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.[1]References
- Translational inefficiency of acid beta-glucosidase mRNA in transgenic mammalian cells. Xu, Y.H., Grabowski, G.A. Mol. Genet. Metab. (1998) [Pubmed]
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