| Project/Area Number |
16590381
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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 |
Virology
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
KANDA Teru Hokkaido Univ., Inst.for Genetic Med., Lec., 遺伝子病制御研究所, 助手 (50333472)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 2004: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | EB virus / latent infection / EBNA1 / epitope tagging / immunofluorescence / FISH / bacterial artificial chromosome / 遺伝子置換 / 転写調節 |
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| Research Abstract |
Epstein-Barr virus (EBV)-encoded protein EBNA1 is always expressed in latently-infected cells. EBNA1 plays various roles, including extrachromosomal maintenance of EBV genomes and transcriptional regulation of viral gene expressions. In order to analyze intranuclear localizations of EBNA1 protein with high resolution, we generated a recombinant EBV expressing an epitope-tagged EBNA1 protein.
The EBNA1 gene of Akata strain EBV genome was replaced with a transgene encoding hemagglutinin (HA)-tagged EBNA1 (on its C terminus) by using a bacterial artificial chromosome (BAC) system. The resultant BAC clone DNA was introduced into Akata cells, and cells harboring only the recombinant EBV were obtained. The established cells expressed HA-tagged EBNA1 protein, and the recombinant EBV genomes were stably maintained as episomes. Immunofluorescence analyses using anti-HA antibody revealed that HA-tagged EBNA1 protein localized as intranuclear punctate dots. A combined method of fluorescence in situ hybridization (FISH) and immunofluorescence (IF) was developed to visualize the localization of EBV genomes and EBNA1 protein simultaneously. The results revealed that EBNA1 signals overlapped with the signals of EBV genomes in all phases of the cell cycle. Notably, EBNA1 signals were frequently observed as double dots in S and G2 phase-synchronized cells. Moreover, we found that more than 50% of the paired dots of EBNA1 localized symmetrically on sister chromatids in prematurely condensed G2-phase chromosome spreads.
These results indicate that EBNA1 can distribute replicated 'sister viral molecules' evenly to sister chromatids while host chromosomes and viral episomes are synchronously replicating during S phase.
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