| Project/Area Number |
07672354
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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 |
Biological pharmacy
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
KAWASAKI Nobuko College of Medical Tecnology, Kyoto University Professor, 医療技術短期大学部, 教授 (70077676)
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| Co-Investigator(Kenkyū-buntansha) |
KAWASAKI Toshisuke Pharmaceutcal Sciences Kyoto University Professor, 薬学部, 教授 (50025706)
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| Project Period (FY) |
1995 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1995: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Conglutinin / Animal lectin / Collagen-like structure / Bovine serum / Expression control of gene / Luciferase / Promoter / Transcription regulatory sequence / レクチン |
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| Research Abstract |
Conglutinin is a unique bovine plasma protein which mediates the agglutination of the sensitized erythrocyte-solid phase iC3b (conglutination). The protein is identified as a Ca^<2+>-dependent animal lectin specific for N-acetylglucosamine and a member of proteins which have a collagen-like domain (collectins, collagen-like lectins).
In this research, the 5'-regulatory region sequence and the expression control of conglutinin gene were studied to elucidate the biological function of conglutinin with respect to the gene structure.
1) Characterization of 5'-upstream region of the conglutinin gene.
The 5'-flanking region of the conglutinin gene was cloned and sequenced by gene walking using vector (cassette) -ligation mediated PCR.In the promoter region (700bp) of the gene, several putative consensus sequences that may be involved in the expression of conglutinin were identified.
2) Analysis of the transcription regulatory sequences with the reporter gene assay.
A 5'-flanking region of the con
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glutinin gene (700bp) was examined for its promoter activity using luciferase as a reporter gene. To determine the region of the conglitinin promoter responsible for the transcription, a series of 5'-stepwise promoter deletions and site-directed mutations were constructed into the luciferase plasmids. These plamid-promoter DNA constructs were transfected into the human hepatoma cell line HepG2. The luciferase promoter assays revealed that an unknown positively controlling cis-acting elements exists around-200bp. This unknown cis-element acted synergistically with the AP-1 sequence present at-158bp--154bp.
3) Gel-shift assay and DNase I footprint analysis of the transcription regulatory sites.
The bindings of the unknown cis-acting element (around-200bp) and AP-1 (-158--154bp) with the nuclear protein extracts of the HepG2 cell and bovine liver cells were demonstrated with gelmobility shift assays. The DNase I footprint analysis identified the binding sites of the unknown cis-acting element with a trans-acting element in the nuclear protein around-180bp. Less
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