A modified TnphoA useful for single-stranded DNA sequencing.
The TnphoA transposon constructed by Manoil and Beckwith [Proc. Natl. Acad. Sci. USA 82 (1985) 8129-8133] has been modified to permit easy isolation of single-stranded (ss) DNA of target plasmids. The intergenic region (IG) of filamentous phage f1, which consists of the phage origin of replication and packaging signal, was inserted into a nonessential region of TnphoA. This modified transposon should be useful for the analysis of genes cloned in plasmids that lack a filamentous phage IG. Transposition of TnphoA-IG into a plasmid carries the IG with it; subsequently, after infection with a filamentous helper phage, ss plasmid DNA suitable for sequence analysis and useful for oligodeoxyribonucleotide-mediated mutagenesis of TnphoA-generated fusions can be isolated. The utility of TnphoA-IG was confirmed by analysis of 'blue hops' into the bla (encoding beta-lactamase) and pspE (encoding phage shock protein) genes whose products are secreted into the Escherichia coli periplasm.
Mielke
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DL
Rockefeller University, New York, NY 10021.
Russel
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D000469:Alkaline Phosphatase / Q000235:genetics; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D006360:Heat-Shock Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D014779:Virus Replication; D001618:beta-Lactamases / Q000235:genetics
D004251:DNA Transposable Elements; D004277:DNA, Single-Stranded; D006360:Heat-Shock Proteins; D000469:Alkaline Phosphatase; D001618:beta-Lactamases