| Project/Area Number |
02680154
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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 |
代謝生物化学
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| Research Institution | Kanazawa University |
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Principal Investigator |
NAKANISHI Y. Fac. Pharm. Sci., Kanazawa Univ., Associate professor, 薬学部, 助教授 (40172358)
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| Co-Investigator(Kenkyū-buntansha) |
SAKURAI H. Fac. Pharm. Sci., Kanazawa Univ., Assistant professor, 薬学部, 助手 (90196428)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1991: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Adenovirus / Transcriptional Regulation / Replication Regulation / Cell-free System / DNA-binding Proteins / Molecular biology / 生化学 / 分子生物学 / DNA結合タンパク質 |
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| Research Abstract |
Celluar factors that regulate transcription and replication of adenovirus type-12(Adl2)DNA were analyzed.
1 Identification of cellular factors regulating transcription and replication of Adl2 DNA.
(1) Identification of nuclear factor I (NF-I) that stimulates distal transcription of the AD12 E1A gene and replication of Ad DNA.
A factor stimulating Adl2 EIA gene transcription in vitro was purified to near homogeneity from nuclear extracts of Ehrlich ascites tumor cells. A purified factor contained three peptides with molecular masses of 48, 53 and 55 kDa. This factor bound to a region located at the left end of Adl2 DNA where NF-I of, HeLa cells bound. Moreover, a purified factor stimulated replication initiation of Ad2 DNA in'vitro to the same extent as HeLa NF-I did. We thus concluded that a purified factor is mouse NFI. These results indicate that NF-I regulates both transcription and replication of Ad 1 2 DNA - by binding to the left end of the viral genome.
(2) Identification of ESF-1 that stimulates proximal transcription of the Adl2 ElA gene.
A factor stimulating proximal transcription of the Adl2 ElA gene was found in nuclear extracts of HeLa cells. This factor, designated ESF-1, recognizes a sequence 5'-TOTCA-3' located in a region adjacent to a TATA box. Purification of ESF-I is in progress.
2 Autoregulation of Adl2 EIA gene.
ElA protein derived from 13S, but not 12S. MRNA stimulated transcription of its own gene in a DNA transcription assay. This indicates that Adl2 EIA gene transcription is under a positive autoregulation. To elucidate a precise mechanism for this regulation, production of ElA protein is now in progress, usinga yeast expression system.
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