GENETIC STUDY OF RADIATION RESISTANCE

这项建议的长期目标是提供对 细胞抗性的遗传和生化机制 电离辐射。通常采取的方法是使用伽马射线。 敏感的中国仓鼠突变体分离人类补体基因 这些突变体对辐射的敏感性。目前，我们已经分离出 12个对伽马射线致死敏感性增加的突变体。在……里面 在这项建议中，我们将重点隔离一种刻画人类修复的 位于5号染色体上的补充双链DNA断裂的基因 修复其中一个突变体XR-1的缺陷和伽马射线敏感性。 获得两株耐伽马射线的XR-1人杂交瘤细胞系 有限量的2Mb人类DNA。来自这种杂交种的DNA探针 将使用带有引物的聚合酶链式反应(PCR)来产生 针对人类的重复序列。两个都共有的探测器 杂交将被用来筛选酵母人工染色体(YAC)人 DNA文库，获得一组含有插入的人DNA的YAC 变成了抗伽马射线的混合体。这些青年会将被引入到 球体融合的XR-1细胞及其授予能力的检测 耐伽马射线。使用这些YAC作为探针，一组编码 对于插入的人DNA，将通过筛选cDNA文库获得。 将构建全长cDNA，并且候选cDNA包含 转XR-1细胞鉴定抗性基因及抗性筛选 耐伽马射线。将获得该cDNA克隆的DNA序列 以及与其他已知修复基因的序列同源性程度 下定决心。

The long term objective of this proposal is to provide an understanding of the genetic and biochemical mechanisms involved in cellular resistance to ionizing radiation. The general approach taken will be to use gamma-ray sensitive Chinese hamster mutants to isolate human genes which complement the radiation sensitivity of these mutants. Currently, we have isolated 12 mutants which have increased sensitivity to killing by gamma-rays. In this proposal, we will focus on isolating an characterizing a human repair gene located on chromosome 5 which complements the double-strand DNA break repair defect and gamma-ray sensitivity of one of these mutants, XR-1. Two gamma-ray resistant XR-1: human hybrid cell lines have been obtained that have a limited amount 2 Mb of human DNA. DNA probes from this hybrid will be generated using the polymerase chain reaction (PCR) with primers directed to human repetitive sequences. Probes which are common to both hybrids will be used to screen a yeast artificial chromosome (YAC) human DNA library and obtain a set of YACs which contain the human DNA inserted into the gamma-ray resistant hybrid. These YACs will be introduced into the XR-1 cell by spheroplast fusion and tested for their ability to confer gamma-ray resistance. Using these YACs as probes, a set of cDNAs coding for the inserted human DNA will be obtained by screening cDNA libraries. Full length cDNAs will be constructed and candidate cDNAs containing the resistance gene identified by transfection into XR-1 and selection for gamma-ray resistance. The DNA sequence of the cDNA clone will be obtained and the extent of sequence homology with other known repair genes determined.