酿酒酵母核糖核酸酶H(70)的纯化及相应基因的鉴定。

Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene.

作者信息

Frank P, Braunshofer-Reiter C, Karwan A, Grimm R, Wintersberger U

机构信息

Department of Molecular Genetics, Institute of Tumor Biology and Cancer Research, University of Vienna, Austria.

出版信息

FEBS Lett. 1999 May 7;450(3):251-6. doi: 10.1016/s0014-5793(99)00512-8.

DOI:10.1016/s0014-5793(99)00512-8

PMID:10359084

Abstract

We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure. Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide. Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S. cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62849. We therefore renamed this gene RNH70. Immunofluorescence microscopy using a RNH70-EGFP fusion construct indicates nuclear localization of RNase H(70). Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested. Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.

摘要

我们采用优化的色谱纯化程序，将酿酒酵母核糖核酸酶H(70)纯化至同质状态。复性凝胶分析将核糖核酸酶H活性定位到一条70 kDa的多肽上。胰蛋白酶肽段测序确定酿酒酵母基因组第七条染色体上的开放阅读框YGR276c为相应基因，该基因编码一个推定的分子量为62849的多肽。因此，我们将该基因重新命名为RNH70。使用RNH70-EGFP融合构建体进行的免疫荧光显微镜检查表明核糖核酸酶H(70)定位于细胞核。在测试条件下，从酵母基因组中删除RNH70并未导致任何严重的表型。同源性搜索显示与许多真核蛋白质和开放阅读框有显著相似性，其中包括黑猩猩GOR蛋白，它是一种人类自身免疫抗原的同源物，在丙型肝炎病毒感染患者中可引发自身免疫反应。