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Regulation of yeast cell cycle by Cdk family and its application for drug discovery.

Cdk家族对酵母细胞周期的调节及其在药物发现中的应用。

基本信息

批准号：
13660099
负责人：
NIHIZAWA Masafumi
金额：
$ 2.3万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660099/
关键词：
Cell cycle Yeast Cyclin-dependent kinase Carbon metabolism Gene chip サイクリン依存性キナーゼ

项目摘要

A unique Cdk (Cdc28) functions in the progression of yeast cell cycle, but there exists a Cdk family whose members, including Pho85 kinase, function in various cellular events. Yeast cells lacking Pho85 kinase display pleiotropic phaenotypes, including constitutive expression of PHO genes, accumulation of glycogen, abnormal cell morphology, slow growth rate, and inability to grow in the absence of CLN1 and CLN2. Pho85 is known to interact with 10 cyclin-lke proteins, but only a little is known regarding which kinase-cyclin combination is responsible for which cellular function. To clarify cellular targets of respective Pho85-cyclin complex, we constructed ten Pho85-cyclin fusion genes and replace the chromosomal PHO85 locus with respective fusion gene, so that the yeast cell produces unique Pho85-cyclin fusion protein, enabling us to study target genes of the corresponding kinase-cyclin complex using a DNA microarray.First, we studied function of the fusion proteins by their ability to … More complement pho85 mutant phenotypes. We found that cells producing Pho85-Pho80 could repress PHO5, those producing Pho85-Pc11 could grow in the absence of CLN1 and CLN2, and Pho85-Pc18 producing cells did not accumulate glycogen, which indicate that these fusion proteins can function as corresponding complex.To reveal immediate target genes of Pho85 and each Pho85-cyclin complex, we constructed an expression system of PHO85 and PHO85-cyclin fusion genes induced by tetracycline, and introduced the expression plasmid into pko85 pho80 mutant cells. After iduction of the kinase or kinase-cyclin fusion gene, RNA was extracted periodically from 0 h to 4 h, and was converted to cDNA and then to cRNA, which was subjected to expression analysis using a DNA chip (GeneChip). When PHO85 was induced, induction of SWI5, CHS2, EGT2, PCL9, TSL1, PCL7, and GSY1, and repression of MAK11, DRS1, and PRS3 were observed. Induction of PHO85-PHO80 gave similar results, suggesting a possibility that most target genes of PHO85 are regulated by PHO85-PHO80 complex. However, PCL7 was induced by Pho85 but not by Pho85-Pho80, suggesting that PCL7 is regulated by a kinase-cyclin complex (es) other than Pho85-Pho80. Taken together, this approach was proven to be useful to distinguish the targets of ten different Pho85-cyclin complexes. Less

一个独特的Cdk（Cdc 28）在酵母细胞周期的进程中起作用，但存在一个Cdk家族，其成员包括Pho 85激酶，在各种细胞事件中起作用。缺乏Pho 85激酶的酵母细胞显示多效性表型，包括PHO基因的组成型表达、糖原的积累、异常的细胞形态、缓慢的生长速率以及在不存在CLN 1和CLN 2的情况下不能生长。已知Pho 85与10种细胞周期蛋白样蛋白相互作用，但对于哪种激酶-细胞周期蛋白组合负责哪种细胞功能知之甚少。为了明确各Pho 85-cyclin复合物的细胞靶点，我们构建了10个Pho 85-cyclin融合基因，并将染色体上的PHO 85位点替换为各自的融合基因，使酵母细胞产生独特的Pho 85-cyclin融合蛋白，从而使我们能够利用DNA微阵列研究相应激酶-cyclin复合物的靶基因。 ...更多信息补体pho 85突变表型。我们发现产生Pho 85-Pho 80的细胞可以抑制PHO 5，产生Pho 85-Pc 11的细胞可以在没有CLN 1和CLN 2的情况下生长，而产生Pho 85-Pc 18的细胞不积累糖原，这表明这些融合蛋白可以作为相应的复合物发挥作用。我们构建了四环素诱导的PHO 85和PHO 85-cyclin融合基因的表达系统，并将表达质粒导入pko 85 pho 80突变细胞中。在诱导激酶或激酶-细胞周期蛋白融合基因后，从0小时至4小时定期提取RNA，并转化为cDNA，然后转化为cRNA，使用DNA芯片（GeneChip）对其进行表达分析。当诱导PHO 85时，观察到SWI 5、CHS 2、EGT 2、PCL 9、TSL 1、PCL 7和GSY 1的诱导以及MAK 11、DRS 1和PRS 3的抑制。PHO 85-PHO 80复合物的诱导也得到了类似的结果，提示PHO 85的大多数靶基因可能受PHO 85-PHO 80复合物的调控。然而，PCL 7被Pho 85诱导，而不是被Pho 85-Pho 80诱导，这表明PCL 7被Pho 85-Pho 80以外的激酶-细胞周期蛋白复合物调节。总之，这种方法被证明是有用的，以区分10个不同的Pho 85-细胞周期蛋白复合物的目标。少