Deletions affecting the transposition of an antibiotic resistance gene.

Abstract

The structural gene for plasmid-mediated ampicillin resistance [in Escherichia coli] resides upon a 3.2 .times. 106 dalton transposable sequence (TnA) flanked by short inverted repeated sequences that accompany its insertion. TnA was transposed to pMB8, a 1.8 .times. 106 dalton derivative of the colicingenic plasmid ColE1. Random deletions were introduced in the resultant 5 .times. 106 dalton recombinant plasmid by a combination of nuclease treatments in vitro. From this set of deletions a subset was isolated that contained deletions affecting the transposition of TnA. The deletions were mapped by digestion with restriction nucleases [EcoRI, BamHI, HincII and HaeII] and analysis of DNA heteroduplexes by EM and included 1 of the inverted repeated sequences or lay in the central portion of TnA. Complementation experiments were attempted between these plasmids and another compatible plasmid carrying a deletion in TnA that abolished its ampicillin resistance. The results of the deletion data indicate that .apprx. 2 .times. 106 daltons of TnA is required for transposition; the complementation experiments suggest that the terminal inverted repetition and the central region of TnA play different essential roles in TnA transposition.