DEVELOPMENT OF A NEW EASY COMPLEMENTATION ASSAY FOR DNA-REPAIR DEFICIENT HUMAN SYNDROMES USING CLONED REPAIR GENES
DEVELOPMENT OF A NEW EASY COMPLEMENTATION ASSAY FOR DNA-REPAIR DEFICIENT HUMAN SYNDROMES USING CLONED REPAIR GENES
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DOI：
10.1093/carcin/16.5.1003
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发表时间：
1995-05-01
期刊：
影响因子：
4.7
通讯作者：
MEZZINA, M
中科院分区：
文献类型：
--
作者：
CARREAU, M;EVENO, E;MEZZINA, M
到目前为止，通过体细胞融合试验和最近通过显微注射克隆DNA修复基因，已将核苷酸切除修复（NER）缺陷的人类细胞分配到遗传互补组。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载体共转染，用于快速测定ner缺陷型着色性干皮病（XP）， Cockayne综合征（CS）和光敏性毛碘营养不良（TTD）人细胞的互补组。报告基因的表达，无论是氯霉素乙酰转移酶（CAT）还是荧光素酶，都反映了通过引入适当的修复基因而恢复的DNA修复能力。所有具有遗传特征的XP， CS和TTD/XP- d细胞均未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所达到的水平。XP17VI细胞与XPC基因共转染，XP18VI和XP19VI细胞与XPA基因共转染后，两种报告酶活性均有选择性恢复。使用这种方法，我们将从出现典型XP临床症状的患者获得的三个新的ner缺陷人类细胞分配到XP A组（XP18VI和XP19VI）和XP C组（XP17VI）。因此，该技术增加了现有方法的范围，可用于确定新的NER缺陷患者的补充组，与体细胞融合试验或显微注射程序不同，该技术具有简单，快速和廉价的优点。
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 defeciency 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.