Restriction systems in M. methylotrophus

• Main Author: Boyd, Alan Christopher
• Published: University of Leicester 1983
• Subjects: 572; Biochemistry
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.348761

Column chromatography of M. methylotrophus extracts revealed the presence of two restriction endonucleases, Mmel and Mmell. Mmel was active against DNA from normal E. coli strains, but Mmell could only cleave DNA from dam- strains. A general computer method was developed for determining restriction enzyme recognition sequences, and was successfully applied to the M. methylotrophus enzymes. Mmel recognizes the novel sequence 5'-TCCPuAC-3', and Mmell the sequence 5'-GATC-3'. the same as that recognized by the dam methylase of E. coli, and several other restriction enzymes, including Sau3A. The recognition sequence of Mmel was confirmed by comparing the cleavage patterns it produced with sequenced DNAs (0X174, SV40, M13) with those predicted and simulated by computer. The partially-purified preparation of Mmel used always gave incomplete digestion. Mutants lacking Mmell activity (mmeB-) were isolated, and shown to be about one thousand-fold better recipients of unmodified RP4 than wild-type M. methylotrophus. A test for dam methylation in E. coli using this mutant was devised. An unusual Tn5 mutant. CBM22. was isolated while screening for mmeB:: Tn5 mutants. This strain was shown to lack an endonuclease active against dam+ DNA which M. methylotrophus possesses, probably Mmel, but conclusive proof that the missing activity was Mmel was not obtained. The mutant allele in CBM22 was designated endA:: Tn5, after the strain was shown to be mmeB+, CBM22 appears to be a mutator strain: enhanced mutation rates to trimethoprim, tetracycline and nalidixic acid resistance were observed. The mutator phenotype was exploited to aid in the construction of endA mmeB double mutants. A quantitative comparison of the in vivo restriction profiles of endonuclease mutants was carried out using filter mating. Under this regime, endA restriction in vivo was not detectable.