Studies on functional domains of cyclins in the cell cycle.

细胞周期进程是由细胞周期蛋白依赖性激酶（cdks）的活性控制的。在本研究中，我们分析了细胞周期蛋白在体内和体外的功能结构域。(1)We已经表明，非洲爪蟾细胞周期蛋白A1中细胞周期蛋白盒中保守残基的突变、该区域的小的缺失和小的C端截短都消除了p34^和p32^的结合和激活<cdc2><cdk2>。相比之下，细胞周期蛋白A1中最开始的160个氨基酸可以被删除而不丧失活性，除了N-末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，这是由非洲爪蟾卵提取物中升高的Ca^2+触发的。（2）在爪蟾卵提取物中检测外源性p34 ~+与细菌表达的GST-cdc 2之间cyclin A和cyclin B的交换率<cdc2>。细胞周期蛋白A-p34^<cdc2>和细胞周期蛋白B-p34^<cdc2>复合物的半衰期估计分别为4小时和15小时。(3)To为了进一步研究细胞周期蛋白A的结构和功能，我们在GAL 1 -10启动子的调控下，在酿酒酵母中表达了非洲爪蟾细胞周期蛋白A。携带这种低拷贝数质粒的酵母的生长，野生型和不可破坏的细胞周期蛋白A都抑制酵母的生长。表达的cyclin A可以与cdc 28结合，并且cyclin A-cdc 28复合物的激酶似乎在细胞周期期间引起S期阻滞。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.