GENETIC STUDY OF RADIATION RESISTANCE

抗辐射的基因研究

• 批准号: 2098995
• 负责人: THOMAS Dominic STAMATO
• 金额: $ 26.08万
• 依托单位: LANKENAU INSTITUTE FOR MEDICAL RESEARCH
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-15 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2098995
• 关键词: CHO cells; DNA damage; DNA repair; artificial chromosomes; cell fusion; complementary DNA; cytogenetics; gamma radiation; genetic library; human genetic material tag; hybrid cells; ionizing radiation; mutant; nucleic acid probes; nucleic acid repetitive sequence; nucleic acid sequence; polymerase chain reaction; radiation genetics; radiation resistance; restriction mapping; transfection

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.

该提议的长期目标是提供对 涉及细胞抗性的遗传和生化机制 电离辐射。 所采用的一般方法是使用伽马射线 敏感的中国仓鼠突变体隔离人类基因 这些突变体的辐射灵敏度。 目前，我们已经孤立了 12个突变体对被伽马射线杀死的敏感性提高。 在 该建议，我们将专注于隔离人类维修的特征 位于5号染色体上的基因，与双链DNA断裂相互补充 这些突变体之一XR-1的修复缺陷和伽马射线敏感性。 已经获得了两个伽马射线XR-1：人类杂化细胞系 量有限2 MB的人DNA。 DNA探针来自该混合动力车 将使用与引物的聚合酶链反应（PCR）生成 针对人类重复序列。 两者共有的探针 杂种将用于筛选酵母人造染色体（YAC）人类 DNA库并获得一组包含人DNA插入的YAC 进入伽马射线抗性杂种。 这些YAC将被引入 XR-1细胞通过hamphoplast Fusion并测试了其赋予的能力 伽马射线阻力。 使用这些YAC作为探针，一组CDNA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，并包含候选cDNA 通过转染到XR-1并选择的抗性基因 伽马射线阻力。 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的程度 决定。