Fig. 2.

Mdj1 depletion results in loss of functional mtDNA. Doxycycline was added to (Dox +) or, as a control, omitted from a culture (Dox −) of mdj1-Δ [TETr-MDJ1] cells. At the indicated generations, aliquots of cells were collected and subject to one of three types of analysis: A) plated on either glycerol (upper panel)- or glucose (lower panel)-based media, B) pelleted prior to preparation of cellular extracts, which were subjected to immunoblot analysis or D) used to start subcultures for preparation of mtDNA, which was subjected to restriction endonuclease testing. A) Representative plates from samples harvested at the indicated generations are shown. Red colony color on glucose-based media is an indicator of respiratory competence; white colony color is an indicator of respiratory incompetence. B) Immunoblot analysis of extracts prepared from cells at the indicated number of generations after doxycycline addition, using antibodies specific for Mdj1 and, as a loading control, porin. Also included is an extract of wt cells (wt), as a control to indicate the level of expression of Mdj1. C) Plot of the data obtained. Left Y axis (open circles): the relative amount of Mdj1 obtained by quantitative analysis of results shown in (B), setting the level present prior to the addition of doxycycline at 100%. Right Y axis (closed squares): percentage of cells able to respire, calculated as the ratio of red colonies to total number of colonies (red and white) on glucose containing media. X axis: number of generations of growth in the presence of doxycycline. D) One red colony from a control culture, and three white colonies from a doxycycline treated culture from a 20 generation time point were picked and used to start liquid cultures in glucose-based media. After 12 h at 30 °C total cellular DNA was isolated from each culture. mtDNA was prepared and subjected to digestion with EcoRV, separated by agarose gel electrophoresis, stained with ethidium bromide and visualized under UV light. MW, molecular weight DNA standard Mass Ruler™ DNA Ladder (Fermentas).