Empirical verification of evolutionary theories of aging

Results

Dominant polygenic trait extending longevity of each of the 3 long-lived yeast mutants does not affect some key features of early-life fitness and enhance other such features

To empirically verify evolutionary theories of programmed or non-programmed aging, we elucidated if the dominant polygenic trait that extends longevity of each of the 3 selected long-lived yeast mutants affects early-life fitness when each mutant grows and ages alone – i.e. in the absence of a parental WT strain. The following key features of early-life fitness were measured: the exponential growth rate and efficacy of post-exponential growth, fecundity, and resistance to apoptotic and liponecrotic forms of programmed cell death.

We first assessed if the long-lived mutant strains 3, 5 and/or 12 exhibit altered exponential growth rate and/or efficacy of post-exponential growth in media containing 1) a fermentable carbon source - i.e. glucose at the initial concentration of 0.2% [CR conditions] or 2% [non-CR conditions]; and 2) a non-fermentable carbon source - i.e. ethanol at the initial concentration of 1% or glycerol at the initial concentration of 3%. In these experiments, we used the single-gene-deletion mutant strains rpp2BΔ and dbp3Δ as controls. Each of these mutant strains is known to exhibit extended replicative lifespan (RLS) and reduced growth rate on 2% glucose [73]. dbp3Δ is also known to have prolonged CLS [89]. rpp2BΔ lacks a gene encoding ribosomal protein P2 beta, whereas dbp3Δ lacks a gene encoding a DEAD-box family protein involved in ribosomal biogenesis [73]. By monitoring the OD₆₀₀ of cell cultures recovered at different time points as a measure of cell growth, we found that the long-lived mutant strains 3, 5 and 12 do not differ from the parental WT strain BY4742 in the exponential growth rates and post-exponential growth efficacies in medium initially containing 0.2% glucose, 2% glucose, 1% ethanol or 3% glycerol (Figures 1A, 1B, 1C and 1D, respectively). Of note, the control strain rpp2BΔ exhibited a reduced growth rate in medium initially containing any of these four carbon sources, whereas the control strain dbp3Δ displayed a decreased growth rate in medium initially containing 0.2% glucose or 2.0% glucose (Figure 1). Moreover, the control strain rpp2BΔ exhibited a significantly reduced efficiency of post-exponential growth in medium initially containing 3% glycerol (Figure 1D).

Figure 1. The long-lived mutant strains 3, 5 and 12 do not differ from the parental WT strain in the exponential growth rates and post-exponential growth efficacies in medium initially containing fermentable or non-fermentable carbon source. The parental haploid WT strain BY4742, long-lived mutant strains 3, 5 and 12 (each in the BY4742 genetic background), and the single-gene-deletion mutant strains rpp2BΔ and dbp3Δ (each in the BY4742 genetic background) were cultured in YP medium initially containing 0.2% glucose (A), 2.0% glucose (B), 1.0% ethanol (C) or 3.0% glycerol (D). The OD₆₀₀ of cell cultures recovered at different time points was measured. Growth curves are shown; data for growth curves are presented as means (n = 3). For each strain, a doubling time (min) was calculated as T_d = (t₂ – t₁) × log 2/log (OD₂/OD₁), where: t₂ = a given time point; t₁ = an earlier time point; OD₂ = OD₆₀₀ at a given time point; OD₁ = OD₆₀₀ at an earlier time point. Data for the values of T_d are presented as means ± SEM (n = 3; ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

We then elucidated if the long-lived mutant strains 3, 5 and/or 12 exhibit altered efficacy of their sexual reproduction by mating, one of the measures of fecundity. In these experiments, yeast cells of mating type MATa (i.e. the haploid WT strain BY4741) and mating type MATα (i.e. the haploid WT strain BY4742 or the selected long-lived haploid mutant strains 3, 5 or 12, all in the BY4742 genetic background) were pre-grown separately to mid-logarithmic phase in YP medium initially containing 0.2% glucose or 1% ethanol. The efficiency of mating was measured as described in the ″Materials and methods″ section; it was calculated as the number of colonies of MATa/MATα diploids divided by the sum of MATa/MATα diploids plus haploid colonies. Crosses between two WT strains of opposite mating types (i.e. the haploid strain BY4741 [MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0]) and the haploid strain BY4742 [MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0]) were used as controls. We found that the long-lived mutant strains 3, 5 and 12 do not differ from the parental WT strain BY4742 in efficacy of their sexual reproduction by mating if pre-grown in medium initially containing 0.2% glucose or 1% ethanol (Figures 2A and 2B, respectively).

