Noyer-Weidner M, Reiners-Schramm L
Max-Planck-Institut für Molekulare Genetik, Berlin, Germany.
Gene. 1988 Jun 30;66(2):269-78. doi: 10.1016/0378-1119(88)90363-0.
We have developed pBR328-derived vectors which allow highly efficient positive selection of recombinant plasmids. The system is based on the rglB-coded restriction activity of Escherichia coli K-12 directed against 5-methylcytosine (5mC)-containing DNA. The vectors code for cytosine-specific, temperature-sensitive DNA methyltransferases (ts-Mtases), whose specificity elicits RglB restriction. 5mC-free vector DNA - a prerequisite to allow establishment of such plasmids in cells expressing the RglB nuclease activity - can be prepared from cultures grown at 42 degrees C. At 30 degrees C the vector plasmids are vulnerable to RglB restriction due to the expression of suicidal Mtase activity. Cloning a DNA fragment into the ts-Mtase-coding gene disrupts the lethal methylation and thus permits selection of such recombinant plasmids at 30 degrees C. The standard vector used, pBN73, contains unique recognition sites for nine restriction enzymes within the ts-Mtase-coding gene, which can be used independently or in combination for the construction of recombinant plasmids selectable by the rglB-coded activity. Plasmid pBN74, which carries the determinants for both the ts-Mtase and the RglB nuclease, contains seven unique sites within the ts-Mtase-coding gene. While selection of recombinant plasmids derived from pBN73 obligatorily requires the employment of rglB+ strains, selection of pBN74 derivatives can be performed independent of the E. coli-host genotype. It remains to be elucidated whether positive selection of pBN74-derived recombinant plasmids can also be achieved in hosts other than E. coli. Plasmids pBN73, pBN74 and the recombinants are structurally stable. Generally applicable procedures, as developed during the establishment of this vector system, are described; they allow the isolation of ts-Mtases and facilitate the cloning of genes coding for nucleases directed against 5mC-containing DNA.
我们构建了源自pBR328的载体，可实现重组质粒的高效阳性筛选。该系统基于大肠杆菌K-12的rglB编码的针对含5-甲基胞嘧啶（5mC）DNA的限制活性。这些载体编码胞嘧啶特异性、温度敏感的DNA甲基转移酶（ts-Mtases），其特异性引发RglB限制。不含5mC的载体DNA（这是在表达RglB核酸酶活性的细胞中建立此类质粒的先决条件）可从在42℃培养的培养物中制备。在30℃时，载体质粒由于自杀性Mtase活性的表达而易受RglB限制。将DNA片段克隆到ts-Mtase编码基因中会破坏致死性甲基化，从而允许在30℃下筛选此类重组质粒。使用的标准载体pBN73在ts-Mtase编码基因内包含九个限制酶的独特识别位点，可单独或组合用于构建可通过rglB编码活性筛选的重组质粒。携带ts-Mtase和RglB核酸酶决定簇的质粒pBN74在ts-Mtase编码基因内包含七个独特位点。虽然源自pBN73的重组质粒的筛选必然需要使用rglB+菌株，但pBN74衍生物的筛选可以独立于大肠杆菌宿主基因型进行。pBN74衍生的重组质粒在大肠杆菌以外的宿主中是否也能实现阳性筛选仍有待阐明。质粒pBN73、pBN74和重组体在结构上是稳定的。描述了在建立该载体系统过程中开发的通用程序；它们允许分离ts-Mtases，并促进编码针对含5mC DNA的核酸酶的基因的克隆。
