in combination. Chemicals useful for selection include methotrexate, cadmium, PALA, Xyl-A, adenosine, 2′-deoxycoformycin, adenine, azaserine, coformycin, 6-azauridine, pyrazofuran, mycophenolic acid, limiting xanthine, hypoxanthine, aminopterin, thymidine, 5-fluorodeoxyuridine, adriamycin, vincristine, colchicine, actinomycin D, puromycin, cytocholasin B, emetine, maytansine, Bakers' antifolate, aphidicolin, methionine sulfoximine, ⁇ -aspartyl hydroxamate, albiziin, canavanine, ⁇ -difluoromethylornithine, compactin, tunicamycin, borrelidin, ouabain, and derivatives and analogs and combinations of these chemicals.
  • the oncogene transfection efficiency the fraction of live cells transfected by an oncogene, may be indirectly enhanced by chemical selection for a co-transfected marker.
  • An oncogene is a sequence which can predispose, or induce the cell into a pre-neoplastic or neoplastic condition or otherwise enhance the metastatic potential of the cell.
  • Sequences with these properties are referred to as oncogenes and include abl, ahi, akt, bcl, crk, dsi, erb, ets, evi, fes/fps, fim, fis, fgr, flv, fms, fos, gin, gli, int, jun, kit, mas, lck, met, mil/raf, mis, mlv, mos, myb, myc, neu, onc, pim, raf ras, rel, ros, seq, sis, ski, spi, src, tcl, thy, trk, and yes.
  • oncogenes such as ras
  • oncogenes such as myc
  • oncogenes are oncogenic when overexpressed or underexpressed.
  • Many oncogenes represent members of multigene families or homologs families. Homologs are proteins that have similar primary, secondary or tertiary structures. Genes may differ in nucleic acid sequence or encoded peptide sequence and still be homologs when the encoded polypeptides have similar spatial folding.
  • Many oncogenes can be classified into dominant oncogenes and recessive oncogenes. One or more dominant oncogenes can confer a neoplastic or preneoplastic phenotype to a cell.
  • One or more recessive oncogenes when silenced, may also confer a neoplastic or preneoplastic phenotype.
  • Gene silencing is performed by transfecting cells with nucleic acids which cause genetic ablation or by antisense suppression.
  • the preferred oncogenes are those that are normally associated with metastasis such as a metastasis specific gene.
  • Such genes include for example. TGF- ⁇ 1, Cyclin D1 p21, p34, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb or ⁇ -actinin 3.
  • Metastatic-specific genes may be used individually or in combination with other oncogenes.
  • the metastatic potential of a cell may be altered, for example, by gene ablation with of a sequence specific for a recessive oncogene.
  • Recessive oncogenes are those genes which encode products which can suppress oncogenesis and metastasis.
  • a gene ablation sequence can be designed to specifically suppress a recessive oncogene.
  • Ablation may include pre-transcriptional inhibition such as homologous recombination with endogenous recessive oncogenes and post transcriptional inhibition such as the expression of antisense oncogenes to suppress translation.
  • Gene ablation sequences may be targeted towards well known recessive oncogenes such as, for example, the retinoblastoma gene (Rb) or Bcg Bcl.
  • ablating a recessive oncogene may include oncogenesis and metastases.
  • the mammalian cell may be treated with an agent, either before or after transfection, that alters the expression of the cell's nucleic acids.
  • Treatment may comprise contacting the cells with one or more agents which affect the neoplastic predisposition (e.g. neoplastic agents; phorbol esters), metabolization (e.g. metabolis agents), metastasis (e.g. metastatic agents), differentiation (e.g. differentiation agents; retinoic acid), activation or proliferation (e.g. growth factors) of the cell.
  • agents which can alter gene expression include chemicals such as benzanthracene (BA), dimethyl benzanthracene (DMBA) or 5-azacytidine.
  • treatment may also comprise altered conditions such as hypoxia which involves subjecting a cell to a reduced oxygen content, exposable to radiation or other stresses to the cell.
  • Treatment may be in vitro or in vivo and may include for example, direct or indirect induction or suppression of well known oncogenic sequences and genes isolated by the invention such as, for example, TGF- ⁇ 1, Cyclin D1, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb, ⁇ actinin 3, and p34.
  • Gene expression induction includes transfecting expression vectors encompassing coding regions of the gene. Gene repression comprises introducing a gene ablation sequence or a repressor of the gene to the cell.
  • a metastatic suppressor gene may be ablated to prevent inhibition to metastases.
  • a useful gene for ablation is a gene capable of affecting the phenotype and behavior or a cell or tumor.
  • suitable genes include both well known genes and genes isolated by the methods of the invention such as for example, TGF- ⁇ 1, Cyclin D1, p21, p34, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb and ⁇ actinin 3.
  • Genetic ablation (gene knockout) refers to a process of silencing the expression of a particular gene in a cell.
  • the silencing process may include, for example, gene targeting or antisense blocking.
  • Gene targeting refers to a process of introducing a nucleic acid construct into a cell to specifically recombine with a target gene. The nucleic acid construct inactivates the targeted gene. Inactivation may be by introduction of termination codons into a coding region or introduction of a repression site into a regulatory sequence.
  • Antisense blocking refers to the incorporation into a cell of expression sequences which directs the synthesis of antisense RNA to block expression of a target gene. Antisense RNA hybridizes to the mRNA of the target gene to inhibit expression.
  • the host animal is preferably the same species as the implanted cell.
  • the host may be immunocompromised genetically or by treatment with drugs such as immunosuppressants.
  • a host may be immunocompromised genetically by breeding such as with nude mice or severe combined immunodeficient (SCID) mice.
  • SCID severe combined immunodeficient
  • a host may also be immunocompromised by chemical or irradiation methods.
  • An additional route to immunocompromise a host is to use transgenic technology to introduce an immunosuppressing gene or to introduce a foreign antigen gene.
  • An immunosuppressing gene is a gene that affects the efficiency of the immune system such as a gene which inhibits the formation of cells of the B cell or T cell lineage.
  • a foreign antigen gene when expressed, may cause the host to tolerate the antigens in a xenogeneic transplant and not mount an immune response.
  • Cells may be implanted into any primary site in a host animal, such as, for example, subcutaneous implantation, intravenous injection, or implantation into the abdominal cardiac, chest, pulmonary, thoracic or peritoneal cavity. Using techniques known to those of ordinary skill in the art, cells can be placed on or in nearly any organ or tissue. Reasons for choosing a site include ease of implant, proximity of similar tissue type, immunoprivileged position and ease of inspection. Metastasises migrate from the primary site to one or more secondary sites such as, for example, the lung, kidney, liver, lymph nodes, brain, testis, bone, spleen, ovaries or mammary. Preferred sites include the renal capsule, the testes, the prostate and the ovaries.
  • the implant may be placed into a histocompatible host animal. Such problems are generally avoided if the implant and host animal are syngeneic. Alternatively, a non-histocompatible host may be used if the host can be made immunotolerant. Hosts may also be transgenic or immunocompromised animals or genetically matched to the mammalian cells to be introduced. Immunocompromised animals may be derived from established mouse lines such as nude mice or severe combined immuno deficiency (SCID) mice, or by treatments such as radiation, chemical, pharmaceutical or genetic targeting. Sufficiently immunosuppressed animals can be made tolerant to xenogeneic transplants.
  • SCID severe combined immuno deficiency
  • the host animal After implantation the host animal is maintained under normal conditions to develop metastases. Alternatively, the host animal may be subjected to an altered treatment or environmental condition to stimulate or repress metastasis or induce other cellular functions. In metastasis, a subpopulation of cells of the implantation site invade and establish one or more secondary colonies in the host animal. The behavior of the implanted cell will depend on the cell type, the transfected sequence and the implantation location. Typical secondary sites for metastatic colonies include lung, kidney, liver, lymph nodes, brain, testis, spleen, bone, ovary, skin and mammary tissue. Metastatic development times vary from days to weeks even months. Cells with a high metastatic potential tend to progress to metastasis quickly while cells with a low metastatic potential may require very long periods of time that span significant portions of the lifespan of the animal.
  • the host animal may be analyzed for metastatic development weekly, from one week to 20 weeks to six months, nine months or one year after implantation. For animals with longer lifespans such as sheep, the animal may be inspected yearly from one year on up to ten years for metastatic tumors. Metastases can be detected by examinations such as palpation, biopsy, imaging, exploratory surgery, CAT scans, autopsy, X-ray and direct observation. In addition, tissue samples may be taken surgically from the host mammal and subjected to histological or other examinations for the detection of metastases.
  • Expressed sequences include mRNA, rRNA, hnRNA, DNA, cDNA and any nucleic acid sequence that is expressed in the cell. These sequences may be amplified by in situ techniques or by purification of nucleic acid from collected cells. Expressed sequences may be obtained by extracting nucleic acids from cells before implantation, at the primary site or at the secondary site. Cells collected at these sites may optionally be cultured for a time before nucleic acid extraction. The effects of treatment with gene expression modifying agents or environmental conditions can be ascertained by collecting cells before and after treatment. Treatment may be applied to the cells while the cells are in the host mammal or after the cells are excised and in culture. Nucleic acid are collected from cells using techniques that are well known to those of ordinary skill in the art.
  • RNA may be enriched for mRNA using a poly-A RNA enrichment method.
  • RNA enrichment methods include oligo-dT columns, oligo-dT magnetic beads, and oligo-dT cellulose, RNA may be further processed into cDNA before analysis by reverse transcription using reverse transcriptase.
  • the cells or the extracted nucleic acid may be preserved, such as by freezing, and analyzed at a later time.
  • Differential display polymerase chain reactions are performed on the expressed sequences using two variable primers which may contain the same or entirely different sequences or an anchor primer and a variable primer. If an anchor primer is used, one anchor primer and one variable primer create a single or a single set of reaction products for each reaction.
  • a complete profile may include 25 or more different PCR reactions per sample wherein each PCR reaction is performed with the same anchor primer and a different variable primer.
  • DD-PCR may also be performed using anchor and variable primers which contain the same sequence. Whether a particular reaction is used depends on whether a difference exists between the products of two PCR reactions using the same primers. When a significant difference exists between the expression sequences amplified, one pair of PCR reactions may be sufficient and informative.
  • Anchor primers are preferably oligonucleotides with a poly-T sequence at the 5′-terminas terminals and a dinucleotide selected from the group consisting of AA, AG, AC, AT, GA, GG, GC, GT, CA, CG, CC and CT at the 3′-terminas terminals.
  • the sequence may be 5′-TTTTTTAA-3′ or 5′-TTTTTTAG-3′.
  • the length of the poly-T sequence is typically between about 5 to about 30 bases in length and preferably between about 10 to about 20 nucleotides long.
  • the total length of the anchor primer can vary greatly for each experiment but is preferably between about 7 to about 32 and more preferably between about 12 and about 22.
  • Differential diagnostic display polymerase chain reaction may also be performed using an anchor primer of any sequence and a length between about 5 to about 30, preferably between about 5 to about 20 and more preferably between about 7 to about 12 bases.
  • variable primer may comprise a random sequence, or a specific sequence such as, for example, a sequence of SEQ ID NO. 1 to SEQ ID NO. 24.
  • Variable primers preferably are oligonucleotides with a length between about 5 to about 30, preferably between about 5 to about 20, and more preferably between about 7 to about 12 bases in length.
  • the anchor primer or the variable primer, or both may comprise a detectable moiety.
  • detectable moieties include radioactive moieties, phosphorescent moieties, magnetic moieties, luminescent moieties, conjugatable moieties or other detectable moiety.
  • a plurality of detectable moieties may be used to enhance detection or to simplify data analysis.
  • Other detectable moieties include conjugatable moieties and molecules which can bind specifically to other molecules which are themselves detectable.
  • conjugatable moieties include avidin, streptavidin, biotin, antibody, antigen, cell adhesion molecules and other molecules with similar activities.
  • Detectable moieties are preferably labeled nucleotides.
  • a nucleotide may be any natural or synthetic nucleotide or nucleotide analog capable of incorporation into an elongation reaction in a polymerase chain reaction.
  • Labeled nucleotides include nucleotide triphosphates labeled with one or more radioactive atoms such as 32 P, 33 P, 3 H, 14 C and 35 S. Products of DD-PCR reactions are compared to detect the metastatic sequence. Comparisons can be performed between expressed sequences from cells at secondary sites with cells at any stage in the method including untreated mammalian cells, transfected or treated mammalian cells, implanted cells or cells obtained from the primary site in the host animal.
  • DD-PCR products may be analyzed by any method which reliably compares the products of two polymerase chain reactions. Typical analytical methods used for this purpose include polyacrylamide gel electrophoresis, capillary electrophoresis and high pressure liquid chromatography (HPLC).
  • Product produced from DD-PCR may be analyzed in double-stranded or single-stranded forms.
  • the sizes and distribution of the products may be monitored and analyzed by following the labels using a radiation monitor or by autoradiography.
  • DD-PCR performed in the presence of radioactive primers or nucleotide triphosphates can be analyzed by gel electrophoresis, by capillary electrophoresis, or by HPLC. Products are easily monitored by the presence of radioactivity.
  • Another method for analyzing and isolating metastatic sequences is to sequence the amplified nucleic acid sequences. Sequencing may be performed using standard methods well known to those of ordinary skill in the art. The resulting sequence may be compared to a sequence database created or well-known, such as Genbank, for identification or for locating homologs. The sequencing information may be used to calculate the physical characteristics of the nucleic acids such as melting temperature and secondary structure. The primary sequence and the physical characteristic may be used to synthesize optimal nucleic acid probes for the detection or staging of metastasis or conditions that are predictive of the presence or absence of the metastatic condition.
  • Another embodiment of the invention is directed to a method for identifying a metastatic sequence.
  • a mammalian cell is pretreated with a metastatic agent to form a population of cells predisposed to metastasize.
  • the treated cells are introduced into a host mammal at a primary site.
  • the host animal is maintained for a period of time sufficient to develop a metastatis at a secondary site.
  • Expressed sequences of cells at the primary site and cells at the secondary site are treated with a genotoxic agent or subjected to genotoxic conditions.
  • Expressed sequences of the treated cells are amplified by differential display polymerase chain reaction and compared with untreated cells from any previous step to identify the metastasis sequence.
  • the metastatic agent may be a chemical compound, a nucleic acid or a protein that alters the metastatic potential of a cell or relates to or is associated with the metastatic process.
  • Chemical compounds include retinoids such as 4-hydroxyphenyl (4HP).
  • Other agents include the proteins TGF- ⁇ 1, Cyclin D1, p21, p34, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb or ⁇ -actinin 3, or their respective genes.
  • the metastatic agent may be a metastatic stimulant or a metastatic suppressant. Metastatic stimulants may be used to enhance the sensitivity of the metastasis sequence detection method.
  • metastatic suppressants may be used to decrease the sensitivity of the method enabling the selective identification of potent metastatis metastatic sequences or sequences specific to a particular tissue type or metastatic disorder.
  • Treatment may comprise direct contact with the metastatic agent or incubation for a period of time.
  • Metastatic agents enhance the metastatic potential of the implanted cells and increase the sensitivity and the speed of the overall method.
  • the cells at the primary site and the metastatic cells at the secondary site may be treated with a genotoxic agent in vivo or in vitro.
  • In vivo treatment may comprise injecting genotoxic agents directly into the host mammal or specifically applying the agent with, for example, topical formulations.
  • the cells at the primary site and the secondary site may also be isolated from the host animal and treated with the genotoxic agent in culture.
  • Genotoxic agents are chemical compounds, nucleic acids or proteins that alter gene expression by effecting the nucleic acid genome directly by, for example, chemical modification, or indirectly by, for example, altering components associated with gene expression.
  • Such agents include, for example, benzanthracene (BA), dimethyl benzanthracene (DMBA) and 5-azacytidine, and may include metastatic agents as well.
  • the cells may be treated to hypoxic conditions or radiation to alter gene expression. Metastatic sequences identified in these methods may be specific for particular genotoxic agents or conditions.
  • Another embodiment of the invention is directed to the use of a host animal with an altered genotypic or phenotypic predisposition for metastases.
  • a host animal may be screened for endogenous expression of metastases gene.
  • metastatic sequences which may be screened for include sequences isolated by the method of the invention, such as, for example, the sequences listed in FIG. 12 and FIG. 13 .
  • Particularly useful metastatic sequences include TFG- ⁇ .
  • a host animal with reduced levels of a metastatic gene product may be used to isolate novel metastatic genes.
  • Host animals may be screened for reduced levels of metastatic gene expression.
  • transgenic technology may be use to ablate a metastatic gene in the germline of a host animal.
  • Another embodiment of the invention is directed to analysis of a cell line before their use as a starting material to isolate metastatic genes in a particular pathway. Analysis is useful in identifying cells, and consequently sequences specific to these cells, which are particularly susceptible or resistant to metastatic transformation. For example, a cell highly predisposed to metastasis may be especially sensitive for detecting metastatic genes. Conversely, a cell showing high resistance to metastasis can be used to isolate especially potent metastatic sequences.
  • One method to analyze susceptibility to metastasis is to determine the cellular response to growth factors or growth inhibitors. Briefly, a control population and a test population of cells are exposed to a growth factor or a growth inhibitor and the cellular response (e.g. proliferation, metabolism) recorded.
  • Cells showing abnormal responses to the growth factor or growth inhibitor may be used as the starting material for metastatic gene isolation.
  • Cellular response include changes in the rate of cellular division (e.g. thymidine uptake), changes in the expression of RNA or proteins, changes in cellular localization or modification patterns of RNA or proteins, and changes in the rate of uptake, release or metabolism of nutrients.
  • Especially potent or weak metastatic genes may be detected by treating and analyzing the metastatic potential of different cells and selecting a suitable cell type as the starting material.
  • cells may be treated with myc, ras, mutant p53 or combinations thereof and analyzed for cyclin D1 expression which is shown to correlates correlate with metastasis.
  • FIG. 2 shows the in situ analysis of cyclin D1 in primary MPR tumors (FIG. 2A) and in metastatic deposits from the lung of the same animal (FIG. 2 B).
  • the gene expression pattern of cyclin D1 in MPR correlates with that of human prostate tumors (FIG. 3) analyzed with stains specific for cyclin D1 expression.
  • Normal human tissue shows no cyclin D1 expression or staining (FIG. 3 A).
  • cyclin D1 expressing cells are a source of cells with high metastatic potential.
  • cells with low cyclin D1 expression are a source of potentially metastatically metastasis resistant cells.
  • This method may be adjusted for the isolation of metastatic sequences expressed along a particular developmental of differentiation pathway by combining the various treatment and analytical techniques.
  • This approach is schematically represented in FIG. 4 .
  • a mammalian cell may be genetically ablated for TGF- ⁇ 6, Cyclin D1, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb, ⁇ actinin 3, or p34.
  • the genetically altered cell is used in an in vivo mouse prostate reconstitution (MPR) model. Metastatic and nonmetastatic cells isolated from the MPR may be analyzed directly or after induction with an agent such as the TGF- ⁇ gene or its product. Analysis involves the use of differential display polymerase chain reaction to identify differentially expressed bands. Sequences identified may be used for subsequent ablation, transformation or differential analysis.
  • Genetic ablation may be performed after a cell is selected or by selecting a cell comprising a genotype with the proper genetic ablation.
  • Cells already comprising gene ablation may be acquired from a cell depository, from other laboratories or from a transgenic animal.
  • transgenic animals comprise genetically ablated genes in every cell, any tissue from a transgenic animal may be used as the starting material.
  • oncogenes are at least additive and often synergistic.
  • dominant oncogenes may be transfected together or multiple recessive oncogenes ablated together for a stronger effect.
  • both methods may be combined and dominant oncogene transfection may be accompanied by recessive oncogene ablation.
  • the function of the metastatic sequence may be determined by the differential expression pattern. For example, a dominate dominant metastatic gene will be present in a metastatic cell while a recessive metastatic gene is present in a nonmetastatic cell. Metastatic sequences may be detected as bands which are present in the DD-PCR of metastases isolated in secondary sites and yet absent from DD-PCR products of primary cells. These sequences may be dominant metastatic genes whose expression is directly responsible for metastases, or they may be metastasis associated genes whose expression correlates with metastasis. Either are useful for therapy and diagnosis. Conversely, DD-PCR bands which are present in primary site tumors, but absent in secondary metastatic sites, may be dominant metastasis suppression genes.
  • Dominant metastasis suppression genes comprise genes whose expression suppresses metastasis while nonmetastatic genes comprise genes whose expression correlates with non-metastatic tissue. Genes which are highly correlative with either the metastatic phenotype or the non-metastatic phenotype may be isolated. Isolation can be performed by cutting the appropriate nucleic acid in the containing band of from a polyacrylamide gel or by collecting the appropriate fraction in an HPLC or capillary electrophoresis. The nucleic acid may be cloned into a plasmid vector, and sequenced, or synthetically prepared.
  • Another embodiment of the invention is directed to a method for identifying sequences in a metastatic pathway which are responsive or unresponsive to extracellular signals. Such sequences may be used in therapy and diagnosis of metastatic disorders. Implanted cells or cells from a primary site and cells from a secondary site are treated with extracellular signals. RNA sequences from the treated cells are compared with RNA sequences of the untreated cells (FIG. 5 B). Treated cells and untreated cells may be derived from a short term or long term in vitro culture of primary tumors and malignant tumors. Alternatively, a part of a primary tumor and a part of a malignant tumor may be collected before the animal is treated with an extracellular cytokine or other factor.
  • transforming growth factor ⁇ 1 may be used to treat cells before DD-PCR analysis. Proteins encoded by the genes isolated by this method are especially useful for the treatment of cells for the isolation of additional sequences. The identification of one sequence responsive to the extracellular signal pathway allows for identification of additional genes upstream and downstream from that sequence.
  • Metastatic sequences are sequences associated with the presence or absence of a metastasis or related to the metastatic process can be used in the therapeutic treatment of metastasis.
  • Metastatic-related sequences include dominant metastatic sequences, recessive metastatic sequences, metastasis associated sequences, dominant oncogenes, recessive oncogenes and cell cycle genes. These genes encode for example, proteins involved in cell cycle, signal processing, DNA replication, growth regulation, inter and intra cellular signaling transcription control and translation control. Isolated sequences are useful in the treatment and for the detection of metastatic and other disorders.
  • disorders which may be treated comprise diseases involving proteins and sequences which are isolated by interaction with the sequences and proteins isolated by the method of the invention. Both malignant or nonmalignant disorders may be treated. Non malignant disorders include hyperplasia, dysplasia and hypertrophy. Examples of nonmalignant disorders include benign enlargement of the prostate, nodular hyperplasia, and benign prostatic hypertrophy.
  • Treatment may involve gene replacement, gene targeting, antisense inhibition, gene expression or gene suppression.
  • Gene replacement involves replacing a copy of a defective gene with another copy by homologous recombination.
  • Gene targeting involves the disruption of a cellular copy of a gene by homologous recombination.
  • Antisense inhibition exploits the specificity of hybridization reactions between two complementary nucleic acid chains to suppress gene expression.
  • Cloned genes can be engineered to express RNA from only one or the other DNA strands. The resultant RNA hybridizes to the sense RNA and inhibits gene expression.
  • Gene expression and gene suppression involve the introduction of genes whose expression actively inhibits neoplastic transformation and metastasis.
  • nucleic acids which comprise a sequence identified by the methods of the invention.
  • the nucleic acid may be DNA, RNA or PNA and may be used as a diagnostic tool in the treatment of neoplastic disorders and malignant tumors.
  • the nucleic acids may comprise additional sequences such as promoters, for expression of a sense or antisense message, recombination sequences for gene targeting, selectable markers for transfections, or replication origins for passage in a prokaryotic or eukaryotic host such as animal cells, bacteria or yeast.
  • nucleic acids which comprise sequences identified by the method of the invention such as, for example, the caveolin gene, ABP280 (actin binding protein 280), the lysyl oxidase gene, and the nmb gene (clone 29), and other sequences listed in FIG. 12 and FIG. 13 .
  • Nucleic acids comprising a sequence corresponding to these genes may be used in treatment or diagnosis and in diagnostic kits for screening biological samples for the presence or absence of metastasis or metastatic potential. Treatment may involve using the sequences in gene therapy, including gene ablation, gene expression and antisense suppression. Diagnosis may involve genotypic analysis of samples to determine the existence and expression levels of the expressed sequences.
  • Another embodiment of the invention is directed to the ue of caveolin gene and protein in the isolation of oncogenes and in the treatment of neoplastic disorders such as, for example, prostate cancer.
  • Caveolin is an integral membrane protein and a principal component of caveolae. Caveolae are small invaginations at or near the plasma membrane of most smooth muscle cells and may function as a component of specific signal transduction pathways. Surprisingly, caveolin expression increases in metastatic human prostate cells as compared to human primary prostate tumors.
  • caveolin expression correlates with metastasis
  • application of biological technologies designed to block the activity of caveolin or the function of caveolae may have therapeutic benefits for the treatment of neoplastic disorders such as human prostate tumors.
  • Specific treatment approaches using caveolin may include the delivery of antisense or dominant negative caveolin sequences using expression or viral vectors; as well as the use of specific anti-caveolin antibodies. Additional approaches could also target the cavoeolae, but are not specifically based on caveolin function. Additional protein and non-protein components of caveolae could also be targeted for abrogation or the local or systemic administraiton of nutritional or biological agent may also be used.
  • caveolae are extremely rich in cholesterol and disruption or depletion of this molecule may alter the function of caveolae.
  • Another embodiment of the invention is directed to methods for treating a neoplastic disorder comprising administering a pharmaceutically effective amount of composition containing a nucleic acid having a sequence identified according to the methods of this invention, its expression product or fragments of either.
  • the nucleic acid may be in the form of a sense or antisense single-stranded or double-stranded nucleic acid.
  • the composition may be combined with a pharmaceutically acceptable carrier such as water, alcohols, salts, oils, fatty acids, saccharides, polysaccharides administered by injection, pulmonary absorption, topical application or delayed release. More than one carrier may be used together to create a pharmaceutical with desirable properties.
  • Kits comprise sequences isolated according to the methods of the invention and reagents and materials useful in such kits, such as, for example, buffers, salts, preservatives, and carriers, all of which are well known to those of ordinary skill in the art. Kits are useful for the analysis of tissues to screen those for the determination of normal, nonmalignant neoplastic or malignant cells. Kits may comprise additional reagents useful for the extraction of nucleic acids from a tissue sample. Reagents for analyzing the nucleic acid extracted from a tissue sample such as polymerase chain reaction reagents and Southern blots reagents may also be included.
  • UGS tissue was isolated from 17 day old mice embryos. Each isolated UGS was digested with 1% trypsin for three hours at 4° C. The trypsin was inactivated by the addition of fetal calf serum. UGS cells were digested with 0.125% collagenase for 1.5 hours, counted and mixed at the appropriate cell ratios prior to infection with retrovirus in the presence of polybrene. Retroviruses used include Zipras/myc-9. Control experiments were performed using BAGA virus.
  • the infected cells were centrifuged and individual reconstitutions containing 1.5 ⁇ 10 6 cells produced by resuspending the cells in rat tail collagen at a density of 6.0 ⁇ 10 7 cells per ml. Aliquots of the infected UGS cells were placed in (DME) with 10% fetal calf serum overnight at 37° C., 5% CO 2 .
