STUDIES OF FUNCTIONAL DOMAIN STRUCTURE OF THE STEROID RECEPTOR AND CLONING OF RELATED GENES.

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In order to elucidate the mechanisms of steroid hormone action, we took following approaches using rat estrogen receptor cDNA clone. 1. Determination of functional doniain structure of the rat estrogen receptor. 2. Identification and cloning of estrogen target genes. 3. Production of estrogen receptor protein by means of the bacterial and etikaryotic gene expression systems. Results.1. A series of deletion mutant plasmids of the estrogen receptor cDNA were constructed and transfected into COS-7 cells together with an estrogen- responsive reporter plasmid which contained the estrogen responsive element(ERE) and chloramphenicolacetyltransferase(CAT) gene. Transactivation activity of the each niutant receptor was determined by CAT assay. We have identified two transactivation domain in rat estrogen receptor, an N-terminal region and C-terminal region. The C-terminal transactivation domain was localized to 316-341 amino acid from N-terminus. This region was rich in acidic amino acids. The … More N-terinizial domain was located at Ala59 to Glu14O. This region was very hydrophobic similar with transactivation domain of the transcription factors OTF, Oct-2 and Oct-3.2. We have developed the novel method for isolation of estrogen target genes by means of specific binding of the DNA binding domain of estrogen receptor and the DNA containing ERE sequence. The DNA binding domain of rat estrogen receptor produced by E.Coli was mixed with the human genoinic DNA digested to 1-2kb fraginerits. The DNA fragments specifically bind to the protein were trapped on nitrocellulose filter. Several clones were obtained from trapped DNA and some of the clones were sequenced. Almost all of the clones determined were contained ERE sequence and some of the clones also contained the sequence common to the enhancer element. These results suggest that cloned DNA is part of the estrogen responsive genes. For cloning the estrogen target genes, we are now screening the genomic and cDNA libraries using this DNA as the probes.3. The estrogen receptor protein was successfully produced in the E.Coli system and used for picked up the estrogen responsive genes as mentioned above. The eukaryotic expression system using silk worm, however, not satisfactory produced the receptor protein and remained to be improved. Less