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

为了阐明类固醇激素的作用机制，我们利用大鼠雌激素受体cDNA克隆进行了以下研究。1. 大鼠雌激素受体功能结构的测定。2. 雌激素靶基因的鉴定与克隆。3. 通过细菌和真核基因表达系统产生雌激素受体蛋白。Results.1。构建了一系列雌激素受体cDNA缺失突变质粒，并与含有雌激素反应元件（ERE）和氯霉素乙酰转移酶（CAT）基因的雌激素反应报告质粒一起转染到COS-7细胞中。采用CAT法测定各营养素受体的反活化活性。我们在大鼠雌激素受体中发现了两个反激活区域，即n端区域和c端区域。c端转激活域定位在n端316-341个氨基酸上。这个区域富含酸性氨基酸。更多的n -结构域位于Ala59至Glu14O。该区域与转录因子OTF、Oct-2和Oct-3.2的反激活结构域相似。我们开发了通过特异性结合雌激素受体DNA结合域和含有ERE序列的DNA分离雌激素靶基因的新方法。大肠杆菌产生的大鼠雌激素受体DNA结合域与人基因DNA混合，消化成1-2kb的碎片。与蛋白质特异性结合的DNA片段被捕获在硝化纤维素过滤器上。从捕获的DNA中获得了几个克隆，并对其中一些克隆进行了测序。几乎所有克隆均含有ERE序列，部分克隆还含有与增强子元件共有的序列。这些结果表明克隆DNA是雌激素反应基因的一部分。为了克隆雌激素靶基因，我们目前正在筛选基因组和cDNA文库，以该DNA为探针。在大肠杆菌系统中成功制备了雌激素受体蛋白，并用于提取上述雌激素应答基因。然而，利用蚕丝进行的真核表达系统产生的受体蛋白并不理想，有待进一步完善。少

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