  • DMEM Dulbecco's Modified Eagle Medium
  • cytokeratins were fixed in 10% buffered formalin and embedded in paraffin for sectioning and staining with hematoxylin and eosin (H&E) or immunohistochemical staining. Immunohistochemical localization of cytokeratins was detected using polyclonal cytokeratin antiserum A575 (Dake Co.; Carpinteria, Calif.) and Vectastain ABC kit (Vector Laboratories; Burlingame, CA).
  • Total cellular RNA was isolated by ultracentrifugation through cesium chloride. Briefly, up to one gram of cells from culture, tumors or organs was placed into 4 ml of ice-cold GIT buffer (4M guanidine isothiocyanate, 0.025M sodium acetate, 0.1M M ⁇ -mercaptoethanol) and homogenized in a tissue homogenizer (Polytron or equivalent). The homogenate was carefully layered over 4 ml of 5.7M CsCl, 0.024M sodium acetate (1.8 g CSC1 per ml) in a centrifuge tube. The layers were centrifuged at 35,000 RPM for 18 hours in a SW50.1 rotor.
  • DNA was collected from the interface between the cushion and the supernatant, diluted two folds with water, added to 2.5 volumes of ethanol and spooled out on a glass rod.
  • RNA that formed a pellet on the bottom of the CsCl layer was resuspended, and once extracted with an equal volume of phenol:chloroform (1:1), twice with chloroform and precipitated with ethanol and resuspended in diethylpyrocarbonate treated water.
  • concentration of DNA and RNA were be determined by absorption at 260 nanometers.
  • mRNA isolated from primary tumors or metastasis was reverse transcribed with one of the primers and subjected to DD-PCR using the same primer as both the forward and reverse primer.
  • a set of 24 primers comprising short oligonucleotides were used for both the reverse transcription of mRNA into c-DNA and for differential display polymerase chain reaction. The sequence of the primers used are shown in Table 1.
  • PCR was performed using standard conditions with 40 cycles of denaturation at 94° C. for 40 seconds, annealing at 40° C. for 2 minutes, and elongation at 72° C. for 35 seconds. After PCR, the products were analyzed with non-denaturing polyacrylamide gel electrophoresis (PAGE) at 12 watts for 15 hours. Bands which differed between test and control samples were eluted from the gel, subjected to reamplification by PCR and cloned. Polyacrylamide gel electrophoresis of DD-PCRs, and the accompanying RNA blot analysis showing the isolation of sequences with substantial similarity to nmb and TGF- ⁇ is shown in FIG. 6 and FIG. 7 respectively.
  • PAGE polyacrylamide gel electrophoresis
  • the p53 allelotype of a cell sample was determined by PCR. Briefly, nucleic acid is extracted from a tissue sample or a cell culture sample. An aliquot of nucleic acids in placed in 45 ⁇ l aliquot of a master mix which contained a final concentration of 0.2 mM of each dATP, dTTP, dGTP, dCTP, 1.5 mM MgCl 2 , 0.5 unit Taq polymerase, 0.05 ⁇ M of each of two primers set specific for the normal wildtype allel of p53 (5′-GTGTTTCATTAGTTCCCCACCTTGAC-3′, SEQ. ID NO. 25; 5′-AGAGCAAGAATAAGTCAGAAGCCG-3′, SEQ. ID NO.
  • a control set of primers specific for the fibroblast growth factor-7 gene was used to monitor the polymerase chain reaction experiment (5′-ACAGACCGTGCTTCCACCTCGTC-3′, SEQ. ID NO. 27; 5′-CCTCATCTCCTGGGTCCCTTTCA-3′, SEQ. ID NO.28).
  • One ⁇ l of the reaction from the first round of PCR was used as the starting material for a second round of PCR using a second set of wildtype p53 specific primer (5′-GTCCGCGCCATGGCCATATA-3′, SEQ. ID NO. 29; 5′-ATGGGAGGCTGCCAGTCCTAACCC-3′, SEQ. ID NO. 30).
  • This second round of PCR was also monitored using a control set of primers specific for the fibroblast growth factor-7 (5′-ACAGACCGTGCTTCCACCTCGTC-3′. SEQ. ID NO 27; 5′-CCTCATCTCCTGGGTCCCTTTCA-3′, SEQ. ID NO 28).
  • 1481-PA cells were grown overnight in DME supplemented with 10% fetal calf serum overnight at 37° C., and 5% CO 2 . Induction was performed by treatment with TGF- ⁇ 1 at a concentration of 2 nanograms per ml. The treated cells were returned to the incubator and cultured for 12 hours. After induction, cells were washed in phosphate buffered saline and harvested and concentrated by centrifugation.

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Abstract

The invention relates to methods for the identification of metastatic sequences. Cells from a cell line or an animal tissue are treated to form a cell line predisposed to metastasis. Treated cells are implanted in an animal of at a primary site and incubated for a period of time sufficient for the cells to proliferate and develop metastases at secondary sites. Expressed sequences from cells at the primary and secondary sites are amplified by differential display polymerase chain reaction and compared. Differentially expressed sequences are identical identified and can be cloned and sequenced. These sequences can be used as probes in the diagnosis of metastatic disorders, as probes to isolate metastatic sequences and as a therapeutic agent.

Description

More than one reissue application has been filed for the reissue of U.S. Pat. No. 5,783,182. Application Ser. No. 09/469,316, filed Dec. 22, 1999, now abandoned, is a reissue application of U.S. Pat. No. 5,783,182. Application Ser. No. 09/977,371, filed Oct. 16, 2001 is a continuation (and a reissue) of Ser. No. 09/469,316. Application Ser. No. 09/985,799, filed Nov. 16, 2001, is a continuation (and a reissue) of Ser. No. 09/977,371.
REFERENCE TO RELATED APPLICATION
This patent application is a continuation ofThis patent application is a Continuation of U.S. application Ser. No. 09/977,371 filed Oct. 16, 2001, which is a Continuation of U.S. application Ser. No. 09/469,316, filed Dec. 22, 1999, abandoned, which is a broadening Reissue Application of U.S. Pat. No. 5,783,182, issued Jul. 21, 1998. The patent application issuing as U.S. Pat. No. 5,783,182 claims priority on United States provisional patent application, serial number 60/006,838, filed Nov. 16, 1995.
RIGHTS IN THE INVENTION
This invention was made in part with United States Government support under grant number CA350129, awarded by the National Cancer Institute, National Institute of Health and the United States Government has certain rights in the invention.
BACKGROUND
1. Field of the Invention
The present invention relates to methods for the identification and isolation of metastatic sequences, to diagnostic probes and kits which contain metastatic sequences and to therapeutic treatments for neoplastic disorders based on metastatic sequences.
2. Description of the Background
The development of higher organisms is characterized by an exquisite pattern of temporal and spatially regulated cell division. Disruption in the normal physiology of cell division are almost invariably detrimental. One such type of disruption is cancer, a disease that can arise from a series of genetic events.
Cancer cells are defined by two heritable properties, uncontrolled growth and uncontrolled invasion of normal tissue. A cancerous cell can divide in defiance of the normal growth constraints in a cell leading to a localized growth or tumor. In addition, some cancer cells also gain the ability to migrate away from their initial site and invade other healthy tissues in a patient. It is the combination of these two features that make a cancer cell especially dangerous.
An isolated abnormal cell population that grows uncontrollably will give rise to a tumor or neoplasm. As long as the neoplasm remains in a single location, it is said to be benign, and a complete cure may be expected by removing the mass surgically. A tumor or neoplasm is counted as a cancer if it is malignant, that is, if its cells have the ability to invade surrounding tissue. True malignancy begins when the cells cross the basal lamina and begin to invade the underlying connective tissue. Malignancy occurs when the cells gain the ability to detach from the main tumor mass, enter the bloodstream or lymphatic vessels, and form secondary tumors or metastases at other sites in the body. The more widely a tumor metastasizes, the harder it is to eradicate and treat.
As determined from epidermiological and clinical studies, most cancers develop in slow stages from mildly benign into malignant neoplasms. Malignant cancer usually begins as a benign localized cell population with abnormal growth characteristic called a dysplasia. The abnormal cells acquire abnormal growth characteristic resulting in a neoplasia characterized as a cell population of localized growth and swelling. If untreated, the neoplasia in situ may progress into a malignant neoplasia. Several years, or tens of years may elapse from the first sign of dysplasia to the onset of full blown malignant cancer. This characteristic process is observed in a number of cancers. Prostate cancer provides one of the more clear examples of the progression of normal tissue to benign neoplasm to malignant neoplasm.
The walnut-sized prostate is an encapsulated organ of the mammalian male urogenital system. Located at the base of the bladder, the prostate is partitioned into zones referred to as the central, peripheral and transitional zones, all of which surround the urethra. Histologically, the prostate is a highly microvascularized gland comprising fairly large glandular spaces lined with epithelium which, along with the seminal vesicles, supply the majority of fluid to the male ejaculate. As an endocrine-dependent organ, the prostate responds to both the major hormone, testosterone, and the major female hormones, estrogen and progesterone. Testicular androgen is considered important for prostate growth and development because, in both humans and other animals, castration leads to prostate atrophy and, in most cases, an absence of any incidence of prostatic carcinoma.
The major neoplastic disorders of the prostate are benign enlargement of the prostate, also called benign prostatic hyperplasia (BPH), and prostatic carcinoma; a type of neoplasia. BPH is very common in men over the age of 50. It is characterized by the presence of a number of large distinct nodules in the periurethral area of the prostate. Although benign and not malignant, these nodules can produce obstruction of the urethra causing nocturia, hesitancy to void, and difficulty in starting and stopping a urine stream upon voiding the bladder. Left untreated, a percentage of these prostate hyperplasia and neoplasias may develop into malignant prostate carcinoma.
In its more aggressive form, transformed prostatic tissues escape from the prostate capsule and metastasize invading locally and throughout the bloodstream and lymphatic system. Metastasis, defined as tumor implants which are discontinuous with the primary tumor, can occur through direct seeding, lymphatic spread and hematogenous spread. All three routes have been found to occur with prostatic carcinoma. Local invasions typically involve the seminal vesicles, the base of the urinary bladder, and the urethra. Direct seeding occurs when a malignant neoplasm penetrates a natural open field such as the peritoneal, pleural or pericardial cavities. Cells seed along the surfaces of various organs and tissues within the cavity or can simply fill the cavity spaces. Hematogenous spread is typical of sarcomas and carcinomas. Hematogenous spread of prostatic carcinoma occurs primarily to the bones, but can include massive visceral invasion as well. It has been estimated that about 60% of newly diagnosed prostate cancer patients will have metastases at the time of initial diagnosis.
Surgery or radiotherapy is the treatment of choice for early prostatic neoplasia. Surgery involves complete removal of the entire (radical prostatectomy), and often removal of the surrounding lymp nodes, lymphadenectomy. Radiotherapy, occasionally used as adjuvant therapy, may be either external or interstitial using 125I. Encodrine therapy is the treatment of choice for more advanced forms. The aim of this therapy is to deprive the prostate cells, and presumably the transformed prostate cells as well, of testosterone. This is accomplished by orchiectomy (castration) or administration of estrogens or synthetic hormones which are agonists of luteinizing hormone-releasing hormone. These cellular messengers directly inhibit testicular and organ synthesis and suppress luteinizing hormone secretion which in turn leads to reduced testosterone secretion by the testes. Despite the advances made in achieving a pharmacologic orchiectomy, the survival rates for those with late stage carcinomas are rather bleak.
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides new methods for the identification of sequences related to metastasis.
One embodiment of the invention is directed to methods for the identification of a metastatic sequence. One or more oncogenic sequences are transfected into a cell to form a transfected cell. The transfected cell is introduced into a primary site of a host animal to establish a colony which is incubated in the animal for a period of time sufficient to develop both a primary tumor and a metastatic tumor. Expressed sequences are harvested from the primary tumor and the metastasis. Harvested sequences are compared to each other and to non-metastatic cells to identify sequences related to metastasis. Dominant metastatic genes are genes whose expression leads to metastasis. Such genes are typically expressed at high levels in metastatic cells and not significantly expressed in normal or nonmetastatic cells. Recessive metastatic genes, genes whose expression prevents metastatis, may be selectively expressed in normal and nonmetastatic cells and absent in metastatic cells. Dominant and recessive metastatic genes may act directly or act pleiotropically by enhancing or inhibiting the expression or function of other dominant and recessive metastatic genes.
Another embodiment of the invention is directed to methods for identifying metastatic sequences. A mammalian cell is treated with a metastatic agent and the treated cell is implanted into a primary site of a host mammal. The host animal is maintained for a period of time sufficient for the cells to proliferate and to develop a metastatsis metastasis at a secondary cite site. Expressed squences from cells of the primary cite and cells of the secondary site are reverse transcribed into cDNA by differential display polymerase chain reaction to identify differentially expressed sequences.
Another embodiment of the invention is directed to sequences isolated by the methods of the invention. Sequences may be in the form of DNA, RNA or PNA. The nucleic acid may be single-stranded or double-stranded. Single stranded nucleic acid may be in the form of a sense strand or an antisense strand. In addition, the sequence may be part of a homologous recombination vector designed to recombine with another metastatic sequence.
Another embodiment of the invention is directed to a method for treating a neoplastic disorder comprising administering a pharmaceutically effective amount of a metastatic nucleic acid to a patient. The nucleic acid may be single-stranded in the sense or the antisense direction. Alternatively, the nucleic acid may be packaged in a viral vector such as, for example, a retroviral, a vaccinia or an adenoviral vector. Administration may be performed by injection, pulmonary absorption, topical application or delayed release of the nucleic acid along with a pharmaceutically acceptable carrier such as water, alcohols, salts, oils, fatty acids, saccharides, polysaccharides and combinations thereof.
Another embodiment of the invention is directed to a kit for detecting of the presence or absence of a metastatic sequence.
Other objects and advantages of the invention are set forth in part in the description which follows, and in part, will be obvious from this description, or may be learned from the practice of the invention.
DESCRIPTION OF THE DRAWINGS
FIG. 1 Schematic showing two paths in the multistep progression to cancer.
FIG. 2 A-B Staining of primary tumor (A) and metastatic deposite (B) from the lung of the same animal
FIG. 3 A-D Staining of normal human prostate (A), moderately differentiated human prostate tumor (B and C), and poorly differentiated prostate tumor (D).
FIG. 4 Schematic of method for isolating a metastatic gene from a gene ablated mouse strain.
FIG. 5 A-B Schematic showing method to establish a tumor and metastatic transplant from fetal tissue(A) and from cell lines and tumors (b).
FIG. 6 Isolation and characterization of nmb gene expression by DD-PCR and RNA blot in primary and metastatic cells.
FIG. 7 Differential expression of multiple genes is determined by DD-PCR and RNA blot of primary and metastatic cells.
FIG. 8 Caveolin identified as a differentially expressed gene by DD-PCR.
FIG. 9 Differential expression of genes isolated by DD-PCR confirmed by RNA blots.
FIG. 10 RNA blot analysis of total tumor mRNA using clone 29 GADPH probes.
FIG. 11 RNA blot of three independent MPR metastatic tumors and 5 MPR non-metastatic tumors.
FIG. A-RR 12 Nucleotide sequences of metastatic nucleic acids.
FIG. 13 A-D Characterization of metastatic sequences isolated.
FIG. 14 Immunohistological staining of primary and metastatic human prostate tumors using anti-caveolin antibodies.
DESCRIPTION OF THE INVENTION
As embodied and broadly described herein, the present invention is directed to methods for identifying metastatic sequences, to the metastatic sequences identified, to methods for the detection, diagnosis and treatment of disorders related to metastasis, and to diagnostic kits which comprise these sequences.
The ability of cancers to metastasize makes tumors difficult to eradicate by any means. Malignant cancer involves a multistage progression from, for example, normal tissue through hyper plasia, early adenoma, early carcinoma and finally to a metastatic tumor (FIG. 1). Cells of a typical tumor loosen their adhesion to their original cellular neighbors and cross the basal lamina and endothelial lining to enter the body's circulation. Once in circulation, the metastatic cell exits from the circulation to disseminate throughout the body and proliferate in a new environment.
Like the initial oncogenic event, the ability of a cell to metastasize requires additional mutationic or epigenetic changes. An understanding of the molecular mechanisms of metastasis allow for the design of treatments to inhibit metastasis. Knowledge of stage specific gene expression for neoplastic disorders allows for early detection and typing of tumors. With early detection and typing, proper treatment may be administered to a patient with the neoplastic disorder earlier, which will lead to a higher probability of a complete cure.
For human prostate tumors, the study of stage specific tumors is difficult, if not impossible, as cell lines are extremely difficult to grow and it is rare that tissue becomes available from the primary tumor as well as metastatic disease from the same patient. This problem is exacerbated because of the infrequent biopsy of metastatic deposits in conjuntion with isolation of material from the primary tumor. Furthermore, the growth of cell lines from malignant prostates has proved to be problematic over the last few decades. This is evidenced by the lack of cell lines from prostate cancer obtained under any conditions.
One embodiment of the invention is directed to a method for identifying a metastatic sequence. A mammalian cell is transformed into a pre-neoplastic or neoplastic state or phenotype by transfection with one or more oncogenic sequences. Alternatively, or in addition to transfection, the mammalian cell may be treated with an agent or subjected to a condition that potentiates the metastatic character of the cell or predisposes the cell to metastasis. The transfected or treated cell is implanted into a host animal at a primary site and grown for a period of time sufficient to develop a metastasis at a secondary site. Expressed sequences from cells of the primary site and cells at the secondary site are amplified by differential display polymerase chain reactions. PCR products from these reactions are compared and the metastatic sequence identified by alteration in the levels or patterns of the resulting products.
Mammalian cells from a wide variety of tissue types and species are suitable for transection or treatment including surgically obtained or primary or immortalized cells and cell lines. Cells may be from humans or primates, mice, rats, sheep, cows, rabbits, horses, pigs or guinea pigs or from transgenic or xenogenic host mammals. Cells may be obtained from adult, juvenile or fetal tissue, and used directly from the mammal, from cryogenically preserved samples, or after culturing in vitro or in vivo for a period of time. In vitro culturing typically involves tissue culture conditions (e.g. 37° C.; 5% CO2) while in vivo culturing may involve successive passage of cells through host animals such as, for example, mice or rabbits. Cells passed in vivo may be obtained from sites proximal or distal to the site of implantation. The tissue type from which the cells are derived or obtained may be any tissue which is susceptible to transfection or other treatment including, for example, urogenital tissues, epithelial cells, hepatic cells, fibroblasts lymphatic tissues, hematopoietic cells, cells of the immune system, cells of the gastrointestinal system and cells of the nervous system.
Cell types useful for the identification of metastatic sequences related to prostate cancer include cells and cell lines of the fetal prostate lineage from normal or transgenic animals, and cells from normal or reconstituted prostate tissue. One method of generating reconstituted prostate cells is to isolate fetal prostate tissue and microdissect the fetal prostate epithelium away from fetal mesenchyme. Fetal prostate epithelium may be genetically manipulated before reassociation with fetal mesenchyme (FIG. 5A). Genetic manipulation involves treatment or transfection with a metastatic agent or a nucleic acid sequence that affects neoplastic or metastatic potential of the cell. Reassociation of fetal epithelium and mesenchyme is performed by implanting epithelial tissue within a pocket of mesenchymal tissue. After manipulation, cells are reimplanted into a mammalian host in a similar manner as other cells, such as reimplantation into or under the renal capsule.
Mammalian cells may be transfected by a variety of techniques, all of which are well-known to those of ordinary skill. Direct methods involve the introduction of genetic material into the nucleus of a cell by injection. These techniques include high velocity projectile injection, microinjection, and electroporation. Indirect methods, involving the active or passive uptake of the genetic information by the cell, include transduction with recombinant vectors, and chemical or physical treatments such as calcium phosphate uptake, lipofection or dextran sulfate transfection. Chemical techniques rely on chemical carriers to introduce nucleic acids into a cell. These methods, for example, utilize unilamellar phospholipid vesicles (e.g. liposomes) loaded with DNA (or RNA). The approach relies on the fusion of the DNA containing vesicles with the plasma membrane of the recipient cells. After entry, DNA traverse the cytoplasm and enter the nucleus. Another lipofection technique uses a synthetic cationic lipid such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA). DOTMA spontaneously associates with nucleic acids and forms unilamellar vesicles upon sonication. Genetic material is incorporated into these vesicles and subsequently transfected into the cell. Calcium phosphate co-precipitation involves mixing of purified nucleic acid with buffers containing phosphate and calcium chloride which results in the formation of a fine precipitate. Presentation of this precipitate to cells results in incorporation of the nucleic acid into cellular genome. Other chemicals, such as DEAE dextran or polybrene, when present in media with nucleic acids, can also cause the transection of mammalian cells.
Physical methods of transfection rely on electric fields, needles and particles to enable nucleic acids to traverse the cellular membrane. Electric field mediated DNA transfection, commonly called electroporation, is based on the principle that membranes, when subjected to an electric field, undergo a reversible breakdown resulting in pores large enough to permit the passage of nucleic acids. In micro-projectile mediated gene transfer, micro-projectiles of subcellular dimensions are coated with nucleic acid and propelled at high velocity into a cell using a particle gun. The nucleic acid is introduced into the nucleus directly when the particles impinge upon the nucleus. In microinjection, nucleic acid is injected directly into the nucleus of a cell with a needle. Lasers have also been used to introduce minute holes in cellular membrane to allow introduction of nucleic acids. All these methods may be used for transfection and the selection of the method will depend on the cell type, the desired transfection efficiency and the equipment available.
The efficiency of transfection may be monitored and enhanced by the co-transfection of a selectable marker. If a marker is co-transfected with a genetic construct, positively transformed cells may be separated from nontransformed cells by chemical selection. The efficiency of transfection will be increased in most cases because the chemicals will selectively kill non-transfected cells. The number of transfected cells may also be monitored by analyzing the degree of chemical resistance of the transfected cells. Markers commonly used for selection purposes include, for example, nucleic acids encoding dihydrofolate reductase, metallothionein, CAD, adenosine deaminase, adenylate deaminase, UMP synthetase, IMP 5′-dehydrogenase, xanthine-guanine phosphoribosyltransferase, mutant thymidine kinase, mutant HGPRTase, thymi dylate synthetase, P-glycoprotein 170, ribonucleotide reductase, glutamine synthetase, asparagine synthetase, arginosuccinate synthetase, ornithine decarboxylase, HMG-CoA reductase, N-acetylglucosaminyl transferase, theronyl-tRNA synthetase, sodium or potassium dependent ATPase or derivatives or mutants of these nucleic acids. Markers may be used individually or in combination. Chemicals useful for selection include methotrexate, cadmium, PALA, Xyl-A, adenosine, 2′-deoxycoformycin, adenine, azaserine, coformycin, 6-azauridine, pyrazofuran, mycophenolic acid, limiting xanthine, hypoxanthine, aminopterin, thymidine, 5-fluorodeoxyuridine, adriamycin, vincristine, colchicine, actinomycin D, puromycin, cytocholasin B, emetine, maytansine, Bakers' antifolate, aphidicolin, methionine sulfoximine, β-aspartyl hydroxamate, albiziin, canavanine, α-difluoromethylornithine, compactin, tunicamycin, borrelidin, ouabain, and derivatives and analogs and combinations of these chemicals. Some chemicals, such as methotrexate, may be used individually while other chemicals, such as HAT (hypoxanthine, aminopterin and thymidine), need to be used in combination to be effective.
The oncogene transfection efficiency, the fraction of live cells transfected by an oncogene, may be indirectly enhanced by chemical selection for a co-transfected marker. An oncogene is a sequence which can predispose, or induce the cell into a pre-neoplastic or neoplastic condition or otherwise enhance the metastatic potential of the cell. Sequences with these properties are referred to as oncogenes and include abl, ahi, akt, bcl, crk, dsi, erb, ets, evi, fes/fps, fim, fis, fgr, flv, fms, fos, gin, gli, int, jun, kit, mas, lck, met, mil/raf, mis, mlv, mos, myb, myc, neu, onc, pim, raf ras, rel, ros, seq, sis, ski, spi, src, tcl, thy, trk, and yes. Some oncogenes, such as ras, are oncogenic when mutated. Other oncogenes, such as myc, are oncogenic when overexpressed or underexpressed. Many oncogenes represent members of multigene families or homologs families. Homologs are proteins that have similar primary, secondary or tertiary structures. Genes may differ in nucleic acid sequence or encoded peptide sequence and still be homologs when the encoded polypeptides have similar spatial folding. Many oncogenes can be classified into dominant oncogenes and recessive oncogenes. One or more dominant oncogenes can confer a neoplastic or preneoplastic phenotype to a cell. One or more recessive oncogenes, when silenced, may also confer a neoplastic or preneoplastic phenotype. Gene silencing is performed by transfecting cells with nucleic acids which cause genetic ablation or by antisense suppression.
While any oncogene may be used, the preferred oncogenes are those that are normally associated with metastasis such as a metastasis specific gene. Such genes include for example. TGF-β1, Cyclin D1 p21, p34, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb or α-actinin 3. Metastatic-specific genes may be used individually or in combination with other oncogenes.
The metastatic potential of a cell may be altered, for example, by gene ablation with of a sequence specific for a recessive oncogene. Recessive oncogenes are those genes which encode products which can suppress oncogenesis and metastasis. A gene ablation sequence can be designed to specifically suppress a recessive oncogene. Ablation may include pre-transcriptional inhibition such as homologous recombination with endogenous recessive oncogenes and post transcriptional inhibition such as the expression of antisense oncogenes to suppress translation. Gene ablation sequences may be targeted towards well known recessive oncogenes such as, for example, the retinoblastoma gene (Rb) or Bcg Bcl. Other candidates for ablation include metastatic genes previously isolated by the invention such as, for example, TGF-β1, cyclin D1, p21, p34, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb or α-actinin-3. The effects of ablating a recessive oncogene may include oncogenesis and metastases.
Alternatively, or in addition to transfection the mammalian cell may be treated with an agent, either before or after transfection, that alters the expression of the cell's nucleic acids. Treatment may comprise contacting the cells with one or more agents which affect the neoplastic predisposition (e.g. neoplastic agents; phorbol esters), metabolization (e.g. metabolis agents), metastasis (e.g. metastatic agents), differentiation (e.g. differentiation agents; retinoic acid), activation or proliferation (e.g. growth factors) of the cell. Agents which can alter gene expression include chemicals such as benzanthracene (BA), dimethyl benzanthracene (DMBA) or 5-azacytidine. Alternatively, treatment may also comprise altered conditions such as hypoxia which involves subjecting a cell to a reduced oxygen content, exposable to radiation or other stresses to the cell.
Treatment may be in vitro or in vivo and may include for example, direct or indirect induction or suppression of well known oncogenic sequences and genes isolated by the invention such as, for example, TGF-β1, Cyclin D1, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb, α actinin 3, and p34. Gene expression induction includes transfecting expression vectors encompassing coding regions of the gene. Gene repression comprises introducing a gene ablation sequence or a repressor of the gene to the cell.
