OPOCONIAL PROTEIN EXPRESSION IN VESTIBULAR EPITHLIA

前庭上皮中的蛋白表达

• 批准号: 3461770
• 负责人: KENNETH G POTE
• 金额: $ 11.58万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-01 至 1995-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3461770
• 关键词: X ray crystallography; Xenopus; antibody formation; antibody specificity; biochemical evolution; cochlea; complementary DNA; ear hair cell; epithelium; gene expression; genetic models; genetic regulatory element; in situ hybridization; laboratory rat; nucleic acid probes; nucleic acid sequence; otocyst /otolith; posttranslational modifications; protein biosynthesis; protein sequence; protein structure; regeneration; vestibular apparatus

This proposal seeks funding for the application of molecular biological techniques to the study of the hair cell epithelia of the peripheral vestibular system of vertebrates. It proposes the study of the proteins of the macular otoconia to determine the interactions of the hair cell containing epithelia with the apical, extracellular otoconial membrane. This membrane is a necessary component for the normal function of the vestibular end organs. In aging, disintegration of otoconia can cause vertigo. The specific aims are to: isolate cDNA clones coding for these proteins from inner ear libraries I have constructed; use these clones to determine the developmental expression of otoconial proteins from the rat and Xenopus laevis and identify the specific site of the protein expression; use the clones to make constructs which will maintain the fidelity of protein modifications for expression of the protein in amounts suitable for protein structure determination using x-ray crystallography. The approach is to use existing antibodies raised against the otoconial proteins of rat and X. laevis to screen the cDNA libraries constructed in the expression vector lambda ZAP II. Protein sequence will also be determined using a combination of micro Edman sequencing and mass spec sequencing. This sequence information will be used to synthesize oligonucleotide probes to screen the cDNA libraries. These proteins will be used to model the interactions of proteins and calcium carbonate in biominerals. Their study will elucidate the interactions of the hair cell epithelium with its extracellular environment and specifically with the accessory sensory structure, the otoconial membrane.

该提案寻求资助分子生物学的应用 外周毛细胞上皮细胞研究技术 脊椎动物的前庭系统。它提出了对蛋白质的研究 黄斑耳石确定毛细胞的相互作用 含有上皮细胞和顶端细胞外耳圆锥膜。 该膜是维持正常功能的必要组成部分 前庭终末器官。在衰老过程中，耳石的崩解会导致 眩晕。具体目标是： 分离编码这些的 cDNA 克隆 来自我构建的内耳文库的蛋白质；使用这些克隆 确定大鼠耳锥蛋白的发育表达 和非洲爪蟾并鉴定蛋白质的特定位点 表达;使用克隆来构建能够维持 蛋白质表达量的蛋白质修饰保真度 适用于使用 X 射线晶体学测定蛋白质结构。 该方法是使用针对耳圆锥细胞产生的现有抗体 大鼠和 X. laevis 的蛋白质筛选构建的 cDNA 文库 表达载体lambda ZAP II。蛋白质序列也将是 使用微埃德曼测序和质谱相结合确定 测序。该序列信息将用于合成 寡核苷酸探针筛选 cDNA 文库。这些蛋白质会 用于模拟蛋白质和碳酸钙的相互作用 生物矿物质。他们的研究将阐明毛细胞的相互作用 上皮细胞及其细胞外环境，特别是 辅助感觉结构，耳圆锥膜。