A novel yeast-based tool to detect mutagenic and recombinogenic effects simultaneously.
In this work, we describe a new yeast-based assay to allow efficient detection of a comprehensive spectrum of genotoxicity events. The constructed diploid Saccharomyces cerevisiae strain allows the simultaneous monitoring of forward mutations, mitotic recombination events and chromosome loss or non-disjunction by direct selection in an easy and highly reproducible approach. The strain contains a DNA module consisting of a single functional copy of the URA3 gene and the kanMX4 gene inserted at the ADE2 locus on the right arm of chromosome XV. The changes of the genotype within the marker region were primarily selected on 5-fluoroorotic acid (5-FOA) agar plates. Further simple phenotypic tests of the 5-FOA-resistant ura3 clones make it possible to analyze the genetic configuration in detail (e.g. point mutations in URA3, gene conversion, crossing-over and chromosome loss). We demonstrate the successful application of our test system by studying the effects of well-known genotoxic agents (UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, aniline and benomyl). We found that the various agents induced mutations and recombination events with different relative frequencies. The integration of the module has generated a hot spot region of mutation and recombination at the borders of the artificially integrated URA3 kanMX4 cassette, which makes the system more sensitive towards DNA-damaging agents. Unlike other test systems, our S. cerevisiae strain is capable to detect a mutagenic effect caused by aniline.
Schafer
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Department of Biology IV (Microbiology & Genetics), RWTH Aachen University, Worringer Weg, D-52056 Aachen, Germany. bernd.schaefer@rwth-aachen.de <bernd.schaefer@rwth-aachen.de>
Neffgen
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Klinner
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D000465:Algorithms; D000814:Aniline Compounds / Q000633:toxicity; D001542:Benomyl / Q000633:toxicity; D002273:Carcinogens / Q000633:toxicity; D004252:DNA Mutational Analysis / Q000379:methods*; D005656:Fungal Proteins / Q000235:genetics; D005659:Fungicides, Industrial / Q000633:toxicity; D019656:Loss of Heterozygosity; D008769:Methylnitronitrosoguanidine / Q000633:toxicity; D008938:Mitosis / Q000235:genetics; D008954:Models, Biological; D009152:Mutagenicity Tests / Q000379:methods; D009154:Mutation; D030781:Organisms, Genetically Modified; D011995:Recombination, Genetic; D014466:Ultraviolet Rays / Q000009:adverse effects; D015003:Yeasts / Q000235:genetics*
D000814:Aniline Compounds; D002273:Carcinogens; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D008769:Methylnitronitrosoguanidine; C023650:aniline; D001542:Benomyl