WO2001012647A1 - Expression genique modulee en tumorigenese de l'ataxie-telangiectasie - Google Patents

Expression genique modulee en tumorigenese de l'ataxie-telangiectasie Download PDF

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WO2001012647A1
WO2001012647A1 PCT/US2000/022889 US0022889W WO0112647A1 WO 2001012647 A1 WO2001012647 A1 WO 2001012647A1 US 0022889 W US0022889 W US 0022889W WO 0112647 A1 WO0112647 A1 WO 0112647A1
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seq
polypeptide
polynucleotide
sequence
nucleic acid
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PCT/US2000/022889
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English (en)
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Carrolee Barlow
Christopher J. Winrow
Marie Lei A. Callahan
Daniel G. Pankratz
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Digital Gene Technologies, Inc.
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Priority to AU67921/00A priority Critical patent/AU6792100A/en
Publication of WO2001012647A1 publication Critical patent/WO2001012647A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C12Q2600/136—Screening for pharmacological compounds

Definitions

  • Ataxia telangiectasia is a human autosomal recessive multisystem disorder comprising progressive cerebellar ataxia with onset in infancy, progressive oculocutaneous telangiectasia, and unusual susceptibility to progressive bronchopulmonary disease and to lymphoreticular neoplasia.
  • AT is a human autosomal recessive multisystem disorder comprising progressive cerebellar ataxia with onset in infancy, progressive oculocutaneous telangiectasia, and unusual susceptibility to progressive bronchopulmonary disease and to lymphoreticular neoplasia.
  • AT generally includes other progressive neurologic degenerations such as choreoathetosis and oculomotor dysfunction, recurrent sinopulmonary infections secondary to immunodeficiency, lymphoreticular malignancies, growth retardation, incomplete sexual maturation, endocrine abnormalities, and premature aging of the skin and hair.
  • itra-def ⁇ cient mice a paradigm of ataxia telangiectasia, Cell, 86: 159-171.
  • the disease is progressive, and death generally occurs by the second or third decade of life due to neurologic deterioration or lymphoreticular malignancies, which occur in about 10% - 15% of patients.
  • AT has been mapped to human chromosome position 1 lq22-23, and the responsible gene (designated ATM, "AT mutated") has been has been defined by positional cloning.
  • the mouse homolog, Atm has also been identified. Atm shows an 84% amino acid identity and a 91 % similarity with A TM. Sequence comparisons revealed that both ATM and Atm are members of a family of genes involved at several stages in double-stranded DNA breakage repair and cell cycle control.
  • ATM is a key protein for managing cell cycle perturbations in response to DNA damage and plays a role in genetic stability and cancer susceptibility. Inactivation of normal ATM function could thus result in inappropriate replication of damaged DNA, thus contributing to the malignant transformation of cells.
  • Available evidence indicates that ATM may be a critical regulator of many important cellular processes and thus has potential implications for cancer in the general population. Notably, many studies have indicated that loss of ATM function is related to cancer predisposition in AT heterozygotes. In particular, it has been found that women heterozygous for one mutated allele of A TM have a three to fivefold increase in breast cancer risk.
  • ATM is a tumor suppressor gene whose inactivation is a key event in the development of many forms of cancer, particularly in T-cell prolymphocytic leukemias and aggressive forms of B cell chronic lymphocytic leukemia in patients who do not have AT.
  • TOGATM Total Gene Expression Analysis
  • the present invention provides novel polynucleotides and the encoded polypeptides. Moreover, the present invention relates to vectors, host cells, antibodies, and recombinant methods for producing the polynucleotides and the polypeptides. Also provided are diagnostic methods for detecting disorders related to the polypeptides and the polynucleotides encoding them, and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying binding partners of the polypeptides.
  • the tumors that develop in ⁇ 4tw-deficient mice provide the ability to specifically characterize the genes involved in cancer formation as a model of particular forms of human hematopoietic cancer.
  • Cancer cell lines established from several T-cell lymphomas from multiple different Atm deficient animals are useful for the study of human cancers, as well as for the development of suitable diagnostic and therapeutic compositions and methods.
  • An extensive cellular analysis of these tumors has been performed and show that they invariably arise at a stage of development when the TCR ⁇ undergoes rearrangement. It has been specifically found that the translocations occur at the TCR ⁇ locus in ten often tumor cell lines established from these mice.
  • Atm deficient tumor cell lines disclosed herein are useful for characterizing the genes involved in the multi-step process of cancer formation.
  • One embodiment of the invention provides an isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID ⁇ O:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • a polynucleotide chosen from the group consisting of SEQ ID ⁇ O:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
  • an isolated nucleic acid molecule comprising a polynucleotide at least 95% identical to the isolated nucleic acid molecule comprising a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • an isolated nucleic acid molecule at least ten bases in length that is hybridizable to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 under stringent conditions.
  • an isolated nucleic acid molecule of the invention encodes a polypeptide epitope.
  • a polynucleotide of the invention can further comprise sequential nucleotide deletions from either the 5 '-terminus or the 3 '-terminus.
  • a polynucleotide of the invention can be present in a recombinant vector and in a recombinant host cell.
  • Another embodiment of the invention provides an isolated polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • a polynucleotide chosen from the group consisting of SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
  • an isolated nucleic acid molecule encoding these polypeptides. Also provided is an isolated nucleic acid molecule encoding a fragment of a polypeptide encoded by a polynucleotide chosen from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • a polypeptide of the invention has biological activity.
  • a polypeptide of the invention can comprise sequential amino acid deletions from either the C- terminus or the N-terminus.
  • Still another embodiment of the invention provides an isolated antibody that binds specifically to an isolated polypeptide of the invention.
  • the antibody can be a monoclonal antibody or a polyclonal antibody.
  • polypeptide is produced by culturing a recombinant host cell that comprises a polynucleotide of the invention under conditions such that the polypeptide is expressed and isolating the polypeptide.
  • Another embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as ataxia telangiectasia, comprising administering to a mammalian subject a therapeutically effective amount of a polypeptide of the invention or a polynucleotide of the invention.
  • a further embodiment of the invention provides an isolated antibody that binds specifically to the isolated polypeptide of the invention.
  • a preferred embodiment of the invention provides a method for preventing, treating, modulating, or ameliorating a medical condition, such as ataxia telangiectasia, comprising administering to a mammalian subject a therapeutically effective amount of the antibody.
  • An additional embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition in a subject.
  • the method comprises determining the presence or absence of a mutation in a polynucleotide of the invention.
  • a pathological condition or a susceptibility to a pathological condition, such as ataxia telangiectasia is diagnosed based on the presence or absence of the mutation.
  • Even another embodiment of the invention provides a method of diagnosing a pathological condition or a susceptibility to a pathological condition, such as ataxia telangiectasia in a subject.
  • the method comprises detecting an alteration in expression of a polypeptide encoded by the polynucleotide of the invention, wherein the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.
  • the alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.
  • a first biological sample is obtained from a patient suspected of having ataxia telangiectasia and a second sample from a suitable comparable control source is obtained.
  • the amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample.
  • the amount of the polypeptide in the first and second samples is determined.
  • a patient is diagnosed as having ataxia telangiectasia if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.
  • Yet another embodiment of the invention provides a method for identifying a binding partner to a polypeptide of the invention.
  • the polypeptide is contacted with a binding partner. Whether the binding partner effects an activity of the polypeptide is determined.
  • Yet another embodiment of the invention provides a method of identifying an activity of an expressed polypeptide in a biological assay.
  • a polypeptide of the invention is expressed in a cell.
  • the expressed polypeptide is isolated and tested for an activity in a biological assay.
  • the activity of the expressed polypeptide is identified based on the test results.
  • Another embodiment of the invention provides a substantially pure isolated DNA molecule suitable for use as a probe for genes regulated in ataxia telangiectasia.
  • the DNA molecule is chosen from the group consisting of the DNA molecules identified in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l 1, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
  • kits for detecting the presence of a polypeptide of the invention in a mammalian tissue sample comprising a first antibody which immunoreacts with a mammalian protein encoded by a gene corresponding to a polynucleotide or polypeptide of the invention in an amount sufficient for at least one assay and suitable packaging material.
  • the kit can further comprise a labeled or unlabeled second antibody that binds to the first antibody.
  • a label can comprise enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, or bioluminescent compounds.
  • Yet another embodiment of the invention provides a kit for detecting the presence of a genes encoding an protein comprising a polynucleotide of the invention, or fragment thereof having at least 10 contiguous bases, in an amount sufficient for at least one assay, and suitable packaging material.
  • Yet another embodiment of the invention provides a method for detecting the presence of a nucleic acid encoding a protein in a mammalian tissue sample.
  • a polynucleotide of the invention or fragment thereof having at least 10 contiguous bases is hybridized with the nucleic acid of the sample. The presence of the hybridization product is detected.
  • Figure 1 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases GAGC, showing PCR products produced from mRNA extracted from cell lines AT-4 (Fig. 1A), AT-7 (Fig. IB), AT-12 (Fig. 1C), and AT-13 (Fig. ID); the arrow indicates the vertical cursor connecting the position of peaks of length 197 base pairs;
  • Figure 2 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (U88908) corresponding to GAGC 197, showing, hybridization with a band of mRNA at 4.2 kb that is expressed at various levels in cell lines AT-4, AT-7, AT-10, AT-11, AT-13; APT-3, P53-1, 101-7, 292-3, and thymus, but not detectable in cell line AT-12; the image below shows the methylene blue stained gel for quantification of mRNA loading;
  • Figure 3 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases GCTG, showing PCR products produced from mRNA extracted from cell lines AT-4 (Fig. 3A), AT-7 (Fig. 3B), AT-12 (Fig. 3C), and AT-13 (Fig. 3D); the arrow indicates the vertical cursor connecting the position of peaks of length 345 base pairs;
  • Figure 4 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (Z31664) corresponding to GCTG 345, showing, hybridization with bands of mRNA at 4.0 kb and 3.7 kb that are expressed at various levels in cell lines AT-4, AT-11, AT-12, AT-13, 292-3, and polyA (pA) thymus mRNA, but not detectable in cell lines AT-7, AT-10, APT-3, P53-1, 101-7; the image below shows the methylene blue stained gel for quantification of mRNA loading;
  • Figure 5 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases ACAT, showing PCR products produced from mRNA extracted from cell lines AT-4 (Fig. 5 A), AT-7 (Fig. 5B), AT-12 (Fig. 5C), and AT-13 (Fig. 5D); the arrow indicates the vertical cursor connecting the position of peaks of length 367 base pairs;
  • Figure 6 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (U38252) corresponding to ACAT 367, showing, hybridization with a band of mRNA at 4.1 kb that is expressed at various levels in cell lines AT-7, AT-10, AT-12, AT-13 and APT-3, but not detectable in cell lines AT-4, AT-11, P53-1, 101-7, 292-3 and polyA (pA) thymus mRNA; the image below shows the methylene blue stained gel for quantification of mRNA loading;
  • Figure 7 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases ACGG, showing PCR products produced from mRNA extracted from cell lines AT-4 (Fig. 7 A), AT-7 (Fig. 7B), AT-12 (Fig. 7C), and AT-13 (Fig. 7D); the arrow indicates the vertical cursor connecting the position of peaks of length 458 base pairs;
  • Figure 8 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (XI 2822) corresponding to ACGG 458, showing, hybridization with bands of mRNA at 1.7 kb and 1.1 kb that are expressed at various levels in cell lines AT-7 and AT-10, but not detectable in cell lines AT-4, AT-12, AT-1 1, AT- 13; APT-3, P53-1, 101-7, and 292-3, and polyA (pA) thymus (only 1.1 kb) mRNA; the image below shows the methylene blue stained gel for quantification of mRNA loading;
  • Figure 9 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases CCGT, showing PCR products produced from mRNA extracted from cell lines AT-4 (Fig. 9A), AT-7 (Fig. 9B), AT-12 (Fig. 9C), and AT-13 (Fig. 9D); the arrow indicates the vertical cursor connecting the position of peaks of length 151 base pairs; and
  • Figure 10 is a graphical representation of a more detailed analysis of the 151 b.p. PCR product indicated in Figure 9, using the extended TOGA primer G-A-T-C-G-A-A-T-C-C-G-G- C-C-G-T-G-T-G-T-G-C-C-T-T-A-G-G-A-G (SEQ ID NO:32).
