Molecular Microbiology (2001) 39(5), 1166±1173

Aged mother cells of Saccharomyces cerevisiae show

markers of oxidative stress and apoptosis
Peter Laun,1² Alena Pichova,2² Frank Madeo,3² Introduction
JoÈrg Fuchs,4 Adolf Ellinger,5 Sepp Kohlwein,6
Yeast mother cell-specific ageing has been intensively
Ian Dawes,7 Kai-Uwe FroÈhlich3 and Michael
researched and reviewed (Jazwinski, 1999; Johnson et al.,
Breitenbach1*
1 1999; Guarente, 2000) in the last few years. This simple
Department of Genetics, University of Salzburg,
model system for cellular and, perhaps, organismic
Hellbrunnerstr. 34, A-5020 Salzburg, Austria.
2 ageing shows similarity to ageing in higher cells in terms
Institute of Microbiology, Czech Academy of Sciences,
of well-characterized morphological and physiological
Prague, Czech Republic.
3 changes. Old cells are much bigger than young cells
Department of Physiological Chemistry, University of
(Mortimer and Johnston, 1959; Egilmez et al., 1990;
TuÈbingen, Germany.
4 Nestelbacher et al., 1999), the cell cycle as well as protein
Department of Botany, University of Vienna, Austria.
5 synthesis is slowed down (Mortimer and Johnston, 1959;
Department of Histology and Embryology, Ordinariat II,
Motizuki and Tsurugi, 1992), and the cell surface has a
University of Vienna, Austria.
6 loose and wrinkled appearance (Pichova et al., 1997).
Department of Biochemistry, Technical University of
The median life span of most laboratory Saccharomyces
Graz, Austria.
7 cerevisiae strains is about 25±35 generations or about
School of Biochemistry and Molecular Biology, UNSW,
3 days (Jazwinski, 1993). Given the short life span of
Sydney, Australia.
yeast cells and the ease of genetic analysis in yeast, it is
feasible to elucidate general mechanisms of ageing by
Summary studying the yeast ageing process.
In recent years, Sinclair and Guarente (1997) proposed
Recently, we and others have shown that genetic and
the hypothesis that DNA minicircles derived from chro-
environmental changes that increase the load of
mosomal rDNA but lacking centromeres accumulate in
yeast cells with reactive oxygen species (ROS) lead
mother cells and eventually prevent ordered replication
to a shortening of the life span of yeast mother cells.
and cell cycle progression by titrating out an essential
Deletions of yeast genes coding for the superoxide
DNA-binding factor, thus leading to senescence. These
dismutases or the catalases, as well as changes
minicircles have a strong tendency to accumulate in the
in atmospheric oxygen concentration, considerably
mother cell (Sinclair and Guarente, 1997; Heo et al.,
shortened the life span. The presence of the physio-
1999) because they cannot be segregated regularly on
logical antioxidant glutathione, on the other hand,
the mitotic spindle. However, so far, rDNA minicircles
increased the life span of yeast cells. Taken together,
have not been demonstrated in senescent cells of
these results pointed to a role for oxygen in the yeast
multicellular organisms. Many different hypotheses have
ageing process. Here, we show by staining with
been put forward to explain cellular and organismic
dihydrorhodamine that old yeast mother cells iso-
ageing (Finch, 1990) including, for example, that ageing
lated by elutriation, but not young cells, contain ROS
may be caused by the accumulation of somatic mutations
that are localized in the mitochondria. A relatively
in senescent cells. These theories are, of course, not
large proportion of the old mother cells shows
mutually exclusive. Moreover, almost certainly, several
phenotypic markers of yeast apoptosis, i.e. TUNEL
independent causes can contribute to the ageing process.
(TdT-mediated dUTP nick end labelling) and annexin
As we have argued previously, an accumulation of
V staining. Although it has been shown previously
mutations in genomic DNA can be ruled out as a cause
that apoptosis in yeast can be induced by a cdc48
of yeast ageing because of its mother cell specificity
allele, by expressing pro-apoptotic human cDNAs or
(Nestelbacher et al., 2000). On the other hand, it is quite
by stressing the cells with hydrogen peroxide, we are
possible that damaged cellular material other than DNA
now showing a physiological role for apoptosis in
could be decisive for yeast ageing provided that this
unstressed but aged wild-type yeast mother cells.
damaged material is inherited asymmetrically.
Cellular materials can be damaged by oxygen radicals
Accepted 11 December, 2000. *For correspondence. E-mail
Michael.Breitenbach@sbg.ac.at; Tel. (143) 662 8044 5787; Fax or oxidizing molecules originating from a `leaky' respira-
(143) 662 8044 144. ²These authors contributed equally to this work. tory chain when those radicals are no longer detoxified by

Q 2001 Blackwell Science Ltd


the cell (Halliwell and Gutteridge, 1989). This `oxygen
theory of ageing', first formulated by Harman (1962), has
gained increased acceptance based on recent experi-
mental results (Ames et al., 1993; Orr and Sohal, 1994;
Sohal and Weindruch, 1996; Gems, 1999). In particular, it
has been found that longevity genes of the nematode
Caenorhabditis elegans, whose biochemical functions
were previously unknown, are now shown to play a role
in the detoxification of oxygen radicals (Gems, 1999;
Taub et al., 1999).
Recently, we and others have shown that genetic and
environmental changes that increase the burden of
reactive oxygen species (ROS) on cells of the yeast S.
cerevisiae lead to a shortening of the life span of mother
cells. Deletions of yeast genes coding for the superoxide
dismutases (Barker et al., 1999; Wawryn et al., 1999) or
the catalases, as well as changes in atmospheric oxygen
partial pressure and the presence of the physiological
antioxidant glutathione, all had the expected distinct
effects on life span and indicated a role for oxygen in
the yeast ageing process (Nestelbacher et al., 2000). We
therefore sought to show in a more direct way that old
yeast mother cells accumulate oxidizing molecules and
build up internal oxidative stress in the absence of any
external stress condition.
Here, we present biochemical evidence that old mother
cells, but not virgin cells, contain strongly oxidizing
molecules originating from the mitochondria. On standard
media, old cells suffer oxidative stress in the absence of
any external source of oxygen radicals. These cells show
markers of apoptosis, including positive terminal deox-
ynucleotidyl transferase-mediated dUTP end labelling
(TUNEL) and annexin V tests and chromatin fragmen-
tation seen after 4 0 ,6-diamidino-2-phenylindole dihydro-
chloride (DAPI) staining. Apoptosis has been shown to
occur in certain yeast mutants and in wild-type cells when
they are oxidatively stressed (Madeo et al., 1997; 1999;
Frohlich and Madeo, 2000). The present work describes
yeast apoptosis in ageing wild-type cells. This is the first
report of a physiological role for apoptosis in yeast. Thus,
we present another strong parallel between yeast ageing
and the ageing processes of cultured human cells
that have recently been shown to undergo apoptosis
induced by oxidative stress (P. Jansen-DuÈrr, personal
communication).
Results
Isolation of large yeast cells by elutriation allows
enrichment of very old cells
The haploid wild-type yeast strain JC482 (Pichova et al.,
1997) was grown and treated as described in Experi-
mental procedures for the isolation of large and small cells
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1166±1173
Aged yeast mother cells undergo apoptosis 1167
by elutriation centrifugation. The large cells obtained after
1 day (about 10 generations) of growth in 100 ml of YPD
were cultivated again for 1 day in 100 ml of YPD, and
large cells were again isolated by elutriation. Fraction V
contained large undamaged cells, whereas fraction II
contained very uniform small cells. The total yield of large
cells was about 1.6  108 cells. These two fractions were
characterized in detail.
Fraction V cells were oval with a major axis diameter of
10±15 mm, and fraction II cells were rounder with a
diameter of about 5±7 mm, as determined by phase-
contrast microscopy. The median life span of fraction V
cells was 4 ^ 3.5 generations, whereas the median life
span of fraction II cells was 18 ^ 3.8 generations, similar
to the standard life span for this strain of 23 ^ 6
generations (Fig. 1). The small difference between the
life span of fraction II and the standard life span of the
strain is statistically significant. Note that about 70% of
the fraction II cells were virgins, and the rest showed one
or two bud scars, which would account for the difference
in life span. About 35% of the large cells (fraction V)
entered but did not complete a single cell division,
although they were not lysed or damaged as shown by
fluorescence microscopy (see below, Fig. 6) and thin-
section electron microscopy (data not shown). Some of
the cells in fraction V were actually young, as shown by
the `tail' of long-lived cells in the life span analysis of
fraction V cells (Fig. 1). These cells are the few daughters
that did not separate in the last cell cycle but were still
adhering to their mothers.
Figure 2 shows representative views of the fraction V
and fraction II cells after Calcofluor White staining. Some
of the fraction V cells showed a large number of bud scars
(Fig. 2A). Note that only half of each cell's surface can be
Fig. 1. Life spans of elutriated wild-type yeast cell fractions of strain
JC482 were determined as described in Experimental procedures.
The error bars indicate standard deviations from the median. Forty
randomly chosen cells from fraction V and fraction II were
analysed. In fraction V, only one of 40 cells never budded. In
fraction II, six out of 40 cells never budded and were excluded from
the calculation. The standard life span of strain JC482 was
determined as described previously (Pichova et al., 1997).
1168 P. Laun et al.
seen in the micrographs, so the real number of bud scars
is probably about twice as great as the number of bud
scars visible in Fig. 2A. However, it is also clear from
Fig. 2A that some of the large cells have only about 5±10
bud scars. We hypothesize that these are already
senescent, as the standard life span of the strain shows
that a certain fraction of mothers is senescent after every
cell generation. It is also obvious from Fig. 2A that some
of the old cells carry unseparated daughters, which
themselves have several bud scars. This is an indication
of cell cycle irregularities during the last cell divisions of a
mother cell, as can be seen by comparison with the
micrographs shown in our previous papers (Pichova et al.,
1997; Nestelbacher et al., 2000). The cells in fraction II
were mostly daughter cells showing a dark birth scar but
no bud scars, although fraction II also contained some
cells with one or two bud scars (Fig. 2B).

Old yeast mother cells contain ROS in the absence of

external oxidative stress

Figure 3 shows that strongly oxidizing molecules (ROS)

were detected by dihydrorhodamine 123 (DHR) staining in
the mitochondria of fraction V cells (Fig. 3A) but not in
fraction II cells (Fig. 3C). For comparison, the staining
with 2-[4-(dimethylamino)styryl]-1-methylpyridinium iodide
(DASPMI) indicates the location of mitochondria in these
cells (Fig. 3E and G). Double staining with DHR and
DASPMI was not possible for technical reasons. How-
ever, the morphology shown in Fig. 3 is clearly indicative
of a mitochondrial location for the strongly oxidizing
molecules. Mitochondria of young cells do not contain
measurable levels for strongly oxidizing molecules
(Fig. 3C and G). As these cells have been grown in
YPD under standard conditions and have never been
exposed to external oxidative stress apart from that found
in a normal aerobically grown cell, we must assume that
the strongly oxidizing molecules that are detected in
Fig. 3A arise internally, either through accumulation by a
Fig. 3. A. Fraction V cells were stained with DHR (5 mg ml21; stock
solution 2.5 mg ml21 in ethanol) and viewed and photographed
under a confocal laser-scanning fluorescence microscope after
10 min using the rhodamine filter set. The stained cells show
typical mitochondrial morphology.
B. The same sample in phase contrast.
C. Fraction II cells treated as above show only very weak staining.
D. The same sample as in (C) viewed in phase contrast.
E. Fraction V cells stained with the mitochondrial-specific dye,
DASPMI (25 mg ml21), and immediately viewed under a confocal
laser-scanning fluorescence microscope using the fluorescein filter
set.
Fig. 2. A. Fraction V cells after staining with Calcofluor White M2R F. The same sample shown in phase contrast.
and viewing under a fluorescence microscope. The budding pattern G. Fraction II cells stained with DASPMI and viewed under a
on mother cells is typical of a haploid cell. Irregular patterns are fluorescence microscope.
seen on some contaminating daughters (see text). H. The same cells as in (G) shown in phase contrast.
B. Fraction II cells treated exactly as in (A). The majority of these Taken together, Fig. 3 shows that old cells (fraction V), but not
cells are virgins with just one (dark) birth scar. The cells are smaller young cells, exhibit strongly oxidizing molecules and that these
than in (A). molecules are contained in the mitochondria.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1166±1173


Fig. 4. A. Fraction V cells after staining of nuclei with DAPI. Note
diffuse chromatin and, occasionally, multiple nuclei.
B. Fraction II cells treated exactly as above. Note compact well-
defined nuclei.
Fig. 5. A. Fraction V cells were fixed, cell walls were digested and
strand breaks in DNA were detected according to the TUNEL
protocol (Madeo et al., 1997). Nuclei containing large amounts of
DNA strand breaks were stained black by the diaminobenzidine±
H2O2 reaction after incorporation of fluorescein isothiocyanate
(FITC)-labelled dUTP and treatment with anti-FITC antibody Fab
fragment from sheep coupled with horseradish peroxidase. Viewing
500 cells, positive staining was observed in about 20% of them. In
some cases (arrows), mother and daughter cells from a pair were
both TUNEL positive, indicating that the last daughters of old
apoptotic mother cells are sometimes also apoptotic.
B. Fraction II cells were treated and stained as in (A). Practically no
TUNEL-positive staining was observed.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1166±1173
Aged yeast mother cells undergo apoptosis 1169
Fig. 6. A. Fraction V cells were stained for exposed
phosphatidylserine with FITC-conjugated annexin V after digestion
of cell walls with glusulase/lyticase and viewed under a
fluorescence microscope using the fluorescein filter set (Madeo
et al., 1997).
B. The same sample washed and stained with propidium iodide
and viewed using the fluorescein filter set. This control shows that
the annexin-positive cells in (A) are not lysed or damaged.
C. Fraction II cells stained for phosphatidylserine with FITC-
conjugated annexin V and viewed under a fluorescence microscope
using the fluorescein filter set. A sample with a very infrequently
observed annexin-positive cell (arrow) is shown.
D. The same sample stained with propidium iodide showing that
the marked cell is lysed (arrow).
E. The same sample as in (C) and (D) shown in phase contrast.
Also here, it is obvious that the marked cell is lysed (arrow).
`leaky' respiratory chain or triggered by the physiological
process of ageing.
Old yeast mother cells display markers of apoptosis
Figure 4 shows the nuclear morphology of fraction V cells
(Fig. 4A) and fraction II cells (Fig. 4B). The young cells
display compact single nuclei of normal appearance,
whereas the old cells showed diffuse staining indicating
chromatin fragmentation similar to that seen when
apoptosis is induced by hydrogen peroxide in yeast cells
(see Fig. 1 in Madeo et al., 1999). Some of these old cells
showed more than one nucleus as a result of endomitosis
caused by irregular cell division cycles as shown earlier
(Pichova et al., 1997).
Figures 5 and 6 illustrate our analysis of apoptotic
landmarks in fraction V cells with young cells (fraction II)
included as important controls to exclude methodological
artifacts. Positively stained cells were detected using
DHR, TUNEL and annexin V tests in about 20% of the
cells for each method. It is impossible for technical
1170 P. Laun et al.
reasons to perform double staining by these methods;
however, the complete absence of positively staining cells
in fraction II strongly indicates that it is the same
subfraction of old cells that stain positively with all three
fluorescent dyes.
Figure 5A and B shows that old cells and not young
cells are stained by the TUNEL method that detects DNA
strand breaks. Many of the TUNEL-positive cells carry
one or more unseparated daughters that are also TUNEL
positive (arrow), as if those cells could not complete the
last or the last few cell cycles, thus corroborating the
finding that about 35% of the fraction V cells start but do
not complete one cell cycle (Fig. 1).
Figure 6 illustrates the results of fluorescent staining
with the annexin V method. The old cells of fraction V
were frequently stained at the cell periphery after
digestion of the cell wall (Fig. 6A), indicating plasma
membrane inversion (externalized phosphatidylserine,
which is an established marker for early apoptotic events).
The same cells showed no staining with propidium iodide
(the usual control), indicating that these cells are not
unspecifically damaged (Fig. 6B). On the contrary, frac-
tion II cells were not stained by this method (Fig. 6C).
Very infrequently, young cells were observed that were
stained, but they were also stained by propidium iodide,
indicating that they had lysed as a result of unspecific
damage. The damaged cell is indicated by an arrow in
Fig. 6C±E.
Discussion
Here, we present a morphological and physiological
characterization of aged yeast mother cells that were
isolated by a new method. These cells are very close to
the terminal senescent state, as shown by determination
of their remaining median life span. Our data presented
here, together with earlier work (Nestelbacher et al., 2000)
showing that the physiological antioxidant, reduced
glutathione, can substantially increase the life span of
yeast cells under conditions of oxidative stress, strongly
support the notion that ROS originating in the mitochon-
dria are a causative factor in senescence. The present
data also show that senescent yeast mother cells undergo
apoptosis.
The method developed by us for the isolation of old
yeast mother cells depends on only one parameter (cell
size) and is limited by the fact that it is impossible
completely to avoid contamination by some young cells
(virgins) that adhere to their mother cells (Fig. 1). In spite
of this limitation, the life span determinations performed
with the size-fractionated old and young cells showed that
the old cells have a remaining median life span of only
4 ^ 3.5 generations in a strain whose standard median
life span is 23 ^ 6 generations. The young cells isolated
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1166±1173
in the same experiment have a median life span that
closely resembles the standard life span of the strain,
thereby excluding the possibility that the elutriation
method itself has any influence on the life span of the
cells or is grossly altering their physiology. About 35% of
the old cells (fraction V) do not complete the first cell cycle
that they enter. We assume that these are cells under-
going senescence. These cells are not lysed or damaged
by the elutriation method, as shown by light microscopy,
fluorescence microscopy with propidium iodide and
electron microscopy of ultrathin sections (data not
shown). A similar fraction of the old cell preparation
(about 20%) shows the presence of strongly oxidizing
molecules in the mitochondria and markers of yeast
apoptosis. Fraction V contains many cells with more than
20 bud scars (Fig. 2A). This fraction also contains a small
proportion of about 10% of younger cells, as seen by the
`tail' of longer lived cells in the life span determination
(Fig. 1). These are daughters that were still attached to
their mothers during the elutriation as well as an
unavoidable subfraction of young cells with a larger
volume. Taken together, these experimental results
strongly support the conclusion that we have indeed
isolated a population of cells that is very close to
senescence and that the population is sufficiently
enriched in senescent cells to enable a biochemical
study of the phenomenon of senescence, which is crucial
for understanding the ageing process. In order to confirm
that these markers of oxidative stress and apoptosis are
not simply caused by the intensity of respiration, we also
stained cells of strain JC482 growing exponentially on
glycerol as well as on glucose as the only carbon source.
Neither mother nor daughter cells stained positively with
DHR under exactly the same conditions that were used
for staining fraction V (not shown in detail). This is in good
agreement with earlier observations that non-fermentable
carbon sources (glycerol) certainly do not shorten the life
span (Egilmez et al., 1990; Barker et al., 1999).
In order to show that the phenotype of old mother cells
described here does not depend on the genetic back-
ground of the strain, it was important to repeat the
elutriation experiment with a second unrelated haploid
MATa strain (BY4742). It was also seen for this strain that
old mother cells, but not young cells, exhibited ROS in
their mitochondria, diffuse or fragmented nuclei and many
of those cells showed . 20 bud scars (data not shown in
detail).
Other methods for isolating old yeast mother cells have
been published (Egilmez et al., 1990; Woldringh et al.,
1995; Smeal et al., 1996). Using the method presented
here, the residual life span of the mother cells is very short
(only about 10% of the total life span of the strain), and a
substantial fraction of the old cells are post-mitotic and
senescent. Old mother cells and young cells are isolated

from the same initial population of batch-grown cells
(see Experimental procedures) that are only minimally
influenced physiologically by the separation method.
These cells are clearly not killed by an artifact of the
method.
We have used these cells to search for phenotypic
markers of oxidative stress and apoptosis and, in addition,
to characterize them with respect to cell surface and
nuclear morphology. The results have provided strong
support for the theory that oxidative stress is implicated in
the cell ageing process. How do these findings fit into the
general hypotheses that are presently in vogue to explain
ageing in both yeast and higher organisms? Could the
oxygen theory of ageing for which we have provided
evidence here and the theory that is based on the
formation of rDNA minicircles as a primary cause of
ageing be part of the same general picture?
In principle, one possible, but unlikely, explanation
is that the perturbation of replication caused by the
accumulation of rDNA circles leading to a titration out of
essential protein factors (Johnson et al., 1999) could
indirectly lead to a perturbation of gene expression and
therefore to an increase in the load of cells with ROS. This
is improbable, because the correlation of ROS with ageing
is a very general one, as it has also been demonstrated in
cells of higher eukaryotes, whereas rDNA minicircles
have been found only in ageing yeast cells, not in ageing
cells of humans, flies or nematodes. Another, more likely
explanation is that the autogenous oxidative stress
observed by us indirectly leads to the accumulation of
rDNA minicircles. This could be the case, for instance via
an effect of ROS on recombination and repair. Intrachro-
mosomal recombination, the process that leads to rDNA
minicircles, has been shown to be induced by oxidative
mutagens (Brennan et al., 1994).
The two processes could even be interdependent:
oxidative stress could cause a moderate increase in rDNA
minicircles that could lead in a vicious circle to more
oxidative stress and more minicircles in the mother cell.
Finally, the two processes could be independent of each
other and both contribute to yeast ageing.
The morphology of the structures stained with DHR
clearly indicates a mitochondrial location for the ROS.
This fits excellently with a large number of investigations
of oxidative ageing in higher cells and points to a parallel
between the ageing processes of yeast and higher cells. It
has been shown that, in higher cells, ROS (oxidative
stress) are key regulators of apoptosis (Hockenbery et al.,
1993; Kane et al., 1993; Ghibelli et al., 1995; Greenlund
et al., 1995) and that primary human cells in culture
undergo oxidative stress and apoptosis when they enter
senescence (Jansen-DuÈrr, personal communication). Our
observation of about 20% TUNEL-positive and annexin V-
positive cells in the preparation of old, but not young,
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1166±1173
Aged yeast mother cells undergo apoptosis 1171
yeast cells (Figs 5 and 6) therefore further strengthens
the oxygen theory of ageing and the generality and
similarity of yeast and higher cell ageing finally leading to
apoptosis. It is important that apoptosis has now been
found in wild-type cells as a physiological process in the
absence of external oxidative stress. The existence of
apoptosis in yeast is now well established (for a review,
see Ink et al., 1997; Frohlich and Madeo, 2000), but it has
only been described previously in special mutants (cdc48-
S565G) or in wild-type cells incubated in the presence of
H2O2.
A final point concerns the origin of mother cell
specificity of ageing in the context of the findings
presented here. Mother cell specificity of yeast ageing
implies that some `death factor' (Jazwinski, 1990) must be
inherited asymmetrically by the mother cell. In the
framework of the hypothesis presented here, this death
factor could be damaged cellular material. One very
plausible candidate for damaged material would be
damaged mitochondria, which produce an increasing
amount of ROS. Mitochondria producing ROS are found
only in senescent cells, not in virgin cells (Fig. 3).
Consequently, we speculate that old (pre-existing and
damaged) mitochondria should be inherited preferentially
by the mother cell in any cell division, whereas newly
synthesized mitochondria should be segregated to the
daughter cell. The ordered inheritance of mitochondria
in yeast during cell division has been well studied
(Warren and Wickner, 1996; Yaffe, 1999; Catlett and
Weisman, 2000), although the question whether the
mother cell inherits old mitochondrial components is
presently unanswered.
Experimental procedures
Yeast strains and media
A wild-type yeast strain (JC 482; Pichova et al., 1997) was
used for all experiments. Some of the experiments were
repeated using a second strain, BY4742 (Brachmann et al.,
1998). Liquid YPD (2% glucose, 1% yeast extract, 2%
peptone in H2O) was used for growing yeast cells. Life spans
were determined on SC agar (2% agar, 2% glucose, 0.17%
yeast nitrogen base, 0.5% ammonium sulphate). Media
components were from Gibco.
Elutriation
Cells were separated according to their diameter using the
Beckman elutriation system and rotor JE-6B with a standard
elutriation chamber. Before the separation, the cells were
grown in 100 ml of YPD medium at 288C on a rotary shaker
for 24 h. Then, the cells were harvested at 3000 r.p.m. and
resuspended in 1 PBS buffer (8 g of NaCl, 0.2 g of KCl,
1.44 g of Na2HPO4, 0.24 g of KH2PO4, pH 7.4, in a total
volume of 1 l) at 48C. The elutriation chamber was loaded
1172 P. Laun et al.
with 4.2 ml of cell suspension corresponding to about 109
cells. To separate cell fractions with different diameters, the
chamber was loaded at a flow rate of 10 ml min21 and a rotor
speed of 3200 r.p.m. Cells with a diameter , 5 mm were
elutriated (fraction I). To collect fraction II (diameter 5±7 mm),
the flow rate was set to 15 ml min21 and rotor speed to
2700 r.p.m. Fraction III (diameter 7±8.5 mm) was elutriated
at 2400 r.p.m., fraction IV (diameter 8.5±10 mm) at
2000 r.p.m. and, finally, fraction V (diameter 10±15 mm) at
1350 r.p.m. The quality of separation of particular fractions
was verified microscopically.
Calcofluor staining
Calcofluor staining of cell walls was carried out as described
previously (Streiblova et al., 1984), using Calcofluor White
M2R (Sigma F-3397).
DAPI staining
The basic protocol for DAPI staining (Streiblova, 1988) of
nuclei was used. Cells were collected, resuspended in 70%
(v/v) ethanol for brief fixation and permeabilization, stained
with DAPI solution and observed under an epifluorescence
microscope (Zeiss Axioskop).
DHR and DASPMI staining
Free intracellular radicals or strongly oxidizing molecules
(ROS) were detected with DHR (Sigma D1054) as described
previously (Madeo et al., 1999). We noticed that it was
necessary to take pictures after no longer than 10 s laser
irradiation to avoid photodamage. Excitation at 488 nm was
used. Cells were viewed through a rhodamine optical filter.
Mitochondria were visualized with DASPMI (2-[4-(dimethyla-
mino)styryl]-1-methylpyridinium iodide; Sigma-Aldrich 280135)
using the fluorescein filter set described previously (McConnell
et al., 1990).
The samples were viewed using a Leica TCS 4D confocal
laser-scanning microscope, and pictures were taken using a
Hamamatsu video system.
Annexin V and TUNEL
Exposed phosphatidylserine was detected by annexin V
labelling (Madeo et al., 1997). DNA strand breaks were
demonstrated by TUNEL (Madeo et al., 1997) using the
Boehringer Mannheim in situ cell death detection kit (POD)
and observed with a Zeiss Axioskop microscope.
Life span procedure
Life span analysis was performed as described previously
(Pichova et al., 1997).
To determine the remaining life span of fraction II and
fraction V cells, cohorts of 40 randomly chosen cells per
fraction were taken directly after elutriation and, for each cell,
the number of remaining cell cycles was determined by
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1166±1173
micromanipulation. Cells that never budded were excluded
from analysis.
Statistical analysis of life span data
The standard deviations of the median life spans at a
confidence level of 95% were calculated by applying Kaplan±
Meier statistics (Kaplan and Meier, 1958). To decide whether
two given survival distributions were significantly different
at a 95% confidence level, Breslow, Tarone±Ware and log-
rank statistics were used. All statistical calculations were
performed using the software package SPSS 9.0.
Acknowledgements
This work has been supported by grant no. P14574-MOB
(FWF Austria; to M.B.), an ARC IREX grant to I.W.D. and
M.B., and grant GA CR(CZ) GA204/97/0541 to A.P. We are
grateful to the Faculty of Sciences of Salzburg University for
making available the Axioskop fluorescence microscope.
References
Ames, B.N., Shigenaga, M.K., and Hagen, T.M. (1993)
Oxidants, antioxidants, and the degenerative diseases of
aging. Proc Natl Acad Sci USA 90: 7915±7922.
Barker, M.G., Brimage, L.J., and Smart, K.A. (1999) Effect of
Cu,Zn superoxide dismutase disruption mutation on repli-
cative senescence in Saccharomyces cerevisiae. FEMS
Microbiol Lett 177: 199±204.
Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J.,
Hieter, P., and Boeke, J.D. (1998) Designer deletion strains
derived from Saccharomyces cerevisiae S288C: a useful
set of strains and plasmids for PCR-mediated gene
disruption and other applications. Yeast 14: 115±132.
Brennan, R.J., Swoboda, B.E., and Schiestl, R.H. (1994)
Oxidative mutagens induce intrachromosomal recombina-
tion in yeast. Mutat Res 308: 159±167.
Catlett, N.L., and Weisman, L.S. (2000) Divide and multiply:
organelle partitioning in yeast. Curr Opin Cell Biol 12: 509±
516.
Egilmez, N.K., Chen, J.B., and Jazwinski, S.M. (1990)
Preparation and partial characterization of old yeast cells.
J Gerontol 45: B9±B17.
Finch, C.E. (1990) Longevity, Senescence and the Genome.
Chicago: University of Chicago Press.
Frohlich, K.U., and Madeo, F. (2000) Apoptosis in yeast ± a
monocellular organism exhibits altruistic behaviour. FEBS
Lett 473: 6±9.
Gems, D. (1999) Putting metabolic theories to the test. Curr
Biol 9: R614±R616.
Ghibelli, L., Nosseri, C., Coppola, S., Maresca, V., and Dini,
L. (1995) The increase in H2O2-induced apoptosis by ADP-
ribosylation inhibitors is related to cell blebbing. Exp Cell
Res 221: 470±477.
Greenlund, L.J., Korsmeyer, S.J., and Johnson, E.M., Jr
(1995) Role of BCL-2 in the survival and function of
developing and mature sympathetic neurons. Neuron 15:
649±661.

Guarente, L. (2000) Sir2 links chromatin silencing, meta-
bolism, and aging. Genes Dev 14: 1021±1026.
Halliwell, B., and Gutteridge, J.M.C. (1989) Free Radicals in
Biology and Medicine, 2nd edn. Oxford: Oxford University
Press.
Harman, D. (1962) Role of free radicals in mutation, cancer,
aging and maintenance of life. Radical Res 16: 752±763.
Heo, S.J., Tatebayashi, K., Ohsugi, I., Shimamoto, A.,
Furuichi, Y., and Ikeda, H. (1999) Bloom's syndrome
gene suppresses premature ageing caused by Sgs1
deficiency in yeast. Genes Cells 4: 619±625.
Hockenbery, D.M., Oltvai, Z.N., Yin, X.M., Milliman, C.L., and
Korsmeyer, S.J. (1993) Bcl-2 functions in an antioxidant
pathway to prevent apoptosis. Cell 75: 241±251.
Ink, B., Zornig, M., Baum, B., Hajibagheri, N., James, C.,
Chittenden, T., and Evan, G. (1997) Human Bak induces
cell death in Schizosaccharomyces pombe with morpholo-
gical changes similar to those with apoptosis in mammalian
cells. Mol Cell Biol 17: 2468±2474.
Jazwinski, S.M. (1990) Aging and senescence of the budding
yeast Saccharomyces cerevisiae. Mol Microbiol 4: 337±
343.
Jazwinski, S.M. (1993) The genetics of aging in the yeast
Saccharomyces cerevisiae. Genetica 91: 35±51.
Jazwinski, S.M. (1999) Molecular mechanisms of yeast
longevity. Trends Microbiol 7: 247±252.
Johnson, F.B., Sinclair, D.A., and Guarente, L. (1999)
Molecular biology of aging. Cell 96: 291±302.
Kane, D.J., Sarafian, T.A., Anton, R., Hahn, H., Gralla, E.B.,
Valentine, J.S., et al. (1993) Bcl-2 inhibition of neural death:
decreased generation of reactive oxygen species. Science
262: 1274±1277.
Kaplan, E.L., and Meier, P. (1958) Nonparametric estimation
from incomplete observations. Am Stat Assoc J 53: 457±
481.
McConnell, S.J., Stewart, L.C., Talin, A., and Yaffe, M.P.
(1990) Temperature-sensitive yeast mutants defective in
mitochondrial inheritance. J Cell Biol 111: 967±976.
Madeo, F., Frohlich, E., and Frohlich, K.U. (1997) A yeast
mutant showing diagnostic markers of early and late
apoptosis. J Cell Biol 139: 729±734.
Madeo, F., Frohlich, E., Ligr, M., Grey, M., Sigrist, S.J., Wolf,
D.H., and Frohlich, K.U. (1999) Oxygen stress: a regulator
of apoptosis in yeast. J Cell Biol 145: 757±767.
Mortimer, R.K., and Johnston, J.R. (1959) Life span of
individual yeast cells. Nature 183: 1751±1752.
Q 2001 Blackwell Science Ltd, Molecular Microbiology, 39, 1166±1173
Aged yeast mother cells undergo apoptosis 1173
Motizuki, M., and Tsurugi, K. (1992) The effect of aging on
protein synthesis in the yeast Saccharomyces cerevisiae.
Mech Ageing Dev 64: 235±245.
Nestelbacher, R., Laun, P., and Breitenbach, M. (1999)
Images in experimental gerontology. A senescent yeast
mother cell. Exp Gerontol 34: 895±896.
Nestelbacher, R., Laun, P., Vondrakova, D., Pichova, A.,
Schuller, C., and Breitenbach, M. (2000) The influence of
oxygen toxicity on yeast mother cell-specific aging. Exp
Gerontol 35: 63±70.
Orr, W.C., and Sohal, R.S. (1994) Extension of life-span by
overexpression of superoxide dismutase and catalase in
Drosophila melanogaster. Science 263: 1128±1130.
Pichova, A., Vondrakova, D., and Breitenbach, M. (1997)
Mutants in the Saccharomyces cerevisiae RAS2 gene
influence life span, cytoskeleton, and regulation of mitosis.
Can J Microbiol 43: 774±781.
Sinclair, D.A., and Guarente, L. (1997) Extrachromosomal
rDNA circles ± a cause of aging in yeast. Cell 91: 1033±
1042.
Smeal, T., Claus, J., Kennedy, B., Cole, F., and Guarente, L.
(1996) Loss of transcriptional silencing causes sterility in
old mother cells of S. cerevisiae. Cell 84: 633±642.
Sohal, R.S., and Weindruch, R. (1996) Oxidative stress,
caloric restriction, and aging. Science 273: 59±63.
Streiblova, E. (1988) Cytological methods. In Yeast ± a
Practical Approach. Campbell, J., and Buffers, J.M. (eds).
Oxford: IRL Press, pp. 9±49.
Streiblova, E., Hasek, J., and Jelke, E. (1984) Septum
pattern in ts mutants of Schizosaccharomyces pombe
defective in genes cdc3, cdc4, cdc8 and cdc12. J Cell Sci
69: 47±65.
Taub, J., Lau, J.F., Ma, C., Hahn, J.H., Hoque, R., Rothblatt,
J., and Chalfie, M. (1999) A cytosolic catalase is needed to
extend adult life span in C. elegans daf-C and clk-1
mutants. Nature 399: 162±166.
Warren, G., and Wickner, W. (1996) Organelle inheritance.
Cell 84: 395±400.
Wawryn, J., Krzepilko, A., Myszka, A., and Bilinski, T. (1999)
Deficiency in superoxide dismutases shortens life span of
yeast cells. Acta Biochim Pol 46: 249±253.
Woldringh, C.L., Fluiter, K., and Huls, P.G. (1995) Production
of senescent cells of Saccharomyces cerevisiae by
centrifugal elutriation. Yeast 11: 361±369.
Yaffe, M.P. (1999) The machinery of mitochondrial inheri-
tance and behavior. Science 283: 1493±1497.