| Project/Area Number |
14360206
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | Mie University |
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Principal Investigator |
OKUMURA Katsuzumi Mie University, Faculty of Bioresources, Professor, 生物資源学部, 教授 (30177183)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥16,600,000 (Direct Cost: ¥16,600,000)
Fiscal Year 2004: ¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 2003: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 2002: ¥6,100,000 (Direct Cost: ¥6,100,000)
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| Keywords | genomic imprinting / chromatin structure / DNA replication / epigenetics / DNA fiber / visualization / nuclear matrix / centromeric heterochromatin / エピジェネティック / ヒストンアセチル化 / チェックポイント / レプリコン / 動物細胞 / インプリンティング / FISH / 核マトリクス / ゲノム |
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| Research Abstract |
Epigenetic modifications of chromatin play important roles in genomic imprinting. In this research, basic techniques for the analysis of DNA replication were developed first. Using new and previous techniques, effects of epigenetic modifications of chromatin on DNA replication were examined. The results obtained are listed as follows :
1.A hypotonic shift method to introduce modified nucleotides into cells was applied for the analysis of DNA replication. In contrast to the beads-loading method, this can be used not only for adherent cells but for floating cells. Other advantages including efficient and homogeneous incorporation of modified nucleotides were also demonstrated.
2.A dynamic molecular combing method that extends DNA molecules precisely and uniformly on a microscope cover slip was established, and applied to analysis of DNA replication. A preparation method of semi-extended DNA fibers from individual cells was also developed. Using this method, asynchronous replication between
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parental alleles in imprinted regions was demonstrated.
3.Direct visualization of DNA fibers showed that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. It was also shown that histone hyperacetylation is tightly associated with changes in chromatin structure, replication timing (origin firing) and fork progression of centromeric heterochromatin regions. S phase checkpoint factors were suggested to be responsible for repression of origin firing and stabilization of replication forks when nuclear DNAs were damaged.
4.Nuclear DNAs generally bind and release to the nuclear matrix just before and after their replication, respectively, although MAR regions consistently bind to it through S phase.
5.Basic researches to examine roles of DNA replication in epigenetics in mammalian development were promoted through replication timing analysis of imprinted domains, visualization of replicating DNA fibers extended from ES cells, and so on. Less
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