MOLECULAR CLONING OF HUMAN DNA REPAIR GENES

人类 DNA 修复基因的分子克隆

• 批准号: 3186107
• 负责人: AUGUSTINUS R RINALDY
• 金额: $ 12.8万
• 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 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3186107
• 关键词: DNA repair; chimeric proteins; clone cells; complementary DNA; cytogenetics; diploidy; drug resistance; fibroblasts; gel electrophoresis; gene complementation; genetic library; genetic mapping; genetic markers; genotype; heterozygote; homozygote; human population genetics; in situ hybridization; ionizing radiation; messenger RNA; molecular cloning; monoclonal antibody; neomycin; neoplastic cell culture for noncancer research; nucleic acid probes; plasmids; radiation carcinogenesis; radiation genetics; radiation sensitivity; transfection; transposon /insertion element; ultraviolet radiation; xeroderma pigmentosum

Maintenance of the genetic material requires a mechanism for repairing damage to the DNA caused by a variety of chemical and physical agents. If DNA repair fails to occur, mutations may arise in the descendants of the affected cells which in turn can be phenotypically associated with neoplastic transformation. A causal relationship between defective repair of ultraviolet light damage to DNA and carcinogenesis is very strong in the disease xeroderma pigmentosum. The objective of this proposal is to clone and characterize the gene coding for the protein which is responsible for the initiation of DNA repair and lacking in xeroderma pigmentosum complementation group A (XP-A). The size class of mRNA containing the XP-A gene in HeLa cells is within the 10.6S pool (Legerski et al., Proc. Natl. Acad. Sci. U.S.A. 81, 5676, 1984). cDNA libraries derived from the pool of this mRNA of an XP-A affected child and the obligate XP-A heterozygous parents have been constructed and used in hybridization competition strategy to screen both the parents' and a normal human adult liver cDNA library. Of the 15 isolated cDNA clones, 14 comprise one gene family. One of these clones has been partially characterized in terms of nucleic acid sequence, the results of which indicate that this is a novel gene. A fragment of this gene was used for 1) Northern blot analysis and 2) TaqI RFLP analyses of the parental and child DNA. The objective of the proposed experiments is to investigate the relationship between this novel gene and the disease state of xeroderma pigementosum. A human cosmid library constructed by using expression cosmid vector pCV108 containing a neomycin- resistance gene marker will be screened. Cosmid clones will be restriction mapped and used for the transformation of the XP-A cell line and selected for both neomycin and UV resistance. XP-A cells which acquire enhanced UV-resistance as a result of cosmid transformation, will be subjected to Southern as well as Northern blot analyses and these results compared with those of a non- transformed XP-A cell line. In addition, this novel gene will be used 1) to study the population genetics of the other XP-A family cell lines by using Northern and RFLP analyses, and 2) for cytogenetic analyses of other XP-A family cell lines in comparison with both aneuploid VA13 repair-proficient cell line (Schultz et al., Proc. Natl. Acad. Sci. U.S.A. 84, 4176, 1987) and normal diploid cell line.

维持遗传物质需要一种机制来 修复由多种化学和化学物质引起的DNA损伤 物理代理。如果DNA修复失败，可能会出现突变 在受影响的细胞的后代中，这反过来可能是 与肿瘤转化相关的表型。因果关系 紫外光损伤修复缺陷的关系 对DNA和致癌作用很强的干皮病 色素沉着症。这项提议的目标是克隆和 确定编码负责蛋白质的基因的特征 对于DNA修复的启动和干皮病的缺乏 色素互补组A(XP-A)。信使核糖核酸的大小类 在HeLa细胞中含有XP-A基因在10.6s池内 (Legerski等人，Proc.娜塔莉。阿卡德。SCI。美国第81,5676,1984)。 从XP-A的这个信使核糖核酸的池中获得的cDNA文库 受影响的孩子和专职的XP-A杂合子父母已经 构建并用于杂交竞争策略 双亲和正常成人肝脏基因的筛选 图书馆。在分离的15个cdna克隆中，有14个包含一个基因。 一家人。这些克隆中的一个已经被部分描述为 核酸序列的术语，其结果表明 这是一个新的基因。该基因的一个片段用于1) Northern杂交分析和亲本TaqI RFLP分析 和儿童DNA。 拟议的实验的目的是调查 该新基因与骨髓瘤病情的关系 色素性干皮病由人类建造的宇宙体库 利用含有新霉素的表达粘粒载体pCV108- 筛选抗性基因标记。科斯米德的克隆人将是 绘制并用于XP-A细胞转化的限制条件 该品系同时具有抗新霉素和抗紫外线的特性。XP-A细胞 由于粘粒的结果，它们获得了增强的紫外线抗性 转型，将受到南方和北方的影响 印迹分析和这些结果与非 转化XP-A细胞系。此外，这种新的基因将是 用于研究另一个XP-A家族的群体遗传学 细胞系通过Northern和RFLP分析，以及2) XP-A家族其他细胞系的细胞遗传学比较分析 与非整倍体VA13修复熟练的细胞系(Schultz et 等，Proc.娜塔莉。阿卡德。SCI。美国84、4176、1987)和正常 二倍体细胞系。