MOLECULAR CLONING OF HUMAN DNA REPAIR GENES

人类 DNA 修复基因的分子克隆

• 批准号: 3186105
• 负责人: AUGUSTINUS R RINALDY
• 金额: $ 7.85万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-01 至 1988-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3186105
• 关键词: DNA repair; beta galactosidase; gene complementation; genetic library; ionizing radiation; messenger RNA; molecular cloning; monoclonal antibody; plasmids; radiation genetics; radiation sensitivity; radiobiology; tissue /cell culture; ultraviolet radiation; xeroderma pigmentosum

A highly efficient mechanism to repair damage to nucleotides in DNA (whether caused by radiation or chemical challenge) is a necessary function of a cell in order to preserve its genetic integrity. A failure to repair these damages or an inaccurate repair mechanism will result in mutations within DNA sequences, which in turn can be associated with direct mutagenesis and ultimately carcinogenesis. There appears to be a strong case for a causal relationship between defective repair of ultraviolet light (UV) damage and carcinogenesis in the human genetic disease, xeroderma pigmentosum (XP). This proposal intends to enhance the understanding of the DNA repair system which is normally responsible for the repair of UV damage in human cells. Novel strategies are proposed for the cloning and expression of one of the genes which is directly associated with the initiation of DNA repair at sites of UV damage in human cells; the deficiency or nonfunctionality of this gene product defines XP complementation group A (XP-A). Relying on the findings of Legerski et al. (1984), that the size class mRNA containing the XP-A gene was 10.6 S, three 10.6 S mRNA cDNA libraries have been constructed: one from an XP-A effected child and two from the obligate XP-A heterozygote parents of the XP-A child. The individual libraries have been used to triplicate screen a fourth human cDNA library in order to identify plaques which are common to both parents but either are not expressed or are overexpressed in the child. Genes which have been identified by these procedures will be further investigated as follows. The gene(s) will be subcloned into a mammalian shuttle vector, transfected into XP-A cells and the cells assayed for enhanced UV survival. The putative DNA repair gene(s) will be used to assay for abundance and any size heterogeneity of mRNAs from other XP-A cells by Northern analyses. In addition, these DNAs will be used to identify restriction fragment length polymorphisms in other XP genomic DNAs. The expression vector Lambdagt11 will be used to produce fusion proteins of Beta-galactosidase and the DNA repair gene product. Antibodies will be raised against the fusion protein and will be used for inhibition of an in vitro thymic dimer excision assay or nicking activities.

一种高效的DNA核苷酸损伤修复机制 （无论是由辐射或化学挑战引起）是必要的功能 以保持其基因的完整性。 无法修复 这些损伤或不准确的修复机制将导致突变 在DNA序列中，这反过来又可以与直接 诱变和最终致癌。 似乎有一个强大的 紫外线修复缺陷与 光（UV）损伤和致癌作用在人类遗传性疾病中， 着色性干皮病（XP）。 这项建议旨在加强 DNA修复系统通常负责 修复人体细胞中的紫外线损伤。 提出了新的策略，用于克隆和表达其中一种 与DNA修复启动直接相关的基因， 人体细胞中紫外线损伤的部位; 该基因产物定义了XP互补组A（XP-A）。 依托 Legerski等人（1984）的发现，即大小类mRNA 含有XP-A基因的cDNA文库的长度为10.6S， 一个来自XP-A效应的孩子，两个来自 XP-A子代的专性XP-A杂合子父母。 个人 文库已用于一式三份筛选第四个人cDNA文库 为了识别父母双方共有的斑块， 在儿童中不表达或过度表达。 基因已经被 这些程序确定的问题将进一步调查如下。 将基因亚克隆到哺乳动物穿梭载体中，转染 转化到XP-A细胞中，并测定细胞的UV存活率。 的 推定的DNA修复基因将用于测定丰度和任何 通过北方分析来自其它XP-A细胞的mRNA的大小异质性。 在 此外，这些DNA将用于鉴定限制性片段长度 其他XP基因组DNA的多态性。 表达载体Lambdagt 11 将用于生产β-半乳糖苷酶和DNA的融合蛋白 修复基因产物。 针对融合蛋白的抗体将会产生 并将用于抑制体外胸腺二聚体切除试验 或偷窃活动。