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PubMed · 1324873

A modified TnphoA useful for single-stranded DNA sequencing.

D L Mielke·M Russel
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Abstract

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.

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Alkaline PhosphataseBacteriophagesBase SequenceCloning, MolecularDNA Transposable ElementsDNA, Single-StrandedEscherichia coliHeat-Shock ProteinsMolecular Sequence DataMutagenesis, InsertionalRegulatory Sequences, Nucleic AcidVirus Replicationbeta-LactamasesJournal ArticleResearch Support, U.S. Gov't, Non-P.H.S.Research Support, U.S. Gov't, P.H.S.

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D L Mielke, M Russel. 1992-09-01. A modified TnphoA useful for single-stranded DNA sequencing.. https://doi.org/10.1016/0378-1119(92)90253-l

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