Cells which have one or more genes ablated may also be used. For example, a metastatic suppressor gene may be ablated to prevent inhibition to metastases. A useful gene for ablation is a gene capable of affecting the phenotype and behavior or a cell or tumor. For example, with prostate tumors, suitable genes include both well known genes and genes isolated by the methods of the invention such as for example, TGF-β1, Cyclin D1, p21, p34, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb and α actinin 3. Genetic ablation (gene knockout) refers to a process of silencing the expression of a particular gene in a cell. The silencing process may include, for example, gene targeting or antisense blocking. Gene targeting refers to a process of introducing a nucleic acid construct into a cell to specifically recombine with a target gene. The nucleic acid construct inactivates the targeted gene. Inactivation may be by introduction of termination codons into a coding region or introduction of a repression site into a regulatory sequence. Antisense blocking refers to the incorporation into a cell of expression sequences which directs the synthesis of antisense RNA to block expression of a target gene. Antisense RNA hybridizes to the mRNA of the target gene to inhibit expression.
The host animal is preferably the same species as the implanted cell. In cases of xenogeneic transplants, the host may be immunocompromised genetically or by treatment with drugs such as immunosuppressants. A host may be immunocompromised genetically by breeding such as with nude mice or severe combined immunodeficient (SCID) mice. A host may also be immunocompromised by chemical or irradiation methods. An additional route to immunocompromise a host is to use transgenic technology to introduce an immunosuppressing gene or to introduce a foreign antigen gene. An immunosuppressing gene is a gene that affects the efficiency of the immune system such as a gene which inhibits the formation of cells of the B cell or T cell lineage. A foreign antigen gene, when expressed, may cause the host to tolerate the antigens in a xenogeneic transplant and not mount an immune response.
Cells may be implanted into any primary site in a host animal, such as, for example, subcutaneous implantation, intravenous injection, or implantation into the abdominal cardiac, chest, pulmonary, thoracic or peritoneal cavity. Using techniques known to those of ordinary skill in the art, cells can be placed on or in nearly any organ or tissue. Reasons for choosing a site include ease of implant, proximity of similar tissue type, immunoprivileged position and ease of inspection. Metastasises migrate from the primary site to one or more secondary sites such as, for example, the lung, kidney, liver, lymph nodes, brain, testis, bone, spleen, ovaries or mammary. Preferred sites include the renal capsule, the testes, the prostate and the ovaries.
To avoid histocompatibility problems, the implant may be placed into a histocompatible host animal. Such problems are generally avoided if the implant and host animal are syngeneic. Alternatively, a non-histocompatible host may be used if the host can be made immunotolerant. Hosts may also be transgenic or immunocompromised animals or genetically matched to the mammalian cells to be introduced. Immunocompromised animals may be derived from established mouse lines such as nude mice or severe combined immuno deficiency (SCID) mice, or by treatments such as radiation, chemical, pharmaceutical or genetic targeting. Sufficiently immunosuppressed animals can be made tolerant to xenogeneic transplants.
After implantation the host animal is maintained under normal conditions to develop metastases. Alternatively, the host animal may be subjected to an altered treatment or environmental condition to stimulate or repress metastasis or induce other cellular functions. In metastasis, a subpopulation of cells of the implantation site invade and establish one or more secondary colonies in the host animal. The behavior of the implanted cell will depend on the cell type, the transfected sequence and the implantation location. Typical secondary sites for metastatic colonies include lung, kidney, liver, lymph nodes, brain, testis, spleen, bone, ovary, skin and mammary tissue. Metastatic development times vary from days to weeks even months. Cells with a high metastatic potential tend to progress to metastasis quickly while cells with a low metastatic potential may require very long periods of time that span significant portions of the lifespan of the animal.
The host animal may be analyzed for metastatic development weekly, from one week to 20 weeks to six months, nine months or one year after implantation. For animals with longer lifespans such as sheep, the animal may be inspected yearly from one year on up to ten years for metastatic tumors. Metastases can be detected by examinations such as palpation, biopsy, imaging, exploratory surgery, CAT scans, autopsy, X-ray and direct observation. In addition, tissue samples may be taken surgically from the host mammal and subjected to histological or other examinations for the detection of metastases.
Expressed sequences include mRNA, rRNA, hnRNA, DNA, cDNA and any nucleic acid sequence that is expressed in the cell. These sequences may be amplified by in situ techniques or by purification of nucleic acid from collected cells. Expressed sequences may be obtained by extracting nucleic acids from cells before implantation, at the primary site or at the secondary site. Cells collected at these sites may optionally be cultured for a time before nucleic acid extraction. The effects of treatment with gene expression modifying agents or environmental conditions can be ascertained by collecting cells before and after treatment. Treatment may be applied to the cells while the cells are in the host mammal or after the cells are excised and in culture. Nucleic acid are collected from cells using techniques that are well known to those of ordinary skill in the art.
Expressed sequences may be used directly for polymerase chain reaction (PCR) analysis using, for example, the technique of reverse transcriptase polymerase chain reaction (RT-PCR). Alternatively, RNA may be enriched for mRNA using a poly-A RNA enrichment method. Numerous poly-A RNA enrichment methods exist and are commercially available. Techniques used for poly-A RNA enrichment include oligo-dT columns, oligo-dT magnetic beads, and oligo-dT cellulose, RNA may be further processed into cDNA before analysis by reverse transcription using reverse transcriptase. The cells or the extracted nucleic acid may be preserved, such as by freezing, and analyzed at a later time.
Differential display polymerase chain reactions (DD-PCR) are performed on the expressed sequences using two variable primers which may contain the same or entirely different sequences or an anchor primer and a variable primer. If an anchor primer is used, one anchor primer and one variable primer create a single or a single set of reaction products for each reaction. A complete profile may include 25 or more different PCR reactions per sample wherein each PCR reaction is performed with the same anchor primer and a different variable primer. DD-PCR may also be performed using anchor and variable primers which contain the same sequence. Whether a particular reaction is used depends on whether a difference exists between the products of two PCR reactions using the same primers. When a significant difference exists between the expression sequences amplified, one pair of PCR reactions may be sufficient and informative.
Anchor primers are preferably oligonucleotides with a poly-T sequence at the 5′-terminas terminals and a dinucleotide selected from the group consisting of AA, AG, AC, AT, GA, GG, GC, GT, CA, CG, CC and CT at the 3′-terminas terminals. For example, the sequence may be 5′-TTTTTTAA-3′ or 5′-TTTTTTAG-3′. The length of the poly-T sequence is typically between about 5 to about 30 bases in length and preferably between about 10 to about 20 nucleotides long. The total length of the anchor primer can vary greatly for each experiment but is preferably between about 7 to about 32 and more preferably between about 12 and about 22. Differential diagnostic display polymerase chain reaction may also be performed using an anchor primer of any sequence and a length between about 5 to about 30, preferably between about 5 to about 20 and more preferably between about 7 to about 12 bases.
The variable primer may comprise a random sequence, or a specific sequence such as, for example, a sequence of SEQ ID NO. 1 to SEQ ID NO. 24. Variable primers preferably are oligonucleotides with a length between about 5 to about 30, preferably between about 5 to about 20, and more preferably between about 7 to about 12 bases in length.
To enhance detection of the PCR product, the anchor primer or the variable primer, or both, may comprise a detectable moiety. Examples of detectable moieties include radioactive moieties, phosphorescent moieties, magnetic moieties, luminescent moieties, conjugatable moieties or other detectable moiety. A plurality of detectable moieties may be used to enhance detection or to simplify data analysis. Other detectable moieties include conjugatable moieties and molecules which can bind specifically to other molecules which are themselves detectable. Examples of conjugatable moieties include avidin, streptavidin, biotin, antibody, antigen, cell adhesion molecules and other molecules with similar activities. Detectable moieties are preferably labeled nucleotides. A nucleotide may be any natural or synthetic nucleotide or nucleotide analog capable of incorporation into an elongation reaction in a polymerase chain reaction. Labeled nucleotides include nucleotide triphosphates labeled with one or more radioactive atoms such as 32P, 33P, 3H, 14C and 35S. Products of DD-PCR reactions are compared to detect the metastatic sequence. Comparisons can be performed between expressed sequences from cells at secondary sites with cells at any stage in the method including untreated mammalian cells, transfected or treated mammalian cells, implanted cells or cells obtained from the primary site in the host animal. DD-PCR products may be analyzed by any method which reliably compares the products of two polymerase chain reactions. Typical analytical methods used for this purpose include polyacrylamide gel electrophoresis, capillary electrophoresis and high pressure liquid chromatography (HPLC). Product produced from DD-PCR may be analyzed in double-stranded or single-stranded forms. When the products of the DD-PCR reaction are labeled the sizes and distribution of the products may be monitored and analyzed by following the labels using a radiation monitor or by autoradiography. For example, DD-PCR performed in the presence of radioactive primers or nucleotide triphosphates, can be analyzed by gel electrophoresis, by capillary electrophoresis, or by HPLC. Products are easily monitored by the presence of radioactivity.
Another method for analyzing and isolating metastatic sequences is to sequence the amplified nucleic acid sequences. Sequencing may be performed using standard methods well known to those of ordinary skill in the art. The resulting sequence may be compared to a sequence database created or well-known, such as Genbank, for identification or for locating homologs. The sequencing information may be used to calculate the physical characteristics of the nucleic acids such as melting temperature and secondary structure. The primary sequence and the physical characteristic may be used to synthesize optimal nucleic acid probes for the detection or staging of metastasis or conditions that are predictive of the presence or absence of the metastatic condition.
Another embodiment of the invention is directed to a method for identifying a metastatic sequence. A mammalian cell is pretreated with a metastatic agent to form a population of cells predisposed to metastasize. The treated cells are introduced into a host mammal at a primary site. The host animal is maintained for a period of time sufficient to develop a metastatis at a secondary site. Expressed sequences of cells at the primary site and cells at the secondary site are treated with a genotoxic agent or subjected to genotoxic conditions. Expressed sequences of the treated cells are amplified by differential display polymerase chain reaction and compared with untreated cells from any previous step to identify the metastasis sequence.
The metastatic agent may be a chemical compound, a nucleic acid or a protein that alters the metastatic potential of a cell or relates to or is associated with the metastatic process. Chemical compounds include retinoids such as 4-hydroxyphenyl (4HP). Other agents include the proteins TGF-β1, Cyclin D1, p21, p34, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb or α-actinin 3, or their respective genes. The metastatic agent may be a metastatic stimulant or a metastatic suppressant. Metastatic stimulants may be used to enhance the sensitivity of the metastasis sequence detection method. Conversely metastatic suppressants may be used to decrease the sensitivity of the method enabling the selective identification of potent metastatis metastatic sequences or sequences specific to a particular tissue type or metastatic disorder. Treatment may comprise direct contact with the metastatic agent or incubation for a period of time. Metastatic agents enhance the metastatic potential of the implanted cells and increase the sensitivity and the speed of the overall method.
The cells at the primary site and the metastatic cells at the secondary site may be treated with a genotoxic agent in vivo or in vitro. In vivo treatment may comprise injecting genotoxic agents directly into the host mammal or specifically applying the agent with, for example, topical formulations. The cells at the primary site and the secondary site may also be isolated from the host animal and treated with the genotoxic agent in culture. Genotoxic agents are chemical compounds, nucleic acids or proteins that alter gene expression by effecting the nucleic acid genome directly by, for example, chemical modification, or indirectly by, for example, altering components associated with gene expression. Such agents include, for example, benzanthracene (BA), dimethyl benzanthracene (DMBA) and 5-azacytidine, and may include metastatic agents as well. In addition to or in place of genotoxic agents, the cells may be treated to hypoxic conditions or radiation to alter gene expression. Metastatic sequences identified in these methods may be specific for particular genotoxic agents or conditions.
Another embodiment of the invention is directed to the use of a host animal with an altered genotypic or phenotypic predisposition for metastases. A host animal may be screened for endogenous expression of metastases gene. Examples of metastatic sequences which may be screened for include sequences isolated by the method of the invention, such as, for example, the sequences listed in FIG. 12 and FIG. 13. Particularly useful metastatic sequences include TFG-β. A host animal with reduced levels of a metastatic gene product may be used to isolate novel metastatic genes. Host animals may be screened for reduced levels of metastatic gene expression. In addition, transgenic technology may be use to ablate a metastatic gene in the germline of a host animal.
Another embodiment of the invention is directed to analysis of a cell line before their use as a starting material to isolate metastatic genes in a particular pathway. Analysis is useful in identifying cells, and consequently sequences specific to these cells, which are particularly susceptible or resistant to metastatic transformation. For example, a cell highly predisposed to metastasis may be especially sensitive for detecting metastatic genes. Conversely, a cell showing high resistance to metastasis can be used to isolate especially potent metastatic sequences. One method to analyze susceptibility to metastasis is to determine the cellular response to growth factors or growth inhibitors. Briefly, a control population and a test population of cells are exposed to a growth factor or a growth inhibitor and the cellular response (e.g. proliferation, metabolism) recorded. Cells showing abnormal responses to the growth factor or growth inhibitor may be used as the starting material for metastatic gene isolation. Cellular response include changes in the rate of cellular division (e.g. thymidine uptake), changes in the expression of RNA or proteins, changes in cellular localization or modification patterns of RNA or proteins, and changes in the rate of uptake, release or metabolism of nutrients.
Especially potent or weak metastatic genes may be detected by treating and analyzing the metastatic potential of different cells and selecting a suitable cell type as the starting material. For example, cells may be treated with myc, ras, mutant p53 or combinations thereof and analyzed for cyclin D1 expression which is shown to correlates correlate with metastasis. FIG. 2 shows the in situ analysis of cyclin D1 in primary MPR tumors (FIG. 2A) and in metastatic deposits from the lung of the same animal (FIG. 2B). The gene expression pattern of cyclin D1 in MPR correlates with that of human prostate tumors (FIG. 3) analyzed with stains specific for cyclin D1 expression. Normal human tissue shows no cyclin D1 expression or staining (FIG. 3A). Moderately differentiated prostate cancers with dispersed (FIG. 3B) or focal positively staining (FIG. 3C) show moderate staining. Advanced poorly differentiated prostate cancer show strong nuclear as well as cytoplasmic staining (FIG. 3D) implying strong expression of cyclin D1. After treatment with myc, ras or mutant p53, cyclin D1 expression shows correlation with the metastatic potential of the cell. Thus, cyclin D1 expressing cells are a source of cells with high metastatic potential. Conversely, cells with low cyclin D1 expression are a source of potentially metastatically metastasis resistant cells.
This method may be adjusted for the isolation of metastatic sequences expressed along a particular developmental of differentiation pathway by combining the various treatment and analytical techniques. This approach is schematically represented in FIG. 4. For example, a mammalian cell may be genetically ablated for TGF-β6, Cyclin D1, mutant p53, lysyl oxidase, caveolin, actin binding protein, ubiquitin activating enzyme E1, nmb, α actinin 3, or p34. The genetically altered cell is used in an in vivo mouse prostate reconstitution (MPR) model. Metastatic and nonmetastatic cells isolated from the MPR may be analyzed directly or after induction with an agent such as the TGF-β gene or its product. Analysis involves the use of differential display polymerase chain reaction to identify differentially expressed bands. Sequences identified may be used for subsequent ablation, transformation or differential analysis.
Genetic ablation (gene knockout) may be performed after a cell is selected or by selecting a cell comprising a genotype with the proper genetic ablation. Cells already comprising gene ablation may be acquired from a cell depository, from other laboratories or from a transgenic animal. As transgenic animals comprise genetically ablated genes in every cell, any tissue from a transgenic animal may be used as the starting material.
The effects of oncogenes are at least additive and often synergistic. Thus, dominant oncogenes may be transfected together or multiple recessive oncogenes ablated together for a stronger effect. Furthermore, both methods may be combined and dominant oncogene transfection may be accompanied by recessive oncogene ablation.
The function of the metastatic sequence may be determined by the differential expression pattern. For example, a dominate dominant metastatic gene will be present in a metastatic cell while a recessive metastatic gene is present in a nonmetastatic cell. Metastatic sequences may be detected as bands which are present in the DD-PCR of metastases isolated in secondary sites and yet absent from DD-PCR products of primary cells. These sequences may be dominant metastatic genes whose expression is directly responsible for metastases, or they may be metastasis associated genes whose expression correlates with metastasis. Either are useful for therapy and diagnosis. Conversely, DD-PCR bands which are present in primary site tumors, but absent in secondary metastatic sites, may be dominant metastasis suppression genes. Dominant metastasis suppression genes comprise genes whose expression suppresses metastasis while nonmetastatic genes comprise genes whose expression correlates with non-metastatic tissue. Genes which are highly correlative with either the metastatic phenotype or the non-metastatic phenotype may be isolated. Isolation can be performed by cutting the appropriate nucleic acid in the containing band of from a polyacrylamide gel or by collecting the appropriate fraction in an HPLC or capillary electrophoresis. The nucleic acid may be cloned into a plasmid vector, and sequenced, or synthetically prepared.
Another embodiment of the invention is directed to a method for identifying sequences in a metastatic pathway which are responsive or unresponsive to extracellular signals. Such sequences may be used in therapy and diagnosis of metastatic disorders. Implanted cells or cells from a primary site and cells from a secondary site are treated with extracellular signals. RNA sequences from the treated cells are compared with RNA sequences of the untreated cells (FIG. 5B). Treated cells and untreated cells may be derived from a short term or long term in vitro culture of primary tumors and malignant tumors. Alternatively, a part of a primary tumor and a part of a malignant tumor may be collected before the animal is treated with an extracellular cytokine or other factor. Long term cultures, or cell lines of primary and malignant cells may also be used as recipients of extracellular growth signal treatment. Suitable signals for each experiment will depend on the cell type. Generally, growth factors, lymphokines, inhibitory factors, migratory factors or hormones may be used. Factors previously isolated by commercial or methods of the invention and factors associated with or causative or suppressive of metastasis are preferred. Thus, transforming growth factor β1 (TFG-β1) may be used to treat cells before DD-PCR analysis. Proteins encoded by the genes isolated by this method are especially useful for the treatment of cells for the isolation of additional sequences. The identification of one sequence responsive to the extracellular signal pathway allows for identification of additional genes upstream and downstream from that sequence.
Another embodiment of the invention is directed to metastatic sequences identified by the methods of the invention. Metastatic sequences are sequences associated with the presence or absence of a metastasis or related to the metastatic process can be used in the therapeutic treatment of metastasis. Metastatic-related sequences include dominant metastatic sequences, recessive metastatic sequences, metastasis associated sequences, dominant oncogenes, recessive oncogenes and cell cycle genes. These genes encode for example, proteins involved in cell cycle, signal processing, DNA replication, growth regulation, inter and intra cellular signaling transcription control and translation control. Isolated sequences are useful in the treatment and for the detection of metastatic and other disorders. Disorders which may be treated comprise diseases involving proteins and sequences which are isolated by interaction with the sequences and proteins isolated by the method of the invention. Both malignant or nonmalignant disorders may be treated. Non malignant disorders include hyperplasia, dysplasia and hypertrophy. Examples of nonmalignant disorders include benign enlargement of the prostate, nodular hyperplasia, and benign prostatic hypertrophy.
Treatment may involve gene replacement, gene targeting, antisense inhibition, gene expression or gene suppression. Gene replacement involves replacing a copy of a defective gene with another copy by homologous recombination. Gene targeting involves the disruption of a cellular copy of a gene by homologous recombination. Antisense inhibition exploits the specificity of hybridization reactions between two complementary nucleic acid chains to suppress gene expression. Cloned genes can be engineered to express RNA from only one or the other DNA strands. The resultant RNA hybridizes to the sense RNA and inhibits gene expression. Gene expression and gene suppression involve the introduction of genes whose expression actively inhibits neoplastic transformation and metastasis.
Another embodiment of the invention is directed to nucleic acids which comprise a sequence identified by the methods of the invention. The nucleic acid may be DNA, RNA or PNA and may be used as a diagnostic tool in the treatment of neoplastic disorders and malignant tumors. The nucleic acids may comprise additional sequences such as promoters, for expression of a sense or antisense message, recombination sequences for gene targeting, selectable markers for transfections, or replication origins for passage in a prokaryotic or eukaryotic host such as animal cells, bacteria or yeast.
Another embodiment of the invention is directed to nucleic acids which comprise sequences identified by the method of the invention such as, for example, the caveolin gene, ABP280 (actin binding protein 280), the lysyl oxidase gene, and the nmb gene (clone 29), and other sequences listed in FIG. 12 and FIG. 13. Nucleic acids comprising a sequence corresponding to these genes may be used in treatment or diagnosis and in diagnostic kits for screening biological samples for the presence or absence of metastasis or metastatic potential. Treatment may involve using the sequences in gene therapy, including gene ablation, gene expression and antisense suppression. Diagnosis may involve genotypic analysis of samples to determine the existence and expression levels of the expressed sequences.
Another embodiment of the invention is directed to the ue of caveolin gene and protein in the isolation of oncogenes and in the treatment of neoplastic disorders such as, for example, prostate cancer. Caveolin is an integral membrane protein and a principal component of caveolae. Caveolae are small invaginations at or near the plasma membrane of most smooth muscle cells and may function as a component of specific signal transduction pathways. Surprisingly, caveolin expression increases in metastatic human prostate cells as compared to human primary prostate tumors.
As caveolin expression correlates with metastasis, application of biological technologies designed to block the activity of caveolin or the function of caveolae may have therapeutic benefits for the treatment of neoplastic disorders such as human prostate tumors. Specific treatment approaches using caveolin may include the delivery of antisense or dominant negative caveolin sequences using expression or viral vectors; as well as the use of specific anti-caveolin antibodies. Additional approaches could also target the cavoeolae, but are not specifically based on caveolin function. Additional protein and non-protein components of caveolae could also be targeted for abrogation or the local or systemic administraiton of nutritional or biological agent may also be used. For example, caveolae are extremely rich in cholesterol and disruption or depletion of this molecule may alter the function of caveolae.
Another embodiment of the invention is directed to methods for treating a neoplastic disorder comprising administering a pharmaceutically effective amount of composition containing a nucleic acid having a sequence identified according to the methods of this invention, its expression product or fragments of either. The nucleic acid may be in the form of a sense or antisense single-stranded or double-stranded nucleic acid. The composition may be combined with a pharmaceutically acceptable carrier such as water, alcohols, salts, oils, fatty acids, saccharides, polysaccharides administered by injection, pulmonary absorption, topical application or delayed release. More than one carrier may be used together to create a pharmaceutical with desirable properties.
Another embodiment of the invention is directed to a kit or diagnostic acid aid for screening biological samples for detection of metastasis, neoplasia or kits metastasis or neoplasia. Kits comprise sequences isolated according to the methods of the invention and reagents and materials useful in such kits, such as, for example, buffers, salts, preservatives, and carriers, all of which are well known to those of ordinary skill in the art. Kits are useful for the analysis of tissues to screen those for the determination of normal, nonmalignant neoplastic or malignant cells. Kits may comprise additional reagents useful for the extraction of nucleic acids from a tissue sample. Reagents for analyzing the nucleic acid extracted from a tissue sample such as polymerase chain reaction reagents and Southern blots reagents may also be included.
The following experiments are offered to illustrate embodiments of the invention and should not be viewed as limiting the scope of the invention.
EXAMPLES Example 1
Production of Mouse Prostate Reconstitution Tumors and Metastasis.
Mouse Urogenital Sinue (UGS) tissue was isolated from 17 day old mice embryos. Each isolated UGS was digested with 1% trypsin for three hours at 4° C. The trypsin was inactivated by the addition of fetal calf serum. UGS cells were digested with 0.125% collagenase for 1.5 hours, counted and mixed at the appropriate cell ratios prior to infection with retrovirus in the presence of polybrene. Retroviruses used include Zipras/myc-9. Control experiments were performed using BAGA virus. After a two-hour infection, the infected cells were centrifuged and individual reconstitutions containing 1.5×106 cells produced by resuspending the cells in rat tail collagen at a density of 6.0×107 cells per ml. Aliquots of the infected UGS cells were placed in (DME) with 10% fetal calf serum overnight at 37° C., 5% CO2.
The next morning each cell/collagen reconstitution was implanted under the renal capsule of an adult male +/+ animal. Reconstitutions were harvested from the mice five weeks later when they showed signs of obvious distress from the tumor burden. Metastasized tumors were isolated from the same mice at sites outside the renal capsule. Isolated tumors and metastasises were either stored in liquid nitrogen or in preservatives such as 10% buffered formalin.
Cell lines were derived from fresh tumors by mincing a small portion of the primary and metastatic tumor and placing each in explant culture in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum. Cells which grow from each explant were propagated in DMEM and 10% fetal calf serum.
For histological analysis, a portion of a fresh tumor was fixed in 10% buffered formalin and embedded in paraffin for sectioning and staining with hematoxylin and eosin (H&E) or immunohistochemical staining. Immunohistochemical localization of cytokeratins was detected using polyclonal cytokeratin antiserum A575 (Dake Co.; Carpinteria, Calif.) and Vectastain ABC kit (Vector Laboratories; Burlingame, CA).
Example 2
Isolation of C-DNA for DD-PCR.
Total cellular RNA was isolated by ultracentrifugation through cesium chloride. Briefly, up to one gram of cells from culture, tumors or organs was placed into 4 ml of ice-cold GIT buffer (4M guanidine isothiocyanate, 0.025M sodium acetate, 0.1M M β-mercaptoethanol) and homogenized in a tissue homogenizer (Polytron or equivalent). The homogenate was carefully layered over 4 ml of 5.7M CsCl, 0.024M sodium acetate (1.8 g CSC1 per ml) in a centrifuge tube. The layers were centrifuged at 35,000 RPM for 18 hours in a SW50.1 rotor. DNA was collected from the interface between the cushion and the supernatant, diluted two folds with water, added to 2.5 volumes of ethanol and spooled out on a glass rod. RNA that formed a pellet on the bottom of the CsCl layer was resuspended, and once extracted with an equal volume of phenol:chloroform (1:1), twice with chloroform and precipitated with ethanol and resuspended in diethylpyrocarbonate treated water. The concentration of DNA and RNA were be determined by absorption at 260 nanometers.
Example 3
Differential Display Polymerase Chain Reaction.
mRNA isolated from primary tumors or metastasis was reverse transcribed with one of the primers and subjected to DD-PCR using the same primer as both the forward and reverse primer. A set of 24 primers comprising short oligonucleotides were used for both the reverse transcription of mRNA into c-DNA and for differential display polymerase chain reaction. The sequence of the primers used are shown in Table 1.
