Development of in vitro replication system of human chromosomal DNA and reconstitution of the system with purified components.
| Project/Area Number |
03044163
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
MURAKAMI Yasufumi Department of Gene Bank Tsukuba Life Science Center Institute of Physical and Chemical Research, ジーンバンク室, 研究員 (90200279)
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| Co-Investigator(Kenkyū-buntansha) |
HURWITZ Jerard Molecular Biology Program Memorial Sloan Kettering Cancer Center, 分子生物学部, 教授
EKI Toshihiko Department of Gene Bank Tsukuba Life Science Center Institute of Physical and Ch, ジーンバンク室, 研究員
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| Project Period (FY) |
1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1991: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | replicon / initiation of DAN synthesis / DNA replication / eukaryote / UV irradiation / emetin / Cell cynchronization / SV40 |
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| Research Abstract |
In order to define replication origin of higher eukaryotic cells, we have tried to establush a method to isolate newly synthesized DNA near the replication origin. To isolate replication origin, although various methods including a method to isolate autonomous replicating sequencies have beeen tried out by various investigators, no one has rearly succeeded to define the general replication origin. To isolate a locus containing replication initiation site, we first analyzed the kinetics of the inhibition of the DNA replication by UV irradiation, as UV irradiation produces pyrimidine dimer in a irradiation dose dependent manner and pyrimidine dimer would inhibits the elongationreaction of DNA replication. As a model experiment, effect of UV irradiflon on SV40 DNA replication in vitro has been analyzed. SV40 DNA was irradiated with UV light and then assayed for DNA replication activity in vitro. In this system, plasmid DNA which has replication origin of SV40 DNA replicates efficiently in
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the presence of SV40 large T antigen. When replication products which has been formed in the presence of [P]-dCTP were analyzed by agarose gel electrophoresis, after restriction digests, , accumulation of origin containing fragments has been observed. This result indicated that initiation stage was specifically inhibited by UV irradiation. Effect of UV irradiation on cellular DNA replication has been analyzed using cultures of HeLa cells and Xeroderma pigmentosis cells. The nascent DNA was labeled by tritiated thymidine extracted and were analyzed by alkaline agarose gel electrophoresis. Alkaline agarose gel electrophoresis experiments indicated that elongation reaction of the chromosomal replication was also inhibity UV irradiation. In addition to these experiments, we also established a method to obtain highly synchronized cell populations by mitotic shake off method. We are currently isolating initiation region of the chromosomal DNA under the condition where elongation reaction is severely inhibited by labelling synchronized cells with BUdR using monoclonals against BUdR. Less
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Report
(1 results)
Research Products
(4 results)
