Question

- [5 pts) Design and describe an in vivo experiment to obtain transposon Tng insertion mutants into a functional lacZ: gene that is carried on an F' plasmid which is within an E. coli strain whose genome is lacZ: (non-polar), lacY+, lacAt. You can use whatever genetic tools you want including generalized transduction, specialized transduction, transformation, conjugation, suicide plasmid vectors, complementation assays, recombination, selective media, chromogenic media, ampicillin enrichment, etc. The microbiology lab is your oyster, go get me a lacZ::Tn9 mutant.

Answer 1

The sites of insertion of 28 independent occurrences of the transposon Tn9 in the final 160 base-pairs of the lacZ gene. The insertions are at 16 different points, and five of these points are represented by multiple occurrences. A comparison of the sequence surrounding each of the insertion sites indicates a preferential, but not obligatory, pattern for the endonucleolytic cleavage at Tn9 insertion sites. The strongest “cutting preference” observed is the presence of a G · C base-pair, without apparent regard to orientation, at each end of the nine base-pair sequence repeated during integration. This feature was observed for 23 of the 28 insertions, and for all the sites of multiple occurrence. Half of the insertions are grouped within a 16 base-pair region that contains a seven base-pair sequence identical to the last seven base-pairs of IS1, raising the possibility that homology with the ends of the element plays a role in insertion site selection.

The conclusion of this and the accompanying paper (Miller et al., 1980) is that the transposition of IS1 and Tn9 occurs more frequently into easily denaturable regions, on a large scale. At the sequence level, a cleavage specificity for the enzyme(s) involved in transposition is revealed, which may act in concert with the presence of sequences homologous to the ends of the element to produce the observed distribution of insertion sites.