Harris S A, Dudov K P, Bowman L H
Department of Biological Sciences, University of South Carolina, Columbia, 29208.
J Cell Biochem. 1992 Oct;50(2):178-89. doi: 10.1002/jcb.240500208.
The sequences required for the maximal expression of the mouse L32 ribosomal protein gene and the binding of nuclear factors to L32 promoter elements were analyzed in mouse myoblasts, fibers, and L cells. Various L32 r-protein promoter sequences were linked to the chloramphenicol acetyltransferase gene (CAT), and the expression of the chimeric genes was measured transiently or after their incorporation into the genome. The sequence requirements for maximal expression of the L32 gene are very similar among the various cells and include the previously identified L32 core promoter from approximately -150 to +75. Only the promoter regions between -45 and +11 displays significant cell type specific differences. Relative to the maximal activity in each cell type, the expression of the L32-CAT gene containing the -45 to +11 region is greater in L cells than in myoblasts or fibers. This difference is correlated with the increased activity of an L cell nuclear factor(s) that binds to this fragment. In addition, our results show that deletion of sequences between -981 and -141 causes a 50-70% reduction of the expression of the L32-CAT gene in myoblasts, fibers and L cells. The transcription of all the L32-CAT genes examined decrease after myoblasts differentiate into fibers in a manner similar to the endogenous L32 gene, but we were unable to distinguish between sequences involved in controlling the expression of the L32 gene during myoblast differentiation and those sequences required for maximal promoter activity. However, gel mobility shift assays showed differences in the binding of myoblast and fiber factors to the four promoter fragments examined. The possible role of these factor binding differences in controlling L32 transcription is discussed.
在小鼠成肌细胞、肌纤维和L细胞中，分析了小鼠L32核糖体蛋白基因最大表达所需的序列以及核因子与L32启动子元件的结合情况。将各种L32核糖体蛋白启动子序列与氯霉素乙酰转移酶基因（CAT）相连，并瞬时测量或在嵌合基因整合到基因组后测量其表达。L32基因最大表达的序列要求在各种细胞中非常相似，包括先前确定的从约-150至+75的L32核心启动子。只有-45至+11之间的启动子区域显示出显著的细胞类型特异性差异。相对于每种细胞类型中的最大活性，包含-45至+11区域的L32-CAT基因在L细胞中的表达高于成肌细胞或肌纤维。这种差异与结合到该片段的L细胞核因子活性增加相关。此外，我们的结果表明，缺失-981至-141之间的序列会导致成肌细胞、肌纤维和L细胞中L32-CAT基因的表达降低50-70%。在成肌细胞分化为肌纤维后，所有检测的L32-CAT基因的转录均以类似于内源性L32基因的方式下降，但我们无法区分在成肌细胞分化过程中控制L32基因表达的序列和最大启动子活性所需的序列。然而，凝胶迁移率变动分析显示，成肌细胞和肌纤维因子与所检测的四个启动子片段的结合存在差异。讨论了这些因子结合差异在控制L32转录中的可能作用。