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • a "secreted" protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a signal sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a "mature" protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.
  • a "polynucleotide” refers to a molecule having a nucleic acid sequence shown in SEQ ID NOs: 1-23.
  • the polynucleotide can contain all or part of the nucleotide sequence of the full length cDNA sequence, including the 5' and 3' untranslated sequences, the coding region, with or without the signal sequence, the secreted protein coding region, as well as fragments, epitopes, domains, and variants of the nucleic acid sequence.
  • a "polypeptide” refers to a molecule having the translated amino acid sequence generated from the polynucleotide as broadly defined.
  • a “polynucleotide” of the present invention also includes those polynucleotides capable of hybridizing, under stringent hybridization conditions, to sequences contained in SEQ ID NOs: 1-23, or the complement thereof, or the cDNA.
  • Stringent hybridization conditions refers to an overnight incubation at 42°C in a solution comprising 50% formamide, 5X SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at about 65°C.
  • nucleic acid molecules that hybridize to the polynucleotides of the present invention at lower stringency hybridization conditions. Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
  • washes performed following stringent hybridization can be done at higher salt concentrations (e.g., 5X SSC).
  • blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
  • the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
  • polynucleotide which hybridizes only to polyA+ sequences (such as any 3' terminal polyA+ tract of a cDNA shown in the sequence listing), or to a complementary stretch of T (or U) residues, would not be included in the definition of "polynucleotide,” since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
  • a polynucleotide of the present invention can be composed of any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • a polynucleotide can be composed of triple- stranded regions comprising RNA or DNA or both RNA and DNA.
  • a polynucleotide may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons.
  • Modified bases include, for example, tritylated bases and unusual bases such as inosine.
  • a variety of modifications can be made to DNA and RNA; thus, "polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
  • the polypeptide of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini.
  • polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formulation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • a polypeptide having biological activity refers to polypeptides exhibiting activity similar, but not necessarily identical to, an activity of a polypeptide of the present invention, including mature forms, as measured in a particular biological assay, with or without dose- dependency. In the case where dose dependency does exist, it need not be identical to that of the polypeptide, but rather substantially similar to the dose-dependence in a given activity as compared to the polypeptide of the present invention (i.e., the candidate polypeptide will exhibit greater activity or not more than about 25-fold less and, preferably, not more than about ten-fold less activity and, most preferably, not more than about three-fold less activity relative to the polypeptide of the present invention).
  • the translated amino acid sequence, beginning with the methionine is identified although other reading frames can also be easily translated using known molecular biology techniques. The polypeptides produced by the translation of these alternative open reading frames are specifically contemplated by the present invention.
  • SEQ ID NOs: 1-23 and the translations of SEQ ID NOs: 1-23 are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. These nucleic acid molecules will also hybridize to nucleic acid molecules in biological samples, thereby enabling a variety of forensic and diagnostic methods of the invention. Similarly, polypeptides identified from the translations of SEQ ID NOs: 1 -23 may be used to generate antibodies which bind specifically to the secreted proteins encoded by the cDNA clones identified.
  • DNA sequences generated by sequencing reactions can contain sequencing errors.
  • the errors exist as misidentified nucleotides, or as insertions or deletions of nucleotides in the generated DNA sequence.
  • the erroneously inserted or deleted nucleotides cause frame shifts in the reading frames of the predicted amino acid sequence.
  • the predicted amino acid sequence diverges from the actual amino acid sequence, even though the generated DNA sequence may be greater than 99.9% identical to the actual DNA sequence (for example, one base insertion or deletion in an open reading frame of over 1,000 bases).
  • the present invention also relates to the genes corresponding to polynucleotides identified in SEQ ID NOs: 1-23, and translations of polynucleotides identified in SEQ ID NOs: 1-23.
  • the corresponding gene can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence and identifying or amplifying the corresponding gene from appropriate sources of genomic material.
  • species homologues may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for the desired homologue.
  • polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • the polypeptides may be in the form of the secreted protein, including the mature form, or may be a part of a larger protein, such as a fusion protein (see below). It is often advantageous to include an additional amino acid sequence which contains secretory or leader sequences, pro-sequences, sequences which aid in purification (such as multiple histidine residues), or an additional sequence for stability during recombinant production.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a polypeptide, including the secreted polypeptide can be substantially purified by the one-step method described in Smith & Johnson, Gene, 67:31-40 (1988).
  • Polypeptides of the invention also can be purified from natural or recombinant sources using antibodies of the invention raised against the secreted protein in methods which are well known in the art.
  • the deduced amino acid sequence of the secreted polypeptide was analyzed by a computer program called Signal P (Nielsen et al., Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • Signal P Neelsen et al., Protein Engineering, 10:1-6 (1997), which predicts the cellular location of a protein based on the amino acid sequence.
  • McGeoch and von Heinje are incorporated.
  • the present invention provides secreted polypeptides having a sequence corresponding to the translations of SEQ. ID NO: 1-23 which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • secreted polypeptides having a sequence corresponding to the translations of SEQ. ID NO: 1-23 which have an N-terminus beginning within 5 residues (i.e., + or - 5 residues) of the predicted cleavage point.
  • cleavage of the signal sequence from a secreted protein is not entirely uniform, resulting in more than one secreted species.
  • the signal sequence identified by the above analysis may not necessarily predict the naturally occurring signal sequence.
  • the naturally occurring signal sequence may be further upstream from the predicted signal sequence.
  • the predicted signal sequence will be capable of directing the secreted protein to the ER.
  • Variants refers to a polynucleotide or polypeptide differing from the polynucleotide or polypeptide of the present invention, but retaining essential properties thereof. In general, variants have close similarity overall and are identical in many regions to the polynucleotide or polypeptide of the present invention.
  • identity is well known to skilled artisans (Carillo et al., SIAM J Applied Math., 48: 1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in "Guide to Huge Computers,” Martin J. Bishop, Ed., Academic Press, San Diego, (1994) and Carillo et al., (1988), Supra. Methods for aligning polynucleotides or polypeptides are codified in computer programs, including the GCG program package (Devereux et al., Nuc. Acids Res.
  • the parameters are set such that the percentage of identity is calculated over the full length of the reference polynucleotide and that gaps in identity of up to 5% of the total number of nucleotides in the reference polynucleotide are allowed.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • sequence includes nucleotide and amino acid sequences.
  • sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences.
  • the result of said global sequence alignment is presented in terms of percent identity.
  • identity to a sequence identified in SEQ ID NOs: 1-23 means that the polynucleotide is identical to a sequence identified in SEQ ID NOs: 1-23 or the cDNA except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the total length (not just within a given 100 nucleotide stretch).
  • polynucleotide having a nucleotide sequence at least 95% identical to polynucleotides identified in SEQ ID NOs: 1-23 up to 5% of the nucleotides in the sequence identified in SEQ ID NOs:l- 23 or the cDNA can be deleted, inserted, or substituted with other nucleotides. These changes may occur anywhere throughout the polynucleotide.
  • a polypeptide having an amino acid sequence having at least, for example, 95% "identity" to a reference polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference polypeptide except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the total length of the reference polypeptide.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the above polypeptides should exhibit at least one biological activity of the protein.
  • polypeptides of the present invention include polypeptides having at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98%, or 99% similarity to an amino acid sequence contained in translations of polynucleotides identified in SEQ ID NOs: 1-23.
  • the variants may contain alterations in the coding regions, non-coding regions, or both.
  • polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide are preferred.
  • Nucleotide variants produced by silent substitutions due to the degeneracy of the genetic code are preferred.
  • variants in which 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or added in any combination are also preferred.
  • Polynucleotide variants can be produced for a variety of reasons. For instance, a polynucleotide variant may be produced to optimize codon expression for a particular host (i.e., codons in the human mRNA may be changed to those preferred by a bacterial host, such as E. coli).
  • Naturally occurring variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (Lewin, B., Ed., Genes II, John Wiley & Sons, New York (1985)). These allelic variants can vary at either the polynucleotide and/or polypeptide level. Alternatively, non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
  • variants may be generated to improve or alter the characteristics of the polypeptides of the present invention.
  • one or more amino acids can be deleted from the N-terminus or C-terminus of the secreted protein without substantial loss of biological function.
  • Ron et al. reported variant KGF proteins having heparin binding activity even after deleting 3, 8, or 27 amino-terminal amino acid residues. (J. Biol. Chem. 268: 2984-2988 (1993)).
  • interferon gamma exhibited up to ten times higher activity after deleting 8-10 amino acid residues from the carboxy terminus of this protein (Dobeli et al., J. Biotechnology, 7:199-216 (1988)).
  • the invention further includes polypeptide variants which show substantial biological activity.
  • variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
  • guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, et al., Science 247: 1306-1310 (1990), wherein the authors indicate that there are two main strategies for studying the tolerance of an amino acid sequence to change.
  • the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, the amino acid positions which have been conserved between species can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions in which substitutions have been tolerated by natural selection indicate positions which are not critical for protein function. Thus, positions tolerating amino acid substitution may be modified while still maintaining biological activity of the protein.
  • the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site-directed mutagenesis or alanine-scanning mutagenesis (the introduction of single alanine mutations at every residue in the molecule) can be used (Cunningham et al., Science 244: 1081- 1085 (1989)). The resulting mutant molecules can then be tested for biological activity.
  • tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin; replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp; and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
  • variants of the present invention include:
  • substitutions with one or more of the non-conserved amino acid residues where the substituted amino acid residues may or may not be one encoded by the genetic code; (ii) substitution with one or more of amino acid residues having a substituent group; (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (e.g., polyethylene glycol); or (iv) fusion of the polypeptide with additional amino acids, such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification.
  • additional amino acids such as an IgG Fc fusion region peptide, a leader or secretory sequence, or a sequence facilitating purification.
  • polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as decreased aggregation.
  • aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity (see, e.g., Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev. Therap. Drug Carrier Sys. 10:307-377 (1993)).
  • a "polynucleotide fragment” refers to a short polynucleotide having a nucleic acid sequence contained in that shown in polynucleotides identified in SEQ ID NOs: 1-23.
  • the short nucleotide fragments are preferably at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length.
  • a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in that shown in polynucleotides identified in SEQ ID NOs: 1-23.
  • These nucleotide fragments are useful as diagnostic probes and primers as discussed herein. Of course, larger fragments (e.g., 40, 50, 150, and greater than 150 nucleotides) are preferred.
  • polynucleotide fragments of the invention include, for example, fragments having a sequence from about nucleotide number 1-40, 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, to the end of polynucleotides identified in SEQ ID NOs: 1-23.
  • “about” includes the particularly recited ranges, larger or smaller by several nucleotides (i.e., 5, 4, 3, 2, or 1 nt), at either terminus or at both termini.
  • these fragments encode a polypeptide which has biological activity.
  • polypeptide fragment refers to a short amino acid sequence contained in the translations of polynucleotides identified in SEQ ID NOs: 1-23. Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region. Representative examples of polypeptide fragments of the invention, include, for example, fragments from about amino acid number 1-20, 21-40, 41-60, or 61 to the end of the coding region. Moreover, polypeptide fragments can be about 20, 30, 40, 50 or 60, amino acids in length. In this context "about” includes the particularly recited ranges, larger or smaller by several amino acids (i.e., 5, 4, 3, 2, or 1), at either extreme or at both extremes.
  • Preferred polypeptide fragments include the secreted protein as well as the mature form.
  • polypeptide fragments include the secreted protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both.
  • any number of amino acids ranging from 1-60 can be deleted from the amino terminus of either the secreted polypeptide or the mature form.
  • any number of amino acids, ranging from 1-30 can be deleted from the carboxy terminus of the secreted protein or mature form.
  • any combination of the above amino and carboxy terminus deletions are preferred.
  • polynucleotide fragments encoding these polypeptide fragments are also preferred.
  • polypeptide and polynucleotide fragments characterized by structural or functional domains, such as fragments that comprise alpha-helix and alpha-helix forming regions, beta-sheet and beta-sheet- forming regions, turn and turn- forming regions, coil and coil- forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions, substrate binding region, and high antigenic index regions.
  • Polypeptide fragments of the translations of polynucleotides identified in SEQ ID NOs: 1-23 falling within conserved domains are specifically contemplated by the present invention.
  • polynucleotide fragments encoding these domains are also contemplated.
  • Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the polypeptide of the present invention.
  • the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
  • epitopes refer to polypeptide fragments having antigenic or immunogenic activity in an animal, especially in a human.
  • a preferred embodiment of the present invention relates to a polypeptide fragment comprising an epitope, as well as the polynucleotide encoding this fragment.
  • a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • an "immunogenic epitope” is defined as a part of a protein that elicits an antibody response.
  • Fragments which function as epitopes may be produced by any conventional means. (See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA, 82:5131-5135 (1985) further described in U.S. Patent No. 4,631,21 1).
  • antigenic epitopes preferably contain a sequence of at least seven, more preferably at least nine, and most preferably between about 15 to about 30 amino acids.
  • Antigenic epitopes are useful to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. (See, e.g., Wilson et al., Cell, 37:767-778 (1984); Sutcliffe et al., Science, 219:660-666 (1983)).
  • immunogenic epitopes can be used to induce antibodies according to methods well known in the art. (See, e.g., Sutcliffe et al., (1983) Supra; Wilson et al., (1984) Supra; Chow et al., Proc. Natl. Acad. Sci. USA, 82:910-914; and Bittle et al., J. Gen. Virol., 66:2347- 2354 (1985)).
  • a preferred immunogenic epitope includes the secreted protein.
  • the immunogenic epitope may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse).
  • the immunogenic epitope may be prescribed without a carrier if the sequence is of sufficient length (at least about 25 amino acids).
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.)
  • antibody As used herein, the term "antibody” (Ab) or “monoclonal antibody” (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to protein. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody. (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, as well as the products of a Fab or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, human, and humanized antibodies.
  • chimeric antibodies e.g., humanized versions of murine monoclonal antibodies.
  • Such humanized antibodies may be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans.
  • a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody.
  • a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody.
  • Procedures for the production of chimeric and further engineered monoclonal antibodies include those described in Riechmann et al.
  • One method for producing an antibody comprises immunizing a non-human animal, such as a transgenic mouse, with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1 -23, whereby antibodies directed against the polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23 are generated in said animal.
  • Procedures have been developed for generating human antibodies in non-human animals.
  • the antibodies may be partially human, or preferably completely human.
  • Non-human animals (such as transgenic mice) into which genetic material encoding one or more human immunoglobulin chains has been introduced may be employed. Such transgenic mice may be genetically altered in a variety of ways.
  • the genetic manipulation may result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some (preferably virtually all) antibodies produced by the animal upon immunization.
  • Antibodies produced by immunizing transgenic animals with a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23 are provided herein.
  • mice in which one or more endogenous immunoglobulin genes are inactivated by various means have been prepared.
  • Human immunoglobulin genes have been introduced into the mice to replace the inactivated mouse genes.
  • Antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animal. Examples of techniques for production and use of such transgenic animals are described in U.S. Patent Nos. 5,814,318, 5,569,825, and 5,545,806, which are incorporated by reference herein.
  • Monoclonal antibodies may be produced by conventional procedures, e.g., by immortalizing spleen cells harvested from the transgenic animal after completion of the immunization schedule.
  • the spleen cells may be fused with myeloma cells to produce hybridomas, by conventional procedures.
  • a method for producing a hybridoma cell line comprises immunizing such a transgenic animal with a immunogen comprising at least seven contiguous amino acid residues of a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23; harvesting spleen cells from the immunized animal; fusing the harvested spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23.
  • Such hybridoma cell lines, and monoclonal antibodies produced therefrom, are encompassed by the present invention.
  • Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques.
  • Antibodies may be employed in an in vitro procedure, or administered in vivo to inhibit biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23. Disorders caused or exacerbated (directly or indirectly) by the interaction of such polypeptides of the present invention with cell surface receptors thus may be treated.
  • a therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective for reducing a biological activity induced by a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23.
  • conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23.
  • detectable or therapeutic agent attached to an antibody directed against a polypeptide translated from a nucleotide sequence chosen from SEQ ID NOs: 1-23.
  • agents include but are not limited to diagnostic radionuclides, therapeutic radionuclides, and cytotoxic drugs.
  • the conjugates find use in in vitro or in vivo procedures.
  • any polypeptide of the present invention can be used to generate fusion proteins.
  • the polypeptide of the present invention when fused to a second protein, can be used as an antigenic tag.
  • Antibodies raised against the polypeptide of the present invention can be used to indirectly detect the second protein by binding to the polypeptide.
  • secreted proteins target cellular locations based on trafficking signals, the polypeptides of the present invention can be used as targeting molecules once fused to other proteins.
  • domains that can be fused to polypeptides of the present invention include not only heterologous signal sequences, but also other heterologous functional regions.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • fusion proteins may also be engineered to improve characteristics of the polypeptide of the present invention. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence during purification from the host cell or subsequent handling and storage.
  • peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to facilitate handling of polypeptides are familiar and routine techniques in the art.
  • polypeptides of the present invention can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • fusion proteins facilitate purification and show an increased half-life in vivo.
  • One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature, 331 :84-86 (1988)).
  • Fusion proteins having disulf ⁇ de-linked dimeric structures can also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem., 270:3958-3964 (1995)).
  • EP A 0 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (see, e.g., EP A 0 232 262).
  • deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5
  • Fc portions for the purpose of high- throughput screening assays to identify antagonists of hIL-5.
  • the polypeptides of the present invention can be fused to marker sequences, such as a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth, CA), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein (Proc. Natl. Acad. Sci. USA 86:821-824 (1989)).
  • Another peptide tag useful for purification, the "HA" tag corresponds to an epitope derived from the influenza hemagglutinin protein. (Wilson et al., Cell 37:767 (1984)).
  • any of these above fusions can be engineered using the polynucleotides or the polypeptides of the present invention.
  • the present invention also relates to vectors containing the polynucleotide of the present invention, host cells, and the production of polypeptides by recombinant techniques.
  • the vector may be, for example, a phage, plasmid, viral, or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • the polynucleotide insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, tip, phoA and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating codon at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells, and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, PNH16a, PNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc.
  • eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that the polypeptides of the present invention may, in fact, be expressed by a host cell lacking a recombinant vector.
  • a polypeptide of this invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention can also be recovered from products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • a prokaryotic or eukaryotic host including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
  • N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells. While the N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • the polynucleotides of the present invention are useful for chromosome identification. There exists an ongoing need to identify new chromosome markers, since few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available. Each polynucleotide of the present invention can be used as a chromosome marker.
  • sequences can be mapped to chromosomes by preparing PCR primers
  • Primers can be selected using computer analysis so that primers do not span more than one predicted exon in the genomic DNA. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the polynucleotides identified in SEQ ID NOs:l-
  • somatic hybrids provide a rapid method of PCR mapping the polynucleotides to particular chromosomes. Three or more clones can be assigned per day using a single thermal cycler. Moreover, sublocalization of the polynucleotides can be achieved with panels of specific chromosome fragments.
  • Other gene-mapping strategies that can be used include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • FISH fluorescence in situ hybridization
  • the polynucleotides can be used individually (to mark a single chromosome or a single site on that chromosome) or in panels (for marking multiple sites and/or multiple chromosomes).
  • Preferred polynucleotides correspond to the noncoding regions of the cDNAs because the coding sequences are more likely conserved within gene families, thus increasing the chance of cross-hybridization during chromosomal mapping.
  • Linkage analysis establishes coinheritance between a chromosomal location and presentation of a particular disease.
  • Disease mapping data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library)). Assuming one megabase mapping resolution and one gene per 20 kb, a cDNA precisely localized to a chromosomal region associated with the disease could be one of 50-500 potential causative genes.
  • polynucleotide and the corresponding gene between affected and unaffected individuals can be examined.
  • the polynucleotides of polynucleotides identified in SEQ ID NOs: 1 -23 can be used for this analysis of individuals.
  • a polynucleotide can be used to control gene expression through triple helix formation or antisense DNA or RNA. Both methods rely on binding of the polynucleotide to DNA or RNA. For these techniques, preferred polynucleotides are usually 20 to 40 bases in length and complementary to either the region of the gene involved in transcription (see, Lee et al., Nuc. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456
  • Polynucleotides of the present invention are also useful in gene therapy.
