Study on Nuclear Protein Involved in DNA Replication of Yeast

我已经克隆了基因，NPS 1，它编码一种新的核蛋白参与细胞周期调控酵母酿酒酵母。测定了含有NPS 1的5，211 bp酵母基因组DNA片段的DNA序列。在该片段中存在一个可能的由4，077 bp组成的开放读码框。在DNA和蛋白质数据库中进行同源性检索，发现NPS 1是一个新基因，并在指数生长的酵母细胞总RNA中以4-5 Kb的mRNA形式转录。另一方面，在静止期细胞中检测到很少的转录本。对NPS 1进行了一步基因破坏，发现该基因是酵母细胞生长所必需的。在NPS 1的第一个ATG上游65 bp处构建了一个含有0.8Kb GAL 1启动子/增强子序列的kimeric基因，并转化到DELTAnps 1/NPS 1二倍体中。从上述二倍体的减数分裂分离子中获得了一个单倍体DELTAnps 1条件致死菌株YET 101，并研究了YET 101在NPS 1转录缺失下的表型。YET 101在葡萄糖培养基中停止生长，大多数细胞具有单核的大芽。结果表明，NPS 1参与了细胞分裂周期中S期到M期的调控。MATa/MAT alpha、DELTAnps 1/DELTAnps 1二倍体carint pGAL 1：：NPS 1质粒在半乳糖培养基中正常生长，但在1% K-培养基中形成很少孢子。NPS 1在芽孢形成过程中起重要作用，为了分析NPS 1产物的功能，本研究用纯化的β-半乳糖苷酶-nps 1融合蛋白免疫小鼠，制备了抗nps 1的抗体。用抗nps 1抗体对酵母细胞进行免疫荧光染色，结果显示nps 1定位于细胞核。亲和纯化的nps 1具有DNA结合和蛋白激酶活性。

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.