Mutagenesis by single site-specific arylamine-DNA adducts. Induction of mutations at multiple sites.
Mutagenesis by single site-specific arylamine-DNA adducts. Induction of mutations at multiple sites.
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DOI：
10.1016/s0021-9258(19)47227-x
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发表时间：
1989-11
期刊：
影响因子：
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通讯作者：
P. K. Gupta;D. Johnson;T. Reid;M. Lee;L. Romano;C. King
中科院分区：
文献类型：
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作者：
P. K. Gupta;D. Johnson;T. Reid;M. Lee;L. Romano;C. King
在M13 mp 9病毒DNA负链的6253位碱基处引入了两个相关的致癌物加合物N-（脱氧鸟苷-8-基）-2-氨基芴（AF）或N-（脱氧鸟苷-8-基）-N-乙酰基-2-氨基芴（AAF）。通过首先制备在位置6251-6257处缺失7个核苷酸的有缺口的异源双链体，然后与未修饰的七聚体或与含有AF或AAF加合物的七聚体连接来完成该位点特异性修饰的DNA的构建。将这些位点特异性修饰的模板转染到感受态野生型大肠杆菌细胞（JM 103）和auvrA菌株（SMH 12）中。突变谱通过无色斑块的表型选择（表明β-半乳糖苷酶标记酶有缺陷）和原位杂交程序（检测加合物区域中的单碱基对错配）确定。DNA测序用于表征获得的179个突变体。我们发现，这两种加合物都能够在加合物位点和加合物的局部区域诱导碱基取代突变。在移位位点也观察到特异性移码（+1G）。所有移码突变均发生在修饰的寡核苷酸的连接位点。用未修饰的寡核苷酸进行的对照实验没有显示出该位点突变的增强，表明加合物可能是这些移码的原因。这些加合物诱导的突变谱表明，诱变不仅取决于加合物的结构，但也加合物位于其中的序列和用于突变表达的宿主细胞类型。
Two related carcinogen adducts,N-(deoxyguanosin-8-yl)-2-aminofluorene (AF) orN-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (AAF), were introduced into thelacZ' gene at base position 6253 of the minus strand of M13mp9 viral DNA. The construction of this site-specifically modified DNA was accomplished by first preparing a gapped heteroduplex missing 7 nucleotides at position 6251-6257 followed by ligation with an unmodified heptamer or with a heptamer containing either an AF or AAF adduct. These site-specifically modified templates were transfected into competent wild-typeEscherichia colicells (JM103) and auvrAstrain (SMH12). The mutation spectrum was determined by phenotypic selection of colorless plaques indicating a defective β-galactosidase marker enzyme and by an in situ hybridization procedure to detect single base pair mismatches in the adduct region. DNA sequencing was used to characterize 179 of the mutants obtained. We found that both adducts were capable of inducing base substitution mutations at the adduct site and in the local region of the adduct. A specific frameshift (+1G) was also observed at a displaced site. All of the frameshift mutations occurred at the ligation site of the modified oligonucleotide. Control experiments with an unmodified oligonucleotide did not show an enhancement of mutations at this site, indicating that the adducts may have been responsible for these frameshifts. The mutations spectra induced by these adducts suggest that mutagenesis depends not only on adduct structure but also the sequence in which the adduct is located and the host cell type used for mutation expression.