TABLE 1
Primer No. Sequence Sequence number
1 5′-TGACAATCG-3′ (SEQ. ID. NO. 1) 
2 5′-AGCTAAGGTC-3′ (SEQ. ID. NO. 2) 
3 5′-TCTGCGATCC-3″ (SEQ. ID. NO. 3) 
4 5′-ATACCGTTGC-3′ (SEQ. ID. NO. 4) 
5 5′-TACGAAGGTG-3′ (SEQ. ID. NO. 5) 
6 5′-TGGATTGGTC-3′ (SEQ. ID. NO. 6) 
7 5′-CTTTCTACCC-3′ (SEQ. ID. NO. 7) 
8 5′-GGAACCAATC-3′ (SEQ. ID. NO. 8) 
9 5′-TGGTAAAGGG-3′ (SEQ. ID. NO. 9) 
10 5′-TCGGTCATAG-3′ (SEQ. ID. NO. 10)
11 5′-CTGCTTGATG-3′ (SEQ. ID. NO. 11)
12 5′-GATCAAGTCC-3′ (SEQ. ID. NO. 12)
13 5′-GATCCAGTAC-3′ (SEQ. ID. NO. 13)
14 5′-GATCACGTAC-3′ (SEQ. ID. NO. 14)
15 5′-GATCTGACAC-3′ (SEQ. ID. NO. 15)
16 5′-TTAGCACCTC-3′ (SEQ. ID. NO. 16)
17 5′-ACCTGCATGC-3′ (SEQ. ID. NO. 17)
18 5′-GCTATACTGC-3′ (SEQ. ID. NO. 18)
19 5′-AGTTGCCAGG-3′ (SEQ. ID. NO. 19)
20 5′-AAGCCGTGTC-3′ (SEQ. ID. NO. 20)
21 5′-TCAACGCTCA-3′ (SEQ. ID. NO. 21)
22 5′-TGTTCGAATC-3′ (SEQ. ID. NO. 22)
23 5′-CGAGTCAGAC-3′ (SEQ. ID. NO. 23)
24 5′-TATGAGTCCG-3′ (SEQ. ID. NO. 24)
PCR was performed using standard conditions with 40 cycles of denaturation at 94° C. for 40 seconds, annealing at 40° C. for 2 minutes, and elongation at 72° C. for 35 seconds. After PCR, the products were analyzed with non-denaturing polyacrylamide gel electrophoresis (PAGE) at 12 watts for 15 hours. Bands which differed between test and control samples were eluted from the gel, subjected to reamplification by PCR and cloned. Polyacrylamide gel electrophoresis of DD-PCRs, and the accompanying RNA blot analysis showing the isolation of sequences with substantial similarity to nmb and TGF-β is shown in FIG. 6 and FIG. 7 respectively. Additional sequences isolated by this method show substantial similarity to lysyl oxidase, actin binding protein, ubiquitin activating enzyme E1, α-actinin, and P34 ribosimal binding protein sequence (FIG. 8). Differential expression of caveolin was demonstrated by DD-PCR followed by PAGE (FIG. 9).
Example 4
p53 Allelotype Determination.
The p53 allelotype of a cell sample was determined by PCR. Briefly, nucleic acid is extracted from a tissue sample or a cell culture sample. An aliquot of nucleic acids in placed in 45 μl aliquot of a master mix which contained a final concentration of 0.2 mM of each dATP, dTTP, dGTP, dCTP, 1.5 mM MgCl2, 0.5 unit Taq polymerase, 0.05 μM of each of two primers set specific for the normal wildtype allel of p53 (5′-GTGTTTCATTAGTTCCCCACCTTGAC-3′, SEQ. ID NO. 25; 5′-AGAGCAAGAATAAGTCAGAAGCCG-3′, SEQ. ID NO. 26). A control set of primers specific for the fibroblast growth factor-7 gene was used to monitor the polymerase chain reaction experiment (5′-ACAGACCGTGCTTCCACCTCGTC-3′, SEQ. ID NO. 27; 5′-CCTCATCTCCTGGGTCCCTTTCA-3′, SEQ. ID NO.28). One μl of the reaction from the first round of PCR was used as the starting material for a second round of PCR using a second set of wildtype p53 specific primer (5′-GTCCGCGCCATGGCCATATA-3′, SEQ. ID NO. 29; 5′-ATGGGAGGCTGCCAGTCCTAACCC-3′, SEQ. ID NO. 30). This second round of PCR was also monitored using a control set of primers specific for the fibroblast growth factor-7 (5′-ACAGACCGTGCTTCCACCTCGTC-3′. SEQ. ID NO 27; 5′-CCTCATCTCCTGGGTCCCTTTCA-3′, SEQ. ID NO 28).
After PCR the products were analyzed with non-danturing polyacrylamide gel electrophoresis (PAGE) at 12 watts for 15 hours. Bands which differed between test and control were eluted from the gel, subjected to reamplification by PCR and cloned.
Example 5
Induction of cell lines with TGFβ6 Influence Cellular Gene Expression.
1481-PA cells were grown overnight in DME supplemented with 10% fetal calf serum overnight at 37° C., and 5% CO2. Induction was performed by treatment with TGF-β1 at a concentration of 2 nanograms per ml. The treated cells were returned to the incubator and cultured for 12 hours. After induction, cells were washed in phosphate buffered saline and harvested and concentrated by centrifugation.
RNA was extracted from treated and untreated cells and subjected to DD-PCR. Differentially expressed bands detected by DD-PCR were cloned and differential expressions were confirmed using RNA blots (FIG. 10). Subsequent cloning and sequencing identified the bands as ABP280 or filamin.
One gene isolated showed differential expression in cells induced by TGF-β (FIG. 11, clone 29), while a control probe on the same cell line showed no difference in expression levels (FIG. 11, GAPDH).
Example 6
Metastatic Sequences Isolated.
Using the methods of Examples 1, 2, 3, 4, and 5, a plurality of metastatic sequences were isolated and sequenced. The expression of the metastatic sequences in primary cells and in metastatic cells were determined using RNA blots. The nucleic acid sequences of other isolated sequences are listed in FIG. 12. Sequence analysis and expression analysis was performed on the isolated cloned and the results of these studies are summarized in FIG. 13.
Example 7
Caveolin Immunoassay in Human Prostate Cancers.
Primary site human prostate tumors and metastases were isolated and analyzed for caveolin expression by immunoassay. The results of the assay is shown in Table 3. Metastases shows higher levels of caveolin proteins in metastases than in primary tumors. Immunohistology of tissue sections reveals both elevated levels and distinct distribution of caveolin protein in metastatic human prostate when compared to a primary human prostate tumor (FIG. 14).
TABLE 3
Patients Primary-site Metastases in lymph node
1 + ++
2 ++ +++
3 ++ +++
4 ++ ++
5 + +
6 ++ ++
7 ++ +++
8 + +
9 − −
10 + +
11 + +
12 ++ ++
13 + +
14 ++ +++
Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. The specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(iii) NUMBER OF SEQUENCES: 175
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TGACAATCG 9
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GATCACGTAC 10
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GATCTGACAC 10
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TTAGCACCTC 10
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ACCTGCATGC 10
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GCTATACTGC 10
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AGTTGCCAGG 10
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AAGCCGTGTC 10
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TCAACGCTCA 10
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TGTTCGAATC 10
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CGAGTCAGAC 10
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TATGAGTCCG 10
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GTGTTTCATT AGTTCCCCAC CTTGAC 26
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AGAGCAAGAA TAAGTCAGAA GCCG 24
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ACAGACCGTG CTTCCACCTC GTC 23
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CCTCATCTCC TGGGTCCCTT TCA 23
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GTCCGCGCCA TGGCCATATA 20
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(C) STRANDEDNESS: single
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ATGGGAGGCT GCCAGTCCTA ACCC 24
(2) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
AATTTTTTTT TTCGACGGCC CAACGGAATT TTTTTTTTCG ACGGCCCAAC GGAATTTTTT 60
TTTTCGACGG CCCAACGGGA ATTCGGCTTA GCTAAGGTCA CCCAGACTTC ATGGACTTGT 120
CTATTTTCTT GCCCAAAGGG ATAGTTCCTC AGGTATTTGG GGACAGCATT CACCTCTTGC 180
AGGAGCTATG CCTGTGTGTT TGTGCTAAGT TGATACTTTC TGCGATGATC TCAC 234
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
TACCATCGGA GAAAGAAGAC CAAGCAAGGC TCAGGCAGCC ACCGCCTGCT TCGCACTGAG 60
CCTCCTGACT CAGACTCAGA GTCCAGCACA GACGAAGAGG AATTTGGAGA ATTGGAAATC 120
GCTCTCGTTT TGTCAAGGGA GACTATCCCG ATGCTGCAAG ATCTGCTGTC CCTCTGGCCT 180
TTGTCATCCT CGCGCCTGCG TTGTGGCCTC TGTGGGCTTG GTGTGGAGCA AATGGCTCTC 240
AAGGAGGACT GAGTCTCAAG GAAATT 266
(2) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
AGCTAAGGTC AGGAGGTGTC TGAAGAATTG GCTGATGCAT GGCAGGGATG TTGTTGACCT 60
GCTTTTAGAA CAATACTTCC ATTTAATTAT AGCATATCTT ATGTGTGTAT TAAAGCAGAG 120
CCGATCTGGT GGGGCTCATT AAGTAAATGT ACTTACTGCA AAAGGTTCAA CTGGTGACCC 180
CAGTTTTCCC CAGAAGCAAT ATGATAGGAC AGAGGCGACT CCTGCAAGTT GTCTCAGACT 240
TCACACATAC ATTGTGACAT TCTCTGAGCA TGTGCACTGT ACATGATATG ACACTATCAA 300
(2) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 312 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
AGCTAAGGTC CACTACCTTG TGAAGATGTA TAAACACCTG AAATGTAGAA GCGATCCGTA 60
TGTCAAGATC GAGGGGAAGG ACGCTGACGA CTGGCTGTGT GTGGACTTTG GGAGTATGGT 120
GATCCATTTG ATGCTTCCAG AAACCAGAGA AACCTATGAA TTAGAGAAAC TATGGACTCT 180
ACGTTCTTTT GATGACCTTA GCTAAGCCGA ATCAGCACAC TGGCGGCGTT ACTAGTGGAT 240
CGAGCTCGTA CAGCTGATGC ATAGCTTGAG TATCTATAGG TTACTAATAG CTGGCTATCA 300
TGTCAAGCGT TC 312
(2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
AGCTAAGGTC AAAATAAAAG CTCAAGATGA CATCAGTCCC ATTTGTCCTA AGTCCTGGTG 60
TTGTATGGAT GGTAAGCAGC AGCCAATTAT GGTGACAGGT GATAGATCCA ATTTGTTAAC 120
ATTTCTCCAT CTCTAAGCCA TCCTTAAAGA AAATCATGAA TGGAGTCACA CCATCTTCAC 180
GGTAGTCCAG GAGAGCAACC ATACCATCTG GATTCATGTT TCACCAATAA AAACTGGTAG 240
TTATTGAATT AGCAAGGATG TGCTACTCTC TGCAGCTCAG C 281
(2) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
AGCTAAGGTC TCATGCAATG GAACTTAATT CTTAGAACTG TAAGAATTAC ATCAAACATA 60
AAAGCCTCCC TATTAATGTA GTCCACAAAA CTGGCAGGTA TATATGCCTT CTGAATTTGT 120
CTCCAGTGAC TTTGGTAAAT CTAACTAAAT TTTTAAAAAT TCTTAATGAA TTTATCGTCA 180
ACAACAACCA CCTCTTGGAA AATTAACCCT TGCAGTGTCT GTGTTAGACT CAGAAGTCAA 240
(2) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 203 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
GAATTCGGCT TAGCTAAGGT CAGCGTGAAG TTTAAGCAGA CATGAGTCTG AAACAGTCTC 60
ATGACACATC TGATAGGATT TTTTAAGACT GCCTGGCTTA GTCTTACTGC TGTTAGTGTA 120
TATTAGGTGT TGTACACATT ATAAAGAAAA TTATGTCTCA TTATCTTGTT TAAGTCAAGG 180
AAAATAGAGA ACTTTGGTCA AAT 203
(2) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
GAATTCGGCT TAGCTAAGGT CAGCGTGAAG TTTAAGCAGA CATGAGTCTG AAACAGTCTC 60
ATGACACATC TGATAGGATT TTTTAAGACT GCCTGGCTTA GTCTTACTGC TGTTAGTGTA 120
TATTAGGTGT TGTACACATT ATAAAGAAAA TTATGTCTCA TTATCTTGTT TAAGTCAAGG 180
AAAATAGAGA ACTT 194
(2) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 230 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
GAATTCGGCT TAGCTAAGGT CAAAATACAC GGATTGCAAT CACTTTTCTA AACAAAAGAA 60
ACAAAGTAAC TGCTGAGGTT AGCAAAGATG AGTTCTCGTC ATACTGCCTT GTACTGTTTT 120
GTGAACTGTG TTATTAAAAA TCTGAGCTTA ACAAAATCTT TACAAGTCAC CTCATGAAAA 180
CAGCATTTGG CCAATAAGAG TTTAATTCCA CACCAGTGAG ACCTTAGCCT 230
(2) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GAATTCGGCT TTCTGCGATC CACTCTTTGA AGCTATTGGC AAGATATTCA GCAACATCCG 60
CATCAGCACG CAGAAAGAGA TATGAGGGAC ATTTCAAGGA TGAAAGGTTT TTTTCCCCCC 120
TTACTATTTC CTTGGTGCCA ATTCCAAGTT GCTCTCGCAG CAGCAAATTT ATGAATGGTT 180
TGTCTTGATC AAGAACAAAG AATTCATTCC CACCATTCTC ATATATACTA CTTTCTCTTC 240
TT 242
(2) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
GAATTCGGCT TTCTGCGATC CACTCTTTGA AGCTATTGGC AAGATATTCA GCAACATCCG 60
CATCAGCACG CAGAAAGAGA TATGAGGGAC ATTTCAAGGA TGAAAGGTTT TTTTCCCCCC 120
TTACTATTTC CTTGGTGCCA ATTCCAAGTT GCTCTCGCAG CAGCAAATTT ATGAATGGTT 180
TGTCTTGATC AAGAACAAAG AATTCATTCC ACCATTCTCA TATATCTACG TCTCTTCTAG 240
(2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 154 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GAATTCGGCT TTCTGCGATC CTAGAGCAGG TAAGTGAAGA AGGCCAGTAA GTTTTAAGGA 60
TGGCCTTGTT GCCTTCTATC AAGTTCTCTG GGACTTTGTA ATTTTGATTA CTACTATTGA 120
TACATGGTTA TGGTCAGAAG GCCTCTTCTC CCTT 154
(2) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
AGCTAAGGTC CGGACTCTAT GGCATGACCC CAAAAACATT GGCTGGAAAG ATTACACTGC 60
CTACAGGTGG CACCTGATTC ACAGGCCTAA GACAGGCTAC ATGAGAGTCT TAGTGCATGA 120
AGGAAAGCAA GTCATGGCTG ACTCAGGACC AATTTATGAC CAAACCTACG CTGGTGGACG 180
GCTGGGCTGT TTGTCTTCTC CAAGAGATGG TCTATTCTCG GACCTCAAGT ATGAGTGCAG 240
AGATGCTAGA GAGCAGGCTC AGTCTCAGCA 270
(2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 285 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
TGACCATCGA GTGCATCAGC CTCATCGGGC TGGCCGTCGG GAAGGAGAAA TTCATGCAGG 60
ATGCTTCAGA TGTGATGCAG CTATTGTTGA AGACACAGAC AGACTTCAAT GATATGGAAG 120
ATGACGACCC CCAGATTTCT TACATGATCT CAGCATGGGC CAGGATGTGC AAAATCTTGG 180
GAAAGAATTC CAGCAGTACC TTCCCGTGGT TATGGGGCCG CTGATGAAGA CTGCTTCAAT 240
TAAGTCCTGA GTGCCTCTAG ACACCAGGAC ATGAGATATG AGGTA 285
(2) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
TGACCATCGT GTAGTTGGTG TGCTTGTTGT CGAAGATGAG GGCCTCCTGG ATGAGCTGGT 60
GCTGCTGCTC CAGCAGGTCC AGGCTGGGCT TGTAGTCCAC GATGCTGCGC TCGTACTGCT 120
TCAGGTGGCT CAGCTGGTCT TCCAGAGTCC CGTTCATCTC AATGGAGATG CGCCCGATCT 180
CCTCCATCTT AGTCTGGATC CACGGCCCCA CCATATTGGC TTGGCTGGCG AACTGTCGGC 240
GAAGGCTGCA TTGGATTGCT 260
(2) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 283 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
TGACCATCGA ACACCCCAAC ACTCTCCACT ACCTGCCATT TCTTCCAGCC TTATCCACAC 60
CACCCCGTTT CTCCTGAAGA CTGATTTGCT TAGCAACTGC ACTGAGCCAA CCCTGAAGAC 120
ACATGATTAT TGGTTGGGCT CCATTAAACA ACAAGCCTAG TGCTTGGGAA GGGGGGTGGG 180
GAGGGGAAGA GACGTGAGAA GCATGTTGGC GTAGACCTTG AGGCATGGAT GAAGCATCTG 240
CCGGCCTGAC CTGGTACAGG TGGCATCTGC ACTGCAGCAA GGC 283
(2) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 277 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
TGACCATCGA AGTGCAAAGG AAATGACTTG ATTTCATGAA GTATCTCCAG AAGTAACGCT 60
TTGTTTTCTG CATCCTGAAC TTTATTCCCA GTGAAGAGCT GAAAATCTGG ACGCTCAAAA 120
AATGGAAGCA CTTTGGAGAG AGCCCTTAAC TCTATCAGGT ACAGGAAGTA CAAGTTCCTC 180
AGCCTTCGTG GGCCTTCTCC TTCAGTCAGA ATCCATCAAA GGTGCTGGAA CTCTGTGACA 240
TTGTGACCCA TTCTTTCAGC CAGTATCTGT AAGATAC 277
(2) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:
GGGAACGAAT GATCTGGAAC TGTGGCTTGT AGACAACCCA AATATCTTAG GTAGGTAAGA 60
AATTCCAGCA TCACACTATA TAGGAAATAC TGTGCGAAAC TGACAGTTAA CTGTGCACAA 120
AGTTCAATGG CTTCAAAATA ATGTATAAAG GATAAGAAGA AACCAGTTTA CCATTTTGGT 180
ATTATTTTGG TTGCTTTGTA TAACTTCAAT AATTT 215
(2) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
GGGAACGAAT GATCTGGAAC TGTGGCTTGT AGACAACCCA AATATCTTAG GTAGGTAAGA 60
AATTCCAGCA TCACACTATA TAGGAAATAC TGTGCGAAAC TGACAGTTAA CTGTGCACAA 120
AGTTCAATGG CTTCAAAATA ATGTATAAAG GATAAGAAGA AACCAGTTTA CCATTTTGGT 180
ATTATTTTGG TTGCTTTGTA TAACTTCAAT AATTT 215
(2) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
GACGTAAGCC 10
(2) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 189 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:
CCACAAAGCA AGCTTCTGTC TGGAGTACAG CTCCTGTGAC TATGGGTACC ACAGGGCCTT 60
TGCGTGCACT GCACACACAC AGGGATTGAG TCCTGGATGT TATGACACCT ATGCGGCAGA 120
CATAGACTGC CAGTGGATTG ATATTACAGA TGTACAACCT GGAAACTACA TTCTAAAGGT 180
CAGTGTAAA 189
(2) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 227 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
CTATCAATGA AGGGGGAGAT CACTGGGTAA GTTCGAATGC CCTCAGGCAA GGTGGCCCAG 60
CCTTCCATTA CTGAATTCAA AGATGGCACT GTTACTGTAC GTTACTCACC CAGTGAAGCT 120
GGCCTGCATG AAATGGACAT TCGCTATGAC AATATGCATA TCCCAGGAAG CCCTCTGCAG 180
TTCTATGTTG ATTATGTCAA CTGTGGCCAC ATCACTGCTT ATGGTCC 227
(2) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 373 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
TTAGCACCTC GACCACGAAA TGAGGAAGAT GCAACAGACG TGGTGGGCCT GGCTCAGGCT 60
GTAAACGCTC GGTCCCCACC TTCAGTAAAA CAGAACAGCT TGGATGAAGA CCTTATTCGG 120
AAGCTAGCTT ATGTTGCTGC TGGGGACCTG GCACCCATAA ATGCTTTCAT TGGGGGCCTT 180
GCTGCCCAGG AAGTCATGAA GGCCTGCTCT GGAAAGTTTA TGCCCATCAT GCAGTGGTTG 240
TACTTTGATG CTCTTGAATG TCTCCCAGAA CGGACAAAGA GGCTCTGACA GAGGAGAGTG 300
CCTCCCACGT CAGAACCGTT ACGATGGGCA GGTAGCTGTA TTGGTCAGAC TTCAGGAGAA 360
GCTGAGAAGC AAA 373
(2) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 257 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
TTAGCACCTC CAATGGCTGG GTACCAGCCA GCCGCAATGT CCGCTCCACA AATTTGGAGT 60
CTGTGAGGTA CTGATTAACA TTTTCTGCTG GCTGCTTGAA AAGGCCTTCA AATTCATCCC 120
GGGCCCACTG AAGAGTGTGT TCGATGGCAT TGGGAAAGTT TTTCAGGGTA CAAATGGGGA 180
TGGATTTCTC TGGTGGATCC TGGCTAGACG TGATGGATTC TGTCAGGAAG GGGATTACCA 240
CCTGCACGTT GCCCTTT 257
(2) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 298 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
TTAGCACCTC ACACTCACAT GCCCTTCTAC ATAGAGACTG GTTAAACAGC CCTCCCTCCC 60
TTGTCCCGAC TTGACTTCCA GGCCCCTCTG CTTTCCTCTC ACAACCACAC CAGGTCTGAT 120
GGAGTCCAGT GCCTGCAGTG ACCCAACATA GACTGCACTT TCACCTACCT ACTGGATGGT 180
CCTGCAGCCC AGACGGCTGC TCTTCTTTCT CATGGAGTTT CTCTCCTGCC TGAGATATGC 240
TATCTGGTCT GCCCCTGTGT AGCTCCCATG GGATCCCTTA AAATCGATCC TTTTTTAA 298
(2) INFORMATION FOR SEQ ID NO: 56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:
TTAGCACCTC GTGAGGAGAC TGTTGTCCAC AGGCCAGCTA GTGGTACCCT ACTGAGAAGT 60
TGGGTTTTGG TTTTGTTTCC CTTGAAGGGT CGCTGTTAGA GGATGGAAGT AACTTCTAAT 120
TCTTGATCTG TTTGTTGGTC TTGTTTTCAG TACTTTTTGC CAGTTGTATA CACTTGGAGA 180
GGGAATTTGT ATGCCTGTAA TCTTGTTCTT GAGGTCAGAA ATTCAAAACA TTGGGAGCTT 240
TTGTTGTAAA GGTTAAACTG TGAATCCATA TAGCAAATGC AGATCCTTTT ACAGTGTAAA 300
CCACATTTCC TGCCTCAGCC TAAAGCACTG GTCATTT 337
(2) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 333 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:
ACCTGCATGC CTAAAGGAGT AGGCTTAGGG GTGGGGAGAG AGAAGGCATA GGCTTTTCTA 60
GTTATACAAA GCTGTGTAAG GCAAGGTTCC TTTCTACTAA ATGGTCAGCT GTCACTACAT 120
TTATACTTTT GTATGTCATA AACCCTTTCT TTCATTCCTC CCTGGGTAAC CAGGACAATC 180
GGAGGGCAGT GTGTTACTGG GATTAGAGGA CTAGCAATAC TGGGTAACCC GCCTAAGCTG 240
GAAGGTGACG TAATACGTTT CTTTAAAGAT TCAGTCAGTC AAGCAGTTTA GCAATATCAA 300
AATGTCTGGC TGTTTGGTCC AGTGTACACT GTT 333
(2) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 296 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
GCTATCTGCG AAACTACAGA AAGGAAGACA GCTTGGCCCA GCGCGGTGAA GTTCAGAATT 60
CACTAGGTAG TTGTTGTTGG TTGACTTGGA GGTAGCTGGG TAATCAACAG CTTTCACTTT 120
AGATTCAATG TGAACCGCAG AGTTACTCAT GACCAAGAGT CTGGCAAACT CATTAATGCT 180
GTTTAATACT TGTTTGATAT TTTTTCACCT TTTGAGCCCT TTTCCCAAAG AATTCAATAT 240
CAGTTTAGTA GCAACAGTAC AGTTGCCATT TAAATTGGTT TAGTTGCAGT ATAGCA 296
(2) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 296 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:
GCTATCTGCG AAACTACAGA AAGGAAGACA GCTTGGCCCA GCGCGGTGAA GTTCAGAATT 60
CACTAGGTAG TTGTTGTTGG TTGACTTGGA GGTAGCTGGG TAATCAACAG CTTTCACTTT 120
AGATTCAATG TGAACCGCAG AGTTACTCAT GACCAAGAGT CTGGCAAACT CATTAATGCT 180
GTTTAATACT TGTTTGATAT TTTTTCACCT TTTGAGCCCT TTTCCCAAAG AATTCAATAT 240
