Figure 2.—
Reversion frequencies of the various trp5 mutant strains when treated with different mutagens. All strains were derived from the S288C derivative SJR828a (from Sue Jinks-Robertson), MATα his3Δ200 ura3-52 leu2Δ1. Gene deletion and mutagenesis was done using the pCORE plasmid and delitto perfetto strategy (Storici et al. 2001). The TRP5 gene was first deleted from its normal location in the genome; a PCR product of the TRP5 gene was then used to replace RNQ1, located near ARS306, in either orientation, after which the desired trp5 mutations were introduced. For measuring revertants, after mutagenesis strains were plated on SD media lacking tryptophan (SD-trp). Usually <1–2 × 108 cells/100-mm-diameter plate were plated, but plating at twice that density did not seem to interfere with the number of revertant colonies obtained. For measurement of UV mutagenesis, strains were plated on SD-trp plates at a density of ∼5 × 107 cells/100-mm-diameter plate and then exposed to 20 J/m2 UV light in an Ultra-Lum ultraviolet crosslinker (for an average survival of 74 ± 9%). For 5-AZ mutagenesis, cultures were grown overnight in YPD medium and ∼2 × 107 cells were transferred to SD-complete medium containing 5 mg/ml 5-AZ, grown overnight, and then plated on SD-trp plates. For the MMS and EMS mutagenesis assays, cells were washed and resuspended three times in the original culture volume of 50 mm potassium phosphate buffer, pH 7. At that point, EMS was added to a final concentration of 2.0% (for an average survival of 79 ± 11%) or MMS was added to a final concentration of 0.5% (for an average survival of 11 ± 4%) and cells were incubated at room temperature for 1 hr before being plated onto SD-trp plates, after inactivation of the mutagen by the addition of an equal volume of sterile 10% sodium thiosulfate. Mutagenesis with HAP was as described (Shcherbakova et al. 1996), using HAP at a concentration of 100 μg/ml. In all cases, YPD plates for determination of viable cells were counted after 2 days of incubation, and SD-trp plates were counted after 5 days. YPD plates for growth of trp5 strains were routinely supplemented with 40 mg/liter tryptophan. Reversion frequencies were determined from at least three cultures per strain and were averaged. If there were >10 revertants per culture, the standard deviation was calculated and is indicated with error bars on the graph. Forward orientation of the TRP5 gene is indicated by and reverse orientation by . In the forward orientation, TRP5 is oriented left to right on chromosome III and is to the left of ARS306.