A novel bacterial reversion and forward mutation assay based on green fluorescent protein
A novel bacterial reversion and forward mutation assay based on green fluorescent protein
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DOI：
10.1016/s1383-5718(98)00043-6
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发表时间：
1998-05-11
影响因子：
1.9
通讯作者：
Casey, WM
中科院分区：
文献类型：
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作者：
Cariello, NF;Narayanan, S;Casey, WM
我们报告了首次使用绿色荧光蛋白（GFP）进行突变检测。我们构建了一个基于质粒的细菌系统，其中突变细胞发出荧光，而非突变细胞不发出荧光。使用简单的手持式紫外灯监测荧光；不需要额外的辅助因子或操作。为了开发逆转系统，我们在 GFP 编码区引入了 fl DNA 移码突变，所得蛋白质在大肠杆菌中不发出荧光。用 ICR-191 处理含有 +1 移码载体的细菌会产生荧光集落，表明已发生向野生型序列的回复。使用定点诱变将额外的胞嘧啶插入到质粒 pBAD-GFPuv 中 GFP 编码区的天然 CCC 序列中，将序列扩展为 CCCC。当用 ICR-191（一种主要诱导移码突变的试剂）处理细菌时，观察到荧光菌落与剂量相关的增加。测试的ICR-191最高剂量为16μg/ml，在重复实验中产生16×10(-5)和8.8×10(-5)的突变部分。逆转系统对 MNNG 没有反应，MNNG 是一种主要产生单碱基取代的试剂。为了开发正向系统，我们使用了阿拉伯糖 P-BAD 启动子控制下的 GFP；在没有阿拉伯糖的情况下，GFP 表达受到抑制，并且没有观察到荧光集落。当用 MNNG 或 ENNG 处理细胞时，观察到荧光集落呈剂量依赖性增加，表明阿拉伯糖控制区域发生了突变，从而解除了启动子的抑制。在重复实验中，用100μg/ml MNNG处理细菌诱导的突变分数高达82×10(-5)和30×10(-5)。在单次实验中用150μg/ml ENNG处理细菌诱导了2.1×10(-5)的突变分数。 (C) 1998 Elsevier Science B.V. 保留所有权利。
We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a fl DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 mu g/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose P-BAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 mu g/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 30 x 10(-5) in duplicate experiments. Treating bacteria with 150 mu g/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment. (C) 1998 Elsevier Science B.V. All rights reserved.