A novel assay for replicative lifespan in Saccharomyces cerevisiae
Stefanie Jarolima,1, Jonathan Millenb,1, Gino Heerena, Peter Launa,
David S. Goldfarbb, Michael Breitenbacha,*
aDepartment of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria
bDepartment of Biology, University of Rochester, Rochester, NY 14627, USA
Received 16 March 2004; received in revised form 8 June 2004; accepted 9 June 2004
First published online 20 August 2004
The replicative lifespan of Saccharomyces cerevisiae is determined by both genetic and environmental factors. Many of the same
factors determine the lifespan of metazoan animals. The lack of fast and reliable lifespan assays has limited the pace of yeast aging
research. In this study we describe a novel strategy for assaying replicative lifespan in yeast, and apply it in a screening of mutants
that are resistant to pro-oxidants. The assay reproduces the lifespan-shortening effects of deleting SIR2 and of growth in the pres-
ence of paraquat, a pro-oxidant. The lifespan-increasing activity of resveratrol is also reproduced. Compared to current assays, this
new strategy promises to significantly increase the possible number of replicative-lifespan determinations.
? 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Saccharomyces cerevisiae; Yeast; Longevity; Aging; Replicative lifespan
Vegetatively dividing Saccharomyces cerevisiae cells
are mortal. Mother cells produce a limited number of
daughter cells before they begin to exhibit signs of func-
tional senescence, cease replicating and, ultimately, die
. Mortimer and Johnston  were the first to count
the maximum number of daughters produced by indi-
vidual mother cells, using micromanipulation to remove
the daughters before they themselves replicated. In this
fashion it was determined, and later verified, that the
number of daughter cells a population of mother cells
produce is empirically described by the Gompertz sur-
vival function, which is commonly used to describe the
increased tendency of older animals to die (reviewed in
). Interest in the replicative lifespan of S. cerevisiae in-
creased when it became apparent that the aging of fungi,
worms, and flies held some promise for revealing the
molecular mechanisms of cellular aging of humans
[4,5]. Another approach to the study of aging in yeast,
called chronological aging, measures the length of time
stationary-phase cells can survive in depleted medium.
In contrast, replicative aging does not depend on calen-
dar time. This property is a boon to experimentalists
who can refrigerate the cells overnight without affecting
their replicative capacity . The relative merits of repli-
cative and chronological aging assays, and their respec-
tive relevance to human aging, have been discussed
elsewhere [7–9]. Chronological and replicative aging in
yeast are related because events in stationary phase af-
fect subsequent replicative lifespan in rich medium and
many of the same genes affect both modes of aging
Mother cell-specific replicative aging of yeast cells is
based on the asymmetric division of mother and daugh-
1567-1356/$22.00 ? 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
*Corresponding author. Tel.: +43 662 8044 5787;
fax: +43 662 8044 144.
E-mail address: firstname.lastname@example.org (M. Breitenbach).
1Both authors contributed equally.
FEMS Yeast Research 5 (2004) 169–177
ter cells. The clock for daughters is generally reset to
zero, although daughters of older mothers, which repli-
cate more slowly, have reduced lifespans . Presuma-
bly, mother cells age and die because they become
damaged. The chemical agents of the damage, the
macromolecular targets, and the cell processes that are
directly and indirectly affected are incompletely under-
stood. Oxidative stress has been proposed as having a
significant effect on lifespan [12,13]. Down-regulation
of glucose signalling pathways also increases oxidative
stress resistance by mechanisms dependent on proteins
such as superoxide dismutases and catalases. These en-
zymes normally promote growth and increase protection
against oxidative stress and the mutation of their genes
in yeast decreases replicative lifespan [14–17]. Tied into
these pathways are genes controlling the mitochondrial
retrograde response pathway [18,19]. The disruption of
the signalling pathways that control caloric restriction
in yeast increases lifespan by a mechanism which
according to some authors depends on the histone
deacetylase SIR2 , but according to others does
not . Sir2p is a conserved NAD+-dependent histone
deacetylase that plays an important role in gene silenc-
ing, and promotes long life in S. cerevisiae and Caenor-
habditis elegans . Sir2p activity appears to be
controlled by enzymes that regulate the concentration
of nicotinamide and/or NADH, both of which act as
Sir2p inhibitors. Pnc1p positively regulates Sir2p-medi-
ated silencing and longevity by preventing the accumu-
lation of nicotinamide during stress [20,23,24]. Others
argue that it is the concentration of the inhibitor NADH
that regulates Sir2p activity via the NAD+/NADH ratio
. Extrachromosomal DNA minicircles (ERCs) as
well as damaged mitochondrial macromolecules like
protein carbonyls, which are products of oxidative
stress, are candidate ‘‘death factors’’ since they accumu-
late in older mothers and are not inherited by their
daughters, except in sir2 mutants [12,26].
The replicative lifespan of a yeast strain is typically
described by the median lifespan (‘‘n’’), which can vary
widely among laboratory strains, but is typically around
20 generations. In any population of logarithmically
replicating cells, only a small fraction (1/2n) is senescent.
