JP2001527397A - Novel polypeptides of the growth factor superfamily - Google Patents

Abstract

  (57) [Summary] The present invention relates to Tango-67 polypeptides, nucleic acid molecules encoding Tango-67, and uses thereof. Tango-67 is associated with a number of growth factors, particularly members of the connective tissue growth factor family.

Description

DETAILED DESCRIPTION OF THE INVENTION               Novel polypeptides of the growth factor superfamily Background of the Invention   The size and differentiation characteristics of the cell compartment are partly due to extracellular growth factors. Is controlled by utilizing These growth factors are responsible for cell replication and cell production. , As well as the function of differentiated cells. Generation of special cell types The ability to control length in vivo has demonstrated clinical utility, Good examples are erythropoietin and granulocyte colony stimulating factor Stimulation of blood cell and leukocyte production. Cures other human diseases (eg, neurodegeneration) The availability of growth factors for treatment is under investigation, and in some cases, exogenous growth To stop or reverse the progress of degenerative disease by intensifying Or provide more rapid functional recovery in case of acute tissue damage (Eg, wound healing) is desired.   Secreted growth factors also play an important role in early development. Details of this process are complete Are not understood and vary considerably from species to species, but Analysis has revealed an important role for growth factors in early embryo differentiation. This One such molecule, twisted gastrulation, is a mutation that causes a defect in embryogenesis.  gastrulation) gene (TSG). TSG messenger RNA (mRNA) Present in early embryos and important in specializing the fate of cells in the backline. TSG is a molecule Is a secreted protein that is rich in cysteine, Shows homology to growth factor (CTGF). CTGF is the fibroblast mitogen itself Yes, they share a common antigenic determinant with platelet-derived growth factor (PDGF).                                Summary of the Invention   The present invention relates to the discovery and characterization of a novel soluble growth factor, Tango-67. The form of Tango-67 described herein is 224 amino acids and has a cysteine. Abundant polypeptide. Northern blot analysis of Tango-67 mRNA was performed using Tango-6 7 has been shown to be present at various concentrations in a wide variety of tissues.   The invention features an isolated nucleic acid molecule that encodes a Tango-67 polypeptide. For example, the isolated nucleic acid molecule is secreted human Tango-67, ie, the sequence of SEQ ID NO: 2. Small It encodes a polypeptide having a sequence that is at least 85% identical. In other aspects Claim 1 is a nucleotide encoding the amino acid sequence of SEQ ID NO: 2. 182 to 850 nucleotides of SEQ ID NO: 1 including.   In another embodiment, the isolated nucleic acid molecule encoding Tango-67 is SEQ ID NO: 1 or the like. With a nucleic acid molecule having the sequence of nucleotides 182 to 850 of the complementary strand of To In other embodiments, the hybridization is under stringent conditions. Occurs.   In other embodiments, the isolated nucleic acid molecule encoding Tango-67 is capable of cell growth and / or Encodes a Tango-67 polypeptide that can promote differentiation.   The present invention also provides an isolated nucleic acid molecule encoding Tango-67, a nucleic acid vector (eg, Expression vector), vector containing regulatory elements; cytomegalovirus hCMV immediate early Gene, SV40 adenovirus early promoter, SV40 adenovirus late promoter motor,lacsystem,trpsystem,TACsystem,TRCSystem, lambda phage Major operator and promoter regions, fd coat protein control region, 3-phosphoglycerate kinase promoter, acid phosphatase promoter And a regulator selected from the group consisting of yeast α-mating factor promoters Vector containing an offspring; vector containing a regulatory element directing tissue-specific expression, reporter Vector containing gene; β-lactamase, chloramphenicol acetyl tiger Shunt erase (CAT), adenosine deaminase (ADA), aminoglycoside Suhotransferase (neo^r, G413^r), Dihydrofolate reductase (DHFR) , Hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK ), LacZ (encoding β-galactosidase) and xanthine guanine phos Reporter selected from the group consisting of horibosyltransferase (XGPRT) A vector containing a gene; a vector that is a plasmid, a vector that is a virus, It features a host cell comprising a vector that is a retrovirus.   In another aspect, the invention provides a substantially pure Tango-67 polypeptide, e. Tango-67 polypeptide soluble under physiologic conditions, Ta containing signal sequence ngo-67 polypeptide, Tango-67 polypeptide that stimulated cell growth and / or differentiation A peptide which is at least 85% identical to the amino acid sequence of SEQ ID NO: 2. A Tango-67 polypeptide, comprising at least 90% identical to the amino acid sequence of SEQ ID NO: 2 A Tango-67 polypeptide comprising an amino acid sequence; Tango-67 polypeptide comprising an amino acid sequence that is at least 95% identical, SEQ ID NO: : Features a Tango-67 polypeptide comprising an amino acid sequence identical to the amino acid sequence of 2 Sign.   In another aspect, the invention provides a method comprising: a first portion comprising a Tango-67 polypeptide; A substantially pure polypeptide, comprising a second portion comprising a functional marker. I do.   The present invention also provides antibodies (eg, monoclonals) that selectively bind to a Tango-67 polypeptide. Clonal antibody).   The invention also features a pharmaceutical composition that includes a Tango-67 polypeptide.   A method for detecting Tango-67 in a sample, comprising:   (A) obtaining a biological sample;   (B) Under stringent conditions to form a Tango-67-antibody complex, Contacting a biological sample with an antibody that specifically binds to Tango-67;   (C) When a complex is present, the complex is used as an indicator of the presence of Tango-67 in the sample. The step of detecting is also included in the present invention.   In another aspect, the present invention provides a method for identifying a compound that modulates Tango-67 expression. A method comprising: removing Tango-67 in cells in the presence and absence of a selected compound. Expression in the presence and absence of the selected compound, including the step of comparing the degree of expression A method wherein the degree of difference indicates that the selected compound modulates Tango-67 expression And   In another aspect, the present invention provides a method for identifying a compound that modulates the activity of Tango-67. A method comprising: removing Tango-67 in cells in the presence and absence of a selected compound. Comparing the degree of activity, in the presence and absence of the selected compound. Differences in the degree of activity indicate that the selected compound modulates Tango-67 activity Features the law.   In another aspect, the invention relates to a disease associated with overexpression or activity of Tango-67. A method for treating a patient afflicted with a disease, comprising inhibiting the expression or activity of Tango-67. A method comprising administering to a patient an offending compound.   The present invention also affected a disease associated with insufficient expression or activity of Tango-67. A method for treating a patient, comprising a compound that increases Tango-67 expression or activity. A method comprising administering an article to a patient.   The present invention also provides a method for diagnosing a disease associated with abnormal expression of Tango-67. And obtaining a biological sample from the patient, and determining the presence of Tango-67 in the biological sample. Measuring the expression, and expressing the Tango-67 in the biological sample compared to a control. Increase or decrease indicates that the patient has a disease associated with abnormal expression of Tango-67. The method is characterized by showing that   In another aspect, the invention diagnoses a disease associated with abnormal activity of Tango-67 Obtaining a biological sample from a patient; Measuring the activity of Tango-67 in a biological sample compared to a control. Diseases in which increased or decreased ngo-67 activity is associated with abnormal Tango-67 activity Characterized by a method of indicating that the subject is suffering from.   The present invention relates to isolated nucleic acid molecules encoding Tango-67 or fragments thereof, Tango, a vector containing a molecule, a cell carrying a recombinant DNA encoding Tango-67 Tango, a transgenic animal expressing Tango-67, a fusion protein containing -67 Includes recombinant knockout animals that do not express -67. Poly shown in Figure (SEQ ID NO: 2) Nucleic acid molecules encoding the peptide are particularly preferred.   The invention includes nucleic acids having a sequence that is substantially identical to a Tango-67 nucleic acid sequence. A nucleic acid that is substantially identical to a given reference nucleic acid molecule is referred to herein as a given group. At least 85%, preferably at least 85%, of the sequence of the subnucleic acid molecule, eg, the nucleic acid sequence of SEQ ID NO: 1 90%, more preferably 95%, 98%, 99% or more sequences having identity Is defined as a nucleic acid having   A polypeptide that is “substantially identical” to a given reference polypeptide molecule is a subject polypeptide. In the specification, the sequence of the given reference polypeptide sequence, for example, the polypeptide of SEQ ID NO: 2, At least 85% with the peptide sequence, preferably 90%, more preferably 95%, 98%, It is defined as a polypeptide having a sequence with 99% or more identity.   The nucleic acid molecules of the invention may be transcribed and / or stimulated to promote expression of the insert as follows. Or into a translation vector. Nucleic acid molecules and they encode The polypeptide can be used directly as a diagnostic or therapeutic agent, or ( Used (in the case of polypeptides) to produce antibodies that are therapeutically useful. Can be. Thus, expression vectors having the nucleic acid molecules of the invention, these vectors Transfected cells, expressed polypeptide and full-length polypeptide Antibodies formed against the tide or antigenic fragments thereof are included in a preferred embodiment. It is.   Transfected cells can be purified by recombinant DNA techniques using the polypeptides of the invention. Cells (eg, Tango-67 polypeptide) into which any nucleic acid has been introduced (eg, Or its ancestors). An isolated nucleic acid molecule is located 5 'immediately adjacent to the organism's natural genome. A nucleic acid molecule that is separated from the flanking and 3 'coding sequences. An isolated nucleic acid molecule Non-naturally occurring nucleic acid molecules, such as nucleic acid molecules produced by recombinant DNA techniques including.   The term “nucleic acid molecule” refers to cDNA, genomic DNA and synthetic (eg, chemically Includes both RNA and DNA, including DNA (synthesized). In the case of single strand, nucleic acid is Or the antisense strand.   The present invention also relates to Tango-67 polysaccharide, preferably under stringent conditions. A nucleic acid molecule encoding a peptide (for example, a nucleic acid having the sequence shown in SEQ ID NO: 1) Molecule or nucleic acid having the sequence of Tango-67 encoding a portion of the sequence of SEQ ID NO: 1 Molecule) that hybridize with a nucleic acid molecule. Preferably hybridize A nucleic acid molecule consists of 400 nucleotides, more preferably 200 nucleotides.   Preferred hybridizing nucleic acid molecules include, for example, cell growth and / or It has the activity that Tango-67 has, such as the ability to increase the chemical.   The present invention also provides Tango-67 equivalents of various functional regions or fragments thereof. Comprising, substantially pure, or isolated Tango-67 polypeptide. The present invention Is substantially identical to the amino acid sequence shown in Figure (SEQ ID NO: 2) Includes amino acid sequence.   The polypeptides of the present invention can also be synthesized chemically, or Can be purified from naturally expressed tissues by biochemical purification methods .   