van Duin M, Vredeveldt G, Mayne L V, Odijk H, Vermeulen W, Klein B, Weeda G, Hoeijmakers J H, Bootsma D, Westerveld A
Department of Cell Biology and Genetics, Erasmus University Rotterdam, The Netherlands.
Mutat Res. 1989 Mar;217(2):83-92. doi: 10.1016/0921-8777(89)90059-1.
The human DNA excision repair gene ERCC-1 complements the ultraviolet light (UV) and mitomycin C (MMC) sensitivity of CHO mutants of complementation group 1. We have investigated whether ERCC-1 is the mutated gene in cell lines from xeroderma pigmentosum (XP) complementation groups A through I by analyzing the endogenous gene in XP cells and by introduction of the gene followed by repair assays. Our studies show that ERCC-1 is not deleted or grossly rearranged in representative cell lines of 9 XP groups. Furthermore, Northern blot analysis revealed correct transcription of ERCC-1 in all groups. The cloned human ERCC-1 gene was introduced into immortalized XP cells by DNA transfection (groups A, C, D, E and F). The presence of the integrated transfected sequences was verified on Southern blots and by selection for 2 dominant marker genes that flank the ERCC-1 gene on the transfected cos43-34 DNA. ERCC-1 failed to confer a normal UV survival and UV-induced unscheduled DNA synthesis (UDS) to transfected populations. In the case of the remaining XP complementation groups (B, G, H and I), nuclear microinjection was used to introduce an ERCC-1 cDNA construct driven by an SV40 promoter into primary fibroblasts. Coinjection of the SV40 large T gene and analysis of its expression served as a control for the injection. The ERCC-1 cDNA failed to induce increased levels of UDS in the microinjected fibroblasts. We infer from these experiments that ERCC-1 is not the mutated gene in the 9 XP complementation groups examined. From a similar type of experiments we conclude that ERCC-1 is not the defective gene in UV-sensitive Cockayne's syndrome cells.
人类DNA切除修复基因ERCC - 1可弥补互补组1的CHO突变体对紫外线（UV）和丝裂霉素C（MMC）的敏感性。我们通过分析着色性干皮病（XP）互补组A至I细胞系中的内源基因，并在导入该基因后进行修复试验，来研究ERCC - 1是否为这些细胞系中的突变基因。我们的研究表明，在9个XP组的代表性细胞系中，ERCC - 1没有缺失或发生大规模重排。此外，Northern印迹分析显示所有组中ERCC - 1转录正确。通过DNA转染（A、C、D、E和F组）将克隆的人类ERCC - 1基因导入永生化的XP细胞。通过Southern印迹以及对转染的cos43 - 34 DNA上位于ERCC - 1基因两侧的2个显性标记基因进行选择，验证了整合的转染序列的存在。ERCC - 1未能使转染群体获得正常的UV存活率和UV诱导的非定标DNA合成（UDS）。对于其余的XP互补组（B、G、H和I），使用核显微注射将由SV40启动子驱动的ERCC - 1 cDNA构建体导入原代成纤维细胞。共注射SV40大T基因并分析其表达作为注射的对照。ERCC - 1 cDNA未能在显微注射的成纤维细胞中诱导UDS水平升高。我们从这些实验推断，ERCC - 1不是所检测的9个XP互补组中的突变基因。从类似类型的实验中我们得出结论，ERCC - 1不是对UV敏感的科凯恩综合征细胞中的缺陷基因。
