GENETICS OF HUMAN DNA REPAIR

人类遗传性色素性干皮病(XP) 这是由于DNA切除修复基因的先天错误造成的。 有缺陷的DNA修复和 致癌。XP患者表现出2000倍的增长 表皮肿瘤的发生和每年10倍的增长 内脏肿瘤的发生。频率很高的 表皮肿瘤是由于无法修复DNA损伤所致 由暴露在阳光下引起的。高患病率 内脏肿瘤可能与敏感度升高有关。 XP细胞对饮食致癌物的影响。九大互补群 已在XP中定义。在所有小组中，XP互补性 A组(XPA)细胞DNA修复能力最低。喜欢 其他DNA修复能力极低的患者，XPA患者 通常有相关的神经病变，表明在 神经组织中的XPA基因产物。该计划的目标 目前的建议旨在了解 DNA切除修复的机制。转型的XPA DNA介导的基因互补的成纤维细胞 人成纤维细胞c DNA表达文库的转移 在这个实验室里获得的。含有该基因的质粒 互补的cDNA将从沙门氏菌基因组DNA中分离 用质粒挽救法转化XPA细胞。核苷酸 将确定该基因的序列，并测定该基因的序列 编码的蛋白质将从cDNA序列中推断出来。这个 将搜索蛋白质和核酸序列数据库 与XPA蛋白同源的蛋白质，以努力鉴定 XPA基因产物的功能。该基因将被用作 一种用于确定XPA细胞中分子缺陷的探针。这将是 关于XPA基因产物的区域的产量信息 对它的功能至关重要。XPA特异性mRNA在XPA中的表达 将检查细胞的数量或质量变化 用斑点杂交法进行杂交分析。删除的站点和 在XPA细胞的XPA特异性mRNAs中的插入将是 由S1保护映射定义。点突变影响 蛋白质的功能将通过克隆和测序来确定 CDNA转化为突变的XPA mRNAs。大量缺失的存在 以及XPA特异性基因的插入和重排 XPA细胞将通过XPA的印迹杂交分析进行鉴定 细胞基因组DNA。正常的DNA修复基因将被分离出来 并以此为特征。啮齿动物细胞含有一种活动，可以 补充XPA细胞。目前尚不清楚这项活动是否为 与XPA基因产物相同的蛋白质。脊椎动物的系统发育 将通过斑点杂交来研究DNA修复基因的 不同脊椎动物基因组DNA的分析 与cdna探针结合。保护的程度将是 通过改变杂交的严格性与 人cDNAs探针。组织分布和水平 同源DNA修复基因在小鼠体内的表达 通过与RNAs的斑点杂交分析确定 人cDNAs探针。

The human hereditary syndrome Xeroderma pigmentosum (XP) results from inborn errors in the genes for DNA excision repair. A strong correlation exists between defective DNA repair and carcinogenesis. XP patients exhibits a 2000-fold increase in the occurrence of epidermal neoplasms and a 10-fold increase in the occurrence of internal neoplasms. The high frequency of epidermal neoplasms is due to an inability to repair DNA damage caused by exposure to sunlight. The elevated prevalence of internal neoplasms is probably linked to the heightened sensitivity of XP cells to dietary carcinogens. Nine complementation groups have been defined in XP. Of all the groups, XP complementation group A (XPA) cells have the lowest DNA repair capability. Like others with extremely low DNA repair capability, XPA patients often have associated neuropathy indicating an essential role for the XPA gene product in neural tissue. The objectives of the current proposal are aimed at gaining an understanding of the mechanisms of DNA excision repair. Transformed XPA fibroblasts which have been complemented by DNA mediated gene transfer of a human fibroblast cDNA expression library have been obtained in this laboratory. The plasmid containing the complementing cDNA will be isolated from the genomic DNA of the transformed XPA cells by plasmid rescue. The nucleotide sequence of the cDNA will be determined and the sequence of the encoded protein will be inferred from the cDNA sequence. The protein and nucleic acid sequence data banks will be searched for proteins homologous to the XPA protein in an effort to identify the function of the XPA gene product. The cDNA will be used as a probe to define the molecular defects in XPA cells. This will yield information on regions of the XPA gene product which are critical to its function. XPA specific mRNA expressed in XPA cells will be examined for quantitative or qualitative changes from normal by blot hybridization analyses. Sites of deletions and insertions in the XPA specific mRNAs of XPA cells will be defined by S1 protection mapping. Point mutations affecting protein function will be identified by cloning and sequencing cDNAs to mutant XPA mRNAs. The presence of large deletions and insertions in, and rearrangement of, the XPA specific gene in XPA cells will be identified by blot hybridization analyses of XPA cell genomic DNAs. The normal DNA repair gene will be isolated and characterized. Rodent cells contain an activity which can complement XPA cells. It is unknown whether this activity is the same protein as the XPA gene product. The vertebrate phylogeny of the DNA repair gene will be investigated by blot hybridization analyses of genomic DNAs from various vertebrate organisms with the cDNA probe. The degree of conservation will be estimated by varying the stringency of hybridization with the human cDNA probe. The tissue distribution and levels of expression of the homologous DNA repair gene in mice will ascertained by blot hybridization analyses of RNAs with the human cDNA probe.