EXERCISE HYPERTROPHY AND CONTROL OF MYOSIN INDUCTION

描述(申请人摘要)：机械过载导致 成人横纹肌增大和两种肌球蛋白的明显变化 表型和收缩特性。仍然存在的关键问题 成体阶段肌肉生物学涉及确定dna调控元件(S) 和转录诱导β-肌球蛋白的核蛋白因子(S) 机械性超负荷足底肌重链的表达及检测 它们在纤维特异性表达中的可能作用。我们建议进行实验 进行两个层次的调查。精细程度包括：1)识别 核因子(S)与脱氧核糖核酸(S)的相互作用 机械诱导体内β-肌球蛋白重链所必需 超载，使用迁移率变化、DNase I足迹和甲基化 干扰分析；2)分离编码核因子的基因(S)(S) 参与机械超负荷诱导β-肌球蛋白重链 表达克隆。Northern分析将确定发育、组织 以及这些核因子的纤维特异性表达模式(S)。这个 总水平涉及转基因小鼠的产生和分析 蕴藏：1)携带DNA定点突变的转基因 参与机械诱导β肌球蛋白重链的元件(S) 超载。转基因启动子的活性将通过检测 氯霉素乙酰转移酶比活力测定 超负荷和含有各种纤维类型的肌肉中，以及2)转基因 特异靶向核因子的过表达基因S(S) 横纹肌。Northern分析将评估过度表达的影响 核因子对横纹肌表型的影响。拟议的工作将 核因子(S)和β-肌球蛋白的体内功能鉴定与检测 肌球蛋白重链纤维中的重链启动子(S) 机械过载诱导的特异性表达。活体内 核因子的过度表达(S)将确定它们在 其他基因的调控(S)，肌肉的增大和发育。这些 预计实验将确定潜在的蛋白质/DNA靶点 旨在为肌肉错乱提供对策的疗法 肌肉疾病的表型和生长模式。

DESCRIPTION (Applicant's abstract): Mechanical overload leads to enlargement of adult striated muscle and distinct changes in both myosin phenotype and contractile properties. Remaining critical questions in adult-stage muscle biology involve determining the DNA regulatory element(s) and nuclear protein factor(s) which transcriptionally induce beta myosin heavy chain expression in mechanical overloaded plantaris muscle and to test their possible role in fiber specific expression. We propose experiments for two levels of inquiry. The fine level involves: 1) identifying interactions between nuclear factor(s) and DNA elements(s) shown to be necessary for in vivo induction of beta myosin heavy chain by mechanical overload, using mobility shift, DNAse I footprinting and methylation interference assays, and 2) isolating cDNA(s) encoding nuclear factor(s) involved in beta myosin heavy chain induction by mechanical overload using expression cloning. Northern analysis will determine developmental, tissue and fiber specific expression patterns of these nuclear factor(s). The gross level involves the generation and analysis of transgenic mice harboring: 1) transgenes carrying PCR site-directed mutations of DNA element(s) involved in beta myosin heavy chain induction by mechanical overload. Transgene promoter activity will be measured by assaying for chloramphenicol acetyltransferase specific activity following mechanical overload and in muscles containing various fiber types, and 2) transgenes overexpressing cDNA(s) encoding nuclear factor(s) specifically targeted to striated muscle. Northern analysis will assess the impact of overexpression of nuclear factors on striated muscle phenotype. The proposed work will identify and test the in vivo function of nuclear factor(s) and beta myosin heavy chain promoter element(s) involved in beta myosin heavy chain fiber specific expression and induction by mechanical overload. In vivo overexpression of nuclear factor(s) will identify their potential roles in regulation of other gene(s), muscle enlargement and development. These experiments are expected to identify potential protein/DNA targets for therapies aimed at providing countermeasures to derangements in muscle phenotypes and growth patterns attributable to muscle diseases.