FEMS Yeast Research 3 (2003) 201^209 www.fems-microbiology.org Chronological and replicative lifespan of polyploid Saccharomyces cerevisiae (syn. S. pastorianus) Dawn L. Maskell a , Alan I. Kennedy b , Je¡ A. Hodgson b , Katherine A. Smart a a; School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane, Headington, Oxford OX3 0BP, UK b Scottish Courage Brewing Limited, Technical Centre, Sugarhouse Close, 160 Canongate, Edinburgh EH8 8DD, UK Received 31 May 2002 ; received in revised form 12 November 2002; accepted 13 November 2002 First published online 12 December 2002 Abstract Chronological lifespan may be defined as the result of accumulation of irreversible damage to intracellular components during extended stationary phase, compromising cellular integrity and leading to death and autolysis. In contrast, replicative lifespan relates to the number of divisions an individual cell has undertaken before entering a non-replicative state termed senescence, leading to cell death and autolysis. Both forms of lifespan have been considered to represent models of ageing in higher eukaryotes, yet the relation between chronologically and replicatively aged populations has not been investigated. In this study both forms of lifespan have been investigated in Saccharomyces cerevisiae (Syn. S. pastorianus) to establish the relationship between chronological and replicative ageing. 1 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Ageing; Chronological lifespan ; Replicative lifespan; Saccharomyces pastorianus; Starvation 1. Introduction It has recently been suggested that the yeast Saccharomyces cerevisiae can be considered to have two distinct lifespans: replicative and chronological [1,2]. Replicative lifespan in Saccharomyces species is determined by the number of divisions completed by each individual cell, and is genetically controlled [3^7] but in£uenced by environment [5,6,8^13]. Once the cell has divided a predetermined number of times it enters the state of cellular senescence, permanently losing the capacity to replicate, and eventually dies [6]. The number of divisions completed prior to entering senescence, or yeast replicative lifespan, is strain-speci¢c [3,5,10^12,14,15] but usually conserved within the range of 9^33 divisions [9]. However, the progression from youth to old age is invariably accompanied by several biomarkers (Table 1) including increasing bud scar number [6,14,16,17,19], increasing cell size [6,14,16,17,19] (Fig. 1), granulation [14], wrinkling of the cell surface [6,14,21] (Fig. 1) and increased generation time [6,14,19]. * Corresponding author. Tel. : +44 (1865) 483248; Fax : +44 (1865) 483242. E-mail address : [email protected] (K.A. Smart). Over 20 genes have been implicated in the ageing mechanism of S. cerevisiae (reviewed by Jazwinski [22] ; Table 2). Some of these genes have been identi¢ed as being involved in sensing of nutritional status [26], stress response [23], DNA repair [31] and the cell cycle [24,32], whereas the function of other genes remains unknown [22,39]. In contrast, chronological lifespan has been de¢ned as the long-term survival of cells maintained in stationary phase and has been postulated to represent a valuable tool for the monitoring of long-term macromolecular damage and mortality [1,2,40^44]. In this case, cell cycle progression is interrupted during G1 due to nutrient limitation, and entry into G0 (stationary phase) results [45^ 47]. G0 is characterised by a cessation of net increase in population concentration (cell number) [48^51]; entry into this phase is accompanied by the accumulation of storage carbohydrates such as glycogen and trehalose, which may be subsequently utilised as a source of carbon or as a stress protectant, respectively [52^56]. In some cases chronologically ageing cells may remain viable for many months [13,57^59]. Progression through chronological lifespan has been poorly de¢ned; however, many intermediate stages of cellular deterioration appear to occur [13] (Fig. 2). Indeed chronological ageing of polyploid strains of S. cerevisiae (ale yeast) demonstrates an initial reversible physiological 1567-1356 / 02 / $22.00 1 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-1356(02)00199-X FEMSYR 1536 27-2-03 202 D.L. Maskell et al. / FEMS Yeast Research 3 (2003) 201^209 