Transpositional properties and organisation of Tn7.

The intermolecular transposition of Tn7 was investigated and the products identified as simple inserts. Replicon fusions mediated by recA and recF independent homologous recombination were also recovered. The frequency of transposition was dependent upon the presence or absence of a cloned hotsite (a specific and preferred point of integration in the E. coli chromosome which is also active when cloned into plasmids) in the target replicon. Transposition to hotsites was more efficient than transposition to plasmids (cold sites). However, the context of the donor (whether Tn7 was integrated in a hotsite or a cold site) also affected the frequency of transposition to hotsites though transposition to cold sites was independent of the donor context or copy number. Transposition from a hotsite to a hotsite was less frequent than transposition from a cold site to a hotsite. The copy number of the donor may influence the magnitude of this effect. cis-acting and trans-acting functions required for transposition have been defined. Only the ends of the transposon were required in cis for efficient transposition. Three trans-acting functions were mapped and found to be required absolutely for transposition to both hotsites and cold sites. Two further trans-acting functions were localised; one was required for transposition to the hotsite while the other was required for transposition to cold sites. This result confirmed that two different, though probably related, transposition mechanisms are employed for Tn7 transposition. These trans-acting functions were correlated with polypeptides using minicells. Almost all of the available coding capacity of Tn7 which is essential for transposition is required to encode these polypeptides. Finally, an E. coli strain with a mini-Tn7 inserted at the chromosomal hotsite was used to study the integration of Tn7 into secondary chromosomal sites. A preliminary analysis of ten insertions provided ho evidence for the presence of preferred secondary sites. The failure to observe such sites was disscussed.