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 journalArticle

12 Citations (Scopus)

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

Fingerprint

Transgenic Mice
mutation
Assays
Genes
Cells
assay
Cell Line
Mutation
gene
Base Pairing
Substitution reactions
substitution
Ethylnitrosourea
Mutagens
protein
Linear regression
mutagenicity
mutant
Proteins
Throughput

Keywords

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

ASJC Scopus subject areas

  • Environmental Science(all)
  • Environmental Chemistry
  • Genetics
  • Genetics(clinical)
  • Toxicology
  • Health, Toxicology and Mutagenesis

Cite this

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

In: Environmental and Molecular Mutagenesis, Vol. 39, No. 1, 2002, p. 55-68.

Research output: Contribution to journalArticle

Valentine, Carrie R. ; Montgomery, Beverly A. ; Miller, Scott G. ; Delongchamp, Robert R. ; Fane, Bentley A ; Malling, Heinrich V. / 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. In: Environmental and Molecular Mutagenesis. 2002 ; Vol. 39, No. 1. pp. 55-68.
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AB - 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.

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