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 转录。研究人员实验室最近的工作 表明在胎鼠心肌细胞的原代培养物中， 细胞内cAMP升高导致α MHC上调 β MHC mRNA表达下调。使用多个删除 在瞬时转染试验中，研究者 鉴定了一个31个碱基对的片段，它含有一个杂合基序， 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。的 识别这种元素的蛋白质似乎与 先前表征的TEF-1转录因子。申请人的 因此，目标是（1）表征与 大鼠α MHC基因启动子中的cAMP诱导元件（EM）， 一种分离和鉴定编码心脏的cDNA的方法， 与EM元件结合的特异性TEF-1同种型，（2）克隆和 描述了一个假定的伴侣蛋白的cDNA， 相互作用，（3）研究cAMP依赖的磷酸化因子 与EM元件的结合（4）定义了磷酸化在 与DNA结合并识别DNA中其他蛋白质的蛋白质因子。 蛋白质-蛋白质相互作用，和（5）验证体外研究，通过在 通过构建转基因小鼠和通过靶向 通过从129个基因组中分离心脏特异性同源基因， SV小鼠据了解，拟议的实验将进一步 阐明信号通路在偶联的作用， 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.