TRANSPORT COMPONENTS FROM EUKARYOTES

来自真核生物的转运成分

• 批准号: 3270141
• 负责人: DALE L OXENDER
• 金额: $ 18.01万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270141
• 关键词: Ehrlich's tumor; Escherichia coli; active transport; aminoacid transport; bioenergetics; cell fusion; cell type; eukaryote; fluorescent dye /probe; gel electrophoresis; gene mutation; genetic manipulation; genetic mapping; genetic recombination; hamsters; human tissue; hybrid cells; laboratory mouse; laboratory rabbit; membrane permeability; membrane proteins; molecular cloning; molecular genetics; monoclonal antibody; mutant; natural gene amplification; phospholipids; protein biosynthesis; protein sequence; radiotracer; temperature sensitive mutant; tissue /cell culture

Mammalian cells contain several distinct transport systems for maintaining their levels of neutral amino acides. These internal levels are maintained by balancing the transport activity and the biosynthesis with the cellular requirements for protein synthesis and energy metabolism. The long-rang goal of this project is the under standing of the molecular basis of active transport of amino acids in mammalian cells. These amion acid transport systems have been characterized in Chinese hamster ovary (CHO) cells, and they include the Na+-dependent A, ASC, and P Systems, as well as the Na+-independent L System. We are applying genetic approaches and recombinant DNA techniques in an attempt to clone the human genes responsible for amion acid transport. The strategy being used is to obtain transport mutants in CHO cells, and then complement these mutants by transferring human DNA sequences that code for the amino acid transport activity. Using genetic approaches, CHO mutants have been isolated that are defective in system L transport and its regulation. These mutants are being characterized and used for identifying human transport genes. The transfer of human DNA will be carried out be either cell fusion techniques or direct transformation with a human genomic cosmid library. The human transport genes will be rescued from the transformants by in vitro packaging techniques. To identify the transpot components, we will attempt to increase their expression by gene amplification of the transport genes, and then sperating and identifying the proteins by two-dimensional gel electrophoresis and by fluorescent reagent labeling. We also plan to raise anti-idiotypic antibodies to the transport receptors to aid in the identification of the transporters.

哺乳动物细胞包含几种不同的运输系统 保持其中性氨基囊的水平。 这些内部 通过平衡运输活动和 生物合成具有蛋白质合成的细胞需求 和能量代谢。 这个项目的远程目标是 在氨基的主动转运的分子基础上 哺乳动物细胞中的酸。 这些氨酸转运系统 已经在中国仓鼠卵巢（CHO）细胞中表征 它们包括Na+依赖性A，ASC和P系统，作为 以及Na+独立的L系统。 我们正在应用遗传 方法和重组DNA技术试图 克隆负责氨酸转运的人类基因。 这 所使用的策略是在Cho细胞中获取转运突变体， 然后通过转移人DNA来补充这些突变体 序列为氨基酸转运活性编码。 使用 遗传方法，已经孤立了Cho突变体 系统L运输及其调节缺陷。 这些突变体 被描述和用于识别人类运输 基因。 将进行人DNA的转移。 细胞融合技术或人类直接转化 基因组宇宙库。 人类运输基因将是 通过体外包装技术从转化体中救出。 为了识别转板组件，我们将尝试增加 它们通过基因扩增传输基因的表达，以及 然后通过二维凝胶吐和识别蛋白质 电泳和荧光试剂标记。 我们也计划 提出对传输受体的抗二元型抗体以帮助 在转运蛋白的识别中。