Table 1 Biomarkers for ageing in S. cerevisiae (adapted from Powell et al. [9]) Characteristic Observed change Reference Cell size Bud scar number Vacuole size Protein synthesis Generation time Cell shape Cell turgor Cell wall chitin Wrinkling of cell surface Granular appearance Telomere length ERCs Speci¢c gene expression rRNA levels Protein synthesis Ribosome activity Cellular rRNA concentration increase increase increase decrease increase altered decrease increase increase [6,14,17^19] [6,14,16,17,19] [19] [20] [6,14,19]. [14] [21] [19] [6,14,21] [14] [22] [23] [3] [20] [20] [20] [20] none increase altered increase decrease decrease decrease deterioration, characterised by a loss of cellular vigour and the accumulation of damaged cellular macromolecules [13,60]. This phase tends to be accompanied by the utilisation of intracellular [61] and wall [13,60,62,63] carbohydrates. Prolonged chronological ageing results in irreversible modi¢cations to phenotype that may be lethal or result in the formation of permanent phenotypic alterations in the cell population [63], including modi¢cations in cell wall structure and composition [62]. In the event that no nutrients are provided at this stage of deteriora- Fig. 1. Cell lineages of S. pastorianus strain SCB2 demonstrating the increase in cell size from newly budded to ageing mother. Lineages were produced using micromanipulation. The divisional age of each cell is indicated. The size bar represents 10 Wm. The photograph has been digitally manipulated to align the individual yeast cells and for clarity. tion, the cell enters a state where replicative potential is permanently lost, yet metabolic activity is retained [13]. Cells exhibiting this post-mitotic phase are ‘senescent’ since they cannot re-enter the cell cycle even when supplied with nutrients [13]. It has been suggested that replicative and chronological yeast lifespan may not be distinct [1], since suspension in stationary phase may be correlated with a reduction in replicative lifespan [64,65] and cells which pass through replicative or chronological lifespan enter a post-mitotic or ‘senescent’ state [13]. Furthermore, replicative and chronological ageing have only been investigated for strains belonging to the species S. cerevisiae. Recent reclassi¢cation of brewing yeast strains based upon physiological and nutritional attributes, biochemical features and DNA homology [66] has led to the assignment of ale strains to the species S. cerevisiae and lager strains to the species S. cerevisiae (syn. pastorianus) [67]. Table 2 Genes associated with longevity in S. cerevisiae Gene Function Reference AGE BUD1 CDC25 CDC35 CDC7 FOB1 GPA2 GPR1 LAG1 LAG2 PHB1 PHB2 RAD52 RAD9 RAS1 RAS2 RTG2/3 SGS1 stress response G-protein, cell polarity GDP^GTP exchange factor for RAS adenylate cyclase cell cycle control replication block G-protein glucose-binding protein unknown unknown mitochondrial protein homologue of PHB1 DNA repair cell cycle nutritional status nutritional status unknown retrograde response DNA recombination DNA helicase unknown N-myristoylprotein rDNA repair gene silencing unknown transcriptional silencing Kennedy et al. [23] Jazwinski et al. [24] Lin et al. [25] Sun et al. [26] ; Lin et al. [25] Jazwinski et al. [24] Defossez et al. [27] Lin et al. [25] Lin et al. [25] D’mello et al. [28] Jazwinski [29] Coates et al. [30] Coates et al. [30] Park et al. [31] Kennedy et al. [32] Sun et al. [26] Sun et al. [26] Kirchman et al. [33] Sinclair et al. [34] Sinclair and Guarente [35] Ashra¢ et al. [36] Kennedy et al. [23] Kennedy et al. [23] Kennedy et al. [37] Roy and Runge [38] SIP2 SIR2/3/4 SIR4 UTH4 ZDS1/2 Adapted from Jazwinski [22]. FEMSYR 1536 27-2-03 D.L. Maskell et al. / FEMS Yeast Research 3 (2003) 201^209 Stress Cell Replication Stress Response Go Entry Loss of Replicative Capacity Lifespan Progression Post-Mitotic State Senescence Death Fig. 2. Mortality model for Saccharomyces species (adapted from Smart [13]). Here the relationship between chronological and replicative longevity for the species S. cerevisiae (syn. S. pastorianus) is demonstrated. 2. Materials and methods 2.1. Yeast strains Four lager production strains of S. cerevisiae (syn. S. pastorianus) designated SCB1, SCB2, SCB3 and SCB4 were obtained from Scottish Courage Technical Centre, Edinburgh. 