MOLECULAR GENETICS OF A LENS INTRINSIC MEMBRANE PROTEIN

晶状体固有膜蛋白的分子遗传学

• 批准号: 6195171
• 负责人: ROBERT L CHURCH
• 金额: $ 26.17万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6195171
• 关键词: DNA footprinting; cataract; cell differentiation; electron microscopy; fiber cell; gel mobility shift assay; gene expression; gene mutation; gene targeting; genetic regulatory element; genetically modified animals; laboratory mouse; lens proteins; membrane proteins; methylation; molecular genetics; polymerase chain reaction; transcription factor

DESCRIPTION: (Investigator's Abstract). The ocular lens is an avascular, semisolid organ whose function is to refract incoming light upon the retina. Concomitant with this function is a necessity for the lens to remain transparent. A loss of transparency of the lens is usually permanent and is termed a cataract. Cell and organ functions, such as obtaining nutrients necessary for maintaining the healthy, transparent lens state, for handling of cell waste removal, and for maintenance of overall membrane stability are carried out through select and specific proteins or groupings of proteins within the lens cell membrane. Proper controls on expression of these proteins and their subsequent function is essential for lens survival, and maintenance of transparency. The mechanisms regulating these membrane proteins in the lens are presently unknown. The goal of this project is to understand the regulation of the lens fiber cell membrane protein MP19 gene (LIM2). To achieve this goal, the P.I. will investigate the following topics: a). To characterize the mouse To cataract mutant and the human congenital nuclear cataract and their relationship to the lens membrane LIM2 gene. Using transgenic mouse technology to mimic the To cataract in order to study the function of MP19. b). To determine the region(s) responsible for transcriptional activity of the human LIM2 gene (cis elements), using CAT reporter gene constructs in transient transfection assays. c). Identify protein:DNA interactions (trans factors) associated with the LIM2 basal promoter region and upstream cis elements using gel mobility shift and DNA foot printing procedures. d). To investigate the methylation patterns of LIM2 upstream regions to determine if methylation of critical cis elements serves to control LIM2 gene function. These studies will give a better understanding of the role of MP19 in the maintenance of lens transparency and its possible role in cataract formation.

描述：（研究者的摘要）。眼晶状体是一种无血管的半固体器官，其功能是将入射光折射到视网膜上。与此功能相伴随的是透镜必须保持透明。晶状体透明度的丧失通常是永久性的，被称为白内障。细胞和器官功能，例如获取维持健康、透明晶状体状态、处理细胞废物清除以及维持整体膜稳定性所需的营养物质，是通过晶状体细胞膜内的选择性和特定蛋白质或蛋白质组来实现的。正确控制这些蛋白质的表达及其后续功能对于晶状体的存活和透明度的维持至关重要。目前尚不清楚调节晶状体中这些膜蛋白的机制。该项目的目标是了解晶状体纤维细胞膜蛋白 MP19 基因 (LIM2) 的调控。为了实现这一目标，P.I.将研究以下主题：a).表征小鼠To白内障突变体和人类先天性核性白内障及其与晶状体膜LIM2基因的关系。利用转基因小鼠技术模拟To白内障，以研究MP19的功能。 b).为了确定负责人类 LIM2 基因转录活性的区域（顺式元件），在瞬时转染测定中使用 CAT 报告基因构建体。 c).使用凝胶迁移率变化和 DNA 足迹打印程序识别与 LIM2 基础启动子区域和上游顺式元件相关的蛋白质：DNA 相互作用（反式因子）。 d).研究 LIM2 上游区域的甲基化模式，以确定关键顺式元件的甲基化是否有助于控制 LIM2 基因功能。这些研究将有助于更好地了解 MP19 在维持晶状体透明度方面的作用及其在白内障形成中的可能作用。