A "functional polypeptide" having one or more of the biological functions or activities of Tango-67 Are also included in the present invention. These functions are normally associated with proteins that bind to Tango-67. Includes the ability to bind some or all. Functional polypeptide specific to Tango-67 When acting as an antigen to produce antibodies that bind specifically, functional polypeptides Are also considered to be within the scope of the present invention. In many cases, a functional polypeptide Carries one or more regions present in the native polypeptide.   Functional polypeptides have been modified from those disclosed herein. It may have the following amino acid sequence: Preferably, such changes are noted herein. It is a conservative amino acid substitution as described.   The terms "protein" and "polypeptide" are used herein to refer to chains. Any length, regardless of length or post-translational modification (eg, glycosylation or phosphorylation) Used to describe the amino acids in the chain. Thus, "Tango-67 polypeptide" The term refers to the full-length native Tango-67 protein (with or without a signal sequence). No), as well as full-length native Tango-67 protein or native protein Polypeptides corresponding to specific regions or parts and produced by recombination or synthesis Including The term also refers to mature Tango-67 with an added amino-terminal methionine. (Useful for expression in prokaryotic cells).   The term "purified" as used herein refers to cellular material, virus Materials or culture media if produced by recombinant DNA techniques, or chemical When synthesized to be substantially free of chemical precursors or other chemicals, Refers to nucleic acids or peptides.   The polypeptide or other compound of interest is at least one compound of interest. "Substantially pure" when present in a formulation that is 60% by weight (dry weight) It is said. Preferably, the formulation is at least 75% by weight of the compound of interest, and Preferably it is at least 90% by weight, most preferably at least 99% by weight. Accuracy can be measured, for example, by column chromatography, polyacrylamide gel electrophoresis, etc. Or by any suitable standard method such as HPLC analysis.   The polypeptide or nucleic acid molecule has a sequence that is less than the sequence of the reference polypeptide or nucleic acid molecule. At least 85%, preferably at least 90%, more preferably at least 95%, 98% Or, if it has a sequence that is 99% identical to the reference polypeptide or nucleic acid molecule, Substantially the same. "   A particular polypeptide is said to have the same proportions as a reference polypeptide of a defined chain length. If so, the percentage identity is relative to the reference peptide. Therefore, 100 amino acids A peptide that is 50% identical to a reference polypeptide of chain length is a 50 amino acid polypeptide. Is exactly the same as the 50 amino acid chain portion of the reference polypeptide. Also, A 100 amino acid long polypeptide that is 50% identical to the reference polypeptide over its entire length May be used. Of course, many other polypeptides are subject to similar criteria. Will be exposed.   In the case of a polypeptide sequence having less than 100% identity with the reference sequence, The preferred position is, but not necessarily, a conservative substitution of the reference sequence. Conservative storage Interchanges generally include substitutions within the following groups: glycine and alanine; valine, iso- Leucine and Leucine; Aspartic acid and Glutamic acid; Asparagine and Glutami Serine and threonine; lysine and arginine; and phenylalanine and tyro. Shin.   For polypeptides, the chain length of the reference polypeptide sequence is generally at least 16 amino acids. Acid, preferably at least 20 amino acids, more preferably at least 25 amino acids , Most preferably 35 amino acids, 50 amino acids or 100 amino acids. For nucleic acids The length of the reference nucleic acid sequence is generally at least 50 nucleotides, preferably at least At least 60 nucleotides, more preferably at least 75 nucleotides, most preferably Or 100 or 300 nucleotides.   Sequence identity can be determined by sequence analysis software (eg, Genetics Computer). Sequence analysis software package of the Genetics Computor Group J (Sequence Analysis Software Package), University of Wisconsin Biotechno University of Wisconsin Biotechnology Center, 1710 Uni Using Varsity Avenue, Madison, Wisconsin 53705) Can be measured using the default parameters specified in.   The nucleic acid molecule of the present invention is inserted into a vector that promotes expression of the insert as follows. can do. Nucleic acid molecules and the polypeptides they encode are diagnostic agents Alternatively, it can be used directly as a therapeutic agent, or as a therapeutic or diagnostic agent. To form antibodies that are useful on the floor (in the case of polypeptides directly Can be used indirectly). Therefore, a vector having the nucleic acid of the present invention. Vector, cells transfected with these vectors, expressed polypeptides And full-length polypeptides or antigens formed against antigenic fragments thereof The body is in a preferred embodiment.   As used herein, the term "transformed cell" refers to a recombinant A nucleic acid molecule encoding the polypeptide of the present invention has been introduced by DNA technology. Cell (or cell ancestor).   The present invention also provides, for example, monoclonal antibodies, polybodies that specifically bind to Tango-67. It features antibodies such as clonal and engineered antibodies. "Specifically bind `` Recognizes and recognizes a particular antigen, such as a Tango-67 polypeptide of the invention. But substantially eliminates other molecules in the sample containing Tango-67, such as biological samples. An antibody that does not recognize or bind.   The invention also provides for inhibiting or increasing one or more of the functions or activities of Tango-67, respectively. Can be characterized by Tango-67 antagonists and agonists . Preferred antagonists are small molecules (ie, molecules having a molecular weight of less than about 500) , Macromolecules (ie, molecules with a molecular weight greater than about 500), (as described below Antibodies that bind to and "neutralize" Tango-67, e.g., A polypeptide that antagonizes native Tango-67 to bind to protein, and Tango-67 Nucleic acid molecules that interfere with transcription (eg, antisense nucleic acid molecules and ribozymes) including. Tango-67 agonists are also small molecules, large molecules, and non-neutralizing antibodies. Including the body.   The invention also relates to Tang (eg, by affecting transcription or translation). It features a molecule capable of increasing or decreasing o-67 expression. Small molecule That is, a molecule with a molecular weight of less than about 500), a macromolecule (ie, a Large molecules) and nucleic acids that can be used to inhibit Tango-67 expression Molecules (eg, antisense and ribozyme molecules) or increase their expression Nucleic acid molecules that can be used to control Expression constructs into which nucleic acid sequences encoding Tango-67 have been introduced) and Tang A transgenic animal expressing the o-67 transgene.   The present invention also provides for functional interaction with Tango-67, such as the Tango-67 receptor. Characterizes substantially pure polypeptides used and the nucleic acid molecules that encode them And   The present invention relates to treating diseases associated with abnormal expression or activity of Tango-67. Including methods. Therefore, the present invention relates to diseases associated with overexpression or activity of Tango-67. And methods for treating. Such a method may reduce the expression or activity of Tango-67. It requires administration of a decreasing compound. The present invention also highlights the lack of Tango-67 And methods for treating diseases associated with high expression. These methods are based on Tango-67 It is necessary to administer a compound that increases the expression or activity of   The invention also features a method for detecting a Tango-67 polypeptide. This Such methods include obtaining a biological sample and providing conditions that allow for specific binding. Contacting the sample with an antibody that specifically binds to Tango-67, Detecting any antibody-Tango-67 complex formed.   The present invention also provides diagnostic evaluation of diseases associated with inappropriate expression or activity of Tango-67. And methods and compositions for titration, classification and prognosis. For example, a nucleic acid molecule of the invention For example, to detect inappropriate expression of Tango-67 or mutation of Tango-67 gene Can be used as a diagnostic hybridization probe. This Such methods can be used to classify cells according to the degree of Tango-67 expression. Wear.   Alternatively, the nucleic acid molecule can be a gene variant, an allelic variant, and a Tango-67 gene. Can be used as a primer in diagnostic PCR analysis to identify regulatory deficiencies Wear. The invention further provides a diagnostic kit for performing such a method. .   The present invention provides for the expression of Tango-67 in the presence and absence of a selected compound. Or a compound that regulates Tango-67 expression or activity by assessing its activity. It is characterized by a method of identifying an object. In the presence and absence of the selected compound The difference in the degree of Tango-67 expression or activity between the selected Or that the activity can be modulated. Expression is by techniques well known to those of skill in the art. The degree of gene expression (eg, by measuring mRNA) or protein It can be evaluated by either the degree of expression of quality. The activity of Tango-67 is functional, That is, by assaying the ability of the compound to inhibit bone marrow cell proliferation. Can be valued.   Preferred methods and materials are for the purpose of illustrating the invention and for the purpose of limiting it. Not described in the following examples. Those skilled in the art will appreciate that Similar or equivalent to those used in practicing or testing the present invention Will recognize the methods and materials that can be used.   Unless otherwise specified, all technical terms and science used herein The terms are to be understood by those of ordinary skill in the art to which this invention belongs. Has the same meaning. Methods and methods similar or equivalent to those described herein And materials can be used in practicing or testing the present invention, but are preferred Methods and materials are described herein. In addition, the materials, methods, and examples are illustrative only. And is not intended to be limiting.   Other features and advantages of the invention will be apparent from the following detailed description of the invention and the claims. It is clear from the box.BRIEF DESCRIPTION OF THE FIGURES   The figure shows the nucleotide sequence encoding Tango-67 and the 3 ′ and 5 ′ untranslated sequences. (SEQ ID NO: 1) and the amino acid sequence of Tango-67 (SEQ ID NO: 2).                                Detailed description   Tango-67 is a new member of the growth factor superfamily. protein At the sequence level of Tango-67, the Drosophila twist gut gene product and human Related to connective tissue growth factor.   Tango-67 Nucleic acid molecule   The Tango-67 nucleic acid molecule of the present invention is cDNA, genomic DNA, synthetic DNA or RNA. May be double-stranded or single-stranded (ie, sense or antisense strand). You may. Fragments of these molecules are also considered to be within the scope of the invention, for example, Re Treat with merase chain reaction (PCR) or one or more restriction endonucleases. And it can be produced. Ribonucleic acid molecules (RNA) are obtained by in vitro transcription. Can be produced. Preferably, the nucleic acid molecule is usually Encodes a polypeptide that is soluble under physiological conditions.   A nucleic acid molecule of the invention can be a native sequence, or a sequence that differs from a native The same polypeptide (eg, the polypeptide of SEQ ID NO: 2) May be provided. These nucleic acid molecules also encode polypeptides. Is not limited to sequences that only load Or some or all of the non-coding sequences.   The nucleic acid molecules of the invention can be synthesized (eg, by phosphoramidite-based synthesis). Or may be obtained from biological cells, such as mammalian cells. Therefore, nucleic acids Are human, mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, , Dog or cat nucleic acid. Combinations in these types of nucleic acids Or nucleotide modifications.   