Genetic structure, function and regulation of the transposable element IS21
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The IncP plasmid R68.45 and other plasmids carrying tandem repeats of the insertion sequence IS21 [=(IS21)2] produce replicon fusions via transposition at high frequencies in Escherichia coli and other gram-negative bacteria, whereas plasmids with a single IS21 copy, e.g. R68, give replicon fusions rarely. The 2131 bp nucleotide sequence of IS21 was determined; at the ends there were 11 bp inverted repeats with one mismatch. Two adjacent open reading frames, istA and istB, were located on one DNA strand of IS21. In E. coli maxicells, polypeptides of 46 kDa (the istA gene product) and 30 kDa (the istB gene product) were expressed by (IS21)2 plasmids, but not by IS21 plasmids. Genetic analysis of (IS21)2 plasmids indicates that the IS21-IS21 junctions form a promoter, which initiates transcription of the istAB operon in one of the two IS21 elements. A single IS21 element fused to an inducible external tac promoter expressed both proteins after induction, but did not promote effective replicon fusion, unless an IS21-IS21 junction (the preferred site for IS21 transposase action) was also present on the plasmid carrying the tac-IS21 construct. The sequences located between the IS21 elements in (IS21)2, 3 bp in R68.45 or 2 bp in pME28, were not recovered in the replicon fusion products. Homologous recombination between the directly oriented IS21 elements in the fusion products led to plasmids with a single IS21 insertion. Analysis of the latter showed that IS21 had a low, but not totally random specificity of insertion and created target duplications of 4 bp (occasionally 5 bp). These and previous results support the following model: plasmids like R68.45 are opened by IS21 transposase at the IS21-IS21 junction and then integrated into a target replicon by simple insertion, with loss of the 2–3 bp junction sequence.
Key wordsIS21 Insertion sequence R68.45 Tandem duplication Replicon fusion
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