MOLECULAR CLONING OF HUMAN DNA REPAIR GENES

一种高效修复DNA核苷酸损伤的机制 (无论是由辐射还是化学挑战引起)是一种必要的功能 为了保持细胞的遗传完整性。未能修复的问题 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 诱变并最终致癌。似乎有一种强烈的 紫外线修复缺陷之间存在因果关系的情况 光(UV)损伤和人类遗传病的致癌作用， 着色性干皮病(XP)。这项建议意在加强 了解DNA修复系统，它通常负责 人体细胞紫外线损伤的修复。 提出了克隆和表达其中一个基因的新策略 与启动DNA修复直接相关的基因 紫外线损伤人体细胞的部位；缺乏或不起作用 该基因产物定义了XP互补组A(XP-A)。依赖于 Legerski等人的发现。(1984)，认为SIZE类基因 含有XP-A基因的有10.6个S、3个10.6个S的mRNA文库 已构建：一个来自受XP-A影响的子项，两个来自 专职XP--XP-A儿童的杂合子父母。个人 文库已被用来三倍筛选第四个人类cDNA库 为了确定父母双方都有共同的斑块，但 在孩子身上没有表达或过度表达。基因已经被 将对这些程序确定的情况进行如下进一步调查。 该基因(S)将被亚克隆到哺乳动物穿梭载体中，并进行转基因 进入XP-A细胞，并对这些细胞进行增强紫外线存活的检测。这个 可能的DNA修复基因(S)将被用于检测丰度和任何 Northern分析其他XP-A细胞中mRNAs的大小异质性。在……里面 此外，这些DNA将用于识别限制片段长度 其他XP基因组DNA的多态性。表达载体Lambdagt11 将用于生产β-半乳糖苷酶和DNA的融合蛋白 修复基因产物。将会产生针对融合蛋白的抗体 并将用于抑制体外胸腺二聚体切除试验 或抓人的行为。

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.