在DNA合成过程中通过掺入O6-甲基鸟嘌呤对噬菌体T7进行体外诱变。
Mutagenesis of bacteriophage T7 in vitro by incorporation of O6-methylguanine during DNA synthesis.
作者信息
Dodson L A, Foote R S, Mitra S, Masker W E
出版信息
Proc Natl Acad Sci U S A. 1982 Dec;79(23):7440-4. doi: 10.1073/pnas.79.23.7440.
Abstract
An in vitro system in which bacteriophage T7 DNA is replicated and efficiently packaged into procapsids to form viable phage has been used to examine mutagenesis. The fidelity of replication was assayed both by measuring reversion of an amber mutation in an essential gene and by generation of temperature-sensitive mutants among the phage produced in vitro. Under standard reaction conditions, the fidelity of DNA replication is about equal to that normally found in vivo. However, when O6-methyldeoxyguanosine triphosphate is included in the reaction, O6-methylguanine is incorporated into newly synthesized DNA and the mutation frequencies increase 10- to 70-fold over the control. These experiments demonstrate in vitro mutagenesis with the T7 DNA replication-packaging system and provide more direct evidence for the premutagenic role of O6-methylguanine.
摘要
一种体外系统已被用于研究诱变作用，在该系统中，噬菌体T7 DNA进行复制并高效包装进原衣壳以形成有活力的噬菌体。通过测量一个必需基因中琥珀突变的回复突变以及通过在体外产生的噬菌体中生成温度敏感突变体来检测复制的保真度。在标准反应条件下，DNA复制的保真度与体内通常发现的保真度大致相当。然而，当反应中包含O6-甲基脱氧鸟苷三磷酸时，O6-甲基鸟嘌呤会掺入新合成的DNA中，并且突变频率比对照增加10至70倍。这些实验证明了利用T7 DNA复制-包装系统进行的体外诱变，并为O6-甲基鸟嘌呤的诱变前作用提供了更直接的证据。
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