Characterization of mutant spectra generated by a forward mutational assay for gene A of ΦX174 from ENU-treated transgenic mouse embryonic cell line PX-2

Carrie R. Valentine, Beverly A. Montgomery, Scott G. Miller, Robert R. Delongchamp, Bentley A. Fane, Heinrich V. Malling

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

The sensitivity of in vivo transgenic mutation assays benefits from the sequencing of mutations, although the large number of possible mutations hinders high throughput sequencing. A forward mutational assay exists for ΦX174 that requires on altered, functionol ΦX174 protein and therefore should have fewer torgets (sense, base-pair substitutions) than forward assays that inactivate a protein. We investigated this assay to determine the number of targets and their suitability for detecting o known mutagen, N-ethyl-N-nitrosourea (ENU). We identified 25 target sites and 33 different mutations in ΦX174 gene A after sequencing over 350 spontaneous and ENU-induced mutants, mostly from mouse embryonic cell line PX-2 isolated from mice transgenic for ΦX174 am3, cs70 (line 54). All six types of base-pair substitution were represented omong both the spontaneous and ENU-treated mutant spectra. The mutant spectra from cells treated with 200 and 400 μg/ml ENU were both highly different from the spontaneous spectrum (P < 0.000001) but not from each other. The dose trend was significant (P < 0.0001) for a linear regression of mutant frequencies (R2 = 0.79), with a ninefold increase in mutant frequency at the 400 μg/ml dose. The spontaneous mutant frequency was 1.9 × 10-5 and the spontaneous spectrum occurred at 11 target base pairs with 15 different mutations. Thirteen mutations at 12 torgets were identified only from ENU-treated cells. Seven mutations had highly significant increases with ENU treotment (P < 0.0001) and 15 showed significant increases. The results suggest that the ΦX174 forward assay might be developed into a sensitive, inexpensive in vivo mutagenicity assay.

Original languageEnglish (US)
Pages (from-to)55-68
Number of pages14
JournalEnvironmental and Molecular Mutagenesis
Volume39
Issue number1
DOIs
StatePublished - 2002

Keywords

  • ENU
  • Gene A
  • Mouse cell
  • PhiX174
  • Transgenic

ASJC Scopus subject areas

  • Epidemiology
  • Genetics(clinical)
  • Health, Toxicology and Mutagenesis

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