Reversion Frequency and Canavanine Resistance

For reversion frequency of Trp-phenotype and the occurrence of Canavanine resistant clones three independent cultures of the considered strains were cultivated at 28 °C in SD medium and auxotrophic requirement as needed. For each culture, three plates of selective medium lacking tryptophan or containing L-canavanine (3 µg/ml Sigma-Aldrich S.r.l. Milan, Italy) were inoculated and incubated at 28 °C for 3–5 days. The frequency of the Trp + reversion and of Canavanine resistant mutants was normalized to the number of viable cells in each experiment. Viability at day 1 was determined with the microcolonies test [25 (link)], while for the following days a standard colony forming units on YPD plates was employed. To measure RNA oxidation and Trp + reversion rate in the presence of antioxidant compounds 1 mM ALC and 26 mg/mL apple extracts [21 (link)] were added to the Kllsm4Δ1cell cultures. Total RNAs were extracted after 1 day of growth and analysed by Northwestern as described. Trp + reversion frequency was determined in the absence or in the presence of antioxidants.