JP2001512003A - Tango-78, Tango-79 and Tango-81 polypeptides, nucleic acid molecules encoding Tango-78, Tango-79 and Tango-81 and uses thereof - Google Patents

Abstract

  (57) [Summary] The present invention relates to Tango-78, Tango-79 or Tango-81 polypeptides, nucleic acid molecules encoding Tango-78, Tango-79 or Tango-81, and uses thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the discovery and characterization of genes encoding Tango-78, Tango-79 and Tango-81.

[0002] The present invention relates to an isolated nucleic acid molecule encoding Tango-78, Tango-79 or Tango-81, a Tango-78, Tango-79 or Tango-81 protein sequence described herein (SEQ ID NO: : 2, an isolated nucleic acid molecule encoding a polypeptide substantially identical to SEQ ID NO: 4 or SEQ ID NO: 6), as well as Tango-78, Tango-79 as described herein.
Or, under stringent conditions, to the protein-encoding portion of the Tango-81 nucleic acid molecule,
Characterized by isolated nucleic acid molecules that hybridize.

[0003] The present invention relates to isolated nucleic acid molecules encoding Tango-78, Tango-79 or Tango-81, nucleic acid vectors (eg, expression vectors; vectors containing regulatory elements; cytomegalovirus hCMV immediate-early genes, SV40, early promoter of SV40 adenovirus
Adenovirus late promoter, lac , trp , TAC , TRC, phage λ
A vector containing a regulatory element selected from the group consisting of a major operator and promoter region of fd, a control region of fd coat protein, a promoter of 3-phosphoglycerate kinase, a promoter of acid phosphatase and a promoter of yeast α-mating factor; tissue a vector comprising a regulatory element that directs the specific expression; beta-Rakutama over Ze, chloramphenicol acetyl transferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neo ^r, G41
8 ^r ), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding β-galactosidase) and xanthine guanine phosphoribosyltransferase (XGPR
A vector containing a reporter gene selected from the group consisting of T); a vector being a plasmid; a vector being a virus; and a host cell containing a vector being a retrovirus.

In another embodiment, the invention relates to substantially pure Tango-78, Tango-79 or Tang-79.
o-81 polypeptides (eg, Tango-78, Tango-79 or Tango-81 polypeptides that are soluble under physiological conditions; Tango-78, Tango-79 or Tang containing a signal sequence)
o-81 polypeptide; a Tango-78 polypeptide that is at least 85%, 90%, 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2; at least 85%, 90% of the amino acid sequence of SEQ ID NO: 4 A Tango-79 polypeptide that is at least 85%, 90%, 95% or 100% identical to the amino acid sequence of SEQ ID NO: 6.
79 polypeptides.

[0005] In another embodiment, the present invention provides a substantially pure form comprising a first portion comprising a Tango-78, Tango-79 or Tango-81 polypeptide and a second portion comprising a detectable marker. Characteristic polypeptides.

[0006] The invention also relates to a polypeptide of the invention (Tango-78, Tango-79 or Tango-81).
An antibody such as a monoclonal antibody that selectively binds to

[0007] The invention also features a pharmaceutical composition comprising Tango-78, Tango-79, or Tango-81.

[0008] The present invention also provides a method for diagnosing a disease associated with abnormal expression of Tango-78, comprising obtaining a biological sample from a patient and measuring the expression of Tango-78 in the biological sample. Wherein the increase or decrease in the expression of Tango-78 in the biological sample relative to the control is indicative of the patient being afflicted with a disease associated with abnormal expression of Tango-78. .

The present invention also provides a method for diagnosing a disease associated with abnormal expression of Tango-79, comprising obtaining a biological sample from a patient and measuring the expression of Tango-79 in the biological sample. Wherein the increase or decrease in expression of Tango-79 in the biological sample relative to the control is indicative of the patient having a disease associated with abnormal expression of Tango-79. .

[0010] The present invention also provides a method for diagnosing a disease associated with abnormal expression of Tango-81, comprising obtaining a biological sample from a patient and measuring the expression of Tango-81 in the biological sample. Wherein the increase or decrease in expression of Tango-78 in the biological sample as compared to the control is indicative of the patient having a disease associated with aberrant expression of Tango-81. .

The present invention relates to an isolated nucleic acid molecule encoding Tango-78, Tango-79 or Tango-81 or a polypeptide fragment thereof; a vector having these nucleic acid molecules;
cells having recombinant DNA encoding ngo-78, Tango-79 or Tango-81; Tango-7
8, a fusion protein comprising Tango-79 or Tango-81; Tango-78, Tango-79 or Tango
Transgenic animals that express -81; including recombinant knockout animals that cannot express Tango-78, Tango-79 or Tango-81.

[0012] The invention includes nucleic acids having a sequence that is substantially identical to the nucleic acid sequence of Tango-78, Tango-79 or Tango-81. A nucleic acid sequence that is substantially identical to a given reference nucleic acid sequence is referred to herein as the sequence of the given reference nucleic acid sequence, such as, for example, the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. It is defined as a nucleic acid having a sequence having at least 85%, preferably at least 90%, more preferably 95%, 98%, 99% or more identity.

[0013] The present invention includes polypeptides having a sequence substantially identical to the amino acid sequence of Tango-78, Tango-79 or Tango-81. A polypeptide that is “substantially identical” to a given reference polypeptide is at least 85% identical to the sequence of the given reference polypeptide sequence, eg, the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6. %
, Preferably a polypeptide having a sequence having at least 90%, more preferably 95%, 98%, 99% or more identity.

[0014] The nucleic acid molecule of the present invention can be inserted into a vector that promotes expression of the insert as follows. Nucleic acid molecules and the polynucleotides they encode can be used directly as diagnostic or therapeutic agents, or (in the case of polypeptides) can be used to produce antibodies that are therapeutically useful. Thus, expression vectors having the nucleic acid molecules of the invention, cells transfected with these vectors, the expressed polypeptide and (either against the full-length polypeptide or antigenic fragments thereof) formed. Antibodies are included in preferred embodiments.

The transformed cell may be any cell into which a nucleic acid encoding a polypeptide of the present invention (eg, a Tango-78, Tango-79 or Tango-81 polypeptide) has been introduced by recombinant DNA techniques. (Or their ancestors).

An isolated nucleic acid molecule is a nucleic acid molecule that has been separated from the 5 ′ and 3 ′ coding sequences located immediately next to the naturally occurring genome of the organism. The isolated nucleic acid molecule is
Includes non-naturally occurring nucleic acid molecules, for example, nucleic acid molecules formed by recombinant DNA techniques.

Nucleic acid molecules include cDNA, genomic DNA, and synthetic (eg, chemically synthesized) DNA.
And both RNA and DNA. If single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand.

The present invention also relates to Tango-78, Ta, preferably under stringent conditions.
A nucleic acid molecule encoding an ngo-79 or Tango-81 polypeptide (eg, SEQ ID NO:
1, the nucleic acid molecule that hybridizes to SEQ ID NO: 3 or the polypeptide coding portion of SEQ ID NO: 5). Preferably, the hybridizing nucleic acid molecule is 400 nucleotides, more preferably 200 nucleotides. Preferred nucleic acid molecules that hybridize have the biological activity of Tango-78, Tango-79 or Tango-81.

The present invention also relates to substantially pure or isolated, including those corresponding to the various functional domains of Tango-78, Tango-79 or Tango-81 or fragments thereof.
It is characterized by a Tango-78, Tango-79 or Tango-81 polypeptide.

The polypeptides of the present invention, whether produced by recombinant gene expression or chemically synthesized, can be purified from tissues in which they are naturally expressed using standard biochemical purification methods. May be done.

[0021] Functional polypeptides having one or more of the biological functions or activities of Tango-78, Tango-79 or Tango-81 are also included in the invention. These features are usually included in the Tango-78
, Tango-79 or the ability to bind to some or all of the proteins that bind to Tango-81. A functional polypeptide is considered to be within the scope of the present invention if it acts as an antigen to produce an antibody that specifically binds to Tango-78, Tango-79 or Tango-81. In many cases, a functional polypeptide will retain one or more domains present in the native polypeptide.

[0022] A functional polypeptide may have an altered primary amino acid sequence from that disclosed herein. Preferably, these changes are conservative amino acid substitutions, as described herein.

The terms “protein” and “polypeptide” are interchangeable herein to describe any amino acid chain, regardless of chain length or post-translational modification (eg, glycosylation or phosphorylation) Used as Therefore, "Tango-78,
The term "Tango-79 or Tango-81 polypeptide" refers to the full-length naturally occurring
Tango-78, Tango-79 or Tango-81 protein; full length naturally occurring Tango-78,
It comprises a recombinantly or synthetically produced polypeptide corresponding to Tango-79 or Tango-81; or comprises a specific domain or portion of a naturally occurring protein.
The term also includes mature Tango-78, Tango-79 or Tango-81 to which an amino-terminal methionine (useful for expression in prokaryotic cells) has been added.

As used herein, the term “purified” refers to a cellular component, a viral component, or a culture medium when produced by recombinant DNA techniques, or a chemical when chemically synthesized. A nucleic acid or peptide substantially free of precursors or other chemicals.

[0025] The polypeptide or other compound of interest is at least one compound of interest.
When present in a formulation that is 60% by weight (dry weight), it is said to be "substantially pure." Preferably, the formulation is at least 75% by weight of the compound of interest, more preferably at least 90% by weight, most preferably at least 9% by weight.
9%. Purity can be measured by any appropriate standard method, such as, for example, column chromatography, polyacrylamide gel electrophoresis or HPLC analysis.

