Translational inefficiency of acid beta-glucosidase mRNA in transgenic mammalian cells.
The cDNA for acid beta-glucosidase, the Gaucher disease enzyme, was overexpressed in a variety of mammalian cells and in Sf9 insect cells. Whether overexpressed from the MFG-GC retrovirus or the tetracycline transactivator system, there was a large discrepancy between the amounts of mRNA (>750-fold) and acid beta-glucosidase protein (approximately 6- to 14-fold) produced in mammalian cells. This was not observed in Sf9 insect cells. Quantitative evaluation of translation of this mRNA in intact mammalian cells indicated a 55- to 135-fold inefficiency in cell lines compared to normal human skin fibroblasts. In vitro translation efficiency with acid beta-glucosidase mRNAs from overexpressing mammalian or insect cells was similar to that from normal human fibroblasts. A cytoplasmic, heat labile protein was suggested as inhibitory to in vitro translation of these RNAs. North-Western blots and cytoplasmic depletion experiments showed this to be an 80-kDa cytoplasmic mRNA-binding protein that recognized acid beta-glucosidase coding sequences. The cytoplasmic protein was not detected in insect cells. These results implicate acid beta-glucosidase coding sequences and a heat labile cytoplasmic protein in modulating the translation of overexpressed mRNA in transgenic cell lines.
Division of Human Genetics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45229, USA.
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Grabowski
D000818:Animals; D030801:Animals, Genetically Modified; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D015870:Gene Expression / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D005962:Glucosylceramidase / Q000235:genetics*; D006801:Humans; D014176:Protein Biosynthesis / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D014162:Transfection / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D016662:Virus Integration / Q000235:genetics
D012333:RNA, Messenger; D005962:Glucosylceramidase