Protein and gene involved in myocyte differentiation

Abstract

A novel gene was unexpectedly isolated in an attempt to isolate a gene specifically expressed in immortalized cells via antibody screening using an antibody raised against a protein occurring specifically in immortalized cells. The gene thus isolated shares no sequence homology with the entries deposited in the database and was strongly expressed in skeletal muscles and undifferentiated cells. The protein encoded by this gene inhibits the differentiation of myoblasts into myotubes. It also inhibits the transactivation function of p 53 , a transcription factor involved in tumor suppression.

What is claimed is:  
     
         1 . A substantially pure polypeptide comprising an amino acid sequence at least 40% identical to SEQ ID NO:1, wherein the polypeptide inhibits the differentiation of myoblasts into myotubes.  
     
     
         2 . The polypeptide of  claim 1 , wherein the amino acid sequence is at least 60% identical to SEQ ID NO:1.  
     
     
         3 . The polypeptide of  claim 1 , wherein the amino acid sequence is at least 80% identical to SEQ ID NO:1.  
     
     
         4 . The polypeptide of  claim 1 , wherein the amino acid sequence is at least 90% identical to SEQ ID NO:1.  
     
     
         5 . A substantially pure polypeptide comprising the sequence of SEQ ID NO:1.  
     
     
         6 . A substantially pure polypeptide comprising the amino acid sequence of SEQ ID NO:1, with up to 30 conservative amino acid substitutions, wherein the polypeptide inhibits the differentiation of myoblasts into myotubes.  
     
     
         7 . A substantially pure polypeptide encoded by a nucleic acid that hybridizes under high stringency conditions to a probe the sequence of which consists of SEQ ID NO:2, wherein the polypeptide inhibits the differentiation of myoblasts into myotubes.  
     
     
         8 . An isolated nucleic acid encoding the polypeptide of  claim 1 .  
     
     
         9 . An isolated nucleic acid encoding the polypeptide of  claim 5 .  
     
     
         10 . An isolated nucleic acid encoding the polypeptide of  claim 6 .  
     
     
         11 . An isolated nucleic acid comprising a strand that hybridizes under high stringency conditions to a single stranded probe, the sequence of which consists of SEQ ID NO:2 or the complement of SEQ ID NO:2.  
     
     
         12 . The isolated nucleic acid of  claim 11 , wherein the nucleic acid encodes a polypeptide that inhibits the differentiation of myoblasts into myotubes.  
     
     
         13 . The nucleic acid of  claim 12 , wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:1.  
     
     
         14 . The nucleic acid of  claim 11 , wherein the strand is at least 15 nucleotides in length.  
     
     
         15 . The nucleic acid of  claim 14 , wherein the nucleic acid is an antisense nucleic acid that inhibits expression of a polypeptide comprising SEQ ID NO:1.  
     
     
         16 . The nucleic acid of  claim 15 , wherein the nucleic acid is at least 15 nucleotides in length.  
     
     
         17 . A vector comprising the nucleic acid of  claim 8 .  
     
     
         18 . A vector comprising the nucleic acid of  claim 9 .  
     
     
         19 . A vector comprising the nucleic acid of  claim 10 .  
     
     
         20 . A vector comprising the nucleic acid of  claim 11 .  
     
     
         21 . A vector comprising the nucleic acid of  claim 12 .  
     
     
         22 . A cultured host cell comprising the nucleic acid of  claim 8 .  
     
     
         23 . A cultured host cell comprising the nucleic acid of  claim 9 .  
     
     
         24 . A cultured host cell comprising the nucleic acid of  claim 10 .  
     
     
         25 . A cultured host cell comprising the nucleic acid of  claim 11 .  
     
     
         26 . A cultured host cell comprising the nucleic acid of  claim 12 .  
     
     
         27 . An antibody that specifically binds to the polypeptide of  claim 1 .  
     
     
         28 . A method of producing a polypeptide, the method comprising culturing the cultured host cell of  claim 22  in a culture, expressing the polypeptide in the cultured host cell, and isolating the polypeptide from the culture.  
     
     
         29 . A method of screening for a compound that binds to the protein according to  claim 1 , the method comprising the steps of: 
 a) contacting a test sample with said protein or a partial peptide thereof;    b) detecting the binding activity of the test sample to said protein or a partial peptide thereof; and    c) selecting a compound binding to said protein or a partial peptide thereof.    
     
     
         30 . A compound binding to the protein according to  claim 1 , wherein said compound can be isolated using the method according to  claim 29 .  
     
     
         31 . A method of screening for a compound that promotes or inhibits the activity of the protein according to  claim 1 , the method comprising the steps of: 
 a) contacting myoblasts with said protein in the presence of a test sample;    b) detecting the differentiation of the cells into myotubes; and    c) selecting a compound which can increase or decrease the inhibitory activity of the protein, compared with its inhibitory activity as detected in the absence of said test sample.    
     
     
         32 . A method of screening for a compound that promotes or inhibits the activity of the protein according to  claim 1 , said method comprising the steps of: 
 a) providing p53-deficient cells with a vector expressing said protein, a vector expressing p53, and a vector expressing a reporter gene in response to p53;    b) contacting a test sample with said cells;    c) detecting the reporter activity in said cells; and    d) selecting a compound that can reduce or increase the reporter activity compared with the activity in the cells without contact with said test sample (control).    
     
     
         33 . A compound that promotes or inhibits the activity of the protein according to  claim 1 , wherein said compound can be isolated using the method according to  claim 31  or  32 .