  • One goal of gene therapy is to insert a normal gene into an organism having a defective gene, in an effort to correct the genetic defect.
  • the polynucleotides disclosed in the present invention offer a means of targeting such genetic defects in a highly accurate manner.
  • Another goal is to insert a new gene that was not present in the host genome, thereby producing a new trait in the host cell.
  • the polynucleotides are also useful for identifying individuals from minute biological samples.
  • the United States military for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymorphism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identifying personnel.
  • This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the polynucleotides of the present invention can be used as additional DNA markers for RFLP.
  • the polynucleotides of the present invention can also be used as an alternative to RFLP, by determining the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, individuals can be identified because each individual will have a unique set of DNA sequences. Once an unique ID database is established for an individual, positive identification of that individual, living or dead, can be made from extremely small tissue samples.
  • DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc.
  • body fluids e.g., blood, saliva, semen, etc.
  • gene sequences amplified from polymorphic loci such as DQa class II
  • HLA gene are used in forensic biology to identify individuals. (Erlich, Ed., PCR Technology,
  • polynucleotides of the present invention can be used as polymorphic markers for forensic purposes.
  • reagents capable of identifying the source of a particular tissue. Such need arises, for example, in forensics when presented with tissue of unknown origin.
  • Appropriate reagents can comprise, for example, DNA probes or primers specific to particular tissue prepared from the sequences of the present invention. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
  • the polynucleotides of the present invention can be used as molecular weight markers on Southern gels; as diagnostic probes for the presence of a specific mRNA in a particular cell type; as a probe to "subtract-out" known sequences in the process of discovering novel polynucleotides; for selecting and making oligomers for attachment to a "gene chip” or other support; to raise anti-DNA antibodies using DNA immunization techniques; and as an antigen to elicit an immune response.
  • polypeptides identified herein can be used in numerous ways. The following description should be considered exemplary and utilizes known techniques.
  • a polypeptide of the present invention can be used to assay protein levels in a biological sample using antibody-based techniques.
  • protein expression in tissues can be studied with classical immunohistological methods.
  • Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable antibody assay labels include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 112 In), and technetium ( 99m Tc)
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • proteins can also be detected in vivo by imaging.
  • Antibody labels or markers for in vivo imaging of protein include those detectable by X-radiography, nuclear magnetic resonance (NMR) or ESR.
  • suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject.
  • suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by labeling of nutrients for the relevant hybridoma.
  • a protein-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (e.g., 13 I I, " 2 In, 99m Tc), a radio- opaque substance, or a material detectable by NMR, is introduced (e.g., parenterally, subcutaneously, or intraperitoneally) into the mammal.
  • a radioisotope e.g., 13 I I, " 2 In, 99m Tc
  • a radio- opaque substance e.g., " 2 In, 99m Tc
  • a material detectable by NMR e.g., a material detectable by NMR
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein.
  • In vivo tumor imaging is described in Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments” (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, Burchiel and Rhodes, Eds., Masson Publishing Inc. (1982).)
  • the invention provides a diagnostic method of a disorder, which involves (a) assaying the expression of a polypeptide of the present invention in cells or body fluid of an individual; and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a disorder.
  • polypeptides of the present invention can be used to treat disease.
  • patients can be administered a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin); to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B); to inhibit the activity of a polypeptide (e.g., an oncogene); to activate the activity of a polypeptide (e.g., by binding to a receptor); to reduce the activity of a membrane bound receptor by competing with it for free ligand (e.g., soluble TNF receptors used in reducing inflammation); or to bring about a desired response (e.g., blood vessel growth).
  • a polypeptide of the present invention in an effort to replace absent or decreased levels of the polypeptide (e.g., insulin); to supplement absent or decreased levels of a different polypeptide (e.g., hemoglobin S for hemoglobin B); to inhibit the activity of a polypeptide (
  • antibodies directed to a polypeptide of the present invention can also be used to treat disease.
  • administration of an antibody directed to a polypeptide of the present invention can bind and reduce overproduction of the polypeptide.
  • administration of an antibody can activate the polypeptide, such as by binding to a polypeptide bound to a membrane (receptor).
  • the polypeptides of the present invention can be used as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well-known to those of skill in the art.
  • Polypeptides can also be used to raise antibodies, which in turn are used to measure protein expression from a recombinant cell, as a way of assessing transformation of the host cell.
  • the polypeptides of the present invention can be used to test the following biological activities.
  • polynucleotides and polypeptides of the present invention can be used in assays to test for one or more biological activities. If these polynucleotides and polypeptides do exhibit activity in a particular assay, it is likely that these molecules may be involved in the diseases associated with the biological activity. Thus, the polynucleotides and polypeptides could be used to treat the associated disease.
  • a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of neuroblasts, stem cells or glial cells. Also, a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the central nervous system or peripheral nervous system by activating or inhibiting the mechanisms of synaptic transmission, synthesis, metabolism and inactivation of neural transmitters, neuromodulators and trophic factors, and by activating or inhibiting the expression and incorporation of enzymes, structural proteins, membrane channels and receptors in neurons and glial cells.
  • the etiology of these deficiencies or disorders may be genetic, somatic (such as cancer or some autoimmune disorder), acquired (e.g., by chemotherapy or toxins), or infectious.
  • a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular nervous system disease or disorder.
  • the disorder or disease can be any of Alzheimer's disease, Pick's disease, Binswanger's disease, other senile dementia, Parkinson's disease, parkinsonism, obsessive compulsive disorders, epilepsy, encephaolopathy, ischemia, alcohol addiction, drug addiction, schizophrenia, amyotrophic lateral sclerosis, multiple sclerosis, depression, and bipolar manic-depressive disorder.
  • the polypeptide or polynucleotide of the present invention can be used to study circadian variation, aging, or long-term potentiation, the latter affecting the hippocampus. Additionally, particularly with reference to mRNA species occurring in particular structures within the central nervous system, the polypeptide or polynucleotide of the present invention can be used to study brain regions that are known to be involved in complex behaviors, such as learning and memory, emotion, drug addiction, glutamate neurotoxicity, feeding behavior, olfaction, viral infection, vision, and movement disorders.
  • a polypeptide or polynucleotide of the present invention may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
  • Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
  • the etiology of these immune deficiencies or disorders may be genetic, somatic (such as cancer or some autoimmune disorders), acquired (e.g., by chemotherapy or toxins), or infectious.
  • a polynucleotide or polypeptide of the present invention can be used as a marker or detector of a particular immune system disease or disorder.
  • a polynucleotide or polypeptide of the present invention may be useful in treating or detecting deficiencies or disorders of hematopoietic cells.
  • a polypeptide or polynucleotide of the present invention could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells.
  • immuno logic deficiency syndromes include, but are not limited to: blood protein disorders (e.g.
  • agammaglobulinemia dysgammaglobulinemia
  • ataxia telangiectasia common variable immunodeficiency
  • Di George's Syndrome HIV infection
  • HTLV-BLV infection leukocyte adhesion deficiency syndrome
  • lymphopenia phagocyte bactericidal dysfunction
  • severe combined immunodeficiency SCIDs
  • Wiskott-Aldrich Disorder anemia, thrombocytopenia, or hemoglobinuria.
  • a polypeptide or polynucleotide of the present invention could also be used to modulate hemostatic (bleeding cessation) or thrombolytic activity (clot formation).
  • a polynucleotide or polypeptide of the present invention could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
  • a polynucleotide or polypeptide of the present invention that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting.
  • a polynucleotide or polypeptide of the present invention may also be useful in the treatment or detection of autoimmune disorders. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue.
  • the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T- cells, may be an effective therapy in preventing autoimmune disorders.
  • autoimmune disorders examples include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus
  • allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by a polypeptide or polynucleotide of the present invention.
  • these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
  • a polynucleotide or polypeptide of the present invention may also be used to treat and/or prevent organ rejection or graft-versus-host disease (GVHD).
  • Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
  • an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
  • the administration of a polypeptide or polynucleotide of the present invention that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
  • a polypeptide or polynucleotide of the present invention may also be used to modulate inflammation.
  • the polypeptide or polynucleotide may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
  • These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1.)
  • SIRS systemic inflammatory response syndrome
  • a polypeptide or polynucleotide can be used to treat or detect hyperproliferative disorders, including neoplasms.
  • a polypeptide or polynucleotide of the present invention may inhibit the proliferation of the disorder through direct or indirect interactions.
  • a polypeptide or polynucleotide of the present invention may proliferate other cells which can inhibit the hyperproliferative disorder.
  • hyperproliferative disorders can be treated.
  • This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • decreasing an immune response may also be a method of treating hyperproliferative disorders, such as by administering the polypeptide or polynucleotide as a chemotherapeutic agent.
  • hyperproliferative disorders that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic region, skin, soft tissue, spleen, thoracic region, and urogenital system.
  • neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic region, skin, soft tissue, spleen, thoracic region,
  • hyperproliferative disorders can also be treated or detected by a polynucleotide or polypeptide of the present invention.
  • hyperproliferative disorders include, but are not limited to hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases may be treated.
  • the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • the polypeptide or polynucleotide of the present invention may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention.
  • viruses include, but are not limited to the following DNA and RNA viral families Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia
  • Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to arthritis, bronchiollitis, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
  • parasitic agents causing disease or symptoms that can be treated or detected by a polynucleotide or polypeptide of the present invention include, but are not limited to the following families Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
  • These parasites can cause a variety of diseases or symptoms, including, but not limited to, Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), Malaria, pregnancy complications, and toxoplasmosis.
  • a polypeptide or polynucleotide of the present invention can be used to treat or detect any of these symptoms or diseases.
  • treatment using a polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy).
  • the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
  • a polynucleotide or polypeptide of the present invention can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues (see, Science 276:59-87
  • tissue damaged by congenital defects trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery (including cosmetic plastic surgery), fibrosis, reperfusion injury, or systemic cytokine damage.
  • Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue.
  • organs e.g., pancreas, liver, intestine, kidney, skin, endothelium
  • muscle smooth, skeletal or cardiac
  • vascular including vascular endothelium
  • nervous hematopoietic
  • hematopoietic skeletal tissue
  • skeletal bone, cartilage, tendon, and ligament
  • a polynucleotide or polypeptide of the present invention may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage.
  • a polynucleotide or polypeptide of the present invention could also be used prophylactically in an effort to avoid damage. Specific diseases that could be treated include of tendinitis, carpal tunnel syndrome, and other tendon or ligament defects.
  • tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
  • nerve and brain tissue could also be regenerated by using a polynucleotide or polypeptide of the present invention to proliferate and differentiate nerve cells.
  • Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stroke).
