activation domain A of GCN4 by generating a GCN4A PCR product using GCN4salAF and GCN4ascAR primers with plasmid pGCN4 (a gift from S. Hahn) as a template. The PCR product was digested with SalI and AscI and subcloned into pUI- tTA-ADH1term-URA3 digested with the same enzymes to create pUI-tTA-GCN4A-ADH1term-URA3. The tTA-GCN4A-AD- H1term-URA3 cassette was integrated into a neutral site on Chromosome I (coordinates 17030-17205) by one-step integration of a PCR product using the primers URA3-tTA-intCHRIF and URA3-tTA-intCHRIR. Subsequent strain construction to gener- ate haploid and diploid MEP strains with the appropriate genotypes was performed by standard methods. Pedigree analysis Pedigree analysis was performed as previously described [11]. Briefly, daughter cells born to individual mothers were microma- nipulated to new positions on a YEPD plate and allowed to form colonies. Colonies were scored for mitochondrial function by replica plating to YEP+glycerol. Liquid aging and LOH assay Diploid cells were grown to saturation overnight in YC media lacking adenine and methionine. Cells were used to inoculate YEPD cultures, which were grown to log phase by incubation with shaking, 30uC for 3 hours. Cells were counted and used to inoculate 25 ml YEPD +1 mM estradiol cultures at 26104 cells/ml and incubated with shaking, 30uC for 95 hours. At indicated times, samples were harvested, washed, and plated to lead nitrate media. Sample volumes were adjusted appropriately to maintain a colony density of ,500–1000 colonies/150 mm plate. Plates were incubated at 30uC for 3 days and colonies were counted using a Geldoc XR+ imaging system (Biorad). Plates were further incubated at room temperature for 2–3 days for color develop- ment before scoring for half sectors. Reciprocal and non- reciprocal colonies were counted separately, and rates of total half sectors were calculated as (2*reciprocal + non-reciprocal/total colonies) [17]. Western blot analysis Lysates were prepared using NaOH lysis followed by TCA precipitation [66]. TCA pellets were resuspended in SUME buffer (1% SDS, 8 M Urea, 10 mM MOPS, pH 6.8, 10 mM EDTA) and total protein concentration was determined using the BCA protein assay (Thermo Scientific). 10 mg total protein per lane was run on 10% tris-glycine polyacrylamide gels (PAGEr gold, Lonza) and transferred to Immobilon P membrane (Millipore) using a semi-dry transfer apparatus. Western blot analysis was performed by standard methods and developed with Supersignal West Pico (Thermo Scientific). Antibodies: Goat a-Sir2 (yN-19; Santa Cruz Biotechnology), mouse a-Sir2 (sc-25753; Santa Cruz Biotechnol- ogy) mouse a-Vma2 (Invitrogen), mouse a-Pkc1 (Invitrogen), and HRP-conjugated secondary antibodies (Jackson Immunoresearch Laboratories). Quantitation of Western blots was performed by densitometry using ImageJ comparing exposures that fell within a linear range of detection. Purification of aged populations Log phase cultures of cells were harvested and labeled with NHC-Biotin as previously described [16]. Labeled populations were transferred to YEPD and incubated for 30uC, 2 hours to ensure that most labeled cells were mothers (have completed at least one cell division). Cells were counted and used to inoculate 1.5 liter cultures of YEPD +1 mM estradiol +100 mg/ml ampicillin Declining rDNA Stability in Aging Yeast at a density of 26104 cells/ml. Cultures were incubated at 30uC, shaking at 100 rpm for the indicated times before purification. Cells were harvested by centrifugation, resuspended at 66108 cells/ml in RNAlater (Ambion) and fixed at room temperature, 45 minutes. Cells were harvested by centrifugation and resuspended at 26108 cells/ml in PBS +2 mM EDTA and incubated with 1/20 volume streptavidin beads (Miltenyi Biotec), 4uC, 30 minutes. Cells were harvested by centrifugation and resuspended in 4 ml 40 mM Tris HCl pH 7.4 and layered onto Percoll Plus gradients (GE Healthcare). Gradients were spun at 4uC, 20 minutes at 2000 RPM in a GS-6R tabletop centrifuge (Beckman). A brown, flocculent layer of cell debris was removed from the top of the gradients with a pipette, and the remainder of the gradient was pooled with 40 ml PBS +2 mM EDTA. Cells were harvested by centrifugation, resuspended in 45 mL PBS +2 mM EDTA and purified on an Automacs Pro separator system (Miltenyi Biotek). ERC Southern blot analysis Genomic DNA was isolated from purified aged populations by standard methods. 1 mg of genomic DNA was digested overnight with BamHI + RecBCD (a gift from G. Smith, FHCRC) and separated by gel electrophoresis (0.8% agarose, 2 V/cm for 36 hours). DNA was visualized by staining with ethidium bromide and transferred to nitrocellulose membranes by standard methods. Membranes were hybridized with a 32P labeled double-stranded probe specific to the rDNA generated with oligos RDN5S-2 and RDN25S-2 using plasmid pDL05 as a template, visualized on a Typhoon phosphorimager (GE Health Sciences), and quantified using ImageJ. For normalization of DNA samples, 1 mg of genomic DNA was digested with BamHI, separated by gel electrophoresis (0.8% agarose, 10 V/cm, 3 hours), transferred and hybridized with a probe specific to NPR2 generated with oligos 5_NPR2 and 3_NPR2. TET-fob1 time courses Because over expression of Fob1 also increases rDNA recombination rates [67], we generated a weaker version of the tet-activator by replacing the VP16 activation domain with activation domain A of Gcn4 [27,68]. By carefully titrating Fob1 expression levels using a single copy of both TETOP2-Fob1 and the tTA-GCN4A activator in diploid cells, we could express FOB1 at normal levels in the absence of doxycycline, while effectively suppressing expression in the presence of 20 mg/ml doxycycline (Figure 5A). Diploid cells were grown to saturation overnight in YC media lacking adenine and methionine. Cells were used to inoculate YEPD +/- 20 mg/ml doxycycline cultures, which were incubated with shaking, 30uC for 5 hours. Cells were counted and used to inoculate 40 ml YEPD +1 mM estradiol +/- 20 mg/ml doxycycline cultures at 26104 cells/ml and incubated with shaking, 30uC for 95 hours. Samples were harvested and plated as described above for the LOH assay. To shift FOB1 expression from repression to activation, 15 ml of the + doxycycline culture was harvested at the 65 hour time point, washed, and resuspended in 15 ml fresh YEPD +1 mM estradiol. Supporting Information Figure S1 Loss of respiratory function is less frequent in MEP strains. A) Examples of inheritance of respiratory function during pedigree analysis of UCC5185. 68% of mothers generate pedigrees with all daughters maintaining respiratory function (upper panel). 32% of mothers lose the ability to produce daughters with respiratory function (small colonies; lower panel). PLoS Genetics | www.plosgenetics.org 11 March 2011 | Volume 7 | Issue 3 | e1002015