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首页> 外文期刊>Journal of Virology >Selective cloning of a DNA single-strand initiation determinant from phi X174 replicative-form DNA.
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Selective cloning of a DNA single-strand initiation determinant from phi X174 replicative-form DNA.

机译：从PHI X174复制形式DNA选择性克隆DNA单链开始判定素。

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摘要

An M13 phage deletion mutant, M13 delta E101, developed as a vector for selecting DNA sequences that direct DNA strand initiation on a single-stranded template, has been used for cloning restriction enzyme digests of phi X174 replicative-form DNA. Initiation determinants, detected on the basis of clear-plaque formation by the chimeric phage, were found only in restriction fragments containing the unique effector site in phi X174 DNA for the Escherichia coli protein n' dATPase (ATPase). Furthermore, these sequences were functional only when cloned in the orientation in which the phi X174 viral strand was joined to the M13 viral strand. A 181-nucleotide viral strand fragment containing this initiation determinant confers a phi X174-type complementary-strand replication mechanism on M13 chimeras. The chimeric phage is converted to the parental replicative form in vivo by a mechanism resistant to rifampin, a specific inhibitor of the normal RNA polymerase-dependent mechanism of M13. In vitro, the chimeric single-stranded DNA promotes the assembly of a functional multiprotein priming complex, or primosome, identical to that utilized by intact phi X174 viral strand DNA. Chimeric phage containing the sequence complementary to the 181-nucleotide viral strand sequence shows no initiation capability, either in vivo or in vitro.
机译：M13噬菌体缺失突变体M13 Delta E101作为用于选择直接DNA链在单链模板上的DNA序列的载体的载体开发，已用于克隆PHI X174重复形式DNA的限制酶消化。在嵌合噬菌体的透明噬菌体的基础上检测到的起始决定簇仅在含有PHI X174 DNA中的唯一效应部位的限制片段中，用于大肠杆菌蛋白N'DATP酶（ATP酶）。此外，这些序列仅在克隆到其中PHI X174病毒链与M13病毒链中的取向克隆时是功能性的。含有该引发的181-核苷酸病毒链片段在M13嵌合体上赋予PHI X174型互补股线复制机制。嵌合噬菌体通过对利福平的机制转化为父母的复制形式，其抗利福平的机制，M13的正常RNA聚合酶依赖性机制的特异性抑制剂。在体外，嵌合单链DNA促进功能性多蛋白引发复合物的组装，或主要的，与完整的PHI X174病毒链DNA所使用的功能。含有与181-核苷酸病毒链序列互补的序列的嵌合噬菌体显示在体内或体外中没有引发能力。

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