  • diseases associated with peripheral nerve injuries, peripheral neuropathy (e.g., resulting from chemotherapy or other medical therapies), localized neuropathies, and central nervous system diseases e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome
  • Alzheimer's disease Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and Shy-Drager syndrome
  • Chemotaxis A polynucleotide or polypeptide of the present invention may have chemotaxis activity.
  • a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T- cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hyperproliferation.
  • the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
  • a polynucleotide or polypeptide of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds. It is also contemplated that a polynucleotide or polypeptide of the present invention may inhibit chemotactic activity. Such molecules could also be used to treat a variety of disorders. Thus, a polynucleotide or polypeptide of the present invention could be used as an inhibitor of chemotaxis.
  • a polypeptide of the present invention may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds.
  • the binding of the polypeptide and the molecule may activate (i.e., an agonist), increase, inhibit (i.e., an antagonist), or decrease activity of the polypeptide or the molecule bound.
  • Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors), or small molecules.
  • the molecule is closely related to the natural ligand of the polypeptide, e.g., a fragment of the ligand, or a natural substrate, a ligand, a structural or functional mimetic (see, Coligan et al., Current Protocols in Immunology 1(2), Chapter 5 (1991)).
  • the molecule can be closely related to the natural receptor to which the polypeptide binds or, at least, a fragment of the receptor capable of being bound by the polypeptide (e.g., an active site). In either case, the molecule can be rationally designed using known techniques.
  • the screening for these molecules involves producing appropriate cells which express the polypeptide, either as a secreted protein or on the cell membrane.
  • Preferred cells include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the polypeptide or the molecule.
  • the assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide.
  • the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support, chemical libraries, or natural product mixtures.
  • the assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard.
  • an ELISA assay can measure polypeptide level or activity in a sample (e.g., biological sample) using a monoclonal or polyclonal antibody.
  • the antibody can measure polypeptide level or activity by either binding, directly or indirectly, to the polypeptide or by competing with the polypeptide for a substrate.
  • All of these above assays can be used as diagnostic or prognostic markers.
  • the molecules discovered using these assays can be used to treat disease or to bring about a particular result in a patient (e.g., blood vessel growth) by activating or inhibiting the polypeptide/molecule.
  • the assays can discover agents which may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues.
  • the invention includes a method of identifying compounds which bind to a polypeptide of the invention comprising the steps of: (a) incubating a candidate binding compound with a polypeptide of the invention; and (b) determining if binding has occurred.
  • the invention includes a method of identifying agonists/antagonists comprising the steps of: (a) incubating a candidate compound with a polypeptide of the invention, (b) assaying a biological activity, and (c) determining if a biological activity of the polypeptide has been altered.
  • a polypeptide or polynucleotide of the present invention may also increase or decrease the differentiation or proliferation of embryonic stem cells from a lineage other than the above described hemopoietic cells.
  • a polypeptide or polynucleotide of the present invention may also be used to modulate mammalian characteristics, such as body height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, size, and shape (e.g., cosmetic surgery).
  • a polypeptide or polynucleotide of the present invention may be used to modulate mammalian metabolism affecting catabolism, anabolism, processing, utilization, and storage of energy.
  • a polypeptide or polynucleotide of the present invention may be used to change a mammal's mental state or physical state by influencing biorhythms, circadian rhythms, depression (including depressive disorders), tendency for violence, tolerance for pain, the response to opiates and opioids, tolerance to opiates and opioids, withdrawal from opiates and opioids, reproductive capabilities (preferably by activin or inhibin-like activity), hormonal or endocrine levels, appetite, libido, memory, stress, or other cognitive qualities.
  • a polypeptide or polynucleotide of the present invention may also be used as a food additive or preservative, such as to increase or decrease storage capabilities, fat content, lipid, protein, carbohydrate, vitamins, minerals, cofactors or other nutritional components.
  • a polynucleotide of the invention is down-regulated and exacerbates a pathological condition, such as ataxia telangiectasia
  • the expression of the polynucleotide can be increased or the level of the intact polypeptide product can be increased in order to treat, prevent, ameliorate, or modulate the pathological condition. This can be accomplished by, for example, administering a polynucleotide or polypeptide of the invention to the mammalian subject.
  • a polynucleotide of the invention can be administered to a mammalian subject by a recombinant expression vector comprising the polynucleotide.
  • a mammalian subject can be a human, baboon, chimpanzee, macaque, cow, horse, sheep, pig, horse, dog, cat, rabbit, guinea pig, rat or mouse.
  • the recombinant vector comprises a polynucleotide shown in
  • the recombinant vector comprises a variant polynucleotide that is at least 80%, 90%, or 95% identical to a polynucleotide comprising SEQ ID NOs: 1-23.
  • a polynucleotide or recombinant expression vector of the invention can be used to express a polynucleotide in said subject for the treatment of, for example, cancer or ataxia telangiectasia.
  • Expression of a polynucleotide in target cells including but not limited to neurons, tumors, and muscle cells, would effect greater production of the encoded polypeptide.
  • a naked polynucleotide can be administered to target cells.
  • Polynucleotides and recombinant expression vectors of the invention can be administered as a pharmaceutical composition.
  • Such a composition comprises an effective amount of a polynucleotide or recombinant expression vector, and a pharmaceutically acceptable formulation agent selected for suitability with the mode of administration.
  • Suitable formulation materials preferably are non-toxic to recipients at the concentrations employed and can modify, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. See Remington 's Pharmaceutical Sciences (18 th Ed., A.R. Gennaro, ed., Mack Publishing Company 1990).
  • the pharmaceutically active compounds i.e., a polynucleotide or a vector
  • the pharmaceutical composition comprising a polynucleotide or a recombinant expression vector may be made up in a solid form (including granules, powders or suppositories) or in a liquid form (e.g., solutions, suspensions, or emulsions).
  • the dosage regimen for treating a disease with a composition comprising a polynucleotide or expression vector is based on a variety of factors, including the type or severity of the cancer or AT, the age, weight, sex, medical condition of the patient, the route of administration, and the particular compound employed. Thus, the dosage regimen may vary widely, but can be determined routinely using standard methods. A typical dosage may range from about 0.1 mg/kg to about 100 mg/kg or more, depending on the factors mentioned above.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the polynucleotide or vector in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
  • the cells of a mammalian subject may be transfected in vivo, ex vivo, or in vitro.
  • Administration of a polynucleotide or a recombinant vector containing a polynucleotide to a target cell in vivo may be accomplished using any of a variety of techniques well known to those skilled in the art.
  • U.S. Patent No. 5,672,344 describes an in vivo viral- mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector.
  • compositions of polynucleotides and recombinant vectors can be transfected in vivo by oral, buccal, parenteral, rectal, or topical administration as well as by inhalation spray.
  • parenteral as used herein includes, subcutaneous, intravenous, intramuscular, intrasternal, infusion techniques or intraperitoneally.
  • nucleic acids and/or vectors of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more vectors of the invention or other agents.
  • the therapeutic agents can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.
  • Another delivery system for polynucleotides of the invention is a "non- viral" delivery system.
  • Techniques that have been used or proposed for gene therapy include DNA-ligand complexes, adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO 4 precipitation, gene gun techniques, electroporation, lipofection, and colloidal dispersion (Mulligan, R., (1993) Science, 260 (5110):926-32). Any of these methods are widely available to one skilled in the art and would be suitable for use in the present invention. Other suitable methods are available to one skilled in the art, and it is to be understood that the present invention may be accomplished using any of the available methods of transfection. Several such methodologies have been utilized by those skilled in the art with varying success (Mulligan, R., (1993) Science, 260 (5110):926-32).
  • a polynucleotide of the invention is up-regulated and exacerbates a pathological condition in a mammalian subject, such as ataxia telangiectasia
  • the expression of the polynucleotide can be blocked or reduced or the level of the intact polypeptide product can be reduced in order to treat, prevent, ameliorate, or modulate the pathological condition.
  • This can be accomplished by, for example, the use of antisense oligonucleotides or ribozymes.
  • drugs or antibodies that bind to and inactivate the polypeptide product can be used.
  • Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence.
  • an antisense oligonucleotide is at least 1 1 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used.
  • Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of gene products of the invention in the cell.
  • Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.
  • Modifications of gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of a gene of the invention. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons.
  • An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent nucleotides, can provide sufficient targeting specificity for mRNA.
  • each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length.
  • Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length.
  • One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular polynucleotide sequence.
  • Antisense oligonucleotides can be modified without affecting their ability to hybridize to a polynucleotide of the invention. These modifications can be internal or at one or both ends of the antisense molecule.
  • intemucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Modified bases and/or sugars such as arabinose instead of ribose, or a 3', 5'-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide.
  • modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al., (1992) Trends Biotechnol, 10:152-158; Uhlmann et al., (1990) Chem. Rev., 90:543-584; Uhlmann et al., (1987) Tetrahedron. Lett, 215:3539-3542.
  • Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, (1987) Science, 236:1532-1539; Cech, (1990) Ann. Rev. Biochem., 59:543-568; Cech, (1992) Curr. Opin. Struct. Biol, 2:605-609; Couture & Stinchcomb, (1996) Trends Genet, 12:510-515. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al., U.S. Patent 5,641,673).
  • ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.
  • the coding sequence of a polynucleotide of the invention can be used to generate ribozymes which will specifically bind to mRNA transcribed from the polynucleotide.
  • Methods of designing and constructing ribozymes which can cleave RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. (1988) Nature, 334:585-591).
  • the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme.
  • the hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, e.g., Gerlach et al., EP 321 ,201).
  • Specific ribozyme cleavage sites within a RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NOs: 1-23 and their complements provide sources of suitable hybridization region sequences.
  • hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.
  • Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease polynucleotide expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art.
  • a ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.
  • ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.
  • Pathological conditions or susceptibility to pathological conditions such as ataxia telangiectasia, can be diagnosed using methods of the invention. Testing for expression of a polynucleotide of the invention or for the presence of the polynucleotide product can co ⁇ elate with the severity of the condition and can also indicate appropriate treatment.
  • the presence or absence of a mutation in a polynucleotide of the invention can be determined and a pathological condition or a susceptibility to a pathological condition is diagnosed based on the presence or absence of the mutation.
  • an alteration in expression of a polypeptide encoded by a polynucleotide of the invention can be detected, where the presence of an alteration in expression of the polypeptide is indicative of the pathological condition or susceptibility to the pathological condition.
  • the alteration in expression can be an increase in the amount of expression or a decrease in the amount of expression.
  • a first biological sample from a patient suspected of having a pathological condition is obtained along with a second sample from a suitable comparable control source.
  • a biological sample can comprise saliva, blood, cerebrospinal fluid, amniotic fluid, urine, feces, or tissue, such as gastrointestinal tissue.
  • a suitable control source can be obtained from one or more mammalian subjects that do not have the pathological condition.
  • the average concentrations and distribution of a polynucleotide or polypeptide of the invention can be determined from biological samples taken from a representative population of mammalian subjects, wherein the mammalian subjects are the same species as the subject from which the test sample was obtained.
