REGULATION OF MYOSIN HEAVY-CHAIN ALPHA GENE BY CAMP

拟议的实验是我们对 心脏正常发育和改变时心脏表型的决定因素 心脏的功能状态。由于夏令营在 心肌收缩能力，我们将研究其在α-肌球蛋白调节中的作用 重链(MHC)的表达。我们的初步研究表明 Forsklin诱导的稳态水平增加了几倍 胎鼠原代培养细胞经血清处理后MHC-αmRNA的表达 而心脏中MHC-β的丰度没有变化 文字记录。因此，我们将主要关注转录 α-MHC基因的调控。我们将首先完成正在进行的 比较培养的心肌细胞对cAMP的反应 稳定的信使核糖核酸水平及其转录速率和通过评估信使核糖核酸 稳定性。接下来，描述血清诱导cAMP的机制(S) 转录调控，我们将使用 原代培养心肌细胞的瞬时转染法。这两个 调控序列及其同源转录因子(S)负责 对于血清诱导的cAMP效应将进行研究，其可能的 已评估交互。因此，我们的具体目标是：1.调查 CAMP对MHC-α基因表达的调节水平。A.分析 CAMP对MHC-α基因表达的转录调控。B.确定 CAMP对血管紧张素转换酶的影响 MHC-αmRNA的稳定性。2.研究基因启动子的功能 夏令营。A.描绘cAMP反应元件(AP-2E)和血清反应 MHC-α启动子内的元件(SRE)。B.研究各自的作用 转录因子AP-2和SRF对MHC-α基因表达的影响。

The proposed experiments are a continuation of our studies on the determinants of cardiac phenotype during normal development and in altered functional states of the heart. Since cAMP plays an important role in cardiac contractility, we will study its role in modulation of alpha-myosin heavy chain (MHC) expression. Our preliminary studies have shown that forskolin induces a several fold increase in the steady state level of MHC-alpha mRNA in serum-pretreated primary cultures derived from fetal rat hearts while no change was observed in the abundance of MHC-beta transcript. Therefore, we will focus primarily on the transcriptional regulation of the alpha-MHC gene. We will first complete our ongoing characterization of the cAMP response in cultured myocytes by comparing the steady level of mRNA with its transcription rate and by evaluating the mRNA stability. Next, to delineate the mechanism(s) of serum induced cAMP transcriptional regulation, we will study the promoter activity using transient transfection assays of primary myocyte cultures. Both the regulatory sequences and their cognate transcription factor(s) responsible for the serum induced cAMP effect will be investigated and their possible interaction evaluated. Thus, our specific aims are to: 1. Investigate the level of regulation of MHC-alpha gene expression by cAMP. a. Analyze the cAMP transcriptional regulation of MHC-alpha gene expression. b. Determine the effect of cAMP on MHC-alpha mRNA stability. 2. Study the promoter function in response to cAMP. a. Delineate the cAMP responsive element (AP-2E) and serum response element (SRE) within MHC-alpha promoter. b. Study the role of respective transcription factors, AP-2 and SRF on the MHC-alpha gene expression.