Ellison A R, Nouspikel T, Jaspers N G, Clarkson S G, Gruenert D C
Laboratory Medicine and Stromatology, University of California San Francisco, San Francisco, California, 94143, USA.
Exp Cell Res. 1998 Aug 25;243(1):22-8. doi: 10.1006/excr.1998.4147.
Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40). The new cell lines, summation operatorXPCS1LVo- and summation operatorXPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The summation operatorXPCS1LVo- and summation operatorXPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene. Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the summation operatorXPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was evident in the XPG RNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-type XPG cDNA was used to correct UV-induced damage in a beta-galactosidase reporter gene.
着色性干皮病（XP）和科凯恩综合征（CS）是人类遗传性疾病，在细胞水平上的特征是无法修复某些类型的DNA损伤。通常，XP和CS在临床和遗传上是不同的。然而，在罕见情况下，已证明CS患者在先前与XP发生相关的基因中存在突变。XP和CS之间的联系一直难以研究，因为从XP/CS患者中建立的永久细胞系很少。为了生成永久细胞系，用复制起点缺陷型猿猴病毒40（SV40）转化了两名表现出与CS相关特征且属于XP互补组G的患者的原代成纤维细胞培养物。对新的细胞系，即∑XPCS1LVo-和∑XPCS1ROo-，进行了表型和基因型特征分析，以验证原代细胞的特性在转化后得以保留。这些细胞系在培养中生长迅速，通过免疫染色显示表达SV40 T抗原。∑XPCS1LVo-和∑XPCS1ROo-细胞系对紫外线高度敏感，并且重新激活紫外线照射的报告基因的能力受损。通过聚合酶链反应（PCR）扩增和限制性酶切，显示∑XPCS1ROo-细胞在XPG位置2972处保留纯合T缺失。这种突变也表征了亲代原代细胞，并且在XPG RNA中很明显。最后，为了表征这些细胞系中的XPG DNA修复缺陷，使用含有野生型XPG cDNA的附加型表达载体来纠正β-半乳糖苷酶报告基因中的紫外线诱导损伤。
