MOLECULAR CLONING OF HUMAN DNA REPAIR GENE(S)

人类 DNA 修复基因的分子克隆

• 批准号: 3186114
• 负责人: AUGUSTINUS R RINALDY
• 金额: $ 10.26万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3186114
• 关键词: DNA repair; DNA replication; 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; northern blottings; 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.

遗传物质的维持需要一种机制， 修复各种化学物质对DNA造成的损伤， 物理代理 如果DNA修复失败， 在受影响细胞的后代中， 表型上与肿瘤转化相关。 的因果 紫外线损伤修复缺陷与 对DNA和致癌作用很强的疾病是干皮病 色素沉着 这项建议的目的是克隆和 表征负责蛋白质编码的基因 启动DNA修复和缺乏干皮病 色素互补A组（XP-A）。 mRNA的大小类别 在HeLa细胞中含有XP-A基因的DNA在10.6S库内 （Legerski等人，Proc. Natl. Acad. Sci. U.S.A.81，5676，1984）。 从XP-A的这种mRNA库衍生的cDNA文库 受影响的孩子和专性XP-A杂合子父母已经 构建并用于杂交竞争策略， 筛选父母和正常成人肝脏cDNA 图书馆 在15个分离的cDNA克隆中，14个包含一个基因 家人 其中一个克隆的部分特征在于， 核酸序列的术语，其结果表明， 这是一个新的基因。 该基因的片段用于1） 亲本的北方印迹分析和2）TaqI RFLP分析 孩子的DNA 所提议的实验的目的是调查 这个新基因与疾病状态之间的关系 着色性干皮病 构建的人粘粒文库， 使用含有新霉素的表达粘粒载体pCV 108， 筛选抗性基因标记。 宇宙体克隆体将会 限制性作图并用于XP-A细胞的转化 品系，并选择新霉素和UV抗性。 XP-A细胞 其由于粘粒而获得增强的UV抗性， 转型，将受到南方以及北方 印迹分析，并将这些结果与非- 转化的XP-A细胞系。 此外，这种新基因将被 用于1）研究其他XP-A家族的群体遗传学 细胞系通过使用北方和RFLP分析，和2） 比较其他XP-A家族细胞系的细胞遗传学分析 用非整倍体VA 13修复熟练细胞系（Schultz et 例如，Proc. Natl. Acad. Sci. U.S.A. 84，4176，1987）和正常 二倍体细胞系