Also, the isolated nucleic acid molecules of the present invention include fragments that are not found in the natural state. Therefore The present invention provides a nucleic acid molecule (eg, an isolated nucleic acid molecule encoding Tango-67) comprising a vector -(Eg, a plasmid or viral vector) or the genome of a heterologous cell (or Or a genome at a different position from the natural chromosomal locus of allogeneic cells). Of recombinant molecules. Recombinant nucleic acid molecules and their uses are discussed in detail below.   Events Encoded by the Nucleic Acid Molecules of the Invention, That is, Act as Antisense Molecules So, they can be used to regulate the translation of Tango-67, for example . Techniques related to detecting or regulating Tango-67 expression are well known to those of skill in the art and Can be used to diagnose and / or treat inflammation or disease associated with it can. These nucleic acid molecules are discussed further below in their clinical use section. I have.   The present invention also provides for the use of Tango-67 polypeptides under stringent conditions. Includes nucleic acid molecules that hybridize to the encoding nucleic acid molecule. As described herein The cDNA sequence (SEQ ID NO: 1), for example, encodes a homologous polypeptide of another species. Nucleic acids and human or other mammalian Tango-67 gene splice variants. Including Can be used to identify these nucleic acids. Therefore, the present invention It features a method of identifying and isolating these nucleic acid molecules. Use these methods Sample (eg, a nucleic acid library such as a cDNA or genomic library) Tango-67-specific probes (eg, sequences with a nucleotide length of at least 12) (No. 1 fragment) is contacted (ie, "screened"). probe Selectively binds to nucleic acids encoding related polypeptides (or their complementary sequences) To hybridize. The polypeptide encoded by Tango-67 is compatible with other C-C chemokines As relevant, the term "selectively hybridize" means that the probe It binds to nucleic acids encoding other CC chemokines (or their complements). Nucleic acids encoding Tango-67 (or their complements) Sequence). At least 12 nucleotides (eg Probes that can have, for example, 15, 25, 50, 100 or 200 nucleotides) Can be found in several standard methods (eg, Ausubel et al., "Molecular Biology"). Protocols in Molecular Biology ”, Volume I, Green Publishing Associates, Inc .) And John Wiley & Sons, Inc., New York 1989) can be employed. For example, Using oligonucleotide primers, as described in Example 1 below, It can be used as a probe to screen libraries, To detect nucleic acid molecules (within the library) that hybridize with the probe Tango-67-specific nucleic acid sequences (eg, encoding a chemokine-like region) Probes can be made using PCR amplification methods that amplify .   A single-stranded nucleic acid is combined with another single-stranded nucleic acid if a double helix is formed between them. It is said to be hybridized. This is because one nucleic acid has the opposite orientation to the other strand. Occurs when you have a sequence that is complementary (this same sequence is A natural interaction with the antisense strand occurs, forming the basis for a "double helix" structure. ). For the double helix to be formed, the perfect complementarity of the hybridizing region Sex is not required. Under the hybridization conditions used, the paired bases It is only necessary that the number be sufficient to maintain a double helix.   Generally, hybridization conditions are low to moderately stringent. Sea. These conditions favor specific interactions between perfectly complementary sequences. However, some non-specific interactions between sequences that do not perfectly correspond You. After hybridization, moderate to high stringency conditions Nucleic acid "washed" underneath and bound by somewhat non-specific interactions Can dissociate the double helix (thus forming such a double helix Nucleic acids are not completely complementary).   As is well known in the art, optimal cleaning conditions are empirically and often are Is determined by gradually increasing the agency's ency. Stringency The parameters that can be changed to affect the Includes salt concentration. In general, the lower the salt concentration and the higher the temperature, the higher the stringency -High. Washing should be equivalent to or equal to the salt concentration of the hybridization solution. Start at low temperature (eg, room temperature) using a solution containing a lower salt concentration Can be Subsequent washes had the same salt concentration and the temperature was gradually increased It can be performed using a solution. Alternatively, reduce salt concentration and wash The temperature may be maintained during the stage, or the salt concentration may be reduced and the temperature increased. Another Can be changed. For example, destabilizing agents such as formamide The use of changes stringent conditions.     A certain level of stringency in the reaction of hybridizing nucleic acids The conditions used to obtain the key are different. For example, all nuclei that are 85% identical to each other The conditions for forming a double helix between the acids are not one set. Hybridization Also depends on the unique characteristics of each nucleic acid. Sequence length, sequence composition (for example, Pyrimidine-like nucleotide content relative to phosphorus-like nucleotide content) and core The type of acid (eg, DNA or RNA) affects hybridization. Another possibility is that one of the nucleic acids is immobilized (eg, on a filter). Or not.   An example of changing stringent conditions from low to high is as follows: is there. It has a relatively sufficient salt content of SSC (sodium chloride and citric acid). A salt solution containing sodium; 2 × SSC is 10 times stronger than 0.2 × SSC). Nucleic acids 42 Hybridized in 2 × SSC / 0.1% SDS (sodium dodecyl sulfate; detergent) And then at room temperature, 0.2 × SSC / 0.1% SDS (for low stringency) ); 0.2 x SSC / 0.1% SDS (for moderate stringency) at 42 ° C; And at 68 ° C. with 0.1 × SSC (for high stringency). Cleaning is described Can be implemented using only one of the conditions (For example, washing in the order described above for 10 to 15 minutes, respectively). Any of the washing or Everything can be repeated. As mentioned above, the optimum conditions change and Can be   The second set of conditions, considered "stringent", is hybridization. At 50 ° C. with Church buffer (7% SDS, 0.5% NaHPO_Four, 1M EDTA, 1% BSA), with washing at 50 ° C. in 2 × SSC.   Once detected, nucleic acid molecules can be obtained using a number of standard techniques (eg, Sambrook ( Sambrook et al., "Molecular Cloning, A Labor. atory Manual), 2nd edition, Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press), Cold Spring, NY G. Haber, 1989).   The present invention also provides (a) a Tango-67-related coding sequence as described above and / or Expression vectors containing any of the complementary sequences (ie, "antisense" sequences) (B) functional for regulatory elements that direct the expression of the coding sequence, examples of which are shown below. An expression vector containing any of the above Tango-67 related coding sequences that binds to ー; (c) In addition to the sequence encoding the Tango-67 polypeptide, a reporter or Unrelated to the nucleic acid sequence encoding Tango-67, such as a molecule encoding a marker An expression vector containing a nucleic acid sequence; and (d) any of the above expression vectors. Genetically expressing the nucleic acid molecule of the present invention in a host cell. Includes engineered host cells.   The recombinant nucleic acid molecule comprises a sequence encoding a soluble Tango-67 polypeptide, mature Tang o-67, Tango-67 with signal sequence, or chemokine-like domain of Tango-67 May be contained. Full length Tango-67 polypeptides, domains of Tango-67 or their The fragment may be fused to another polypeptide, as described below. Similarly, The light nucleic acid molecule encodes mature Tango-67 or a polypeptide that promotes secretion Can be coded. In the latter case, the polypeptide is generally a pro- Called protein, for example, by removing the signal sequence in the host cell It can be converted to the active form. Proprotein removes inactivating sequences Can be converted into an active form of the protein.   The regulatory elements described above include inducible and non-inducible promoters, enhancers, Operators, and other, well known to those of skill in the art, that drive or regulate gene expression. Including but not limited to elements. Such modulators may be cytomegalo Ils hCMV immediate early gene, SV40 adenovirus early or late promoter,lac system,trpsystem,TACsystem,TRCSystem, major operation of A phage Region, fd coat protein regulatory region, 3-phosphog Lyserate kinase promoter, acid phosphatase promoter, and And yeast α-mating factor promoters, including but not limited to.   Similarly, nucleic acids are more often than sequences that act as markers or reporters, for example. Form part of a hybrid gene encoding another polypeptide sequence be able to. Examples of marker or reporter genes are β-lactamase, Loramphenicol acetyltransferase (CAT), adenosine deamina (ADA), aminoglycoside phosphotransferase (neo^r, G418^r), Hydrofolate reductase (DHFR), hygromycin-B-phosphotransferer (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase ) And xanthine guanine phosphoribosyltransferase (XGPRT) No. As with many standard approaches relevant to the practice of the present invention, those skilled in the art will appreciate the For example, another sequence of another marker that can serve as a marker or reporter You will notice useful reagents. Generally, the hybrid polypeptide will be a first part And a second part; the first part is a Tango-67 polypeptide and the second part Is, for example, a constant region of a reporter or immunoglobulin described above.   Expression systems that can be used for the purposes of the present invention include the nucleic acid molecules of the present invention. Recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector Bacteria transformed with the bacterium (eg, E. coli and B. subt. il a nucleic acid molecule of the present invention (preferably, (SEQ ID NO: 1 A recombinant yeast expression vector containing a nucleic acid sequence encoding Tango-67) Yeast (eg, Saccharomyces) and Pichia); a recombinant virus expression vector containing the nucleic acid molecule of the present invention -Insect cell lines infected with (eg, baculovirus); recombinant virus expression Vectors such as cauliflower mosaic virus (CaMV) and tobacco mosaic Virus (TMV)) or contains the Tango-67 nucleotide sequence. Plants transformed with a recombinant plasmid expression vector (eg, Ti plasmid) Cell lines; or promoters derived from the genome of mammalian cells (eg, Promoter derived from the tarothionine promoter) or a mammalian virus (Eg, the adenovirus late promoter and vaccinia virus 7.5K A mammalian cell line harboring a recombinant expression construct containing a promoter (eg, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38 and NIH 3T3 cells) It is not limited to these.   In bacterial systems, there are many expression vectors depending on the intended use in which the gene product will be expressed. Can be advantageously selected. For example, containing a Tango-67 polypeptide To produce pharmaceutical compositions or to produce antibodies against these polypeptides. If you plan to produce large quantities of such proteins to produce Vectors that can direct the expression of high levels of fusion protein products to be purified May be desirable. Such a vector would form a fusion protein. The coding sequence of the insert is aligned with the lacZ coding region of the vector. The E. coli expression vector pUR278 (Ruzer (Rut her) et al., EMBO J .; 2: 1791, 1983), pIN vectors (Inouye and Noue (Inouye), Nucleic Acids Res. 13: 3101-3109, 1985; Fan Heke (Van Heeke) and Schuster, J.M. Biol. Chem. 264: 5503-5509 , 1989) and the like. When using the pGEX vector, Foreign polypeptide as a fusion protein with Tathione S-transferase (GST) Pide can be expressed. Generally, such fusion proteins are soluble, Glutathione-adsorbed on agarose beads and then in the presence of free glutathione Stay By elution, it can be easily purified from lysed cells. pGEX vector Can release the cloned target gene product from the GST moiety. Designed to contain thrombin or factor Xa protease cleavage sites I have.   