2.2. Media and growth conditions Each strain was maintained on YPD (2% w/v glucose, 2% w/v bacteriological peptone and 1% w/v yeast extract). Where required 1.2% agar was added. When stationaryphase cultures were required cells were grown in 100 ml YPD at 25‡C and 250 rpm for 72 h. Length of time to stationary phase for each strain was determined by growth curves on YPD and con¢rmed by the optical density at 600 nm. The speci¢c growth rates obtained con¢rmed those previously reported for these strains [12]. 2.3. Micromanipulation YPD plates no more than 5 mm thick were inoculated with a single yeast colony and incubated for 48 h at 25‡C. The resultant microcolony was then examined using a Zeiss Axioscope microscope with a long working-distance 40U objective lens, by viewing through the Petri dish and agar. Cells were manipulated by using a micromanipulation glass needle. Virgin cells were isolated by the separation of newly formed buds away from midsized mother cells. Careful monitoring of cell cycle progression and subsequent separation of newly generated daughter cells al- 203 lowed the development from virgin to aged mother cells to be investigated. Plates were incubated at 25‡C during the day and 4‡C overnight to decrease growth rate and prevent excessive division. Where necessary, ¢lter paper soaked in sterile deionised water was placed in the lid of each Petri dish to prevent desiccation of the media. More than 65 cells were monitored for each strain [6]. Light micrographs were obtained using an Axioscope ¢xed stage microscope (Carl Zeiss, Germany) with a Yashica Multiprogramme 109 camera attachment. 2.3.1. Data analysis The data obtained from the micromanipulation were expressed as the mean, maximum, and maximum10% (mean Hay£ick limit of the longest-lived upper decile of the population) according to the method of Barker and Smart [6]. In addition to this, the standard deviation for each sample group was calculated. Replicative and chronological mortality pro¢les were statistically analysed and signi¢cance was demonstrated using one-way analysis of variance followed by the Tukey^Kramer test for Gaussian populations. Where the populations were not Gaussian (or a Gaussian approximation) non-parametric analysis was used (Kruskal^Wallis test) followed by Dunn’s multiple comparison test. Signi¢cance was demonstrated when the probability was less than 5%. 2.4. Chronological ageing Chronological ageing was induced by the inoculation of stationary-phase cells (grown for 72 h in YPD), which were washed twice in phosphate-bu¡ered saline (PBS), into 100 ml sterile deionised water in 250-ml Erlenmeyer £asks to a ¢nal cell concentration of 1U106 cells ml31 . The £asks were incubated in orbital shakers at 25‡C and 250 rpm for 50 days; £asks were sampled every 5 days for viability according to the method of Ashra¢ et al. [64]. 2.5. Production of sucrose gradients Gradients were prepared in 50-ml skirted centrifuge tubes; 22.5 ml of 10% (w/v) sucrose were layered upon 22.5 ml of 30% (w/v) sucrose. These gradients were stored at 4‡C for 48 h to produce 45-ml linear 10^30% sucrose gradients. 2.5.1. Production of virgin and non-virgin populations Stationary-phase cells were sonicated and washed twice in 0.1 M PBS. Cells were resuspended to an optimum cell concentration of 5U108 cells ml31 in PBS. Final cell concentration was determined in triplicate using a haemocytometer. Of this suspension 1-ml aliquots were layered onto the surface of the sucrose gradients. Following centrifugation at 1300 rpm for 5 min (4‡C) banding could be observed within the gradient. The upper portion contained FEMSYR 1536 27-2-03 D.L. Maskell et al. / FEMS Yeast Research 3 (2003) 201^209 the virgin population; the lower portion consisted of mother cells of varying ages. These portions were then washed twice and resuspended in PBS. Purity of the population was determined by confocal microscopy (LSM 410 inverted laser scanning confocal microscope) using £uorescein isothiocyanate-labelled wheatgerm agglutinin (SigmaAldrich, Poole, UK) at a concentration of 1 mg ml31 according to the method of Powell et al. [68], to enumerate the bud scars present on the cell surface and con¢rm the age of the population. Viability (%) 204 100 90 80 70 60 50 40 30 20 10 0 SCB1 SCB2 SCB3 SCB4 0 10 20 30 40 50 60 Divisional age at senescence Fig. 3. Replicative mortality pro¢les of four lager brewing yeast strains grown on YP with 2% (w/v) glucose. Adapted from Maskell et al. [12]. 