Study of NPS1, a novel CDC gene of cerevisiae

NPSL基因是酿酒酵母新发现的细胞分裂周期(CDC)基因，编码160kd的核蛋白，为细胞生长所必需。我对NPS1基因在细胞周期中的功能进行了研究，得到了以下结果：1)我构建了一个突变的NPS1基因，在蛋白激酶Conennsus基序的一个可疑的ATP结合位点上有一个谷氨酸残基，而不是赖氨酸残基(Lys792)，发现Lys792是NPS1基因产物在体内发挥功能所必需的。2)通过对只能在半乳糖介质中表达NPS1基因的条件致死菌株的表型进行研究，发现NPS1基因作用于酿酒酵母的G2期，传递DNA复制完成的信号。3)NPS1基因产物定位于酿酒酵母核囊中，具有DNA结合活性。4)我成功地在携带NPS1基因的重组杆状病毒转化的SF9细胞中表达了NPS1蛋白。转化的SF9细胞的大规模培养和NPS1蛋白的纯化正在进行中。5)为了鉴定与NPS1相关的基因，我进行了以下实验：i)分离NPS1基因高剂量表达抑制生长的突变体；ii)筛选NPS1缺失引起的高拷贝致死抑制因子；iii)筛选NPS1基因高剂量表达导致合成致死的基因。对上述实验获得的突变体和基因的分析目前正在进行中。

The NPSl gene is a novel cell division cycle (CDC) gene of Saccharomyces cerevisiae enconding a 160 kd nuclear protein essenntial for cell growth. I investigated the functions of NPS1 gene in cell cycle and obtained the following results.1) I constructed a mutant nps1 gene which has a glutamic acid residue at position 792 instead of lysin residue (Lys792) at a suspected ATP-binding site of protein kinase consennsus motief, and found that Lys792 is indispensable for in vivo function of NPS1 gene product.2) By investigating the phenotypes of conditional lethal strain which can express NPS1 gene only in galactose medium, I found that nps1 gene works in g2 phase of S.cerevisiae transducing the signal of completion of DNA replication.3) The NPS1 gene product was found to locate in nuclear saffold of S.cerevisiae and possess DNA-binding activity.4) I succeeded to produce the Nps1 protein in SF9 cell transformed with recombinant baculovirus carrying NPS1 gene. Large scaled culture of transformed SF9 cells and purification of Nps1 protein are now on going.5) In order to identify genes acting in relation to NPS1, I carried out the following experiments : i) isolate mutant whose growth is suppressed by the high dosage-expression of NPS1 gene ; ii) screen the high copy suppressor of lethality caused by NPS1 depletion ; iii) screen the gene which cause synthetic lethality with high-dosage expression of NPS1 gene. The analyzes of mutants and genes obtained by the above experiments are now on going.