CONSTRUCTION OF PLASMIDS CONTAINING A UNIQUE ACETYLAMINOFLUORENE ADDUCT LOCATED WITHIN A MUTATION HOT SPOT - A NEW PROBE FOR FRAMESHIFT MUTAGENESIS
CONSTRUCTION OF PLASMIDS CONTAINING A UNIQUE ACETYLAMINOFLUORENE ADDUCT LOCATED WITHIN A MUTATION HOT SPOT - A NEW PROBE FOR FRAMESHIFT MUTAGENESIS
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DOI：
10.1016/0022-2836(89)90259-3
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发表时间：
1989-05-20
影响因子：
5.6
通讯作者：
FUCHS, RPP
中科院分区：
文献类型：
--
作者：
KOEHL, P;BURNOUF, D;FUCHS, RPP
n -2-乙酰氨基芴（AAF）是一种强效的大鼠肝癌致癌物，主要与鸟嘌呤残基的C-8位点结合。在细菌正向突变试验中，超过90%由-AAF加合物诱导的突变是位于特定位点的移码突变：即所谓的突变热点。我们特别感兴趣的是一类-2移码突变发生在一个特定的序列，NarI序列。NarI位点GGCGCC包含三个鸟嘌呤残基，它们对-AAF取代的反应大致相同。为了进一步研究-AAF加合物在该位点诱导突变的机制，我们设计了一种新的质粒探针。在本文中，我们描述了该探针pSM14的构建及其有效性，该探针为检测NarI位点内的移码突变提供了一种简单的表型测试。还描述了在NarI位点的三个位置中每个位置都具有单个-AAF加合物的质粒的结构和特征。所使用的构建策略涉及将NarI位点上含有单个加合物的寡核苷酸连接到间隙双工pSM14质粒中。通过连接在5‘和3’端成功整合寡核苷酸的质粒通过CsCl梯度离心纯化。这些结构已用于单加合物突变研究。
N-2-acetylaminofluorene (AAF), a potent rat liver carcinogen, binds primarily to the C-8 position of guanine residues. In a bacterial forward mutation assay, more than 90% of the mutations induced by -AAF adducts are frameshift mutations located at specific sites: the so-called mutation hot spots. We are particularly interested in a class of -2 frameshift mutations occurring within a specific sequence, the NarI sequence. The NarI site, GGCGCC, contains three guanine residues that are approximately equally reactive toward -AAF substitution. To study further the mechanism by which mutations are induced by -AAF adducts at this site, we designed a new plasmid probe. In this paper we describe the construction and the effectiveness of this probe, pSM14, which provides a simple phenotypic test for detecting frameshift mutations within the NarI site. The construction and the characterization of plasmids with a single -AAF adduct in each of the three positions of the NarI site are also described. The strategy of construction that was used involves the ligation of oligonucleotides containing a single adduct in a NarI site into a gapped-duplex pSM14 plasmid. Plasmids that have successfully integrated the oligonucleotides by ligation at both the 5'' and the 3'' ends were purified by centrifugation on CsCl gradients. These constructs have been used in single adduct mutation studies.