Xu Y H, Grabowski G A
Division of Human Genetics, University of Cincinnati, College of Medicine, Cincinnati, Ohio 45229, USA.
Mol Genet Metab. 1998 Jun;64(2):87-98. doi: 10.1006/mgme.1998.2709.
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.
戈谢病酶酸性β-葡萄糖苷酶的互补DNA（cDNA）在多种哺乳动物细胞和Sf9昆虫细胞中过表达。无论从MFG-GC逆转录病毒还是四环素反式激活系统过表达，哺乳动物细胞中产生的信使核糖核酸（mRNA）量（>750倍）与酸性β-葡萄糖苷酶蛋白量（约6至14倍）之间存在很大差异。在Sf9昆虫细胞中未观察到这种情况。对完整哺乳动物细胞中这种mRNA翻译的定量评估表明，与正常人皮肤成纤维细胞相比，细胞系中的效率低55至135倍。来自过表达哺乳动物或昆虫细胞的酸性β-葡萄糖苷酶mRNA的体外翻译效率与来自正常人成纤维细胞的相似。一种细胞质、热不稳定蛋白被认为对这些RNA的体外翻译具有抑制作用。蛋白质印迹法和细胞质去除实验表明，这是一种识别酸性β-葡萄糖苷酶编码序列的80 kDa细胞质mRNA结合蛋白。在昆虫细胞中未检测到这种细胞质蛋白。这些结果表明，酸性β-葡萄糖苷酶编码序列和一种热不稳定细胞质蛋白参与调节转基因细胞系中过表达mRNA的翻译。