In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) Can be used as a vector for expressing a foreign gene. The virus is sport It grows in Spodoptera frugiperda cells. Insert Coding sequences are individually defined in non-essential regions of the virus (eg, the polyhydrin gene). Can be cloned, the AcNPV promoter (eg, polyhydrin Motor). Upon successful insertion of the coding sequence, When the recombinant gene has been inactivated and is not closed (ie, A virus without the protein coat encoded by the drin gene) is produced. Next Spodoptera expressing the inserted gene using these recombinant viruses Infect Frugiperda (Spodoptera frugiperda) cells. (For example, Smith (Smith) et al. Virol. 46: 584, 1983; Smith, U.S. Patent No. 4,215,051. No.).   In mammalian host cells, a number of viral expression systems are available. You. When an adenovirus is used as an expression vector, the nucleic acid molecule of the present invention may be used. The adenovirus transcription / translation control complex, such as the late promoter and tripartite It can be linked to a leader sequence. Next, this chimeric gene was Can be inserted into the adenovirus genome by or in vivo recombination . Insertion into non-essential regions of the viral genome (eg, the E1 or E3 region) can A recombinant window capable of surviving in the host after staining and expressing the Tango-67 gene product Ruth is made (eg, Logan and Shenk, Proc . Natl. Acad. Sci. USA 81: 3655-3659,1984). Efficiency of inserted nucleic acid molecules Specific translation may require a specific initiation signal. These The signal contains the ATG start codon and adjacent sequences. Start codon and adjacent sequence If the entire gene or cDNA containing is inserted into an appropriate expression vector, additional Translation control signals may not be required. But only part of the code array Insert If present, exogenous translational control signals, possibly including the ATG initiation codon Need to provide. In addition, to ensure translation of the entire insert, start Must be aligned with the reading frame of the desired coding sequence. These extrinsic factors Sex translational control signals and initiation codons can be of various origins, both natural and synthetic. Good. Expression efficiency includes appropriate transcription enhancer elements, transcription terminators, etc. (See, for example, Bittner et al., Method in Enzymol. 153: 516-544, 1987).   It may also regulate the expression of the inserted sequence or produce the gene product in a specific manner desired. A host cell strain that modifies and processes E. coli can be selected. Protein products Modifications (eg, glycosylation) and processing (eg, cleavage) such as It can be important for quality function. For different host cells, proteins and Characteristic and specific mechanisms for post-translational processing and modification of gene products Have. Ensure proper modification and processing of expressed foreign protein For this purpose, a suitable cell line or host system can be selected. To do this, Cells for proper processing of primary transcription, glycosylation and phosphorylation of gene products Eukaryotic host cells with equipment can be used. The above mammalian cell types are preferred Included among those that may serve as suitable host cells.   For long-term, high-yield production of recombinant proteins, stable expression is required. Is preferred. For example, a cell line that stably expresses the above Tango-67 sequence You can manipulate the command. Use an expression vector with viral origin of replication Instead, the host cell is treated with appropriate expression control elements (eg, promoters, enhancer sequences). Sequence, transcription terminator, polyadenylation site, etc.) Can be transformed with a suitable marker. Cells created after introduction of foreign DNA Can be grown for 1-2 days in an enriched media, and then transferred to a selective media. Recombination The selectable marker in the rasmid confers resistance to selection, and cells Chromosomes are stably integrated into the chromosome, propagated, cloned, and It becomes possible to form a growth foci that can be established. This method uses Tango- It can be advantageously used to engineer cell lines expressing 67. like this An engineered cell line screens for compounds that affect the endogenous activity of the gene product. Lee Can be particularly useful in training and evaluation.   Numerous selection systems can be used. For example, herpes simplex virus thimi Gin kinase gene (Wigler et al., Cell 11: 223, 1977), Hipoki Santin-guanine phosphoribosyltransferase gene (S. varsica (S zybalska) and Szybalski, Proc. Natl. Acad. Sci. USA 48: 2026, 1962), and the adenine phosphoribosyltransferase gene ( Lowry et al., Cell 22: 817, 1980)^-Cells, hgprt^-cell Or aprt^-Can be used for cells. In addition, anti-metabolic resistance Can be used as a basis for selecting offspring: resistance to methotrexate Dhfr conferring properties (Wigler et al., Proc. Natl. Acad. Sci. USA 77:35). 67, 1980; O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527, 1981) Gpt (Mulligan) which confers resistance to mycophenolic acid; Berg, Proc. Natl. Acad. Sci. USA 78: 2072, 1981); aminoglyco Neo (Colberre-Garapi) confers resistance to Sid G-418 n) et al. Mol. Biol. 150: 1, 1981); and resistance to hygromycin Hygro (Santerre et al., Gene 30: 147, 1984).   Alternatively, by utilizing an antibody specific for the expressed fusion protein, Any fusion protein can be easily purified. For example, Junknecht The system described by (Janknecht) et al. Sex fusion protein can be easily purified (Proc. Natl. Acad. Sci. USA 88: 8972-8976, 1991). In this system, open reading of genes The frame is translationally fused to an amino-terminal label consisting of six histidine residues. Subclonin into the vaccinia recombination plasmid, as To Extract from cells infected with recombinant vaccinia virus²⁺-Nitrilo Apply to acetic acid-agarose column and histidine in buffer containing imidazole The labeled protein is eluted selectively.   Tango-67 nucleic acid molecules are useful for diagnosing diseases associated with abnormal expression of Tango-67. You. Tango-67 nucleic acid molecules are useful in gene mapping.   Tango-67 Polypeptide   The Tango-67 polypeptides described herein can be any of the above nucleic acid molecules. And includes Tango-67 fragments, variants, truncations and fusion proteins. No. Tango-67 can regulate the activity or expression of other cellular gene products or As an agent in diagnostic assays to identify compounds, and abnormal Tango-67 Production of antibodies as agents useful for treating diseases associated with expression or activity Prepare these polypeptides for various uses, including but not limited to Can be   A preferred polypeptide is the full length signal sequence of human Tango-67 polypeptide. Substantially, including those corresponding to secreted polypeptides, It is a pure Tango-67 polypeptide. Soluble under normal physiological conditions Polypeptides are particularly preferred.   The invention also includes polypeptides that are functionally equivalent to Tango-67. these Polypeptides can perform one or more of the functions of Tango-67 in biological systems. In that it is equivalent to Tango-67. Preferred Tango-67 polypeptides are Has 20%, 40%, 50%, 75%, 80% or 90% of the activity of full-length mature human Tango-67 . Such comparisons generally involve the use of equal concentrations of the polypeptides and the comparison of the polypeptides. Product activity assay. This comparison is also the maximum stimulus that can be obtained Can also be based on the amount of polypeptide required to reach 50% of the polypeptide.   Functionally equivalent proteins include, for example, added or substituted amino acid residues. You may have. Substitution depends on the polarity, charge, solubility, and hydrophobicity of the residues involved It can be carried out on the basis of similarity, hydrophilicity and / or amphipathic similarity. In general, amino acids that are considered to provide conservative substitutions for one another are The point is clearly specified.   A polypeptide functionally equivalent to Tango-67 (SEQ ID NO: 2) is known to those of skill in the art. Can be generated using a known random mutation generation method (obtained mutant Tango- 67 proteins can be tested for activity). However, such a port Lipeptides can be made by site-directed mutagenesis (using techniques well known to those of skill in the art). More likely to be made. These polypeptides have an increased function, namely Can have a greater ability to inhibit cell growth or elicit an inflammatory response Come You. Such polypeptides may be subject to the effects of chemotherapy and / or radiation therapy. Can be used to protect stem cells.   Designing functionally equivalent polypeptides requires conservative and variable positions. It is useful to identify the location. This is because Tango-67cD obtained from various organisms This can be done by aligning the sequences of NA. Those skilled in the art will It will be appreciated that no acid residues are more likely to be required for conservation of function. Therefore, it is preferred that conservative residues are not changed.   Mutations in the Tango-67 coding sequence are more suitable for expression in a selected host cell Can be implemented to make Tango-67. For example, N-linked glycosylation Altering or removing sites, for example, hyperglycosylating the N-linked site Performs expression of homogeneous products that are more easily recovered and purified from known yeast hosts can do. For this, the resulting glycosylation recognition sequence (N-X-S or N -X--) at one or both of one or more of the first or third amino acid positions Various amino acid substitutions, and / or any one or more second amino acids of such recognition sequences. Deletion of an amino acid at the position indicated in the above results in glycosylation of the modified tripeptide sequence. (Eg, Miyajima et al., EMBO J. 5: 1193, 1986).   The polypeptides of the present invention may be other polypeptides, such as marker polypeptides. That is, it can be expressed by fusion with a fusion partner. For example, a polypeptide Hex-histidine labeling to facilitate the purification of bacterially expressed proteins or Is a hemagglutinin label to facilitate purification of proteins expressed in eukaryotic cells Can be fused with intellect.   The polypeptides of the present invention can be chemically synthesized (eg, Kraton (Creighton), "Proteins: Structures and M. olecular Principles), Freeman & Co., New York 1983), or perhaps more advantageously, the recombinant DN described herein. It can be made by A technology. Another guidance is that those skilled in the art (Ausubel) et al. (Supra) and Sambrook et al. . (See “Molecular Cloning, A Laboratory Manual ) ", Cold Spring Harbor Press, New York -Yo Cold Spring Harbor, KY, 1989), and especially examples of chemical synthesis As edited by Gate (MJ) (“Oligonucleoti de Synthesis) ", IRL Press, Oxford, 1984).   The present invention also interacts with Tango-67, thereby altering the function of Tango-67. Polypeptides (and the genes encoding them). Interaction The polypeptide can be identified using methods well known to those skilled in the art. 1 One preferred method for detecting protein interactions in vivo is the "2-hi Brid System "(Chien et al., Proc. Natl. Acad. Sci. USA , 88: 9578, 1991). Kits for performing this method are available from Clont ech) (Palo Alto, CA).   Tango-67 polypeptides are useful for promoting growth. So they are wound healing, It has applications in tissue repair, graft fixation and stimulation of bone growth.   Transgenic animals   Tango-67 polypeptide can also be expressed in transgenic animals . These animals are caused by or overexpressing Tango-67 To study diseases that are exacerbated by the disease and the expression or activity of Tango-67. A model system for developing modulating therapeutics.   Transgenic animals are domestic animals (pigs, goats, sheep, cows, horses, rabbits, etc.) ), Rodents (such as rats, guinea pigs and mice), and non-human primates (eg, Baboons, monkeys and chimpanzees) and domestic animals (eg, dogs and And cats). Transgenic mice are particularly preferred.   The Tango-67 transgene is introduced into the animal using any technique known in the art. , A transgenic animal of the founder strain can be produced. Such techniques Is a pronuclear microinjection (US Pat. No. 4,873,191); -Mediated gene transfer into the germ line (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148, 1985); Gene targets for embryonic stem cells Ting (Thompson et al., Cell 56: 313, 1989); Crotropolation (Lo, Mol. Cell. Biol. 3: 1803, 1983), It is not limited to these.   