SINGLE-STRANDED SHUTTLE PHAGEMID FOR MUTAGENESIS STUDIES IN MAMMALIAN-CELLS - 8-OXOGUANINE IN DNA INDUCES TARGETED G.C -] T.A TRANSVERSIONS IN SIMIAN KIDNEY-CELLS
SINGLE-STRANDED SHUTTLE PHAGEMID FOR MUTAGENESIS STUDIES IN MAMMALIAN-CELLS - 8-OXOGUANINE IN DNA INDUCES TARGETED G.C -] T.A TRANSVERSIONS IN SIMIAN KIDNEY-CELLS
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DOI：
10.1073/pnas.90.3.1122
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发表时间：
1993-02-01
影响因子：
11.1
通讯作者：
MORIYA, M
中科院分区：
文献类型：
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作者：
MORIYA, M
为了研究哺乳动物细胞中的translesional事件，已经开发了单链穿梭载体。该载体被设计为允许在新霉素抗性基因及其启动子之间位点特异性引入限定的DNA损伤。通过测定引入经修饰的载体后分别回收的新霉素和氨苄青霉素抗性菌落的数量，建立了猴肾细胞（COS）和大肠杆菌中translesional合成的效应。通过分析对应于损伤位点的区域中的子代噬菌粒DNA的核苷酸序列来评价跨损伤合成的保真度。该实验系统，能够检测致突变性和非致突变性的事件在和邻近的病变部位，被用来建立一个单一的8-氧代鸟嘌呤残基在DNA中的致突变潜力。当含有损伤的单链DNA在COS细胞或E.杆菌观察到的主要突变（>78%）是靶向病变部位的G -> T颠换。COS细胞的突变频率为2.5-4.8%，E.杆菌它的结论是，单链穿梭载体，结合位点特异性的方法，可用于调查translesional哺乳动物细胞和细菌中的事件。
A single-stranded shuttle vector has been developed for the purpose of investigating translesional events in mammalian cells. The vector is designed to permit site-specific introduction of defined DNA lesions between a gene for neomycin resistance and its promoter. Efficiencies of translesional synthesis in simian kidney cells (COS) and Escherichia coli are established by determining the number of neomycin- and ampicillin-resistant colonies recovered, respectively, after introduction of a modified vector. Fidelity of translesional synthesis is evaluated by analyzing the nucleotide sequence of progeny phagemid DNA in the region corresponding to the lesion site. This experimental system, capable of detecting mutagenic and nonmutagenic events at and adjacent to the lesion site, was used to establish the mutagenic potential of a single 8-oxoguanine residue in DNA. This modified base, produced by attack of reactive oxygen species on cellular DNA, did not cause a decrease in the number of transformants when single-stranded DNA containing the lesion replicated in COS cells or E. coli. The predominant mutations observed (>78%) were G --> T transversions targeted to the site of the lesion. The mutation frequencies for this event were 2.5-4.8% in COS cells and 1.8% in E. coli. It is concluded that a single-stranded shuttle vector, utilized in conjunction with a site-specific approach, can be used to investigate translesional events in mammalian cells and in bacteria.