Sister Chromatids Segregate at Mitosis Without
Mother–Daughter Bias in Saccharomyces cerevisiae
Brice E. Keyes,* Kenneth D. Sykes,†,1Courtney E. Remington,†and Daniel J. Burke†,2
†Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, University of Virginia,
Charlottesville, Virginia 22906, and*Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New
York, New York 10021-6399
ABSTRACT There is evidence accumulating for nonrandom segregation of one or more chromosomes during mitosis in different cell
types. We use cell synchrony and two methods to show that all chromatids of budding yeast segregate randomly and that there is no
mother–daughter bias with respect to Watson and Crick-containing strands of DNA.
during development and was postulated to be especially im-
portant for stem cells (Cairns 1975). According to the model,
when stem cells undergo asymmetric cell division, one daugh-
ter (the self-renewing stem cell) selectively retains the older
template DNA strand from each chromosome, avoiding mu-
tations introduced during DNA replication (Cairns 1975;
Rando 2007; Tajbakhsh 2008). The model has been tested
in a large number of cells from yeast to humans with mixed
results and much debate (Neff and Burke 1991; Booth et al.
2002; Merok et al. 2002; Potten et al. 2002; Karpowicz et al.
2005; Conboy et al. 2007; Lansdorp 2007; Rando 2007;
Fei and Huttner 2009; Walters 2009; Escobar et al. 2011;
Schepers et al. 2011; Yadlapalli et al. 2011). The experi-
ments often utilize halogenated deoxyribonucleotides to label
DNA and determine if the label is retained over successive
divisions. This protocol was applied to yeast by labeling cells
for several generations with 5-bromo-deoxyuridine (BrdU),
followed by two rounds of cell division in the presence of
unlabeled thymidine to obtain cells in the second mitosis with
HE immortal strand hypothesis was proposed by J.
Cairns as a mechanism to preserve genome integrity
one unlabeled chromatid and one hemi-labeled chromatid
(Neff and Burke 1991). Immunoflourescence was used to fol-
low the fate of the hemi-labeled chromatids after the second
mitosis. The immortal strand hypothesis predicts that the old-
est (labeled) DNA strands would be segregated to the same
daughter; therefore, half of the cells would be labeled and
half unlabeled. Random segregation predicts that all of the
cells are labeled with each cell containing half as much BrdU.
Our results were consistent with random segregation in that all
the cells were labeled and the amount of BrdU per cell de-
creased by half between the first and second division. Sister-
chromatid recombination was minimal, and the data could
not be explained by nonrandom segregation coupled with
sister-chromatid exchange (Neff and Burke 1991).
More recently, a different model for nonrandom chromo-
some segregation on a chromosome-by-chromosome basis
was proposed and called “strand-specific imprinting and pat-
terned segregation” (SSIS) (Klar 2007). The model proposes
that epigenetic imprinting during DNA replication marks the
sister chromatids as different and that differential inheri-
tance of the imprinted chromatids results in different cell
fates in the daughter cells (Klar 2007; Tajbakhsh 2008).
Chromatid imprinting during DNA replication underlies
mating-type switching in Schizosaccharomyces pombe (Klar
1987, 2007; Yamada-Inagawa et al. 2007). SSIS was pro-
posed as the explanation for nonrandom chromosome seg-
regation in mouse embryonic stem cells where chromosome
7 segregates nonrandomly in a cell-type-specific manner
that is dependent on a dynein motor protein (Armakolas
and Klar 2006, 2007; Klar 2007; Armakolas et al. 2010).
Supporting evidence for nonrandom segregation of a subset
Copyright © 2012 by the Genetics Society of America
Manuscript received September 6, 2012; accepted for publication September 26, 2012
Supporting information is available online at http://www.genetics.org/lookup/suppl/
1Present address: Department of Pharmacology, 412 Preston Research Bldg.,
Vanderbilt University School of Medicine, 23 Ave. South and Pierce, Nashville,
2Corresponding author: Department of Biochemistry and Molecular Genetics,
University of Virginia School of Medicine, 1300 Jefferson Park Ave., Charlottesville VA
22908-0733. E-mail: email@example.com
Genetics, Vol. 192, 1553–1557December 2012
of chromosomes in intestinal crypt cells was demonstrated
using a fluorescence in situ hybridization strategy and is
consistent with SSIS operating on a subset of chromosomes
in intestinal cells (Falconer et al. 2010). Previous experi-
ments to test the Cairns hypothesis in yeast had insufficient
resolution to detect SSIS (Neff and Burke 1991). Selective
nonrandom segregation of a single yeast chromosome, es-
pecially one of the smaller chromosomes, would have been
difficult to distinguish from completely random segregation
solely on the basis of immunofluorescence. Sister chroma-
tids of yeast chromosome 5 are randomly segregated in
mitosis but that cannot be said with certainty for the other
15 chromosomes (Chua and Jinks-Robertson 1991).
