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Cloning, subcellular localization and functional expression of human RNase HII
P Frank1, C Braunshofer-Reiter, A Pöltl
1Department of Molecular Genetics, Institute of Tumor Biology and Cancer Research, University of Vienna, Austria.
Biological Chemistry
|January 23, 1999
Summary
Researchers identified a novel human RNase H enzyme, homologous to the main bacterial RNase HI. This enzyme, designated human RNase HII, is active and primarily located in the cell nucleus.
Area of Science:
- Molecular Biology
- Enzymology
Background:
- The major mammalian RNase H (RNase HI) shows evolutionary links to prokaryotic RNase HII.
- RNase HII is considered a minor activity in E. coli.
Purpose of the Study:
- To investigate the existence of a human RNase H homologous to the primary bacterial RNase HI.
- To clone, sequence, express, and characterize this potential human enzyme.
Main Methods:
- Sequence analysis of expressed sequence tags (ESTs).
- Polymerase Chain Reaction (PCR) amplification of human cDNA.
- Recombinant protein expression and purification (His(6)-tag, Ni(2+)-chelating chromatography).
- Activity assays (renaturation gel assay).
- Subcellular localization studies (green fluorescent protein-fusion construct).
Main Results:
- A human open reading frame encoding a 32 kDa protein was identified and expressed.
- The recombinant protein demonstrated active RNase H enzymatic function.
- Enzymatic properties suggest identity with previously purified human RNase HII.
- Subcellular localization studies confirmed the protein's presence in the nucleus.
Conclusions:
- A human RNase H enzyme, homologous to bacterial RNase HI and likely identical to human RNase HII, has been characterized.
- This nuclear-localized enzyme plays a role in cellular processes involving RNA processing or degradation.