Isolation and genetic analysis of deletion mutants of colicin E1 plasmids carrying a TnA insertion

Abstract

Deletions of colicin El (colEl) plasmid deoxyribonucleic acid (DNA) carrying the TnA transposon have been isolated. All except two were generated by nuclease digestion of plasmid DNA from its EcoRI-sensitive site. A plasmid containing about 16% of the ColEl DNA (6.5 x 105 daltons) was generated that also contained the part of the TnA transposon conferring ampicillin resistance. The extents of different deletions were determined by analysis of restriction endonuclease fragments generated by the restriction endonucleases HaeII, BamHI, and HincII. NaCl plus 0.015 M sodium citrate).Enzymatic digestions of DNA. The endonucleases used were EcoRI, HaeII, BamHI, and HincII, which were obtained from New England Biolabs. Most HaeII used was a generous gift of H. Ohmori and J. Tomizawa. DNA digestions were conducted overnight at 37°C. EcoRI digestions of DNA were 321 on July 31, 2020 by guest

ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility

“…The origins and properties of most plasmids used are also reported in the accompanying paper (8). Plasmids derived from CoIEl are: pDMS630, carrying the Tn3 transposon, which confers ampicillin (Ap) resistance (8,12); pDMS6641 (Apr) (8,13); and ColEl::Tn5, which confers kanamycin (Kni) resistance (1). pHSG1 is a temperature-sensitive replication mutant derived from pSC101 (9).…”

“…3), we expected the deletion in pHSG211 to extend further into the HaeII B fragment than that of pHSG206. The nucleotide sequence of a pDMS630 deletion mutant we previously isolated, pDMS6641 (8,13), has been found to contain only 684 base pairs (bp) to the left (-) (25) of the ColEl replication origin (H. Ohmori and J. Tomizawa, personal communication). That plasmid is also incompatible with ColEl::Tn5 (9; Table 2).…”

ColE1 copy number mutants

“…1. pDMS630 is ColEl containing a Tn3 transposon which confers ampicillin resistance (5). pDMS66 (13) is a mutant of pDMS630 in which the end of the Tn3 transposon associated with the ,3lactamase gene is fused to the ColEl DNA 684 bp upstream from the ColEl replication origin (20) and 129 bp upstream from the promoter that initiates transcription of the RNA primer of ColEl replication (10). pDMS6642 was formed by constructing a plasmid that contains only two HaeII-generated DNA fragments observed in pDMS66 ( Fig.…”

“…One contains a part of the ColEl DNA that extends from 185 to 684 bp upstream from the replication origin and is fused to a (approximately 1,200 bp) portion of the Tn3 transposon that includes the P-lactamase gene and a portion of the tnpR gene (8,12,13,20). The other fragment is the 360-bp HaeII fragment E that contains the ColEl replication origin (12,13,20). These two fragments were ligated to form pDMS6642.…”

“…They can only be joined in one way to generate a functional plasmid (11). The location of the BamHI-and PstI-sensitive sites in the Tn3 portion of the plasmid has previously been identified (8,10,13,24). pDMS1005 (Fig.…”