Study on Nuclear Protein Involved in DNA Replication of Yeast

我已经克隆了NPS1基因，它编码了一种参与酵母细胞周期调节的新型核蛋白。测定了含有NPS1的521bp酵母基因组DNA片段的DNA序列。在该片段中，存在一个可能的开放死帧，其长度为4077 bp。在DNA和蛋白质数据库中对序列进行相似性搜索，发现NPS1是一个新基因。从指数生长的酵母细胞制备的总RNA中发现NPS1转录物为4-5 Kb mRNA。另一方面，在静止期细胞中很少检测到转录本。对NPS1进行了一步基因破坏，发现该基因对酵母细胞生长至关重要。在NPS1的第一个ATG上游65 bp处构建了一个具有0.8 Kb GAL1启动子/增强子序列的kimeric基因，并将其转化为DELTAnps1/NPS1二倍体。从上述二倍体的减数分裂分离体中获得了DELTAnps1单倍体条件致死菌株YET101，并研究了NPS1转录缺失情况下YET101的表型。YET101在葡萄糖培养基中停止生长，在葡萄糖培养基中发现大多数细胞有一个单核大芽。结果提示NPS1参与细胞分裂周期中S期到M期的调控。MATa/MATalpha、DELTAnps1/DELTAnpsl二倍体carint pGAL1:: NPS1质粒在半乳糖培养基中生长正常，在1% k -乙酸盐培养基中很少形成孢子。发现NPS1在孢子形成过程中起重要作用。为了分析NPS1产物的功能，我用纯化的β -半乳糖苷酶- NPS1融合蛋白免疫小鼠制备了NPS1抗体。用抗nps1抗体对酵母细胞进行免疫荧光染色，发现nps1在细胞核中定位。亲和纯化的nps1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.