Cloning, subcellular localization and functional expression of human RNase HII
| Autor: | Christa Braunshofer-Reiter, Klaus Holzmann, Angelika Pöltl, Peter Frank |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
DNA
Complementary RNase P Sequence analysis Recombinant Fusion Proteins Clinical Biochemistry Molecular Sequence Data Ribonuclease H Biochemistry Open Reading Frames Complementary DNA Escherichia coli Humans Amino Acid Sequence Cloning Molecular RNase H Molecular Biology Expressed sequence tag Microscopy Confocal biology Base Sequence Sequence Homology Amino Acid Chemistry Molecular biology Protein subcellular localization prediction Open reading frame biology.protein Nucleic acid HeLa Cells Subcellular Fractions |
| Zdroj: | Biological chemistry. 379(12) |
| ISSN: | 1431-6730 |
| Popis: | Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett. 421, 23-26, 1998), an enzyme described to be a minor activity in E. coli. As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E. coli enzyme, RNase HI. Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein. Purification of the recombinant His(6)-tagged protein from E. coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H. The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res. 22, 5247-5254, 1994). Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus. |
| Databáze: | OpenAIRE |
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