Carreau M, Eveno E, Quilliet X, Chevalier-Lagente O, Benoit A, Tanganelli B, Stefanini M, Vermeulen W, Hoeijmakers J H, Sarasin A
Laboratory of Molecular Genetics, UPR 42 CNRS IFC1 Institu de Recherches sur le Cancer, Villejuif, France.
Carcinogenesis. 1995 May;16(5):1003-9. doi: 10.1093/carcin/16.5.1003.
Nucleotide excision repair (NER)-deficient human cells have been assigned so far to a genetic complementation group by a somatic cell fusion assay and, more recently, by microinjection of cloned DNA repair genes. We describe a new technique, based on the host cell reactivation assay, for the rapid determination of the complementation group of NER-deficient xeroderma pigmentosum (XP), Cockayne's syndrome (CS) and photosensitive trichothiodystrophy (TTD) human cells by cotransfection of a UV-irradiated reporter plasmid with a second vector containing a cloned repair gene. Expression of the reporter gene, either chloramphenicol acetyltransferase (CAT) or luciferase, reflects the DNA repair ability restored by the introduction of the appropriate repair gene. All genetically characterized XP, CS and TTD/XP-D cells tested failed to express the UV-irradiated reporter gene, this reflecting their NER deficiency whereas cotransfection with the repair plasmid expressing a gene specific for the given complementation group increased the enzyme activity to the level reached by normal cells. Selective recovery of both reporter enzyme activities was observed after cotransfection with the XPC gene for the XP17VI cells and with the XPA gene for both XP18VI and XP19VI cells. Using this method, we assigned three new NER-deficient human cells obtained from patients presenting clinical symptoms described as classical XP to either XP group A (XP18VI and XP19VI) and XP group C (XP17VI). Therefore, this technique increases the range of methods now available to determine the complementation group of new NER deficient patients with the advantage, unlike the somatic cell fusion assay or the microinjection procedure, of being simple, rapid, and inexpensive.
到目前为止，通过体细胞融合试验，以及最近通过显微注射克隆的DNA修复基因，已将核苷酸切除修复（NER）缺陷的人类细胞归入一个遗传互补组。我们描述了一种基于宿主细胞再激活试验的新技术，通过将紫外线照射的报告质粒与含有克隆修复基因的第二个载体共转染，快速确定NER缺陷的着色性干皮病（XP）、科凯恩综合征（CS）和光敏性毛发硫营养不良（TTD）人类细胞的互补组。报告基因氯霉素乙酰转移酶（CAT）或荧光素酶的表达反映了通过引入适当的修复基因恢复的DNA修复能力。所有经过基因特征分析的XP、CS和TTD/XP-D细胞均未能表达紫外线照射的报告基因，这反映了它们的NER缺陷，而与表达特定互补组基因的修复质粒共转染可将酶活性提高到正常细胞达到的水平。在用XPC基因转染XP17VI细胞以及用XPA基因转染XP18VI和XP19VI细胞后，观察到两种报告酶活性的选择性恢复。使用这种方法，我们将从表现出经典XP临床症状的患者身上获得的三个新的NER缺陷人类细胞归入XP A组（XP18VI和XP19VI）和XP C组（XP17VI）。因此，与体细胞融合试验或显微注射程序不同，该技术具有简单、快速且廉价的优点，增加了目前可用于确定新的NER缺陷患者互补组的方法范围。
