Studies on functional domains of cyclins in the cell cycle.

细胞周期的进展是由周期蛋白依赖激酶（cdks）的活性控制的。在本研究项目中，我们分析了细胞周期蛋白在体内和体外的功能域。(1)我们已经证明，Xenopus cyclin A1中cyclin box中保守残基的突变，该区域的小缺失，以及c端小截断都破坏了p34^<cdc2>和p32^<cdk2>的结合和激活。相比之下，细胞周期蛋白A1中最重要的160个氨基酸可以被删除而不丧失活性，除了n端破坏盒的突变阻止了细胞周期蛋白A在减数分裂II中期退出时的破坏，这是由爪蟾卵提取物中Ca^<2+>升高引起的。(2)测定了外源p34^<cdc2>与细菌表达GST-cdc2之间细胞周期蛋白A和细胞周期蛋白B的交换率。周期蛋白A-p34^<cdc2>和周期蛋白B-p34^<cdc2>复合物的半衰期估计分别为4和15小时。(3)为了进一步分析细胞周期蛋白A的结构和功能，我们在GAL1-10启动子控制下，在酿酒酵母芽殖中表达了Xenopus cyclin A。酵母的生长携带如此低拷贝数的质粒，无论是野生型还是不可破坏的细胞周期蛋白a都抑制了酵母的生长。表达的细胞周期蛋白A可以与cdc28结合，细胞周期蛋白A-cdc28复合物的激酶似乎在细胞周期的S期引起阻滞。我们正在利用该系统进一步分析细胞周期蛋白A的结构和功能。

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.