Deletions affecting the transposition of an antibiotic resistance gene

Abstract

The structural gene for plasmid-mediated ampicillin resistance resides upon a 3.2 X 10(6) dalton transposable sequence (TnA) flanked by short inverted repeated sequences that accompany its insertion. TnA was transposed to pMB8, a 1.8 X 10(6) dalton derivative of the colicingenic plasmid ColE1. Random deletions were introduced in the resultant 5 X 10(6) 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 and electron microscopic analysis of DNA hetero-duplexes and were found to include one of the inverted repeated sequences or lie 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 approximately 2 X 10(6) 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.