CAGTTTAGTA GCAACAGTAC AGTTGCCATT TAAATTGGTT TAGTTGCAGT ATAGCA 296
(2) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 273 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
GCTATACTGC AACTAAACCA ATTTAAATGG CAACTGTACT GTTGCTACTA AACTGATATT 60
GAATTCTTTG GGAAAAGGGC TCAAAAGGTG AAAAAATATC AAACAAGTAT TAAACAGCAT 120
TAATGAGTTT GCCAGACTCT TGGTCATGAG TAACTCTGCG GTTCACATTG AATCTAAAGT 180
GAAAGCTGTT GATTACCCAG CTACCTCCAA GTCAACCAAC AACAACTACC TAGTGAATTC 240
TGAACTTCAC CGCGCTGGGC CAAGCTGTCT TCC 273
(2) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 322 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
GCTATACTGC CCACCACATT GCCACACTCG GAATGACATT TCTATATTTT CACCTCCCCA 60
GATTTCCATT TCTTCATCGT AACTTCCAAT GTGCTCAAAA TATTTTTTAG ATATAGAAAA 120
AAGGCCTCCT GCAAAGGTGG GGGTCTTAAT TGGGTAGGTT TCATCTTTCC TTCTTTGCTT 180
CTCATGATCA GGAAGTGACT CCCAGCCAAA GGAAAGGCTC CAGTCAAAAT TTCCACGGTT 240
ATGGTTGCTT CCGTACGGAG AAGGCTTGTT GAATTCAAAT GTGTTTAGAT CTATGGATGC 300
GATGTCTGGA CTCACCACGG CA 322
(2) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
GCTATACTGC TGAAGGAGAT CATTTTGGTG GATGATGCTA GTGTAGACGA CTACCTGCAT 60
GAAAAGCTGG AGGAATACAT AAAACAGTTT TCTATTGTGA AAATAGTCAG GCAGCAAGAA 120
AGGAAAGGCC TGATCACCGC GCGGTTGCTA GGGGCAGCTG TAGCAACTGC CGAGACGCTC 180
ACGTTCTTAG ATGCTCACTG TGAGTGCTTC TATGGCTGGC TGGAACCTCT GCTGGCCAGG 240
ATAGCTGAGA ACTACACTGC CG 262
(2) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 295 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
AGTTGCCAGG GGGCAGCTCA CGGCGCAGCT CATCCTCTGT GATGTAATTC TTATCTCCAG 60
CCAGGATCTT GAAGGAAGCC ATGACCTGAT CTGCAGTATC AGTATCTGCC GTCTCTCGGG 120
ACATAAAGTC GATGAAGGCC TGGAACGTCA CTACCCCCAA GCGGTTGGGG TCTACAATGC 180
TCATGATTCG GGCAAACTCT GCCTCTCCCA TGTTGTAACC CATGGAGATA AGGCAGGCGC 240
GGAAATCGTC TGTGTCCATC ATGCCCGTCT TCTTCCGGTC AAAGTGGTTG AAAGA 295
(2) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 287 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
AAGCCGTGTC GCTGAACTGG GAGGACACAC TGCTCACCCT AGAAGGCTCT GGCTGACCCT 60
CCGCCCGGTT AAACAGGGAC TTTGTGGCCA TGTGCTGGCG ACACAGGTCC TGGTACTCAA 120
AAGTAGTGTC ACCATGGGCC CCCTCCGGCC CCAGCGCTGC CAGGCGTCCT TATCCCGCTG 180
TCTCGAATGA TGGCGCATAC CAAGGCCACT GAAAGCCACT AGCAGCCCAG CGACGCCTGC 240
CAGGGCCACT AGAGTAAGCA GCACTGAGCG CATGGGAGAT ATGCCAT 287
(2) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
AAGCCGTGTC TGGACGTCCG TGTGTCCGGC TCTTGCTCAC GCAGTCATGG CCTCCGGAAC 60
GCGCAAATCG GAAAGTCGGC TCCTGACTTC ACGGCCACAG CGGTGGTGGA TGGTGCCTTC 120
AAGGAAATCA AGCTTTCGGA CTACAGAGGG AAGTACGTTG TCCTCTTTTT CTACCCACTG 180
GACTTCACTT TTGTTTGCCC CACGGAGATC ATCGCTTTTA GCGACCATGC TGAGGACTTC 240
CGAAAGCTAG GCTGCGAGGT GCTGGGAGTG TCTGTGGACT CTCAGTTCAC CCACCTGGCG 300
TGGATCAATA CCCCACGGAA AGAGGGAGGC TT 332
(2) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 331 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
AAGCCGTGTC GGAGGGCACC AAGGCTGTCA CCAAGTACAC CAGCTCCAAG TGAGTGCTCA 60
AGACTCAGCT CTTAACCCAA AGGCTCTTTT CAGAGCCACT CAAGACTTCA AAATTGGAGC 120
TTTAATGCTG ACTTAGTGAC TACCGGGAAA ATAACTGACT TCATCTGCAG GATTGTGTAC 180
AAACACTTAT GGTTTAGTAA ATCGAAAAGA TAGACATTGC CCATCAGTTC TGTCTGGTCC 240
ACTTAAATAT GCTTTTTTCT TAGAAGTTCT AAGAACCCTG TCAATAACCT ATCTAGGTCC 300
AGTCCTTGAG TTCAAAGGCC AAATACCAAT G 331
(2) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 359 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
CAACGCTCAG GATGTAAGCT GTTTCCAGCA CCTGGTTCAA GCGAATGTAA GAAATAAGAA 60
GGTGTTGAAA GATGCCGTGA ATAACATTAC AGCAAAGGGG ATCACAGATT ACAAGAAAGG 120
CTTTAGCTTT GCCTTCGAAC AGCTACTTAA TTATAATGTT TCCAGAGCTA ATTGCAATAA 180
GATTATCATG TTATTCACGG ATGGAGGAGA AGAGAGAGCC CAGGAGATAT TTGCCAAATA 240
CAATAAAGAC AAAAAAGTCC GTGTGTTTAC ATTTTCCGTC GGTCAACATA ATTATGACAG 300
AGGACCTATT CAGTGGATGG CTTGTGAAAT AAAGGTTACT ATTATGAGAT TCCTCCATT 359
(2) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
TCAACGCTCA TCACACCAAG AATCAACTGG TTCTTCAAGT TTGTCTTATT TTCAGATTGG 60
CCAGTGACGT TGAAGACTGG TAGAGTTCCA GTAATGACAA GTCCCAGTTC CAGGGCATCC 120
AAATACACAT TTGTCCATTG AACTTGCTTC GCTTTGTCAC CAGCTAAAAC CATTGGTCTT 180
CCCAGAACAT CTAGATATTC CTGAGTATTG ATTCTTATTG CACCAATGGA GGGAATCTCA 240
TAATAGTAAC CTTTATTTTC ACAAGCCATC CACTGAATAG GTCTCTGTCA TAATTATGTT 300
GACCGACGGA AATGTAA 317
(2) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
TAACGCTCAG GAGAAGAATA GGAATGCAGA GAACTCTGCC ACAGCCCCCA CGCTCCCGGG 60
CAGCACCTCA GCCACCACCG CAACCACCAC CCCTGCTGTA GATGAAAGCA AGCCTTGGAA 120
CCAGTATCGC TTGCCTAAGA CTCTTATACC TGACTCCTAC CGGGTGATCT TGAGACCCTA 180
CCTCACCCCC AACAATCAGG GCCTGTACAT CTTCCAAGGC AACAGTACTG TTCGCTTTAC 240
CTGCAACCAG ACCACGGATG TCATTATCAT CCACAGCAAA AAGCTCAACT ACACCCTCAA 300
AGGAAACCAC AGGGTGG 317
(2) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 287 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:
CGAGTCAGAC GGCTTCAGCA TCGAGACCTG TAAGATCATG GTGGACATGC TGGATGAAGA 60
TGGGAGTGGC AAGCTTGGCC TGAAGGAGTT CTACATCCTC TGGACGAAGA TTCAGAAATA 120
CCAAAAAATC TACCGGGAAA TCGATGTGGA CAGGTCTGGA ACTATGAATT CCTACGAGAT 180
GCGGAAAGCA CTGGAAGAAG CAGGTTTCAA GCTGCCCTGT CAACTCCATC AAGTCATCGT 240
TGCCCGGTTT GCAGACGACG AGCTAATCAT CGACTTTGAC AATTTTG 287
(2) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 311 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:
CGAGTCAGAC AACCTGTTCA AGTGGGGTGG GGACCATCCA CGGAGCAGCC GGCACCGTAT 60
ATGAAGACCT GAGGTACAAA CTCTCCCTAG AGTTCCCCAG CGGCTACCCT TACAACGCAC 120
CCACAGTGAA GTTCCTCACA CCCTGCTACC ACCCCAACGT GGACACCCAG GGCAACATCT 180
GCCTGGACAT CCTCAAGGAT AAGTGGTCTG CACTATATGA TGTCAGGACT ATCTTGCTCT 240
CTATCCAGAG CCTGCTAGGA GAACCCAACA TCGATAGCCT TTGAACACAC ACGCTGCGGA 300
ACTCTGGAAA A 311
(2) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 352 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:
TATGAGTCCG GAGCGACGGC TACGAGTGTG AACTGTTCCA GCCCCGAGCG ACACACCAGA 60
AGTTATGACT ACATGGAAGG AGGGGATATA AGGGTGAGAA GACTGTTCTG TCGCACCCAG 120
TGGTACCTGA GGATTGACAA ACGAGGCAAA GTGAAAGGGA CCCAGGAGAT GAAGAACAGC 180
TACAACATCA TGGAAATCAG GACCGTGGCA GTTGGAATTG TGGCAATCAA AGGGGTGGAA 240
AGTGAATACT ATCTTGCCAT GAACAAGGAA GGGAAACTCT ATGCAAAGAA AGAATGCAAT 300
GAGGATTGCA ACTTCAAAGA ACTGATTCTG GAAAACCATT ATAACACCTA TG 352
(2) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 317 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
TATGAGTCCG AGGAGGAGCA CAATGCTGGG AGTGTGGAAA GCCAGGTTGT CCCCAGCACA 60
CACCGAGTGA CCGATTCCAA GTTCCATCCA CTCCATGCCA AGATGGATGT CATCAAAAAA 120
GGCCACGCCA GGGACAGCCA GCGCTACAAA GTTGACTATG AGTCTCAAAG CACAGACACC 180
CAGAACTTCT CCTCCGAGTC TAAGCGGGAG ACAGAATACG GTCCCTGCCG CAGAGAAATG 240
GAGGACACAC TGAATCATCT GAAGTTCCTC AATGTGCTGA GTCCAGAGTC TCACATCCAA 300
ACTGTGACAA GAAGGGG 317
(2) INFORMATION FOR SEQ ID NO: 74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 247 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
TCGCCCGGGA CTTCATGCGA TTGAGAAGAT TGTCTACCAA ATATAGAACA GAAAAGATTT 60
ATCCCACAGC CACTGGAGAA AAAGAAGAAA ATGTTAAAAA GAACAGATAT AAGGACATAC 120
TGCCATTTGA TCACAGCCGA GTTAAGTTGA CTTTGAAGAC TCCATCCCAA GATTCAGATT 180
ATATCAATGC AAATTTTATT AAGGGTGTGT ATGGGCCAAA AGCATATGTG GCAACCCAAG 240
GGCCTTT 247
(2) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 256 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
TGTGGAAAGC CAGGTTGTCC CCAGCACACA CCGAGTGACC GATTCCAAGT TCCATCCACT 60
CCATGCCAAG ATGGATGTCA TCAAAAAAGG CCACGCCAGG GACAGCCAGC GCTACAAAGT 120
TGACTATGAG TCTCAAAGCA CAGACACCCA GAACTTCTCC TCCGAGTCTA AGCGGGAGAC 180
AGAATACGGT CCCTGCCGCA GAGAAATGGA GGACACACTG AATCATCTGA AGTTCCTCAA 240
TGTGCTGAGT CCAGAG 256
(2) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 383 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
TGACCATCGA AGTGCAAAGG AAATGACTTG ATTTCATGAA GTATCTCCAG AAGTAACGCT 60
TTGTTTTCTG CATCCTGAAC TTTATTCCCA GTGAAGAGCT GAAAATCTGG ACGCTCAAAA 120
AATGGAAGCA CTTTGGAGAG AGCCCTTAAC TCTATCAGGT ACAGGAAGTA CAAGTTCCTC 180
AGCCTTCGTG GGCCTTCTCC TTCAGTCAGA ATCCCATCAA AGCGCTGCTG GAACTCTGTG 240
ACATTGTGAC CCCATTTCTT TTCCAGCCAA GTATCTTGTA AAAGATACCT TGCACTCAAA 300
TGCACATTAA TGCTTGCGTG CAGGCCAGAT ATAAGTCTGT AGAATCGCTC TTTCTACACA 360
GAGGCCTTCT AGCCAGTTGT AAA 383
(2) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 400 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:
CTGCTTGATG CTAAGCCCGG CAGCCTGTGT TTCATCTACA GGATGCACAA CATAAAAGAA 60
AAGATCTGAT TCCCGCAGGT TCTCTTCTGA CCTACACACA CACACACTAA AATAACATTT 120
AAAAATATGT GCCAAATTAT ATTTGTTCGG GTGCCACCTT CCACCAGCTT ACCACTACGG 180
TAGAACTGTC AAATTCATCT CCCTGAATTT GTCTTAAAGG GGTGTCCATG CACAGGCCCA 240
AGAGTCACCT CCAATGAAAT AAATGTAATA CTGAAGTATG CCATGATGTT TGTTGTTTTC 300
TTTCATCGTA AGCCTGTAAG CAGGAAAAAT AGTAATAGAT AGAATAGAGA CTTACCAGTG 360
GTCGATGGCC TGGTCAGTCT GTGCGGTGAC TAGGACCAGG 400
(2) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 343 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:
ACCTGCATGC CGAGTGTGAC GCCTTTGAGG AGAAGATCCA GGCTGCCGGA GGGATCGAAC 60
TCTTTGTCGG AGGCATTGGC CCCGATGGAC ACATTGCCTT CAATGAGCCA GGCTCCAGCC 120
TGGTGTCCAG GACCCGTGTG AAGACTCTGG TTATGGACAC CATCCTGGCC AACGCTAGGT 180
TCTTTGATGG TGATCTTGCC AAGGTGCCCA CCATGGCCCT GACAGTGGGT GTCGGCACTG 240
TCATGGATGC TAAAGAGGTG ATGATCCTCA TCACAGGCGC TCACAAGGCC TTTGCTCTGT 300
ACAAAGCCAT CGATGGAGGC GTGAACCACA TGTGGACGGT GTG 343
(2) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
GCTATACTGC AATGTTAGGG GAATGAACGC GTTTTCCTAC TGCACTGGGG ACTTTTAGAT 60
AGGTTAATGA AAGGCCTTTT ATTCTGTTAC TGGACACGAA AACTTTGTCT AATTTCTTAT 120
ACTCTATTGT ACGTTTACAG TCGCAGCACT AAAATGGAAG ACATCAAACA TTTTTAACAG 180
AAAAAAAAAA AGATGTAAAA ACTAACTAAG GACTATTTAT TGATAATGTT TTGCTACTCC 240
TGTCAGACAA TGGCTATAAA CTGAATTAGG CAGTCTTAAA AAAAAAAAAA GAAAAAAAAG 300
AAAAAAGAAA AAAAGAAAAG AAAAGAAAAA AAACTGG 337
(2) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 371 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
AGCTAAGGTC GGGTACTCTG ATACTTCAGA GTTTAAAATC ATCAGCCCTT GTAGATCTAT 60
TCCTAAATCT TATGAAAATG CTCAGATGTT TACACAGCTG TGAAACAGGG TCAGTTCAGA 120
TCGCTGATGG CTTGAGAATG TGTTTCTTGT TGACATCAGG AACTGGAAAT GTTTACTTCC 180
CGTCATTTAT GAGTCATCAA GTATCTCGGC TCTTTTAAGA GCGCAAGATA AAACAAGCTT 240
AAACCAGGTG ATAAGAGCAG AGTCCACTTG AGTCTGAGCT CACCCGAGAA CTTGCTATCG 300
AGGACATTTG GAATGGGAGT GTGCAGGCTT CCTTCAGTTA CTGAATGAGT CCATCTGCTA 360
GTCACCTTGA C 371
(2) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 319 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
AGCTAAGGTC CAGGGGGCAA AGCGGTGACG TGTGCACATC GATATGAGAA ACGGCAGCAC 60
GTCAACACGA AGCAGGAGTC GCGGGATATC TTTGGAAGAT GTTATGTCCT AAGTCAGAAT 120
CTCAGAATTG AAGATGATAT GGACGGAGGA GACTGGAGTT TCTGCGATGG CCGGTTGAGA 180
GGCCATGAAA AGTTTGGCTC CTGTCAGCAA GGAGTAGCGG CTACTTTCAC TAAGGACTTT 240
CATTACATTG TTTTTGGAGC CCCAGGGACT TACAACTGGA AAGGGATCGT CGTGTAGAAC 300
AAAAGAATAA CACTTTTTT 319
(2) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 368 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
AAGCCGTGTC TGTGCTCAAG GAAGAAACCC ACTGGACCAA CTTCTGTCAG AAAGGAAAAC 60
CTTGTTCAAA GTTTCAGGAC CCTGTTCTTT GCTTATTTGC ACATGGTCAC CTTGGTCTGA 120
GCTAGCCACC ATTGTCACCC ACAGCTGCAA AGAAAGCAGA CCTTAGGAAA CACTGTCACG 180
GCTGAGTGTG ACTGCCTTGT TCATCCCCTG GACTGGTACT GTGTTGCCTG CAGTACCATT 240
GGGATCCCAT AGCAAGAGAG GGAGAGGGAG ATGTTAGTTA GCCTTTGCTA CGAACCAAGC 300
TGTCCCAAGT CTCAACAGCT AAACAGGTAT TCATTTACCA TGATTCTATG GTTAGCTAAG 360
CTCTTGAG 368
(2) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 340 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
CTTTCTACCC TGGAGGATGT GCTTGAGGCA CACTGCTCCT GTGCTCTCCA CTTGAGGCAT 60
AAGCCCAGTC AGTTGTGCAT AGATGATTAA CCTCTGACCC CTAAAGATGG TAAGTTGCTC 120
TGGAGAAAGC ATTTTAACAG ACAAACCAGG AGGCAAATCC CAACTTAGAG AGATGTTATC 180
CACTGCACAC TGTAGAGCAA ACTTGAGAGA CCCAAGAGCC TTGGTCTGCA TCCTGTCCTT 240
GCCTGTGATA AACACTCGAG TACCCCCTGA TACCGGGCGA TATTTTTGAT TAACTGGTCG 300
AGGCTCCTTG TCCAATTCCA AAAGAGAACA TCTGTGTTTC 340
(2) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 252 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
TGGTAAAGGG CATCTGTAAA TACACTCTAT GAGGAAATTA AAACTTGAAC ATGGCAGTCT 60
GACATTGCAA AACAAAACAA AACAAAACTG ACCCTCCAAT AGCAGCGAAA ACAACGTGAA 120
AGATACAAAG CAATGAGAAT CTGGTTCTGA ACGCCTGGGA TCCTGGGAGT CATCGGTAGC 180
AGCGCCATGA GAGGAGCCGT GGCCTGTCCC ATGTGGTCCC ACCTTCACCT CTTCCCTCAC 240
ATCCCTCTTA AG 252
(2) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 348 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
TGGTAAAGGG GGCAAGGGCA AAGGCACGGG AGACAGAGGC CACTGCATCT GTACCCACAT 60
CAGACATGTT TGTCCATTTT CTCTCATTTG GCCTTAGACC ATTGGCAAGA GTAAATGCTC 120
TTAGTCCCGT TATCTAGAAA TTTCTTCCTT TGGGGAGAAC CACTTATAGA CAATATCAGC 180
TCTCTACAAA TAACACGAAA GGTCGTAACA CAGCAAGTGA CCAGAAAGTG CCCGTCCTTG 240
CGGCTCTGAT CCACGTGGCT CTCCGTAGAC AAATTGTTTT TTCTTGTAGG GATATCTGTT 300
TTGCTTCTGA ACTTTCTTAC AAGTGTTTGG GACTCTTCGG GTGGCGTT 348
(2) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 351 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
TGGTAAAGGG TCAAGTGTTC GATCAGAGTG GAGCTCCATT ACCGAATGTA ATCGTGGAAG 60
TCCAAGACAG AAAGCATATC TGCCCGTTTA GAACCAACAA GCTTGGAGAA TACTATCTGC 120
TTCTGCTGCC CGGGTCCTAC GTGATCAATG TTACAGTCCC TGGACACGAC TCCTACCTCA 180
CGAAGCTTAC TATTCCAGGG AAATCCCAGC CCTTCAGTGC TCTTAAAAAG GATTTTCACC 240
TCCCGCTGCG ATGGCAGCCG GATTCCATCT CCGTATCCAA TCCTTCGTGC CGATGATTCC 300
GCTGTACAAA TTCATGCCAA GCCACTCGGC TGCCACAAAG CCTAGTCTGG G 351
(2) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 242 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
GAATTCGGCT TTCTGCGATC CACTCTTTGA AGCTATTGGC AAGATATTCA GCAACATCCG 60
CATCAGCACG CAGAAAGAGA TATGAGGGAC ATTTCAAGGA TGAAAGGTTT TTTTCCCCCC 120
TTACTATTTC CTTGGTGCCA ATTCCAAGTT GCTCTCGCAG CAGCAAATTT ATGAATGGTT 180
TGTCTTGATC AAGAACAAAG AATTCATTCC CACCATTCTC ATATATACTA CTTTCTCTTC 240
TT 242
(2) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
GAATTCGGCT TTCTGCGATC CACTCTTTGA AGCTATTGGC AAGATATTCA GCAACATCCG 60
CATCAGCACG CAGAAAGAGA TATGAGGGAC ATTTCAAGGA TGAAAGGTTT TTTTCCCCCC 120
TTACTATTTC CTTGGTGCCA ATTCCAAGTT GCTCTCGCAG CAGCAAATTT ATGAATGGTT 180
TGTCTTGATC AAGAACAAAG AATTCATTCC ACCATTCTCA TATATCTACG TCTCTTCTAG 240
(2) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 687 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
ACGAGGGGAA ACCTCCTCAG AGCCTGCAGC CAGCCACGCG CCAGCATGTC TGGGGGCAAA 60
TACGTAGACT CCGAGGGACA TCTCTACACT GTTCCCATCC GGGAACAGGG CAACATCTAC 120
AAGCCCAACA ACAAGGCCAT GGCAGACGAG GTGACTGAGA AGCAAGTGTA TGACGCGCAC 180
ACCAAGGAGA TTGACCTGGT CAACCGCGAC CCCAAGCATC TCAACGACGA CGTGGTCAAG 240
ATTGACTTTG AAGATGTGAT TGCAGAACCA GAAGGGACAC ACAGTTTCGA CGGCATCTGG 300
AAGGCCAGCT TCACCACCTT CACTGTGACA AAATATTGGT TTTACCGCTT GTTGTCTACG 360
ATCTTCGGCA TCCCAATGGC ACTCATCTGG GGCATTTACT TTGCCATTCT CTCCTTCCTG 420
CACATCTGGG CGGTTGTACC GTGCATCAAG AGCTTCCTGA TTGAGATTCA GTGCATCAGC 480
CGCGTCTACT CCATCTACGT CCATACCTTC TGCGATCCAC TCTTTGAAGC TATTGGCAAG 540
ATATTCAGCA ACATCCGCAT CAGCACGCAG AAAGAGATAT GAGGGACATT TCAAGGATGA 600
AAGGTTTTTT TCCCCCCTTA CTATTTCCTT GGTGCCAATT CCAAGTTGCT CTCGCAGCAG 660
CAAATTTATG AATGGTTTGT CTTGATC 687
(2) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 560 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
Met Glu Cys Leu Tyr Tyr Phe Leu Gly Phe Leu Leu Leu Ala Ala Arg
1 5 10 15
Leu Pro Leu Asp Ala Ala Lys Arg Phe His Asp Val Leu Gly Asn Glu
20 25 30
Arg Pro Ser Ala Tyr Met Arg Glu His Asn Gln Leu Asn Gly Trp Ser
35 40 45
Ser Asp Glu Asn Asp Trp Asn Glu Lys Leu Tyr Pro Val Trp Lys Arg
50 55 60
Gly Asp Met Arg Trp Lys Asn Ser Trp Lys Gly Gly Arg Val Gln Ala
65 70 75 80
Val Leu Thr Ser Asp Ser Pro Ala Leu Val Gly Ser Asn Ile Thr Phe
85 90 95
Ala Val Asn Leu Ile Phe Pro Arg Cys Gln Lys Glu Asp Ala Asn Gly
100 105 110
Asn Ile Val Tyr Glu Lys Asn Cys Arg Asn Glu Ala Gly Leu Ser Ala
115 120 125
Asp Pro Tyr Val Tyr Asn Trp Thr Ala Trp Ser Glu Asp Ser Asp Gly
130 135 140
Glu Asn Gly Thr Gly Gln Ser His His Asn Val Phe Pro Asp Gly Lys
145 150 155 160
Pro Phe Pro His His Pro Gly Trp Arg Arg Trp Asn Phe Ile Tyr Val
165 170 175
Phe His Thr Leu Gly Gln Tyr Phe Gln Lys Leu Gly Arg Cys Ser Val
180 185 190
Arg Val Ser Val Asn Thr Ala Asn Val Thr Leu Gly Pro Gln Leu Met
195 200 205
Glu Val Thr Val Tyr Arg Arg His Gly Arg Ala Tyr Val Pro Ile Ala
210 215 220
Gln Val Lys Asp Val Tyr Val Val Thr Asp Gln Ile Pro Val Phe Val
225 230 235 240
Thr Met Phe Gln Lys Asn Asp Arg Asn Ser Ser Asp Glu Thr Phe Leu
245 250 255
Lys Asp Leu Pro Ile Met Phe Asp Val Leu Ile His Asp Pro Ser His
260 265 270
Phe Leu Asn Tyr Ser Thr Ile Asn Tyr Lys Trp Ser Phe Gly Asp Asn
275 280 285
Thr Gly Leu Phe Val Ser Thr Asn His Thr Val Asn His Thr Tyr Val
290 295 300
Leu Asn Gly Thr Phe Ser Leu Asn Leu Thr Val Lys Ala Ala Ala Pro
305 310 315 320
Gly Pro Cys Pro Pro Pro Pro Pro Pro Pro Arg Pro Ser Lys Pro Thr
325 330 335
Pro Ser Leu Gly Pro Ala Gly Asp Asn Pro Leu Glu Leu Ser Arg Ile
340 345 350
Pro Asp Glu Asn Cys Gln Ile Asn Arg Tyr Gly His Phe Gln Ala Thr
355 360 365
Ile Thr Ile Val Glu Gly Ile Leu Glu Val Asn Ile Ile Gln Met Thr
370 375 380
Asp Val Leu Met Pro Val Pro Trp Pro Glu Ser Ser Leu Ile Asp Phe
385 390 395 400
Val Val Thr Cys Gln Gly Ser Ile Pro Thr Glu Val Cys Thr Ile Ile
405 410 415
Ser Asp Pro Thr Cys Glu Ile Thr Gln Asn Thr Val Cys Ser Pro Val
420 425 430
Asp Val Asp Glu Met Cys Leu Leu Thr Val Arg Arg Thr Phe Asn Gly
435 440 445
Ser Gly Thr Tyr Cys Val Asn Leu Thr Leu Gly Asp Asp Thr Ser Leu
450 455 460
Ala Leu Thr Ser Thr Leu Ile Ser Val Pro Asp Arg Asp Pro Ala Ser
465 470 475 480
Pro Leu Arg Met Ala Asn Ser Ala Leu Ile Ser Val Gly Cys Leu Ala
485 490 495
Ile Phe Val Thr Val Ile Ser Leu Leu Val Tyr Lys Lys His Lys Glu
500 505 510
Tyr Asn Pro Ile Glu Asn Ser Pro Gly Asn Val Val Arg Ser Lys Gly
515 520 525
Leu Ser Val Phe Leu Asn Arg Ala Lys Ala Val Phe Phe Pro Gly Asn
530 535 540
Gln Glu Lys Asp Pro Leu Leu Lys Asn Gln Glu Phe Lys Gly Val Ser
545 550 555 560
(2) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2669 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
CAGATGCCAG AAGAACACTG TTGCTCTTGG TGGACGGGCC CAGAGGAATT CAGAGTTAAA 60
CCTTGAGTGC CTGCGTCCGT GAGAATTCAG CATGGAATGT CTCTACTATT TCCTGGGATT 120
TCTGCTCCTG GCTGCAAGAT TGCCACTTGA TGCCGCCAAA CGATTTCATG ATGTGCTGGG 180
CAATGAAAGA CCTTCTGCTT ACATGAGGGA GCACAATCAA TTAAATGGCT GGTCTTCTGA 240
TGAAAATGAC TGGAATGAAA AACTCTACCC AGTGTGGAAG CGGGGAGACA TGAGGTGGAA 300
AAACTCCTGG AAGGGAGGCC GTGTGCAGGC GGTCCTGACC AGTGACTCAC CAGCCCTCGT 360
