REPAIR OF X-RAY INDUCED DNA DAMAGE: GENETIC BASIS

为了了解X射线和EMS抗性的遗传基础，我们 建议克隆编码这些表型的人类DNA序列， 性质并表征其结构和活性。 EM9细胞是突变的中国仓鼠卵巢细胞系， 对X射线和烷基化剂的杀伤作用过敏。 EM9细胞 对X射线的敏感度大约高出1.4倍， 对EMS的杀伤敏感性（用DO值评价）高于亲本 中国仓鼠卵巢系（AA8）。 碱洗分析法修复 EM9细胞显示它们缺乏修复单链的能力， DNA的断裂 通过DNA介导的基因转移，我们已经产生了转基因EM9细胞， 含有人类DNA，并显示出对X射线和EMS的增强抵抗力。 的 本文提出的研究目的是克隆和表征 负责EM9细胞逆转的假定人类修复基因。 为了实现这一目标，我们提出了产生和筛选粘粒 来自含有人DNA的回复突变体细胞系的文库。 将通过人类DNA杂交技术和 标记基因（PSV 2neo）。 被筛选为阳性的Cosmetics，以及 因此可能含有修复基因，将进一步筛选 通过DNA介导的基因转移到EM9细胞中用于表型活性。 的 将通过限制性内切酶分析克隆的人序列的结构 映射. 含有修复的敏感细胞的表型活性 基因将通过细胞存活、DNA单链和双链分析 断裂修复能力将通过碱性和中性洗脱来确定， 分别 我们的初步工作表明，有一种人类基因， 对敏感仓鼠细胞系的抗性。 拟议的研究将 提供了对X射线细胞杀伤抗性的更深入的了解 在人身上。

In order to understand the genetic basis of X-ray and EMS resistance, we propose to clone the human DNA sequence coding for these phenotypic properties and to characterize its structure and activity. EM9 cells are a mutant Chinese hamster ovary cell line that is hypersensitive to killing by X-rays and alkylating agents. EM9 cells are approximately 1.4 times more sensitive to X-rays and 17 times more sensitive to killing by EMS (evaluated by Do values) than the parental Chinese hamster ovary line (AA8). Alkaline elution analysis of repair in EM9 cells shows them to be deficient in the ability to repair single strand breaks in DNA. By DNA mediated gene transfer, we have produced transgenic EM9 cells that contain human DNA and display an enhanced resistance to X-ray and EMS. The objective of the research proposed here is to clone and characterize the putative human repair gene responsible for the reversion of the EM9 cells. To accomplish this objective, we propose to generate and screen cosmid libraries from revertant transformant cell lines containing human DNA. Cosmids will be screened by hybridization techniques for human DNA and the marker gene (PSV2neo). Cosmids that are screened as positive, and therefore potentially containing the repair gene, will be further screened by DNA mediated gene transfer into EM9 cells for phenotypic activity. The structure of the cloned human sequences will be analyzed by restriction mapping. The phenotypic activity of sensitive cells containing the repair gene will be analyzed by cell survival, and DNA single and double strand break repair capability will be determined by alkaline and neutral elution, respectively. Our preliminary work suggests that there is a human gene that confers X-ray resistance to a sensitive hamster cell line. The proposed research will provide a more intimate understanding of resistance to X-ray cell killing in man.