Figure 2. The long-lived mutant strains 3, 5 and 12 do not differ from the parental WT strain in efficacy of their sexual reproduction by mating, a measure of fecundity and a key trait of early-life fitness. Yeast cells of mating type MATa (i.e. the haploid WT strain BY4741) and mating type MATα (i.e. the haploid WT strain BY4742 or the selected long-lived haploid mutant strains 3, 5 or 12, each in the BY4742 genetic background) were pre-grown separately to mid-logarithmic phase in YP medium initially containing 0.2% glucose (a fermentable carbon source; CR conditions) (A) or 1% ethanol (a non-fermentable carbon source) (B). The efficiency of mating was measured as described in the ″Materials and methods″ section; it was calculated as the number of colonies of MATa/MATα diploids divided by the sum of MATa/MATα diploids plus haploid colonies. Data are presented as means ± SEM (n = 3; ns, not significant difference).

We then investigated if the long-lived mutant strains 3, 5 and/or 12 display altered efficacy of their sexual reproduction by sporulation, another measure of fecundity. In these experiments, each of the four diploid strains formed between cells of the haploid WT strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and cells of the haploid WT strain BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) or cells of each of the selected long-lived haploid mutant strains 3, 5 or 12 (each in the BY4742 genetic background) were pre-grown to mid-logarithmic phase in YP medium initially containing 0.2% glucose or 1% ethanol. The efficiency of sporulation of each of the four diploid strains was then measured at various time points since the beginning of a sporulation assay as described in the ″Materials and methods″ section; it was calculated as the percentage of tetrads and dyads produced by a diploid strain, relative to the total number of cells. We found that the long-lived mutant strains 3, 5 and 12 do not differ from the parental WT strain BY4742 in efficacy of their sexual reproduction by sporulation when cells of the hybrid each of them formed with the haploid WT strain BY4741 of opposite mating type were pre-grown in medium initially containing 0.2% glucose or 1% ethanol (Figures 3A and 3B, respectively).

Figure 3. The long-lived mutant strains 3, 5 and 12 do not differ from the parental WT strain in efficacy of their sexual reproduction by sporulation, a measure of fecundity and a key trait of early-life fitness. Each of the four diploid strains formed between cells of the haploid WT strain BY4741 (MATa) and cells of the haploid WT strain BY4742 (MATα) or cells of each of the selected long-lived haploid mutant strains 3, 5 or 12 (each MATα in the BY4742 genetic background) were pre-grown to mid-logarithmic phase in YP medium initially containing 0.2% glucose (a fermentable carbon source; CR conditions) (A) or 1% ethanol (a non-fermentable carbon) (B). The efficiency of sporulation of each of the four diploid strains was measured at various time points since the beginning of a sporulation assay as described in the ″Materials and methods″ section; it was calculated as the percentage of tetrads and dyads produced by a diploid strain, relative to the total number of cells. Data are presented as means ± SEM (n = 3; ns, not significant difference). At each time point, sporulation efficiencies of the WT × 3, WT × 5 and WT × 12 diploid strains were statistically insignificant in comparison with sporulation efficiency of the WT × WT diploid strain.

We also assessed if the dominant polygenic trait that extends longevity of each of the 3 long-lived mutant strains affects two other essential aspects of early-life fitness, namely 1) cell susceptibility to a mitochondria-controlled apoptotic form of death triggered by a brief exposure to exogenous hydrogen peroxide [48,90–99]; and 2) cell susceptibility to a ″liponecrotic″ form of death elicited by a short-term exposure to exogenous palmitoleic acid [48,100–103]. We found that the long-lived mutant strains 3, 5 and 12 exhibit enhanced (as compared to the parental WT strain BY4742) susceptibilities to 1) mitochondria-controlled apoptotic death of yeast cells pre-grown in media initially containing 0.2% glucose or 1% ethanol (Figures 4A and 4B, respectively); and 2) liponecrotic death of yeast cells pre-grown in media initially containing 0.2% glucose or 1% ethanol (Figures 5A and 5B, respectively).

