REGULATION OF MYOSIN HEAVY CHAIN ALPHA GENE BY CAMP

描述：该应用程序是对 CAMP诱导α-MHC基因调控的机制 抄写。调查员实验室最近的工作是 证明在原代培养的胎鼠心肌细胞中 细胞内cAMP升高导致α-MHC上调 下调β-MHC基因的表达。使用多个删除 在瞬时转染实验中，研究人员已经 鉴定出一个31个碱基对的片段，其中包含一个与 E-box和MCAT box(EM元件)的重叠序列 肌肉特异性和cAMP诱导的基因表达。这个 识别这一元素的蛋白质似乎与 先前鉴定的TEF-1转录因子。申请人的 因此，目标是(1)表征与之相互作用的蛋白质 大鼠α-MHC基因启动子中cAMP诱导元件(EM) 心脏编码基因的分离和鉴定方法 与EM元件结合的特异性TEF-1亚型，(2)克隆和 鉴定一种可能的伙伴蛋白的cdna。 相互作用，(3)研究cAMP依赖的因子磷酸化 与EM元件(4)的结合定义了磷酸化在 与DNA结合并识别细胞中其他蛋白质的蛋白质因子 蛋白质-蛋白质相互作用，以及(5)通过In 构建转基因小鼠和靶向接种的活体实验 从129例患者中分离心脏特异性同源基因消融选定的基因 小白鼠。据了解，拟议的实验将进一步 阐明信号通路在细胞周期蛋白偶联中的作用 血流动力学负荷对心脏代偿性生长的影响。

DESCRIPTION: The application is a continuation of studies on the mechanism involved in the cAMP-induced regulation of alpha MHC gene transcription. Recent work in the investigator's laboratory has demonstrated that in primary cultures of fetal rat heart myocytes the elevation of intra-cellular cAMP results in up-regulation of alpha MHC and down-regulation of beta MHC mRNA expression. Using several deletion constructs in transient transfection assay, the investigator has identified a 31 base-pair fragment, which contains a hybrid motif with overlapping sequence of E-box and MCAT box (EM element) which confers both muscle specific and cAMP-inducible expression of the gene. The protein that recognizes this element appears to be indistinguishable from the previously characterized TEF-1 transcription factor. The applicant's goal, therefore, is (1) to characterize the protein which interacts with the cAMP-inducible element (EM) in the rat alpha MHC gene promoter by way of isolating and characterizing the cDNA encoding the cardiac specific TEF-1 isoform binding to the EM element, (2) clone and characterize the cDNA of a putative partner protein involved in the interaction, (3) study the cAMP-dependent phosphorylation of factors binding to the EM element (4) define the role of phosphorylation in protein factors which bind to DNA and recognize other proteins in the protein-protein interaction, and (5) verify the in vitro studies by in vivo experiments by constructing transgenic mice and by targeted ablation of selected loci by isolating cardiac specific isogenes from 129 S.V. mouse. It is understood that the proposed experiments will further elucidate the role of signaling pathways in the coupling of the hemodynamic load to the compensatory growth of the heart.