DETERMINATION AND DIFFERENTIATION OF CARDIAC MYOCYTES

控制心源性分化和心脏的机制-- 具体的基因表达尚不清楚。我们之前的数据显示， 心脏承担的时间和心脏的胚胎期 基因在发育中的心脏中被激活。我们的数据表明， 心脏中肌肉基因表达的激活可能涉及独特的 中胚层最初转化为肌细胞后未见的机制。 最后，两个基因，心肌肌钙蛋白I(TnI)和心室肌球蛋白重 链1(VMHC1)，在启动心肌生成时被激活 进行了分化鉴定。心脏TnI的表达是真实的 心脏特异性方式，而VMHC1在所有横纹肌源性组织中表达 细胞在肌源性分化的开始，但随后是 立即局限于心室肌细胞。我们建议确定 指导这两种心脏基因表达的调控序列 基因。我们将：1)分离和鉴定5‘端和近端 心脏TnI和VMHC1基因序列，2)转染人 与PTMG载体LacZ报告基因融合的调控序列 心源性中胚层、分化的心肌细胞、骨骼肌源性 细胞和非肌源性细胞，以及3)识别特定的区域 利用DNA足迹研究调控元件中的DNA/蛋白质相互作用 和电泳迁移率改变分析。我们的假设是 心脏TnI基因包含的调控元件赋予心脏 特定的表达，而VMHC1基因包含引导 一种独特的普遍存在的表达阶段，紧随其后的是严格的心脏 特定阶段。我们目前的目标是确定哪些序列 通过比较和对比假定的监管来指导这些活动 基因元件，从而有助于确定 调控心脏特异基因表达的分子机制 心源性分化。

The mechanisms which govern cardiogenic differentiation and cardiac-- specific gene expression are unknown. Our previous data have shown the time of cardiogenic commitment and the embryonic period when cardiac genes are activated in the developing heart. Our data suggest that the activation of muscle gene expression in the heart may involve unique mechanisms not seen after the initial conversion of mesoderm to myocyte. Finally, two genes, cardiac troponin I (TnI) and ventricular myosin heavy chain one (VMHC1) Which are activated at the initiation of cardiomyogenic differentiation were identified. Cardiac TnI is expressed in a truly cardiac-specific manner while VMHC1 is expressed in all striated myogenic cells at the initiation of myogenic differentiation but then is immediately restricted to ventricular myocytes. We propose to identify regulatory sequences which direct the expression of these two cardiac genes. We will: 1) isolate and characterize the 5' and proximal sequences of the cardiac TnI and VMHC1 genes, 2) transfect putative regulatory sequences fused to the lacZ reporter gene of pTMG vector into cardiogenic mesoderm, differentiated cardiac myocytes, skeletal myogenic cells and non-myogenic cells, and 3) identify specific regions of DNA/protein interaction in the regulatory elements using DNA footprinting and electrophoretic mobility shift assays. Our hypothesis is that the cardiac TnI gene contains regulatory elements which confer cardiac specific expression, while the VMHC1 gene contains sequences which direct a unique ubiquitous phase of expression followed by a strict cardiac specific phase. Our present goal is to identify the sequences which direct these activities by comparing and contrasting putative regulatory elements of the genes and thereby aid in the determination of the molecular mechanisms which govern cardiac-specific gene expression and cardiogenic differentiation.