  • the amount of at least one polypeptide encoded by a polynucleotide of the invention is determined in the first and second sample.
  • the amounts of the polypeptide in the first and second samples are compared.
  • a patient is diagnosed as having a pathological condition if the amount of the polypeptide in the first sample is greater than or less than the amount of the polypeptide in the second sample.
  • nucleic acid molecule comprising a nucleotide sequence which is at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% identical to a sequence of at least about 40 contiguous nucleotides in the nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of polynucleotides identified in SEQ ID NOs:l-23 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the clone sequence and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the start codon and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for polynucleotides identified in SEQ ID NOs: 1-23.
  • nucleic acid molecule wherein said sequence of contiguous nucleotides is included in the nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23 in the range of positions beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for polynucleotides identified in SEQ ID NOs: 1-23.
  • nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 150 contiguous nucleotides in the nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23.
  • nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least about 500 contiguous nucleotides in the nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23.
  • a further preferred embodiment is a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23 beginning with the nucleotide at about the position of the 5' nucleotide of the first amino acid of the signal peptide and ending with the nucleotide at about the position of the 3' nucleotide of the clone sequence as defined for polynucleotides identified in SEQ ID NOs: 1-23.
  • a further preferred embodiment is an isolated nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to the complete nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23. Also prefened is an isolated nucleic acid molecule which hybridizes under stringent hybridization conditions to a nucleic acid molecule, wherein said nucleic acid molecule which hybridizes does not hybridize under stringent hybridization conditions to a nucleic acid molecule having a nucleotide sequence consisting of only A residues or of only T residues.
  • a further prefened embodiment is a method for detecting in a biological sample a nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a sequence of at least 40 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23, which method comprises a step of comparing a nucleotide sequence of at least one nucleic acid molecule in said sample with a sequence selected from said group and determining whether the sequence of said nucleic acid molecule in said sample is at least 95% identical to said selected sequence.
  • step of comparing sequences comprises determining the extent of nucleic acid hybridization between nucleic acid molecules in said sample and a nucleic acid molecule comprising said sequence selected from said group.
  • step of comparing sequences is performed by comparing the nucleotide sequence determined from a nucleic acid molecule in said sample with said sequence selected from said group.
  • the nucleic acid molecules can comprise DNA molecules or RNA molecules.
  • a further prefened embodiment is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting nucleic acid molecules in said sample, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 40 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23.
  • Also prefened is a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene, which method comprises a step of detecting in a biological sample obtained from said subject nucleic acid molecules, if any, comprising a nucleotide sequence that is at least 95% identical to a sequence of at least 40 contiguous nucleotides in a sequence selected from the group consisting of a nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23.
  • the method for diagnosing a pathological condition can comprise a step of detecting nucleic acid molecules comprising a nucleotide sequence in a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 40 contiguous nucleotides in a sequence selected from said group.
  • composition of matter comprising isolated nucleic acid molecules wherein the nucleotide sequences of said nucleic acid molecules comprise a panel of at least two nucleotide sequences, wherein at least one sequence in said panel is at least 95% identical to a sequence of at least 40 contiguous nucleotides in a sequence selected from the group consisting of: a nucleotide sequence of polynucleotides identified in SEQ ID NOs: 1-23.
  • the nucleic acid molecules can comprise DNA molecules or RNA molecules.
  • an isolated polypeptide comprising an amino acid sequence at least 90% identical to a sequence of at least about 10 contiguous amino acids in an amino acid sequence translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • polypeptide wherein said sequence of contiguous amino acids is included in amino acids in an amino acid sequence translated from polynucleotides identified in SEQ ID NOs:l-23, in the range of positions beginning with the residue at about the position of the first amino acid of the secreted portion and ending with the residue at about the last amino acid of the open reading frame.
  • an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence of at least about 30 contiguous amino acids in an amino acid sequence translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • an isolated polypeptide comprising an amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least amino acid sequence at least amino acid sequence at least amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence at least one amino acid sequence
  • an isolated polypeptide comprising an amino acid sequence at least 95% identical to amino acids in an amino acid sequence translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • a method for detecting in a biological sample a polypeptide comprising an amino acid sequence which is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from polynucleotides identified in SEQ ID NOs: 1-23, which method comprises a step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group and determining whether the sequence of said polypeptide molecule in said sample is at least 90% identical to said sequence of at least 10 contiguous amino acids.
  • step of comparing an amino acid sequence of at least one polypeptide molecule in said sample with a sequence selected from said group comprises determining the extent of specific binding of polypeptides in said sample to an antibody which binds specifically to a polypeptide comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • step of comparing sequences is performed by comparing the amino acid sequence determined from a polypeptide molecule in said sample with said sequence selected from said group.
  • Also prefened is a method for identifying the species, tissue or cell type of a biological sample which method comprises a step of detecting polypeptide molecules in said sample, if any, comprising an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • a method for diagnosing in a subject a pathological condition associated with abnormal structure or expression of a gene comprises a step of detecting in a biological sample obtained from said subject polypeptide molecules comprising an amino acid sequence in a panel of at least two amino acid sequences, wherein at least one sequence in said panel is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • the step of detecting said polypeptide molecules includes using an antibody.
  • nucleic acid molecule comprising a nucleotide sequence which is at least 95% identical to a nucleotide sequence encoding a polypeptide wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to a sequence of at least 10 contiguous amino acids in a sequence selected from the group consisting of amino acid sequences translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • nucleic acid molecule wherein said nucleotide sequence encoding a polypeptide has been optimized for expression of said polypeptide in a prokaryotic host.
  • nucleic acid molecule encodes a polypeptide comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from polynucleotides identified in SEQ ID NOs: 1-23.
  • a method of making a recombinant vector comprising inserting any of the above isolated nucleic acid molecule into a vector. Also prefened is the recombinant vector produced by this method. Also prefened is a method of making a recombinant host cell comprising introducing the vector into a host cell, as well as the recombinant host cell produced by this method.
  • Also preferred is a method of making an isolated polypeptide comprising culturing this recombinant host cell under conditions such that said polypeptide is expressed and recovering said polypeptide. Also preferred is this method of making an isolated polypeptide, wherein said recombinant host cell is a eukaryotic cell and said polypeptide is a secreted portion of a human secreted protein comprising an amino acid sequence selected from the group consisting of amino acid sequences translated from polynucleotides identified in SEQ ID NOs: 1-23. The isolated polypeptide produced by this method is also preferred.
  • a method of treatment of an individual in need of an increased level of a secreted protein activity comprises administering to such an individual a pharmaceutical composition comprising an amount of an isolated polypeptide, polynucleotide, or antibody of the claimed invention effective to increase the level of said protein activity in said individual.
  • the present invention also includes a diagnostic system, preferably in kit form, for assaying for the presence of the polypeptide of the present invention in a body sample, such as brain tissue, cell suspensions or tissue sections, or body fluid samples such as CSF, blood, plasma or serum, where it is desirable to detect the presence, and preferably the amount, of the polypeptide of this invention in the sample according to the diagnostic methods described herein.
  • a nucleic acid molecule can be used as a probe (i.e., an oligonucleotide) to detect the presence of a polynucleotide of the present invention, a gene conesponding to a polynucleotide of the present invention, or a mRNA in a cell that is diagnostic for the presence or expression of a polypeptide of the present invention in the cell.
  • the nucleic acid molecule probes can be of a variety of lengths from at least about 10 contiguous bases, suitably about 10 to about 5000 nucleotides long, although they will typically be about 20 to 500 nucleotides in length. Hybridization methods are extremely well known in the art and will not be described further here.
  • PCR primers are utilized in pairs, as is well known, based on the nucleotide sequence of the gene to be detected.
  • the nucleotide sequence is a portion of the nucleotide sequence of a polynucleotide of the present invention.
  • Particularly prefened PCR primers can be derived from any portion of a DNA sequence encoding a polypeptide of the present invention, but are preferentially from regions which are not conserved in other cellular proteins.
  • PCR primer pairs useful for detecting the genes conesponding to the polynucleotides of the present invention and expression of these genes are described in the Examples, including the conesponding Tables. Nucleotide primers from the conesponding region of the polypeptides of the present invention described herein are readily prepared and used as PCR primers for detection of the presence or expression of the conesponding gene in any of a variety of tissues.
  • the diagnostic system includes, in an amount sufficient to perform at least one assay, a subject polypeptide of the present invention, a subject antibody or monoclonal antibody, and/or a subject nucleic acid molecule probe of the present invention, as a separately packaged reagent.
  • a diagnostic system preferably in kit form, is contemplated for assaying for the presence of the polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention in a body fluid sample.
  • Such diagnostic kit would be useful for monitoring the fate of therapeutically administered polypeptide of the present invention or an antibody immunoreactive with the polypeptide of the present invention.
  • the system includes, in an amount sufficient for at least one assay, a polypeptide of the present invention and/or a subject antibody as a separately packaged immunochemical reagent.
  • a package refers to a solid matrix or material such as glass, plastic (e.g., polyethylene, polypropylene or polycarbonate), paper, foil and the like capable of holding within fixed limits a polypeptide, polyclonal antibody or monoclonal antibody of the present invention.
  • a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide or antibody or it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide or antibody have been operatively affixed, (i.e., linked) so as to be capable of being immunologically bound by an antibody or antigen, respectively.
  • Instructions for use typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/ sample admixtures, temperature, buffer conditions and the like.
  • a diagnostic system of the present invention preferably also includes a label or indicating means capable of signaling the formation of an immunocomplex containing a polypeptide or antibody molecule of the present invention.
  • the word "complex” as used herein refers to the product of a specific binding reaction such as an antibody-antigen or receptor-ligand reaction. Exemplary complexes are immunoreaction products.
  • label and "indicating means” in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in an expressed protein, polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel proteins methods and/or systems.
  • the labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer.
  • Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like.
  • fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), 5-dimethylamine-l- naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate
  • the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, or the like.
  • HRP horseradish peroxidase
  • glucose oxidase or the like.
  • additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed.
  • additional reagents for HRP include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine.
  • An additional reagent useful with glucose oxidase is 2,2'-amino-di-(3-ethyl- benzthiazoline-G-sulfonic acid) (ABTS).
  • Radioactive elements are also useful labeling agents and are used illustratively herein.
  • An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions.
  • Elements which themselves emit gamma rays represent one class of gamma ray emission-producing radioactive element indicating groups. Particularly prefened is 125 I.
  • Another group of useful labeling means are those elements such as ' 'C, 18 F, 15 O and 13 N which themselves emit positrons. The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body.
  • a beta emitter such 111 indium or 3 H.
  • antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component in the culture medium. See, e.g., Galfre et al., Meth. Enzymol, 73:3-46 (1981).
  • the techniques of protein conjugation or coupling through activated functional groups are particularly applicable. See, e.g., Aurameas, et al., Scand. J. Immunol, Vol. 8 Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Pat. No. 4,493,795.
