HEART-SPECIFIC CREATINE KINASE REGULATORY ELEMENTS

本提案的总体目标是确定 肌酸激酶的两种基因亚型（MCK和 BCK）在哺乳动物心脏细胞中受到调节。 具体 目的是：（1）确定顺式作用的DNA元件内的 小鼠MCK基因是组织和/或心脏 MCK的细胞类型特异性表达;（2）鉴定顺式作用 MCK基因内的DNA元件， 其表达的各种生理调控;（3）克隆 小鼠BCK基因的5 ′侧翼和第一内含子部分， 并识别相似的细胞类型和生理调节 （4）确定并开始纯化 与这些DNA区域结合的反式作用因子， 选择性地刺激或抑制M-和 BCK启动子区;和（5）衍生永久细胞系 它们是各种心肌细胞类型的代表 （心房、心室和传导）以及心脏祖细胞 细胞 从这些研究中获得的基本知识可能特别重要。 关于组织特异性的一般问题的信息 基因在发育过程中的作用 我们的方法将允许 直接比较两种表达方式 进化相关的基因在一组 心脏和骨骼中与发育相关的细胞类型 肌肉细胞谱系 此外，由于肌酸激酶发挥着重要作用， 在心脏高能磷酸盐穿梭中的关键作用 肌肉细胞，而且由于CK水平似乎会响应 心脏肥大，这些研究应该提供信息 这与心脏病的一般问题有关。 最后， 永久性细胞系的可用性代表不同的 心肌细胞类型对几乎所有 心脏细胞功能的研究。 主要方法包括：基因克隆，修饰， 转染，DNA酶足迹法，凝胶阻滞，体外 转录测定，克隆细胞培养，和自发， 癌基因和病毒诱导的转化。

The overall goal of this proposal is to determine how the expression of two genetic isoforms of creatine kinase (MCK and BCK) is regulated in mammalian heart cells. The specific objectives are: (1) to identify cis-acting DNA elements within the mouse MCK gene which are required for tissue- and/or cardiac cell type-specific expression of MCK; (2) to identify cis-acting DNA elements within the MCK gene which are required for various physiological modulations of its expression; (3) to clone the 5'-flanking and first intron portion of the mouse BCK gene, and to identity similar cell type and physiological regulatory elements within it; (4) to identify and begin purification of the trans-acting factors that bind to these DNA regions or that selectively stimulate or repress transcription from the M- and BCK promoter regions; and (5) to derive permanent cell lines which are representative of the various cardiac muscle cell types (atrial, ventricular, and conducting), as well as cardiac progenitor cells. Basic knowledge derived from these studies may be particularly informative with respect to the general problem of tissue-specific gene regulation during development. Our approach will permit direct comparisons between how the expression of two evolutionarily-related genes is regulated within a set of developmentally-related cell types in the cardiac and skeletal muscle cell lineages. In addition, since the creatine kinases play a critical role in the high-energy phosphate shuttle of cardiac muscle cells, and since CK levels appear to change in response to cardiac hypertrophy, these studies should provide information which is relevant to general problems of heart disease. Finally, the availability of permanent cell lines representing different cardiac muscle cell types would be of great value to virtually all studies of heart cell function. Major methodologies include: gene cloning, modification, transfection, DNAase foot-printing, gel retardation, in vitro transcription assays, clonal cell culture, and spontaneous, oncogene-, and viral-induced transformation.