The present invention relates to a transgene having the Tango-67 transgene in all its cells. Transgenic to nick animals and some but not all of their own cells. Providing an animal having a skin. That is, the present invention provides a mosaic animal. G The transgene can be a single transgene or, for example, head-to-head or Can be incorporated in concatemers such as head-to-tail. Transgene Can also be selectively introduced into specific cell types and activated (Lasko Proc. Natl. Acad. Sci. USA 89: 6232, 1992). Such cell type-specific activities The regulatory sequences required for sex depend on the particular cell type of interest and are obvious to those of skill in the art. There will be.   Integrate the Tango-67 transgene at the chromosomal location of the endogenous Tango-67 gene. If is desired, gene targeting is preferred. Briefly, If such techniques must be used, homologous sets using chromosomal sequences Purpose of disrupting the function of the nucleotide sequence of an endogenous gene through integration Has several nucleotide sequences homologous to the endogenous Tango-67 gene Design a vector. Transgenes can be selectively introduced into specific cell types, Therefore, it is also possible to inactivate the endogenous Tango-67 gene of only that cell type (G (Gu) et al., Science 265: 103, 1984). Required for such cell type-specific inactivation The exact regulatory sequences will depend on the particular cell type of interest and will be apparent to those skilled in the art. this Their technique is to prepare a "knockout" without a functional Tango-67 gene Useful.   Once transgenic animals are created, expression of the recombinant Tango-67 gene is Assays can be performed using standard techniques. The first screening is Performed by Southern blot analysis or PCR techniques to incorporate the transgene It is determined whether the error has occurred. Northern blot of tissue samples from animals Analysis, in situ hybridization analysis and RT-PCR Transgenic animals, using techniques that are not limited to Gene mRNA expression can also be assayed. Tango-67 transgene products Of Tango-67 gene-expressing tissue using an antibody specific for Can be evaluated biologically.   Techniques that can be used to create and evaluate transgenic animals For those of skill in the art, Gordon (Intl. Rev. Cytol. 115: 171). 229, 1989), for example: Hogan et al., Mouse Manipulating the Mouse Embryo ”(Cold Spring Harbor Press (Cold Spring Harbor Press), Cold Spring, NY, 19 86; Krimpenfort et al., Bio / Technology 9:86, 1991; Palmiter et al., Cell 41: 343, 1985; Kraemer et al. Genetic Manipulation of the Early Mammalian Embryo ) ", Cold Spring Harbor Press, New York -Cold Spring, York, 1985; Hammer et al., Nature 315: 680, 1985; Purcel et al., Science, 244: 1281, 1986; Wagner et al. And US Patent No. 5,175,385; and Krimpenfort et al., US No. 5,175,384 (the last two are incorporated herein by reference). Different guidance can be obtained.   Anti-Tango-67 antibody   A human Tango-67 polypeptide (or immunogenic fragment or analog) Such polypeptides can be used to generate particularly useful antibodies; Can be made by recombinant techniques or synthesized (eg, “solid phase peptides”). Synthesis (Solid Phase Peptide Synthesis) ", supra; Ausubel et al., See above). In general, the peptides are described in Ausubel et al., Supra. Binds to described carrier protein such as KLH and mixes with adjuvant And can be injected into a host mammal. Antibody is peptide antigen affinity It can be purified by chromatography.   In particular, injection of Tango-67 protein or polypeptide Animals can be immunized. Host animals include rabbits, mice, guinea pigs and Including Various adjuvants that can be used to increase the immune response Depending on the host species, Freund's adjuvant (complete and incomplete), hydroxylation Mineral gels such as aluminum, lysolecithin, pluronic polyols, Lianion, peptide, oil emulsion, keyhole limpet hemocyanin And surfactants such as dinitrophenol. A potentially useful Toadjuvant is BCG (bacille Calmette-Guerin) and Corynebacterium pa Rubum (Corynebacterium parvum). Polyclonal antibodies are A heterogeneous population of antibody molecules contained in the serum of the product.   Thus, the antibodies of the present invention include polyclonal antibodies and monoclonal antibodies. Body, humanized or chimeric antibody, single chain antibody, Fab fragment, F (ab ')_TwoFragment and Fab expression Includes molecules made using the library.   The Tango-67 protein described above and standard hybridoma technology (eg, Coff Kohler et al., Nature 256: 495, 1975; Kohler et al., Eur. J. Imm unol. 6: 511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; c Hammerling et al., "Monoclonal Antibodies and T Cell Hybridomas (M onoclonal Antibodies and T cell Hybridomas), Elsevier, New York, 1981; Ausubel et al., Supra, see) Monoclonal antibodies can be prepared that are the same population of antibodies to the antigen .   In particular, monoclonal antibodies are described in Kohler et al., Nature 256: 495, 1975. And continuous cell lines in culture, as described in U.S. Patent No. 4,376,110 Any technique that provides for the production of antibody molecules by the human B-cell hybridoma technique (t he human B-cell hybridoma technique), Kosbor et al., Immunology T oday 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA 80: 2026, 1983 ) And the EBV-hybridoma technique (Cole et al., "Monoclonal Antibodies and Cancer Therapy (Monoclonal Antibodies and Cancer Therapy) ", Alan Earl Squirrel (Alan R. Liss, Inc.), pp. 77-96, 1983). Such antibodies include IgG, IgM, IgE, IgA, IgD and any subclass thereof. And of any immunoglobulin class. MAb producing hive of the present invention The lidoma can be cultured in vitro or in vivo. High antibody titer The ability to produce mAbs in vivo makes this a preferred method of production of the present invention. You.   The polyclonal or monoclonal antibodies produced can be C) by standard methods, as described in Ausubel et al., Supra. E Test for specific Tango-67 recognition by stump blot or immunoprecipitation analysis I do. Antibodies that specifically recognize and bind to Tango-67 are useful in the present invention. For example, such antibodies can be used in immunoassays to produce Tango-67 produced by mammals. Levels can be monitored (eg, the amount or subcellular location of Tango-67) Decision).   Preferably, the antibodies of the present invention are outside of the highly conserved region and have May be antigenic, depending on criteria such as frequency of group occurrence Produced using fragments of the Tango-67 protein. In one specific example, Such a fragment is produced by standard PCR techniques, and then a pGEX expression vector (Ausubel et al., Supra). Ausubel (Ausub el) et al., supra, the fusion protein is E. coli. And purified using a glutathione agarose affinity substrate. You.   In some cases, problems arise that reduce the affinity or specificity of the antiserum It may be desirable to minimize the possibilities. In such situations, each tamper Two or three fusions can be made for a protein, with at least Can also be injected into two rabbits. The antiserum is preferably administered at least three times. It can be made by continuous injection, including booster injections.   The antiserum also contains recombinant Tango-67 protein or glucocorticoid receptor, CA Investigate the ability to immunoprecipitate regulatory proteins such as T or luciferase Can be   For example, when detecting Tango-67 in biological samples as part of a diagnostic assay The antibodies of the present invention can be used. Indication for Tango-67 expression or localization Antibodies should also be used in screening assays to determine the effects of complement compounds. Can be. Also, for example, prior to introduction into a patient, a conventional and / or In conjunction with the described gene therapy techniques to evaluate Tango-67 expressing cells Such antibodies can be used. In addition, such an antibody is It can also be used in methods to inhibit normal activity.   In addition, a gene for a mouse antibody molecule having appropriate antigen specificity is By splicing together with the gene of a human antibody molecule having A technique developed to produce "antibodies" (Morrison et al., Proc. Na tl. Acad. Sci. USA, 81: 6851, 1984; Neuberger et al., Nature. , 312: 604, 1984; Takeda et al., Nature, 314: 452, 1984). Can be. The chimeric antibody is composed of a variable region derived from mouse mAb and human immunoglobulin. Molecules derived from animals that differ by part, such as those with constant regions of It is.   Alternatively, the techniques described for producing single chain antibodies (US Pat. No. 4,946,7 Nos. 78 and 4,704,692) against Tango-67 protein or polypeptide Can be adjusted to produce a single-chain antibody. Fv region heavy and light chain breaks By joining the pieces through amino acid bonds to form a single-chain polypeptide. To form a single-chain antibody.   Antibody fragments that recognize and bind to specific antigenic determinants are made by well-known techniques be able to. For example, such fragments are produced by pepsin digestion of antibody molecules. F (ab ') that can be produced_TwoFragment and F (ab ')_TwoReduce fragment disulfide bonds Include, but are not limited to, Fab fragments that can be produced by Absent. Alternatively, rapid and easy production of monoclonal Fab fragments with the desired specificity Fab expression library that allows for accurate identification (Huse (H use) et al., Science, 246: 1275, 1989).   Using techniques well known to those skilled in the art (eg, Greenspan et al., F. ASEB J. 7: 437, 1993: Nissinoff, J.C. Immunol. 147: 2429, 1991 )) Anti-idio similar to a part of Tango-67 using antibodies against Tango-67 Type antibodies can be made. For example, combining with Tango-67, Using antibodies that competitively inhibit ligand binding, the ligand-binding region of Tango-67 To produce anti-idiotypes that bind to and neutralize Tango-67 ligands Can be manufactured. Such a neutralizing anti-idiotype antibody or Fab fragments of anti-idiotype antibodies, such as, can be used for therapy.   Antibodies can be humanized by methods well known in the art. For example, Monoclonal antibodies with high binding specificity have been commercialized as humanized Scotgene, Scotland; Oxford Molecular (Ox ford Molecular), Palo Alto, California). Transgenic animals Fully human antibodies, such as those expressed in (Green) en) et al., Nature Genetics 7: 13-21, 1994; also incorporated herein by reference. See also U.S. Patent Nos. 5,545,806 and 5,569,825.   The method described herein using an anti-Tango-67 antibody, for example, is described below. Conveniently used in clinical settings to diagnose patients with symptoms of the described disease At least one specific Tango-67 nucleic acid described herein. By using pre-packaged diagnostic kits containing the nucleotide sequences or antibody reagents Can be implemented.   Antisense nucleic acid   Therapies based on the “antisense” method use oligos complementary to Tango-67 mRNA. Includes designing nucleotides (either DNA or RNA). These ori The gonucleotide binds to a complementary Tango-67 mRNA transcript and blocks translation. Perfect complementarity is preferred but not required. As referred to herein, as part of the RNA "Complementary" sequences are capable of hybridizing to RNA to form a stable double helix. Means a sequence having sufficient complementarity to be able to: double-stranded antisense nucleic acid In this case, one strand of a double helix can be tested, or You can essay. The ability to hybridize depends on the complementarity of the antisense nucleic acid. And the chain length. Generally, the longer the hybridizing nucleic acid It has a double helix (or triple helix that it has and is still stable ), The base mismatch with RNA increases. The person skilled in the art By using standard techniques to determine the melting point of the The acceptable degree of match can be ascertained.   For example, a message containing the AUG start codon and 5 'untranslated sequence up to this position. Oligonucleotides complementary to the 5 'end of the sage are most efficient at blocking translation Should work in a proper way. However, the sequence complementary to the 3′-side untranslated sequence of mRNA is also It has recently been shown to be effective in blocking the translation of mRNA (Wagner, Nature 372: 333, 1984). Therefore, 5 'or 3' non-translated non-coding of the Tango-67 gene. Oligonucleotide complementary to either of the nucleic acid regions, for example, the human gene shown in the figure. Is Can be used in antisense methods to block the translation of endogenous Tango-67 mRNA. I think it will work. Oligonucleotides complementary to the 5 'untranslated region of the mRNA It should include the complement of the start codon.   