MYOSIN HEAVY CHAIN EXPRESSION IN MUSCLE CELL LINE

肌肉发生是研究导致肌肉瘤的过程的有利系统。 将多能前体细胞转化为具有 将其发育潜力限制在肌源性谱系。 由这一事件产生的成肌细胞反过来又具有 有可能表达肌肉特异性基因。然而，为了 触发此表达式需要在其中触发第二个事件 对环境细胞的反应，导致表达 分化的表型。基于这些原因，从长远来看 这个项目的目标仍然是加深我们对 导致肌源性细胞产生的分子事件 细胞谱系，肌源性细胞对不可逆的承诺 有丝分裂后状态与肌肉的调控表达 特定的基因。将追求三个主要的具体目标：i)。 使用高效的基因转移方法和 敏感的可选择标记，我们将分离和鉴定cdna 和基因组序列，能够将多潜能细胞转化为 肌源性通路。此方法将在此和 以下部分基于两个事实：a)未甲基化 真核DNA(在细菌中生长)在转入 有能力的细胞和，b)有几个多能细胞能够 在被激活时转换为肌源性血统 用5-去甲基胞苷去甲基化似乎是单一的 吉恩。这些基因的表型将被分析并强制 在各种细胞中的表达研究确定了基础 观察到在许多不同的 表型。Ii)。使用类似的方法和适当的单元 已经有线，我们将分离和鉴定基因(S) 负责肌源性细胞对终末的承诺和 不可逆分化表型。三)。使用DNA 已经准备好的序列和表达结构，我们将 进一步表征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， 限制该基因在分化肌肉中的表达 细胞。 预计拟议的工作将改善我们的 了解导致经济衰退的决策事件 细胞谱系的产生和最终表达完整的 分化的表型。

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.