GENETIC STUDIES OF THE MEMBRANE SKELETON

膜骨架的遗传学研究

• 批准号: 2415197
• 负责人: RONALD R DUBREUIL
• 金额: $ 15.53万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415197
• 关键词: Drosophilidae; ankyrins; bioassay; cell membrane; chromosome aberrations; fluorescent dye /probe; gene complementation; genetic mapping; genetically modified animals; immunoprecipitation; laboratory rabbit; membrane biogenesis; membrane proteins; polymerase chain reaction; protein biosynthesis; protein structure function; sodium channel; sodium potassium exchanging ATPase; spectrin; tissue /cell culture; western blottings

The long range goal of the proposed research is to understand the contributions of ankyrin and spectrin (the membrane skeleton) to plasma membrane assembly and function in higher organisms. Human genetic studies have made an important contribution to our understanding of the membrane skeleton as a link between transmembrane proteins and actin. But these studies are constrained to specialized protein isoforms that have a limited tissue distribution. Genetic studies of the more widely expressed membrane skeleton isoforms have not been possible in mammals. Drosophila will be used as a model system where powerful molecular and genetic tools are available for the study of protein function in a complex developing organism. We have identified and partially characterized a Drosophila ankyrin homolog that is conserved in size, sequence, and spectrin interaction, relative to its human counterpart. Mutations in Drosophila ankyrin will be identified and characterized. The phenotype of ankyrin null mutants will reveal the cellular consequences of ankyrin deficiency in a range of different cell types. Molecular analysis of ankyrin mutations will provide new information on functional sites within the ankyrin molecule. A transgene rescue approach with ankyrin cDNA constructs will be established to verify that defects in ankyrin explain the observed phenotypes of mutants. Rescue experiments with modified ankyrin transgenes will be used to test the functional contribution of selected sites in the ankyrin molecule. A unique advantage of Drosophila as a model system is the availability of mutants that are defective in several of the proteins that interact with ankyrin, such as the sodium pump sodium channels and cell adhesion molecules. Simple bioassays that reflect the functional levels of these proteins will be used to study the consequences of membrane skeleton defects. The levels of these interacting proteins will also be manipulated in order to study their effects on membrane skeleton assembly. These experiments will provide the first direct tests of the predicted functions of ankyrin that have emerged from descriptive studies in mammalian systems. Studies of erythrocyte ankyrin have established the importance of this membrane skeleton protein in human disease. Current questions concern the unknown role of ankyrins in non-erythroid cells. The results of the proposed experiments will provide new insights into the role of non- erythroid ankyrins and will significantly extend the current understanding of ankyrin function in humans.

拟议研究的长期目标是了解 锚蛋白和血影蛋白（膜骨架）对血浆的贡献 高等生物中的膜组装和功能。人类遗传学研究 为我们对膜的理解做出了重要贡献 骨架作为跨膜蛋白和肌动蛋白之间的连接。但这些 研究仅限于具有特定蛋白质异构体 组织分布有限。更广泛表达的基因研究 膜骨架亚型在哺乳动物中是不可能的。果蝇 将被用作模型系统，其中强大的分子和遗传工具 可用于复杂发育中蛋白质功能的研究 生物。我们已经鉴定并部分表征了果蝇 大小、序列和血影蛋白保守的锚蛋白同源物 交互，相对于人类对应物。果蝇突变 锚蛋白将被鉴定和表征。锚蛋白的表型 无效突变体将揭示锚蛋白缺陷的细胞后果 在一系列不同的细胞类型中。锚蛋白的分子分析 突变将提供有关功能位点的新信息 锚蛋白分子。使用锚蛋白 cDNA 构建体的转基因拯救方法 将建立验证锚蛋白缺陷解释观察到的现象 突变体的表型。改良锚蛋白转基因的拯救实验 将用于测试所选站点的功能贡献 锚蛋白分子。果蝇作为模型系统的独特优势是 几种蛋白质有缺陷的突变体的可用性 与锚蛋白相互作用，例如钠泵钠通道和 细胞粘附分子。反映功能的简单生物测定 这些蛋白质的水平将用于研究后果 膜骨架缺陷。这些相互作用的蛋白质的水平将 也可以被操纵以研究它们对膜骨架的影响 集会。这些实验将提供对 从描述性研究中得出的锚蛋白的预测功能 在哺乳动物系统中。 对红细胞锚蛋白的研究已经证实了这一点的重要性 人类疾病中的膜骨架蛋白。当前的问题涉及 锚蛋白在非红细胞中的作用未知。结果 拟议的实验将为非非物质的作用提供新的见解 红系锚蛋白并将显着扩展当前的理解 人类中锚蛋白的功能。