GENETIC STUDY OF RADIATION RESISTANCE

本提案的长期目标是提供对以下内容的了解： 涉及细胞抵抗的遗传和生化机制， 电离辐射 一般采用的方法是使用伽马射线 敏感的中国仓鼠突变体分离人类基因， 这些突变体的辐射敏感性 目前，我们已经分离出 12种突变体对伽马射线杀伤的敏感性增加。 在 这个建议，我们将集中在分离一个表征人类修复 位于5号染色体上的一种基因，补充双链DNA断裂 修复缺陷和伽马射线敏感性的突变体之一，XR-1。 获得了两个抗γ射线的XR-1：人杂交细胞系 只有有限的2 Mb人类DNA 这个杂交体的DNA探针 将使用聚合酶链反应（PCR）和引物生成 涉及人类重复序列。 两者共有的探针 杂交将用于筛选酵母人工染色体（YAC）人类 DNA文库，并获得一组含有插入的人DNA的YAC 植入抗伽马射线的混合体 这些青年活动中心将被引入 通过原生质球融合XR-1细胞，并测试它们赋予 伽马射线抗性 使用这些YAC作为探针，一组编码 将通过筛选cDNA文库获得插入的人DNA。 将构建全长cDNA，并将含有本发明的cDNA的候选cDNA与全长cDNA进行比较。 通过转染到XR-1中鉴定抗性基因并选择用于 伽马射线抗性 将获得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 测定

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.