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A modified TnphoA useful for single-stranded DNA sequencing.

D L Mielke1, M Russel

  • 1Rockefeller University, New York, NY 10021.

Gene
|September 1, 1992
PubMed
Summary
This summary is machine-generated.

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Researchers modified the TnphoA transposon to enable easy isolation of single-stranded DNA from plasmids. This new TnphoA-IG tool aids gene analysis and mutagenesis, particularly for genes lacking a filamentous phage origin of replication.

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • The TnphoA transposon is a tool for genetic analysis.
  • Isolating single-stranded DNA from plasmids is crucial for sequencing and mutagenesis.
  • Filamentous phage intergenic regions (IG) facilitate single-stranded DNA production.

Purpose of the Study:

  • To modify the TnphoA transposon for efficient single-stranded DNA isolation from target plasmids.
  • To create a versatile tool for analyzing genes cloned into plasmids lacking a filamentous phage IG.
  • To facilitate oligodeoxyribonucleotide-mediated mutagenesis of transposon-generated fusions.

Main Methods:

  • Insertion of the filamentous phage f1 intergenic region (IG) into a nonessential region of the TnphoA transposon.

Related Experiment Videos

  • Transposition of the modified TnphoA-IG into target plasmids.
  • Infection with a filamentous helper phage to induce single-stranded DNA production.
  • Analysis of transposition events ('blue hops') into specific genes (bla and pspE).

    • Main Results:

    • The modified TnphoA-IG transposon successfully facilitated the isolation of single-stranded plasmid DNA.
    • The utility of TnphoA-IG was demonstrated through successful transposition and analysis of gene fusions.
    • The method proved effective for genes encoding periplasmic proteins like beta-lactamase and phage shock protein.

    Conclusions:

    • The modified TnphoA-IG transposon is a valuable tool for genetic analysis, enabling single-stranded DNA isolation and mutagenesis.
    • This system is particularly useful for plasmids that do not inherently possess a filamentous phage IG.
    • The TnphoA-IG system enhances the study of gene function and protein localization in bacteria.