When a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined chain length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. . Thus, a peptide that is 50% identical to a reference polypeptide that is amino acid chain 100 can be a 50 amino acid polypeptide that is completely identical to the 50 amino acid chain portion of the reference polypeptide. It may be a 100 amino acid chain polypeptide that is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rules apply to nucleic acid molecules.

For polypeptides, the chain length of the reference polypeptide chain is generally at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably at least 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 60 nucleotides,
More preferably it is at least 75 nucleotides, most preferably 100 nucleotides or 300 nucleotides.

In the case of polypeptide sequences having less than 100 identity to a reference sequence, the non-identical portions are preferably, but not necessarily, conservatively substituted portions relative to the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and Tyrosine.

[0029] Sequence identity can be determined by sequence analysis software (eg, the Sequence Analysis So
ftware Package of Genetics Computer Group, University of Wisconsin Biote
chnology Center, 1710 University Avenue, Madison, Wisconsin 530
705) and can be measured using the default parameters specified there.

[0030] The invention also features antibodies, such as monoclonal, polyclonal, and engineered antibodies that specifically bind to Tango-78, Tango-79, or Tango-81. "Specifically binds" means, for example, Tango-78, Tango-79 or Tago of the present invention.
An antibody that recognizes and binds to a particular antigen, such as an ngo-81 polypeptide, but does not substantially recognize and bind to other molecules in a sample, such as a biological fluid, containing the polypeptide.

The present invention also provides an antagonist or agonist of Tango-78, Tango-79 or Tango-81 that inhibits or enhances one or more of the biological activities of Tango-78, Tango-79 or Tango-81, respectively. It is characterized by. Preferred antagonists are small molecules (ie,
Antibodies that bind and "neutralize" Tango-78, Tango-79 or Tango-81 (as described below); macromolecules (ie, molecules with a molecular weight greater than about 500); A polypeptide in which native Tango-78, Tango-79 or Tango-81 competes for binding to a functional binding partner of the native protein; and Tango-78, Tango-79 or
It may include nucleic acid molecules that suppress transcription of Tango-81 (eg, antisense nucleic acid molecules and ribozymes). Tango-78, Tango-79 or Tango-81 agonists also include small and large molecules and antibodies other than neutralizing antibodies.

The present invention also relates to Tang (eg, by affecting transcription or translation).
It is characterized by a molecule that increases or decreases the expression of o-78, Tango-79 or Tango-81. It can be used to inhibit the expression of small molecules (ie, molecules with a molecular weight less than about 500), macromolecules (ie, molecules with a molecular weight greater than about 500) and Tango-78, Tango-79 or Tango-81. Nucleic acid molecules (eg, antisense and ribozyme molecules) or nucleic acid molecules that can be used to increase their expression (eg, binding to a Tango-78, Tango-79 or Tango-81 transcription regulatory sequence,
Molecules that increase transcription).

The invention also features substantially pure polypeptides that functionally interact with Tango-78, Tango-79, or Tango-81, and nucleic acid molecules that encode them.

The present invention relates to a protein of the present invention (ie, Tango-78, Tango-79 or Tango-81).
And methods for treating diseases associated with abnormal expression or activity of Therefore,
The invention includes a method for treating a disease associated with overexpression or activity of a protein of the invention. Such methods include administering a compound that reduces the expression of a protein of the invention. The invention also includes a method for treating a disease associated with insufficient expression or activity of a protein of the invention. These methods include administering a compound that increases the expression or activity of a protein of the invention.

The invention also features a method for detecting a protein of the invention. Such methods involve obtaining a biological sample, contacting the sample with an antibody that specifically binds to the protein under conditions that permit specific binding, and any antibody-protein formed. Detecting the complex.

The invention also includes methods and compositions for the diagnostic evaluation, classification and prognosis of diseases associated with inappropriate expression or activity of Tango-78, Tango-79 or Tango-81. For example, nucleic acid molecules of the invention can be used, for example, to detect inappropriate expression of Tango-78, Tango-79 or Tango-81, or diagnostics for detecting mutations in the Tango-78, Tango-79 or Tango-81 gene. It can be used as a hybridization probe.
Such methods can be used to classify cells according to Tango-78, Tango-79 or Tango-81 expression levels.

Accordingly, the present invention provides a method for diagnosing a disease associated with the abnormal activity of the protein of the present invention, comprising obtaining a biological sample from a patient and measuring the activity of the protein in the biological sample. Wherein the increase or decrease in activity in the biological sample compared to the control is
It features a method of indicating that a patient has a disease associated with abnormal activity of a protein of the invention.

The nucleic acid molecule can be used as a primer in a diagnostic PCR analysis to identify genetic mutations, allelic variations and regulatory sequence deletions in the Tango-78, Tango-79 or Tango-81 gene. . The present invention further provides a diagnostic kit for performing such a method.

The present invention features a method for identifying a compound that regulates the expression or activity of a protein of the present invention by evaluating the expression or activity of the protein in the presence and absence of the selected compound. . A difference in the level of protein expression or activity in the presence or absence of the selected compound indicates that the selected compound can modulate protein expression or activity. Expression can be assessed at either the gene expression level (eg, by measuring mRNA) or the protein expression level by techniques well known to those of skill in the art.

Preferred methods and materials are described in the following examples, which are intended to illustrate, but not limit, the invention. One skilled in the art will recognize that the methods and materials are similar or equivalent to those described herein, and can be used in practicing or testing the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. If there is a contradiction,
It shall be governed by this specification, including definitions. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0042] Other features and advantages of the invention will be apparent from the detailed description and from the claims.

DETAILED DESCRIPTION Tango-78, Tango-79, and Tango-81 Nucleic Acid 27 Molecules The Tango-78, Tango-79, and Tango-81 nucleic acid molecules of the present invention can be cDNA, genomic DNA, synthetic DNA or RNA. And may be double-stranded or single-stranded (ie, either the sense strand or the antisense strand). Fragments of these molecules that are considered to be within the scope of the invention can be made, for example, by the polymerase chain reaction (PCR), or by treatment with one or more restriction endonucleases. Ribonucleic acid (RNA) molecules can be made by in vitro transcription.

[0044] The nucleic acid molecules of the present invention can include naturally occurring sequences or sequences that differ from the naturally occurring sequences, but that encode the same polypeptide due to the degeneracy of the genetic code. Furthermore, these nucleic acid molecules are not limited to sequences encoding only the polypeptide, and thus can include some or all of the non-coding sequences that are upstream or downstream of the coding sequence.

The nucleic acid molecules of the invention can be synthesized (eg, by phosphoramidite-based synthesis) or obtained from a biological cell, such as a mammalian cell. Thus, the nucleic acid may be a human, mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, dog, or cat nucleic acid. Also included are combinations or modifications of nucleotides in these types of nucleic acids.

In addition, the isolated nucleic acid molecules of the present invention include fragments that are not found in the natural state. Thus, the invention provides nucleic acid sequences (eg, Tango-78, Tango-79, or
The isolated nucleic acid molecule encoding Tango-81) is integrated into a vector (eg, a plasmid or viral vector) or the genome of a heterologous cell (or the genome of a homologous cell at a location other than the natural chromosomal locus). Such recombinant molecules are included. Recombinant nucleic acid molecules and their uses are described in detail below.

If the nucleic acid molecules of the invention encode or act as antisense molecules, they can be used, for example, to regulate the transcription of the nucleic acid molecules of the invention. With respect to modulation of Tango-78, Tango-79, or Tango-81 mRNA translation, such techniques can be used to diagnose and / or treat disorders associated with apoptotic cell death. These nucleic acids are further described in the text.

The present invention also provides nucleic acid molecules encoding Tango-78, Tango-79 or Tango-81 polypeptides under stringent conditions (eg, SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 3). 5 protein-encoding portion). The cDNA sequences described herein include, for example, nucleic acids encoding homologous polypeptides of other species and Tango-78, Ta in humans and other animals.
They can be used to identify these nucleic acids, including splices of the ngo-79 or Tango-81 genes. Accordingly, the invention features methods for detecting and isolating these nucleic acid molecules. Using these methods, a sample (eg, a nucleic acid library, such as a cDNA or genomic library) is prepared using Tango-78, Tang-78.
Contact (ie, "screen") with an o-79 or Tango-81-specific probe. Probes selectively hybridize to nucleic acids that encode the relevant polypeptides (or their complements). Using any of several standard methods, at least 25 nucleotides (eg, nucleotides
Probes can be made that can have 25, 50, 100 or 200 (see, for example, Ausubel et al., "Current Protocols in Molecular Biology, V
ol. 1 ", Green Publishing Associates, Inc., and John Wiley & Sons, Inc., N
Y, 1989). For example, a Tango-78, Tango-79 or Tango-81-specific can be used as a probe to screen a nucleic acid library and thereby detect nucleic acid molecules (within the library) that hybridize to the probe. Probes can be made using PCR amplification methods that use oligonucleotide primers to amplify nucleic acid sequences.

When a double strand occurs, one single-stranded nucleic acid is said to hybridize to the other. This occurs when one nucleic acid has a sequence that is opposite and complementary to the other (this same sequence results in a natural interaction between the sense and antisense strands of the genomic DNA, and the "double helix" Is the basis of the structure). Full complementarity of the hybridizing region is not required for duplex formation; the number of bases in the pair will maintain the duplex under the hybridization conditions used. It only needs to be enough.

Typically, hybridization conditions are low to moderate stringency conditions. These conditions primarily result in specific interactions between perfectly complementary sequences, but also nonspecific interactions between sequences that are not perfectly matched. After hybridization, the nucleic acids are "washed" under moderately or highly stringent conditions to remove some non-specific interactions (
Thus, the nucleic acids that form these duplexes are not completely complementary) and can break the associated duplexes.

