INSERTION SITE-SPECIFICITY OF THE TRANSPOSON TN3

The Tn3-deletion method [Davies and Hutchison, Nucleic Acids Res. 19, 5731-5738, (1991)] was used to sequence a 9.4 kb DNA fragment. Transpo sitional 'warm' spots were not a limiting factor but a 935 bp 'cold' s pot was completed using a synthetic oligonucleotide primer. Two hundre d and twenty three miniTn3 insertion sites from three sequencing proje cts were aligned and a 19 bp asymmetric consensus site was identified. There is no absolute sequence requirement at any position in this con sensus, so insertion occurs promiscuously (similar to 37% of sites are potential targets). In our sequencing projects, multiply targeted sit es always closely matched the consensus, although not all close matche s were targeted frequently The 935 bp cold spot showed no unusual feat ures when analysed with the consensus sequence. The consensus can be u sed to accurately predict likely insertion sites in a new sequence. Sy nthetic oligonucleotides based on the consensus and a known hot spot f or Tn3 were mutagenised. These sequences were not hot spots in our vec tors, suggesting that the primary sequence alone is not sufficient to create an insertional hot spot. We conclude that some other factor suc h as DNA secondary structure, also plays an important role in target s ite selection for the transposon Tn3.