EXERCISE HYPERTROPHY AND CONTROL OF MYOSIN INDUCTION

描述（申请人摘要）：机械过载导致 成年横纹肌增大，肌球蛋白 表型和收缩特性。 剩余的关键问题 成年期肌肉生物学包括确定DNA调节元件 和转录诱导β肌球蛋白的核蛋白因子 重链在机械负荷跖肌中表达及检测 它们在纤维特异性表达中的可能作用。 我们提出实验 两个层次的调查。 精细化层面涉及：1）识别 核因子和DNA元件之间的相互作用被证明是 机械诱导β肌球蛋白重链所必需的 超负荷，使用迁移率改变，DNA酶I足迹和甲基化 干扰测定，和2）分离编码核因子的cDNA， 参与β肌球蛋白重链诱导的机械过载， 表达克隆 北方分析将确定发育，组织 以及这些核因子的纤维特异性表达模式。 的 总体水平涉及转基因小鼠的产生和分析 携带：1）携带DNA的PCR定点突变的转基因 参与机械诱导β肌球蛋白重链的元素 超载。 转基因启动子活性将通过测定 氯霉素乙酰转移酶比活性 超负荷和含有各种纤维类型的肌肉，和2）转基因 过表达编码特异性靶向 横纹肌 北方分析将评估过表达的影响 核因子对横纹肌表型的影响。 拟议的工作将 鉴定和测试核因子和β肌球蛋白的体内功能 参与β肌球蛋白重链纤维重链启动子元件 特异性表达和机械过载诱导。 体内 核因子的过表达将确定它们在以下方面的潜在作用： 其他基因的调节，肌肉的扩大和发展。 这些 实验预计将确定潜在的蛋白质/DNA靶点， 旨在提供肌肉紊乱对策的疗法 可归因于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.