As is known in the art, optimal cleaning conditions have been determined empirically,
Often determined by gradually increasing stringency. Parameters that can be altered to affect stringency include primarily temperature and salt concentration. In general, the lower the salt concentration and the higher the temperature, the higher the stringency. Washing can be initiated at a low temperature (eg, room temperature) using a solution containing a salt concentration equal to or lower than the salt concentration of the hybridization solution. Subsequent washing can be performed using a solution having the same salt concentration and gradually increasing temperature. Alternatively, the salt concentration may be reduced and the temperature maintained during the washing step, or the salt concentration may be reduced and the temperature increased. Other parameters may be changed. For example, the use of a destabilizing agent, such as formamide, alters stringency conditions.

[0052] In the reaction of hybridizing nucleic acids, the conditions used to achieve a given level of stringency vary. For example, the conditions for forming a duplex between all nucleic acids that are 85% identical to each other are not one set; hybridization also depends on the unique characteristics of each nucleic acid. Sequence length, sequence composition (eg,
Purine-like nucleotide content versus pyrimidine-like nucleotide content) and the type of nucleic acid (eg, DNA or RNA) affect hybridization. It is also contemplated whether one of the nucleic acids is immobilized (eg, on a filter).

When the salt content is indicated as a relative SSC concentration (a salt solution containing sodium chloride and sodium citrate; 2 × SSC is 10 times more concentrated than 0.2 × SSC):
An example of a transition from lower stringency to higher stringency is as follows. The nucleic acids are hybridized in 2 × SSC / 0.1% SDS (sodium dodecyl sulfate; detergent) at 42 ° C., then 0.2 × SSC at room temperature
Wash in 0.2 × SSC / 0.1% SDS at 42 ° C. (moderate stringency conditions); and 0.1 × SSC at 68 ° C. (high stringency conditions) in 0.1% SDS (low stringency conditions). Washing may be performed using only one of the indicated conditions, or each of the conditions may be used (eg, washing in the above order for 10-15 minutes each). Any or all of the washing may be performed repeatedly. As described above, the optimal conditions vary and can be determined empirically.

A second set of conditions, considered “stringent conditions”, consisted of hybridizing at 50 ° C. with Church buffer (7% SDS, 0.5% NaHPO ₄ , 1 M EDTA, 1%
BSA) and washing is performed in 2 × SSC at 50 ° C.

[0055] Once detected, the nucleic acid molecule can be isolated by any of a number of standard techniques (eg, Sambrook et al., "Molecular Cloning, A Labor").
atory Manual, 2nd edition, Col Spring Harbor Laboratory Press, Cold Spring H
arbor, New York, 1989).

The invention also has (a) any of the foregoing Tango-78, Tango-79 or Tango-81-related coding sequences and / or their complements (ie, “antisense” sequences). An expression vector; (b) a Tango-78, Tango-79 or Tango-81 as described above operably linked to regulatory elements that direct the expression of the coding sequence, examples of which are shown below.
-An expression vector having any of the relevant coding sequences; (c) a nucleic acid sequence unrelated to the nucleic acid sequence encoding Tango-78, Tango-79 or Tango-81, such as a molecule encoding a reporter or marker; -78, an expression vector having, in addition to the sequence encoding the Tango-79 or Tango-81 polypeptide; and (d) having any of the foregoing expression vectors, thereby allowing the nucleic acid of the invention to be expressed in a host cell. Includes genetically engineered host cells that express the molecule.

The recombinant nucleic acid molecule may be a soluble Tango-78, Tango-79 or Tango-81 polypeptide: mature Tango-78, Tango-79 or Tango-81; or a signal sequence-added or homologous signal. Tango-78, Tango-79 or Tango-81 with sequence
May be provided. Full length Tango-78, Tango-79 or Tango-81 polypeptide; a domain of Tango-78, Tango-79 or Tango-81; or a fragment thereof fused to another polypeptide as described below. Is also good. Similarly, a nucleic acid molecule of the invention may encode mature Tango-78, Tango-79 or Tango-81, or a form encoding a polypeptide that promotes secretion. In the latter case, the polypeptide, commonly referred to as a protein, can be converted to an active form, for example, by removing a signal sequence in a host cell. The protein can be converted to an active protein by removing the inactive chain.

The above regulatory elements are well known to those of skill in the art and include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements that drive or regulate gene expression. Such regulatory elements, the cytomegalovirus hCMV immediate gene, the early or late promoters of SV40 adenovirus, la c system, trp system, TAC system, TRC system, the major operator and promoter regions of phage lambda, the control of fd coat protein Region, 3-phosphoglycerate kinase promoter, acid phosphatase promoter and yeast α-mating factor promoter.

Similarly, a nucleic acid may form part of a hybrid gene encoding another polypeptide sequence, for example, a sequence that acts as a marker or reporter. Examples of marker or reporter genes are β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA
), Aminoglycoside phosphotransferase (neo ^r, G418 ^r), dihydro-leaf acid reductase (DHFR), hygromycin -B- phosphotransferase (HPH)
, Thymidine kinase (TK), lacZ (encoding β-galactosidase) and xanthine guanine phosphoribosyltransferase (XGPRT). As with many of the standard approaches involved in practicing the present invention, one skilled in the art will be aware of other useful reagents, such as, for example, markers or other sequences that can perform the function of a reporter. ing. Generally, the hybrid polypeptide is Tango-78, Ta
It includes a first portion that is an ngo-79 or Tango-81 polypeptide and a second portion that is, for example, a reporter or immunoglobulin constant region described above.

Expression systems that can be used for the purpose of the present invention include bacteria (eg, E. coli) transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector having the nucleic acid molecule of the present invention. ) And microorganisms such as B. subtilis; yeast (Saccharomyces and Pichia) transformed with a recombinant yeast expression vector having a nucleic acid molecule of the present invention; Insect cell lines infected with a recombinant viral expression vector (eg, a baculovirus); a recombinant viral expression vector having a Tango-78, Tango-79 or Tango-81 nucleotide sequence (eg, a cauliflower mosaic virus (
CaMV) and tobacco mosaic virus (TMV))
plant cell lines transformed with a recombinant plasmid expression vector (eg, a Ti plasmid) having an ango-78, Tango-79 or Tango-81 nucleotide sequence; or a mammalian cell genome (eg, a metallothionein promoter) or a mammal Mammalian cell lines (eg, COS, CHO, BHK, 293, VERO, Hela, MDCK, WI38) with recombinant expression constructs having promoters derived from viruses (eg, adenovirus late promoter and vaccinia virus 7.5K promoter).
, And NIH 3T3 cells).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the gene product being expressed. For example, large quantities of such proteins are produced to produce pharmaceutical compositions containing Tango-78, Tango-79 or Tango-81 polypeptides, or to form antibodies against these polypeptides. If so, a vector that can direct the expression of large quantities of the fusion protein product that is easily purified may be desirable. Such vectors provide an E. coli expression vector pUR278 (Ruther et al.) That can individually ligate the coding sequence of the insert into the vector in frame with the lacZ coding sequence so that a fusion protein is produced. EMBO J. 2: 1791, 1983); pIN vector (Inoue and Inoue, Nucleic acid Res. 13: 3101-3109, 1985; Van Heeke and Schuster,
J. Biol. Chem. 264: 5503-5509, 1989) and the like.
The pGEX vector can also be used to express a heterologous polypeptide as a fusion protein with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be readily purified from thawed cells by adsorption to glutathione-agarose beads and then elution in the presence of free glutathione. The pGEX vector is designed to include a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa Californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express heterologous genes. The virus propagates in Spodoptera frugiperda cells. The coding sequence for the insert can be cloned individually into non-essential regions of the virus (eg, the polyhedron gene) and placed under the control of an AcNPV promoter (eg, the polyhedron promoter). Successful insertion of the coding sequence inactivates the polyhedron gene and produces a non-occluded recombinant virus (ie, a virus that lacks the proteinaceous coat encoded by the polyhedron gene).
These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (For example, Smi
th et al., J. Virol. 46: 584, 1983; see Smith, US Pat. No. 4,215,051).
.

[0063] In mammalian host cells, a number of viral-based expression systems may be used.
When an adenovirus is used as an expression vector, the nucleic acid molecule of the present invention is
Adenovirus transcription, eg, late promoter and tripartite leader sequence
/ Translation control complex. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Recombinant that can survive in the host after infection and express a Tango-78, Tango-79 or Tango-81 gene product by insertion into a non-essential region of the viral genome (eg, the E1 or E3 region) Viruses are obtained (eg Logan and Shenk
Natl. Acad. Sci. USA 81: 3655-3659, 1984). Specific initiation signals may be required for efficient translation of the inserted nucleic acid molecule. These signals include the ATG start codon and adjacent sequences. If the entire gene or cDNA, including the initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translation control signals may be required. However, if only a portion of the coding sequence is inserted, a heterologous translational control signal, possibly including the ATG start codon, must be provided. In addition, the start codon must be in the same reading frame as the desired coding sequence to ensure translation of the entire insert. These heterologous translational control signals and initiation codons can be of various origins, both natural and synthetic. Expression efficiency is determined by appropriate transcription enhancer elements,
It can be increased by adding a transcription terminator or the like (Bittner et al., Met
hods in Enzymol. 153: 516-544, 1987).

