MYOSIN HEAVY CHAIN EXPRESSION IN MUSCLE CELL LINE

肌发生是研究导致肌纤维化的过程的有利系统。 将多能前体细胞转化为具有 限制了其肌源性谱系的发育潜力。 反过来，由这一事件产生的成肌细胞具有 表达肌肉特异性基因的潜力。 但为了 trigger this expression第二个事件需要被触发 细胞对环境的反应，导致表达 分化的表型。 由于这些原因，长期 这个项目的目标仍然是进一步了解 导致肌原性细胞生成的分子事件 细胞谱系，肌原细胞的承诺，以不可逆转的 有丝分裂后状态和肌肉的调节表达 特定基因 三个主要的具体目标将被追求：一）。 使用有效的基因转移方法和 敏感的选择标记，我们将分离和表征cDNA 和基因组序列，能够将多能细胞转化为 生肌途径 这种方法将用于本和 以下部分基于两个事实：a）未甲基化的 真核DNA（生长在细菌中）在转染到 感受态细胞，和，B）存在几种多能细胞， 在激活后转化为肌源性谱系， 用5-agacytidine去甲基化似乎是一个单一的 基因 这些基因的表型将被分析，其迫使 在各种细胞中的表达研究，以确定基础 在许多不同的国家之间观察到的相互排斥 表型 II）。使用类似的方法和适当的细胞 线已经在手，我们将分离和表征基因（S） 负责肌原细胞向终末的定向， 不可逆分化表型 III）。使用DNA 序列和表达构建体已经准备好，我们将 进一步描述序列和反式作用因子 负责胚胎的肌肉特异性表达 肌球蛋白重链基因。 具体的兴趣将集中在 负调控序列的作用，当存在时， 将该基因的表达限制在分化的肌肉中 细胞 预计拟议的工作将改善我们的 了解导致 细胞谱系的产生和最终完全表达 分化表型

Myogenesis is a favorable system to study the processes leading to the conversion of a multipotent precursor cell into one that has restricted its developmental potential to the myogenic lineage. The myoblasts resulting from this event, in turn, have the potential to express muscle-specific genes. However, in order to trigger this expression a second event needs to be triggered in response to environmentals cells, that leads to the expression of the differentiated phenotype. For these reasons, the long-term goal of this project continues to be to further our understanding of the molecular events leading to the production of the myogenic cell lineage, the commitment of myogenic cells to an irreversible post-mitotic state and the regulated expression of the muscle specific genes. Three main specific aims will be pursued: I). Using a combination of efficient gene transfer methods and sensitive selectable markers, we will isolate and characterize cDNA and genomic sequence, able to convert multipotent cells to the myogenic pathway. This approach to be used in this and the following section is based on two facts: a) unmethylated eukaryotic DNA (grown in bacteria) is active when transfected into competent cells and, b) there are several multipotent cell able to convert to the myogenic lineage upon the activation by demethylation with 5-agacytidine of what appears to be a single gene. The phenotype of these genes will be analyzed and its forced expression in a variety of cell studied to determine the basis for the mutual exclusivity observed between many differentiated phenotypes. II). Using similar approaches and the appropriate cell lines already at hand, we will isolate and characterize the gene(s) responsible for the commitment of myogenic cells to a terminal and irreversible differentiated phenotype. III). Using the DNA sequences and expression constructs already prepared we will further characterize the sequences and trans-acting factors responsible for the muscle-specific expression of the embryonic myosin heavy chain gene. Specific interest will be focussed on the role of a negative regulatory sequence that, when present, restricting the expression of this gene to differentiated muscle cells. It is expected that the work proposed will improve our understanding of the decision making events leading to the production of cell lineages and the eventual expression of a fully differentiated phenotype.