Target sequence specificity of transposon Tn5 in the absence of major hotspots in the plasmid pBR322: identification of a new hotspot.
The plasmid pLB11-1 is a pBR322 derivative in which part of the tetracycline resistance (tet) gene (basepair coordinates 23 to 375), containing five hotspots for Tn5 insertion (D. E. Berg et al., Genetics 105, 813-828, 1983), has been replaced with a 5.1-kb fragment of Escherichia coli genomic DNA encoding an osmoregulatory function. Restriction mapping of 40 pLB11-1::Tn5 derivatives, chosen at random from 240 Tn5 insertion derivatives with an unaltered osmotolerant phenotype, placed Tn5 in the vector portion of 20 clones. The majority of these insertions (16/20) were located in a 0.09-kb region immediately downstream of the beta-lactamase (bla) gene. Nucleotide (nt) sequence analysis of seven insertions from this group revealed an identical site of insertion within pBR322, representing a hitherto unidentified hotspot for Tn5 insertion. The target sequence, 5'GTCTGACGC, was found to be duplicated in these cases.
Boyd
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LA
Plant Biotechnology Institute, National Research Council of Canada, Saskatoon, Saskatchewan.
Woytowich
A
A
Selvaraj
G
G
D001483:Base Sequence; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011815:R Factors; D015183:Restriction Mapping; D013753:Tetracycline Resistance / Q000235:genetics*; D014158:Transcription, Genetic
D004251:DNA Transposable Elements