Also, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Protein Products Such modifications (eg, glycosylation) and processing (eg, cleavage) are important for the function of the protein. Different host cells have characteristic and specific post-translational processing and modification mechanisms for proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the appropriate modification and processing of the heterologous protein expressed. To this end, eukaryotic host cells having the appropriate processing of primary transcripts, intracellular devices for glycosylation and phosphorylation of the gene product can be used. The mammalian cell types described above are among those which would act as suitable host cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, a cell line that stably expresses the above Tango-78, Tango-79 or Tango-81 can be prepared. Transforming host cells with a selectable marker and DNA controlled by appropriate expression control elements (eg, promoter, enhancer sequence, transcription terminator, polyadenylation site, etc.) without using an expression vector having a viral origin of replication can do. Following the introduction of the foreign DNA, the engineered cells can be grown for 1-2 days in an enriched media, and then transferred to a selective media. The selectable marker in the recombinant plasmid is resistant to selection, allowing cells to stably transfer the plasmid into the chromosome and proliferate to form colonies. Plasmids are cloned and expanded in cell lines. Advantageously, this method can be used to generate cell lines that express Tango-78, Tango-79 or Tango-81. Such engineered cell lines can be particularly useful in screening and evaluating compounds that affect the endogenous activity of the gene product.

A number of selection systems can be used. For example, herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybaska and Szybalski, Proc. Natl. A
.. cad Sci USA 48: 2026 , 1962) and adenine phosphoribosyltransferase (Lowy et al., Cell 22: 817, 1980) genes tk ^-, hgprt ^- or aprt ^- can be used in the respective cells. Also, antimetabolite resistance can be used as a basis for selecting the following genes: dhfr (Wigler et al., Proc. Natl. Acad. Sci. USA 77: 3567, 1980), which is resistant to methotrexate. ; O'Hare et al., P
roc. Natl. Acad. Sci. USA 78: 1527, 1981); gpt showing resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78: 2072, 19).
81); neo showing resistance to aminoglycoside G-418 (Colberre-Garapin et al.,
J. Mol. Biol. 150: 1, 1981); and shows resistance to hygromycin (Santerre et al., Gene 30: 147, 1984).

The nucleic acid molecules of the invention are useful for diagnosing a disease associated with abnormal expression of Tango-78, Tango-79 or Tango-81. Tango-78, Tango-79 or Tango-81 nucleic acid molecules are also useful for gene mapping and chromosome identification.

Tango-78, Tango-79 or Tango-81 polypeptides The Tango-78, Tango-79 or Tango-81 polypeptides described herein are those encoded by any of the nucleic acid molecules described above. Tango-78, Tango-79
Or Tango-81 fragments, variants, truncations and fusion proteins. These polypeptides can be used as agents in diagnostic assays to identify Tango-78, Tango-79 or other cellular gene products or compounds that can modulate the activity or expression of Tango-81 or Tango-78, Tango-78, Pharmaceutical agents useful for treating diseases associated with aberrant expression or activity of -79 or Tango-81 can be made for a variety of uses, including but not limited to antibody formation .

Preferred polypeptides include substantially pure Tango-78, Tango-79, including those having the full-length signal sequence and those corresponding to the secreted polypeptides.
Or a Tango-81 polypeptide.

The present invention also includes polypeptides that are functionally equivalent to Tango-78, Tango-79 or Tango-81. These polypeptides are found in biological systems as Tango-78, Tango-79 or
It is equivalent to Tango-78, Tango-79 or Tango-81 in that it can perform one or more of the functions of Tango-81. Preferred Tango-78, Tango-79 or Ta
ngo-81 provides 20%, 40%, 50%, 75%, 80% or even 90% of one or more of the biological activities of mature human Tango-78, Tango-79 or Tango-81 in full length. Have. Such comparisons are generally based on assays of biological activity using equal concentrations of the polypeptides and comparing. The comparison is also based on the amount of polypeptide required to reach 50% of the maximum stimulus that can be obtained.

A functionally equivalent protein may have, for example, amino acid residue additions or substitutions. Substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues involved. Generally, amino acids that are believed to confer conservative substitutions with one another are specified in the Summary of the Invention.

[0072] Polypeptides that are functionally equivalent to Tango-78, Tango-79 or Tango-81 can be made using random mutation techniques well known to those skilled in the art. However, such polypeptides are more likely to be formed by site-directed mutagenesis (using techniques well known to those skilled in the art). These polypeptides may have increased functions or reduced functions.

To design a functionally equivalent polypeptide, it is useful to distinguish between conserved and variable positions. This can be done by aligning the amino acid sequence of a protein of the invention from one species with homologs of another species. One of skill in the art recognizes that conserved amino acid residues are likely to be required for maintenance of function. Therefore, it is preferred that the conserved residues remain unchanged.

To create a mutant gene that is more suitable for expression in a selected host cell,
Mutations in the coding sequence of the nucleic acid molecules of the invention can be made. For example, the N-linked glycosylation sites can be altered or eliminated to effect expression of homologous products that are more easily recovered and purified from yeast hosts well known, for example, to hyperglycosylate N-linked sites. To this end, various amino acid substitutions at one or both of any one or more of the first or third amino acid positions of the resulting glycosylation recognition sequence and / or any one or more of the second or any second amino acid positions of such a recognition sequence. Amino acid deletion at position prevents glycosylation of the modified tripeptide sequence (eg, Mi
yajima et al., EMBO J. 5: 1193, 1986).

A polypeptide of the invention may be expressed in fusion with another polypeptide, such as a marker polypeptide or a fusion partner. For example, a polypeptide can be fused with a hexa-histidine tag to facilitate purification of a bacterially expressed protein, or fused with a hemagglutinin tag to facilitate purification of a protein expressed in prokaryotic cells. it can.

[0076] The fusion protein can be easily purified by using an antibody specific for the fusion protein to be expressed. For example, the system described by Janknecht et al. Allows easy purification of native fusion proteins expressed in human cell lines (Proc. Natl. Acad. Sci. USA 88: 8972). -8976, 1991). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is fused by translation to an amino-terminal tag consisting of six histidine residues. The extract from cells infected with the recombinant vaccinia virus is applied to a Ni ²⁺ nitriloacetic acid-agarose column, and the histidine-tagged protein is selectively eluted with a buffer containing imidazole.

The polypeptides of the present invention may be chemically synthesized (eg, Creighton, “P
roteins: Structures and Molecular Principles, WH Freeman & Co., New York, 1983), or, more advantageously, may be produced by recombinant DNA techniques described herein. For further guidance, those skilled in the art will appreciate that Ausube et al. (Supra), Sambrook et al. (Molecular Cloning, A Laboratoty Manual, Cold Spring Harbor Press,
Cold Spring Harbor, New York, 1989) and especially as examples of chemical synthesis, edited by Gait, MJ, ("Oligonucleotide Synthesis", IRL Press, Oxford
, 1984).

The present invention also interacts with Tango-78, Tango-79 or Tango-81 (and the genes encoding them), thereby altering the function of Tango-78, Tango-79 or Tango-81 Characterized by a polypeptide. Interacting polypeptides can be identified using methods well known to those skilled in the art. One preferred method is to use the "two-hybrid system" to detect protein interactions in vivo.
stem) "(Chien et al., Proc. Natl. Acad. Sci. USA 88: 9578, 1991). Kits for performing this method are available from Clontech (Palo Alto, CA).

Transgenic Animals Tango-78, Tango-79 or Tango-81 polypeptides can also be expressed transgenic. These animals can be Tango-78, Tango-79 or Tang
A model system to study diseases caused or exacerbated by over- or under-expression of o-81 and to develop therapeutics that modulate the expression or activity of Tango-78, Tango-79 or Tango-81 .

Transgenic animals include livestock (pigs, goats, sheep, cows, horses, rabbits, etc.), rodents (rats, guinea pigs, mice, etc.), non-human primates (eg, baboons, monkeys, chimpanzees). And companion animals (eg dogs and cats)
It may be. Transgenic mice are particularly preferred.

Using any technique known in the art, Tango-78, Tango-79, or Tang
The o-81 transgene can be introduced into an animal to create a first generation of transgenic animals. Such techniques include germline microinjection (US Pat. No. 4,873,191); germline retroviral-mediated gene transfer (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82 : 6148, 19
85); Gene targeting in fetal stem cells (Thompson et al.,
Cell 56 : 313, 1989); and electroporation of embryos (Lo, Mol Cell Biol. 3 :
1803, 1983).

The present invention provides transgenic animals that have the Tango-78, Tango-79, or Tango-81 transgene in all of their cells, and animals that have the transgene in some, but not all, of the cells. . That is, the present invention provides a mosaic animal. The transgene can be integrated as a single transgene or as a concatamer, eg, head-to-head or head-to-tail tandem. The transgene can be selectively introduced into specific cell types and activated (Lasko et al., Proc. Natl. Acad. Sci.
USA 89 : 6232, 1992). The regulatory sequences required for such cell type-specific activation include
Depending on the particular cell type of interest, it will be apparent to one of skill in the art.

If it is desired that the Tango78, Tango-79, or Tango-81 transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when using such techniques, endogenous Tango-78, Tango-79,
Alternatively, a vector containing a nucleotide sequence homologous to the Tango-81 gene is integrated into an endogenous gene through chromosomal sequence and homologous recombination, and is designed for the purpose of inhibiting the function of the nucleotide sequence of the endogenous gene. In addition, the transgene is selectively introduced into a particular cell type, whereby the endogenous Tango-78, Ta
The ngo-79 or Tango-81 gene can be inactivated (Gu et al., Science 265 : 103, 1984). The regulatory sequences required for such cell type-specific inactivation will depend on the particular cell type of interest and will be apparent to those skilled in the art. These techniques are useful for creating “knockouts” that lack a functional gene.

