| Project/Area Number |
63570110
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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| Research Institution | HOKKAIDO UNIVERSITY (1989)
The University of Tokyo (1988) |
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Principal Investigator |
SAKAI Masaharu HOKKAIDO UNIVERSITY SCHOOL OF MEDICINE, ASSOCIATE PROFFESOR, 医学部, 助教授 (50162269)
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| Co-Investigator(Kenkyū-buntansha) |
IMAKADO Sumihisa UNIVERSITY OF TOKYO, FACULTY OF MEDICINE INSTRUCTOR, 医学部, 助手 (60168507)
近藤 滋 東京大学, 医学部第一生化学, 特別研究員
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1989: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1988: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | STEROID HORMONE / ESTROGEN RECEPTOR / DELETION MUTANTS / ESTROGEN TARGET GENE / FUNCTIONAL DOMAIN STRUCTURE / GENE CLONING / TRANSCRIPTIONAL REGULATION / エストロゲン受容体の機能的ドメイン構造 / トランス作用活性 / DNA結合領域 / ステロイド結合領域 / 大腸菌による大量産生 / カイコ多角体病ウイルスベクター |
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| Research Abstract |
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
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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
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