HEAT-SENSITIVE MUTANTS IN ENDOCYTIC FUNCTION

这项建议的总体目标是研究分子机制。 参与内吞作用的过程。我们的方法是孤立和 描述缺乏内吞功能的动物细胞突变体。 此外，我们特别关注携带条件性致死基因的突变体。 缺陷。我们用来分离突变体的试剂是非常有效的 需要内吞功能才能表达其致命性的蛋白质毒素 活动。我们的具体目标有三个方面： 1.中国仓鼠卵巢细胞突变体G.7.1的进一步研究 内噬细胞功能的条件性致死性缺陷：我们迄今已分离出 两个在高温(39.5摄氏度)下失去活力的突变体和 对蛋白质毒素产生抵抗力。我们已经研究了其中的一个 突变体，称为G.7.1，更详细，结果表明它 含有影响空泡酸化的热敏性损伤。我们 建议更准确地识别该突变体中的病变，并使用 以突变体为模型研究病变对内吞细胞的影响 流程。 2.更多突变体的分离和鉴定：我们的选择程序 能够拯救许多不同种类的突变体，包括 内分泌功能中的条件致命性缺陷。我们打算搜索一下 对更多的突变体进行分析和描述。 3.利用体细胞技术对突变体的遗传特性进行研究 从遗传学上讲，我们将确定我们突变体中的损伤是否表达 显性的或隐性的。含有隐性损伤的突变体将是 被分成互补组。我们还建议隔离和 描述突变体的返回型。

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.