  • the diagnostic systems can also include, preferably as a separate package, a specific binding agent.
  • a "specific binding agent” is a molecular entity capable of selectively binding a reagent species of the present invention or a complex containing such a species, but is not itself a polypeptide or antibody molecule composition of the present invention.
  • Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A, and the like.
  • the specific binding agent binds the reagent species when that species is present as part of a complex.
  • the specific binding agent is labeled.
  • the agent is typically used as an amplifying means or reagent.
  • the labeled specific binding agent is capable of specifically binding the amplifying means when the amplifying means is bound to a reagent species-containing complex.
  • the diagnostic kits of the present invention can be used in an "ELISA" format to detect the quantity of the polypeptide of the present invention in a sample.
  • ELISA refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen present in a sample.
  • a description of the ELISA technique is found in Sites et al., Basic and Clinical Immunology, 4th Ed. Chap. 22, Lange Medical Publications, Los Altos, CA (1982) and in U.S. Patent No. 3,654,090; U.S. Patent No. 3,850,752; and U.S. Patent No. 4,016,043, which are all inco ⁇ orated herein by reference.
  • a polypeptide of the present invention an antibody or a monoclonal antibody of the present invention can be affixed to a solid matrix to form a solid support that comprises a package in the subject diagnostic systems.
  • a reagent is typically affixed to a solid matrix by adso ⁇ tion from an aqueous medium, although other modes of affixation applicable to proteins and polypeptides can be used that are well known to those skilled in the art. Exemplary adso ⁇ tion methods are described herein.
  • Useful solid matrices are also well known in the art. Such materials are water insoluble and include the cross-linked dextran available under the trademark SEP HAD EX from Pharmacia Fine Chemicals (Piscataway, NJ), agarose, polystyrene beads of about 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers, (e.g., Abbott Laboratories, Chicago, IL), polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs (sheets, strips or paddles) or tubes, plates or the wells of a microtiter plate, such as those made from polystyrene or polyvinylchloride.
  • SEP HAD EX Pharmacia Fine Chemicals
  • agarose agarose
  • polystyrene beads of about 1 micron ( ⁇ m) to about 5 millimeters (mm) in diameter available from several suppliers, (e.g., Abbott Laboratories, Chicago,
  • the reagent species, labeled specific binding agent, or amplifying reagent of any diagnostic system described herein can be provided in solution, as a liquid dispersion or as a substantially dry power, e.g., in lyophilized form.
  • the indicating means is an enzyme
  • the enzyme's substrate can also be provided in a separate package of a system.
  • a solid support such as the before-described microtiter plate and one or more buffers can also be included as separately packaged elements in this diagnostic assay system.
  • packaging materials discussed herein in relation to diagnostic systems are those customarily utilized in diagnostic systems.
  • SKY Spectral Karvotvping
  • FISH Fluorescent In situ Hybridization
  • Atm deficient tumor cell lines Ten different Atm deficient tumor cell lines were established.
  • the Atm deficient tumor cell lines have been analyzed with a variety of methods and shown to maintain their clonality in culture even with continuous passage. These cell lines were used as a source of mRNA and were characterized further.
  • the genetic alterations in these cell lines were characterized using spectral karyotyping (SKY) and fluorescent in situ hybridization (FISH).
  • SKY spectral karyotyping
  • FISH fluorescent in situ hybridization
  • Consistent abnormalities of chromosome 14 and chromosome 12 similar to those found in human leukemias and lymphomas, were observed in all tumors studied. Examples of such abnormalities are shown in Table 1.
  • the observed abnormalities included insertions (ins6:14) as well as translocations (tl2;10, tl4;15 and tl4;3).
  • ATM function is therefore critical for maintaining appropriate recombination pathways and in the absence of ATM, both humans and mice develop aggressive tumors.
  • a similar pathway of tumorigenesis is also likely to occur in the absence of ATM in both human and mouse. Further studies have involved the transformation of the normal T cell to a malignant cell as described below.
  • Cytoplasmic RNA was prepared from cells from four tumor cell lines: AT-4, AT-7, AT- 12 and AT-13 that had the chromosomal abnormalities summarized in Table 1.
  • the isolated RNA is enriched to form a starting polyA-containing mRNA population by methods known in the art.
  • the TOGA method further comprises an additional Polymerase Chain Reaction ("PCR") step performed using four 5' PCR primers in four separate reactions and cDNA templates prepared from a population of antisense cRNAs.
  • PCR Polymerase Chain Reaction
  • a final PCR step that used 256 5' PCR primers in separate reactions produced PCR products that were cDNA fragments that conesponded to a 3'-region of the starting mRNA population.
  • the produced PCR products were then identified by a) the initial 5' sequence comprising the sequence remainder of the recognition site of the restriction endonuclease used to cut and isolate the 3 ' region plus the sequence of the preferably four parsing bases immediately 3' to the remainder of the recognition site, preferably the sequence of the entire fragment, and b) the length of the fragment. These two parameters, sequence and fragment length, were used to compare the obtained PCR products to a database of known polynucleotide sequences. Since the length of the obtained PCR products includes known vector sequences at the 5' and 3' ends of the insert, the sequence of the insert provided in the sequence listing is shorter than the fragment length that forms part of the digital address.
  • the method yields Digital Sequence Tags (DSTs), that is, polynucleotides that are expressed sequence tags (ESTs) of the 3' end of mRNAs. DSTs that showed changes in relative levels when comparing 1 of 4 samples, or showed paired differences in 2 of 4 samples or were highly expressed and located on mouse chromosomes involved in the translocations were selected for further study. The intensities of the laser-induced fluorescence of the labeled PCR products were compared across samples isolated from the four tumor cell lines AT-4, AT-7, AT-12 and AT-13. The results are presented in Tables 2 and 3.
  • double-stranded cDNA is generated from poly(A)-enriched cytoplasmic RNA extracted from the tissue samples of interest using an equimolar mixture of all 48 5'- biotinylated anchor primers of a set to initiate reverse transcription.
  • One such suitable set is G- A-A-T-T-C-A-A-C-T-G-G-A-A-G-C-G-G-C-C-C-G-C-A-G-G-A-A-T-T-T-T-T-T-T-T-T-T-T-T-T-T-T-V-N-N (SEQ ID NO:24), where V is A, C or G and N is A, C, G or T.
  • One member of this mixture of 48 anchor primers initiates synthesis at a fixed position at the 3' end of all copies of each mRNA species in the sample, thereby defining a 3' endpoint for each species, resulting in biotinylated double stranded cDNA.
  • Each biotinylated double stranded cDNA sample was cleaved with the restriction endonuclease Mspl, which recognizes the sequence CCGG.
  • the 3' fragments of cDNA were then isolated by capture of the biotinylated cDNA fragments on a streptavidin-coated substrate.
  • Suitable streptavidin-coated substrates include microtitre plates, PCR tubes, polystyrene beads, paramagnetic polymer beads and paramagnetic porous glass particles.
  • a prefened streptavidin- coated substrate is a suspension of paramagnetic polymer beads (Dynal, Inc., Lake Success, NY).
  • the cDNA fragment product was released by digestion with Notl, which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • Notl which cleaves at an 8-nucleotide sequence within the anchor primers but rarely within the mRNA-derived portion of the cDNAs.
  • the 3' Mspl-Notl fragments which are of uniform length for each mRNA species, were directionally ligated into CJal-, Notl-cleaved plasmid pBC SK + (Stratagene, La Jolla, CA) in an antisense orientation with respect to the vector's T3 promoter, and the product used to transform Escherichia coli SURE cells (Stratagene).
  • the ligation regenerates the Notl site, but not the Mspl site leaving CGG as the first three bases of the 5' end of all PCR products obtained.
  • Each library contained in excess of 5 x 10 5 recombinants to ensure a high likelihood that the 3' ends of all mRNAs with concentrations of 0.001 % or greater were multiply represented. Plasmid preps (Qiagen) were made from the cDNA library of each sample under study.
  • each library was digested with Mspl, which effects linearization by cleavage at several sites within the parent vector while leaving the 3' cDNA inserts and their flanking sequences, including the T3 promoter, intact.
  • the product was incubated with T3 RNA polymerase (MEGAscript kit, Ambion) to generate antisense cRNA transcripts of the cloned inserts containing known vector sequences abutting the Mspl and Notl sites from the original cDNAs.
  • T3 RNA polymerase MEGAscript kit, Ambion
  • each of the cRNA preparations was processed in a three-step fashion.
  • 250 ng of cRNA was converted to first-strand cDNA using the 5' RT primer (A-G-G- T-C-G-A-C-G-G-T-A-T-C-G-G (SEQ ID NO:25).
  • step two 400 pg of cDNA product was used as PCR template in four separate reactions with each of the four 5' PCR primers of the form G-G-T-C-G-A-C-G-G-T-A-T-C-G-G-N (SEQ ID NO:26), each paired with a "universal" 3' PCR primer G-A-G-C-T-C-C-A-C-C-G-C-G-G-T (SEQ ID NO:27) to yield four sets of PCR reaction products ("Nl reaction products").
  • step three the product of each subpool was further divided into 64 subsubpools (2ng in 20 ⁇ l) for the second PCR reaction.
  • This PCR reaction comprised adding 100 ng of the fluoresceinated "universal" 3' PCR primer (SEQ ID NO: 27) conjugated to 6-FAM and 100 ng of the appropriate 5' PCR primer of the form C-G-A-C-G-G-T-A-T-C-G-G-N-N-N-N (SEQ ID NO: 28), and using a program that included an annealing step at a temperature X slightly above the T m of each 5' PCR primer to minimize artifactual mispriming and promote high fidelity copying.
  • Each polymerase chain reaction step was performed in the presence of TaqStart antibody (Clonetech).
  • the products (“N4 reaction products”) from the final polymerase chain reaction step for each of the tissue samples were resolved on a series of denaturing DNA sequencing gels using the automated ABI Prizm 377 sequencer. Data were collected using the GeneScan software package (ABI) and normalized for amplitude and migration.
  • Complete execution of this series of reactions generated 64 product subpools for each of the four pools established by the 5' PCR primers of the first PCR reaction, for a total of 256 product subpools for the entire 5' PCR primer set of the second PCR reaction.
  • Table 2 is a summary of the expression levels of 1136 mRNAs determined from cDNA.
  • cDNA molecules are identified by their digital address, that is, a partial 5' terminus nucleotide sequence coupled with the length of the molecule, as well as the relative amount of the molecule produced at different time intervals after treatment.
  • the 5' terminus partial nucleotide sequence is determined by the recognition site for Mspl and the nucleotide sequence of the parsing bases of the 5' PCR primer used in the final PCR step.
  • the digital length of the fragment was determined by inte ⁇ olation on a standard curve, and as such, may vary plus or minus 1 or 2 base pairs from the actual length as determined by sequencing.
  • the entry in Table 2 that describes a DNA molecule identified by the digital address Mspl GAGC 197 is further characterized as having a 5' terminus partial nucleotide sequence of CGGGAGC and a digital address length of 197 b.p.
