Analysis of initiatin and its regulation in in vitro replication system of plasmid DNA
| Project/Area Number |
61580165
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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 |
YAMAGUCHI Kazuo Institute for Gene Research, Kanazawa University, 遺伝子実験施設, 助教授 (00019879)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIURA Shigeki Institute for Gene Research, Kanazawa University, 遺伝子実験施設, 助手 (40179130)
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| Project Period (FY) |
1986 – 1987
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| Project Status |
Completed (Fiscal Year 1987)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1987: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1986: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | Plasmid / pSC101 / DNA replication / Replication origin / DNA binding protein / 遺伝子発現 / DNA複製開始 |
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| Research Abstract |
The plasmid pSC101 genome codes a proten (Rep) which functions specifically in initiation of its own DNA. We have developed on overproducing system of the protein using gene manipulation technique. In this study, Rep protein was purified and characterized as follows.
1. Reo protein was highly purified with various kinds of column chromatography from the over-producer. In vitro DNA replication was tried with the Fraction II from the E. coli crude extract. However, no replication of pSC101 DNA dependent on Rep protein was observed while oriC plasmid containing the replication origin of the E. coli chromosome was replicated in the same fraction, suggesting that replication machinaries of the fraction were active.
2. Binding loci of Rep protein in the replication origin (ori) of pSC101 were determined with gel-retardatin assay, DNase I-footprinting method and exonuclease III-digestion method. In any experiment, it was observed that Rep protein binded preferentially to inverted repeat sequences overlapping to the promoter region of the rep gene at low concentrations as Rep dimer/DNA molar ratios were 1 to 3. Rep protein could bind to the repeated sequences in the ori region, which were indispensable to replication initiation, at more than 50 of molar ratio of Rep dimer to DNA.
3. Rep protein could repress specifically in vitro transcription dependent on the rep promoter at the lower concentration.
These results lead us a conclution that Rep protein purified in this study retains the function as a repressor for rep gene expression but not as an initiator of DNA replication. To do so, the protein might have to change its conformation or form a complex with other factor(s) from host cells.
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Report
(2 results)
Research Products
(5 results)