Figure 4. The long-lived mutant strains 3, 5 and 12 exhibit enhanced (as compared to the parental WT strain) susceptibilities to a mitochondria-controlled apoptotic form of cell death, one of the traits of early-life fitness. The parental WT strain BY4742 and long-lived mutant strains 3, 5 and 12 (each in the BY4742 genetic background) were cultured in YP medium initially containing 0.2% glucose (a fermentable carbon source; CR conditions) (A) or 1% ethanol (a non-fermentable carbon source) (B). Cell aliquots were recovered from various growth phases and then treated for 2 h with 2.5 mM hydrogen peroxide to induce mitochondria-controlled apoptosis. The % of viable cells was calculated as described in in the ″Materials and methods″ section. D, diauxic growth phase; L, logarithmic growth phase; PD, post-diauxic growth phase; ST, stationary growth phase. Data originate are presented as means ± SEM (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5. The long-lived mutant strains 3, 5 and 12 exhibit enhanced (as compared to the parental WT strain) susceptibilities to a liponecrotic form of cell death, one of the traits of early-life fitness. The parental WT strain BY4742 and long-lived mutant strains 3, 5 and 12 (each in the BY4742 genetic background) were cultured in YP medium initially containing 0.2% glucose (a fermentable carbon source; CR conditions) (A) or 1% ethanol (a non-fermentable carbon source) (B). Cell aliquots were recovered from various growth phases and then exposed for 2 h to 0.2 mM palmitoleic acid to induce liponecrosis. The % of viable cells was calculated as described in in the ″Materials and methods″ section. D, diauxic growth phase; L, logarithmic growth phase; PD, post-diauxic growth phase; ST, stationary growth phase. Data originate are presented as means ± SEM (n = 3; *p < 0.05; **p < 0.01; ***p < 0.001).

Of note, our recent study revealed that the long-lived mutant strains 3, 5 and 12 show enhanced resistance to chronic oxidative, thermal and osmotic stresses (Gomez-Perez et al., submitted). Akin to cell susceptibility to apoptotic and liponecrotic forms of cell death, such resistance to acute stresses is one of the key traits of early-life fitness [12,48,58,61,65,99,100,104–109].

In sum, findings presented in this section and elsewhere (Gomez-Perez et al., submitted) imply that the dominant polygenic traits extending longevities of the long-lived mutant strains 3, 5 and 12 do not affect such key features of early-life fitness as the exponential growth rate, efficacy of post-exponential growth and fecundity. Moreover, these longevity-extending polygenic traits enhance such features of early-life fitness as susceptibility to chronic exogenous stresses, and the resistance to apoptotic and liponecrotic forms of programmed cell death.

Development and validation of a quantitative assay for assessing the relative fitness of a long-lived mutant strain that competes for nutrients with a parental WT strain

