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一种利用杀伤性质粒快速高效克隆II型限制修饰系统基因的方法。

A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid.

作者信息

Mruk Iwona, Kaczorowski Tadeusz

机构信息

Department of Microbiology, University of Gdansk, Kladki 24, Gdansk, Poland.

出版信息

Appl Environ Microbiol. 2007 Jul;73(13):4286-93. doi: 10.1128/AEM.00119-07. Epub 2007 Apr 27.

DOI:10.1128/AEM.00119-07
PMID:17468281
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1932789/
Abstract

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.

摘要

我们提出了一种基于使用致死质粒（pKILLER）克隆限制-修饰（R-M）系统的方法。该质粒携带一个与目标R-M系统具有相同DNA特异性的限制性内切酶功能基因。第一步是制备具有代表性的质粒携带基因组文库的标准步骤。然后用杀伤质粒转化该文库。唯一存活的细菌是那些携带指定保护性DNA甲基转移酶基因的细菌。从概念上讲，这种体内选择方法类似于早期的方法，即在体外通过用合适的限制性内切酶消化来选择质粒文库，但它比那些方法效率高得多。该新方法已成功用于克隆两个R-M系统，来自嗜热脂肪芽孢杆菌14P的BstZ1II和来自柠檬酸杆菌属菌株RFL231的Csp231I，它们都与原型HindIII R-M系统等特异性。

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REBASE--enzymes and genes for DNA restriction and modification.REBASE——DNA 限制与修饰的酶及基因
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Genetic organization and molecular analysis of the EcoVIII restriction-modification system of Escherichia coli E1585-68 and its comparison with isospecific homologs.大肠杆菌E1585-68的EcoVIII限制修饰系统的遗传组织与分子分析及其与同特异性同源物的比较。
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C.EcoO109I, a regulatory protein for production of EcoO109I restriction endonuclease, specifically binds to and bends DNA upstream of its translational start site.C.EcoO109I是一种用于产生EcoO109I限制内切酶的调控蛋白，它特异性地结合并弯曲其翻译起始位点上游的DNA。
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