Regulation of Skeletal Myofiber-Type Gene Expression

骨骼肌纤维型基因表达的调控

• 批准号: 9020998
• 负责人: Robert Wade
• 金额: $ 24万
• 依托单位: University of Maryland at Baltimore
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9020998&HistoricalAwards=false
• 关键词: Regulation; Skeletal; Myofiber; Type; Gene

The study of vertebrate muscle development presents exceptional opportunities for the investigation of gene regulation during complex ontogenetic processes. During myogenesis both tissue- specific and developmental stage-specific gene switching occur, culminating in the formation of the two major types of skeletal muscle: fast-twitch and slow-twitch myofibers. Each of the muscle fiber types is characterized by the expression of unique combinations is contractile protein isoforms. The establishment of muscle fiber type arises from the interplay of factors such as cell lineage, type of innervation the myofiber receives, and the level of circulating hormones. Whereas much progress has been made in understanding the molecular mechanisms responsible for muscle-specific gene expression, almost nothing is known about differential myofiber-type-specific gene expression. Dr Wade proposes to investigate the mechanisms regulating myofiber-type specific gene expression through the study of the human troponin I gene family. The adult slow-fiber TnI gene is differentially expressed during embryonic muscle fiber formation, and can serve as a model for the developmental regulation of fiber-type- specific gene expression. He has cloned the human skeletal muscle TnI slow gene, and has identified multiple cis-acting sequences required for its muscle-specific expression by the use of in vitro transient expression assays. These studies have identified a muscle specific enhancer which is not trans- activated by the myogenic regulatory factor MyoD1. He will further characterize the muscle-specific regulatory elements and extend these studies to examine muscle fiber-type-specific gene activity. The mechanisms underlying fiber-type-specific gene expression will be investigated in vivo by the novel approach of directly introducing TnI gene constructs into specific fast and slow twitch muscles in mice and rats, and by the development of transgenic mice carrying hybrid TnI gene constructs. The in vivo expression of these TnI gene constructs will be monitored by CAT activity assays, RNase protection assay,and by in situ hybridization of gene specific probes to cross sections of muscle tissue. These in vitro and in vivo expression studies will assess whether specific cis-acting sequences can confer muscle fiber-type-specific gene expression and evaluate the role of trans-acting factors in the process. The putative trans-acting factors regulating fiber-type-specific gene expression will be characterized by gel mobility shift assays, and DNA footprinting techniques. If such factors are found, he will clone representative cDNAs from expression libraries. These studies will be among the first to examine the molecular mechanisms regulating muscle fiber type development and maintenance.

对脊椎动物肌肉发育的研究 研究基因调控的机会， 复杂的个体发育过程 在肌生成过程中，两种组织- 发生特异性和发育阶段特异性基因转换， 最终形成了两种主要的骨骼 肌肉：快收缩和慢收缩肌纤维。 每个 肌纤维类型的特点是表达独特的 组合是收缩蛋白同种型。 建立 肌纤维类型的变化是由多种因素的相互作用引起的， 细胞谱系，肌纤维接受的神经支配类型，以及 循环激素水平。 尽管取得了很大进展， 在理解负责的分子机制 肌肉特异性基因表达，几乎一无所知 差异肌纤维类型特异性基因表达。 韦德医生 建议研究调节肌纤维类型的机制， 通过研究人肌钙蛋白的特异性基因表达 我是基因家族。 成人慢纤维肌钙蛋白I基因是差异 在胚胎肌纤维形成过程中表达， 作为一个模型的发展规律的纤维型- 特异性基因表达 他克隆了人类骨骼 肌钙蛋白I慢基因，并已确定多个顺式作用 序列所需的肌肉特异性表达， 体外瞬时表达试验。 这些研究 确定了一种肌肉特异性增强子， 由生肌调节因子MyoD1激活。 他将 进一步表征肌肉特异性调节元件， 将这些研究扩展到检测肌纤维类型特异性基因 活动 纤维类型特异性基因的作用机制 将通过以下新方法在体内研究表达： 将TnI基因构建体直接导入特异性快速和 小鼠和大鼠的慢颤肌，以及 携带杂交TnI基因构建体的转基因小鼠。 体内 这些TnI基因构建体的表达将通过CAT监测 活性测定，RNA酶保护测定，并通过原位 基因特异性探针与肌肉横切面杂交 组织. 这些体外和体内表达研究将 评估特定的顺式作用序列是否可以赋予肌肉 纤维类型特异性基因表达和评估的作用， 过程中的反作用因素。 假定的反式作用 调节纤维类型特异性基因表达的因素将是 通过凝胶迁移率变动分析和DNA足迹分析 技术. 如果发现这样的因素， 来自表达文库的代表性cDNA。 这些研究 将是第一批研究 调节肌纤维类型的发育和维持。