Base changes in the replication of DNA induced by nucleotide analogs and the use for in vitro mutagenesis

核苷酸类似物诱导的DNA复制的碱基变化及其在体外诱变中的应用

• 批准号: 61480430
• 负责人: HAYATSU Hikoya
• 金额: $ 3.65万
• 依托单位: Okayama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1986
• 资助国家: 日本
• 起止时间: 1986 至 1987
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61480430/
• 关键词: N^4-Aminocytidine; transition mutation; replication error; M13mp2,FM3A cell; V79 cell; トランジション変異; 複製; 【N^4】ーアミノシチジン; 【N^4】ーアミノデオキシシチジン; 変異原性; 複製エラー; ONAシークエンス; 金属イオン; N^4-Aminocytidine; transition mutation; replication error; M13mp2,FM3A cell; V79 cell; トランジション変異; 複製; 【N^4】ーアミノシチジン; 【N^4】ーアミノデオキシシチジン; 変異原性; 複製エラー; ONAシークエンス; 金属イオン

The moecular mechanism of N^4-aminocytidine-mediated mutagenesis was investigated. A summary of findings achieved is as follows. (1) A study of in vitro DNA synthesis using E. coil DNA polymerase I large fragment as the enzyme showed that N^4-aminodeoxycytidine 5'-triphosphate (dC^<am>TP) can serve as a substrate for the enzyme and can be incorporated int onewly formed DNA. The efficiency of incorporation was about 50% of the natural substrate dCTP, and this nucleotide analog can be used as a substitute for dCTP and dTTP. (2) Use of dC^<am>TP in the M13mp2 DNA synthesizing system, followed by transfection of the resulting DNA to E. coli allowed production of mutant phages. More than 100 mutant clones were spearated, the DNA was isolated and their nucleotide sequences determined. The results indicated that all possible transitions, A to G, G to A, C to T and T to C, and only these transitions, have occurred. This result is consistent with a mechanism in which tautomeric shift in N^4-aminocytosine between the amino and the imino forms is the cause of the mutation. (3) Mutations were induced in Drosophila when the larve were fed with N^4-aminocytidine. (4) An improved method to synthesize N^4-aminocytidine was established.

研究了N^4-氨基细胞丁胺介导的诱变的含量机制。所达到的发现的摘要如下。 （1）使用E. coil DNA聚合酶I大片段对体外DNA合成的研究表明，N^4-氨基氧基胞苷5'-三磷酸（DC^<AM> tp）可以用作酶的底物，并且可以掺入酶促的intwly形成的DNA。掺入的效率约为天然底物DCTP的50％，该核苷酸类似物可用作DCTP和DTTP的替代品。 （2）在M13MP2 DNA合成系统中使用DC^<AM> TP，然后将所得DNA转染至大肠杆菌允许产生突变噬菌体。将超过100个突变的克隆螺旋性，分离出DNA并确定其核苷酸序列。结果表明，所有可能的过渡，即g，g至a，c至t和t到c，并且仅发生了这些过渡。该结果与一种机制一致，在这种机制中，氨基和伊米诺形式之间n^4-氨基胞菌素中的互变异转移是突变的原因。 （3）当幼虫用N^4-氨基丁胺喂食时，在果蝇中诱导了突变。 （4）建立了一种改进的合成N^4-氨基丁胺的方法。