GGGCTCAAAT ATAACATTTG CGGTGAACCT GATATTCCCT AGATGCCAAA AGGAAGATGC 420
CAATGGCAAC ATAGTCTATG AGAAGAACTG CAGAAATGAG GCTGGTTTAT CTGCTGATCC 480
ATATGTTTAC AACTGGACAG CATGGTCAGA GGACAGTGAC GGGGAAAATG GCACCGGCCA 540
AAGCCATCAT AACGTCTTCC CTGATGGGAA ACCTTTTCCT CACCACCCCG GATGGAGAAG 600
ATGGAATTTC ATCTACGTCT TCCACACACT TGGTCAGTAT TTCCAGAAAT TGGGACGATG 660
TTCAGTGAGA GTTTCTGTGA ACACAGCCAA TGTGACACTT GGGCCTCAAC TCATGGAAGT 720
GACTGTCTAC AGAAGACATG GACGGGCATA TGTTCCCATC GCACAAGTGA AAGATGTGTA 780
CGTGGTAACA GATCAGATTC CTGTGTTTGT GACTATGTTC CAGAAGAACG ATCGAAATTC 840
ATCCGACGAA ACCTTCCTCA AAGATCTCCC CATTATGTTT GATGTCCTGA TTCATGATCC 900
TAGCCACTTC CTCAATTATT CTACCATTAA CTACAAGTGG AGCTTCGGGG ATAATACTGG 960
CCTGTTTGTT TCCACCAATC ATACTGTGAA TCACACGTAT GTGCTCAATG GAACCTTCAG 1020
CCTTAACCTC ACTGTGAAAG CTGCAGCACC AGGACCTTGT CCGCCACCGC CACCACCACC 1080
CAGACCTTCA AAACCCACCC CTTCTTTAGG ACCTGCTGGT GACAACCCCC TGGAGCTGAG 1140
TAGGATTCCT GATGAAAACT GCCAGATTAA CAGATATGGC CACTTTCAAG CCACCATCAC 1200
AATTGTAGAG GGAATCTTAG AGGTTAACAT CATCCAGATG ACAGACGTCC TGATGCCGGT 1260
GCCATGGCCT GAAAGCTCCC TAATAGACTT TGTCGTGACC TGCCAAGGGA GCATTCCCAC 1320
GGAGGTCTGT ACCATCATTT CTGACCCCAC CTGCGAGATC ACCCAGAACA CAGTCTGCAG 1380
CCCTGTGGAT GTGGATGAGA TGTGTCTGCT GACTGTGAGA CGAACCTTCA ATGGGTCTGG 1440
GACGTACTGT GTGAACCTCA CCCTGGGGGA TGACACAAGC CTGGCTCTCA CGAGCACCCT 1500
GATTTCTGTT CCTGACAGAG ACCCAGCCTC GCCTTTAAGG ATGGCAAACA GTGCCCTGAT 1560
CTCCGTTGGC TGCTTGGCCA TATTTGTCAC TGTGATCTCC CTCTTGGTGT ACAAAAAACA 1620
CAAGGAATAC AACCCAATAG AAAATAGTCC TGGGAATGTG GTCAGAAGCA AAGGCCTGAG 1680
TGTCTTTCTC AACCGTGCAA AAGCCGTGTT CTTCCCGGGA AACCAGGAAA AGGATCCGCT 1740
ACTCAAAAAC CAAGAATTTA AAGGAGTTTC TTAAATTTCG ACCTTGTTTC TGAAGCTCAC 1800
TTTTCAGTGC CATTGATGTG AGATGTGCTG GAGTGGCTAT TAACCTTTTT TTCCTAAAGA 1860
TTATTGTTAA ATAGATATTG TGGTTTGGGG AAGTTGAATT TTTTATAGGT TAAATGTCAT 1920
TTTAGAGATG GGGAGAGGGA TTATACTGCA GGCAGCTTCA GCCATGTTGT GAAACTGATA 1980
AAAGCAACTT AGCAAGGCTT CTTTTCATTA TTTTTTATGT TTCACTTATA AAGTCTTAGG 2040
TAACTAGTAG GATAGAAACA CTGTGTCCCG AGAGTAAGGA GAGAAGCTAC TATTGATTAG 2100
AGCCTAACCC AGGTTAACTG CAAGAAGAGG CGGGATACTT TCAGCTTTCC ATGTAACTGT 2160
ATGCATAAAG CCAATGTAGT CCAGTTTCTA AGATCATGTT CCAAGCTAAC TGAATCCCAC 2220
TTCAATACAC ACTCATGAAC TCCTGATGGA ACAATAACAG GCCCAAGCCT GTGGTATGAT 2280
GTGCACACTT GCTAGACTCA GAAAAAATAC TACTCTCATA AATGGGTGGG AGTATTTTGG 2340
TGACAACCTA CTTTGCTTGG CTGAGTGAAG GAATGATATT CATATATTCA TTTATTCCAT 2400
GGACATTTAG TTAGTGCTTT TTATATACCA GGCATGATGC TGAGTGACAC TCTTGTGTAT 2460
ATTTCCAAAT TTTTGTATAG TCGCTGCACA TATTTGAAAT CATATATTAA GACTTTCCAA 2520
AGATGAGGTC CCTGGTTTTT CATGGCAACT TGATCAGTAA GGATTTCACC TCTGTTTGTA 2580
ACTAAAACCA TCTACTATAT GTTAGACATG ACATTCTTTT TCTCTCCTTC CTGAAAAATA 2640
AAGTGTGGGA AGAGACAAAA AAAAAAAAA 2669
(2) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
AAGGTGAAAG ATGTGTATGT GATAACAGAT CAGATCCCTG TATTCGTGAC CATGTCCCAG 60
AAGAATGACA GGAACTTGTC TGATGAGATC TTCCTCAGAG ACCTCCCCAT CGTCTTCGAT 120
GTCCTCATTC ATGATCCCAG CCACTTCCTC AACGACTCTG CCATTTCCTA CAAGTGGAAC 180
TTTGGGGACA ACACTGGCCT GTTTGTCTCC AACAATCACA CTTTGAATCA CACTTATGTG 240
CTCAATGGAA CCTTCAACCT TAACCTCACC GTGCAAACTG CAGTGCCCGG GCCATGCCCT 300
CCCCCTTCGC CTTCGACTCC GCCTCCACCT TCGTA 335
(2) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 262 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
AAGGTGAAAG ATGTGTATGT GATAACAGAT CAGATCCCTG TATTCGTGAC CATGTCCCAG 60
AAGAATGACA GGAACTTGTC TGATGAGATC TTCCTCAGAG ACCTCCCCAT CGTCTTCGAT 120
GTCCTCATTC ATGATCCCAG CCACTTCCTC AACGACTCTG CCATTTCCTA CAAGTGGAAC 180
TTTGGGGACA ACACTGGCCT GTTTGTCTCC AACAATCACA CTTTGAATCA CACTTATGTG 240
CTCAATGGAA CCTTCAACCT TA 262
(2) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
AAGGTGAAAG ATGTGTATGT GATAACAGAT CAGATCCCTG TATTCGTGAC CATGTCCCAG 60
AAGAATGACA GGAACTTGTC TGATGAGATC TTCCTCAGAG ACCTCCCCAT CGTCTTCGAT 120
GTCCTCATTC ATGATCCCAG CCACTTCCTC AACGACTCTG CCATTTCCTA CAAGTGGAAC 180
TTTGGGGACA ACACTGGCCT GTTTGTCTCC AACAATCACA CTTTGAATCA CACTTATGTG 240
CTCAATGGAA CCTTCAACCT TAACCTCACC GTGCAAACTG CAGTGCCCGG GCCATGCCCT 300
CCCCCTTCGC CTTCGACTCC GCCTCCACCT TCGTA 335
(2) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 190 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
TACGAAGGTG GAGGCGGAGT CGAAGGCGAA GGGGGAGGGC ATGGCCCGGG CACTGCAGTT 60
TGCACGGTGA GGTTAAGGTT GAAGGTTCCA TTGAGCACAT AAGTGTGATT CAAAGTGTGA 120
TTGTTGGAGA CAAACAGGCC AGTGTTGTCC CCAAAGTTCC ACTTGTAGGA AATGGCAGAG 180
TCGTTGAGGA 190
(2) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
AAGGTGAAAG ATGTGTATGT GATAACAGAT CAGATCCCTG TATTCGTGAC CATGTCCCAG 60
AAGAATGACA GGAACTTGTC TGATGAGATC TTCCTCAGAG ACCTCCCCAT CGTCTTCGAT 120
GTCCTCATTC ATGATCCCAG CCACTTCCTC AACGACTCTG CCATTTCCTA CAAGTGGAAC 180
TTTGGGGACA ACACTGGCCT GTTTGTCTCC AACAATCACA CTTTGAATCA CACTTATGTG 240
CTCAATGGAA CCTTCAACCT TAACCTCACC GTGCAAACTG CAGTGCCCGG GCCATGCCCT 300
CCCCCTTCGC CTTCGACTCC GCCTCCACCT TCGTA 335
(2) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 74 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
Arg Arg Trp Arg Arg Ser Arg Arg Arg Arg Gly Arg Ala Trp Gly His
1 5 10 15
Cys Ser His Gly Val Lys Val Gly Ser His Ser Val Ser Val Val Gly
20 25 30
Asp Lys Ala Ser Val Val Lys Val Val Gly Asn Gly Arg Val Val Val
35 40 45
Ala Gly Met Asn Asp Asp Asp Gly Val Ser Asp Arg Val Val Gly His
50 55 60
Gly His Tyr Arg Asp Cys Tyr His His His
65 70
(2) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
Lys Val Lys Asp Val Tyr Val Thr Asp Val Val Thr Met Ser Lys Asn
1 5 10 15
Asp Arg Asn Ser Asp Arg Asp Val Asp Val His Asp Ser His Asn Asp
20 25 30
Ser Ala Ser Tyr Lys Trp Asn Gly Asp Asn Thr Gly Val Ser Asn Asn
35 40 45
His Thr Asn His Thr Tyr Val Asn Gly Thr Asn Asn Thr Val Thr Ala
50 55 60
Val Gly Cys Ser Ser Thr Ser
65 70
(2) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 75 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
Tyr Gly Gly Gly Gly Val Gly Gly Gly Gly His Gly Gly Thr Ala Val
1 5 10 15
Cys Thr Val Arg Arg Lys Val Ser Thr Val Lys Val Thr Asn Arg Val
20 25 30
Ser Lys His Met Ala Ser Arg Lys Trp Gly Ser Met Arg Thr Ser Lys
35 40 45
Thr Met Gly Arg Ser Arg Lys Ser Ser Asp Lys Ser Trp Asp Met Val
50 55 60
Thr Asn Thr Gly Ser Val Thr Tyr Thr Ser Thr
65 70 75
(2) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 376 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
Met Cys Tyr Tyr Gly Ala Ala Arg Asp Ala Ala Lys Arg His Asp Val
1 5 10 15
Gly Asn Arg Ser Ala Tyr Met Arg His Asn Asn Gly Trp Ser Ser Asp
20 25 30
Asn Asp Trp Asn Lys Tyr Val Trp Lys Arg Gly Asp Met Arg Trp Lys
35 40 45
Asn Ser Trp Lys Gly Gly Arg Val Ala Val Thr Ser Asp Ser Ala Val
50 55 60
Gly Ser Asn Thr Ala Val Asn Arg Cys Lys Asp Ala Asn Gly Asn Val
65 70 75 80
Tyr Lys Asn Cys Arg Asn Ala Gly Ser Ala Asp Tyr Val Tyr Asn Trp
85 90 95
Thr Ala Trp Ser Asp Ser Asp Gly Asn Gly Thr Gly Ser His His Asn
100 105 110
Val Asp Gly Lys His His Gly Trp Arg Arg Trp Asn Tyr Val His Thr
115 120 125
Gly Tyr Lys Gly Arg Cys Ser Val Arg Val Ser Val Asn Thr Ala Asn
130 135 140
Val Thr Gly Met Val Thr Val Tyr Arg Arg His Gly Arg Ala Tyr Val
145 150 155 160
Ala Val Lys Asp Val Tyr Val Val Thr Asp Val Val Thr Met Lys Asn
165 170 175
Asp Arg Asn Ser Ser Asp Thr Lys Asp Met Asp Val His Asp Ser His
180 185 190
Asn Tyr Ser Thr Asn Tyr Lys Trp Ser Gly Asp Asn Thr Gly Val Ser
195 200 205
Thr Asn His Thr Val Asn His Thr Tyr Val Asn Gly Thr Ser Asn Thr
210 215 220
Val Lys Ala Ala Ala Gly Cys Arg Ser Lys Thr Ser Gly Ala Gly Asp
225 230 235 240
Asn Ser Arg Asp Asn Cys Asn Arg Tyr Gly His Ala Thr Thr Val Gly
245 250 255
Val Asn Met Thr Asp Val Met Val Trp Ser Ser Asp Val Val Thr Cys
260 265 270
Gly Ser Thr Val Cys Thr Ser Asp Thr Cys Thr Asn Thr Val Cys Ser
275 280 285
Val Asp Val Asp Met Cys Thr Val Arg Arg Thr Asn Gly Ser Gly Thr
290 295 300
Tyr Cys Val Asn Thr Gly Asp Asp Thr Ser Ala Thr Ser Thr Ser Val
305 310 315 320
Asp Arg Asp Ala Ser Arg Met Ala Asn Ser Ala Ser Val Gly Cys Ala
325 330 335
Val Thr Val Ser Val Tyr Lys Lys His Lys Tyr Asn Asn Ser Gly Asn
340 345 350
Val Val Arg Ser Lys Gly Ser Val Asn Arg Ala Lys Ala Val Gly Asn
355 360 365
Lys Asp Lys Asn Lys Gly Val Ser
370 375
(2) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2669 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
CAGATGCCAG AAGAACACTG TTGCTCTTGG TGGACGGGCC CAGAGGAATT CAGAGTTAAA 60
CCTTGAGTGC CTGCGTCCGT GAGAATTCAG CATGGAATGT CTCTACTATT TCCTGGGATT 120
TCTGCTCCTG GCTGCAAGAT TGCCACTTGA TGCCGCCAAA CGATTTCATG ATGTGCTGGG 180
CAATGAAAGA CCTTCTGCTT ACATGAGGGA GCACAATCAA TTAAATGGCT GGTCTTCTGA 240
TGAAAATGAC TGGAATGAAA AACTCTACCC AGTGTGGAAG CGGGGAGACA TGAGGTGGAA 300
AAACTCCTGG AAGGGAGGCC GTGTGCAGGC GGTCCTGACC AGTGACTCAC CAGCCCTCGT 360
GGGCTCAAAT ATAACATTTG CGGTGAACCT GATATTCCCT AGATGCCAAA AGGAAGATGC 420
CAATGGCAAC ATAGTCTATG AGAAGAACTG CAGAAATGAG GCTGGTTTAT CTGCTGATCC 480
ATATGTTTAC AACTGGACAG CATGGTCAGA GGACAGTGAC GGGGAAAATG GCACCGGCCA 540
AAGCCATCAT AACGTCTTCC CTGATGGGAA ACCTTTTCCT CACCACCCCG GATGGAGAAG 600
ATGGAATTTC ATCTACGTCT TCCACACACT TGGTCAGTAT TTCCAGAAAT TGGGACGATG 660
TTCAGTGAGA GTTTCTGTGA ACACAGCCAA TGTGACACTT GGGCCTCAAC TCATGGAAGT 720
GACTGTCTAC AGAAGACATG GACGGGCATA TGTTCCCATC GCACAAGTGA AAGATGTGTA 780
CGTGGTAACA GATCAGATTC CTGTGTTTGT GACTATGTTC CAGAAGAACG ATCGAAATTC 840
ATCCGACGAA ACCTTCCTCA AAGATCTCCC CATTATGTTT GATGTCCTGA TTCATGATCC 900
TAGCCACTTC CTCAATTATT CTACCATTAA CTACAAGTGG AGCTTCGGGG ATAATACTGG 960
CCTGTTTGTT TCCACCAATC ATACTGTGAA TCACACGTAT GTGCTCAATG GAACCTTCAG 1020
CCTTAACCTC ACTGTGAAAG CTGCAGCACC AGGACCTTGT CCGCCACCGC CACCACCACC 1080
CAGACCTTCA AAACCCACCC CTTCTTTAGG ACCTGCTGGT GACAACCCCC TGGAGCTGAG 1140
TAGGATTCCT GATGAAAACT GCCAGATTAA CAGATATGGC CACTTTCAAG CCACCATCAC 1200
AATTGTAGAG GGAATCTTAG AGGTTAACAT CATCCAGATG ACAGACGTCC TGATGCCGGT 1260
GCCATGGCCT GAAAGCTCCC TAATAGACTT TGTCGTGACC TGCCAAGGGA GCATTCCCAC 1320
GGAGGTCTGT ACCATCATTT CTGACCCCAC CTGCGAGATC ACCCAGAACA CAGTCTGCAG 1380
CCCTGTGGAT GTGGATGAGA TGTGTCTGCT GACTGTGAGA CGAACCTTCA ATGGGTCTGG 1440
GACGTACTGT GTGAACCTCA CCCTGGGGGA TGACACAAGC CTGGCTCTCA CGAGCACCCT 1500
GATTTCTGTT CCTGACAGAG ACCCAGCCTC GCCTTTAAGG ATGGCAAACA GTGCCCTGAT 1560
CTCCGTTGGC TGCTTGGCCA TATTTGTCAC TGTGATCTCC CTCTTGGTGT ACAAAAAACA 1620
CAAGGAATAC AACCCAATAG AAAATAGTCC TGGGAATGTG GTCAGAAGCA AAGGCCTGAG 1680
TGTCTTTCTC AACCGTGCAA AAGCCGTGTT CTTCCCGGGA AACCAGGAAA AGGATCCGCT 1740
ACTCAAAAAC CAAGAATTTA AAGGAGTTTC TTAAATTTCG ACCTTGTTTC TGAAGCTCAC 1800
TTTTCAGTGC CATTGATGTG AGATGTGCTG GAGTGGCTAT TAACCTTTTT TTCCTAAAGA 1860
TTATTGTTAA ATAGATATTG TGGTTTGGGG AAGTTGAATT TTTTATAGGT TAAATGTCAT 1920
TTTAGAGATG GGGAGAGGGA TTATACTGCA GGCAGCTTCA GCCATGTTGT GAAACTGATA 1980
AAAGCAACTT AGCAAGGCTT CTTTTCATTA TTTTTTATGT TTCACTTATA AAGTCTTAGG 2040
TAACTAGTAG GATAGAAACA CTGTGTCCCG AGAGTAAGGA GAGAAGCTAC TATTGATTAG 2100
AGCCTAACCC AGGTTAACTG CAAGAAGAGG CGGGATACTT TCAGCTTTCC ATGTAACTGT 2160
ATGCATAAAG CCAATGTAGT CCAGTTTCTA AGATCATGTT CCAAGCTAAC TGAATCCCAC 2220
TTCAATACAC ACTCATGAAC TCCTGATGGA ACAATAACAG GCCCAAGCCT GTGGTATGAT 2280
GTGCACACTT GCTAGACTCA GAAAAAATAC TACTCTCATA AATGGGTGGG AGTATTTTGG 2340
TGACAACCTA CTTTGCTTGG CTGAGTGAAG GAATGATATT CATATATTCA TTTATTCCAT 2400
GGACATTTAG TTAGTGCTTT TTATATACCA GGCATGATGC TGAGTGACAC TCTTGTGTAT 2460
ATTTCCAAAT TTTTGTATAG TCGCTGCACA TATTTGAAAT CATATATTAA GACTTTCCAA 2520
AGATGAGGTC CCTGGTTTTT CATGGCAACT TGATCAGTAA GGATTTCACC TCTGTTTGTA 2580
ACTAAAACCA TCTACTATAT GTTAGACATG ACATTCTTTT TCTCTCCTTC CTGAAAAATA 2640
AAGTGTGGGA AGAGACAAAA AAAAAAAAA 2669
(2) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 376 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
Met Cys Tyr Tyr Gly Ala Ala Arg Asp Ala Ala Lys Arg His Asp Val
1 5 10 15
Gly Asn Arg Ser Ala Tyr Met Arg His Asn Asn Gly Trp Ser Ser Asp
20 25 30
Asn Asp Trp Asn Lys Tyr Val Trp Lys Arg Gly Asp Met Arg Trp Lys
35 40 45
Asn Ser Trp Lys Gly Gly Arg Val Ala Val Thr Ser Asp Ser Ala Val
50 55 60
Gly Ser Asn Thr Ala Val Asn Arg Cys Lys Asp Ala Asn Gly Asn Val
65 70 75 80
Tyr Lys Asn Cys Arg Asn Ala Gly Ser Ala Asp Tyr Val Tyr Asn Trp
85 90 95
Thr Ala Trp Ser Asp Ser Asp Gly Asn Gly Thr Gly Ser His His Asn
100 105 110
Val Asp Gly Lys His His Gly Trp Arg Arg Trp Asn Tyr Val His Thr
115 120 125
Gly Tyr Lys Gly Arg Cys Ser Val Arg Val Ser Val Asn Thr Ala Asn
130 135 140
Val Thr Gly Met Val Thr Val Tyr Arg Arg His Gly Arg Ala Tyr Val
145 150 155 160
Ala Val Lys Asp Val Tyr Val Val Thr Asp Val Val Thr Met Lys Asn
165 170 175
Asp Arg Asn Ser Ser Asp Thr Lys Asp Met Asp Val His Asp Ser His
180 185 190
Asn Tyr Ser Thr Asn Tyr Lys Trp Ser Gly Asp Asn Thr Gly Val Ser
195 200 205
Thr Asn His Thr Val Asn His Thr Tyr Val Asn Gly Thr Ser Asn Thr
210 215 220
Val Lys Ala Ala Ala Gly Cys Arg Ser Lys Thr Ser Gly Ala Gly Asp
225 230 235 240
Asn Ser Arg Asp Asn Cys Asn Arg Tyr Gly His Ala Thr Thr Val Gly
245 250 255
Val Asn Met Thr Asp Val Met Val Trp Ser Ser Asp Val Val Thr Cys
260 265 270
Gly Ser Thr Val Cys Thr Ser Asp Thr Cys Thr Asn Thr Val Cys Ser
275 280 285
Val Asp Val Asp Met Cys Thr Val Arg Arg Thr Asn Gly Ser Gly Thr
290 295 300
Tyr Cys Val Asn Thr Gly Asp Asp Thr Ser Ala Thr Ser Thr Ser Val
305 310 315 320
Asp Arg Asp Ala Ser Arg Met Ala Asn Ser Ala Ser Val Gly Cys Ala
325 330 335
Val Thr Val Ser Val Tyr Lys Lys His Lys Tyr Asn Asn Ser Gly Asn
340 345 350
Val Val Arg Ser Lys Gly Ser Val Asn Arg Ala Lys Ala Val Gly Asn
355 360 365
Lys Asp Lys Asn Lys Gly Val Ser
370 375
(2) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 247 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
CTGACCAGGA ACCCACTCTT CTGTGCATGT ATGTGAGCTG TGCAGAAGTA TGTGGCTGGG 60
AACTGTTGTT CTCTAAGGAT TATTGTAAAA TGTATATCGT GGCTTAGGGA GTGTGGTTAA 120
ATAGCATTTT AGAGAAGAAA AAAAAAAAAA AAAAAACTCG AGAGTACTTC TAGAGCGGCC 180
GCGGCGCCAT CGATTTTCCA CCCGGGTGGG GTACCAGGTA AGTGTACCCA ATTCGCCTAT 240
AGTGAGT 247
(2) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
AGGACAAGCC AAGGACACTC TAAGTCTTTG GCCTTCCCTC TGACCAGGAA CCCACTCTTC 60
TGTGCATGTA TGTGAGCTGT GCAGAAGTAT GTGGCTGGGA ACTGTTGTTC TCTAAGGATT 120
ATTGTAAAAT GTATATCGTG GCTTAGGGAG TGTGGTTAAA TAGCATTTTA GAGAAGACAT 180
GGGAAGACTT AGTGTTTCTT CCCATCTGTA TTGTGGTTTT TACACTGTTC GTGGGGTGGA 240
CACGCTGTGT CTGAAGGGGA GGTGGGGGTC ACTGCTACTT AAGGTCCTAG GTTAACTGGG 300
GGAGATACCA CAGATGCTCA GCTTTCCACA TAACATGGGC ATGAACCAGC TAATCACACT 360
GAA 363
(2) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 524 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
GGATCCTTCT CCTGGTCTCC TCGGAAGAAC GGGGCTTTCG CGTGACTGAG GAGAACACTC 60
AGGCCCTTGC CCTTGACCGT GTTCCTGGGG CAGTTTCCTA TTGGCTTGTA CGCCTTGTGT 120
TTTTTGTACA GCAAGATGGT AACCATGGTG ACAAGCACAG CCAGGCAGCC GATGGAGATC 180
AGGACACCAT TCACTGCTCT CAGAGGGAGT CTGGGTCTTT GCCAGGGATA GAGATCAGGG 240
TGCTGGTGAG GGCCAGGCTT CGATCATCTC CCAGAGTGAA ATTCACACAG TAGGTGCCAG 300
ACCCATTGAA GGCTCTTCTC ACAGACAGCA GCACAGCCCA TCCACAGCCA CAGGGCTGCA 360
GACCCGGTTC TGGGCGATCT GGCAGGTGGG GTCGGAGATG ATCGTACAGG CTTCCATGGG 420
GGTGGCCCCT TTGCAGGTCA CAGTGAAGTC CATCAGGGAG TTGGCAGGCT GCGGTGTGGG 480
CATGGGGACA TCTGCTATCT GCATGATGCT GACTTCCAGG ATCC 524
(2) INFORMATION FOR SEQ ID NO: 106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 309 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
TAGCAGATGT CCCCATGCCC ACACCGCAGC CTGCCAACTC CCTGATGGAC TTCACTGTGA 60
CCTGCAAAGG GGCCACCCCC ATGGAAGCCT GTACGATCAT CTCCGACCCC ACCTGCCAGA 120
TCGCCCAGAA CCGGGTCTGC AGCCCTGTGG CTGTGGATGG GCTGTGCTGC TGTCTGTGAG 180
AAGAGCCTTC AATGGGTCTG GCACCTACTG TGTGAATTTC ACTCTGGGAG ATGATCGAAG 240
CCTGGCCCTC ACCAGCACCC TGATCTCTAT CCCTGGCAAA GACCCAGACT CCCTCTGAGA 300
GCAGTGAAT 309
(2) INFORMATION FOR SEQ ID NO: 107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 292 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
GGATCCTTCT CCTGGTCTCC TCGGAAGAAC GGGGCTTTCG CGTGACTGAG GAGAACACTC 60
AGGCCCTTGC CCTTGACCGT GTTCCTGGGG CAGTTTCCTA TTGGCTTGTA CGCCTTGTGT 120
TTTTTGTACA GCAAGATGGT AACCATGGTG ACAAGCACAG CCAGGCAGCC GATGGAGATC 180
AGGACACCAT TCACTGCTCT CAGAGGGAGT CTGGGTCTTT GCCAGGGATA GAGATCAGGG 240
TGCTGGTGAG GGCCAGGCTT CGATCATCTC CCAGAGTGAA ATTCACACAG TA 292
(2) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 263 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:
TTTTTTTTTT TTTTTTTTAG ACTGCCTTTT TAATGAGTAG AATATGTACA CACACGCACC 60
ATACACAAAG CCCGGGCCCA TTATAATTTT GTCAGGAGCT CAGGCATGCT CAGTGAGTTG 120
GAAGGCAGAT GAAGCATGCC TTCAGGTGGT GATTAGCTGG GTTCATGCCC ATGTTATCGT 180
GGAAAGCTGA GGCATCTGTG GTATCTCCCC CAGTTAACCT AGGACCTTAA GTAGCAGTGA 240
CCCACCTCCC TTCAGACACA GCG 263
(2) INFORMATION FOR SEQ ID NO: 109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:
GGATCCTGGA AGTCAGCATC ATGCAGATAG CAGATGTCCC CATGCCCACA CCGCAGCCTG 60
CCAACTCCCT GATGGACTTC ACTGTGACCT GCAAAGGGGC CACCCCCATG GAAGCCTGTA 120
CGATCATCTC CGACCCCACC TGCCAGATCG CCCAGAACCG GGTCTGCAGC CCTGTGGCTG 180
TGGATGGGCT GTGCTGCTGT CTGTGAGAAG AGCCTTCAAT GGGTCTGGCA CCTACTGTGT 240
GAATTTCACT CTGGGAGATG ATCGAAGCCT 270
(2) INFORMATION FOR SEQ ID NO: 110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 239 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
TTTTTTTTTT TTTTTTTTTC TTCTCTAAAA TGCTATTTAA CCACACTCCC TAAGCCACGA 60
TATACATTTT ACAATAATCC TTAGAGAACA ACAGTTCCCA GCCACATACT TCTGCACAGC 120
TCACATACAT GCACAGAAGA GTGGGTTCCT GGTCAGAGGG AAGGCCAAAG ACTTAGAGTG 180
TCCTTGGCTT GTCTGGAGCA ATGGATCCTT CTCCTGGTCT CCTCGGAAGA ACGGGCTTT 239
(2) INFORMATION FOR SEQ ID NO: 111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:
AAACTGCAGT GCCCGGGCCA TGCCCTCCCC CTTCGCCTTC GACTCCGCCT CCACCTTCAA 60
CTCCGCCCTC ACCTCCGCCC TCACCTCTGC CCACATTATC AACACCTAGC CCCTCTTTAA 120
TGCCTACTGG TTACAAATCC ATGGAGCTGA GTGACATTTC CAATGAAAAC TGCCGAATAA 180
ACAGATATGG CTACTTCAGA GCCACCATCA CAATTGTAGA GGGGATCCTG GACGCAGCAT 240
CATGCAGATA GCAGATGTCC CATGCCCACA CCGCAGCCGT CCAACTCCTG ATGGACTTCA 300
CTGTGACCTC AAGGGCACCC ATGGAAGCTG TCAGA 335
(2) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 217 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
CCTCAACGAC TCTGCCATTT CCTACAAGTG GAACTTTGGG GACAACACTG GCCTGTTTGT 60
CTCCAACAAT CACACTTTGA ATCACACTTA TGTGCTCAAT GGAACCTTCA ACCTTAACCT 120
CACCGTGCAA ACTGCAGTGC CCGGGCCATG CCCTCCCCCT TCGCCTTCGA CTCCGCCTCC 180
ACCTTCAACT CCGCCCTCAC CTCCGCCCTC ACCTCTG 217
(2) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 620 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
CCTCAACGAC TCTGCCATTT CCTACAAGTG GAACTTTGGG GACAACACTG GCCTGTTTGT 60
