Conditional dihydrofolate reductase deficiency due to transposon Tn5tac1 insertion downstream from the folA gene in Escherichia coli

Andrew F. Neuwald, B. Rajendra Krishnan, Patricia M. Ahrweiler, Carl Frieden, Douglas E. Berg

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Transposon Tn5 tac 1 can generate conditional mutations by virtue of an outward-facing tac promoter, which is regulated by the lac repressor and isopropyl-β-D-thiogalactopyranoside (IPTG). We report here on a Tn5 tac 1 insertion in Escherichia coli that results in a conditional (IPTG-elicited) folA mutant phenotype: During aerobic growth, IPTG caused decreased synthesis of dihydrofolate reductase (DHFR; encoded by the folA gene) and hypersensitivity to trimethoprim (a DHFR inhibitor); during anaerobic growth, IPTG elicited auxotrophy that was satisfied by thymine or glycine or threonine. The Tn5 tacl insertion was downstream from folA, with the tac promoter pointing into the gene (antisense direction). Complementation tests indicated that the conditional folA deficiency was a cis effect of transcription from the tac promoter, perhaps due to head-to-head collision between converging RNA polymerases.

Original languageEnglish
Pages (from-to)69-73
Number of pages5
JournalGene
Volume125
Issue number1
DOIs
StatePublished - Mar 15 1993

Keywords

  • Mutagenesis
  • RNA polymerase collision
  • antisense inhibition
  • lac promoter
  • transposon
  • trimethoprim

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