| Project/Area Number |
08680747
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Molecular biology
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
ARAKI Eiichi KUMAMOTO UNIVERSITY,SCHOOL OF MEDICINE,LECTURER, 医学部・附属病院, 講師 (10253733)
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| Co-Investigator(Kenkyū-buntansha) |
MIYAMURA Nobuhiro KUMAMOTO UNIVERSITY,SCHOOL OF MEDICINE,ASSISTANT PROFESSOR, 医学部・附属病院, 助手 (40274716)
SHIROTANI Tetsuya KUMAMOTO UNIVERSITY,SCHOOL OF MEDICINE,ASSISTANT PROFESSOR, 医学部・附属病院, 助手 (30274715)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1997: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1996: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | insulin / promoter / IRS-1 / gene / C / EBP / E box |
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| Research Abstract |
The results are summarized as follows.
IRS-1 is one of the major substrates of insulin receptor tyrosine kinase and mediates multiple insulin signals downstream.Tp elucidate the molecular mechanisms of the tissue specific regulation of IRS-1, we have studied the cis-acting elements and trans-acting factors in CHO and HepG2 cells.
Using the CAT (Chloramphenicol acetyltransferase) assay with the various deletion mutants of the IRS-1 promoter-CAT fusion plasmids, several regions responsible for positive or negative regulation in each cells were identified.A region from-1645 to-1585 bp which regulated expression negatively in CHO cells and positivity in HepG2 cells, was further analyzed.Within this region a fragment from-1645 to-1605bp up-regulated the IRS-1 promotor only in HepG2 cells, whereas a fragment from-1605 to-1585bp down-regulated only in CHO cells.
In the gel mobility shift assay, several nucler proteins which bind to these fragments were detected, and among them, two nuclear proteins which bind to a potential E box (nt-1635--1630) , and two nuclear proteins which bind to a potential C/EBP binding site (nt-1599--1591) were identified in HepG2 and CHO cells, respectively.
CAT assays using promoters mutated at the E box or at the C/EBP binding site revealed that these sequences were responsible for cell spcific regulation of the IRS-1 gene.
We therefore concluded that the two nulclear proteins which bind to the E box regulate IRS-1 gene expression positively in HepG2 cells, and the two nuclear proteins which bind to the C/EBP binding site regulate it negatively in CHO cells.
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