Antisense oligonucleotides complementary to the mRNA coding region are less efficient Although not a translation inhibitor, it is believed that it can be used in accordance with the present invention. Ta Designed to hybridize to the 5 ', 3' or coding region of ngo-67 mRNA Antisense nucleic acids, whether or not Oligonucleotides, preferably 6 to about 50 nucleotides in length. Is. In specific aspects, the oligonucleotide is at least 10 nucleotides At least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.   Regardless of the choice of target sequence, in vitro studies initially involve antisense oligonucleotides. Quantification of the ability of the nucleotide to block gene expression is preferably performed. New These studies demonstrate that oligonucleotide antisense gene blocking and nonspecific It is preferred to use a control that discriminates between the biological and biological effects. These studies are Compare the amount of target RNA or protein with that of the internal control RNA or protein. Are also preferred. Also, the results obtained using antisense oligonucleotides Comparing to that obtained using a control oligonucleotide . The control oligonucleotide is approximately the same length as the test oligonucleotide. The nucleotide sequence of the oligonucleotide Antisense is not required to prevent specific hybridization Preferably, the sequence is different from the sequence of the sequence.   The oligonucleotide may be DNA or RNA, or a chimeric mixture thereof or It may be a derivative or a modified form, and may be single-stranded or double-stranded. Oh Ligonucleotides improve, for example, molecular stability, hybridization properties, etc. To improve, the base moiety, sugar moiety or phosphate backbone may be modified. Oligo Nucleotides can be used to target peptides (eg, host cell receptors in vivo). Or other agents that facilitate transport across cell membranes (eg, See, for example, Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553 , 1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA 84: 648, 1987 PCT Publication No. WO 88/09810), or blood -Agents that promote passage through the brain barrier (eg, PCT Publication No. WO 89/10134) ) Or hybridization-stimulated cleavage agents (eg, Krol et al., Bi). oTechniques 6: 958, 1988), or intercalating agents (eg, Zon, Pharm. Res. . 5: 539, 1988). For this reason, oligonucleotides, for example, Peptide, hybridization-stimulated cross-linking agent, transport agent or hybridization Can be conjugated to another molecule, such as a stimulatory cleavage agent.   Antisense oligonucleotides include 5-fluorouracil, 5-bromouracil , 5-chlorouracil, 5-indouracil, hypoxanthine, xanthine, 4-acetone Tylcytosine, 5- (carboxyhydroxymethyl) uracil, 5-carboxymethy Ruaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, Dihydrouracil, β-D-galactosylkeosin, inosine, N6-isopentenyl Adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-meth Tiladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6- Adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamido Nomethyl-2-thiouracil, β-D-mannosylkeosin, 5'-methoxycarboxy Methyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenyl Uracil-5-oxyacetic acid (v), wybutoxosine, shoe Douracil, keosine, 2-thiocytosine, 5-methyl-2-teourashi (Theourasil), 2-thiouracil, 4-thiouracil, 5-methyluracil, ura Syl-5-oxyacetic acid methyl ester, 5-methyl-2-thiouracil, 2- (3-amino-3- N-2-carboxypropl uracil, (acp3) w and 2,6-dia At least one selected from the group including, but not limited to, minopurine It may include a modified base moiety.   Antisense oligonucleotides also include arabinose, 2-fluoroarabino Selected from the group including, but not limited to, glucose, xylulose and hexose. It may include at least one selected modified sugar moiety.   In yet another aspect, the antisense oligonucleotide comprises a phosphorothioate , Phosphorodithioate, phosphoramidothioate, phosphoramidate G, phosphole diamidate, methyl phosphonate, alkyl phosphotriester And formacetal, or analogs of any of these skeletons. At least one modified phosphate skeleton selected from the group consisting of   In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligo. Nucleotides. α-anomeric oligonucleotides are different from normal β-units. Form a specific double-stranded hybrid with complementary RNA whose strands are parallel to each other (Gautier et al., Nucl. Acids. Res. 15: 6625, 1987). Oligonu Nucleotides are 2'-O-methyl ribonucleotides (Inoue et al., Nucl. Acid s Res. 15: 6131, 1987) or chimeric RNA-DNA analogs (Inoue et al., FEBS Lett. 215: 327, 1987).   The antisense oligonucleotide of the present invention can be produced by, for example, (Biosearch (Bios earch), available from Applied Biosystems, etc. The use of an automated DNA synthesizer (such as those described in Can be synthesized by a conventional method. As an example, phosphorothioate oligos Nucleotides can be prepared by the method of Stein et al. (Nucl. Acids Res. 16: 3209, 1988). ), And the methylphosphonate oligonucleotide is controlled Can be prepared by using a porous glass polymer support (Sarin et al., Proc. Natl. Acad. Sci. USA 85: 7448, 1988).   Using antisense nucleotides complementary to the Tango-67 coding region sequence It is believed that those complementary to the transcribed untranslated region are most preferred.   Antisense molecules include, for example, cells of the heart, skeletal muscle, thymus, spleen and small intestine. Must be delivered to cells expressing Tango-67 in vivo. A Numerous methods have been developed to deliver antisense DNA or RNA to cells For example, the antisense molecule can be injected directly into a tissue site, or Modified antisense molecules designed to target new cells (eg, targeting cells). Peptide or antibody that specifically binds to a receptor or antigen expressed on the cell surface (Antisense binding to) can be administered systemically.   However, intracellular concentrations of antisense molecules are not sufficient to suppress endogenous mRNA translation. It is often difficult to get it right. Therefore, the preferred method is antisense Oligonucleotides placed under control of strong pol III or pol II promoter Use the recombinant DNA constructs that have been used. Transfect patient target cells By using such a construct to complement the endogenous Tango-67 transcript A sufficient number of base pairs to form a unique base pair, thereby blocking the translation of Tango-67 mRNA The strand RNA is transcribed. For example, when a vector is taken up by a cell, The vector can be introduced in vivo to direct transcription of the vector. this Such vectors are transcribed to produce the desired antisense RNA, It may remain sosome or may be integrated into the chromosome.   Such vectors are prepared by recombinant DNA technology methods standard in the art. Can be built. The vector can be a plasmid, virus, or mammalian cell May be others well known in the art used for replication and expression in E. coli. Expression of the sequence encoding the antisense RNA is expressed in mammalian, preferably human, cells. And any promoter known in the art to act. This Such a promoter may be inducible or constitutive. Such a professional The motor uses the SV40 early promoter region (Bernoist et al., Nature  290: 304,1981); a promoter contained in the 3 'long terminal repeat of Rous sarcoma virus. -(Yamamoto et al., Cell 22: 787-797, 1988); Herpes thymidine Nase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441, 1981); or the regulatory sequence of the metallothionein gene (Brynstar (Br inster) et al., Nature 296: 39, 1988).   Ribozyme   Ribozyme molecules designed to catalytically cleave Tango-67 mRNA transcripts Can be used to block Tango-67 mRNA translation and Tango-67 expression Yes (eg, PCT Publication No. WO 90/11364; Saraver) Et al., Science 247: 1222, 1990). MRNA in site-specific recognition sequence Various Ribozymes That Cleave Tango-67 mRNA Can Be Used to Disrupt Tango-67 mRNA Although possible, hammerhead ribozymes are preferred. Hammerhead ribozyme Inserted by flanking a region that forms a complementary base pair with the target mRNA Position To cleave the mRNA. The only requirement is that the target mRNA has the following two base sequence: 5'-UG- 3 '. Construction and production of hammerhead ribozymes is a matter of skill in the art. It is well known (Haseloff et al., Nature 334: 585, 1988). Human Tango- Cleavage within the nucleotide sequence of the 67 cDNA by the hammerhead ribozyme There are numerous examples of potential sites. Preferably, the cleavage recognition site is Tango-67 m Located near the 5 'end of the RNA, i.e., increase the efficiency of non-functional mRNA transcripts Manipulate ribozymes to minimize intracellular accumulation.   The ribozymes of the present invention also include Tetrahymena Thermor. mophila) and naturally occur in Cech et al. (Zaug et al., Science 224). : 574, 1984; Zaug et al., Science, 231: 470, 1986; Zug et al., Natu. re 324: 429, 1986; PCT Application No. WO 88/04300; and Bean n) et al., Cell 47: 207, 1986) (IVS or L-1). 9 RNA endoribonucleases (hereinafter known as IVSRNA) ch) -type ribozyme). Cech-type ribozymes are Has an eight base pair sequence that hybridizes to the sequence and subsequently cleaves the target RNA. I do. The present invention targets eight base pair active site sequences present in Tango-67. And Cech-type ribozymes such as   As in the antisense method, ribozymes contain modified oligonucleotides. (Eg, to improve stability, targeting), eg, heart, skeleton Cells expressing Tango-67 in vivo, such as muscle, thymus, spleen and small intestine Must be transported to A preferred delivery method is post-transfection. The cells destroy enough of the endogenous Tango-67 message and have sufficient resources to block translation. Strong bodily constitutive pol III or pol II promoter It involves using a DNA construct that "encodes" the ribozyme under control. Unlike antisense molecules, ribozymes are catalyzed, so they are more efficient The cell concentration required is lower.   Tango-67 Other methods for reducing the expression of   Expression of the endogenous Tango-67 gene can also be determined using targeted homologous recombination to Inactivating or "knocking out" a gene or its promoter Thus, it can be reduced (see, for example, US Pat. No. 5,464,764). . For example, the endogenous Tango-67 gene (coding region or regulatory region of the Tango-67 gene) Mutated non-functional Tango-67 flanked by DNA homologous to either (ie, Unlinked DNA sequence) is replaced with a selectable marker and / or a negative selectable marker. Cells expressing Tango-67 in vivo with or without a car Cells can be transfected. Of DNA constructs by targeted homologous recombination The insertion inactivates the Tango-67 gene. Such a method is called ES (embryo stem) Cell progeny can be used to produce progeny of animals with inactive Tango-67. Particularly suitable for use in the agricultural sector. But a suitable virus Using a vector, the recombinant DNA construct is directly injected into the required site in vivo. If given or targeted, the method may be adjusted for use in humans. it can.   Alternatively, the regulatory regions of the Tango-67 gene (ie, the Tango-67 promoter and / Or enhancer) to target a deoxyribonucleotide sequence Forms triple helical structure that blocks Tango-67 gene transcription in target cells in vivo Can reduce the expression of the endogenous Tango-67 gene (here (Helene) Anticancer Drug Res. 6: 569, 1981; Helene et al., Ann. N . Y. Acad. Sci. 660: 27, 1992; and Maher, Bioassays 14: 807, 1992).   Of course, in some situations involving many certain stages of the above conditions, examples For example, use immune cells to eliminate primary infections or mediate antitumor responses. In some cases, it may be desirable to enhance the capabilities of Tango-67.   