Construction of a functional cDNA clone of the hamster ERCC2 DNA repair and transcription gene.
Construction of a functional cDNA clone of the hamster ERCC2 DNA repair and transcription gene.
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构建仓鼠 ERCC2 DNA 修复和转录基因的功能 cDNA 克隆。
DOI：
10.1007/bf02369437
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Weber,CA
中科院分区：
文献类型：
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作者：
Kadkhodayan,S;Salazar,EP;Lamerdin,JE;Weber,CA
完整的仓鼠ERCC 2cDNA构建在质粒载体中，从三个重叠的逆转录/聚合酶链反应扩增片段的克隆中，使用重叠区域内的独特限制性内切酶识别位点。然后将此完整的cDNA插入片段克隆到哺乳动物表达载体pcD 2 E中，并通过赋予ERCC 2突变体CHO细胞系UV 5 UV抗性的能力来测试其功能。采用定点突变法分别引入G347→A和G1844→A突变，导致先前在UV 5和UVL-13（也是ERCC 2突变CHO细胞系）中鉴定的Cys 116→Tyr和Gly 615 →Glu突变。116 Tyr和615 Glu质粒均未能分别赋予UV 5或UVL-13细胞UV抗性，表明鉴定的变化确实是UV 5和UVL-13中的致病突变。
The complete hamsterERCC2cDNA was constructed in a plasmid vector from clones of three overlapping reverse transcribed/polymerase chain reaction amplified fragments using unique restriction enzyme recognition sites within the regions of overlap. This complete cDNA insert was then cloned into a mammalian expression vector, pcD2E, and tested for function by the ability to confer UV resistance to theERCC2mutant CHO cell line UV5. Site-specific mutagenesis was used to introduce the G347→A and G1844→A changes resulting in the Cys 116→Tyr and Gly615→Glu mutations previously identified in UV5 and UVL-13 (also anERCC2mutant CHO cell line), respectively. The 116Tyrand 615Gluplasmids each failed to confer UV resistance to UV5 or UVL-13 cells, respectively, demonstrating that the changes identified are indeed the causative mutations in UV5 and UVL-13.