Nucleic acid molecule encoding a novel estrogen receptor beta variant

Abstract

This invention relates to an isolated nucleotide fragment of a novel estrogen receptor, in particular, a novel ERβ variant protein and isolated nucleic acid fragment comprising the coding regions of the genes encoding such variant proteins. Also provided are vectors, host cells, and methods for producing the novel ERβ variant protein. The invention further relates to method of obtaining such nucleotide fragment and the method of determining the presence of such ERβ variant protein in a sample.

1 . An isolated nucleic acid fragment encoding an estrogen receptor variant polypeptide selected from the group consisting of:
 (a) an isolated nucleic acid fragment encoding SEQ ID NO:1;   (b) an isolated nucleic acid fragment encoding an amino acid sequence having at least 95% identity with the SEQ ID NO:1;   (c) an isolated nucleic acid molecule that hybridizes with the isolated nucleic acid fragment of (a) under hybridization conditions of 6×SSC (1M NaCl), 45 to 50% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.; and   (d) an isolated nucleic acid fragment that is complementary to (a), (b) or (c).   
     
     
         2 . The isolated nucleic acid fragment of  claim 1  as set forth in SEQ ID NO:2. 
     
     
         3 . The isolated nucleic acid fragment of  claim 1  wherein the isolated polynucleotide is DNA. 
     
     
         4 . The isolated nucleic acid fragment of  claim 1  wherein the isolated polynucleotide is RNA. 
     
     
         5 . The nucleic acid fragment of  claim 1  wherein the nucleic acid fragment comprises SEQ ID NO:4 
     
     
         6 . The nucleic acid fragment of  claim 1  wherein the nucleic acid fragment encodes a polypeptide which is involved in estrogen signaling pathway. 
     
     
         7 . A polypeptide encoded by the isolated nucleic acid fragment of  claim 1 . 
     
     
         8 . A polypeptide of  claim 7  as set forth in SEQ ID NO:1. 
     
     
         9 . The polypeptide of  claim 7  further characterized by estrogen receptor dominant negative activity. 
     
     
         10 . The polypeptide of  claim 7  further characterized by ERβ dominant negative activity. 
     
     
         11 . The polypeptide of  claim 7  wherein the polypeptide comprises SEQ ID NO:3. 
     
     
         12 . A polypeptide comprising SEQ ID NO:3 further characterized by estrogen receptor dominant negative activity. 
     
     
         13 . A polypeptide comprising SEQ ID NO:3 further characterized by ERβdominant negative activity. 
     
     
         14 . A chimeric construct comprising the isolated nucleic acid fragment of any of  claims 1 ,  2 , or  5  operatively linked to suitable regulatory sequences. 
     
     
         15 . A host cell transformed with the chimeric construct of  claim 14 . 
     
     
         16 . The host cell of  claim 15  wherein the host cell is selected from the group consisting of an eukaryotic, a prokaryotic cell, and a multicellular organism. 
     
     
         17 . The host cell of  claim 15  wherein the host cell is a mammalian cell. 
     
     
         18 . The host cell of  claim 15  wherein the host cell is selected from the group consisting of a human hepatoma cell (HepG2), a chinese hamster ovary cell (CHO), a monkey COS-1 cell line, a mammalian cell CV-1, an amphibian cells, a yeast cell, and an insect cell. 
     
     
         19 . The host cell of  claim 15  wherein the host cell is selected from the group consisting of a  Saccharomyces cerevisiae , a  Schizosaccharomyces pombe , and a  Pichia pastoris.    
     
     
         20 . A vector comprising the nucleic acid fragment of  claim 1 . 
     
     
         21 . The vector of  claim 20 , wherein the vector is a plasmid. 
     
     
         22 . The vector of  claim 21 , wherein the plasmid is pERβcx2. 
     
     
         23 . A transformed cell comprising the vector of  claim 20 . 
     
     
         24 . The transformed cell of  claim 23 , wherein the host microorganism is selected from the group consisting of a human hepatoma cell (HepG2), a chinese hamster ovary cell (CHO), a monkey COS-1 cell line, a mammalian cell CV-1 an amphibian cells, a yeast cell, and an insect cell. 
     
     
         25 . The transformed cell of  claim 23 , wherein the host microorganism is selected from the group consisting of a  Saccharomyces cerevisiae , a  Schizosaccharomyces pombe , and a  Pichia pastoris.    
     
     
         26 . The transformed cell of  claim 23 , wherein the host microorganism is  E. coli.    
     
     
         27 . An isolated nucleic acid fragment selected from the group consisting of:
 (a) an isolated nucleic acid fragment as set forth in SEQ ID NO:11;   (b) an isolated nucleic acid molecule that hybridizes with the isolated nucleic acid fragment of (a) under hybridization conditions of 6×SSC (1M NaCl), 45 to 50% formamide, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.; and   (c) an isolated nucleic acid fragment that is complementary to (a), or (b).   
     
     
         28 . A method of obtaining a nucleic acid fragment encoding the polypeptide of  claim 7 , the method comprising:
 (a) probing a genomic library with all or a portion of a nucleic acid fragment as set forth in SEQ ID NO:2;   (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of step (a); and   (c) determining the sequence of the nucleic acid fragment that comprises the DNA clone identified in step (b).   
     
     
         29 . A method of detecting the presence of a nucleic acid fragment of any of  claims 1  or  27  in a sample, the method comprising:
 (a) contacting said sample with the oligonucleotide of any of  claims 1  or  27 ; and   (b) determining whether the oligonucleotide detects the nucleic acid fragment.