5-Fluorouracil forward mutation assay in Salmonella: Determination of mutational target and spontaneous mutational spectra
- 作者：Warren E. Glaab ; Laurie S. Mitchell ; Judith E. Miller ; Katerina Vlasakova and Thomas R. Skopek
- 刊名：Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
- 出版年：2005
- 期刊代码：115_00275107
- 类别：bio
- 出版时间：2005
- 卷：578
- 期：1-2
- 页码：238-246
- 文件大小：300 K
摘要
A forward mutation assay in Salmonella typhimurium that selects for 5-fluoruracil (FU) resistance has been developed. The two genes possibly involved in FU resistance, the uracil phosphoribosyl transferase gene (upp) and the uracil transport protein (uraA), have been cloned from S. typhimurium and sequenced. One hundred percent of FU-resistant clones display sequence changes in the upp gene, indicating that its loss is the major mechanism involved in FU resistance. The spontaneous mutational spectra at the upp locus were then determined in two S. typhimurium strains, FU100 and FU1535, that differ only in the presence of pKM101 plasmid. The pKM101 plasmid provides error-prone replicative bypass of DNA lesions and renders FU100 more susceptible to induced mutagenesis. Fluctuation analysis of FU-resistant clones demonstrated a 10-fold higher spontaneous mutation rate at the upp locus in FU100 relative to FU1535. Over 300 independent FU-resistant clones were then used to generate the spectra at the upp locus in both the strains. Approximately 40%of all the mutations were base substitutions, present at the same relative percentage in both the strains. Frameshift mutations also accounted for approximately 40%of the total; however, their incidence was slightly elevated in FU100. The remaining mutations were larger insertions and deletions, which were both slightly elevated in FU1535. pKM101 significantly elevated the rate of all classes of mutations at the upp locus, with profound effects on A:T to T:A transversions and −2-base frameshift mutations. These initial mutational spectra at the upp locus reveal 147 mutable sites, or 23%of the total 627-base coding sequence and suggest that the target can detect a diverse spectrum of mutagenic events.