Toward understanding of regulation of mRNA capping and functions of the cap structure.

酵母mRAN盖帽酶由α亚基和β亚基组成，分别具有鸟苷基转移酶和三磷酸氢酶活性。在这项研究中，我们分离了10个编码α亚基的CEG1基因的隐性温度敏感突变，并确定了突变位点。其中9个突变（ceg1-1至ceg1-9）在单拷贝质粒上分离，其余一个突变（ceg1-10）在多拷贝质粒上分离。ceg1-10在多个拷贝中的存在对于携带突变的细胞的生存是必不可少的，并且转移到限制性温度导致ceg1-10细胞的快速生长停止，而其他突变体在温度升高时逐渐降低生长速率。突变的成对组合未观察到基因内互补。通过测定共价Ceglp-GMP复合物的形成来检测每个突变蛋白的guanylytransferase活性。虽然大部分突变蛋白表现出明显的热不稳定性，但Ceg1-8p和Ceg1-9p在高温培养过程中保持了大量的活性。对分离的突变蛋白在大肠杆菌细胞中表达进行了进一步的鉴定。目前，基因外抑制子的分离以及对ceg1突变细胞的详细分析正在进行中。

The yeast mRAN capping enzyme is composed of alpha and beta subunits possessing guanylyltransferase and triphoshatase activities, respectively. In this study, we have isolated 10 recessive temperature-sensitive mutations of the CEG1 gene encoding the alpha subunit and determined the mutation sites. Nine of the mutations(ceg1-1 to ceg1-9)were isolated on a single-copy plasmid-and the remaining one(ceg1-10)on a multi-copy plasmid. The presence of ceg1-10 in multiple copies is essential for the viability of cells carrying the mutation, and a shift to the restrictive temperature resulted in quick growth arrest of ceg1-10 cells while other mutants decreased growth rates gradually upon the temperature up-shift. Intragenic complementation was not observed for pairwise combinations of the mutations.Guanylyltransferase activity was examined for each mutant protein by assaying of a covalent Ceglp-GMP complex formation. While most of the mutant protein showed clear heat-lability, Ceg1-8p and Ceg1-9p maintained substantial activities during incubation at a high temperature. Further characterization was carried out for isolated mutant proteins expressed in E.coli cells.Isolation of extragenic suppressors as well as detailed analyzes of the ceg1 mutant cells are in progress currently.