【摘要】 为探讨酿酒酵母(S.cerevisiae)S期检查点通路上,web2(wantsE1Abadly2,web2)基因与rad53基因的上下游位置及相互作用关系,利用羟基脲(hydroxyurea,HU)分别阻断web2突变株和野生株细胞的DNA合成.采用pHA-rad53质粒救助实验测定质粒源性Rad53蛋白是否能救助web2突变株对羟基脲的敏感性;Western印迹及免疫共沉淀反应检测Rad53蛋白表达及磷酸化.结果显示,pHA-rad53质粒可以救助web2突变株的存活;Western印迹检测到web2突变株内质粒源性Rad53蛋白表达增强而且至少Rad53部分蛋白为磷酸化蛋白.说明在HU作用下,过表达并磷酸化的质粒源性Rad53蛋白可以救助web2突变株的S期检查点功能缺陷,在酿酒酵母S期检查点通路上web2基因位于rad53基因上游,可能直接参与将检查点信号传递至Rad53蛋白.更多还原

【Abstract】 To investigate the location and interaction between web2 gene and rad53 gene in S checkpoint pathway of S.cerevisiae,both web2 mutant and wild type cells were treated with hydroxyurea(HU). HU sensitivity, expressions and phosphorylation of Rad53 were studied by means of pHA-rad53 plasmid rescue test, Western blot and immunoprecipitation. The result of rescue test showed Rad53 from plasmid could rescue web2 mutant’s survival from HU treatment. The results of immunoblots showed increased and partly phosphorylated Rad53 from plasmids in web2 mutant. The results suggested that overexpression and phosphorylation of Rad53 from plasmid improved its kinase activity, which at least partly restore the S checkpoint function of web2 mutant, web2 gene performs functions upstream of rad53 gene,and in some way directly signaling to Rad53 in S checkpoint pathway in response to HU.更多还原