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.

拟议研究的远距离目标是了解 Ankyrin和Spectrin（膜骨架）对等离子体的贡献 较高生物体中的膜组装和功能。人类遗传研究 为我们对膜的理解做出了重要贡献 骨骼作为跨膜蛋白与肌动蛋白之间的联系。但是这些 研究被限制在具有A的专用蛋白质同工型 组织分布有限。更广泛表达的遗传研究 在哺乳动物中不可能使用膜骨架同工型。果蝇 将用作强大分子和遗传工具的模型系统 可用于研究复杂发育中的蛋白质功能 生物。我们已经确定并部分表征了果蝇 脚尖林同源物在大小，序列和光谱上保守 相对于人类对应物的互动。果蝇突变 Ankyrin将被识别和表征。氨基林的表型 无效突变体将揭示脚踝缺乏的细胞后果 在各种不同的单元格中。氨基林的分子分析 突变将提供有关功能站点的新信息 Ankyrin分子。用脚尖林cDNA构建体的转基因救援方法 将建立以验证Ankyrin中的缺陷解释所观察到的 突变体的表型。修改后的Ankyrin转基因的救援实验 将用于测试选定位点在 Ankyrin分子。果蝇作为模型系统的独特优势是 在几种蛋白质中有缺陷的突变体的可用性 它与安氨酸相互作用，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 膜骨骼缺陷。这些相互作用蛋白的水平将 也可以操纵以研究其对膜骨架的影响 集会。这些实验将提供第一个直接测试 从描述性研究中出现的Ankyrin的预测功能 在哺乳动物系统中。 红细胞安氨酸的研究确定了这一点的重要性 人类疾病中的膜骨架蛋白。当前问题涉及 Ankyrins在非果蝇细胞中的未知作用。结果 拟议的实验将为非 - 的作用提供新的见解 红细胞骨蛋白肌动蛋白，将大大扩展当前的理解 人类的Ankyrin功能。