CTCCAACAAT CACACTTTGA ATCACACTTA TGTGCTCAAT GGAACCTTCA ACCTTAACCT 120
CACCGTGCAA ACTGCAGTGC CCGGGCCATG CCCTCCCCCT TCGCCTTCGA CTCCGCCTCC 180
ACCTTCAACT CCGCCCTCAC CTCCGCCCTC ACCTCTGCCC ACATTATCAA CACCTAGCCC 240
CTCTTTAATG CCTACTGGTT ACAAATCCAT GGAGCTGAGT GACATTTCCA ATGAAAACTG 300
CCGAATAAAC AGATATGGCT ACTTCAGAGC CACCATCACA ATTGTAGAGG GGATCCTGGA 360
AGTCAGCATC ATGCAGATAG CAGATGTCCC CATGCCCACA CCGCAGCCTG CCAACTCCCT 420
GATGGACTTC ACTGTGACCT GCAAAGGGGC CACCCCCATG GAAGCCTGTA CGATCATCTC 480
CGACCCCACC TGCCAGATCG CCCAGAACCG GGTCTGCAGC CCTGTGGCTG TGGATGGGCT 540
GTGCTGCTGT CTGTGAGAAG AGCCTTCAAT GGGTCTGGCA CCTACTGTGT GAATTTCACT 600
CTGGGAGATG ATGCAAGCCT 620
(2) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:
GGATCCCCTC TACAATTGTG ATGGTGGCTC TGAAGTAGCC ATATCTGTTT ATTCGGCAGT 60
TTTCATTGGA AATGTCACTC AGCTCCATGG ATTTGTAACC AGTAGGCATT AAAGAGGGGC 120
TAGGTGTTGA TAATGTGGGC AGAGGTGAGG GCGGAGGTGA GGGCGGAGTT GAAGGTGGAG 180
GCGGAGTCGA AGGCGAAGGG GGAGGGCATG GCCCGGGCAC TGCAGTTTGC ACGGTGAGGT 240
TAAGGTTGAA GGTTCCATTG AGCACATAAG TGTGATTCAA AGTGTGATTG TTGGAGACAA 300
ACAGGCCAGT GTTGTCCCAA AGTTCCACTT GTAGGAATGG CAGAGTCGTT GAGG 354
(2) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 473 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
CCTCAACGAC TCTGCCATTT CCTACAAGTG GAACTTTGGG GACAACACTG GCCTGTTTGT 60
CTCCAACAAT CACACTTTGA ATCACACTTA TGTGCTCAAT GGAACCTTCA ACCTTAACCT 120
CACCGTGCAA ACTGCAGTGC CCGGGCCATG CCCTCCCCCT TCGCCTTCGA CTCCGCCTCC 180
ACCTTCAACT CCGCCCTCAC CTCCGCCCTC ACCTCTGCCC ACATTATCAA CACCTAGCCC 240
CTCTTTAATG CCTACTGGTT ACAAATCCAT GGAGCTGAGT GACATTTCCA ATGAAAACTG 300
CCGAATAAAC AGATATGGCT ACTTCAGAGC CACCATCACA ATTGTAGAGG GGATCCTGGA 360
AGTCAGCATC ATGCAGATAG CAGATGTCCC CATGCCCACA CCGCAGCCTG CCAACTCCCT 420
GATGGACTTC ACTGTGACCT GCAAAGGGGC CACCCCCATG GAAGCCTGTA CGA 473
(2) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 223 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116:
GAAGGTGGAG GCGGAGTCGA AGGCGAAGGG GGAGGGCATG GCCCGGGCAC TGCAGTTTGC 60
ACGGTGAGGT TAAGGTTGAA GGTTCCATTG AGCACATAAG TGTGATTCAA AGTGTGATTG 120
TTGGAGACAA ACAGGCCAGT GTTGTCCCCA AAGTTCCACT TGTAGGAAAT GGCAGAGTCG 180
TTGAGGAAGT GGCTGGGATC ATGAATGAGG ACATCGAAGA CGA 223
(2) INFORMATION FOR SEQ ID NO: 117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 247 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:
GAATTCGCAC GAGGGGAGTC AGAGTCAAGC CCTGACTGGT TGCAGGCGCT CGGAGTCAGC 60
ATGGAAAGTC TCTGCGGGGT CCTGGGATTT CTGCTGCTGG CTGCAGGACT GCCTCTCCAG 120
GCTGCCAAGC GATTTCGTGA TGTGCTGGGC CATGAACAGT ATCCCGATCA CATGAGAGAG 180
CACAACCAAT TACGTGGCTG GTCTTCGGAT GAAAATGAAT GGGTTCCAAT ATCACTTTTG 240
TGGTGAA 247
(2) INFORMATION FOR SEQ ID NO: 118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:
GAATTCGGCA CGAGGAAGGA GGCCGTGTGC AGGCAGTCCT GACCAGTGAC TCACCGGCTC 60
TGGTGGGTTC CAATATCACT TTTGTGGTGA ACCTGGTGTT CCCCAGATGC CAGAAGGAAG 120
ATGCTAATGG CAATATCGTC TATGAGAAGA ACTGCAGGAA TGATTTGGGA CTGACATCTG 180
ACCTGCATGT CTACAACTGG ACTGCAGGGG CAGATGATGG TGACTGGGAA GATGGCACCT 240
(2) INFORMATION FOR SEQ ID NO: 119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:
GAAGGTGGAG GCGGAGTCGA AGGCGAAGGG GGAGGGCATG GCCCGGGCAC TGCAGTTTGC 60
ACGGTGAGGT TAAGGTTGAA GGTTCCATTG AGCACATAAG TGTGATTCAA AGTGTGATTG 120
TTGGAGACAA ACAGGCCAGT GTTGTCCCCA AAGTTCCACT TGTAGGAAAT GGCAGAGTCG 180
TTGAGGAAGT GGCTGGGATC ATGAATGAGG ACATCGAAGA CGATGGGGAG GTCTCTGAGG 240
AAGATCTCAT CAGACAAGTT 260
(2) INFORMATION FOR SEQ ID NO: 120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
GAATTCGGCA CGAGGTCAAG CCCTGACTGG TTGCAGGCGC TCGGAGTCAG CATGGAAAGT 60
CTCTGCGGGG TCCTGGGATT TCTGCTGCTG GCTGCAGGAC TGCCTCTCCA GGCTGCCAAG 120
CGATTTCGTG ATGTGCTGGG CCATGAACAG TATCCCGATC ACATGAGAGA GCACAACCAA 180
TTACGTGGCT GGTCTTCGGA TGAAAATGAA TGGATGAACA CCTTGTATCC A 231
(2) INFORMATION FOR SEQ ID NO: 121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 286 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:
AAGGGGGAGG GCATGGCCCG GGCACTGCAG TTTGCACGGT GAGGTTAAGG TTGAAGGTTC 60
CATTGAGCAC ATAAGTGTGA TTCAAAGTGT GATTGTTGGA GACAAACAGG CCAGTGTTGT 120
CCCCAAAGTT CCACTTGTAG GAAATGGCAG AGTCGTTGAG GAAGTGGCTG GGATCATGAA 180
TGAGGACATC GAAGACGATG GGGAGGTCTC TGAGGAAGAT CTCATCAGAC AAGTTCCTGT 240
CATTCTTCTG GGACATGGTC ACGAATACAG GGATCTGATC TGTTAT 286
(2) INFORMATION FOR SEQ ID NO: 122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 224 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:
GAATTCGGCA CGAGCCGACA CTGTGACTCC TGGTGGATGG GACTGGGGAG TCAGAGTCAA 60
GCCCTGACTG GTTGCAGGCG CTCGGAGTCA GCATGGAAAG TCTCTGCGGG GTCCTGGGAT 120
TTCTGCTGCT GGCTGCAGGA CTGCCTCTCC AGGCTGCCAA GCGATTTCGT GATGTGCTGG 180
GCCATGAACA GTATCCCGAT CACATGAGAG AGCACAACCA ATTA 224
(2) INFORMATION FOR SEQ ID NO: 123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123:
AAGGTGAAAG ATGTGTATGT GATAACAGAT CAGATCCCTG TATTCGTGAC CATGTCCCAG 60
AAGAATGACA GGAACTTGTC TGATGAGATC TTCCTCAGAG ACCTCCCCAT CGTCTTCGAT 120
GTCCTCATTC ATGATCCCAG CCACTTCCTC AACGACTCTG CCATTTCCTA CAAGTGGAAC 180
TTTGGGGACA ACACTGGCCT GTTTGTCTCC AACAATCACA CTTTGAATCA CACTTATGTG 240
CTCAATGGAA CCTTCAACCT TAACCTCACC GTGCAAACTG CAGTGCCCGG GCCATGCCCT 300
CCCCCTTCGC CTTCGACTCC GCCTCCACCT TCGTA 335
(2) INFORMATION FOR SEQ ID NO: 124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:
TACCATCGGA GAAAGAAGAC CAAGCAAGGC TCAGGCAGCC ACCGCCTGCT TCGCACTGAG 60
CCTCCTGACT CAGACTCAGA GTCCAGCACA GACGAAGAGG AATTTGGAGA ATTGGAAATC 120
GCTCTCGTTT TGTCAAGGGA GACTATCCCG ATGCTGCAAG ATCTGCTGTC CCTCTGGCCT 180
TTGTCATCCT CGCGCCTGCG TTGTGGCCTC TGTGGGCTTG GTGTGGAGCA AATGGCTCTC 240
AAGGAGGACT GAGTCTCAAG GAAATT 266
(2) INFORMATION FOR SEQ ID NO: 125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:
AGCTAAGGTC AGGAGGTGTC TGAAGAATTG GCTGATGCAT GGCAGGGATG TTGTTGACCT 60
GCTTTTAGAA CAATACTTCC ATTTAATTAT AGCATATCTT ATGTGTGTAT TAAAGCAGAG 120
CCGATCTGGT GGGGCTCATT AAGTAAATGT ACTTACTGCA AAAGGTTCAA CTGGTGACCC 180
CAGTTTTCCC CAGAAGCAAT ATGATAGGAC AGAGGCGACT CCTGCAAGTT GTCTCAGACT 240
TCACACATAC ATTGTGACAT TCTCTGAGCA TGTGCACTGT ACATGATATG ACACTATCAA 300
(2) INFORMATION FOR SEQ ID NO: 126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 312 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126:
AGCTAAGGTC CACTACCTTG TGAAGATGTA TAAACACCTG AAATGTAGAA GCGATCCGTA 60
TGTCAAGATC GAGGGGAAGG ACGCTGACGA CTGGCTGTGT GTGGACTTTG GGAGTATGGT 120
GATCCATTTG ATGCTTCCAG AAACCAGAGA AACCTATGAA TTAGAGAAAC TATGGACTCT 180
ACGTTCTTTT GATGACCTTA GCTAAGCCGA ATCAGCACAC TGGCGGCGTT ACTAGTGGAT 240
CGAGCTCGTA CAGCTGATGC ATAGCTTGAG TATCTATAGG TTACTAATAG CTGGCTATCA 300
TGTCAAGCGT TC 312
(2) INFORMATION FOR SEQ ID NO: 127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 281 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127:
GCTGAGCTGC AGAGAGTAGC ACATCCTTGC TAATTCAATA ACTACCAGTT TTTATTGGTG 60
AAACATGAAT CCAGATGGTA TGGTTGCTCT CCTGGACTAC CGTGAAGATG GTGTGACTCC 120
ATTCATGATT TTCTTTAAGG ATGGCTTAGA GATGGAGAAA TGTTAACAAA TTGGATCTAT 180
CACCTGTCAC CATAATTGGC TGCTGCTTAC CATCCATACA ACACCAGGAC TTAGGACAAA 240
TGGGACTGAT GTCATCTTGA GCTTTTATTT TGACCTTAGC T 281
(2) INFORMATION FOR SEQ ID NO: 128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 295 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128:
AGCTAAGGTC AGAGCCAATA GTATCATGAG AACTGAAGAA GTAATAAAGC AACTTCTCCA 60
GAAATTTAAG ATTGAGAATA GCCCTCGGGA TTTCGCTCTT TACATTATTT TTGGGACAGG 120
AGAGCAGAGA AAGCTAAAGA AGACCGATGT CCACTGCTGC AGAGGTTACT ACAAGGACCA 180
TCCAAAAGCA ATGCTCGGAT CTCTCATGGA TAAAGATGCA GAAGAATCAC GAGAGATGTG 240
GCTCGTACAT TATTTCACTT TCTTCTGATC ATACTCAAGA TAGATGAGAG AGAAT 295
(2) INFORMATION FOR SEQ ID NO: 129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 240 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129:
TTGACTTCTG AGTCTAACAC AGACACTGCA AGGGTTAATT TTCCAAGAGG TGGTTGTTGT 60
TGACGATAAA TTCATTAAGA ATTTTTAAAA ATTTAGTTAG ATTTACCAAA GTCACTGGAG 120
ACAAATTCAG AAGGCATATA TACCTGCCAG TTTTGTGGAC TACATTAATA GGGAGGCTTT 180
TATGTTTGAT GTAATTCTTA CAGTTCTAAG AATTAAGTTC CATTGCATGA GACCTTAGCT 240
(2) INFORMATION FOR SEQ ID NO: 130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 196 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130:
AAGGTGAATC CCCGACGGCT CTGGGCCCGA GGAGAAGCGT CGCCGTGGCA AATTGGCACT 60
GCAGGAGAAG CCCTCCACAG GTACTTGGAA AAACTGGTCT CTGAGGCCAA GGCCAGCTCC 120
GAGACATTCA GGACTTCTGG ATCAGCCTCC AGGGACACTG TGCAGTGAGA AGATGGCCAT 180
GAGTCCTGCC AGTGAG 196
(2) INFORMATION FOR SEQ ID NO: 131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 187 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131:
AATTTTTTTT TTCGACGGCC CAACGGGGGC TTGGTGGATG GAAATATGGT TTTGTGAGTT 60
ATTGCACTAC CTGGAATATC TATGCCTCTT ATTTGCGTGT ACTGTTGCTG CTGATCGTTT 120
GGTGCTGTGT GAGTGAACCT ATGGCTTAGA AAAACGACTT TGTCTTAAAC TGAGTGGGTG 180
TTCAGGG 187
(2) INFORMATION FOR SEQ ID NO: 132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132:
CACCTGATTT AAAGGAAAAG CATTCTGACG TAAGAAGCTG AAAGGCGGCC CTTGCGTGCT 60
TTGAACTTTC TTATACAGCA CAGTCATCTG AAGCTTCCTG TGTGACCAAG ACAAGAACGC 120
GTGCACAAGA CTGAGAAACA GCAAGAAACA ACCCGGCATT CTACTTTCTC AACACTATCA 180
TACTTTAAAC CTTTCAC 197
(2) INFORMATION FOR SEQ ID NO: 133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:
CTAGCTTACG CTAGTCCCCC ATGCATAAAG ACTGATCGCT TTTCCTTAGA AAGGTGAGAG 60
GGTTAGGACA AGGCCGTGTG GTAACAACAC CCGCAGCTCG AAAAACCAAT GGCTTGTTAA 120
CGTGTCAGTG AGGCACTGTA CGGACGTCCA TAGTCCACAT CTTCAAATTC CCGCAGAAGG 180
CTTCCTATTC TTAAACTCTA 200
(2) INFORMATION FOR SEQ ID NO: 134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:
CTACATTTCT GTATCCATTC CTCTGTTGAA GGCTCTGGTT CTTTCCAGCT TCTGGCTATT 60
ATAAATAAGG CTGCTATAAA CACAGTGGAG GCATGTGTCC TTGTTATATT TTGGAGCATC 120
TTTTGGGTAT ATGCCCAGAA GTGCTATAGC TGGTTCCTCA GGTAGTACTA TGTCGAATTT 180
TCTGAGGAAC TGCCAGACTG ATTTCCAGAG TGGTTGTACC AGCTTGCAAT CCCACCAGCA 240
ATAGAGGAGT GTTCCTCTTT CTCTATATTC TTGCCAACAT CTGCTGTCAC CTGAGTGTTT 300
(2) INFORMATION FOR SEQ ID NO: 135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:
TGGTAAAGGG GGAATGATGT CGAGGCCATC CTGGGCTGTA GAGCCAGGCC CTGGCTTGGG 60
GAGTGGGCAT TGTTAACTTG TTGCTGACTT TGTGTTGACC CCTGCATCAG CAACTATTTC 120
CTTAAATCCA GGATACAACT TGTTAAGTGT GACAGCTTTC CTTTACACAC CATTTTTGTG 180
GGTGTATATA TATATTTGAC TTGGGGAGAA TTATTTTTTA CAAAAATACA AAATAGCTTT 240
TAA 243
(2) INFORMATION FOR SEQ ID NO: 136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:
AGCTAAGGTC CGGACTCTAT GGCATGACCC CAAAAACATT GGCTGGAAAG ATTACACTGC 60
CTACAGGTGG CACCTGATTC ACAGGCCTAA GACAGGCTAC ATGAGAGTCT TAGTGCATGA 120
AGGAAAGCAA GTCATGGCTG ACTCAGGACC AATTTATGAC CAAACCTACG CTGGTGGACG 180
GCTGGGCTGT TTGTCTTCTC CAAGAGATGG TCTATTCTCG GACCTCAAGT ATGAGTGCAG 240
AGATGCTAGA GAGCAGGCTC AGTCTCAGCA 270
(2) INFORMATION FOR SEQ ID NO: 137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:
TGACCTACGT GTAGTTGGTG TGCTTGTTGT CGAAGATGAG GGCCTCCTGG ATGAGCTGGT 60
GCTGCTGCTC CAGCAGGTCC AGGCTGGGCT TGTAGTCCAC GAGTCTGCGC TCGTACTGCT 120
TCAGGTGGCT CAGCTGGTCT TCCAGAGTCC CGTTCATCTC AATGGAGATG CGCCCGATCT 180
CCTCCATCTT AGTCTGGATC CACGGCCCCA CCATATTGGC TTGGCTGGCG AACTGTCGGC 240
GAAGGCTGCA TTGGATTGCT 260
(2) INFORMATION FOR SEQ ID NO: 138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 187 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138:
AATTTTTTTT TTCGACGGCC CAACGGGGGC TTGGTGGATG GAAATATGGT TTTGTGAGTT 60
ATTGCACTAC CTGGAATATC TATGCCTCTT ATTTGCGTGT ACTGTTGCTG CTGATCGTTT 120
GGTGCTGTGT GAGTGAACCT ATGGCTTAGA AAAACGACTT TGTCTTAAAC TGAGTGGGTG 180
TTCAGGG 187
(2) INFORMATION FOR SEQ ID NO: 139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 197 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:
CACCTGATTT AAAGGAAAAG CATTCTGACG TAAGAAGCTG AAAGGCGGCC CTTGCGTGCT 60
TTGAACTTTC TTATACAGCA CAGTCATCTG AAGCTTCCTG TGTGACCAAG ACAAGAACGC 120
GTGCACAAGA CTGAGAAACA GCAAGAAACA ACCCGGCATT CTACTTTCTC AACACTATCA 180
TACTTTAAAC CTTTCAC 197
(2) INFORMATION FOR SEQ ID NO: 140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140:
CTAGCTTACG CTAGTCCCCC ATGCATAAAG ACTGATCGCT TTTCCTTAGA AAGGTGAGAG 60
GGTTAGGACA AGGCCGTGTG GTAACAACAC CCGCAGCTCG AAAAACCAAT GGCTTGTTAA 120
CGTGTCAGTG AGGCACTGTA CGGACGTCCA TAGTCCACAT CTTCAAATTC CCGCAGAAGG 180
CTTCCTATTC TTAAACTCTA 200
(2) INFORMATION FOR SEQ ID NO: 141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:
CTACATTTCT GTATCCATTC CTCTGTTGAA GGCTCTGGTT CTTTCCAGCT TCTGGCTATT 60
ATAAATAAGG CTGCTATAAA CACAGTGGAG GCATGTGTCC TTGTTATATT TTGGAGCATC 120
TTTTGGGTAT ATGCCCAGAA GTGCTATAGC TGGTTCCTCA GGTAGTACTA TGTCGAATTT 180
TCTGAGGAAC TGCCAGACTG ATTTCCAGAG TGGTTGTACC AGCTTGCAAT CCCACCAGCA 240
ATAGAGGAGT GTTCCTCTTT CTCTATATTC TTGCCAACAT CTGCTGTCAC CTGAGTGTTT 300
(2) INFORMATION FOR SEQ ID NO: 142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142:
TGGTAAAGGG GGAATGATGT CGAGGCCATC CTGGGCTGTA GAGCCAGGCC CTGGCTTGGG 60
GAGTGGGCAT TGTTAACTTG TTGCTGACTT TGTGTTGACC CCTGCATCAG CAACTATTTC 120
CTTAAATCCA GGATACAACT TGTTAAGTGT GACAGCTTTC CTTTACACAC CATTTTTGTG 180
GGTGTATATA TATATTTGAC TTGGGGAGAA TTATTTTTTA CAAAAATACA AAATAGCTTT 240
TAA 243
(2) INFORMATION FOR SEQ ID NO: 143:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 270 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143:
AGCTAAGGTC CGGACTCTAT GGCATGACCC CAAAAACATT GGCTGGAAAG ATTACACTGC 60
CTACAGGTGG CACCTGATTC ACAGGCCTAA GACAGGCTAC ATGAGAGTCT TAGTGCATGA 120
AGGAAAGCAA GTCATGGCTG ACTCAGGACC AATTTATGAC CAAACCTACG CTGGTGGACG 180
GCTGGGCTGT TTGTCTTCTC CAAGAGATGG TCTATTCTCG GACCTCAAGT ATGAGTGCAG 240
AGATGCTAGA GAGCAGGCTC AGTCTCAGCA 270
(2) INFORMATION FOR SEQ ID NO: 144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144:
TGACCTACGT GTAGTTGGTG TGCTTGTTGT CGAAGATGAG GGCCTCCTGG ATGAGCTGGT 60
GCTGCTGCTC CAGCAGGTCC AGGCTGGGCT TGTAGTCCAC GAGTCTGCGC TCGTACTGCT 120
TCAGGTGGCT CAGCTGGTCT TCCAGAGTCC CGTTCATCTC AATGGAGATG CGCCCGATCT 180
CCTCCATCTT AGTCTGGATC CACGGCCCCA CCATATTGGC TTGGCTGGCG AACTGTCGGC 240
GAAGGCTGCA TTGGATTGCT 260
(2) INFORMATION FOR SEQ ID NO: 145:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 255 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145:
TGACCATCGA TAAGTTTAAT AACTACAGAC TTTTCCCAAG ACTACAAAAG CTTCTTGAAA 60
GTGACTACTT TAGATATTAC AAGGTGAACT TGAAGAAGCC TTGTCCTTTC TGGAATGACA 120
TCAACCAGTG TGGAAGAAGA GACTGTGCCG TCAAACCCTG CCATTCTGAT GAAGTTCCTG 180
ATGGAATTAA GTCTGCCGAG CTACAAGTAT TCTGAGGAAG CCCAACCGCA TTGAAGAATG 240
TGAGCAAGCT GAGCG 255
(2) INFORMATION FOR SEQ ID NO: 146:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 236 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146:
AACTCTGTGA ACCGTGCCTT TCTCTGTGGA GGTGGAGGTG TCGGTTGAAG ACAAGCGAGG 60
TCCTCCAAGG GGCTGTGTCT TATGTTGCCA TCTCCCCTTG TAGCTTGGCT GCCCACCCTC 120
CAGACTGTGC GCCATGGCTC CAAGGCTGTG ACCCGCCACT GGAGTCATGC ACTTCCAGCG 180
GCAGAAGCTG ATGCTATAAC TGAGTATATT CCTCCAAACC TGCCATCAAC CCGAGA 236
(2) INFORMATION FOR SEQ ID NO: 147:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 291 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147:
ACTTCTCCAG AGAATTTAAG ATTGAGAATA GCCCTCGGGA TTTCGCTCTT TACATTATTT 60
TTGGGACAGG AGAGCAGAGA AAGCTAAAGA AGACCGATGT CCCACTGCTG CAGAGGTTAC 120
TACAAGGACC ATCCAAAAGC AATGCTCGGA TCTTCCTCAT GGATAAAGAT GCAGAAGAAA 180
TCAGCAGAGA TGTGGCTCCG TACATTAATT TCACTTTTCT TTCTTGGATC CATCCTTCAA 240
GATTAGATGA AGAAGAGAAA TGGAGATTGA GAGAATATGC AATCATACCG A 291
(2) INFORMATION FOR SEQ ID NO: 148:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 255 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148:
AGGGTTACTT CAGGCTAAGG CAATAGAAAT CCATTTTAAG ATGGTGTGCT AAAGGCTTGA 60
TGGATGTTCA TCGTCTGTCT AAAGGAGAAT GAAGTCATCA ACAGGATGTC AGGGGAAAGT 120
GAGATCATCG CAGAAAGTAT CAACTTAGCA CAAACACACA GGCATAGCTC CTGCAAGAGG 180
TGAATGCTGT CCCCAAATAC CTGAGGAACT ATCCCTTTGG GCAAGAAAAT AGACAAGTCC 240
ATGAAGTCTG GGTGA 255
(2) INFORMATION FOR SEQ ID NO: 149:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 284 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149:
GACCAGGTAC ACTTGAGCAA AGCACCCAGT ATTTAATTCC TTACAGAAAG GAGAGGAAAG 60
GTCTGCAGTT GGACTGATGG TATGCTAACA CCGCAAATGA CTGTCATTTG ATCTCAGAAG 120
TTCAGGATTG ATTGCTATGT TTTAGCTCTA ATTGTGAGAA ACAGTAGTCA TTTTAGTCTT 180
AAATTTTGCC CTCAGGAAAT TCAGGGAGAC TGAGCCTTCC TTCCCCCACC TTCGTAAAGC 240
CGAATTCCAG CACACGGCGG CCGTTACTAG TGGATCCGAG CTCG 284
(2) INFORMATION FOR SEQ ID NO: 150:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150:
TACAAGGTGG GATGGCAGGA ACTGAAGGCT TCTGTAAATC CAGTTTTGGC TCTCTCTCTG 60
GTCTTTCTTT CTCTTCTGTT CTGTTTGGAA GGGTTTCTGG TCTTTCAGGA GGTATTTTTT 120
TAATTTCATG TTTTCTCTCT GTGGTACCTG CCCCTTGTTT GACGACAGGA GCTGATGGAG 180
GTGGCGGTTT CTTGGGTCTA TTCCCTTCCT TGTCAAAGTC CGATGGAAGT AACTTCACGA 240
AGTTGTCAGG AAACACGCCT CGTCTGCCAT TGAGTTCTCC TTCCCACCAG CCTACGCGAT 300
GCAGTCTTAT TGATGAGAGT CACTATATCT CCTTA 335
(2) INFORMATION FOR SEQ ID NO: 151:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 254 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151:
TCACCCATGA CTTCTATGGA CTTGTCTATT TTCTTGCCCA AAGGGATAGT TCCTCAGGTA 60
TTTGGGGACA GCATTCACCT CTTGCAGGAG CTATGCCTGT GTGTTTGTGC TAAGTTGATA 120
CTTTCTGCGA TGATCTCACT TTCCCCTGAC ATCCTGTTGA TGACTTCATT CTCCTTTAGA 180
CAGACGATGA ACATCCATCA GGCCTTTATG CACACCATCT TAAAATGGAT TTCTATTGCC 240
TTAGCCTGAA GTCC 254
(2) INFORMATION FOR SEQ ID NO: 152:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 241 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152:
CCCATAGAGA TAGGTTTGCT CCAGAACCTG CAGCATTTGC ACATCACAGG GAACAAGGTG 60
GACATTCTGC CAAAACAGTT GTTTAAGTGC GTGAAGTTGA GGACTTTGAA CCTGGGGCAG 120
AACTGTATCG CCTCCCTGCC TGAGAAAATC AGTCAGCTCA CCCAGCTCAC TCAGCTGGAG 180
CTGAAGGGCA ACTGCCTAGA CCGCCTGCCA GCCCAGCTGG CAGTGTCGAT GCTCAAGAAG 240
A 241
(2) INFORMATION FOR SEQ ID NO: 153:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 256 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153:
CAATAATCCA GGTAAAATAG AGTAAAATAG TCTGCTAGCA GCAAGTTCCT ACCATACTTT 60
CAACAACACT CACGAGATAC GGAATGATTA CAGCATTAAG AATATTTCAG AAATGACAGG 120
TAGGTGTGGT GGACAGGTGG CTCACATTCA AGACTCAAGT CTACTTAAAA AAGAAAATCT 180
CACTAGCACT AGATTCTAGC TCCTTTGTTT CCCCCTTTCT TTTGGTTTCA AAGGCGTTTC 240
TACAACCCAT AAGAGG 256
(2) INFORMATION FOR SEQ ID NO: 154:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 404 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154:
GCCAAGCTAT TATGACACTA TAGATACTCA ACGTATCGAT CAACGTTGGT ACCGAGCTCG 60
GATCCACTAG TAACGGCCGC CAGTGTGCTG GAATTCGGCT TGGATTGGTC AGAGCAGTGT 120
GCAATATGAT CCAACTAAGT CTCCTCCCTT GGCCCCTCCC CAAAATGTTT GCAGTGTTAT 180
