The bacterial Tn9 chloramphenicol resistance gene: an attractive DNA segment for Mos1 mariner insertions

Abstract

The eukaryotic mariner transposons are currently thought to have no sequence specificity for integration other than to insert within a TA contained in a degenerated [TA](1-4) tract, either in vitro or in vivo. We have investigated the properties of a suspected hotspot for the integration of the mariner Mos1 element, namely the Tn9 cat gene that encodes a chloramphenicol acetyl transferase. Using in vitro and bacterial transposition assays, we confirmed that the cat gene is a preferential target for MOS1 integration, whatever its sequence environment, copy number or chromosomal locus. We also observed that its presence increases transposition rates both in vitro and in bacterial assays. The structural and sequence features that constitute the attractiveness of cat were also investigated. We first demonstrated that supercoiling is essential for the cat gene to be a hot spot. In contrast to the situation for Tc1-like elements, DNA curvature and bendability were not found to affect integration target preferences. We found that Mos1 integrations do not occur randomly along the cat gene. All TA dinucleotides that are preferred for integration were found within either TATA or TA x TA motifs. However, these motifs are not sufficient to constitute an attractive dinucleotide, since four TATA and TA x TA sites are cold spots.

Physical properties of DNA components affecting the transposition efficiency of the mariner Mos1 element

“…It contained the pBR322 tetracycline-resistance gene without its own promoter cloned between the XbaI and HindIII restriction sites of the multi-cloning site of the pBC vector, in reverse orientation to that of chloramphenicol and LacZ genes. Thus, it was unable to confer resistance to E. coli cells in tetracycline concentrations over 10 g/ml (Crénès et al 2009). This gene was Xanked by two 3ЈITR cloned between KpnI/ SacI and SalI/NotI sites.…”

A Simple Topological Filter in a Eukaryotic Transposon as a Mechanism To Suppress Genome Instability

“…Indeed, the effects of supercoiling on the rate of mariner transposition and target site selection have been previously noted (11,18,20). However, one might reasonably wonder about the biological significance of these observations.…”

Target Capture during Mos1 Transposition

“…We demonstrated here for the first time that the TA dinucleotide plays a crucial role in target capture and DNA strand transfer. Previous studies revealed that the local DNA sequences immediately surrounding the TA dinucleotide confer little target specificity, although they show a slight preference for TA dinucleotides that are flanked by A-T base pairs (10,22,23). It is tempting to imagine that the bending of the TA-rich targets is energetically more favorable for setting up the strand transfer reactions.…”

“…Moreover, some elements have specific (attTn7 for Tn7) (13) or preferred (HisG1 for Tn10, G(CT)(CT) (CT)(AT)(AG)(AG)(AG)C for Tn5) (20,21) targets, whereas mariner elements display an essentially random integration pattern. However, it has been shown that Mos1 may have preferred integration spots such as the one in the chloramphenicol resistance gene of Tn9 (22). No structural features explaining the preferential integration of Mos1 have yet been identified (23).…”