Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene.
Analysis at the sequence level of mutations induced by the ultimate carcinogen N-acetoxy-N-2-acetylaminofluorene.
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DOI：
10.1289/ehp.8349135
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发表时间：
1983-03
影响因子：
10.4
通讯作者：
Daune MP
中科院分区：
文献类型：
--
作者：
Fuchs RP;Schwartz N;Daune MP
最终致癌物与DNA碱基或磷酸基团的共价结合产生突变前损伤，该损伤在体内通过修复、复制和重组酶进行处理，并最终可能转化为突变。感兴趣的方式，一个初始的premutational事件转化为一个稳定的遗传突变，我们已经测序了稳定的突变基因，已形成共价加合物在体外与N-乙酰氧基-N-2-乙酰氨基芴（N-AcO-AAF，模型的最终代谢产物的大鼠肝脏致癌物2-乙酰氨基芴，AAF）。体内研究表明，AAF及其衍生物在细菌和真核生物系统中都具有致突变性。N-AcO-AAF在体外与DNA反应，主要形成鸟嘌呤加合物，N-2-（脱氧鸟苷-8-基）-乙酰氨基芴（80%）和至少三种次要加合物。本课题组的研究表明，N-AcO-AAF与DNA的结合导致了C-8加合物周围DNA螺旋的局部扭曲（插入-变性模型）。我们在这里描述的正向突变诱导的pBR 322的四环素耐药基因的化学反应，直接到一个小的限制性片段（BamHI-SalI）内的耐药基因的分析。针对氨苄青霉素（Ap）抗性和四环素（Tc）敏感性选择突变体。分析这些突变体的质粒DNA中已定向AAF结合的片段中的序列变化。我们在这里表明，突变主要是涉及GC碱基对的移码，并且某些碱基对（热点）以高频率受到影响。
The covalent binding of an ultimate carcinogen to the DNA bases or phosphate groups creates a premutational lesion that in vivo is processed by the repair, replication and recombination enzymes, and eventually may be converted into a mutation. Being interested in the way that an initial premutational event is converted into a stable heritable mutation, we have sequenced stable mutations in a gene that has formed covalent adducts in vitro with N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF, a model for the ultimate metabolite of the rat liver carcinogen 2-acetylaminofluorene, AAF). In vivo studies have shown the mutagenicity of AAF and its derivatives in both bacterial and eukaryotic systems. N-AcO-AAF reacts in vitro with DNA leading mainly to the formation of a guanine adduct, N-2-(deoxyguanosin-8-yl)-acetylaminofluorene (80%) and to at least three minor adducts. Studies by our group showed that binding of N-AcO-AAF to DNA resulted in a local distortion of the DNA helix around the C-8 adduct (the insertion-denaturation model). We describe here the analysis of forward mutations induced in the tetracycline-resistance gene of pBR322 by directing the chemical reaction of the carcinogen to a small restriction fragment (BamHI-SalI) inside the antibiotic-resistance gene. Mutants are selected for ampicillin (Ap) resistance and tetracycline (Tc) sensitivity. The plasmid DNA of such mutants was analyzed for sequence changes in the fragment where the AAF binding had been directed. We show here that the mutations are mainly frameshifts involving GC base pairs and that certain base pairs (hotspots) are affected at high frequencies.