FIGURE 2: Mutation frequency after multiple EMS mutagenesis rounds. Two mutants, TDA1 and TDA3, selected after 210 min of EMS mutagenesis of the S288c strain, were submitted to three additional EMS mutagenesis rounds. In each round, the mutation frequency was assessed after 0, 90, 150, 210 and 360 min of incubation with EMS. It was based on temperature sensitivity (white bars), auxotrophic mutations on eight drop-out media (grey bars) and respiratory deficiency (black bars). The time period of incubation with EMS for each selected mutant is underlined in each mutagenesis round. For both mutants, no viable colonies were obtained after 360 min of EMS treatment in the second mutagenesis round and at the other time points at which no bars are present. Both mutant lines became temperature sensitive in round 2 and TDA3 acquired auxotrophic mutations for methionine and histidine in round 3.