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

FEBS Letters

Braunshofer-Reiter, Christa¹  Karwan, Anneliese¹  Grimm, Rudolf²  Frank, Peter¹  Wintersberger, Ulrike¹ 

[1] Department of Molecular Genetics, Institute of Tumor Biology and Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria;Hewlett-Packard, Waldbronn Analytical Division, Hewlett-Packard-Str. 8., D-76337 Waldbronn, Germany

关键词: Ribonuclease H;    RNA-DNA hybrid;    Saccharomyces cerevisiae;    DNA replication;    Nuclease;    Evolution;    PCR;    polymerase chain reaction;    RNase H;    ribonuclease H;    SDS-PAGE;    sodium dodecyl sulfate polyacrylamide gel electrophoresis;    HPLC;    high performance liquid chromatography;    EST;    expressed sequence tag;    ORF;    open reading frame;    PMSF;    phenylmethylsulfonyl fluoride;    PEG;    polyethylene glycol;    EGFP;    enhanced green fluorescent protein;    DAPI;    4′;    6-diamino-2-phenylindole;    TPCK;    N-tosyl-L-phenylalanine chloromethylketone;    TLCK;    Nα-p-tosyl-L-lysine chloromethylketone;    pI;    isoelectric point;   

DOI  :  10.1016/S0014-5793(99)00512-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 62 849. 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.

【 授权许可】

Unknown