Once transgenic animals have been produced, recombinant Tango-78, Tango-79
Alternatively, the expression of the Tango-81 gene can be assayed using standard techniques. Initial screening can be performed by Southern blot analysis or PCR techniques to determine if transgene introduction has occurred. The transgene mRNA expression level in the tissue of the transgenic animal is also assessed using techniques including, but not limited to, Northern blot analysis, in situ hybridization analysis, and RT-PCR of a tissue sample obtained from the animal. be able to. Biological samples can also be assessed immunocytochemically using antibodies specific for the Tango-78, Tango-79 or Tango-81 transgene products.

To refer to techniques that can be used to make and evaluate transgenic animals, one of skill in the art should see Gordon (Intl. Rev. Cytol. 115: 1
71-229, 1989), for example, Hogan et al., “Manipulating the Mouse Embryo” (Cold Spring Harbor Press, Col.
d Spring Harbor, New York, 1986); Krimpenfort
Bio / Technology 9: 86, 1991; Palmiter et al., Cell 41: 343,
1985; Kraemer et al., "Genetic Manipulation of Early Mammal Embryos (Genetic Man
ipularion of the Early Mammalian Embryo, "Cold Spring Harbor Press, Cold Spring Harbor, NY, 1985; Hammer et al., Nature 315: 680, 1985; Purcel et al., Science. , 244: 1281, 1986; Wagner et al., US Patent No.
No. 5,175,385; and Krimpenfort et al., US Pat. No. 5,17.
Additional guidance can be obtained from US Pat. No. 5,384, the latter two of which are incorporated herein by reference.

Anti-Tango-78, Tango-79 or Tango-81 Antibodies Human Tango-78, Tango-79 or Tango-81 polypeptides (or immunogenic fragments or analogs) form antibodies useful in the present invention. Such polypeptides may be produced by recombinant techniques or may be synthesized (eg, “Solid Phase Peptide”, supra;
Ausbel) see above). Generally, the peptide can be conjugated to a carrier protein such as KLH, mixed with an adjuvant, and injected into a host mammal, as described by Ausbel et al. Antibodies can be purified by peptide antigen affinity chromatography.

In particular, various host animals can be immunized by injecting a Tango-78, Tango-79 or Tango-81 polypeptide. Host animals include rabbits, mice, guinea pigs and rats. The various adjuvants that can be used to boost the immune response depend on the host species, including Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, Contains surfactants such as peptides, oil emulsions, keyhole limpet hemocyanin and dinitrophenol. Human adjuvants that may be useful are BCG (bacille Calmette-Guerin) and Corynebacterium parvum (Coryn).
ebacterium parvum). Polyclonal antibodies are heterogeneous populations of antibody molecules contained in the serum of immunized animals.

Thus, the antibodies of the present invention are prepared using polyclonal antibodies, and further using monoclonal antibodies, humanized or chimeric antibodies, single-chain antibodies, Fab fragments, F (ab ′) ₂ fragments, and Fab expression libraries. Containing molecules.

Monoclonal antibodies, which are a homogeneous population of antibodies to a particular antigen, are described in Tang, above.
o-78, Tango-79 or Tango-81 polypeptides and standard hybridoma technology (eg, Kohler et al., Nature 256: 495, 1975; Kohler et al., Eur. J. Immonol. 6: 511,
1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling et al., Monoclonal Antibodies and T cell Hybridomas.
omas) ", Elsevier, New York, 1981; see Ausubel et al., supra).

In particular, monoclonal antibodies are described in Kohler et al., Nature 256: 495, 197.
5 and any technique for producing antibody molecules by continuous cell lines in culture as described in US Pat. No. 4,376,110; human B-cell hybridoma technique (Kosber
Et al., Immunology Today 4: 72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA 80: 2.
026, 1983), and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy), Alan R. Liss, In
c., pp. 77-96, 1983). Such an antibody may be an IgG
, IgM, IgE, IgA, IgD and any subclass thereof. Hybridomas producing the mAbs of the invention can be cultured in vitro or in vivo. High titer mA in vivo
The ability to produce bs makes the present invention a particularly useful production method.

[0091] Once produced, polyclonal or monoclonal antibodies can be made to specific Tango-antibodies by Western blot or immunoprecipitation analysis by standard methods, eg, as described by Ausubel et al., Supra. 78, Tango-79
Or tested for Tango-81 recognition. Antibodies that specifically recognize and bind to Tango-78, Tango-79 or Tango-81 are useful in the present invention. For example, using such antibodies in immunoassays, Tango-78 produced by a mammal (e.g., to determine the amount or location of Tango-78, Tango-79 or Tango-81) in a mammal. Ta
The amount of ngo-79 or Tango-81 can be monitored.

Preferably, the antibodies of the present invention are outside of the highly conserved regions, and are likely to be antigenic by criteria such as the frequency of charged residues.
, Tango-79 or Tango-81 protein. In one specific example, such fragments are produced by standard PCR techniques and then cloned into a pGEX expression vector (Ausubel et al., Supra). The fusion protein is E. coli (E.
coli and purified using a glutathione agarose affinity matrix as described by Ausubel et al., supra.

[0093] It may be desirable to minimize the potential for problems with low affinity or specificity of the antisera. In such a situation, two or three fusions may be formed for each protein, and each fusion may be injected into at least two rabbits. Antisera can be formed by continuous injection, preferably involving at least three booster injections.

The antisera may be tested for the ability to immunoprecipitate a recombinant Tango-78, Tango-79 or Tango-81 protein or control protein such as the glucocorticoid receptor, CAT or luciferase.

For example, as part of a diagnostic assay, Tango-78, Tango-79 or Tango-79 in a biological sample
Antibodies can be used to detect Tango-81. Antibodies can also be used in screening assays to determine the effect of a candidate compound on Tango-78, Tango-79 or Tango-81 expression or localization. Also, for example, to evaluate normal Tango-78, Tango-79 or Tango-81 expressing cells and / or genetically engineered Tango-78, Tango-79 or Tango-81 expressing cells prior to introduction into a patient. Such antibodies can be used in combination with the gene therapy techniques described in.

In addition, it has been developed for the production of “chimeric antibodies” by splicing genes from mouse antibody molecules with appropriate antigen specificity together with genes from human antibody molecules with appropriate biological activity. Natl. Acad. Sci. USA 81 : 6851, 1984; Neuberger et al., Nature 312 : 604, 1984; Takeda et al. Natu
re 314 : 452, 1984). A chimeric antibody is a molecule in which different portions are derived from different animal species, such as an antibody having a variable region derived from a mouse mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single-chain antibodies (US Pat. Nos. 4,946,778 and 4,704,692) can be used to generate single-chain antibodies to Tango-78, Tango-79, or Tango-81 or a polypeptide. Can be applied to produce. Single-chain antibodies are formed by combining the heavy and light chain fragments of the Fv region by amino acid crosslinking to form a single-chain polypeptide.

[0098] Antibody fragments that recognize and bind to a specific epitope can be generated by known techniques. For example, such fragments include F (ab ') ₂ fragments that can be made by pepsin digestion of antibody molecules, and Fabs that can be made by reducing the disulfide bridges of the F (ab') ₂ fragment. Fragments, including but not limited to. Alternatively, a monoclonal Fab fragment with the desired specificity can be quickly and easily identified by constructing an Fab expression library (Huse et al., Science 246 : 1275, 1989).

[0099] Antibodies to Tango-78, Tango-79 or Tango-81 can be used to form anti-idiotype antibodies similar to portions of the protein using techniques well known to those of skill in the art ( See, for example, Greenspan et al., FASEB J. 7: 437, 1993; Nissinoff, J. Immu.
nol. 147: 2429, 1991). For example, using an antibody that binds to the protein and competitively inhibits the binding of the protein binding partner, similar to the binding domain of the protein binding partner,
Accordingly, it is possible to produce an anti-idiotype that neutralizes by binding to a protein binding partner. Such neutralizing anti-idiotype antibodies or Fab fragments of such anti-idiotype antibodies can be used in therapeutic methods.

[0100] Antibodies can be humanized by methods known in the art. For example,
Monoclonal antibodies with the desired binding specificities can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA). Fully human antibodies, such as those expressed in transgenic animals, are also a feature of the invention (Green et al., Nature Genetics 7 : 13-21, 1994; both incorporated herein by reference). U.S. Patent Nos. 5,545,806 and 5,569
, No. 825).

The methods described herein in which an anti-Tango-78, Tango-79, or Tango-81 antibody is used, for example, are associated with abnormal expression of Tango-78, Tango-79, or Tango-81 It can be conveniently used in a clinical setting to diagnose patients exhibiting symptoms of the disease, for example, at least one specific Tango-78, Tango-79, or Tango-81
This can be done by utilizing a pre-packaged diagnostic kit containing the antibody reagent.

Antisense Nucleic Acids Therapeutics based on the “antisense” method include Tango-78, Tango-79 or Tango-79.
Including making oligonucleotides (either DNA or RNA) that are complementary to 81 mRNA. These oligonucleotides may be Tango-78, Tango-79 or Tango-79.
Binds to the complementary transcript of 81 mRNA and inhibits translation. Absolute complementarity is preferred, but not required. A sequence "complementary" to a portion of the RNA referred to herein means a sequence that has sufficient complementarity to hybridize to the RNA and form a stable duplex; In the case of double-stranded antisense nucleic acids, one strand of double-stranded DNA can be tested, ie, the formation of triple strands can be assayed. The ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. In general, the longer the nucleic acid to hybridize, the more base mismatches of the RNA that it has and still forms a stable duplex (or, in some cases, triplex). One skilled in the art can ascertain the tolerable degree of mismatch by using standard procedures for determining the melting point of the hybridized complex.

