VIMENTINS REGULATORY FACTORS INVOLVED IN DEVELOPMENT

肌肉发生需要肌肉特有基因的协调表达 以及肌肉不需要的其他物质的失活 表型。尽管存在大量关于什么控件的信息 骨骼基因表达，对控制心脏的因素知之甚少 表达，而几乎没有关于非肌肉特异性基因是如何 在肌肉发生过程中被关闭，尽管基因失活 对保持肌肉表型同样重要。细胞骨架 蛋白质，波形蛋白和结蛋白，提供了一个很好的模型系统 确定基因在发育过程中的差异表达方式。 波形蛋白的合成首先在中胚层的轮廓处被检测到。 许多细胞类型从这个谱系中分化出来，并继续 合成波形蛋白。其他的，如肌肉，使波形蛋白基因失活 和“开机”的Desmin。这项提案的目标是勾勒出 波形蛋白基因在肌肉发生过程中的特异性失活及其机制 异常表达可能会影响发育程序。显然， 这一决定中的任何偏差都可能导致损害发展。 在分析波形蛋白基因的表达时，我们发现了唯一的阳性和 控制下调监管的负面监管因素 波形蛋白基因。在鸡骨骼肌母细胞中，DNA结合活性 负面(消音器)系数低，正面(抗消音器) 蛋白质含量高，波形蛋白mRNA丰富。随着肌肉发生的进行 消音器因子的活性急剧增加，而 抗沉默活性几乎消失，波形蛋白mRNA水平 急剧减少。因此，这两个因素的相互作用 对于确定生理上正确表达的 波形蛋白基因。在心脏中(第14天)，我们发现 比骨骼(乳房)肌中的消音器因子。因此，我们得出结论 在心肌形成过程中，必须有类似的程序发生。 在这项提案中，我们的目标是进一步定义该机制。我们会 继续描述和克隆这两个独特的 消音器和抗消音器因子。我们把这种蛋白质称为一种 反消音器，因为它只能与消音器协同工作 元素本身并不有助于基因表达，这与 典型的增强子蛋白。随着核苷酸和蛋白质的获取 序列信息，我们计划改变这些细胞的内容 正常细胞和肌源性细胞中的因子。我们将确定其效果 这些基因改变对波形蛋白基因表达的影响等 重要的是，骨骼和心肌细胞中的生肌程序。一个 基因组数据库的屏幕显示了几个基因(从鸡到人) 具有同源消音剂和抗消音剂元素。作为抗体或 Cna探测器可用，我们将检查这些键的表达式 胚胎发育过程中的调控因素。最后，我们将确定 这些调控因子调控血管内皮生长因子表达的机制 波形蛋白和其他基因。希望通过了解如何 这些基因控制有助于发育，我们可以确定如何 心脏可能会出现异常。

Myogenesis requires the coordinate expression of genes unique to muscle and the inactivation of others which are not required for the muscle phenotype. Although considerable information exists about what controls skeletal gene expression, less is known about what governs cardiac expression, and virtually nothing about how non-muscle-specific genes are "turned-off" during myogenesis, despite the fact that gene inactivations equally important to maintaining the muscle phenotype. The cytoskeletal proteins, vimentin and desmin, provide an excellent model system for determining how genes are differentially expressed during development. Vimentin synthesis is first detected at the delineation of the mesoderm. A number of cell types differentiate from this lineage and continue to synthesize vimentin. Others, like muscle, inactivate the vimentin gene and "turn-on" desmin. The goal of this proposal is to delineate how the vimentin gene is specifically inactivated during myogenesis and how aberrant expression could affect the developmental program. Obviously, any deviation in this decision could result in compromised development. In analyzing vimentin gene expression, we have found unique positive and negative regulatory factors which control the downregulation of the vimentin gene. In chick skeletal myoblasts, the DNA binding activity of the negative (silencer) factor is low, the positive (antisilencer) protein is high, and vimentin mRNA is abundant. As myogenesis proceeds the activity of the silencer factor increases dramatically, whereas antisilencer activity virtually disappears and the level of vimentin mRNA decreases dramatically. Therefore, the interplay of these two factors is crucial for determining the physiologically correct expression of the vimentin gene. In heart (day 14) we find a 3-fold higher level of the silencer factor than in skeletal (breast) muscle. Therefore, we conclude that a similar program must be occurring during cardiac myogenesis. In this proposal we aim to further define the mechanism. We will continue with the characterization and cloning of both the unique silencer and antisilencer factor. We refer to this protein as an antisilencer, because it only functions in concert with the silencer element and does not contribute to gene expression on its own unlike the typical enhancer protein. With the acquisition of nucleotide and protein sequence information, we plan to alter the cellular content of these factors in both normal and myogenic cells. We will determine the effect of these genetic alterations on vimentin gene expression and more importantly the myogenic program in both skeletal and cardiac cells. A screen of the genomic data base reveals several genes (from chick to man) with homologous silencer and antisilencer elements. As antibodies or cRNA probes become available, we will examine the expression of these key regulatory factors during embryogenesis. Finally, we will determine the mechanism by which these regulatory factors control expression of vimentin as well as other genes. It is hoped that by understanding how these genetic controls contribute to development, we can determine how abnormalities may develop in the heart.