To investigate if the dominant polygenic trait that extends longevity of each of the 3 selected long-lived yeast mutants influences the relative fitness of the mutant when it competes for nutrients and other environmental resources with a parental WT strain, we developed a direct competition assay. In this assay (Figure 6), the WT strains BY4739 (MATα leu2Δ0 lys2Δ0 ura3Δ0) and BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0), the single-gene-deletion mutant strain dbp3Δ (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 dbp3Δ::kanMX4) in the BY4742 genetic background, and the long-lived mutant strains 3, 5 and 12 (each in the BY4742 genetic background) were grown separately in the liquid nutrient-rich YP medium initially containing 0.2% glucose, 2% glucose or 1% ethanol as carbon source until mid-exponential phase. The single-gene-deletion mutant strain dbp3Δ was used as a control mutant strain because it is known to exhibit 1) extended CLS [89] and RLS [73]; 2) a decreased growth rate on 0.2% glucose (see above), 2% glucose [73] and 1% ethanol (see above); and 3) a reduced relative fitness when it is co-cultured with a parental WT strain in medium initially containing 2% glucose [73,89]. Cells of the WT strain BY4739 were mixed with the same number of cells of BY4742, dbp3Δ or a selected long-lived mutant strain (i.e. mutant strain 3, 5 or 12) in liquid YP medium initially containing 0.2% glucose, 2% glucose or 1% ethanol as carbon source. After culturing the cell mixture for 7 days, an aliquot of cell suspension was diluted and plated on solid YP medium supplemented with 2% glucose. Following 2 days of incubation, colonies on each plate were replicated onto plates with the synthetic minimal YNB medium without amino acids and nucleotides supplemented with 2% glucose. One of these plates contained leucine, lysine, uracil and histidine (hereafter it is called a ″His⁺″ plate), whereas the other plate contained leucine, lysine and uracil (hereafter it is called a ″His^-″ plate). After 2 days of incubation at 30^oC, the number of CFU on ″His⁺″ and ″His^-″ plates was counted. The relative fitness of each His⁺ strain (i.e. the BY4742, dbp3Δ, 3, 5 or 12 strain) in direct competition with the His^- strain BY4739 was calculated as log₂ [(CFU^x_mutant/CFU^x_WT/(CFU⁰_mutant/CFU⁰_WT)], where: CFU^x is the colony count at the end of week x, whereas CFU⁰ is the colony count at initial inoculation of a mixed culture (Figure 6). In every experiment for measuring relative fitness, the direct competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.

Figure 6. Quantifying the relative fitness of a long-lived mutant strain in a direct competition assay with a parental WT strain. His⁺ and His^- strains used in the direct fitness competition experiment are first cultured separately in the complete YP medium rich in amino acids, nucleotides and other nutrients until mid-exponential phase. Cells of the His⁺ strain are then mixed with the same number of cells of the His^- strain in liquid YP medium. After culturing the cell mixture for 7 days, an aliquot of cell suspension is diluted and plated on a solid YP medium. Following 2 days of incubation, colonies on each plate are replicated onto plates with the synthetic minimal YNB medium without amino acids and nucleotides. One of these plates contains leucine, lysine, uracil and histidine (it is called a His⁺ plate), whereas the other plate contains leucine, lysine and uracil (it is called a ″His^-″ plate). After 2 days of incubation at 30^oC, the number of CFU on ″His⁺″ and ″His^-″ plates is counted. The relative fitness of each His⁺ strain in a direct competition with the His^- is calculated as log₂ [(CFU^x_mutant/CFU^x_WT/(CFU⁰_mutant/CFU⁰_WT)], where: CFU^x is the colony count at the end of week x, whereas CFU⁰ is the colony count at initial inoculation of a mixed culture. The direct competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.

To validate this assay in a control experiment, we compared the fitness of the WT strain BY4742 (His^-; the parental strain of the long-lived mutant strains 3, 5 and 12) to that of the WT strain BY4739 (His⁺, but otherwise isogenic to BY4742). We found that even after 6 consecutive 7-days incubations BY4742 (His^-) exhibits similar relative fitness in a direct competition assay with BY4739 (His⁺) co-cultured in YP medium initially containing the following carbon source: 1) 0.2% glucose, after cell transfer from 0.2% glucose (Figure 7A); 2) 2% glucose, after cell transfer from 2% glucose (Figure 7B); 3) 1% ethanol, after cell transfer from 0.2% glucose (Figure 7C); 4) 1% ethanol, after cell transfer from 2% glucose (Figure 7D); or 5) 1% ethanol, after cell transfer from 1% ethanol (Figure 7E). Based on these findings, we concluded that the developed direct competition assay outlined in Figure 6 accurately reproduces the expected equal fitness of each of the two WT strains used, i.e. BY4739 (His⁺) and BY4742 (His^-). Moreover, this assay also accurately reproduces the reduced fitness [73,89] of the mutant strain dbp3Δ (which is isogenic to the WT strain BY4742) in direct competition with the parental WT strain BY4739 (His⁺) (Figure 7).