2.6. Viability assessment 2.6.1. Citrate methylene violet Citrate methylene violet (Sigma, Aldrich, UK) solution was prepared according to the method of Smart et al. [69] ; 0.5 ml of yeast suspension was mixed by vortexing with 0.5 ml of citrate methylene violet and examined microscopically after 5 min. 2.6.2. Bis 1,3-dibutylbarbituric acid trimethine oxonol (DiBAC4 , oxonol) Oxonol (Molecular Probes, Eugene, OR, USA) solution was prepared according to the method of Lloyd and Dinsdale [70] ; 0.5 ml of yeast suspension was mixed by vortexing with one drop of oxonol and examined using a £uorescence microscope (Carl-Zeiss, Germany) after 5 min. 2.6.3. Plate counts Of a 1U103 cells ml31 yeast suspension 0.1 ml was spread on YPD agar plates and incubated for 48 h at 25‡C. After 48 h the plates were removed and the number of colony-forming units were evaluated. 3. Results and discussion 3.1. Replicative lifespan of lager brewing yeast Although yeast replicative lifespan has been demonstrated to be strain-dependent [3^5,55,71] previous investigations have indicated that S. pastorianus lager brewing yeast strains exhibit similar mean replicative lifespans due to their common ancestry and therefore closely related genotypes [9,10]. The four lager strains investigated in this study, however, demonstrated unique ageing pro¢les (Table 3, Fig. 3) the mean and maximum10% lifespans of which were signi¢cantly di¡erent (P s 0.05), with the exception of the comparison between SCB2 and SCB4. 3.2. The chronological lifespan of lager brewing yeast Previous chronological ageing studies using haploid strains have employed either plate counts [1,40,42,43] or the LIVE/DEATH test (FUN1) (Molecular Probes, OR, USA) [44] and thus have not demonstrated the permanent loss of replicative capacity which is characteristic of both replicative and chronological ageing. In this study the occurrence of the chronological senescence state was considered using three determinants of life potential: the citrate methylene violet assay [69], the oxonol exclusion assay [70] and plate counts. Methylene violet determines viability through the reduction of the dye by metabolically active cells, oxonol is an anionic £uorescent dye, which is excluded by functional plasma membranes and therefore re£ects membrane integrity, and plate counts represent a quantitative method of determining the replicative capacity of cells within a population. Independent of strain it was observed that replicative capacity was reduced by up to 90% during the ¢rst 5 days of chronological ageing. This reduced level of replicative capacity was maintained for between 10 and 35 days depending on the strain assessed and then declined until no replicative capacity remained (Fig. 4a). However, cellular reduction capacity (Fig. 4c) and membrane integrity (Fig. 4b) exceeded replicative capacity (P 6 0.05) indicating the generation of viable cells that exhibited a permanent deactivation of replicative potential and were therefore termed ‘senescent’ due to their post-mitotic phenotype. The state of senescence is poorly understood in Saccha- Table 3 Mean and maximum replicative lifespans for four strains of lager brewing yeast grown on YP with 2% (w/v) glucose Strain Total cells Mean lifespan Maximum10% lifespan Maximum lifespan SCB1 SCB2 SCB3 SCB4 122 165 98 125 15.51 U 7.20 9.54 U 4.83 26.43 U 11.60 12.36 U 5.45 27.83 U 1.70 18.17 U 3.91 45.40 U 5.46 20.46 U 3.23 32 29 55 28 Data are expressed with standard deviation as appropriate. Adapted from Maskell et al. [12]. FEMSYR 1536 27-2-03 D.L. Maskell et al. / FEMS Yeast Research 3 (2003) 201^209 a % Replication Competent 1000 SCB1 SCB2 SCB3 SCB4 100 10 1 0 10 20 30 40 50 60 Chronological Age (Days) % Membrane Integrity b 1000 SCB1 SCB2 SCB3 SCB4 100 10 1 0 10 20 30 40 50 60 Chronological Age (Days) % Reduction Competent c 1000 SCB1 SCB2 SCB3 SCB4 100 10 0 10 20 30 40 50 60 Chronological Age (Days) Fig. 4. Changes in viability during chronological ageing of four strains of lager brewing yeast demonstrated by their (a) replication competence, (b) membrane integrity and (c) intracellular