Analysis of N-acetoxy-N-2-acetylaminofluorene-induced frame-shifts in pBR322

K. Yoon, V. N. Shelegedin, N. R. Kallenbach

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We present a simple system which can be used to study directly directly the sequence change and the cellular repair functions involved in frame-shift mutagenesis by a covalently reactive mutagen. Positive (+S) and negative (-S) alterations in the number of base pairs of the Tc gene of pBR322 were generated and particular clones with ApRTcS phenotypes were selected for mutagenesis experiments. Exposure of these frame-shifted plasmid DNAs to a potent carcinogen, N-acetoxy-N-2-acetylaminofluorene (AAAF), in vitro, caused covalent alterations to DNA sequence and resulted in a number of revertants (ApRTcR) not observed in the untreated controls. The dose curve indicated an exponential response suggesting single-hit kinetics. Differential inactivation of the Ap gene was observed among various E. coli strains. The wild-type AB1157 and AB2463 (yrecA) showed a similar dose curve while AB1886 (uvrA) showed a marked decrease in ApR clones at the same dose. Both addition (+S) and deletion (-S) plasmids exhibited similar dose curves on inactivation of Ap gene. The reversion frequency, however, of -S plasmid was a factor of 10 times higher than +S plasmid. The reversion frequency also increase markedly with uvrA host but not with recA host. 2 types of deletion revertants of the +S plasmid were found. 1 revertant has a single GC base-pair deletion in GC-rich region which is likely to be a target for AAAF reaction. The other showed a deletion of 4 base pairs (TCGA) at the tandem repeating sequence TCGATCGA which may represent a hot spot for frame-shift mutation.

Original languageEnglish (US)
Pages (from-to)273-283
Number of pages11
JournalMutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Issue number2
StatePublished - Apr 1982

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Health, Toxicology and Mutagenesis


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