  • the DNA molecule identified as Mspl GAGC 197 was further characterized as being expressed at the lowest level in cell line AT-12, at higher levels in cell lines AT-7 and AT-4 and at the highest level in the cell line AT-13.
  • Mspl GAGC 197 shown in Table 2 and Figure 1 were generated with a 5'-PCR primer (C-G-A-C-G-G-T-A-T-C-G-G-G-A-G-C, SEQ ID NO:29) paired with the "universal" 3' primer (SEQ ID NO: 27) labeled with 6-carboxyfluorescein (6FAM, ABI) at the 5' terminus.
  • PCR reaction products were resolved by gel electrophoresis on 4.5% acrylamide gels and fluorescence data acquired on ABI377 automated sequencers. Data were analyzed using GeneScan software (Perkin-Elmer).
  • the other DNA molecules identified in Table 2 by their Mspl digital addresses are further characterized by the pattern of the level of gene expression in the different cell lines. Further examples of TOGA analysis are shown in Figures 3, 5, 7, and 9. Some PCR products, which were also differentially represented, appeared to migrate in positions that suggest that the products were novel based on comparison to data extracted from GenBank. Sequence alignments are shown for selected DSTs in Table 3. Table 3 summarizes the alignment information from a BLAST report generated upon comparing the cloned sequence to those in the GenBank/EST databases. In these cases, the PCR product is isolated, cloned into a TOPO vector (Invitrogen) and sequenced on both strands.
  • TOPO vector Invitrogen
  • PCR primers are designed based on the determined sequence and PCR is performed using the cDNA produced in the first PCR reaction as substrate. Oligonucleotides were synthesized conesponding to the 5' PCR primer in the second PCR step extended at the 3' end with an additional 14 nucleotides from the cloned sequence.
  • the primer sequences are shown in Table 4.
  • TOGA analysis figures 1, 3, 5, and 7 are based on data from a single library. These experiments were replicated with a second library from the same source. Any differences between the length of the DST in the digital addresses in the single library versus the duplicate libraries are no more than two base pairs.
  • the digital addresses reported in Table 2, the information in Tables 3 and 4, and Figures 9 and 10 are the product of an analysis that uses data obtained from both libraries.
  • Figure 3 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases GCTG, showing PCR products produced from mRNA extracted from (top to bottom panels) cell lines AT-4 (Fig. 3 A), AT-7 (Fig. 3B), AT-12 (Fig. 3C), and AT-13 (Fig. 3D); the anow indicates the vertical cursor connecting the position of peaks of length 345 base pairs.
  • Figure 5 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases ACAT, showing PCR products produced from mRNA extracted from (top to bottom panels) cell lines AT-4 (Fig. 5A), AT-7 (Fig. 5B), AT-12 (Fig. 5C), and AT-13 (Fig. 5D); the anow indicates the vertical cursor connecting the position of peaks of length 367 base pairs.
  • Figure 7 shows the results of another example of TOGA analysis, in this case using a 5' PCR primer with parsing bases ACGG, showing PCR products produced from mRNA extracted from (top to bottom panels) cell lines AT-4 (Fig. 7 A), AT-7 (Fig. 7B), AT-12 (Fig. 7C), and AT-13 (Fig. 7D); the anow indicates the vertical cursor connecting the position of peaks of length 458 base pairs.
  • FigurelO illustrates the verification process, in which the cDNA produced in the first PCR reaction was used as template, and the PCR product obtained using an extended PCR primer designed based on the determined sequence was compared to products obtained using the original PCR primers. Oligonucleotides were synthesized conesponding to the 5' PCR primer in the second PCR step for each candidate extended at the 3' end with an additional 14 nucleotides from the cloned sequence. This clone specific 5' PCR primer (SEQ ID NO:32) was paired with a fluorescent labeled universal 3' PCR primer (SEQ ID NO:27) in PCR reactions using the cDNA produced in the first PCR reaction or substrate. Primers designed for such studies of cloned DSTs are shown in Table 4.
  • Figure 9 is a graphical representation of the results of TOGA analysis using a 5' PCR primer with parsing bases CCGT showing PCR products produced from mRNA extracted from cell lines AT-4 (Fig. 9A), AT-7 (Fig. 9B), AT-12 (Fig. 9C), and AT-13 (Fig. 9D); the anow indicates the vertical cursor connecting the position of peaks of length 151 base pairs.
  • the length of the PCR fragment amplified with an extended primer based on the cloned sequence was compared to the length of the original PCR product as shown in Figure 10.
  • the upper panel (A) shows the extended primer reaction product created with a 5' PCR primer (SEQ ID NO:32) and the 3' PCR primer (SEQ ID NO:27).
  • Figure 10B shows the PCR products produced using the original PCR primers.
  • Figure IOC the traces from the top and middle panels are overlaid, demonstrating that the PCR product produced using an extended primer based on the cloned sequence is the same length as the original PCR product.
  • BAR1 20 maps to chromosome 13
  • BAR1_24 maps to chromosome 7
  • BAR1 27 maps to chromosome 14
  • 20.5 cM maps to chromosome 14
  • BAR1 28 maps to chromosome 15; 43.3 cM.
  • RNA and multiple northern blots have been prepared from various cell lines, including approximately 20 different tumor cell lines with mutations in Atm, p53 or combinations of Atm and p53. These cell lines have been characterized using flow cytometry and the majority are T-cell lymphomas. These tumors are being characterized or have been characterized using SKY and FISH.
  • Genes that are abnormally regulated among AT cell lines may be uniquely associated with AT tumorigenesis (as compared to other mechanisms of tumorigenesis) due to genetic abnormalities caused by AT deficiency. These may include phenomena such as translocations of lymphoid specific promoters or other regulatory sequences in or near genes promoting cell growth, or inactivation or deletion of genes that may regulate or suppress lymphoid tumor growth. Thus, the examples shown here were also chosen in part on the basis of their potential location in or near chromosomal abenations among the AT cell lines studied. The genes identified in these examples may relate to specific biological aspects found both in the mouse model and in human AT tumorigenesis (e.g., the development of particularly aggressive forms of lymphomas).
  • Poly A enriched mRNA was extracted from four tumor cell lines, AT-4, AT-7, AT-12, and AT-13, as described above.
  • the mRNA samples were electrophoresed through an agarose gel, blotted, and probed using well-known methods. Briefly, 20 ⁇ g of total RNA or 2 ⁇ g of poly A+ mRNA was electrophoresed through a 1.2% agarose gel containing formamide along with the appropriate molecular weight standards. The gel was blotted overnight using nylon membrane to transfer the RNA. The membrane was prehybridized for one hour at 42°C in hybridization buffer (5X SSPE, 5X Denhardt's solution, 50% formamide, 0.2% SDS, 100 ⁇ g/ml salmon sperm DNA, and water).
  • hybridization buffer 5X SSPE, 5X Denhardt's solution, 50% formamide, 0.2% SDS, 100 ⁇ g/ml salmon sperm DNA, and water.
  • the probe DNA (50ng) was labeled with 32 [P]-dCTP and 32 [P]-dATP using asymmetric PCR labeling.
  • the membrane was probed with radiolabeled DNA (2-5 x 10 6 cpm/ ml) overnight at 42°C in hybridization buffer.
  • the northern blots were probed with radiolabeled cyclophylin DNA to normalize the amount of mRNA in each sample. Band intensities of the probed mRNA samples were quantitated using a Phosphoimager SI and normalized to the hybridization signal of cyclophilin.
  • Figure 2 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (GenBank Accession Number U88908) mapping to mouse Chromosome 9, FISH mapped to band A2 (cytogenic) (see Liston, P., (1997) Genomics, 46(3):495-503) conesponding to GAGC 197, showing, in the image in the upper panel, hybridization with a band of mRNA at 4.2 kb that is expressed at various levels in cell lines AT-4, AT-7, AT-10, AT-11, AT-13, APT-3, P53-1, 101-7, 292-3 and thymus, but not detectable in cell line AT 12; the image in the lower panel shows the methylene blue stained gel for quantification of mRNA loading.
  • Figure 4 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (GenBank Accession Number Z31664) human homolog (ACVRL1- GenBank accession Z22533) maps to 12ql l-ql4, this region is syntenic to mouse chromosome 15 between 56.8 cM-64.0 cM, conesponding to GCTG 345, showing, in the image in the upper panel, hybridization with bands of mRNA at 4.0 kb and 3.7 kb that are expressed at various levels in cell lines AT-4, AT-11, AT-12, AT-13, 292-3, and poly A + (pA) thymus mRNA, but not detectable in cell lines AT-7, AT-10, APT-3, P53-1, 101- 7; the image in the lower panel shows the methylene blue stained gel for quantification of mRNA loading.
  • a candidate gene GeneBank Accession Number Z31664
  • human homolog ACVRL1- GenBank accession Z22533
  • Figure 6 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (GenBank Accession Number U38252) mapping to mouse Chromosome 5, 65.0 cM conesponding to ACAT 367, showing, in the image in the upper panel, hybridization with a band of mRNA at 4.1 kb that is expressed at various levels in cell lines AT-7, AT-10, AT-12, AT-13 and APT-3, but not detectable in cell lines AT-4, AT-11, P53-1, 101-7, 292-3 and polyA (pA) thymus mRNA; the image in the lower panel shows the methylene blue stained gel for quantification of mRNA loading.
  • Figure 8 is a graphical representation of the results of Northern Blot analysis using radiolabeled probe derived from a candidate gene (GenBank Accession Number XI 2822) mapping to mouse Chromosome 14, 20.5 cM conesponding to ACGG 458, showing, in the image in the upper panel, hybridization with bands of mRNA at 1.7 kb and 1.1 kb that are expressed at various levels in cell lines AT-7 and AT-10, but not detectable in cell lines AT-4, AT-12, AT-11, AT-13; APT-3, P53-1, 101-7, and 292-3, and polyA (pA) thymus (only 1.1 kb) mRNA; the image in the lower panel shows the methylene blue stained gel for quantification of mRNA loading.
  • a candidate gene GeneBank Accession Number XI 2822
  • FISH FISH is used to map the chromosomal location of the gene.
  • Candidate genes that fall within the region that is reananged in the particular cell line are identified as a gene whose change in expression is essential for tumorigenesis.

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Abstract

L'invention concerne des poynucléotides, des polypeptides, des trousses et des procédés se rapportant à des gènes régulés caracteristiques de la tumorgénèse de l'ataxie-télangiectasie.
PCT/US2000/022889 1999-08-19 2000-08-18 Expression genique modulee en tumorigenese de l'ataxie-telangiectasie WO2001012647A1 (fr)

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* Cited by examiner, † Cited by third party
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Patent Citations (1)

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US4874845A (en) * 1984-06-13 1989-10-17 Massachusetts Institute Of Technology T lymphocyte receptor subunit

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