MOLECULAR ANALYSIS OF RAD SENSITIVITY IN MAMMALIAN CELLS

哺乳动物细胞辐射敏感性的分子分析

• 批准号: 2087014
• 负责人: MARK L MEUTH
• 金额: $ 28.18万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-09-30 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2087014
• 关键词: ADP ribosylation; DNA damage; DNA directed DNA polymerase; DNA repair; Mammalia; ataxia telangiectasia; complementary DNA; enzyme activity; gene complementation; gene mutation; genetic strain; human genetic material tag; human tissue; intermolecular interaction; ionizing radiation; mutant; nucleic acid sequence; phenotype; phenylamide; radiation genetics; radiation immunosuppression; radiation sensitivity; tissue /cell culture; transfection; western blottings

Although great strides have been made in definition of the genes involved in nucleotide excision repair, relatively little is known regarding the genes that govern sensitivity to ionizing radiation. The long term goal of our work is to identify and characterize genes that govern radiation sensitivity in mammalian cells. Recent work from our laboratory has demonstrated that the radiation sensitive phenotype of the hamster mutant irs-2 is suppressed in strains transfected with the human poly(ADP- ribose) polymerase (PARP) cDNA or selected for resistance to 3- aminobenzamide (3-AB, an inhibitor of PARP). These observations suggest that PARP may play an important role in the response of cells to ionizing radiation and suggest PARP as the candidate gene that is altered in irs- 2. The specific aims of the work proposed here are: i) to determine whether mutations of PARP are responsible for the radiation sensitive phenotype of irs-2 and the mechanism by which It confers this sensitivity. This will be pursued by both DNA sequence analysis of the PARP coding sequences in irs-2 and 3-AB resistant strains and biochemical analysis of PARP activity by Western and activity blots. To further confirm that mutations of PARP cause suppression of the radiation sensitive phenotype, mutants of irs-2 resistant to other more specific inhibitors of PARP will be selected. We will also attempt to suppress the radiation sensitive phenotype of irs-2 by transfecting wild type and mutant forms of PARP. To investigate the mechanism by which these mutations confer sensitivity, the interaction of PARP with other proteins will be examined. ii) to determine whether the suppression of radiation sensitivity is specific for irs-2. Radiation-sensitive strains deficient in double stand break repair, base excision repair and strains proficient in these pathways will be transfected with the human PARP cDNA to determine whether they show a similar suppression of radiation sensitivity. iii) to determine whether the human PARP cDNA complements the radiation sensitive phenotype of cells from Ataxia-telangiectasia (A- T) patients. Considering that irs-2 is the most A-T like of the hamster radiation sensitive mutantS, we will attempt to complement the radiation sensitive phenotype of A-T cells by transfecting them with the human PARP cDNA. iv) to identify and clone the human gene complementing or suppressing the radiation sensitive phenotype of irs-2. This will be accomplished by successive rounds of transfection of either purified human DNA or metaphase chromosomes to-complement the irs-2 phenotyPe followed by cloning of human specific sequences. Alternatively we will transfect expressing cDNA libraries followed by rapid recovery of the complementing cDNA. The significance of this work lies in the identification of genes that govern sensitivity to ionizing radiation. This may ultimately enable us to predict and alter the response of tumor cells to radiation therapy.

尽管在涉及的基因的定义方面取得了长足的进步 在核苷酸切除修复中，关于 控制对电离辐射的敏感性的基因。长期目标 我们的工作是识别和表征控制辐射的基因 哺乳动物细胞的敏感性。我们实验室的最近工作 证明仓鼠突变体的辐射敏感表型 IRS-2在用人聚（ADP-）转染的菌株中抑制 核糖）聚合酶（PARP）cDNA或选择以抗3- 氨基苯甲酰胺（3-AB，PARP的抑制剂）。这些观察表明 该PARP可能在细胞对电离的响应中起重要作用 辐射并建议PARP作为IRS-改变的候选基因 2。此处提出的工作的具体目的是：i）确定 PARP突变是否负责辐射敏感 IRS-2的表型及其同意的机制 灵敏度。这两个DNA序列分析都将通过 IRS-2和3-AB抗性菌株和生化的PARP编码序列 通过西方和活动印迹分析PARP活性。进一步 确认PARP的突变会导致辐射的抑制 敏感表型，IRS-2的突变体对其他更具体的抗性 将选择PARP的抑制剂。我们还将尝试压制 通过转染野生型和 PARP的突变形式。调查这些机制 突变赋予灵敏度，PARP与其他蛋白质的相互作用 将被检查。 ii）确定是否抑制辐射 灵敏度针对IRS-2。辐射敏感性不足 在双支架休息修复中，基础切除修复和压力熟练 在这些途径中，将用人Parp cDNA转染至 确定它们是否显示出类似的辐射抑制 灵敏度。 iii）确定人PARP cDNA是否补充 来自共济失调 - 凝血症的细胞的辐射敏感表型（a- t）患者。考虑到IRS-2是仓鼠最喜欢的A-T 辐射敏感突变体，我们将尝试补充辐射 A-T细胞的敏感表型通过用人PAR转染 cDNA。 iv）识别和克隆人类基因补充或 抑制IRS-2的辐射敏感表型。这将是 通过连续转染任何一种纯化的回合来完成 人类DNA或中期染色体融合IRS-2表型 然后是克隆人特异性序列。或者我们会的 转染表达cDNA文库，然后快速恢复 补充cDNA。这项工作的意义在于 识别控制对电离辐射敏感性的基因。 这可能最终使我们能够预测和改变肿瘤的反应 细胞进行放射治疗。