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

类固醇受体功能域结构的研究及相关基因的克隆。

基本信息

批准号：
63570110
负责人：
SAKAI Masaharu
金额：
$ 1.34万
依托单位：
HOKKAIDO UNIVERSITY (1989)The University of Tokyo (1988)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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

为了阐明类固醇激素的作用机制，我们采用以下方法利用大鼠雌激素受体cDNA克隆。1.大鼠雌激素受体功能区结构的测定。2.雌激素靶基因的鉴定与克隆。3.利用细菌和病原菌基因表达系统生产雌激素受体蛋白。1.研究结果构建了一系列雌激素受体cDNA的缺失突变质粒，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COS-7细胞。用CAT法测定各突变体受体的反式激活活性。我们在大鼠雌激素受体中鉴定了两个反式激活结构域，一个是N端区域，一个是C端区域。C端反式激活结构域定位于距N端316-341个氨基酸。该区域富含酸性氨基酸。的 ...更多信息 N-末端区位于Ala 59-Glu 14 O之间。该区域与转录因子OTF、Oct-2和Oct-3.2的反式激活结构域非常疏水。利用雌激素受体的DNA结合域与含有ERE序列的DNA特异性结合，建立了分离雌激素靶基因的新方法。将大肠杆菌产生的大鼠雌激素受体结合区DNA与人基因组DNA混合，酶切成1-2kb片段。将与蛋白质特异性结合的DNA片段捕获在硝酸纤维素滤膜上。从捕获的DNA获得几个克隆，并对一些克隆进行测序。几乎所有测定的克隆都含有ERE序列，一些克隆还含有增强子元件共有的序列。这些结果表明，克隆的DNA是雌激素反应基因的一部分。为了克隆雌激素靶基因，我们正在以该DNA为探针筛选基因组文库和cDNA文库.在大肠杆菌系统中成功地生产了雌激素受体蛋白，并用于提取上述雌激素应答基因。然而，利用家蚕的真核表达系统不能令人满意地产生受体蛋白，有待于改进。少