Abstract
RNA helicase A is an abundant nuclear enzyme of HeLa cells that unwinds double-stranded RNA in a 3' to 5'direction (Lee, C. G., and Hurwitz, J. (1992) J. Biol. Chem. 267, 4398-4407). A complementary DNA (cDNA) clone expressing RNA helicase A was isolated by screening a human cDNA library with polyclonal antibodies produced against the purified protein. The deduced amino acid sequence from this clone showed that RNA helicase A is a member of the DEAH family of proteins thought to be helicases. Sequence comparison among all known proteins of the DEAH family revealed that the highest homology was between RNA helicase A and the maleless protein (MLE) of Drosophila. There was 49% identity and 85% similarity throughout the overall primary sequences of both proteins, suggesting that RNA helicase A is the human counterpart of Drosophila MLE. Polyclonal antibodies against Drosophila MLE recognized RNA helicase A in crude nuclear extracts of HeLa cells as well as the purified protein. A recombinant RNA helicase A containing 6 histidine residues at the NH2 terminus was expressed in Sf9 cells using a baculovirus vector. The protein isolated from insect cells and the enzyme purified from HeLa cells exhibited identical RNA helicase and RNA-dependent ATPase activities.
Highlights
From the Graduate Program in Mo&cular Biology, Memorial S’loan-Kettering Cancer Center, Sloan-Kettering Institute, New York,New York 10021
The deduced amino acid sequence from this clone showed that RNA helicase A is a member of the DEAH family of proteins thought to behelicases
There was no differencein the immunoreactivitybetween the recombinant helicase and the native protein. These results indicate that the cDNA obtained by screening the X ZAP11 library with polyclonal antibodies represented the full-length gene of RNA helicase A, and provide the firstexperimental evidence that aneukaractivities of the native and expressedproteins and to rule out yotic DEAH protein catalyzes the unwinding of dsRNA
Summary
Large T antigen [4], most of these RNA helicases share extensive homology in their primaraymino acid sequences, defining a superfamily of RNA (or DNA helicases) and/or RNA- or DNA-dependent ATPases, called the DEAD or DEAH family (1, 2, 5 ). The partial nucleotide sequences of 5different clones containing cDNA inserts larger than 4 kb were determined using the dideoxy chain termination method employing a Sequenase Version 2.0 kit The positive recombinant clone was selected by DNA sequencing using the dideoxy chain termination method as described above This recombinant DNA was cut with BamHIand EcoRI enzymes, rendered blunt-ended with the Klenow fragment, and ligated into thebaculovirus expression vector pBlueBac. 5 plaques contained positive recombinant viruses harboring the RNA helicase A gene, which was determined by dot hybridization with the complete 32P-labeledclone 1cDNA (see above) as theprobe under the condition described for the Northernanalysis. Fractions enriched in recombinant RNA helicase A, which were analyzed by SDS-PAGE, were pooled (0.2 mg, 7 ml) and directly loaded onto a heparin-SepharoseCL-4B column (0.5 ml) equilibrated with buffer A containing 0.1 M NaCI, 1 mM Dl-", and 10% glycerol. They all were truncated with respect to clone 1, with deletions coveringthe first
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