Expression of human aspartyl-tRNA synthetase in COS cells.
Mammalian aspartyl-tRNA synthetase (DRS) occurs in a multi-enzyme complex of aminoacyl-tRNA synthetases, while DRS exists as free soluble enzymes in bacteria and yeast. The properties of human DRS transient expressed in COS cells were examined. After transfection of COS cells with the recombinant plasmids pSVL-63 that contained hDRS cDNA coding and non-coding sequences, and pSV-hDRS where the non-coding sequences were deleted, DRS in the transfected COS cells significantly increased compared to mock transfected cells. COS cells transfected with pSV-hDRS delta 32 that contained N-terminal 32 residue-coding sequence deleted hDRS cDNA showed no increase in DRS activity. Northern blot analysis showed that concentrations of corresponding mRNAs of hDRS and hDRS delta 32 were greatly enhanced in transfected cells. The increases in the level of the transcripts were much higher than those of the corresponding proteins. Gel filtration analysis showed that hDRS in pSV-hDRS transfected cells expressed as a low molecular weight form of hDRS and pSV-hDRS delta 32 transfected cells did not. Epitope tagging and indirect immunofluorescence microscopy was used to localize hDRS. Both hDRSmyc and hDRS delta 32myc were localized in the cytoplasm and showed diffused patterns. These results showed that hDRS has little tendency to aggregate in vivo and suggested that the N-terminal extension in hDRS was not involved in the expression and sub-cellular localization of hDRS, but may play a role in the maintenance of enzymatic activity of hDRS in COS cells.
Escalante
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Department of Chemistry, Georgetown University, Washington, DC 20057.
Qasba
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Yang
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D000818:Animals; D001226:Aspartate-tRNA Ligase / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis; D017384:Sequence Deletion / Q000502:physiology; D014162:Transfection
D046911:Macromolecular Substances; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D001226:Aspartate-tRNA Ligase