MEMBRANE BIOSYNTHESIS IN NORMAL AND DYSTROPHIC RETINA

正常和营养不良视网膜中的膜生物合成

• 批准号: 3263612
• 负责人: DAVID S PAPERMASTER
• 金额: $ 24.29万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-07-01 至 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3263612
• 关键词: Anura; cell biology; congenital eye disorder; density gradient ultracentrifugation; gene expression; genetic transcription; immunocytochemistry; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; membrane reconstitution /synthesis; physical separation; retina; retina degeneration; retina disorder; rhodopsin; tissue /cell culture; transport proteins; visual photoreceptor

Specific Aims and Long Term Objectives: The biosynthesis of retinal cell membranes is being explored in frogs by subcellular fractionation and by evaluation of gene transcription and translation in mouse and rat retinas from animals bearing various forms of inherited retinal dystrophy. These studies are directed to understanding the cellular basis of sorting of membrane proteins from sites of synthesis to sites of function and the genetic controls that regulate these processes. They should contribute to understanding of retinal causes of blindness and to the organization of cells in tissues in normal and pathologic states. Experimental Design and Methods: Frog retinas will be incubated with [35S]-methionine and after homogenization, sucrose density gradient subcellular fractions bearing newly synthesized opsin and other outer segment proteins will be isolated. Preliminary studies have indicated the presence of a highly labeled low-density fraction with kinetics of labeling suggesting it contains the post-Golgi vesicles. This fraction will be used to determine the structure of the vesicles transporting opsin from the Golgi to the outer segment. Rabbit and mouse monoclonal antibodies will be generated to these partially purified fractions to characterize their composition. The vesicles will then be purified further by density-shift sucrose gradient fractionation by using gold- antibody conjugates bound to the membranes of the fraction. These studies are designed to determine if the vesicles carry unique molecules bearing an "address" to specifically sort newly synthesized opsin to the appropriate site in the photoreceptor in other cells tranfected with the opsin gene. Using renal tubular epithelial cell cultures transfected with the opsin gene, we are exploring polarity of expression. These cells provide a model system for studying photoreceptor cell polarity. Extensive EM studies of rats and mice bearing genes for inherited retinal dystrophies have demonstrated a uniform feature: the loss of the polarized distribution of opsin on the rod plasma membrane as the outer segment becomes damaged or fails to form. We are, therefore, examining the molecular control of gene expression of opsin and other proteins in these dystrophic rodents to evaluate alternative models to account for the altered localization of these molecules and the residual visual sensitivity in these retinas that lack outer the altered localization of these molecules and the residual visual sensitivity in these retinas that lack outer segments. Using cDNA radiolabelled probes, containing the opsin gene, we are evaluating the effects of retinal degeneration in C3H (rd) and O20/A (rds) mice and RCS rats on levels of gene transcription by quantitating mRNA expression throughout the light cycle and as the animals age. We have demonstrated at least five mRNA transcripts of the opsin gene exist in normal and dystrophic mice and four transcripts in rats in contrast to the single transcript in bovine and human retinas. These transcripts are differentially expressed as the animals age. We also have determined that expression of opsin genes in O20/A mice bearing the rds dystrophy is nearly normal and that translation rates of the mRNA are also nearly normal despite the presence of only 3% of the retinal opsin content when compared to age-matched control mice. Since outer segment structure is distorted in this dystrophy such that disks are not assembled, our data indicate that the gene defect interferes with disk morphogenesis rather than opsin synthesis.

特定目标和长期目标：视网膜细胞的生物合成 在蛙类中，通过亚细胞分离和 小鼠和大鼠视网膜中基因转录和翻译的研究 来自携带各种形式遗传性视网膜营养不良的动物。这些 研究旨在了解分选的细胞学基础 膜蛋白从合成部位到功能部位 控制这些过程的基因控制。他们应该为 了解视网膜致盲的原因和组织 处于正常和病理状态的组织中的细胞。 实验设计和方法：青蛙视网膜将与 [35S]-蛋氨酸和均质后，蔗糖密度梯度 含有新合成的视蛋白和其他外膜的亚细胞组分 片段蛋白将被分离出来。初步研究表明， 高标记低密度组分的存在与动力学 标签显示它含有高尔基体后的小泡。这个分数 将被用来确定囊泡运输的结构 视神经从高尔基体到外节。兔和小鼠的单抗 这些部分纯化的组份将产生抗体以 描述它们的组成。然后，小泡将被提纯 进一步采用密度移位蔗糖梯度分级法，用金- 抗体结合物结合到组分的膜上。这些研究 被设计用来确定这些小泡是否携带独特的分子 专门将新合成的视蛋白分类到 在其他细胞中光感受器中的适当位置 视蛋白基因。用转基因的肾小管上皮细胞培养 视蛋白基因，我们正在探索表达的极性。这些细胞 为研究感光细胞的极性提供了一个模型系统。 携带遗传性视网膜基因的大鼠和小鼠的广泛EM研究 营养不良症表现出一个统一的特征： 视蛋白在杆状质膜上的极化分布 数据段损坏或无法形成。因此，我们正在研究 视蛋白等蛋白基因表达的分子调控 这些营养不良的啮齿动物评估替代模型来解释 这些分子的局部改变和残留的视觉 这些视网膜的敏感性缺乏外部改变的定位 这些分子和视网膜中残留的视觉敏感度 缺少外部节段。使用cdna放射性标记的探针，包含 视蛋白基因，我们正在评估视网膜变性对C3H的影响 (RD)和O20/A(Rds)小鼠和RCS大鼠基因转录水平的变化 量化整个光周期的mRNA表达，并作为 动物会变老。我们已经展示了至少五个mRNA转录本 视蛋白基因在正常和营养不良小鼠中存在，在 与牛和人视网膜中的单一转录本形成对比的是大鼠。 随着动物年龄的增长，这些转录本会有不同的表达。我们 还检测了视蛋白基因在O20/A小鼠体内的表达。 RDS营养不良几乎是正常，而且mRNA的转译率 也几乎是正常的，尽管只有3%的视网膜 当与年龄匹配的对照组小鼠相比时，视蛋白含量。自外 在这种营养不良中，节段结构被扭曲，从而使盘不 综合起来，我们的数据表明，基因缺陷干扰了磁盘 形态发生而不是视素合成。