Abstract

We describe the isolation of a strain of Escherichia coli bearing a deletion/insertion (i.e., a substitution mutation) in the dam gene (dam-16). The mutagenesis protocol used should be applicable to any cloned non-essential gene of E. coli. The substitution mutation confers resistance to kanamycin and can easily be transferred to other strains by standard genetic techniques. The amount of Dam methyltransferase (MTase) in dam-16 strains as determined either in vitro or in vivo is below the level of detection. We conclude that the Dam MTase is not required for viability of E. coli

Author Keywords: Methyltransferases; genes; bacteria; recombinant DNA; methylation; conjugation

Abbreviations: aa, amino acids; Ap, ampicillin; 2-AP, 2-amino-purine; bp, base pair(s); Cm, chloramphenicol; CGSC, E. coliGenetic Stock Center, Department of Biology, Yale University, New Haven CT 06510 USA; dam, gene coding for Dam; Dam, DNA adenine methyltransferase; Del or Δ, deletion; Hfr, highfrequency chromosome transfer; kb, kilobase(s) or 1000 bp; Km, kanamycin; m⁶A, N⁶-methyladenine; MNNG, N-methyl-N′nitro-JV-nitrosoguanidine; MTase, methyltransferase; Mud, defective Mu phage; Oc, ochre; oriC, origin of E. coli chromosome replication; PO, point of origin of Hfr transfer; ^R, resistance; ^s, sensitivity; SDS, sodium dodecyl sulfate; Sm, streptomycin; SSC, 0.15 M NaCl,0.0015 M Na₃· citrate,pH 7.6; Tn, transposon; ::, novel joint (fusion); [], designates plasmidcarrier state

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