Research in the field of yeast mother cell-specific aging
has been hampered by the fact that old cells are rare
in any population and there is no direct and easy way
to efficiently separate old (terminally senescent) mother
cells from young cells . The purification methods
that were tried include gradient purification, immobili-
zation on magnetic beads [27–29] and elutriation centrif-
ugation . However, in most cases the preparation of
old cells still contains young cells which adhere to their
mothers due to the peculiarities of yeast cytokinesis .
An even more efficient purification would be necessary
for the construction of a genetic selection system for
long-lived mutants. The development of faster, higher-
throughput, and less labor-intensive replicative lifespan
assays would greatly facilitate the study of aging in
yeast. In this study we describe such an assay and apply
it in a screen for long-lived mutants that are resistant to
2. Materials and methods
2.1. Yeast strains
Mutant strain K6001 in W303 background is marked
MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15,
ura3::URA3 GAL-ubiR-CDC6 (at URA3) . K6001
sir2D was produced by replacing the SIR2 gene with a
kanamycin resistance marker (kanR) cassette.
SIR2 plasmid: SIR2 was amplified via PCR from
genomic DNA isolated from K6001 using primers with
EcoRI and HindIII restriction sites. The PCR product
was integrated between the HindIII and EcoRI restric-
tion site in plasmid pRK2 . All purification steps
were done with Qiagen PCR purification kit.
Yeast strains were grown on YPGlucose containing
2% (w/v) glucose, 1% (w/v) yeast extract, 2% (w/v) pep-
tone or on YPGalactose with same components as
YPGlucose but containing 3% (w/v) galactose instead
of glucose. For resistance and sensitivity tests on differ-
ent oxidants galactose minimal medium (SC) containing
3% (w/v) galactose, 0.5% ammonium sulphate, 0.017%
yeast nitrogen base with amino acids (0.002% Arg,
0.001% His, 0.006% Ile, 0.006% Leu, 0.004% Lys,
Trp, 0.001% Ade, 0.004% Ura, 0.005% Tyr (Sigma))
was used. Agar plates were made by adding 2% (w/v)
agar to the media.
2.3. Lifespan assays
Standard lifespan assays were performed as described
previously . All lifespans were determined on defined
SCGlucose or SCGalactose media for a cohort of at
least 40 cells. In a previous review we have discussed
that the median of a lifespan distribution, and its stand-
ard deviation at a 95% level, are in practice the best
parameters to describe lifespan (Figs. 1(c), 2 and 3) .
Lifespan assay with spectrophotometer: cells were
grown to logarithmic phase in YPGalactose media,
washed twice with water and transferred to liquid
YPGlucose or SCGlucose media. Optical density at
600 nm at the beginning was between 0.1 and 0.3. Cells
were shaken at 28 ?C for two days and optical density
S. Jarolim et al. / FEMS Yeast Research 5 (2004) 169–177
 Gershon, H. and Gershon, D. (2000) Paradigms in aging research:
a critical review and assessment. Mech. Ageing Dev. 117, 21–28.
 Muller, I., Zimmermann, M., Becker, D. and Flomer, M. (1980)
Calendar life span versus budding life span of Saccharomyces
cerevisiae. Mech. Ageing Dev. 12, 47–52.
 MacLean, M., Harris, N. and Piper, P.W. (2001) Chronological
lifespan of stationary phase yeast cells; a model for investigating
the factors that might influence the ageing of postmitotic tissues in
higher organisms. Yeast 18, 499–509.
 Jazwinski, S.M. (2002) Growing old: metabolic control and yeast
aging. Annu. Rev. Microbiol. 56, 769–792.
 Fabrizio, P. and Longo, V.D. (2003) The chronological life span
of Saccharomyces cerevisiae. Aging Cell 2, 73–81.
 Longo, V.D. and Finch, C.E. (2003) Evolutionary medicine: from
dwarf model systems to healthy centenarians. Science 299, 1342–
 Ashrafi, K., Sinclair, D., Gordon, J.I. and Guarente, L. (1999)
Passage through stationary phase advances replicative aging in
Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 96, 9100–
 Aguilaniu, H., Gustafsson, L., Rigoulet, M. and Nystrom, T.
(2003) Asymmetric inheritance of oxidatively damaged proteins
during cytokinesis. Science 299, 1751–1753.
 Laun, P., Pichova, A., Madeo, F., Fuchs, J., Ellinger, A.,
Kohlwein, S., Dawes, I., Fro ¨hlich, K.U. and Breitenbach, M.
(2001) Aged mother cells of Saccharomyces cerevisiae show
markers of oxidative stress and apoptosis. Mol. Microbiol. 39,
 Barker, M.G., Brimage, L.J. and Smart, K.A. (1999) Effect of
Cu,Zn superoxide dismutase disruption mutation on replicative
senescence in Saccharomyces cerevisiae. FEMS Microbiol. Lett.
 Van Zandycke, S.M., Sohier, P.J. and Smart, K.A. (2002) The
impact of catalase expression on the replicative lifespan of
Saccharomyces cerevisiae. Mech. Ageing Dev. 123, 365–373.