For example, oligonucleotides that include the AUG start codon and are complementary to the 5 ′ end of the message, such as the 5 ′ untranslated sequence up to the AUC start codon, are most efficient at inhibiting translation. Should work. However, it has been reported that a sequence complementary to the 3 'untranslated sequence of mRNAs is similarly effective in inhibiting the translation of mRNAs (Wagner, Nature 372: 333, 1984). Thus, the 5 'or 3' untranslated gene,
Oligonucleotides complementary to non-coding regions may be used in antisense methods to inhibit translation of endogenous mRNA. Oligonucleotides complementary to the 5 'untranslated region of the mRNA should include the complement of the AUG start codon.

Antisense oligonucleotides complementary to the mRNA coding region are not efficient translation inhibitors, but could be used according to the invention. Whether designed to hybridize to the 5 ', 3' or coding region of the mRNA, the antisense nucleic acid should be at least six nucleotides in length, preferably at least six nucleotides in length. 6 to about 50 oligonucleotides. In specific aspects, the oligonucleotide is at least 10 nucleotides, is at least 17 nucleotides, is at least 17 nucleotides, is at least 25 nucleotides, or is at least 50 nucleotides.

Regardless of the choice of target sequence, in vitro studies preferably first quantify the ability of the antisense oligonucleotide to inhibit gene expression.
These studies preferably employ controls that distinguish between inhibition of the gene by antisense and nonspecific biological effects of the oligonucleotide. In these studies, it is also preferable to compare the amount of the target RNA or protein with the amount of the internal control RNA or protein. It is also contemplated to compare the results obtained using the antisense oligonucleotide with the results obtained using the control oligonucleotide. Preferably, the control oligonucleotide is about the same length as the test oligonucleotide, and the nucleotide sequence of the oligonucleotide differs only to the extent that it does not inhibit specific hybridization of the antisense sequence with the target sequence.

The oligonucleotide may be DNA or RNA or a chimeric mixture or a derivative or modification thereof, single-stranded or double-stranded. Oligonucleotides may be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve molecular stability, hybridization, and the like. The oligonucleotide is
Peptides (eg, to target host cell receptors in vivo) or agents that facilitate transport across cell membranes (eg, Letsinger et al., Proc. Nat.
l. Acad. Sci. USA 86: 6553, 1989; Lemaitre et al., Proc. Natl. Acad. Sci. USA.
84: 648, 1987; described in PCT Publication No. WO 88/09810), or agents that facilitate transport across the blood-brain barrier (eg, PCT Publication No.
88/09810) or cleavage agents that induce hybridization (
See, for example, Krol et al., BioTechniques 6: 958, 1988), or intercalating agents (
For example, Zon, Pharm. Res. 5: 539, 1988). To this end, the oligonucleotide may be conjugated to another molecule such as, for example, a peptide, a hybridization triggered cross-linking agent, a transport agent or a hybridization triggered cleavage agent.

Antisense oligonucleotides include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxyl Methyl)
Uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylcuosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine , 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl- 2-thiouracil, beta-D-mannosylcuosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),
Wybutoxosine, pseudouracil, cuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (
v), 5-methyl-2-thiouracil, 2- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine, It may include at least one modified base moiety.

[0108] The antisense oligonucleotide can also include at least one modified sugar moiety selected from, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotides include, but are not limited to, phosphorothioic acid, phosphorodithioic acid, phosphoramidothioic acid, phosphoramidoic acid, phosphorodiamidic acid, methylphosphonic acid, alkyl phosphotriester, and At least one modified phosphate backbone selected from the group consisting of formacetal or any analog of these backbones.

[0110] In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. The α-anomeric oligonucleotide forms a specific double-stranded hybrid with complementary RNA whose strands are parallel to each other, contrary to the normal β-unit (Gautier et al., Nucl. Acids Res. 15 ： 6625
, 1987). Oligonucleotides are 2'-o-methyl ribonucleotides (Inoue (I
Noue) et al., Nucl. Acids. 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 used, for example, in an automatic DNA synthesizer (for example, Biosearch, Applied Biosystems).
(available from systems) can be synthesized by conventional methods known in the art. For example, phosphorothioic acid oligonucleotides can be synthesized by the method of Stein et al. (Nucl. Acids. Res. 16 : 3209, 1988), and methylphosphoric acid oligonucleotides can be synthesized from glass with controlled pores. It can be prepared by utilizing a polymer support or the like (Sarin et al., Proc. Natl. Acad. Sci. USA 85 : 7448, 1988).

The antisense molecules need to be delivered to cells that express Tango-78, Tango-79, or Tango-81 in vivo. Many methods have been developed to deliver antisense DNA or RNA to cells; for example, antisense molecules can be injected directly into a tissue site or modified to be directed to the desired cell. Antisense molecules (eg, antisense conjugated to a peptide or antibody that specifically binds to a receptor or antigen expressed on the surface of a target cell) can be administered systemically.

However, it is often difficult to achieve sufficient intracellular concentrations of antisense molecules to suppress translation of endogenous mRNA. Thus, a preferred method utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. Utilizing such constructs to transfect patient target cells, endogenous Tango-78, Tang-
Sufficient transcription of single-stranded RNA that forms a complementary base pair with o-79 or Tango-81 transcripts occurs, thereby preventing translation of endogenous mRNA. For example, the vector can be introduced in vivo such that the vector is taken up by cells and transcribes the antisense RNA. Such a vector contains the desired antisense RN
As long as A can be transcribed to produce, it may remain episomal or integrate into the chromosome.

[0114] Such vectors can be constructed by recombinant DNA technology, a method standard in the art. Vectors can be plasmids, viruses or others known in the art used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be driven by a promoter known in the art to act in mammalian, preferably human cells. Such a promoter may be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernoist et al., Nature 290: 304, 1981), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22: 787-797, 1980). , Herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.
USA 78 : 1441, 1981), or the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296 : 39, 1982).

Ribozymes Tango-78, Tango-79 or Tango-81 mRNA The Tango-78, Tango-79 or Tango-81 mRNA using ribozyme molecules made to catalyze the cleavage of the transcript
Translation can be suppressed. (Eg, PCT Publication No. 90/11354; Sar
aver et al., Science 247: 1222, 1990). M at the site-specific recognition sequence
Tango-78, Tango-79 or Tango-81m using various ribozymes that cleave RNA
Although RNAs can be destroyed, the use of hammerhead-type ribozymes is preferred. Hammerhead-type ribozymes cleave mRNAs at locations indicated by flanking regions that form complementary base pairs with the target mRNA. The only requirement is the target m
The RNA has the following two nucleotide sequence: 5'-UG-3 '. The construction and production of hammerhead-type ribozymes is well known in the art (Haseloff et al., Nature 334: 585,
1988). There are many examples in the nucleotide sequence of human Tango-78, Tango-79 or Tango-81 cDNA which are likely to be cleavage sites for hammerhead-type ribozymes. Preferably, ribozymes are generated so that the cleavage recognition site is located near the 5 'end of the mRNA, ie, to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts.

[0116] The ribozymes of the present invention may also include Tetrahymena Ther
mophila) (known as IVS or L-19 IVS RNA) and Cech (Cech)
(Zaug et al., Science 224: 574, 1984; Zaug et al., Science, 231: 470, 1986; Z)
ug, et al., Nature 324: 429, 1986; PCT Application No. WO 88/04300; and RNs, such as those described extensively by Been et al., Cell 47: 207, 1986).
A endoribonuclease (hereinafter referred to as "Cech-type ribozyme"). Cech-type ribozymes have an eight base pair sequence that hybridizes to a target RNA sequence, followed by cleavage of the target RNA. The present invention, Tango-78
Such Cech-type ribozymes that target the eight base pair active site sequence present in Tango-79 or Tango-81.

As with the antisense method, ribozymes may include modified oligonucleotides (eg, to improve stability, targeting, etc.) and must be delivered to cells expressing TLC in vivo. The preferred method of delivery is to use a strong constitutive pol III or pol II promoter so that the transfected cells produce sufficient ribozyme to disrupt endogenous messages and prevent translation. Under control, using a DNA construct that "encodes" the ribozyme. Ribozymes, unlike antisense molecules, have a catalytic effect and therefore can be used at lower intracellular concentrations.

Other Methods to Reduce the Expression of Tango-78, Tango-79 or Tango-81 Expression of an endogenous Tango-78, Tango-79 or Tango-81 gene can be accomplished using targeted homologous recombination. It can also be reduced by inactivating the promoter (see, for example, US Pat. No. 5,464,764). For example, the endogenous Tango-78, Tango-79 or Tango-81 gene (Tango-78, Tango-79 or Tango-79
Mutant flanking DNA that is homologous to either the coding or regulatory region of the 81 gene, and is a non-functional Tango-78, Tango-79 or Tango-81 gene (or completely unrelated
DNA sequences) with or without a selectable marker and / or a minus-strand selectable marker, in vivo, using Tango-78, Tang-
Cells expressing o-79 or Tango-81 can be transfected. By introducing a DNA construct by targeted homologous recombination, Tango-78, Tango-79 or Tango-8
One gene is inactivated. Such methods can be used in agriculture where the use of ES (embryonic stem) cells can produce Tango-78, Tango-79 or Tango-81 offspring in inactive animals. Particularly preferred. However, the method can be adapted for use in humans, provided that the recombinant DNA construct is administered or targeted directly to the required site in vivo using an appropriate viral vector.

