| Project/Area Number |
03454186
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Virology
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| Research Institution | The University of Tokyo |
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Principal Investigator |
YAMAGUCHI Nobuo The Institute of Medical Science, The University of Tokyo, Department of Virology, Professor, 医科学研究所, 教授 (90012723)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | HCMV / DNA replication / PCR / Microinjection / サイトメガロウイルス / 潜伏感染 / ウイルスDNA複製 |
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| Research Abstract |
We developed a sensitive assay for DNA replication in mammalian cells that enable us to detect replicated DNA fragments of human cytomegalovirus (HCMV). A 1/100 portion of DNA extracted from 1000 microinjected cells was subjected to polymerase chain reaction (PCR) after digestion with appropriate restriction enzymes to differentiate replicated from nonreplicated plasmids. A portion of the amplified DNA was electrophoresed to detect replicated DNA. Subfragments of the HindIII fragment A of HCMV strain Towne, which contains a previously identified origin of DNA replication (oriLyt), were analyzed with the assay system. A 4.3-kb subfragment (AatII-SacI) replicated as efficiently as the HindIII fragment A. Efficient replication ability was lost with a 1.3-kb deletion from the AatII end or a 0.9-kb deletion from SacI end. These results suggest that the boundaries of oriLyt of HCMV strain Towne lie within the 1.3- and the 0.9- regions of the 4.3-kb fragment.
We analyzed intermediates of DNA replication initiated from HCMV oriLyt by a novel method which consists of a combination of microinjection of template DNA into cells, 0.15% agarose gel electrophoresis, and PCR in the fraction of the gel to detect a small amount of replicated DNA. A plasmid containing oriLyt replicated in the HCMV-infected cells and produced DNA larger than the viral genome. Heterogeneous tandem polymers of a unit length plasmid constituted the replicating intermediates of the plasmid. These results suggest that HCMV oriLyt is involved in a rolling circular mode of DNA replication during the lytic phase of infection.
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