Cloning of the cDNA encoding the large subunit of human RNase HI, a homologue of the prokaryotic RNase HII.
Two RNases H of mammalian tissues have been described: RNase HI, the activity of which was found to rise during DNA replication, and RNase HII, which may be involved in transcription. RNase HI is the major mammalian enzyme representing around 85% of the total RNase H activity in the cell. By using highly purified calf thymus RNase HI we identified the sequences of several tryptic peptides. This information enabled us to determine the sequence of the cDNA coding for the large subunit of human RNase HI. The corresponding ORF of 897 nt defines a polypeptide of relative molecular mass of 33,367, which is in agreement with the molecular mass obtained earlier by SDS/PAGE. Expression of the cloned ORF in Escherichia coli leads to a polypeptide, which is specifically recognized by an antiserum raised against calf thymus RNase HI. Interestingly, the deduced amino acid sequence of this subunit of human RNase HI displays significant homology to RNase HII from E. coli, an enzyme of unknown function and previously judged as a minor activity. This finding suggests an evolutionary link between the mammalian RNases HI and the prokaryotic RNases HII. The idea of a mammalian RNase HI large subunit being a strongly conserved protein is substantiated by the existence of homologous ORFs in the genomes of other eukaryotes and of all eubacteria and archaebacteria that have been completely sequenced.
Frank
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P
Department of Molecular Genetics, Institute of Tumor Biology and Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria.
Braunshofer-Reiter
C
C
Wintersberger
U
U
Grimm
R
R
Büsen
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D016914:Ribonuclease H / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013950:Thymus Gland / Q000201:enzymology; D014357:Trypsin
D018076:DNA, Complementary; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C091139:ribonuclease HII; D016914:Ribonuclease H; C078856:ribonuclease HI; D014357:Trypsin