Figure 7. Validation of the developed assay for quantifying the relative fitness of a long-lived mutant strain in direct competition with a parental WT strain. The WT strains BY4742 (His^-) and BY4739 (His⁺, but otherwise isogenic to BY4742) were cultured separately in the complete YP medium containing 0.2% glucose, 2% glucose or 1% ethanol glucose until mid-exponential phase. Another pair of strains whose relative fitness was measured, namely the long-lived mutant strain dbp3Δ (His^-; is isogenic to BY4742) and the WT strain BY4739 (His⁺), was also cultured separately in YP medium containing 0.2% glucose 2% glucose or 1% ethanol glucose until mid-exponential phase. Cells of the His⁺ strain were mixed with the same number of cells of the His^- strain and then co-cultured for 7 days in liquid YP medium initially containing different carbon sources. Cells of the His^- and His⁺ strains pre-cultured separately on 0.2% glucose were subjected to direct fitness competition by being cultured together on 0.2% glucose (A) or 1% ethanol (C). Cells of the His^- and His⁺ strains pre-cultured separately on 2% glucose were subjected to direct fitness competition by being cultured together on 2% glucose (B) or 1% ethanol (D). Cells of the His^- and His⁺ strains pre-cultured separately on 1% ethanol were subjected to direct fitness competition by being cultured together on 1% ethanol (E). After culturing the cell mixture for 7 days, an aliquot of cell suspension was used to measure the relative fitness of the His⁺ strain in direct competition with the His^- strain (as described in ″Materials and Methods″). The direct fitness competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.

Dominant polygenic trait extending longevity of each of the 3 long-lived yeast mutants decreases its relative fitness under some laboratory conditions

We used the developed direct competition assay to measure the relative fitness of the long-lived mutant strain 3, 5 or 12 in direct competition with a parental WT strain. Cells of each of these mutant strains were first cultured separately in liquid YP medium containing different concentrations of glucose or ethanol. Cells of each mutant strain were then mixed with the same number of cells of the WT strain BY4739 (His⁺, but otherwise isogenic to the parental WT strain BY4742) and underwent 6 consecutive 7-days incubations together. We found that the dominant polygenic trait extending longevity of the long-lived mutant strain 3, 5 or 12 does not alter its relative fitness in a direct competition assay with the parental WT strain co-cultured in medium initially containing one of the following carbon sources: 1) 0.2% glucose, after cell transfer from 0.2% glucose (Figures 8A, 9A and 10A, respectively); or 2) 2% glucose, after cell transfer from 2% glucose (Figures 8B, 9B and 10B, respectively). In contrast, the dominant polygenic trait extending longevity of the long-lived mutant strain 3, 5 or 12 decreased its relative fitness in a direct competition assay with the parental WT strain co-cultured in medium initially containing 1% ethanol, after cell transfer from any of the following carbon sources: 1) 0.2% glucose (Figures 8C, 9C and 10C, respectively); 2) 2% glucose (Figures 8D, 9D and 10D, respectively); or 3) 1% ethanol (Figures 8E, 9E and 10E, respectively).

Figure 8. Dominant polygenic trait extending longevity of the long-lived yeast mutant 3 decreases its relative fitness in medium initially containing 1% ethanol. The WT strains BY4742 (His^-) and BY4739 (His⁺, but otherwise isogenic to BY4742) were cultured separately in the complete YP medium containing 0.2% glucose, 2% glucose or 1% ethanol glucose until mid-exponential phase. Another pair of strains whose relative fitness was measured, namely the long-lived mutant strain 3 (His^-; selected during lasting exposure of BY4742 to LCA) and the WT strain BY4739 (His⁺), was also cultured separately in YP medium containing 0.2% glucose 2% glucose or 1% ethanol glucose until mid-exponential phase. Cells of the His⁺ strain were mixed with the same number of cells of the His^- strain and then co-cultured for 7 days in liquid YP medium initially containing different carbon sources. Cells of the His^- and His⁺ strains pre-cultured separately on 0.2% glucose were subjected to direct fitness competition by being cultured together on 0.2% glucose (A) or 1% ethanol (C). Cells of the His^- and His⁺ strains pre-cultured separately on 2% glucose were subjected to direct fitness competition by being cultured together on 2% glucose (B) or 1% ethanol (D). Cells of the His^- and His⁺ strains pre-cultured separately on 1% ethanol were subjected to direct fitness competition by being cultured together on 1% ethanol (E). After culturing the cell mixture for 7 days, an aliquot of cell suspension was used to measure the relative fitness of the His⁺ strain in a direct competition with the His^- strain (as described in ″Materials and Methods″). The direct fitness competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.

