GENETICS OF HUMAN DNA REPAIR

人类遗传综合征着色性干皮病（XP） 是由DNA切除修复基因的先天性缺陷引起的。 DNA修复缺陷与 致癌作用 XP患者的血压升高了2000倍， 发生表皮肿瘤和10倍增加， 内部肿瘤的发生。 的高频率 表皮肿瘤是由于无法修复DNA损伤 由于暴露在阳光下。 高发病率 内部肿瘤可能与高敏感性有关 XP细胞对膳食致癌物的反应。 九互补群 在XP中定义。 在所有组中，XP互补 A组（XPA）细胞具有最低的DNA修复能力。 像 其他人的DNA修复能力极低，XPA患者 通常有相关的神经病变，表明 XPA基因在神经组织中的产物。 的目标 目前的建议旨在了解 DNA切除修复机制。 XPA转型 DNA介导的基因互补的成纤维细胞 人成纤维细胞cDNA表达文库的转移已经 在这个实验室里。 质粒含有 互补cDNA将从以下的基因组DNA中分离： 通过质粒拯救转化的XPA细胞。 的核苷酸 将确定cDNA的序列，并且将确定cDNA的序列。 编码的蛋白质将从cDNA序列推断。 的 将搜索蛋白质和核酸序列数据库， 与XPA蛋白同源的蛋白质， XPA基因产物的功能。 cDNA将用作 一种用于确定XPA细胞中分子缺陷的探针。 这将 产生关于XPA基因产物区域的信息， 对它的功能至关重要。 XPA中表达的XPA特异性mRNA 将检查细胞的定量或定性变化 通过印迹杂交分析， 缺失位点和 在XPA细胞的XPA特异性mRNA中的插入将被 由S1保护映射定义。 点突变影响 通过克隆和测序鉴定蛋白质功能 突变的XPA mRNA。 大缺失的存在 以及XPA特异性基因的插入和重排， XPA细胞将通过XPA的印迹杂交分析来鉴定。 细胞基因组DNA。 正常的DNA修复基因将被分离出来 和表征了 啮齿动物细胞含有一种活性物质， 补体XPA细胞。 目前尚不清楚这项活动是否是 与XPA基因产物相同的蛋白质 脊椎动物的生殖 将通过印迹杂交研究DNA修复基因的 各种脊椎动物的基因组DNA分析 cDNA探针。 保护的程度将是 通过改变与 人cDNA探针。 组织分布和水平 同源DNA修复基因在小鼠中的表达将 通过RNA的印迹杂交分析确定， 人cDNA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.