RUI: DNA - Protein Interactions Regulating Transcription of the Skeletal Muscle Actin Gene in Different Myogenic Milieu

RUI：DNA-蛋白质相互作用调节不同生肌环境中骨骼肌肌动蛋白基因的转录

• 批准号: 9305351
• 负责人: Sandra Sharp
• 金额: $ 28.32万
• 依托单位: California State L A University Auxiliary Services Inc.
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-15 至 1997-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9305351&HistoricalAwards=false
• 关键词: RUI; DNA; Protein; Interactions; Regulating

A compelling question is how animals develop specialized organs from a single fertilized egg. One facet of this process is the formation of muscle or myogenesis. When myogenesis is duplicated in cell culture, cells turn on numerous genes whose expression is required for muscle function. One such gene is the skeletal muscle actin gene. Genes are turned on and off by interactions between specialized regulatory proteins and specific DNA sequences in their promoter or control regions. Our experiments will help determine specific protein-DNA interactions in the promoter region of the skeletal muscle actin gene which are important in establishing the different levels of actin gene expression observed among cells which have been programmed in different ways to become muscle. We will transfer into different muscle cells a reporter gene driven by the skeletal muscle actin gene promoter and establish at what level the reporter gene is expressed. We will next compare results using the same reporter but with defined deletions in the actin promoter region. We will also use "in vivo footprinting" to determine more specifically where regulatory proteins are bound to the actin promoter in the DNA of the different cell types. The results will provide pieces to the puzzles of gene regulation and development. %%% A compelling question is how multicellular animals such as mice and humans develop specialized organs from a single fertilized egg. We know that every cell, regardless of its location or function in the body, contains for the most part the same set of instructions as every other cell, but that different kinds of cells follow different subsets of these instructions. These instructions are encoded as genes in DNA. This project is designed to help determine how muscle cells know which instructions to follow. The facet of the overall developmental process which results in formation of muscle is called myogenesis. Some steps in myogenesis can be duplicated with mouse pre-muscle cell lines cultured in the laboratory. During myogenesis in culture, cells turn on numerous genes which code for proteins whose expression is required for muscle function. One such gene is the skeletal muscle actin gene. This gene is expressed at different levels in different muscle cell types. While it is known that the activity of genes is regulated by interactions between specialized regulatory proteins and specific control regions in the DNA, the details of these interactions which are important in establishing different levels of expression of the skeletal muscle actin gene are not completely known. Our experiments will use a combination of genetic engineering and biochemical techniques to help determine these specific protein-DNA interactions and how they might differ among cells which have been programmed in different ways to become muscle. The results will provide pieces to the puzzles of gene regulation and development. Solution of this puzzle is intrinsically attractive because of the undeniable elegance of development. Beyond aesthetics, however, understanding the basic science of molecular mechanisms of muscle gene expression and development may one day lead to applications in understanding and managing muscle system disorders which are related to failures in regulated gene expression.

一个引人注目的问题是动物是如何从一个受精卵发育出专门的器官的。这个过程的一个方面是肌肉的形成或肌肉发生。当肌肉发生在细胞培养中复制时，细胞会开启许多肌肉功能所需的基因表达。其中一个基因是骨骼肌肌动蛋白基因。基因的开启和关闭是通过在其启动子或控制区的特殊调节蛋白和特定DNA序列之间的相互作用。我们的实验将有助于确定骨骼肌肌动蛋白基因启动子区域的特定蛋白质- dna相互作用，这对于建立不同水平的肌动蛋白基因表达是重要的，这些细胞以不同的方式被编程成肌肉。我们将把一个由骨骼肌肌动蛋白基因启动子驱动的报告基因转移到不同的肌肉细胞中，并确定报告基因在什么水平上表达。接下来，我们将比较使用相同报告基因但在肌动蛋白启动子区域有明确缺失的结果。我们还将使用“体内足迹”来更具体地确定不同细胞类型DNA中调节蛋白与肌动蛋白启动子结合的位置。这一结果将为基因调控和发育之谜提供线索。一个引人注目的问题是，像老鼠和人类这样的多细胞动物是如何从一个受精卵发育出专门的器官的。我们知道，每个细胞，无论其在体内的位置或功能如何，在很大程度上都包含与其他细胞相同的一套指令，但不同种类的细胞遵循这些指令的不同子集。这些指令被编码为DNA中的基因。这个项目的目的是帮助确定肌肉细胞如何知道应该遵循哪些指令。导致肌肉形成的整个发育过程的一个方面被称为肌肉发生。在实验室培养的小鼠肌前细胞系可以重复肌发生的一些步骤。在培养的肌肉形成过程中，细胞开启了许多编码蛋白质的基因，这些蛋白质的表达是肌肉功能所必需的。其中一个基因是骨骼肌肌动蛋白基因。该基因在不同的肌肉细胞类型中表达水平不同。虽然已知基因的活性是由DNA中特殊调节蛋白和特定控制区域之间的相互作用来调节的，但这些相互作用的细节对建立骨骼肌肌动蛋白基因不同水平的表达很重要，目前还不完全清楚。我们的实验将结合使用基因工程和生化技术来帮助确定这些特定的蛋白质- dna相互作用，以及它们在以不同方式被编程成肌肉的细胞之间可能有何不同。这一结果将为基因调控和发育之谜提供线索。这个谜题的解决方案本质上是有吸引力的，因为开发是不可否认的优雅。然而，除了美学之外，了解肌肉基因表达和发育的分子机制的基础科学可能有一天会导致应用于理解和管理与受调节基因表达失败相关的肌肉系统疾病。