Alternatively, expression of the endogenous Tango-78, Tango-79 or Tango-81 gene is
, A deoxyribonucleotide sequence complementary to the regulatory region of the Tango-79 or Tango-81 gene (ie, the Tango-78, Tango-79 or Tango-81 promoter and / or enhancer) to target the target cell in vivo. Tango-78,
It can be reduced by forming a triple helix structure that represses transcription of the Tango-79 or Tango-81 gene (Helene Anticancer Drug Res. 6: 569, 1981;
Helene et al., Ann. NY Acad. Sci. 660: 27, 1992; and Maher, Bioassays.
14: 807, 1992).

Detection of Proteins That Bind to Tango-78, Tango-79 or Tango-81 The invention also features polypeptides that interact with Tango-78, Tango-79 or Tango-81. Use any method suitable for detecting protein-protein interactions to identify transmembrane, intracellular or extracellular proteins that interact with Tango-78, Tango-79 or Tango-81 can do. Conventional methods that can be used include cell lysates or proteins obtained from cell lysates to identify proteins that interact with Tango-78, Tango-79 or Tango-81 in the lysate. Concentration gradient or chromatography column simultaneous-immunoprecipitation, cross-linking and simultaneous-
There is purification. In these assays, Tango-78, Tango-79 or Tango-81 is a full-length Tango-78, Tango-79 or Tango-81, Tango-78, soluble extracellular domain of Tango-79 or Tango-81, and others. Some suitable Tango-78, Tango-79 or Tango-
It may be 81. Once isolated, such interacting proteins can be used in conjunction with standard techniques to identify, clone, and identify interacting proteins. For example, at least a portion of the amino acid sequence of a protein that interacts with Tango-78, Tango-79, or Tango-81 is confirmed using techniques well known to those of skill in the art, such as by the Edman degradation technique. be able to. The resulting amino acid sequence can be used as a guide to form an oligonucleotide mixture that can be used to screen for gene sequences encoding interacting proteins. Screening can be performed, for example, by standard hybridization or PCR techniques. Techniques for forming oligonucleotide mixtures and screening techniques are well known. (Ausu
bel, supra; and "PCR Protocols: A Guide to Methods and Application",
Innis et al., Ed., Academic Press, Inc., New York, 1990).

[0121] In addition, methods directly leading to the identification of a gene encoding a protein that interacts with Tango-78, Tango-79 or Tango-81 can be used. These methods are, for example, in a manner similar to the well-known antibody probe technique of the λgt11 library, for example, Tango-78, Tango-79 or Tango-81 polypeptide fused to an enzyme, a fluorescent dye, a photoprotein or an IgFc domain. Or labeled Tango-78, Tango- like domain
Screening an expression library using a 79 or Tango-81 polypeptide or a Tango-78, Tango-79 or Tango-81 fusion protein.

There are also methods by which protein interactions can be detected. A method for detecting protein interactions in vivo is the two hybrid system (Chien et al., Proc. Natl. Acad. Sci. USA 88: 9578, 1991). Kits for performing this method are available from Clonetech (Palo Alto, CA).

Identification of Tango-78, Tango-79 or Tango-81 Receptors Tango-78, Tango-79 or Tango-81 receptors can be identified as follows. First, cells or tissues that bind to Tango-78, Tango-79 or Tango-81 are identified. Expression libraries are made using mRNA isolated from Tango-78, Tango-79 or Tango-81 binding cells. The expression library is used to transfect eukaryotic cells, eg, CHO cells. Tang labeled for detection
Using o-78, Tango-79 or Tango-81, Tango-78, Tango-79 or Tango-81
Identify clones that bind to These clones are isolated and purified. The expression plasmid is then isolated from a Tango-78, Tango-79 or Tango-81 binding clone. These expression plasmids encode putative Tango-78, Tango-79 or Tango-81 receptors.

Cells that form Tango-78, Tango-79 or Tango-81 receptor by exposing detectably labeled Tango-78, Tango-79 or Tango-81 to various cell lines and tissues Alternatively, the tissue can be identified. Alternatively, a microphysiometer can be used to determine whether selected cells respond to the presence of a cell receptor ligand (McConnel et al., Science 25
7: 1906, 1992).

Compounds that Bind Tango-78, Tango-79 or Tango-81 Tango-78, Tango-79 or Tango-81 using any standard binding assay
Can be identified. For example, a candidate compound can be bound to a solid support. The Tango-78, Tango-79 or Tango-81 is then exposed to the immobilized compound and binding is measured (European Patent Application No. 84/03564).

Effective Amounts The toxic and therapeutic efficacy of the polypeptides of the invention and the compounds that modulate their expression or activity is determined by their LD ₅₀ (the dose that kills 50% of the population) and ED ₅₀ (50% of the population). To determine the therapeutically effective dose) can be determined by standard pharmaceutical techniques using either cultured cells or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be Table serial as the ratio LD ₅₀ / ED _50. Polypeptides or other compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects can also be used, but to minimize potential damage to non-diseased cells, and thereby reduce side effects, such compounds can be placed at the site of diseased tissue. Care must be taken to design the transport system to be directed.

[0127] Data from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED ₅₀ with little or no toxicity. The dosage depends on the dosage form used and the route of administration used,
It can be changed within this range. For compounds used in the methods of the invention, the therapeutically effective amount can be estimated initially from cell culture assays.
A dose that achieves a circulating plasma concentration range that includes the IC ₅₀ determined in cell culture (ie, the concentration of the test compound that achieves half-maximal inhibition of symptoms) can be formulated in animal models. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

Formulation and Use The pharmaceutical compositions used according to the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.

Thus, the compounds and physiologically acceptable salts and solvates thereof,
It can be formulated for administration by inhalation or insufflation (either through the mouth or nose) or for oral, buccal, parenteral, or rectal administration.

For oral administration, the pharmaceutical composition may comprise, for example, a binder (eg, pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose); a bulking agent (eg, lactose, microcrystals) cellulose,
Or calcium hydrogen phosphate); lubricants (eg, magnesium stearate,
Prepared by conventional means with pharmaceutically acceptable excipients, such as talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets or capsules. Tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be in the form of a dry preparation for reconstitution with water or other suitable solvent before use. Can be. Such liquid formulations include suspending agents (eg, sorbitol syrup, cellulose derivatives or hardened edible oils); emulsifiers (eg, lecithin or acacia); non-aqueous solvents (eg, almond oil, oily esters, ethyl alcohol or purified oil). Together with pharmaceutically acceptable additives such as preservatives, for example methyl or propyl-p-hydroxybenzoic acid or sorbic acid, and the like. The preparation can also optionally contain buffer salts, flavoring, coloring and sweetening agents. Preparations for oral administration can be suitably formulated to give controlled release of the active compound.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

When administered by inhalation, the compounds used in accordance with the present invention may be prepared using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon monoxide, or other suitable gas. It is conveniently transported from a pressure pack or nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges containing a powder mix of the compound and a suitable powder base such as lactose or starch can be formulated for use in an inhaler or insufflator.

The compounds can be formulated for parenteral administration by injection, eg, by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing and / or dispersing agents. . Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, eg, containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
Thus, for example, the compound may be a suitable polymeric or hydrophobic material (eg,
(As an emulsion in an acceptable oil) or with ion exchange resins, or as a less soluble derivative, for example, as a less soluble salt.

Optionally, the compositions are provided in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack.
The pack or dispenser device can be accompanied by instructions for administration.

The therapeutic compositions of the present invention can also include carriers or excipients, many of which are known to those skilled in the art. Excipients that can be used include buffers (eg, citrate, phosphate, acetate, and bicarbonate buffers), amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (eg, , Serum albumin)
, EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The nucleic acids, polypeptides, antibodies or modulatory compounds of the invention can be administered by standard routes of administration. For example, administration is parenteral,
It may be intravenous, subcutaneous, intramuscular, intracranial, intraorbital, intraocular, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal, buccal, or oral administration. Modulatory compounds can be formulated in various ways according to the corresponding route of administration. For example, liquid solutions can be made for ingestion or injection; gels or powders can be made for ingestion, inhalation or topical application. Methods for preparing such formulations are well known,
For example, it is found in "Remington's Pharmaceutical Sciences." The preferred route of administration is expected to be intravenous.

[0138]

【Example】

T from human bone marrow cDNA library (Clonetech; Palo Alto, CA)
ango-78 cDNA (SEQ ID NO: 1; FIG. 1) was isolated. This Tango-78 cDNA is derived from Tango-78, a novel protein (SEQ ID NO: 2; FIG. 1) having high homology with the mouse nodal protein.
Encodes a 168 amino acid portion (Collignon et al., Nature 381: 155, 1996).

[0139] US patent application Ser. No. 08 / 752,307 (November 1996, incorporated herein by reference)
Tango-78 as described herein using the method described in
cDNA (SEQ ID NO: 1; FIG. 1) was isolated. The Tango-78 protein (SEQ ID NO: 2; FIG. 1) has high homology with the mouse segment protein (Collignon et al., Supra; FIG.

[0140] Tan from a human fetal brain library (Clonetech; Palo Alto, CA)
go-79 cDNA (SEQ ID NO: 3; FIG. 2) was isolated. This Tango-78 cDNA encodes a 615 amino acid protein (SEQ ID NO: 4; FIG. 2) that is homologous to the Drosophila Melanogaster slit protein (Taguchi et al., Br.
ain Res. Mol. Brain Res. 35: 31, 1996).

[0141] US patent application Ser. No. 08 / 752,307 (November 1996, incorporated herein by reference).
Tango-79 as described herein using the method described in
The cDNA (SEQ ID NO: 3; FIG. 2) was isolated. The Tango-79 protein (SEQ ID NO: 4; FIG. 2) is homologous to D45913 (leucine-rich repeat protein) (FIG. 5). Northern blot analysis of Tango-79 mRNA shows that about 3.0 kB and about 3.5 kB transcripts are expressed in the brain. The function of Tango-79 can be examined by overexpressing this protein in the mouse brain.