reductive capacity. Triplicate samples were taken every 5 days for 50 days for analysis. romyces species, yet it is clear that this post-mitotic phase of lifespan is progressive with an altered pattern of gene expression [72,73] including the initiation of death metabolism. In this study chronologically aged senescent populations appeared to demonstrate a loss of membrane integrity prior to a loss of intracellular reducing power. This observation is consistent with the hypothesis that dying cells exhibit residual reducing power due to the retained activity of cellular enzymes and therefore longevities assessed in this manner re£ect enzyme activity rather than cellular viability per se. Thus lifespan is a function of the capacity to divide and a loss of replicative potential equates to senescence onset. 3.3. Is there a correlation between replicative and chronological lifespan? replicative ageing in S. cerevisiae [1,2,40^44], although the only quantitative comparison between chronological and replicative longevity previously reported involved the mutant laboratory haploid strain Bky1-14c. Cells of this strain were starved by plating onto media without nitrogen or carbon sources for 8 days after which an extension in replicative lifespan was observed. These long-lived mutants were more resistant to heat shock and demonstrated an enhanced ability to grow on ethanol [23] ; however the relationship between chronological and replicative ageing in wild-type ‘normal’ strains was not demonstrated. From the literature three haploid strains of S. cerevisiae were identi¢ed for which both chronological and replicative lifespan has been independently determined (Table 4). No correlation between these parameters was apparent (r = 30.36). Therefore in these strains there does not appear to be a relationship between replicative and chronological lifespan despite the fact that both re£ect replicative potential. Comparisons between chronological and replicative longevities of the four lager brewing strains of S. cerevisiae (syn. S. pastorianus) (Table 4) demonstrated a positive but not statistically signi¢cant correlation between chronological longevity and mean (r = 0.938), maximum10% (r = 0.875) or maximum (r = 0.937) replicative lifespans. The reasons for this lack of correlation are not known; however, recent observations suggest that the deletion of genes reducing replicative longevity [30] does not a¡ect chronological longevity [74] in the same strain, implying that the genetic control of both forms of ageing may be unrelated. Interestingly, replicative lifespan potential considers the divisional capacity of virgin cells, whereas chronological lifespan potential has previously been investigated by using age-heterogeneous populations [1,2,23,40^44,74] comprising virgin cells (approximately 50%), one-division-old cells (approximately 25%), two-division-old cells (approximately 12.5%) and so on. It is postulated that older cells may exhibit di¡ering chronological longevities compared to virgin cells, in part determined by their remaining but reduced replicative potential. Table 4 Replicative and chronological lifespans of haploid laboratory strains of S. cerevisiae and brewing strains of S. cerevisiae (syn. S. pastorianus) Strain Chronological lifespan (days) Mean replicative lifespan (divisions) EG103 EG223 W303-1a SCB1 SCB2 SCB3 SCB4 50a 15a 20c 35 25 50 20 18.18b 18.33b 24.2d 15.51 9.54 16.43 12.36 a [24]. [55]. c [1]. d [23]. b Chronological ageing has been suggested as a model for 205 FEMSYR 1536 27-2-03 206 D.L. Maskell et al. / FEMS Yeast Research 3 (2003) 201^209 It has previously been demonstrated that virgin populations of S. cerevisiae may display di¡erent stress tolerances compared to those populations which contain only mother cells or comprise a mixed-age population (Van Zandycke and Smart, unpublished data). It is therefore suggested that previous chronological lifespan studies have not considered the inherent replicative fecundities of mixed-age populations, despite the evidence which suggests that replicative potential is indeed limited in Saccharomyces species [3,5,6,10,14,32,39]. To examine the impact of replicative age on chronological longevity potential, the chronological lifespans of virgin and non-virgin populations were investigated using