Tango-67 For proteins associated with   The invention also features a polypeptide that interacts with Tango-67. Protein Any method suitable for detecting protein-protein interactions can be used to interact with Tango-67. Identify transmembrane, intracellular or extracellular proteins that work Can be used for Some of the traditional methods that can be used include detailed Co-immunoprecipitation, cross-linking and concentration gradient of proteins from lysate or cell lysates Interaction with Tango-67 and co-purification by chromatography or chromatography columns There is the use of Tango-67 to identify proteins in lysates. These Tsu For sequencing, the Tango-67 polypeptide is full-length Tango-67, a soluble form of Tango-67. It may be the extracellular region, or some other suitable Tango-67 polypeptide. The isolated, interacting proteins were identified, cloned, and then labeled. Use in conjunction with standard techniques to identify proteins with which Tango-67 interacts Can be. For example, the amino acid sequence of a protein that interacts with Tango-67 And in part by techniques well known to those skilled in the art, such as by the Edman decomposition technique. You can use and confirm. The resulting amino acid sequence is Oligos that can be used to screen gene sequences encoding proteins It can be used as a guide to make a nucleotide mixture. School Leaning can be accomplished, for example, by standard hybridization or PCR techniques. Can be implemented. Preparation and screening of oligonucleotide mixture Techniques for doing so are well known. (Ausubel, supra and "PCR Protocol (PCR Protocols): A Guide to Methos and Applicati ons), edited by Innis et al., Academic Press Inc. ), New York, 1990).   Also, directly identify genes encoding proteins that interact with Tango-67 Any method that can be used can be used. These methods include, for example, enzymes, fireflies, Tango-67 fused to markers such as photodye, photoprotein or IgFc domain Labeled Tango-67 polypeptide or Tango-, such as a polypeptide or domain Antibodies using well-known techniques, for example, λgt11 library using 67 fusion proteins Includes screening of expression libraries in a manner similar to the probe method.   There are also methods that can detect protein interactions. Imbi A method for detecting protein interactions in vivo is with a two-hybrid system. (Chien et al., Proc. Natl. Acad. Sci. USA 88: 9578, 1991). This A kit for performing the method of Clontech (California) Available from Palo Alto, California.   Briefly, using such a system, two hybrid proteins Construct plasmids encoding: Tango-67, Tango-67 Fusion to a nucleotide sequence encoding a polypeptide or Tango-67 fusion protein Combination Nucleotide sequence encoding the DNA-binding region of a transcriptional activator protein The other plasmid contains the plasmid as part of the cDNA library. Transcriptional activator fused to cDNA encoding unknown protein It contains a nucleotide sequence encoding the activation region of the tibeta protein. DNA- The regulatory region activates transcriptional activation of the binding region fusion plasmid and cDNA library. A reporter gene (eg, HBS or lacZ) that has a binding site for Transformation of yeast Saccharomyces cerevisiae I do. Both hybrid proteins alone activate reporter gene transcription. Cannot be activated: DNA-binding domain hybrid does not provide activation function Since it is not possible, activation region hybrids bind the activator binding site It is impossible because it cannot be localized. Two hybrid proteins Interaction reconstitutes a functional activator protein and produces a reporter gene. Express the reporter gene detected by the product assay.   A two-hybrid system or related method involves the production of "bait" genes. For screening active region libraries for proteins that interact with Can be used for To illustrate, not to limit, Tango-67 can be used as a bait gene product. Up to the whole genome Alternatively, the cDNA sequence is fused to a DNA encoding an activation region. This library Of bait Tango-67 gene product fused to DNA and DNA-binding region Is co-transformed into a yeast reporter strain and the resulting Transformants are screened for those that express a reporter gene. example For example, a bait Tango-67 gene sequence such as Tango-67 or a region of Tango-67 Is translatably fused to the DNA encoding the DNA-binding region of the GAL4 protein. As such, it can be cloned into a vector. Purify these colonies Then, a library plasmid responsible for expression of the reporter gene is isolated. Then, using DNA sequencing, the library plasmid-encoded protein To identify proteins.   Using methods commonly used in the art, bait Tango-67 CDNA library of the cell line where the protein interacting with the progeny product will be detected Can be produced. According to the particular system described herein, The cDNA fragment to be translatably fused to the transcriptional activation region of GAL4. Alternatively, the cDNA fragment can be inserted into a vector. Bait this library it) Together with the Tango-67 gene-GAL4 fusion plasmid, Co-transformation into a yeast strain with a lacZ gene driven by a motor Can be. Coat proteins that interact with the bait Tango-67 gene product. CDNA fused to the GAL4 transcription activation region reconstitutes the active GAL4 protein And thereby drive expression of the HIS3 gene. Next, colonies expressing HIS3 Was purified from these strains and purified from the bay using techniques commonly used in the art. Bait Tango-67 gene-producing and isolating interacting proteins it can.   Tango-67 Receptor identification   The Tango-67 receptor can be identified as follows. First, combine Tango-67 Cells or tissues to be identified. Using mRNA isolated from Tango-67 binding cells Prepare an expression library. Using an expression library, for example, CHO cells Transfect eukaryotic cells such as Tango-67 labeled for detection And clones that bind to Tango-67 are isolated and purified. Then, the expression plasmid Are isolated from the Tango-67-binding clone. These expression plasmids are Tango-67 It encodes what is measured as a receptor.   Exposure of various cell lines and tissues to detectable Tango-67 Thus, cells or tissues having the Tango-67 receptor can be identified. Ma Alternatively, determine whether the selected cells respond to the presence of a cell receptor ligand For this purpose, a microphysiology measuring device can be used (McConnel ) Et al., Science 257: 1906, 1992).   Identify compounds that bind to Tango-67 using any standard binding assay Can be For example, a candidate compound can be bound to a solid support. Next Then, Tango-67 is exposed to the immobilized compound and binding is measured (European Patent Application No. 84 / 03564).   Effective dose   Toxicity of polypeptides of the invention and compounds that modulate their expression or activity And the therapeutic effect depends on cultured cells or LD₅₀(Dose that kills 50% of the population) and And ED₅₀(A dose that produces a therapeutic effect in 50% of the population) It can be determined by standard pharmaceutical techniques used. Toxic and therapeutic effects The dose ratio is the therapeutic index, LD₅₀/ ED₅₀Can be expressed as a ratio of Great treatment Polypeptides or other compounds that exhibit an index are preferred. Compounds showing toxic side effects Can be used, but minimize the potential for damage to uninfected cells, and Target such compounds to the affected tissue site to reduce side effects. Attention must be paid to the design of the transport system.   Use data from cell culture assays and animal studies for human use Can be used in preparing a dose range of The dose of such compounds is Preferably, ED₅₀Concentration in circulating fluid with little or no toxicity Is obtained. The dosage depends on the dosage form used and the route of administration used. Accordingly, it may vary within this range. Any compound used in the method of the present invention Thus, a therapeutically effective amount can be estimated initially from cell culture assays. IC determined by cell culture₅₀(Ie, trials that produce half the maximal inhibition of symptoms Doses to achieve the circulating plasma concentration range in animal models. Can be manufactured. Use this information to determine useful doses in humans. Can be determined more accurately. Plasma concentrations can be determined, for example, by high performance liquid chromatography. It can be measured by fee.   Formulations and uses   According to the invention, using one or more physiologically acceptable carriers or excipients, Pharmaceutical compositions for use can be formulated in conventional manner.   Thus, inhalation or insufflation (oral or nasal) or oral, buccal, parenteral Alternatively, for administration by rectal administration, the compounds of the invention and their physiological Salts and solvates that are well-tolerated can be formulated.   For oral administration, the pharmaceutical composition may include, for example, a binder (eg, Tinned corn starch, polyvinylpyrrolidone or hydroxypro A filler (eg, lactose, microcrystalline cellulose or liquor). Lubricating agents (eg, magnesium stearate, talc Disintegrants (eg potato starch, starch glycolate) Thorium); or a pharmaceutical such as a wetting agent (eg, sodium lauryl sulfate) Tablets or capsules formulated by conventional means using excipients that are It may be in the form of a file. Tablets may be coated by methods well known in the art. it can. Liquid preparations for oral administration include, for example, solutions, syrups or suspensions. Or they can be dissolved at the time of use in water or other suitable substrate. It may be provided as a dry formulation to be dissolved. Such liquid preparations may be used as a suspending agent (eg, For example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); Emulsifiers (eg, lecithin or gum arabic); non-aqueous bases (eg, Oil, oily esters, ethyl alcohol or fractionated vegetable oils); Solvent (eg, methyl or propyl-p-hydroxybenzoate or sorbin) Formulation) by conventional means using pharmaceutically acceptable excipients such as Can be. The formulation may optionally contain buffer salts, flavoring, coloring and sweetening agents. . Formulations for oral administration are suitably formulated to give controlled release of the active compound. be able to.   For buccal administration, the composition may comprise tablets or lozenges formulated in conventional manner It may be in the form of an agent.   For administration by inhalation, the compounds for use according to the invention include, for example, Dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro Using a suitable propellant, such as roethane, carbon dioxide or other suitable gas Easily transported from pressurized fillings or nebulizers in the form of an aerosol spray formulation Sent. In the case of pressurized aerosols, provide a valve to carry a metered amount Will determine the dosage unit. Compounds of the invention and lactose or den Inhalers or insufflators containing a powder mixture with a suitable powder base such as a pour Formulation of capsules and cartridges of eg gelatin for use in be able to.   For parenteral administration by injection, for example, as a single bolus injection or continuous infusion In addition, the compounds of the present invention can be formulated. Formulations for injection include preservatives Attachment For example, in unit dosage form in ampoules or in multidose containers. Can be The composition may be a suspension, solution or emulsion of an oily or aqueous base. Such as a suspending agent, a stabilizing agent and / or a dispersing agent. A formulation agent may be contained. Alternatively, the active ingredient may be sterilized at the time of use, for example, It may be in the form of a powder that dissolves in a suitable base such as water containing no exothermic component.   The compounds of the present invention may also include compounds such as cocoa butter or other glycerides. Formulated in the form of a suppository containing the conventional suppository base or in a rectal composition such as a retention enema Can be   In addition to the formulations described previously, the compounds of the present invention may also be formulated as a depot preparation. can do. Such long acting formulations may be implanted (for example, subcutaneously or Intramuscular) or by intramuscular injection. Therefore, For example, the compounds of the present invention can be prepared using any suitable polymer or hydrophobic material (eg, (Such as an emulsion of possible oils) or ion exchange resins. Or as a slightly solvable derivative, for example a slightly solvable salt Can be   If desired, the compositions of the present invention may contain one or more units containing the active ingredient. Can be provided in the form of a pack or dispenser, which can contain dosage forms it can. Packs can be metal or plastic, for example, blister packs May include foil. The pack or dispenser may be accompanied by instructions for administration.   