Horlick R A, Benfield P A
Central Research and Development, E. I. du Pont de Nemours and Co., Wilmington, Delaware 19880-0328.
Mol Cell Biol. 1989 Jun;9(6):2396-413. doi: 10.1128/mcb.9.6.2396-2413.1989.
A series of constructs that links the rat muscle creatine kinase promoter to the bacterial chloramphenicol acetyltransferase gene was generated. These constructs were introduced into differentiating mouse C2C12 myogenic cells to localize sequences that are important for up-regulation of the creatine kinase gene during myogenic differentiation. A muscle-specific enhancer element responsible for induction of chloramphenicol acetyltransferase expression during myogenesis was localized to a 159-base-pair region from 1,031 to 1,190 base pairs upstream of the transcription start site. Analysis of transient expression experiments using promoters mutated by deletion indicated the presence of multiple functional domains within this muscle-specific regulatory element. A DNA fragment spanning this region was used in DNase I protection experiments. Nuclear extracts derived from C2 myotubes protected three regions (designated E1, E2, and E3) on this fragment from digestion, which indicated there may be three or more trans-acting factors that interact with the creatine kinase muscle enhancer. Gel retardation assays revealed that factors able to bind specifically to E1, E2, and E3 are present in a wide variety of tissues and cell types. Transient expression assays demonstrated that elements in regions E1 and E3, but not necessarily E2, are required for full enhancer activity.
构建了一系列将大鼠肌肉肌酸激酶启动子与细菌氯霉素乙酰转移酶基因相连的载体。将这些载体导入正在分化的小鼠C2C12成肌细胞中，以定位在成肌分化过程中对肌酸激酶基因上调起重要作用的序列。一个负责在肌生成过程中诱导氯霉素乙酰转移酶表达的肌肉特异性增强子元件被定位到转录起始位点上游1031至1190个碱基对的159个碱基对区域。使用通过缺失突变的启动子进行瞬时表达实验分析表明，该肌肉特异性调控元件内存在多个功能域。跨越该区域的一个DNA片段被用于DNA酶I保护实验。来自C2肌管的核提取物保护该片段上的三个区域（命名为E1、E2和E3）不被消化，这表明可能有三个或更多的反式作用因子与肌酸激酶肌肉增强子相互作用。凝胶阻滞分析显示，能够特异性结合E1、E2和E3的因子存在于多种组织和细胞类型中。瞬时表达分析表明，E1和E3区域中的元件对于完全增强子活性是必需的，但E2区域不一定是必需的。