age-synchronised populations of the lager strain SCB2. Age-heterogeneous (Fig. 4), virgin and non-virgin (Fig. 5) populations lost their replicative capacity during chronological ageing yielding postmitotic populations demonstrating senescence. During the ¢rst 5 days of G0 maintenance, replicative capacity was reduced by up to 45%. This reduced level of replicative potential was maintained until day 15 for virgin (Fig. 5a) and day 20 for non-virgin (Fig. 5b) populations, respectively. Replicative capacity for all populations was permanently lost following 50 days chronological ageing. The non-virgin population (Fig. 5b) exhibited impaired membrane integrity following 20 days chronological ageing, a decrease in cellular reduction capacity was not observed until 35 days had been completed. In contrast, vir- Viability (%) a 1000 CMV Oxonol Plate Count 100 10 1 0 10 20 30 40 50 60 Chronological Age (Days) Viability (%) b 1000 CMV Oxonol Plate Count 100 10 1 0 10 20 30 40 50 60 Chronological Age (Days) Fig. 5. Viability of (a) a pure virgin population and (b) a non-virgin population of S. pastorianus, strain SCB2, which were suspended in water for 50 days. Triplicate samples were taken for analysis every 5 days. Viability (%) 3.4. Does replicative age a¡ect chronological longevity ? 100 90 80 70 60 50 40 30 20 10 0 SCB2 Day 0 Day 10 Day 20 Day 30 0 2 4 6 8 10121416182022242628303234363840 Divisional Age at Senescence Fig. 6. Replicative mortality pro¢les of strain SCB2 and chronologically aged populations of SCB following chronological ageing for 0, 10, 20 and 30 days. Replicative longevities were determined using micromanipulation on YP with glucose, 2% (w/v). gin cells did not exhibit impaired membrane integrity (Fig. 5a) or reductive capacity during the incubation period examined, indicating a di¡erent post-mitotic longevity for these cells. It is suggested that the older mother cells exhibit more cellular damage and potentially an impaired defence, compared to their younger counterparts, and that this in turn adversely a¡ects their longevity potential. It is further postulated that younger cells are more tolerant to the starvation stresses imposed during chronological ageing than older mothers. Furthermore a yeast strain demonstrating a high chronological lifespan may also exhibit a greater tolerance to environmental stress, perhaps as a result of a more e⁄cient STRE-activated global stress response or a greater capacity to withstand and repair stressinduced damage. This hypothesis remains the subject of further investigation. 3.5. Do chronologically aged lager brewing yeast cells demonstrate a reduced replicative lifespan? Where a cell has permanently lost replicative potential no re-entry into the cell cycle can occur (Figs. 4 and 5). However, it has also been previously demonstrated by Ashra¢ et al. [64] that chronologically aged haploid cells of a laboratory strain of S. cerevisiae exhibit a reduced replicative capacity. By inference, short-term starvation may not merely temporarily suspend progress through the cell cycle but may also reduce the number of replications permitted once nutrients are restored. To verify this observation in other Saccharomyces species a virgin cell population was obtained from a stationary-phase culture of S. cerevisiae (syn. S. pastorianus) strain SCB2, and subjected to chronological ageing. At 10-day intervals chronologically aged virgin cells were harvested and their replicative longevity potential monitored using micromanipulation to determine mean, maximum10% and maximum longevities. The corresponding heterogeneous population (control) exhibited a unique mortality pro¢le that appeared to di¡er from the mortality pro¢le exhibited by SCB2 virgin populations acquired through FEMSYR 1536 27-2-03 D.L. Maskell et al. / FEMS Yeast Research 3 (2003) 201^209 207 Table 5 Replicative lifespan potential of chronologically aged virgins (strain SCB2) Control Day 0 Day 10 Day 20 Day 30 Total cells Mean lifespan Maximum10% lifespan Maximum lifespan 165 88 83 85 91 9.54 U 4.83 4.07 U 4.48 3.83 U 4.34 2.25 U 2.58 1.714 U 2.95 18.17 U 3.91 13.22 U 2.11 11.75 U 0.88 8.25 U 1.28 8.78 U 1.30 29 17 13 11 11 sucrose gradient separation (Fig. 6). However, statistical analysis demonstrated that they were not signi¢cantly different (P s 0.05). It