Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant.
We constructed a derivative of Tn5, Tn5 ORFlac, that is capable of creating lacZ translational fusions upon transposition. Lac- strains carrying this construct formed red papillae when plated on MacConkey-lactose media. Lac+ cells isolated from independent papillae expressed distinct beta-galactosidase fusion proteins, suggesting that the Lac+ phenotype resulted from transposition. In support of this, analysis of plasmids carrying Tn5 ORFlac prepared from these cells indicated that the Lac+ phenotypes arose as a result of intermolecular rearrangements. Furthermore, a derivative of Tn5 ORFlac that contains an ochre mutation in the transposase gene formed papillae only in a supB strain. Tn5 ORFlac is useful for obtaining mutants that affect Tn5 transposition and for creating lacZ fusions. We used the papillation phenotype to isolate a spontaneous revertant of IS50L that promotes transposition at a 3.6-fold higher rate than IS50R. The mutation altered the amino acid sequence of both transposase and inhibitor.
Krebs
M P
MP
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Reznikoff
W S
WS
D001483:Base Sequence; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis
D004251:DNA Transposable Elements