Cribbs L L, Shimizu N, Yockey C E, Levin J E, Jakovcic S, Zak R, Umeda P K
Department of Medicine, University of Chicago, Illinois 60637.
J Biol Chem. 1989 Jun 25;264(18):10672-8.
We have examined the transcriptional regulation of the rabbit myosin heavy chain (HC) beta gene by using DNA-mediated transfection experiments. To analyze the activity of the myosin HC beta promoter in a myogenic background, cultured myoblasts from 12-day-old chick embryonic breast muscle were transfected with a chimeric gene containing 781 base pairs of the promoter region fused to the gene for chloramphenicol acetyltransferase (CAT). As indicated by the transient expression of chloramphenicol acetyltransferase, the activity of the promoter in myoblast cultures increased at least 32-fold following differentiation and was selectively inhibited when myogenesis was blocked with 5-bromodeoxyuridine. Furthermore, RNase protection experiments showed that the in vivo myosin HC beta transcriptional initiation (or cap) site was utilized in the transfected skeletal muscle cells and also that the regulation of the exogenous promoter was similar to the induction of the endogenous skeletal alpha-actin gene. The results indicated that the exogenous promoter is regulated in a tissue- and stage-specific manner. By creating progressive 5' deletions of the promoter, we showed that only the region extending -294 base pairs upstream from the cap site is necessary for the muscle-specific expression. Linker-scanner mutagenesis of this region indicated that the positive regulation in differentiated skeletal muscle is mediated by at least two distinct elements within the 5'-flanking region of the myosin HC beta gene.
我们通过DNA介导的转染实验研究了兔肌球蛋白重链（HC）β基因的转录调控。为了分析肌球蛋白HCβ启动子在成肌背景下的活性，将来自12日龄鸡胚胎胸肌的培养成肌细胞用一个嵌合基因进行转染，该嵌合基因包含781个碱基对的启动子区域，与氯霉素乙酰转移酶（CAT）基因融合。如氯霉素乙酰转移酶的瞬时表达所示，成肌细胞培养物中启动子的活性在分化后增加了至少32倍，并且当用5-溴脱氧尿苷阻断肌生成时被选择性抑制。此外，核糖核酸酶保护实验表明，体内肌球蛋白HCβ转录起始（或帽）位点在转染的骨骼肌细胞中被利用，并且外源启动子的调控类似于内源性骨骼肌α-肌动蛋白基因的诱导。结果表明，外源启动子以组织和阶段特异性方式受到调控。通过对启动子进行逐步的5'缺失，我们表明只有从帽位点上游延伸-294个碱基对的区域对于肌肉特异性表达是必需的。对该区域进行接头扫描诱变表明，分化骨骼肌中的正调控由肌球蛋白HCβ基因5'侧翼区域内的至少两个不同元件介导。