 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–
 Wawryn, J., Swiecilo, A., Bartosz, G. and Bilinski, T. (2002)
Effect of superoxide dismutase deficiency on the life span of the
yeast Saccharomyces cerevisiae. An oxygen-independent role of
Cu,Zn-superoxide dismutase. Biochim. Biophys. Acta 1570, 199–
 Sekito, T., Thornton, J. and Butow, R.A. (2000) Mitochondria-
to-nuclear signaling is regulated by the subcellular localization of
the transcription factors Rtg1p and Rtg3p. Mol. Biol. Cell 11,
 Kirchman, P.A., Kim, S., Lai, C.Y. and Jazwinski, S.M. (1999)
Interorganelle signaling is a determinant of longevity in Saccha-
romyces cerevisiae. Genetics 152, 179–190.
 Anderson, R.M., Bitterman, K.J., Wood, J.G., Medvedik, O. and
Sinclair, D.A. (2003) Nicotinamide and PNC1 govern lifespan
extension by calorie restriction in Saccharomyces cerevisiae.
Nature 423, 181–185.
 Jiang, J.C., Wawryn, J., Shantha Kumara, H.M. and Jazwinski,
S.M. (2002) Distinct roles of processes modulated by histone
deacetylases Rpd3p, Hda1p, and Sir2p in life extension by caloric
restriction in yeast. Exp. Gerontol. 37, 1023–1030.
 Hekimi, S. and Guarente, L. (2003) Genetics and the specificity of
the aging process. Science 299, 1351–1354.
 Bitterman, K.J., Anderson, R.M., Cohen, H.Y., Latorre-Esteves,
M. and Sinclair, D.A. (2002) Inhibition of silencing and acceler-
ated aging by nicotinamide, a putative negative regulator of yeast
sir2 and human SIRT1. J. Biol. Chem. 277, 45099–45107.
 Gallo, C.M., Smith Jr., D.L. and Smith, J.S. (2004) Nicotinamide
clearance by Pnc1 directly regulates Sir2-mediated silencing and
longevity. Mol. Cell Biol. 24, 1301–1312.
 Lin, S.J., Ford, E., Haigis, M., Liszt, G. and Guarente, L. (2004)
Calorie restriction extends yeast life span by lowering the level of
NADH. Genes Dev. 18, 12–16.
 Breitenbach, M., Madeo, F., Laun, P., Heeren, G., Jarolim, S.,
Fro ¨hlich, K.U., Wissing, S. and Pichova ´, A. (2003) Yeast as a
model for ageing and apoptosis researchModel Systems in Aging
(Nystro ¨m, T. and Osiewacz, H.D., Eds.), Topics in Current
Genetics, vol. 3. Springer-Verlag, Berlin, Heidelberg.
 Kennedy, B.K., Austriaco Jr., N.R. and Guarente, L. (1994)
Daughter cells of Saccharomyces cerevisiae from old mothers
display a reduced life span. J. Cell Biol. 127, 1985–1993.
 Laun, P. (1999) Immobilisierung von Hefezellen durch genetische
Derivatisierung der Zelloberfla ¨che. Diploma thesis, University of
 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.
 Bobola, N., Jansen, R.P., Shin, T.H. and Nasmyth, K. (1996)
Asymmetric accumulation of Ash1p in postanaphase nuclei
depends on a myosin and restricts yeast mating-type switching
to mother cells. Cell 84, 699–709.
 Gietz, R.D. and Sugino, A. (1988) New yeast-Escherichia coli
shuttle vectors constructed with in vitro mutagenized yeast genes
lacking six-base pair restriction sites. Gene 74, 527–534.
 Breitenbach, M., Laun, P., Heeren, G., Jarolim, S. and Pichova ´,
A. (2004) Mother cell-specific ageing In: The Metabolism and
Molecular Physiology of Saccharomyces cerevisiae (Schweizer,
M., Ed.), pp. 20–41. CRC Press, London.
 Piatti, S., Lengauer, C. and Nasmyth, K. (1995) Cdc6 is an
unstable protein whose de novo synthesis in G1 is important for
the onset of S phase and for preventing a ?reductional? anaphase in
the budding yeast Saccharomyces cerevisiae. Embo J. 14, 3788–
 Sil, A. and Herskowitz, I. (1996) Identification of asymmetrically
localized determinant, Ash1p, required for lineage-specific tran-
scription of the yeast HO gene (see comments). Cell 84, 711–722.
 Andrews, B.J. and Herskowitz, I. (1989) Identification of a DNA
binding factor involved in cell-cycle control of the yeast HO gene.
Cell 57, 21–29.
 Guarente, L. (2000) Sir2 links chromatin silencing, metabolism,
and aging. Genes Dev. 14, 1021–1026.
 Howitz, K.T., Bitterman, K.J., Cohen, H.Y., Lamming, D.W.,
Lavu, S., Wood, J.G., Zipkin, R.E., Chung, P., Kisielewski, A.,
Zhang, L.L., Scherer, B. and Sinclair, D.A. (2003) Small molecule
activators of sirtuins extend Saccharomyces cerevisiae lifespan.
Nature 425, 191–196.
S. Jarolim et al. / FEMS Yeast Research 5 (2004) 169–177