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.

具体目标和长期目标：视网膜细胞的生物合成 通过亚细胞分级分离和 小鼠和大鼠视网膜中基因转录和翻译的评价 来自携带各种形式的遗传性视网膜营养不良的动物。这些 研究的目的是了解细胞分类的细胞基础， 膜蛋白从合成位点到功能位点， 调节这些过程的遗传控制。他们应该为 了解视网膜致盲的原因，并组织 正常和病理状态下的组织细胞。 实验设计和方法：将青蛙视网膜与 [35 S]-蛋氨酸和均质化后的蔗糖密度梯度 含有新合成的视蛋白和其他外膜蛋白的亚细胞组分 将分离片段蛋白。初步研究表明， 存在高度标记的低密度级分，动力学为 标签表明它含有高尔基体后囊泡。该馏分 将被用来确定小泡的结构， 从高尔基体到外段的视蛋白。兔和小鼠单克隆 将针对这些部分纯化的级分产生抗体， 描述其组成。这些囊泡将被纯化 进一步通过使用金的密度偏移蔗糖梯度分级， 结合到级分的膜上的抗体偶联物。这些研究 是为了确定囊泡是否携带独特的分子， 一个“地址”，专门将新合成的视蛋白分类到 在其他细胞中的光感受器中的适当位点， 视蛋白基因使用转染了 视蛋白基因，我们正在探索表达的极性。这些细胞 为研究感光细胞极性提供了一个模型系统。 对携带遗传性视网膜病变基因的大鼠和小鼠进行的广泛EM研究 营养不良已经证明了一个统一的功能：的丧失， 视蛋白在视杆细胞质膜上的极化分布 节段损坏或无法形成。因此，我们正在研究 视蛋白和其他蛋白质的基因表达的分子控制， 这些营养不良的啮齿动物来评估替代模型， 这些分子的改变定位和残留的视觉 这些视网膜中缺乏外部敏感性， 这些分子和视网膜中残留的视觉敏感度， 缺少外部部分。使用cDNA放射性标记的探针， 视蛋白基因，我们正在评估C3 H视网膜变性的影响， (rd)和O20/A（rds）小鼠和RCS大鼠的基因转录水平， 在整个光周期中定量mRNA表达， 动物年龄我们已经证明了至少五种mRNA转录本， 正常和营养不良小鼠存在视蛋白基因， 与牛和人视网膜中的单一转录物相比， 这些转录物随着动物年龄的增长而差异表达。我们 还确定了O20/A小鼠中视蛋白基因的表达， RDS营养不良几乎是正常，mRNA的翻译速率 也几乎正常，尽管只有3%的视网膜 与年龄匹配的对照小鼠相比，由于外 在这种营养不良中，节段结构被扭曲， 组装，我们的数据表明，基因缺陷干扰磁盘 形态发生而不是视蛋白合成。