Insertion Mutations Affecting Transposition of Tn3 and Replication of a ColE1 Derivative

active DNA molecules employs random chemical modification of individual base residues. More re-cently, a method for in vitro mutagenesis ol circular DNA molecules has been devised which employs a combination of nuclease treatments to produce small deletions (Shenk et al. 1976). With this method, reces-sive and dominant transposition-defective deletions were generated within the transposon Tn3 (Heffron et al. 1977). These methods suffer from opposing limitations; chemical mutagens generally yield single-base changes which can only be mapped genetically, but deletions, although easily mapped by a variety of modern techniques, frequently affect more than one function. In this paper we present a new approach to mutagenesis which has general advantages over these procedures, It employs as mutagens synthetic oligodeoxyribonucleotides (Scheuer et al. 1977; Bahl etaI. 1977), encoding a restriction endonuclease r cog-nition site. The circular plasmid DNA is linearized by nuclease treatment, ligated to the oligomer, treated with the cognate restriction endonuclease to generate overlapping single-stranded DNA tails, and resealed. This method yields a new circular plasmid DNA into which a single new restriction site has been randomly inserted. Thus, it allows large numbers of mutants to be isolated at defined sites which are readily localized on the physical map through restriction endonuclease mapping. Its utility has been demonstrated by the production and characterization f a large number of mutations in the transposon Tn3. This transposon is 5 kb in length and flanked by short inverted repeats; it includes the structural gone for one class of penicillin-ase and codes for at least some of the proteins that mediate its own transposition (Bennett and Richmon