论文部分内容阅读
酵母ＰＨＯ８５结合蛋白ＰＡＰ１与其它的ＰＨＯ８５结合蛋白ＰＨＯ８０、ＰＣＬ１及ＰＣＬ２有较高的同源性。我们构建了ＰＡＰ１与ＨＡ抗原的融合基因，利用抗－ＨＡ抗体进行免疫沉淀。体外翻译的融合ＨＡ标记肽的ＰＡＰ１与体外翻译的ＰＨＯ８５的免疫共沉淀证明了ＰＡＰ１与ＰＨＯ８５的结合。同时纯化了大肠杆菌表达的ＰＨＯ４蛋白，并构建了ＰＨＯ８５∷ＴＲＰ１缺失株。ＰＡＰ１与ＨＡ的融合基因被置于酵母ＡＤＨ１启动子的控制之下，转入酵母ＹＰＨ４９９和ＰＨＯ８５∷ＴＲＰ１菌株中，抽提总蛋白，利用ＨＡ抗体制备免疫沉淀复合物。ＰＡＰ１的免疫沉淀复合物能够在体外磷酸化ＰＨＯ４蛋白，并且这种磷酸化作用是ＰＨＯ８５依赖的，而与酵母的ＣＤＣ２８蛋白激酶无关。这表明ＰＡＰ１能与ＰＨＯ８５形成细胞周期蛋白－ＣＤＫ复合物，ＰＡＰ１可能是一种细胞周期蛋白。Ｎｏｒｔｈｅｒｎ杂交显示ＰＡＰ１基因的转录在细胞周期的各时相中没有明显的差异。构建了ＰＡＰ１基因的缺失株，酸性磷酸酯酶活力分析表明，ＰＡＰ１在ＰＨＯ系统中没有作用。
The yeast PHO85 binding protein PAP1 has high homology with other PHO85 binding proteins PHO80, PCL1 and PCL2. We constructed a fusion gene for PAP1 with HA antigen and immunoprecipitated with anti-HA antibody. Co-immunoprecipitation of in vitro translated PAP of fusion HA tag peptide with PHO85 translated in vitro demonstrated the binding of PAP1 to PHO85. At the same time, the PHO4 protein expressed in E. coli was purified and the PHO85 :: TRP1 deletion strain was constructed. The fusion gene of PAP1 and HA was placed under the control of the yeast ADH1 promoter and transformed into yeast YPH499 and PHO85 :: TRP1 strains. The total protein was extracted and the HA antibody was used to prepare the immunoprecipitating complex. The immunoprecipitated complex of PAP1 is capable of phosphorylating PHO4 protein in vitro, and this phosphorylation is PHO85-dependent, independent of yeast CDC28 protein kinase. This indicates that PAP1 forms a cyclin-CDK complex with PHO85, and PAP1 may be a cyclin. Northern blotting showed that there was no significant difference in the transcription of the PAP1 gene in each phase of the cell cycle. The deletion of PAP1 gene was constructed, and the activity analysis of acid phosphatase showed that PAP1 had no effect in PHO system.