New indicator Escherichia coli strain for rapid and accurate detection of supF mutations
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RESEARCH
Open Access
New indicator Escherichia coli strain for rapid and accurate detection of supF mutations Ruriko Fukushima†, Tetsuya Suzuki*†
and Hiroyuki Kamiya*
Abstract Background: The supF gene of Escherichia coli is useful for forward mutation analysis in bacterial and mammalian cells used in mutagenesis and DNA repair studies. Indicator E. coli strains, such as KS40/pOF105, have been used to analyze supF mutations. However, KS40/pOF105 is not enough to select supF mutants on nutrient-rich agar plates. Therefore, in this study, a new indicator E. coli strain for rapid and accurate detection of supF mutations was developed. Results: The gyrA and rpsL genes with an amber mutation were integrated into the chromosomal DNA of E. coli KS40 to produce a new indicator strain, RF01. RF01 cells transformed by the wild-type supF gene were sensitive to nalidixic acid and streptomycin on LB agar plates. supF mutant frequencies and mutation spectra in RF01 were similar to those in KS40/pOF105. In addition, some mutations in supF were only detected in RF01. Conclusion: RF01 is a new and useful indicator E. coli strain for analyzing supF mutations. Keywords: supF mutation assay, Indicator Escherichia coli strain, gyrA, rpsL
Background The supF gene of Escherichia coli codes for an amber suppressor transfer RNA (tRNA) that translates the amber codon (UAG) into tyrosine [1, 2]. supF mutations stop translation of genes with an amber mutation. On the basis of this property, supF-bearing plasmids have been developed to study chemical mutagenesis and DNA repair mechanisms by using selectable marker genes with an amber mutation in host bacteria [3–6]. E. coli MBM7070 has an amber mutation in lacZ, and supF mutants can be identified by colorimetric screening using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) [6]. However, it is difficult to select white mutant colonies from a large number of wild-type (WT) blue colonies and accurately determine low-level mutant * Correspondence: [email protected]; [email protected] † Ruriko Fukushima and Tetsuya Suzuki contributed equally to this work. Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
frequencies in MBM7070. Akasaka et al. (1992) established E. coli KS40/pKY241 to overcome this difficulty [7]. KS40 is a nalidixic acid–resistant (gyrA) MBM7070 derivative, and pKY241 is a plasmid bearing the gyrA gene with an amber mutation. WT supF (supF+)-transformed KS40/pKY241 cells are sensitive to nalidixic acid, and supF mutants are selected as white colonies on nalidixic acid- and X-gal-containing agar plates. However, this antibiotic system does not work properly for selecting supF mutants, and false-positive mutant (blue) colonies are found on selection agar plates. To improve this antibiotic system, Obata et al. (1998) developed the indicator strain E. coli KS40/pOF105 for positive screening of supF mutants in order to measure low-level mutant frequencies [4], and KS40/pOF105 has
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