PMC

Bcl-2 delayed viability loss in wild-type yeast. Wild-type yeast (EG103) transformed with pAD4 or pAD4–bcl-2 were grown in complete glucose medium (SDC), and left in the medium (□, ▪), or switched to water at 72 h (○, •). Viability was assayed by plating on YPD plates at the indicated times. The experiment was repeated four times with similar results. A representative experiment is shown. Note that the viability is reported on a log scale. pAD4 transformed (▪, •); pAD4–bcl-2 transformed (□, ○).

Bcl-2 expression increased catalase activity in stationary phase yeast. Protein extracts were prepared from wild-type and sod1Δ cells transformed with pAD4 or pAD4–bcl-2 and grown 72 h (to stationary phase). Catalase activity was measured as described in Materials and Methods. Results are the average of four experiments and are reported as percent of wild-type activity; error bars represent standard deviation. The asterisks indicate data sets that are significantly different (P < 0.05 by the two-sided t test). The difference between wild-type and either sod1Δ strain was also statistically significant. Specific activity for wild type was 770 U/mg protein.

Bcl-2 reversed stationary phase viability loss in strains lacking one or both SODs. Percent viability of sod1Δ (EG118) and sod1Δsod2Δ (EG133) strains with or without Bcl-2 expression at different stages of growth. Data are reported as percent viable cells, or colony forming units (live cells), per total cells (live and dead cells). Total cell number was determined by optical density (OD₆₀₀) and confirmed by hemocytometer counting. Differences between control and Bcl-2–expressing strains at 24 h are significant (P < 0.01). (white bars) sod1Δ; (gray bars) sod1Δ- sod2Δ; (crosshatched pattern) strains expressing Bcl-2; (no pattern) strains harboring plasmid vector (pAD4).

Bcl-2 expression and Mn metabolism. (A) Wild-type (EG103), sod1Δ (EG118), and sod1Δsod2Δ (EG133) cells transformed with pAD4 or pAD4–bcl-2 were inoculated at 10⁶ cells/ml in SD–leu medium with or without 5 mM MnSO₄ added, and incubated at 30°C and 220 rpm. OD₆₀₀ was measured at 48 h. Experiments were performed three times with duplicate samples from two separate transformations. Results are reported relative to growth of wild type without Bcl-2 measured on the same day. (B) The same strains were analyzed for manganese accumulation by atomic absorption at stationary phase (after 3 d of incubation in SDC medium with normal levels of manganese). Experiments were repeated three times. Results are reported as percent of the manganese levels of the wild-type strain (lacking Bcl-2) measured on the same day.

Bcl-2 expression allowed sod1Δ mutants to form colonies in 100% oxygen. sod1Δ (EG118), sod2Δ (EG110), and sod1Δsod2Δ (EG133) cells harboring pAD4–bcl-2 (crosshatched bars) or pAD4 (open bars), were grown to stationary phase and equal numbers of cells were plated on selective plates (SD–leu) and incubated in low aeration (CampyPaks) for 3 d, or in an atmosphere of 100% oxygen for 48 h followed by incubation in air. (Oxygen was replenished after 24 h.) Viability was recorded when colonies became big enough to count—at day 3 (EG118) or day 4 (EG110 and EG133)—and is reported as a percentage of the viability of the same strain in low aeration (relative viability). The experiment was performed with similar results at least four times for each strain, with two separate samples in each experiment. A representative experiment is shown.