FIGURE 3: Confirmation of mtDNA origin of cox23Δ suppressor.
(A) BY4743 cox23Δ suppressors were grown in potassium acetate solution at room temperature to induce tetrad formation. After 4-5 days, tetrads were dissected and the spores were allowed to grow on YPD-Agar. The spores were replica plated into YPGL or SCD minus Lys or Met Agar plates to localize the suppressor DNA.
(B) cox23Δ suppressor spore was mated with cox23Δ haploid of the opposite mating type. Approximately 1000 cells were allowed to grow in SCD minus Lys and Met Agar plate. The diploid colonies were then replica plated into SCGL minus Lys and Met plate to confirm the mitochondrial location of the suppressor.
(C) cox23Δ suppressor spore and cox23Δ haploid cells were exposed to EtBr to induce rhoo status before mating with rho+ cox23Δ and cox23Δ suppressor cells of the opposite mating type, respectively. Four colonies from each mating plate were streaked on YPD and then replica plated in YPGL to confirm the cox23Δ mitochondrial suppressor.