HEAT-SENSITIVE MUTANTS IN ENDOCYTIC FUNCTION

内吞功能中的热敏突变体

• 批准号: 3285028
• 负责人: ROCKFORD Keith DRAPER
• 金额: $ 15.2万
• 依托单位: UNIVERSITY OF TEXAS DALLAS
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-01-01 至 1989-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3285028
• 关键词: adenosinetriphosphatase; antibody formation; autosomal dominant trait; autosomal recessive trait; cell transformation; chromosome complement; electron microscopy; fluorescence; gel electrophoresis; immunofluorescence technique; lethal genes; low density lipoprotein; lysosomes; mixed tissue /cell culture; phagocytosis; pinocytosis; radiotracer; temperature sensitive mutant; transferrin; vesicle /vacuole

The overall objective of this proposal is to study the molecular mechanisms involved in the process of endocytosis. Our approach is to isolate and characterize animal cell mutants that are deficient in endocytic function. Moreover, we focus specifically on mutants that carry conditional-lethal defects. The agents that we use to isolate the mutants are highly potent protein toxins that require endocytic functions to express their lethal activities. Our specific aims are three-fold: 1. Further studies on G.7.1, a Chinese hamster ovary cell mutant carrying a conditional-lethal defect in endocytic function: We have so far isolated two mutants that at elevated temperature (39.5 C) lose viability and in the process become resistant to protein toxins. We have studied one of these mutants, called G.7.1, in more detail and the results suggest that it contains a heat-sensitive lesion affecting vacuolar acidification. We propose to more precisely identify the lesion in this mutant and to use the mutant as a model to investigate what effect the lesion has on endocytic processes. 2. Isolation and characterization of more mutants: Our selection procedure is capable of rescuing many different kinds of mutants containing conditional-lethal defects in endocytic function. We propose to search for, and characterize, additional mutants. 3. Genetic characterization of mutants: With techniques of somatic cell genetics we will determine whether the lesions in our mutants are expressed dominantly or recessively. Mutants that contain recessive lesions will be classified into complementation groups. We also propose to isolate and characterize revertants of the mutants.

这项建议的总体目标是研究分子机制 参与胞吞作用的过程。 我们的方法是隔离和 表征内吞功能缺陷的动物细胞突变体。 此外，我们特别关注携带条件致死性基因的突变体， 缺陷 我们用来隔离变种人的药剂 需要内吞功能以表达其致死性的蛋白质毒素 活动 我们的具体目标有三个方面： 1. G.7.1中国仓鼠卵巢细胞突变株的进一步研究 内吞功能的条件性致死缺陷：迄今为止，我们已经分离出 两种突变体在高温（39.5 ℃）下失去活力， 对蛋白质毒素有抵抗力。 我们研究了其中一个 突变体，称为G.7.1，更详细，结果表明， 含有影响液泡酸化的热敏感病变。 我们 建议更精确地识别这种突变体中的病变，并使用 突变体作为模型来研究病变对内吞 流程. 2.更多突变体的分离和表征：我们的选择程序 能够拯救许多不同种类的变种人， 内吞功能的条件性致死缺陷。 我们建议搜索 寻找并鉴定其他突变体 3.突变体的遗传表征：利用体细胞技术 遗传学，我们将确定我们的突变体中的病变是否表达为 占主导地位或reckless。 含有隐性病变的突变体将被 分类为互补组。 我们还建议隔离和 表征突变体的回复突变体。