CLONING AND ANALYSIS OF TETRAHYMENA SOMATIC CHROMOSOMES

四膜虫体细胞染色体的克隆与分析

• 批准号: 3333493
• 负责人: MARSHA I ALTSCHULER
• 金额: $ 8.46万
• 依托单位: WILLIAMS COLLEGE
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-15 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3333493
• 关键词: Golgi apparatus; Tetrahymena; cell membrane; chromosomes; density gradient ultracentrifugation; enzyme mechanism; gel electrophoresis; gene expression; growth factor; hormone receptor; immunoprecipitation; lysosomes; molecular cloning; protein biosynthesis; protein structure; protein transport; proteolysis; radionuclides; secretion; secretory protein; tissue /cell culture; virus protein

This proposal seeks to identify and characterize the cellular machinery that governs the intracellular transport of plasma membrane, and secretory proteins from their site of synthesis on the rough endoplasmic reticulum (ER) to their site of function. The inability of a cell to direct proteins along the correct intracallular pathway may lead to serious physiological problems. For example, patients that suffer from a number of genetic diseases synthesize biologically active proteins, but fail to deliver them to their site of function. Two unique probes will be used to examine different steps in transport. The first probe is a hybrid protein, GHHA, encoded by a fusion of the cDNA's for rat growth hormone and a fragment of influenza hemagglutinin. GHHA accumulates in a region of the ER tentatively identified as a pre-Golgi subcompartment. This subcompartment will be characterized, in situ and as isolated microsomes, to determine if it is an essential ER subcompartment with unique functions that distinguish it from the rough ER. The effect of GHHA on the transport of other proteins to the Golgi will be assessed by immunofluorescence and by measuring the rate at which these proteins are modified by Golgi enzymes. The relative abundance of various proteins and enzyme activities in the rough ER and pre-Golgi will be determined. The second probe is a hybrid protein, GHG3, encoded by a fusion of the cDNA's for rat growth hormone and a fragment of vesicular stomatitis virus G protein. GHG3 is transported rapidly to lysosomes and degraded. GHG3 will be used to examine mechanisms of transport to the cell surface and lysosomes, by determining whether it contains three distinct sorting signals, or whether it is transported by a previously unrecognized autophagic pathway that directs misfolded proteins to the lysosome for degradation. The role of different subdomains of GHG3 on transport will be determined by using site-directed mutagenesis. The structure of these mutant proteins will be determined by limited proteolysis and measuring binding to growth hormone receptor in order to relate structure of the mutant proteins to their transport properties.

这项提案试图识别和表征细胞 管理细胞内血浆运输的机械 膜和分泌蛋白质从它们的合成部位 粗面内质网(ER)到它们的功能部位。 细胞不能沿着正确的方向引导蛋白质 囊内通路可能导致严重的生理问题。 例如，患有多种遗传性疾病的患者 疾病可以合成生物活性蛋白，但不能 把他们送到他们的工作地点。两个独特的探测器将是 用于检查运输过程中的不同步骤。第一个探测器是一个 大鼠融合基因编码的杂合蛋白GHHA 生长激素和一段流感血凝素。 GHHA在初步确定的内质网区域积聚 作为一个前高尔基小舱。这个小班将是 表征的、原位的和分离的微生物体，以确定 如果它是具有独特功能的基本ER小舱 使它有别于粗糙的急诊室。羟基羟基磷灰石对人体健康的影响 其他蛋白质向高尔基体的运输将通过以下方式进行评估 免疫荧光和通过测量这些细胞 蛋白质被高尔基体酶修饰。相对丰度 粗面内质网中各种蛋白质和酶的活性 之前的高尔基将被确定。第二个探测器是一种混合探测器 大鼠生长所需的融合基因编码的蛋白质GHG3 激素和水疱性口炎病毒G蛋白片段。 GHG3被迅速运输到溶酶体并被降解。GHG3 将被用来检查运输到细胞的机制 表面和溶酶体，通过确定它是否包含三个 不同的排序信号，或者它是否由 先前未知的自噬途径引导 错误折叠的蛋白质进入溶酶体进行降解。的作用 将确定GHG3在运输上的不同亚区 通过使用定点突变。这些东西的结构 突变的蛋白质将通过有限的蛋白质分解和 测量与生长激素受体的结合以关联 突变蛋白的结构对其运输特性的影响。