MOLECULAR MECHANISMS OF CARDIAC ONTOGENY AND HYPERTROPHY

本申请中提出的研究的总体目标继续是 是心脏收缩系统的分子特征， 发展和应对不同的生理和病理 刺激。 这一目标是基于这样一种信念，即为了理解 心肌收缩力及其在发育过程中的调节， 荷尔蒙和工作超负荷，这是至关重要的有一个明确的认识 心脏收缩器官的表型和机制 参与其监管。 为了解决这些问题，我们将继续 心肌肌球蛋白重链（MHC）的表征， α-原肌球蛋白（Alpha-TM）基因核苷酸序列，并将进一步 使用S1-核酸酶阐明它们在各种条件下的表达 映射技术 然而，为了确定因果关系， 这种描述性的方法是不够的。 为此 因此，我们将使用分子遗传技术来确定 假定的调控序列在基因表达中的作用 study. 已经分离的MHC和Alpha-TM基因将在 体外产生良好表征和可操作的小基因。 这些在 然后将体外修饰的基因导入心肌细胞， 培养以及小鼠生殖细胞系及其在培养过程中的表达 发展和响应各种刺激的研究。 进一步 这些结构的修改应该提供对这些结构的详细分析。 参与心脏MHC转录调节的序列和 转录后过程负责产生几个 不同的Alpha-TM蛋白。 因此，该提案是 研究分为三个主要领域：a）生产和 心原性细胞系的表征; B）进一步表征 和MHC基因的体内分析，和c）MHC基因的分子分析， α-TM基因

The general goal of the research proposed in this application continues to be the molecular characterization of the cardiac contractile system during development and in response to different physiological and pathological stimuli. This goal is based on the belief that in order to understand cardiac contractility and its modulation during development, in response to hormones and work overload, it is essential to have a clear understanding of the phenotype(s) of the cardiac contractile apparatus and the mechanisms involved in its regulation. To address these issues we will continue the characterization of the cardiac myosin heavy chain (MHC) and Alpha-tropomyosin (Alpha-TM) gene nucleotide sequences and will further elucidate their expression in a variety of conditions using S1-nuclease mapping techniques. However, in order to establish cause-effect relationships this descriptive approach is not sufficient. For this reason, we will use molecular genetic techniques in order to determine the role of putative regulatory sequences in the expression of the genes under study. The MHC and Alpha-TM genes already isolated will be mutagenized in vitro to generate well characterized and manipulable mini-genes. These in vitro modified genes will then be introduced into cardiac myocytes in culture as well as the mouse germ cell line and their expression during development and in response to a variety of stimuli studied. Further modification of these constructs should provide a detailed analysis of the sequences involved in the transcriptional regulation of cardiac MHC and the post-transcriptional processes responsible for the generation of several different Alpha-TM proteins from a single gene. Therefore, the proposal is divided into three main areas of research: a) Production and characterization of a cardiogenic cell line; b) Further characterization and in vivo analysis of the MHC genes, and c) Molecular analysis of the Alpha-TM gene.