| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1993: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
Cell cycle progression is controlled by the activity of cyclin-dependent kinases (cdks). In this research project, we have analyzed the functional domains of cyclins in vivo and in vitro.
(1)We have shown that mutations of conserved residues in Xenopus cyclin A1 in the cyclin box, small deletions of this region, and small C-terminal truncations all abolish binding to and activation of p34^<cdc2> and p32^<cdk2>. By contrast, the fiest 160 amino acids in cyclin A1 can be deletedwithout loss of the activity, except that mutations in the N-terminal destruction box prevent the destruction of cyclin A at the exit from meiosis II metaphase, which is triggered by elevated Ca^<2+> in Xenopus egg extracts.
(2)The exchange rate of cyclin A and cyclin B between exogenous p34^<cdc2> and bacterially expressed GST-cdc2 was measured in Xenopus egg extracts. The half-lives of the cyclin A-p34^<cdc2> and the cyclin B-p34^<cdc2> complexes are estimated as 4 and 15 hours, respectively.
(3)To allow further analysis of the structure and function of cyclin A,we expressed Xenopus cyclin A in the budding yeast S.cerevisiae under the control of GAL1-10 promoter. The growth of yeast carrying such a low-copy number plamid, both the wild-type and indestructible cyclin A inhibits the growth of yeast. Expressed cyclin A can bind to cdc28, and the kinase of the cyclin A-cdc28 complex appears to cause a block in S phase during the cell cycle. We are using this system to further analyze the structure and function of cyclin A.
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