|Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
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
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