TTTTGTGGGT TTTTTTTTAA CACCCTGACA CCTGTTGTGG ACATTGTCAA CCTTTGTAAG 240
AAAACCCAAA TAAAAATTGA AAAATAAAAT AAAAAGAAAC CCATGAACAT TCGCACCACT 300
TGTGGCTTCT GACTATCTTC CACAGAGGGA AGTTTAAAAC CCAAACTTCC AAAGGTTTGA 360
ACTACCTCAA GACACTTTCG CAGTGGAGTC GTAGACCAAT CCCA 404
(2) INFORMATION FOR SEQ ID NO: 155:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 167 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155:
TAAATAAATT AAAAAACTAT TAAACCTAAA AACGTCCACC AAACCCTAAA ACCATTAAAC 60
AACCAACAAA CCCACTAACA ATTAAACCTA AACCTCCATA AATAGGTGAA GGCTTTAATG 120
CTAACCCAAG ACAACCAACC AAAAATAATG AACTTAAAAC AAAAATA 167
(2) INFORMATION FOR SEQ ID NO: 156:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 212 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156:
GGTAAAGGGG ACCTGGAGAA CGCCTTCCTG AACCTGGTCC AGTGCATCCA GAACAAGCCC 60
CTGTACTTCG CTGACCGGCT GTACGACTCC ATGAAGGGCA AGGGGACTCG AGACAAGGTC 120
TGATTAGAAT CATGGTCTCT CGCAGTGAAG TGGACATGCT GAAAATCAGA TCTGAATTCA 180
AGAGGAATAT GGCAAGTCCT GTACTACTAC AT 212
(2) INFORMATION FOR SEQ ID NO: 157:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157:
AGAGCAGCAG GCCAGCTGTA CTTGGTTTGG CAAGAAAAAG AAGCAGTACA AAGATAAATA 60
TTTGGCAAAG CACAACGCAG TGTTTGATCA ATTAGATCTT GTCACATATG AAGAAGTAGT 120
CAAACTGCCA GCATTCAAAA GGAAAACATT AGTCTTATTA GGTGCACATG GTGTTGGAAG 180
AAGACACATA AAAAATACCC TCATCACAAA GCAC 214
(2) INFORMATION FOR SEQ ID NO: 158:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158:
TCGGTCATAG TAGTAAGGGA AATCTCCCAG GTAAGATGAA TACTGCGGTA GGACGAACAA 60
TCCTCCAGGA TGTTTGTTCC ATATTAAACT GTTACGTGAT ATGTGCTTGA ATATTCTGTC 120
CTGAATAATC TCTAGTGTAG TTAATACAAT CTTCTCAACT GAAGAAAAAT AAGCCTCCCA 180
CAAGAACTGT GTCTGCTGTC TAAGTGCTAG GATTTTATCC TGATGAATAG ACCTGATTGT 240
AGAAGGAATC TGTAATAGCA ATCTCTCATC GCCTATGACC GAAAGCCGAA TTCTGCAGAT 300
ATCCATCACA CTGGCCGGCC GCTCGAGCAT CGATCTAGAG GG 342
(2) INFORMATION FOR SEQ ID NO: 159:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 303 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159:
CTGCTTGATG ACAAAGGGTG TAGTCTTCAT CTTTTCCTGG ATTATTTTGG AAGTGACAGG 60
TGGAAATTCC ATCGTCACGT TTATGTGGTC TGTAAAGCCA ACGATCTCAA ATTCTGGCGG 120
CTCAAGAGGA GCGTTTGCAG GCACGATGTA GTCTGAGCAG CGGCACACGG TCAAGTCCCC 180
TCTGTGCACT ATGACGATGG CGACGACGTA GCTCTCCATG CCCTCCAACC ACTTATCTGT 240
CACGTCACAT GATGACTTCG TGGTATCTGA ACAGTTCTTA ACCTTCGTCA GATTTTCGTC 300
TTT 303
(2) INFORMATION FOR SEQ ID NO: 160:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160:
AAATCGTTGC TTCAGAAAGA CTCAATAACA CTTACTTGTG CCTGGCTGTG CTGACAGTAC 60
ATTCTGTGTC ATTTTCCTTC ATGGGCGGAA CAGTCCACAG AGCTCACCAA CAAGTACTCC 120
AAAACTGAGC AAGAGTTTAA GCTTCGAGAT GCAACCAGAT GAGCTTCTAG AAAAGCCCAT 180
GTCTCCCATG CAGTACGCAC GGTCTGGACT AGGGACAGCA GAGATGAATG GCAAACTCAT 240
AGCTGCAGGT GGTTATAACA GAGAGGAATG TCTTCGAACA GTTGAATGCT ATGATCCACA 300
TACAGATCAC TGGTCCTTCC TTGCTCCCAT GAGAACATCA AGCAG 345
(2) INFORMATION FOR SEQ ID NO: 161:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 315 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161:
CTTTCCGAAG AGCACACCCT CCTCTCAATG AGCTTGTGAG GTCTCTTTCT TCTCTTCCTT 60
CCAACGTGGT GCTAGCTCCA GGCGAGCGAC GTGAGAGTGC CACCTGAGAC AGACACCTTG 120
GTCTCAGTTA GAAGGAAGAT GCAGGTCTAA GAGGAATCCC CGCAGGTCTG TCTGAGCTGT 180
GATCAAGAAT ATTCCGCAAT GTGCCTTTTC TGAGATCGTG TTAGCTCCAA AGCTTTTTCC 240
TATCGCAGAG TGTTCAGTTT GTGTTTGTTT GTTTTTGTTT TGTTTTGTTT TTCCCTTGGC 300
GGATTTCCCG TGTGT 315
(2) INFORMATION FOR SEQ ID NO: 162:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162:
CCTATTGAAC GGTCTTGCAA TGACGAGCAT TCAGATGCTT AAGGAAAGCA TTGCTGCTAC 60
AAATATTTCT ATTTTTAGAA AGGGTTTTTA TGGACCAATG CCCCAGTTGT CAGTCAAAGC 120
CGTTGGTGTT TTCATTGTTT AAAATGTCAC CTATAAAACG GGCATTATTT ATGTTTTTTT 180
TCCCTTTGTT CATATTCTTT TGCATTCCTG ATTATTGTAT GTATCGTGTA AAGGAAGTCT 240
GTA 243
(2) INFORMATION FOR SEQ ID NO: 163:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 243 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163:
CCTATTGAAC GGTCTTGCAA TGACGAGCAT TCAGATGCTT AAGGAAAGCA TTGCTGCTAC 60
AAATATTTCT ATTTTTAGAA AGGGTTTTTA TGGACCAATG CCCCAGTTGT CAGTCAAAGC 120
CGTTGGTGTT TTCATTGTTT AAAATGTCAC CTATAAAACG GGCATTATTT ATGTTTTTTT 180
TCCCTTTGTT CATATTCTTT TGCATTCCTG ATTATTGTAT GTATCGTGTA AAGGAAGTCT 240
GTA 243
(2) INFORMATION FOR SEQ ID NO: 164:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164:
CCTGGGTCCG TCCTCCAACC CCTCACGCCC AAACCCTCCG ACTTTCACTT CTTGAAGTGA 60
TCGGAAAGGG CAGTTTTGGA AAGGTTCTTC TGGCTAGGCA CAAGGCAGAA GAAGTATTCT 120
ATGCAGTCAA AGTTTTACAG AAGAAGCCAT CCTGAAGAAG AAAGGAAGGA AGCATATTAT 180
GTCAGAGCGG AATGTTCTGT TGAAGAATGT GAAGCACCCT TTCCTGGTGG GCCTTCACTT 240
CTCATTCCAG ACCGCTGACA AGCTCT 266
(2) INFORMATION FOR SEQ ID NO: 165:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165:
GATGCTGAAC ACAAAAAGAA AGAAGAAAAG GAAGAGGAGG AGCAAGAGAA GCTGAAGGGA 60
GGGAGCCTTG GCGAAAATCA GATCAAAGAT GAGAAGATTA AAAAGGACAA AGAGCCCAAA 120
GAAGAGTCAA GAGCTTCTTG GATAGAAAGA AAGGATTTAC AGAGTGAGGC GCAGAATGGA 180
GATTCATGAC CCACAAACTT AAAC 204
(2) INFORMATION FOR SEQ ID NO: 166:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 166:
AAAGCCAATT GGTAGAGAAA TTGAAGACAC AAATGCTGGA TCAGGAAGAG CTTCTGGCAT 60
CAACCAGAAG GGATCAAGAT AATATGCAAG CTGAACTGAA TCGCCTCCAA GCAGAAAATG 120
ATGCTTCTAA AGAAGAGTAA AGAGTTTTAC AGGCCTTAGA GGACTGCTGT TAATTATGAT 180
CAGAGTTCAG GAGTTAAGAC 200
(2) INFORMATION FOR SEQ ID NO: 167:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 167:
CTGCTTGATG TCCTGTGTAG CGAATGTCAC AGCGTACAAC ATTGTTAGTG TAGTCTGATT 60
CAGGCACCAG GTAGCTGGGG TTTACACTGA CCTTTAGAAT GTAGTTTCCA GGTTGTACAT 120
CTGTAATATC AATCCACTGG CAGTCTATGT CTGCCGCATA GGTGTCATAA CATCCAGGAC 180
TCAATCCCTG TGTGTGTGCA GTGCACGCAA AGGCCCTGTG GTACCCATAG TCACAGGACG 240
TGTCCTCCAG ACAGAAGCTT GCTTTGTGGC CTTCAGCCAC TCTCCTCTGT GTGTTGGCAT 300
CAACGAGAAG CCGAATTCTC GAGATATCCA TCACACT 337
(2) INFORMATION FOR SEQ ID NO: 168:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 337 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 168:
CTGCTTGATG TCCTGTGTAG CGAATGTCAC AGCGTACAAC ATTGTTAGTG TAGTCTGATT 60
CAGGCACCAG GTAGCTGGGG TTTACACTGA CCTTTAGAAT GTAGTTTCCA GGTTGTACAT 120
CTGTAATATC AATCCACTGG CAGTCTATGT CTGCCGCATA GGTGTCATAA CATCCAGGAC 180
TCAATCCCTG TGTGTGTGCA GTGCACGCAA AGGCCCTGTG GTACCCATAG TCACAGGACG 240
TGTCCTCCAG ACAGAAGCTT GCTTTGTGGC CTTCAGCCAC TCTCCTCTGT GTGTTGGCAT 300
CAACGAGAAG CCGAATTCTC GAGATATCCA TCACACT 337
(2) INFORMATION FOR SEQ ID NO: 169:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 374 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 169:
GATCTGACAC TACAGCATGA GCGTTAGATT TCATAAAATT ATTTTTCTTC TAAATGCTGG 60
AAACTCTAAG GGTTTATTCA GAAAAAAAAC TGGCCAATTT TCAAATGGCT TAGAAGCAGG 120
GTTAATTAAG TATTGAATGA GCCACTGTGA TATCCTGATG ACACCCAGTC ACAATGACAG 180
TTTTGAAGCA TACAACCAAA ACAATTGAGA TCTCAAAACT ATTTTACATC ACTTATGGTA 240
ATGTTATGTA AAAATGAAAA TGCTTTCTGT GGAAGTTACA TTCTTTACCA GGTCTTTAAC 300
ATAAATTAAC ACGACGTCGA GTAAGCCTTT GTTCGGAAGA CAAACTAGTT TGTGAGTTCA 360
GTCAGATCCC AGCT 374
(2) INFORMATION FOR SEQ ID NO: 170:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 334 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 170:
AGTTGCCAGG ACCACCACCA TAGTTGCCAG GTTCATCATA AACAAATCCA ACATCAATCT 60
TAAATTCCCC CATCAGACAA TCTGCCCTCA AAGAATGGGA ATTATAAACC CGGATACTGA 120
TGATCTCATC CATGAGCTCA GAGGGTGTGA TGTGCACATT GTAGAAAAAT AACTCGTCAA 180
AAAACGGATT GTTCCCTCTC TTGATTCTCG TGCGATGCGT CTGACCACAG ATGTGAACTT 240
TCACCACGGG CCTTATGTTG TTGCCGCATA ACTGACGGCC CTCGATCACT CTGACACGGA 300
TCTGGAAATC TGTGGCTTGT TGGACAGCAT CCTT 334
(2) INFORMATION FOR SEQ ID NO: 171:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 380 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 171:
AAGCCGTGTC CCAAAGAATG GATAGAGACG CGATCAGATG CGACAGTGCT GTGGAGAAAG 60
CCCAGGAACC TGCACAATTG CCCTGGTCCA ATGGCTCGTG GATCAGGTTG GGCCACTTCT 120
CTGAAGCTTC AAAGGCAGTG GGTAGCACTT CCCCTTGGCC CAGCACCGTA TAAATCTCAT 180
TCATATTCAT GACAGTGGAG GATGGGCGGA TTGTGCCCAG GCGGTACGGA ATGCCCTCAT 240
CCAGGGTCAT GCCCCAGAAG GCACTGTGGT TCCCAGCCTG CCACCCGTAG TTGCCTCGGT 300
TGATGGCTTT AATCATGTCT GGTCACTAGA CACGGCTTAA GCGAATCTCG AGATATCCAT 360
CACACTGGCG GCGTCGAGAT 380
(2) INFORMATION FOR SEQ ID NO: 172:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 353 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 172:
AAGCCGTGTC TGATGATGGA GGTAGTGGTG GGGGAGGAGG GACTGAGGGT CCTGAGGTGG 60
TGGCCCCTGG AACTGATCCC ACATAGTTAC CCACTGCTAG TTCTGACCCC GTGGACAACG 120
TGCCAGAGGC CATGACTGGC AGTATGGCAA TGTCCCCATC CCCTTTCTTC TTAATTTTAA 180
TGGTCCCTTG TTTCTCCAGT TCGTGAATCT TTTTTTCCAG GGTAGACTGT CTTTGAATGG 240
CTTCTTCCTT TTCTTTGACC ATTTTTCTTA ACGTGTGAAC TTGGGTATTT GCATCTTTGT 300
AGATTTCCGG ACAACATCAG TTCCTTATTC CTCTGCATAA GTTGCTTTCA GTT 353
(2) INFORMATION FOR SEQ ID NO: 173:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 350 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 173:
CGAGTCAGAC ACATGAAAGC AAAACGCGGG CAGATAAAAC GATCGCCTTA CCTTCTAGCA 60
AAAATCTGAA GCTTGTGTCA GAAACAAAGA CTCAGAAAGG TTTGTTTTCA GATGAAGAAG 120
ACTCTGAGGA TTTGTTTTCT TCTCAAAGTT CAAGTAAGCC AAAAAGTGCA TCACTTTCAT 180
CCAGCCAGCC CCCAACATCA GTCTCCCTTT TTGGTGATGA AGATGAAGAG GACAGTCTTT 240
TTGGGAGTGC AGCAGCTAAG AAGCAGACTT CATCTCTACA ACCTCAGAGT CAAGAGAAAG 300
CAAAGCCTTC CGAGCAGCCC TCAAAGAAGA CATCTGCCTT GTTGTTCAGA 350
(2) INFORMATION FOR SEQ ID NO: 174:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 377 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 174:
CGAGTCAGAC TTAATTTAAA AACGAAACAA AACAAAAATA ACATAGTTTA GAAATCAAGG 60
AGAAAGGACA GATAGTCTAA GAAAAAAGAC AACACAAAAG AGGGGCAGGG CGGCCAGCTT 120
GCATCAGGGA TCTTGGCTGG AGACCTGCTT TGAATAGGTT TCTTGCAGGT ATTTCTTAAA 180
TGCTGTGGGG TTTTTCCAGA GTTCCGCAGC GTGTGTGTTC AAAGGGCTAT CGATGTTGGG 240
TTCTCCTAGC AGGCTCTGGA TAGAGAGCAA GATAGTCCTG ACATCATATA GTGCAGACCA 300
CTTATCCTTG AGGATGTCCG GCAGATGTTG CCTGGGTGTC ACGTTGGGGT GGTAGCAGGG 360
TGTGAGGAAC TTCACTG 377
(2) INFORMATION FOR SEQ ID NO: 175:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 326 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: <Unknown>
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 175:
CGAGTCAGAC ACTCCTGGCT CCTGGATTCT TTAGATGCCT CCATCAGACT GGGTACTTTA 60
GATGCCTCCA TCAGACTACT TCGTCATTGT ATTTCTCAGT TCGCTCAGGG CAAGCGGCAG 120
TCTCTGGGCT GCTGTGGCAG GTGCCACCAC TGCATTTAAA AGTTAAAATT TCTTCAAATA 180
TTCCCATCAA GGCCTTGTAG CCTCTGAGAT TGGTTTACTA TTTGCCCAGT TATTTAAAGC 240
TCTCTGCATT CCTTCCTGAT TTAATATTGC TATGGCCAGG ACAATGTGTA GAAGTAAAAA 300
GGATATCATA TTTACAGGTG TAACGC 326

Claims (43)

I claim:
1. A method for identifying a sequence expressed in a metastasis comprising the steps of:
a) transfecting an oncogenic sequence into a mammalian cell to form a population of transfected cells;
b) administering transfected cells to a primary site of a host mammal to form a primary tumor;
c) maintaining said mammal for a period of time sufficient to develop a metastasis at a secondary site;
d) amplifying expressed RNA sequences of the transfected cells and expressed RNA sequences of the metastasis by differential-display PCR; and
e) comparing the amplified expressed RNA sequences of the transfected cells with the amplified expressed RNA sequences of the metastasis and identifying the sequence expressed at a higher level in the metastasis as compared to the expressed RNA sequences of the transfected cells.
2. The method of claim 1 wherein the mammalian cell is transfected by calcium phosphate transfection, viral transduction, lipofection, dextran sulfate transfection or electroporation.
3. The method of claim 1 wherein the oncogenic sequence is a sequence of the gene that erodes encodes the oncoproteins p21, p34, mutant p53, myc, ras or src.
4. The method of claim 1 wherein the oncogenic sequence is a sequence that enhances metastatic potential.
5. The method of claim 4 wherein the oncogenic sequence is a sequence of the gene that encodes cyclin D1, caveolin or TGF-β1.
6. The method of claim 1 wherein the mammalian cell is treated with an agent that alters gene expression prior to the administration of said cell to said host mammal.
7. The method of claim 6 wherein the agent is benzantracene (BA), dimethyl benzanthracene (DMBA) or 5-azacytidine.
8. The method of claim 1 wherein the mammalian cell is a primary cell or an established cell line.
9. The method of claim 1 wherein the mammalian cell is isolate isolated from urogenital sinus tissue.
10. The method of claim 1 wherein the mammalian cell is a fetal cell.
11. The method of claim 1 wherein the mammalian cell contains a gene selected from the group consisting of TGF-β1, cyclin D1, p21, p34, mutant p53, ras, and myc.
12. The method of claim 1 wherein the mammalian cell is isolated from the same species as the host mammal.
13. The method of claim 1 wherein the mammalian cell and the host mammal are histocompatible.
14. The method of claim 1 wherein the mammalian cell and the host mammal are syngeneic.
15. The method of claim 1 wherein the transfected cell is isolated and maintained in vivo or in vitro for a period of time prior to introduction of said cell to the host mammal.
16. The method of claim 1 wherein the expressed sequences of the transfected cells are obtained from a cell line of immortalized transfected cells.
17. The method of claim 1 wherein the transfected cells are administered to the primary site by subcutaneous implantation.
18. The method of claim 1 wherein the host mammal is a mouse, a rabbit or a primate.
19. The method of claim 1 wherein the host mammal is a syngeneic, xenogeneic, immunocompromised or transgenic host mammal.
20. The method of claim 1 further comprising suppressing expression of TGF-β in the host mammal prior to the introduction of transfected cells into said host mammal.
21. The method of claim 1 wherein the primary site is the renal capsule, the prostate or the testis.
22. The method of claim 1 wherein the secondary site is selected from the group of sites consisting of lung, kidney, liver, lymph nodes, brain, bone, testis, spleen, ovaries and mammary.
23. The method of claim 1 wherein differential display PCR is performed with an anchor primer and a variable primer.
24. The method of claim 22 wherein the anchor primer comprises a polythymidine sequence and a dinucleotide sequence connected to a 3′-terminus.
25. The method of claim 24 wherein the polythymidine sequence comprises between about 5 to about 30 thymidines.
26. The method of claim 24 wherein the dinucleotide sequence is selected from the group of sequences consisting of AA, AG, AC, AT, GA, GG, GC, GT, CA, CG, CC and CT.
27. The method of claim 23 wherein the anchor primer or the variable primer comprise a detectable moiety selected from the group consisting of radioactive moieties, phosphorescent moieties, magnetic moieties, luminescent moieties and conjugatable moieties.
28. The method of claim 23 wherein the anchor primer and the variable primer have a common sequence.
29. The method of claim 7 wherein the agent is a retinoid.
30. A method for identifying a sequence expressed in metastasis comprising the steps of:
a) pretreating a mammalian cell with an agent that enhances metastatic potential to form a population of cells predisposed to metastasis;
b) introducing the pretreated cells to a primary site of a host mammal;
c) maintaining said mammal for a period of time sufficient to develop a metastatis at a secondary site;
d) amplifying expressed RNA sequences of pretreated cells and expressed RNA sequences of the metastasis by differential-display PCR; and
e) identifygidentifying the sequence expressed at a higher level in the metastasis as compared to expressed RNA sequences of the pretreated cells.
31. The method of claim 30 further comprising the step of treating cells of the primary or secondary sites with a genotoxic agent prior to amplification.
32. The method of claim 31 wherein the genotoxic agent is benzenthracene (BA), dimethyl benzanthracene (DMBA) or 5-azacytidine.
33. The method of claim 30 further comprising the step of comparing the expressed sequences amplified from the metastasis with expressed sequences amplified from mammalian cells before pretreatment to identify the sequence selectively expressed in the metastasis.
34. The method of claim 30 wherein the chemical compound is a benzenthracene, dimethyl benzanthracene, or 5-azacytidine.
35. The method of claim 30 wherein the mammalian cell is transfected, prior to the administration of said cell to the host mammal, with an oncogenic sequence before or after treatment of said cell with the agent that enhances metastatic potential.
36. The method of claim 30 wherein the mammalian cell is a cell line.
37. The method of claim 30 wherein the mammalian cell is isolated from lymphatic tissue, hematopoietic cells, reproductive tissues or urogenital sinus tissue.
38. The method of claim 30 wherein the mammalian cell is a fetal cell.
39. The method of claim 30 wherein the mammalian cell is isolated from a transgenic animal.
40. The method of claim 30 wherein the primary site is the renal capsule, the prostate or the testis.
41. The method of claim 30 wherein the secondary site is selected from the group of sites consisting of lung, kidney, liver, lymph nodes, brain, bone, testis, spleen, ovaries and mammary.
42. The method of claim 30 wherein differential display PCR is performed using an anchor primer and a variable primer.
43. A method of identifying a sequence expressed in metastatis comprising the steps of:
pretreating a population of mamalian cells with an agent that enhances metastatic potential to form a population of cells predisposed to metastasis;
introducing the pretreated cells to a primary site of a host mammal;
maintaining said mammal for a period of time sufficient to develop a metastasis at a secondary site;
amplifying expressed RNA sequences of the mammalian cells before pretreatment and expressed RNA sequences of the metastasis by differential-display PCR; and
comparing the expressed sequences amplified from the metastasis with expressed sequences amplified from mammalian cells before pretreatment to identify the sequence selectively expressed in the metastasis.
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