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首页> 外文期刊>Nucleic Acids Research >A VIRAL GENOME CONTAINING AN UNSTABLE AFLATOXIN B-1-N7-GUANINE DNA ADDUCT SITUATED AT A UNIQUE SITE
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A VIRAL GENOME CONTAINING AN UNSTABLE AFLATOXIN B-1-N7-GUANINE DNA ADDUCT SITUATED AT A UNIQUE SITE

机译：包含不稳定黄曲霉毒素B-1-N7-鸟嘌呤DNA的病毒基因组在一个独特的位点上发生

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摘要

A problem that has hindered the study of the biological properties of certain DNA adducts, such as those that form at the N7 atoms of purines, is their extreme chemical lability, Conditions are described for the construction of a single-stranded genome containing the chemically and thermally labile 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B-1 (AFB(1)-N7-Gua) adduct, the major DNA adduct of the potent liver carcinogen aflatoxin B-1 (AFB(1)). A 13mer oligonucleotide, d(CCTCTTCGAACTC), was allowed to react with the exo-8,9-epoxide of AFB(1) to form an oligonucleotide containing a single AFB(1)-N7-Gua (at the underlined guanine), This modified 13mer was 5'-phosphorylated and ligated into a gap in an M13 bacteriophage genome generated by annealing a 53mer uracil-containing scaffold to M13mp7L2 linearized by EcoRI, Following ligation, the scaffold was enzymatically removed with uracil DNA glycosylase and exonuclease III, The entire genome construction was complete within 3 h and was carried out at 16 degrees C, pH 6.6, conditions determined to be optimal for AFB(1)-N7-Gua stability, Characterization procedures indicated that the AFB(1)-N7-Gua genome was similar to 95% pure with a small (5%) contamination by unmodified genome, This construction scheme should be applicable to other chemically or thermally unstable DNA adducts.
机译：阻碍某些DNA加合物（例如在嘌呤的N7原子上形成的那些）的生物学特性研究的一个问题是其极端的化学不稳定性。描述了构建包含化学和化学构象的单链基因组的条件。热不稳定的8,9-二氢-8-（N7-胍基）-9-羟基黄曲霉毒素B-1（AFB（1）-N7-Gua）加合物，强肝致癌物黄曲霉毒素B-1（AFB（ 1））。使13mer寡核苷酸d（CCTCTTCGAACTC）与AFB（1）的exo-8,9-环氧反应形成包含单个AFB（1）-N7-Gua的寡核苷酸（带下划线的鸟嘌呤）。修饰的13mer被5'-磷酸化并连接到M13噬菌体基因组的缺口中，该基因是通过将含53mer尿嘧啶的支架退火至EcoRI线性化的M13mp7L2而产生的。基因组的构建在3小时内完成，并在16摄氏度，pH 6.6的条件下进行，确定的条件对于AFB（1）-N7-Gua稳定性最佳。表征过程表明，AFB（1）-N7-Gua基因组是类似于95％的纯度，未修饰的基因组造成的污染很小（5％），该构建方案应适用于其他化学或热不稳定的DNA加合物。

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