| Budget Amount *help |
¥17,320,000 (Direct Cost: ¥15,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2007: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000)
Fiscal Year 2006: ¥9,000,000 (Direct Cost: ¥9,000,000)
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| Research Abstract |
In the present research, we investigated the mechanisms for the cell-cycle control and the role of each histone-chaperone for histone H3-H4 in Xenopue eggs, and found the followings.
Using Xenopus eggs and their cell-free extracts, we found that in Xenopus eggs the APC/C inhibitor protein Erp1 was a downstream substrate of the kinase Mos, and that Mos-dependent phosphorylation of Erp1 by Rsk was crucial for both stabilizing Erp1 and enhancing its activity to suppress the APC/C, thereby establishing meiosis-II arrest. We also found that the Ca^<2+>/calmodulin-dependent protein phosphatase calcineurin was transiently activated immediately after Ca^<2+> addition to CSF-arrested egg extracts, independently of CaMKII, whose activity is essential for cancellation of meiosis-II arrest via Erp1 degradation. Thus, in Xenopus eggs, meiosis-II arrest is established and maintained by Erp1 that has been activated by the Mos-Rsk kinase cascade and is canceled by Erp1 degradation that depends on both
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calcineurin and CaMKII.
We have also examined roles of the histone H3-H4 chaperones CAF-1 and Asf1 in assembling both basic and higher-order chromatin structures, using Xenopus egg extracts that support chromosomal DNA replication in S phase and chromosome condensation in M phase. We found that, by immunodepletion of CAF-1 from extracts, only chromatin assembly instantly after DNA replication was disturbed. In contrast, depletion of Asf1 severely impaired chromatin assembly onto nascent DNA during S phase, resulting in not only replicated chromatin with half-reduced nucleosome density but also less condensed, elongated chromosomes in the subsequent M phase. These results strongly suggested that in Xenopus eggs CAF-1 mediates nucleosome assembly coupled to chromosomal DNA replication while Asf-1 is essential for de novo nucleosome assembly onto replicated chromosomal DNA, and that the histone chaperone-mediated de novo nucleosome assembly during chromatin replication ensures the faithful transmission of chromosomes through contributing to mitotic chromosome condensation. Less
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