| Budget Amount *help |
¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1990: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1989: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
I have cloned the gene, NPS1, which encodes a novel nuclear protein involved in cell cycle regulation in yeast Saccharomyces cerevisiae. DNA sequence of 5,211 bp yeast genomic DNA fragment containing NPS1 was determined. In this fragment, one possible open deading frame composed of 4,077 bp was existed. The sequence was searched for similarity on DNA and protein databases, and NPS1 was found to be a novel gene.
NPS1 transcript was found as 4-5 Kb mRNA in total RNA preparation from exponentially growing yeast cells. On the other hand, little transcript was detected in stationary-phase cells. One step gene disruption was carried out on NPS1, and the gene was found to be essential for yeast cell growth. A kimeric gene which have 0.8 Kb GAL1 promoter/enhancer sequence at 65 bp upstream of first ATG of NPS1 was constructed and transformed to DELTAnps1/NPS1 diploid. A haploid DELTAnps1 conditional lethal strain, YET101, was obtained from the meiotic segregants of the above diploid and phenotyopes of YET101 in the absence of NPS1 transcription was studied. YET101 stopped its growth in glucose medium and most of the cells in this medium was found to have a mononucleated large bud. The result suggested that NPS1 is involved in S to M phase control in cell division cycle. MATa/MATalpha, DELTAnps1/DELTAnpsl diploid carint pGAL1 : : NPS1 plasmid grew normally in galactose medium, but formed little spore in 1% K-asetate. NPS1 was found to play an important role in sporulating process.
In order to analyze the function of NPS1 product, I prepared antibody against nps1 by immunizing mice with purified beta-galactosidase-nps1 fusion protein. Immunofluorecent staining of yeast cells with anti-nps1 antibody revealed the localization of nps1 in nucleus. Affinity purified nps1 possessed both DNA-binding- and protein kinase activities.
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