[0142] Tango-81 cDNA was isolated from a human fetal brain library. This Tango-81 cDNA (
SEQ ID NO: 5; FIG. 3) encodes a protein with 261 amino acids (SEQ ID NO: 6; FIG. 3).

[0143] US patent application Ser. No. 08 / 752,307 (November 1996, incorporated herein by reference)
Tango-81 as described herein using the method described in
cDNA was isolated. Northern analysis of Tango-81 expression shows that Tango-81 is expressed in heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis (FIG. 6).

[Brief description of the drawings]

FIG. 1 shows a nucleic acid sequence of Tango-78 (SEQ ID NO: 1) and a putative amino acid sequence of Tango-78 (SEQ ID NO: 2).

FIG. 2 describes the nucleic acid sequence of Tango-79 (SEQ ID NO: 3) and the putative amino acid sequence of Tango-79 (SEQ ID NO: 4).

FIG. 3 describes a nucleic acid sequence of Tango-81 (SEQ ID NO: 5) and a putative amino acid sequence of Tango-81 (SEQ ID NO: 6).

FIG. 4 shows an alignment of the amino acid sequence of Tango-78 and the amino acid sequence of mouse nodule protein.

FIG. 5 shows an alignment of the amino acid sequence of Tango-79 and D45913 (leucine-rich repeat protein).

FIG. 6 shows the results of Northern blot analysis of Tango-81 expression.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] February 4, 2000 (200.2.4)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0143

[Correction method] Change

[Correction contents]

[0143] US patent application Ser. No. 08 / 752,307 (November 1996, incorporated herein by reference)
Tango-81 as described herein using the method described in
cDNA was isolated. Northern analysis of Tango-81 expression indicates that Tango-81 is expressed in heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis.

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] Brief description of drawings

[Correction method] Change

[Correction contents]

[Brief description of the drawings]

FIG. 1 shows a nucleic acid sequence of Tango-78 (SEQ ID NO: 1) and a putative amino acid sequence of Tango-78 (SEQ ID NO: 2).

FIG. 2 describes the nucleic acid sequence of Tango-79 (SEQ ID NO: 3) and the putative amino acid sequence of Tango-79 (SEQ ID NO: 4).

FIG. 3 describes a nucleic acid sequence of Tango-81 (SEQ ID NO: 5) and a putative amino acid sequence of Tango-81 (SEQ ID NO: 6).

FIG. 4 shows an alignment of the amino acid sequence of Tango-78 and the amino acid sequence of mouse nodule protein.

FIG. 5 shows an alignment of the amino acid sequence of Tango-79 and D45913 (leucine-rich repeat protein).

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Claims (23)

[Claims] 1. a) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, Accession No. CDNA insert of plasmid registered with ATCC as accession number CDNA insert of plasmid registered with ATCC as accession number A nucleic acid molecule comprising a nucleotide sequence that is at least 55% identical to the nucleotide sequence of the cDNA insert of the plasmid registered with the ATCC or its complement, b) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 , Accession number CDNA insert of plasmid registered with ATCC as accession number CDNA insert of plasmid registered with ATCC as accession number A) a nucleic acid molecule comprising a nucleotide sequence containing a fragment of at least 300 nucleotides of the nucleotide sequence of the cDNA insert fragment of the plasmid registered with the ATCC, or its complementary strand; c) the amino acid sequence of SEQ ID NO: 2; Amino acid sequence, amino acid sequence of SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number Amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC, or accession number A) a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence encoded by a cDNA insert of a plasmid registered with the ATCC; d) an amino acid sequence of SEQ ID NO: 2, an amino acid sequence of SEQ ID NO: 4; A nucleic acid molecule encoding a fragment of a polypeptide comprising an amino acid sequence, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number The polypeptide encoded by the cDNA insert of the plasmid registered with the ATCC, accession number The polypeptide encoded by the cDNA insert of the plasmid registered with the ATCC, or the accession number A) a nucleic acid molecule comprising a polypeptide encoded by a cDNA insert of a plasmid registered with the ATCC, e) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number ATCC as
Amino acid sequence encoded by cDNA insert of plasmid registered in Accession No., or accession number Encodes a naturally-occurring allelic form of a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC, under stringent conditions, SEQ ID NO: 1, SEQ ID NO: 3, sequence An isolated nucleic acid molecule selected from the group consisting of a nucleic acid molecule comprising No. 5 or a nucleic acid molecule that hybridizes to the complement thereof. 2. a) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, Accession No. CDNA insert of plasmid registered with ATCC as accession number CDNA insert of plasmid registered with ATCC as accession number A nucleic acid comprising the nucleotide sequence of the cDNA insert of the plasmid registered with the ATCC or its complementary strand, and b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number ATCC as
Amino acid sequence encoded by cDNA insert of plasmid registered in Accession No., or accession number 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule is selected from the group consisting of a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence encoded by a cDNA insert of a plasmid registered with the ATCC. 3. The nucleic acid molecule of claim 1, further comprising a vector nucleic acid sequence. 4. The method of claim 1, further comprising a nucleic acid sequence encoding a heterologous polypeptide.
The nucleic acid molecule according to 1. 5. A host cell having the nucleic acid molecule according to claim 1. 6. The host cell according to claim 5, which is a mammalian host cell. 7. A non-human mammalian host cell having the nucleic acid molecule of claim 1. 8. A) comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6, wherein at least 15 consecutive amino acids of SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 A) a polypeptide fragment comprising; b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number ATCC as
Amino acid sequence encoded by cDNA insert of plasmid registered in Accession No., or accession number A nucleic acid molecule comprising an amino acid sequence encoded by a cDNA insert of a plasmid registered with the ATCC as a nucleic acid and hybridizing under stringent conditions to a nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 Or a naturally occurring allelic form of the polypeptide encoded by its complement; and c) nucleotides that are at least 55% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 An isolated polypeptide selected from the group consisting of a nucleic acid molecule comprising or a polypeptide encoded by the complement thereof. 9. The amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, Accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number Amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC, or accession number 9. The isolated polypeptide according to claim 8, which comprises an amino acid sequence encoded by a cDNA insert of a plasmid registered with the ATCC. 10. The polypeptide of claim 8, further comprising a heterologous amino acid sequence. 11. An antibody which selectively binds to the polypeptide according to claim 8. 12. An amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number AT as
Amino acid sequence encoded by the cDNA insert of the plasmid registered with CC,
Accession number A) a polypeptide comprising the amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC as; b) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number AT as
Amino acid sequence encoded by the cDNA insert of the plasmid registered with CC,
Or accession number A polypeptide comprising a fragment of the amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC,
The fragment has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number Amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC, or accession number A polypeptide comprising at least 15 contiguous amino acids of the amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC; and c) the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 , Accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC, accession number AT as
Amino acid sequence encoded by the cDNA insert of the plasmid registered with CC,
Or accession number A polypeptide comprising an amino acid sequence encoded by a cDNA insert of a plasmid registered with the ATCC as a nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 under stringent conditions A method of producing a polypeptide selected from the group consisting of a naturally occurring allele of a polypeptide encoded by a nucleic acid molecule or its complementary strand that hybridizes to Item 6. A method comprising the step of culturing the host cell according to Item 5. 13. The amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, accession number Amino acid sequence encoded by cDNA insert of plasmid registered with ATCC as accession number ATCC as
Amino acid sequence encoded by cDNA insert of plasmid registered in Accession No., or accession number 13. The isolated polypeptide according to claim 12, comprising the amino acid sequence encoded by the cDNA insert of the plasmid registered with the ATCC. 14. A step of contacting a sample with a compound that selectively binds to the polypeptide according to claim 8, and b) a step of determining whether the compound binds to the polypeptide in the sample. 9. A method for detecting whether the polypeptide according to claim 8 is present in a sample, comprising: 15. The method of claim 14, wherein the compound that binds to the polypeptide is an antibody. 16. A kit comprising a compound that selectively binds to the polypeptide according to claim 8 and an instruction manual for use. 17. a) contacting a sample with a nucleic acid probe or primer that selectively hybridizes to a nucleic acid molecule; and b) determining whether the nucleic acid probe or primer binds to a nucleic acid molecule in the sample. A method for detecting whether or not the nucleic acid molecule according to claim 1 is present in a sample. 18. The method of claim 17, wherein the sample comprises an mRNA molecule and is contacted with a nucleic acid probe. 19. A kit comprising a compound that selectively hybridizes to the nucleic acid molecule according to claim 1 and an instruction manual for use. 20) a) contacting a test compound with the polypeptide of claim 8 or a cell expressing the polypeptide of claim 8, and b) detecting whether the polypeptide binds to the test compound. 9. A method for identifying a compound that binds to the polypeptide according to claim 8, comprising: 21. The test compound binding to the polypeptide comprises: a) detecting binding by directly detecting the test compound / polypeptide binding; b) detecting binding using competitive binding analysis; and c) detecting Tango- 21. The method of claim 20, wherein the method is detected by a method selected from the group consisting of detecting binding using an assay for 72-mediated signaling. 22. The step of contacting the polypeptide of claim 8 or a cell expressing the polypeptide of claim 8, with a compound that binds to the polypeptide at a concentration sufficient to modulate the activity of the polypeptide. 9. A method for modulating the activity of the polypeptide according to claim 8, comprising: 23. a) contacting a test compound with the polypeptide of claim 8, and b) determining the effect of the test compound on the activity of the polypeptide, thereby identifying a compound that modulates the activity of the polypeptide. 9. A method for identifying a compound that modulates the activity of the polypeptide according to claim 8, comprising the steps of: