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
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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.
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