A rapid and efficient method for cloning genes of type II restriction-modification systems by use of a killer plasmid.
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.
Mruk
Iwona
I
Department of Microbiology, University of Gdansk, Kladki 24, Gdansk, Poland.
Kaczorowski
Tadeusz
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002954:Citrobacter / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D015280:DNA Restriction-Modification Enzymes / Q000145:classification* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics
D017931:DNA Primers; D015280:DNA Restriction-Modification Enzymes; D004269:DNA, Bacterial; C090528:DNA modification methylase HindIII; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)