Figure 9. Dominant polygenic trait extending longevity of the long-lived yeast mutant 5 decreases its relative fitness in medium initially containing 1% ethanol. The WT strains BY4742 (His^-) and BY4739 (His⁺, but otherwise isogenic to BY4742) were cultured separately in the complete YP medium containing 0.2% glucose, 2% glucose or 1% ethanol glucose until mid-exponential phase. Another pair of strains whose relative fitness was measured, namely the long-lived mutant strain 5 (His^-; selected during lasting exposure of BY4742 to LCA) and the WT strain BY4739 (His⁺), was also cultured separately in YP medium containing 0.2% glucose 2% glucose or 1% ethanol glucose until mid-exponential phase. Cells of the His⁺ strain were mixed with the same number of cells of the His^- strain and then co-cultured for 7 days in liquid YP medium initially containing different carbon sources. Cells of the His^- and His⁺ strains pre-cultured separately on 0.2% glucose were subjected to direct fitness competition by being cultured together on 0.2% glucose (A) or 1% ethanol (C). Cells of the His^- and His⁺ strains pre-cultured separately on 2% glucose were subjected to direct fitness competition by being cultured together on 2% glucose (B) or 1% ethanol (D). Cells of the His^- and His⁺ strains pre-cultured separately on 1% ethanol were subjected to direct fitness competition by being cultured together on 1% ethanol (E). After culturing the cell mixture for 7 days, an aliquot of cell suspension was used to measure the relative fitness of the His⁺ strain in a direct competition with the His^- strain (as described in ″Materials and Methods″). The direct fitness competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.

Figure 10. Dominant polygenic trait extending longevity of the long-lived yeast mutant 12 decreases its relative fitness in medium initially containing 1% ethanol. The WT strains BY4742 (His^-) and BY4739 (His⁺, but otherwise isogenic to BY4742) were cultured separately in the complete YP medium containing 0.2% glucose, 2% glucose or 1% ethanol glucose until mid-exponential phase. Another pair of strains whose relative fitness was measured, namely the long-lived mutant strain 12 (His^-; selected during lasting exposure of BY4742 to LCA) and the WT strain BY4739 (His⁺), was also cultured separately in YP medium containing 0.2% glucose 2% glucose or 1% ethanol glucose until mid-exponential phase. Cells of the His⁺ strain were mixed with the same number of cells of the His^- strain and then co-cultured for 7 days in liquid YP medium initially containing different carbon sources. Cells of the His^- and His⁺ strains pre-cultured separately on 0.2% glucose were subjected to direct fitness competition by being cultured together on 0.2% glucose (A) or 1% ethanol (C). Cells of the His^- and His⁺ strains pre-cultured separately on 2% glucose were subjected to direct fitness competition by being cultured together on 2% glucose (B) or 1% ethanol (D). Cells of the His^- and His⁺ strains pre-cultured separately on 1% ethanol were subjected to direct fitness competition by being cultured together on 1% ethanol (E). After culturing the cell mixture for 7 days, an aliquot of cell suspension was used to measure the relative fitness of the His⁺ strain in a direct competition with the His^- strain (as described in ″Materials and Methods″). The direct fitness competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.

Our findings revealed that the conditions of pre-culturing of any of the 3 long-lived mutant strains do not influence the extent of its decreased relative fitness during the subsequent co-culturing with the parental WT strain in medium initially containing 1% ethanol (Figures 8C-8E, 9C-9E, 10C-10E). We therefore concluded that none of these long-lived mutant strains keeps a ″memory″ of conditions under which it has been grown prior to being mixed with the parental WT strain in medium supplemented with 1% ethanol for fitness competition.

Acknowledgements

We are grateful to current and former members of the Titorenko laboratory for discussions. We acknowledge the Centre for Structural and Functional Genomics at Concordia University for outstanding service.

Conflicts of Interest

The authors have no conflict of interests to declare.

Funding

This study was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and Concordia University Chair Fund. P.K. was supported by the Fonds québécois de la recherche sur la nature et les technologies Doctoral Research Fellowship Award. V.I.T. is a Concordia University Research Chair in Genomics, Cell Biology and Aging.

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