THE ENDO-BLUE METHOD FOR DIRECT CLONING OF RESTRICTION-ENDONUCLEASE GENES IN ESCHERICHIA-COLI

A new E.coli strain has been constructed that contains the dinD1::LacZ (+) fusion and is deficient in methylation-dependent restriction syste ms (McrA(-), McrBC(-), Mrr(-)). This strain has been used to clone res triction endonuclease genes directly into E.coii. When E.coli cells ar e not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response. The SOS-induced cells form blue colonies on indicator plates containing X-gal. Using this method the genes coding for the thermostable restriction enzymes Taql (5'TCGA3') and Tth111l (5'GACNNNGTC3') have been successfully clo ned in E.coli. The new strain wilt be useful to clone other genes invo lved in DNA metabolism.