was observed that chronological ageing (10, 20 and 30 days) impaired replicative longevity potential (Table 5), yielding signi¢cantly di¡erent (P 6 0.001) mean longevities compared to the control. Thus extended G0 maintenance during chronological ageing shortens replicative lifespan in a progressive manner until no replicative potential remains. This observation implies that replicative potential may be a function of resilience to stress. Therefore it is postulated that a yeast strain demonstrating a high replicative lifespan may also exhibit a greater tolerance to environmental stress. Indeed oxidative stress resistance has been demonstrated to play an important role in replicative [8,15,75^77] and chronological [2,40,42] lifespans. However, the means by which chronological ageing reduces replicative lifespan is not known. Ashra¢ et al. [64] have postulated that chronologically aged mother cells display accelerated replicative ageing despite an apparent cessation in the accumulation of extrachromosomal ribosomal DNA circles (ERC’s). It was proposed that chronologically aged cells either accumulate an alternative senescence factor or become increasingly sensitised to the resumption of ERC accumulation once replicative lifespan continues [64]. From our study it appears that the accumulation of a senescence factor may be the more likely reason for this apparent impairment of replicative potential. Furthermore, we suggest that the apparent acceleration of the replicative ageing process exhibited by chronologically aged cells may be due to the accumulation of cellular damage during G0 maintenance, resulting in a progressively impaired phenotype. In support of this theory, it was observed in this study that the time taken to complete the ¢rst round of replication following chronological ageing increased, although this time period could not be readily quanti¢ed. Iida and Yahara [78] have previously demonstrated that arrests in the cell cycle cause subsequent delays in the onset of DNA synthesis upon the shift back to proliferating conditions. Interestingly, the length of delay appeared to be positively correlated with the duration of cell cycle arrest in both studies, and this would be consistent with a requirement to repair accumulated damage before up-regulating cell cycle progression. 4. Conclusions Replicative and chronological longevities in Saccharomyces species are strain-dependent. Both forms of ageing are characterised by a progressive deterioration in replicative potential that culminates in a post-mitotic phenotype that may be termed senescence. Both forms of post-mitotic cells exhibit surface wrinkling [6,13] and an increased cell size [6,13]. Moreover, chronologically aged cells exhibit impaired replicative longevities and vice versa, although they appear to be distinctly regulated since no statistical correlation between them can be determined. It is postulated that replicative and chronological longevities are related to tolerance to stress and the e¡ectiveness of stress damage repair. Indeed the time required to progress through the cell cycle in the presence of nutrients is related to the duration of G0 maintenance and during the latter stages of replicative lifespan to the divisional age of the mother [6]. The common and disparate mechanisms underlying chronological and replicative longevity remain the subject of further investigation. Acknowledgements Dawn Maskell is funded by the J and J Morison Scholarship and the authors would like to thank Mrs Pamela Morison-Inches for her support. Katherine Smart is the Scottish Courage Reader in Brewing Science and gratefully acknowledges the support of Scottish Courage Brewing Limited. Katherine Smart is a Royal Society Industrial Fellow and is grateful to the Royal Society for supporting her fellowship. The authors are also grateful to the directors of Scottish Courage Brewing Limited for permission to publish this work. References [1] Maclean, M., Harris, N. and Piper, P. (2001) Chronological lifespan of stationary phase yeast cells; a model for investigating the factors that might in£uence the ageing of post mitotic tissues in higher organisms. Yeast 18, 499^509. [2] Harris, N., Maclean, M., Hatzianthis, K., Panaretou, B. and Piper, P.W. 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