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大肠杆菌细胞提取物中mini-R1质粒DNA复制的起源与方向

Origin and direction of mini-R1 plasmid DNA replication in cell extracts of Escherichia coli.

作者信息

Diaz R, Staudenbauer W L

出版信息

J Bacteriol. 1982 Jun;150(3):1077-84. doi: 10.1128/jb.150.3.1077-1084.1982.

DOI:10.1128/jb.150.3.1077-1084.1982
PMID:6281234
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC216325/
Abstract

Replication of the mini-R1 plasmids pKN177 and pKN182 can be carried out efficiently in cell extracts of Escherichia coli and depends on both transcription and translation. Heteroduplex and restriction analyses indicate that both plasmids are derived from the R1 copy mutant pKN104 by Is1-mediated recombination events without involving structural alterations in the replication region. To ascertain whether the in vitro replication of these miniplasmids corresponds to R1 replication in vivo, the origin and direction of replication were analyzed by electron microscopy of replicative intermediates. It was found that replication starts at a unique origin located within the RepA region at R1 coordinate at 82.4 kilobases and proceeds unidirectionally toward the IS/b sequence. The specification of the origin and the direction of in vitro replication are therefore in full agreement with the pattern observed previously for the in vivo replication of the closely related plasmids R100 and R6-5. This agreement provides additional evidence that R1 DNA synthesis in vitro employs the same replication mechanism as it does in vivo.

摘要

微型R1质粒pKN177和pKN182的复制可在大肠杆菌的细胞提取物中高效进行，且依赖于转录和翻译。异源双链和限制性分析表明，这两种质粒均通过Is1介导的重组事件从R1复制突变体pKN104衍生而来，而不涉及复制区域的结构改变。为确定这些微型质粒的体外复制是否与R1在体内的复制相对应，通过对复制中间体进行电子显微镜分析来研究复制起点和方向。结果发现，复制起始于位于R1坐标82.4千碱基处RepA区域内的一个独特起点，并单向朝着IS/b序列进行。因此，体外复制起点和方向的确定与先前观察到的密切相关质粒R100和R6-5体内复制模式完全一致。这种一致性提供了额外证据，表明R1 DNA在体外的合成采用了与体内相同的复制机制。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/747b/216325/94c227aa2274/jbacter00259-0093-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/747b/216325/067715527dc6/jbacter00259-0091-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/747b/216325/94c227aa2274/jbacter00259-0093-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/747b/216325/067715527dc6/jbacter00259-0091-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/747b/216325/94c227aa2274/jbacter00259-0093-a.jpg

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引用本文的文献

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本文引用的文献

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Plasmid. 1981 Jan;5(1):53-75. doi: 10.1016/0147-619x(81)90077-9.
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Runaway replication of plasmid R1 is not caused by loss of replication inhibitor activity of gene cop.质粒R1的失控复制并非由基因cop的复制抑制活性丧失所致。
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Proc Natl Acad Sci U S A. 1983 Nov;80(22):6814-8. doi: 10.1073/pnas.80.22.6814.
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Regulation of plasmid replication.质粒复制的调控
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Initiation of plasmid R1 replication in vitro is independent of transcription by host RNA polymerase.质粒R1在体外的复制起始不依赖于宿主RNA聚合酶的转录。
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The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA.宿主复制蛋白和特定起始序列参与小质粒R1 DNA的体外复制。
Nucleic Acids Res. 1986 Jun 25;14(12):4865-79. doi: 10.1093/nar/14.12.4865.
8
Replication of the streptococcal plasmid pMV158 and derivatives in cell-free extracts of Escherichia coli.链球菌质粒pMV158及其衍生物在大肠杆菌无细胞提取物中的复制
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9
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