The therapeutic compositions of the present invention may contain carriers or excipients, many of which are It is well known. Excipients that can be used include buffers (eg, citrate buffer). Buffer, phosphate buffer, acetate buffer and bicarbonate buffer), amino acid, urea , Alcohol, ascorbic acid, phospholipids, proteins (eg, serum albumin) ), EDTA, sodium chloride, liposomes, mannitol, sorbitol and Contains glycerol. The nucleic acids, polypeptides, antibodies or modifying compounds of the invention can be any It can be administered by any standard route of administration. For example, administration is parenteral, Intravenous injection, subcutaneous, intramuscular, intracranial, intraorbital, ocular, intraventricular, intracapsular, intraspinal, intracisternal May be intraperitoneal, transmucosal or oral. The modifying compound may have a corresponding route of administration. Can be formulated by various methods. For example, liquids are oral or injectable Made for oral, inhalation or topical application It is. Methods for producing such formulations are well known and include, for example, "Remington's Pharmaceutical Chemistry (Remington's Pharmaceutical Sciences). The preferred route of administration is expected to be intravenous injection.                                  Example   Example 1 describes the identification and sequencing of the human Tango-67 gene. Example 2 , Tango-67, especially the expression pattern.   Example 1: Cloning of Tango-67 gene   Human Astrocytes (Clonetics Corpor ation), San Diego, California), at the recommendation of the seller Astrocyte Growth Media (AGN; Clontics ( Clonetics)). When cells reach ~ 80 to 90% confluence , 200 units / ml interleukin 1β (Boehringer Ma nnheim), Indianapolis, IN) and cycloheximide (CHI; 4 (0 microgram / ml) for 4 hours. RNeasy Midi Kit (Qiagen) ; Chatsworth, Calif.) To isolate total RNA and Further expose the poly A + fraction using Oligotex beads (Qiagen). Was purified.   Superscript cDNA Synthesis Kit (Superscript cDNA Synthesis Kit) 3 micrograms using Gibco BRL (Gaithersburg, MD) A cDNA library was synthesized using lamb's poly A + RNA. Plasmid live Complementation using SalI and NotI sites of the polylinker to build the rally The specific DNA was cloned in one direction into the expression plasmid pMET7. Remove the transformant Once expanded, expanded and single pass sequencing was performed. Also, λ phage cDNA live To construct the rally, the astrocyte cDNA was transferred to the ZipLox vector (Gibco o) BRL) bound to Sal I / Not I sites. Copy proteins with homology to TSG Identified partial cDNA clones and screened the phage library further. By isolation, a full-length clone of Tango-67 was isolated. Tango-67 is Based on comparison using the GAP program from GCG (Madison, Wis.) , D. It consists of 223 amino acids that are 36% identical to D. melanogasuter TSG. Encodes a protein.   Example 2: Characterization of Tango-67   The expression pattern of Tango-67 was examined as described below.   Tango-67 Analysis of expression   Analyze Tango-67 expression using Northern blot hybridization did. Use PCR with a 410 bp DNA fragment to use as a probe (Corresponding to nucleotides 234 to 643 of SEQ ID NO: 1). Dealer's Prime-It kit (Stratagene, California, CA) La Jolla, A.) Using DNA³²Radiolabeled with P-dCTP. Human mRNA (claw MTNI and MTNII from Clontech, Palo Alto, CA Using a slightly modified manufacturer's recommendations, In ExpressHyb hybridization solution (Clontech) Probed and washed under high stringency. At high stringency Washing was performed for 2 × 20 minutes at 55 ° C. in 2 × SSC, 0.05% SDS; then 0.1 × SSC, 0.1%  2 × 20 minutes at 55 ° C. in SDS.   Tango-67 was found in all tissues examined (spleen, thymus, prostate, testis, ovary, small intestine, Various concentrations in the colon, PBL, heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas) Expressed in degrees. The Tango-67 gene had two copies, in good agreement with the isolated cDNA clone. Transcript, expressed as ~ 4.4 kilobases (kb) and ~ 2.4 kb mRNA. Two rolls Relative abundance varies by tissue, with the exception of testis, which is significantly richer in 4.4 transcripts It is. In the testes, the amounts of the 4.4 and 2.4 kb mRNAs were about the same, and Soy transcripts are observed at ~ 800 bp.                             Deposit information   A plasmid containing the human full-length nucleotide sequence encoding TANGO 67 is On April 16, 1997, the American Type Culture Collection in Rockville, Maryland (American typ e Culture Collection) and was assigned deposit number 98410.   The cultures of the present invention are entitled according to 37 CFR 1.14 and 35 USC 122. Determined by the Director of the Patent and Trademark Office to ensure that the culture is To Deposited under available conditions. Deposits may be made by the corresponding patent in this application or It can be used at the request of the foreign patent law of the country where the substitute application is filed. However The use of the deposit deviates from the patent rights granted by the government to practice the present invention It does not constitute a right.   In addition, the culture deposit of the present invention may be used in accordance with the terms of the Budapest Treaty on the deposit of microorganisms. Is stored and commonly used. In other words, the latest supply At least 5 years after commissioning, and in any case at least 30 years from the date of deposit Or the implementation of a patent by a person who discloses and can grant the culture Survived and free of contamination for 5 years after the latest request for samples from the period plus the date of deposit The deposit will be stored with all necessary care. Required depending on the condition of the deposit If the depository cannot provide the sample at the time of the contract, the depositor is obliged to exchange the deposit Recognize that there is. All restrictions on the general use of the culture deposit of the present invention Are irreversibly removed upon the grant of the patents that disclose them.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/18 C12N 1/15 16/22 1/19 C12N 1/15 1/21 1/19 C12Q 1 / 68 1/21 C12N 15/00 ZNAA 5/10 5/00 A // C12Q 1/68 A61K 37/24

Claims (1)

[Claims] 1. An isolated nucleic acid molecule encoding a Tango-67 polypeptide. 2. 2. The nucleic acid molecule of claim 1, wherein the molecule encodes mature human Tango-67. 3. 2. The isolated nucleic acid molecule of claim 1, wherein the molecule has a nucleotide sequence that encodes a polypeptide having a sequence that is at least 85% identical to the sequence of SEQ ID NO: 2. 4. 2. The isolated nucleic acid molecule of claim 1, wherein the molecule has a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2. 5. 2. The isolated nucleic acid molecule of claim 1, wherein the molecule has a nucleotide sequence of nucleotides 182 to 850 of SEQ ID NO: 1. 6. 2. The isolated nucleic acid molecule of claim 1, wherein the molecule hybridizes under stringent conditions to a nucleic acid molecule having a sequence of nucleotides 182 to 850 of SEQ ID NO: 1 or its complement. 7. A host cell having the isolated nucleic acid molecule of claim 1. 8. A nucleic acid vector having the nucleic acid molecule according to claim 1. 9. 9. The nucleic acid vector according to claim 8, wherein the vector is an expression vector. Ten. 10. The vector according to claim 9, further comprising a regulatory element. 11． Modulators, cytomegalovirus hCMV immediate early gene, early promoter of SV25 adenovirus, the late promoter of SV25 adenovirus, lac system, t rp system, TAC system, TRC system, the major operator and promoter regions of λ phage, fd coat 11. The vector according to claim 10, which is selected from the group consisting of a protein regulatory region, a 3-phosphoglycerate kinase promoter, an acid phosphatase promoter, and a yeast α-mating factor promoter. 12． 11. The vector of claim 10, wherein the regulatory element directs tissue-specific expression. 13. The vector according to claim 9, further comprising a reporter gene. 14. Reporter genes are β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo ^τ , G403 ^τ ), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH) 14. The vector of claim 13, wherein the vector is selected from the group consisting of: thymidine kinase (TK), lacZ (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). 15． 9. The vector according to claim 8, wherein the vector is a plasmid. 16． 9. The vector according to claim 8, wherein the vector is a virus. 17． 17. The vector according to claim 16, wherein the virus is a retrovirus. 18． Substantially pure Tango-67 polypeptide. 19. 19. The polypeptide according to claim 18, wherein said polypeptide is soluble under physiological conditions. 20. 19. The polypeptide of claim 18, wherein the polypeptide has an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2. twenty one. 19. The polypeptide of claim 18, wherein the polypeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2. twenty two. 19. The polypeptide of claim 18, wherein the polypeptide has the same amino acid sequence as SEQ ID NO: 2. twenty three. A substantially pure polypeptide having a first portion having a Tango-67 polypeptide and a second portion having a detectable marker. twenty four. An antibody that selectively binds to a Tango-67 polypeptide. twenty five. 25. The antibody according to claim 24, wherein said antibody is a monoclonal antibody. 26. A pharmaceutical composition comprising the polypeptide of claim 18. 27. A method for detecting Tango-67 in a sample, comprising: (a) obtaining a biological sample; and (b) under stringent conditions to form a Tango-67-antibody complex. Contacting a biological sample with an antibody that specifically binds to Tango-67; and (c) detecting the complex, if present, as an indicator of the presence of Tango-67 in the sample And a method comprising: 28. A method for identifying a compound that regulates the expression of Tango-67, comprising comparing the degree of expression of Tango-67 in cells in the presence and absence of the selected compound, in the presence of the selected compound. And a method wherein the difference in the degree of expression in the absence is indicative that the selected compound modulates Tango-67 expression. 29. A method for identifying a compound that modulates the activity of Tango-67, comprising the step of comparing the degree of Tango-67 activity in cells in the presence and absence of the selected compound, in the presence of the selected compound. And a method wherein the difference in the degree of activity in the absence is indicative that the selected compound modulates the activity of Tango-67. 30. A method for preparing a medicament for treating a patient suffering from a disease associated with overexpression or activity of Tango-67, comprising a compound that inhibits the expression or activity of Tango-67 and a pharmaceutically acceptable agent. Mixing with a carrier. 31. A method for preparing a medicament for treating a patient suffering from a disease associated with insufficient expression or activity of Tango-67, comprising a compound that increases expression or activity of Tango-67 and a pharmaceutically acceptable agent. Mixing with a carrier to be prepared. 32. A method for diagnosing a disease associated with abnormal expression of Tango-67, comprising obtaining a biological sample from a patient, and measuring the expression of Tango-67 in the biological sample. Wherein the increased or decreased expression of Tango-67 in the biological sample relative to a control indicates that the patient has a disease associated with abnormal expression of Tango-67. 33. A method for diagnosing a disease associated with abnormal Tango-67 activity, comprising obtaining a biological sample from a patient, and measuring the activity of Tango-67 in the biological sample. A method wherein an increase or decrease in the activity of Tango-67 in said biological sample as compared to a control indicates that said patient has a disease associated with abnormal activity of Tango-67. 34. SEQ ID NO: 1 or an isolated nucleic acid molecule that hybridizes to a nucleic acid molecule having a sequence at positions 182 to 850 of the nucleotide of the complement thereof. 35. An isolated nucleic acid molecule having a sequence that is at least 95% identical to the sequence of nucleotides 182-850 of SEQ ID NO: 1. 36. A polypeptide encoded by the nucleic acid molecule according to any one of claims 34 or 35.