Figure 5
Acquired phenotypes of COX assembly mutants. (a) Quantification of mtDNA. Yeast strains were grown overnight in liquid glucose-containing medium. Total DNA was isolated and the copy number of the mitochondrial COX3 gene was related to that of the nuclear GAL4 gene by RT-PCR and calculation of the 2-ΔΔCT value. Error bars indicate standard deviations of triplicate PCR reactions. (b) Complementation test. Δcox10, Δcox16, Δcox19, and Δmss2 strains taken from the MATα yeast deletion library have been transformed with single copy plasmids carrying the respective complementing wild-type alleles under control of their endogenous promoters. Wild-type cells (WT) were transformed with an empty vector. Young cells were grown on complete medium at 30°C overnight before transformation (light bars). Aged cells were incubated on complete medium at room temperature for 14 to 28 days before they were transferred to fresh plates, grown at 30°C overnight, and transformed with complementing plasmids (dark bars). Three days after transformation, colonies were replicated on plates containing fermentable or non-fermentable carbon sources, and the percentage of respiratory-deficient transformants was determined. Error bars indicate standard deviations of three independent experiments. (c) ROS accumulation. Yeast strains were grown for the indicated time periods in liquid glucose-containing medium (YPD), stained by the addition of DHR and analyzed by differential interference microscopy (left panels) and fluorescence microscopy (right panels). All fluorescent micrographs were taken with identical camera settings.