The structure and function of myogenic factors.

生肌因子的结构和功能。

基本信息

批准号：
02454151
负责人：
NABESHIMA Yo-ichi
金额：
$ 3.9万
依托单位：
National Institute of Neuroscience
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1992
项目状态：
已结题

项目摘要

1) In order to analyze the roles of individual members of myogenic regulatory factors in myogenesis, we have investigated their effects on the transcription driven by the promoter of the chicken myosin alkali light chain (MLC1/3) gene. CMD1(MyoD) or c-myogenin, but cMRF4 activated transcription of reporter plasmid containing 3.4kb upstream region of MLC1 gene in primary fibriblasts. The activation by MyoD or myogenin was dependent on the existence of a muscle-specific enhancer located from -2096 to -1743. The distal half of the enhancer, containing a pair of E-boxes, E1 and E2, was found to be a MyoD specific enhancer. In contrast, the activation by myogenin was dependent on the existence of the proximal half of the enhancer, containing three E-boxes, E3,E4,E5. In their E-boxes, the proximal E-box, E5 was essential for trans-activation by myogenin. Although MyoD and myogenin bound to E5 with similar affinity to each other, E5 and its falnking sequence worked as a myogenin-specific enhancer. From the analysis of the effect of chimeric proteins of MyoD and myogenin, the region(s) outside of the basic-helix-loop-helix (b-HLH) domain of myogenin was involved in the specificity of the myogenin-specific enhancer.2) To understand regulatory mechanisms which govern transcriptional activation of the myogenin gene, transgenic mice bearing the lacZ gene driven by the upstream region of the myogenin gene were generated. Stereoscopic visualization of LacZ positive cells of these transgenic mouse embryos revealed that the upstream region of the myogenin gene conferred its transcriptional activation in cells of the skeletal muscle lineages in somites, limb buds, and viceral arches.3) To investigate the molecular function of myogenin gene during muscle development, myogenin gene was desrurpted by homologous recombination. The analysis of effects of myogenin gene knock out is now in progress.

1）为了分析肌生成调节因子各个成员在肌发生中的作用，我们研究了它们对鸡肌球蛋白碱轻链（MLC1/3）基因启动子驱动的转录的影响。 CMD1(MyoD)或c-肌细胞生成素，但cMRF4在原代成纤维细胞中激活含有MLC1基因上游3.4kb区域的报告质粒的转录。 MyoD 或肌细胞生成素的激活取决于位于 -2096 至 -1743 的肌肉特异性增强子的存在。增强子的远端一半包含一对 E 盒 E1 和 E2，被发现是 MyoD 特异性增强子。相反，肌细胞生成素的激活依赖于增强子近端一半的存在，其中包含三个E-盒，E3、E4、E5。在他们的 E-box（近端 E-box）中，E5 对于肌生成素的反式激活至关重要。尽管 MyoD 和肌生成素以彼此相似的亲和力与 E5 结合，但 E5 及其falking 序列充当肌生成素特异性增强子。从MyoD和肌细胞生成素嵌合蛋白的作用分析来看，肌细胞生成素的碱性螺旋-环-螺旋（b-HLH）结构域之外的区域参与了肌细胞生成素特异性增强子的特异性2。）为了了解控制肌生成素基因转录激活的调节机制，产生了带有由肌生成素基因上游区域驱动的lacZ基因的转基因小鼠。对这些转基因小鼠胚胎的 LacZ 阳性细胞进行立体观察，发现肌细胞生成素基因的上游区域在体节、肢芽和副弓的骨骼肌谱系细胞中赋予其转录激活作用。 3）研究肌细胞生成素的分子功能在肌肉发育过程中，肌细胞生成素基因通过同源重组而被破坏。目前正在对肌生成素基因敲除的影响进行分析。