We have tested the SSIS model for mother–daughter bias
and nonrandom segregation of chromatids in budding yeast
using two different strategies. Both depended on a yeast strain
engineered to permit BrdU labeling and on a simple method to
purify mother cells from daughters (Park et al. 2002; Viggiani
and Aparicio 2006). Cells were arrested with a-factor, and the
cell surface was biotinylated. Cells were released into the cell
cycle, allowed to divide, and arrested prior to budding in the
second cell cycle by adding a-factor again to the culture. The
biotinylated mother cells were purified from the unlabeled
daughters using streptavidin-coated magnetic beads. The first
strategy to determine if there was nonrandom segregation of
individual chromosomes is shown in Figure 1A. Cells were
labeled with BrdU in the first cell cycle before separating the
mothers (M) from the daughters (D). The daughter cells were
biotinylated, and both populations were grown for one cell
cycle in the absence of BrdU and arrested with a-factor,
and mothers were separated from daughters (MM, MD and
DM, DD). Figure 1 shows the prediction for the SSIS model
with the hypothesis that the mother cells inherit the parental
Watson-containing strand and the daughter cells inherit the
Figure 1 Watson and Crick-containing chromatids are not
exclusively segregated to mother or daughter cells. (A) The
labeling protocol is shown with the BrdU-containing strands
of DNA in red and the unlabeled DNA in black. The original
Watson (W) and Crick (C) strands are indicated, as are cen-
tromeres (circles). Cells are labeled with BrdU in the first cell
cycle, and mothers and daughters are separated and grown
for a second cell cycle in the presence of unlabeled thymi-
dine (TdR). Mothers derived from the first mother (MM)
are purified from the daughters (MD) and similarly for the
mothers derived from the first daughter (DM) and the
corresponding daughter (DD) after the second cell cycle.
(B) Short and long exposures of the Southwestern blot to
detect BrdU. All experiments were performed in strain
CVY63 MATa ade2-1 trp1-1 can1-100 leu2-3,112, his3-
11,15 bar1::hisG LEU2:BrdU-inc, which is isogenic with
W303a and was kindly supplied by Oscar Aparicio. All
methods are in File S1.
B. E. Keyes et al.
parental Crick-containing strand. The label is expected to be in
two of the four cell types if there is complete nonrandom
segregation of chromatids. We assayed the inheritance by sep-
arating chromosomes in a contour-clamped homogeneous
electric field (CHEF) gel and by detecting the BrdU by South-
western blots (Figure 1B). We saw no evidence of completely
nonrandom segregation of chromatids for any chromosome.
We used an independent method that was highly
quantitative and had sufficient resolution to determine if
there was any mother–daughter bias associated with sister-
chromatid segregation (Figure 2A). Cells were arrested in
a-factor and biotinylated as described above. Cells were re-
leased to the cell cycle, and BrdU was incorporated into
newly synthesized DNA strands (W9 and C9 in Figure 2A).
a-Factor was added to arrest the cells after cell division,
prior to budding in the subsequent cell cycle, and mothers
were separated from daughters. DNAwas purified and dena-
tured, and the BrdU-containing strands were recovered by
immunoprecipitation and eluted by competition with BrdU.
The complementary strand was biotin-labeled in vitro, and
Figure 2 There is no mother–daughter bias in
the segregation of Watson and Crick-containing
chromatids during mitosis. (A) The labeling pro-
tocol is shown with the BrdU-containing strands
of DNA in red and the unlabeled DNA in black.
The original Watson (W) and Crick (C) strands
are indicated, as are centromeres (circles). (B)
Normalized mean raw intensities for individual
genes on the Watson strand (red) or the Crick
strand (blue) of chromosome 5 vs. the position
along the chromosome. (C) The log2 ratios of
intensities for every gene on the Watson strand
(red) and the Crick strand (blue) vs. the position
along the chromosome. (D) Q-Q plot of the log2
ratio for the Watson strand of chromosome 5. (E)
Plot of the distribution of the intensities of log2
ratios for the Watson strand (red) and the Crick
strand (blue) for chromosome 5.
B.?? E.?? Keyes?? et?? al.?? 9?? SI??
Cells were grown at 30 degrees C in 50 mls of YPD to a density of 2x107 cells/ml
and α-factor was added at 1 µM. Cells were incubated for 2.5 hours until greater
than 90% of the cells were unbudded with mating projections. Cells were
collected by centrifugation and washed twice with water and twice with
phosphate buffered saline (PBS) and resuspended in 2 mls of PBS. Cells were
sonicated and 24 mg of EZ-biotin (Sulfo-NHS-LC-Biotin, Thermo-Scientific) was
added and incubated for 15 min at room temperature with gentle mixing. Cells
were washed three times in 50 mls of water and a sample was stained with FITC
streptavidin to confirm biotinylation of the cell surface by fluorescence
microscopy. Cells were resuspended in 50 mls of YPD containing 20 mg of 5-
Bromo-deoxyuridine (Sigma) and incubated in the dark for 1 hour at 30 degrees
C until greater than 95% of the cells were budded at which time α-factor was
added at 1 µM. Cells were incubated for 2 hours until greater than 90% of the
cells were unbudded with mating projections. Cells were washed twice in 50 ml
of water and twice in PBS, sonicated and aliquoted into 1ml aliquots of 108 cells
in eppendorf tubes. Cells were concentrated by centrifugation and resuspended
in 400 µl of PBS-washed Streptavidin dynabeads and incubate with rotation for
15 minutes. The beads were recovered using a magnet and washed three times.
The unbound samples (daughters) were pooled and an aliquot stained with
calcoflour and FITC streptavidin. The cells were greater than 90% daughter cells
as determined by fluorescence microscopy and identifying the fraction of cells
that were stained with both dyes (mothers). In the first protocol, mother and
daughter cells were returned separately to YPD medium and incubated in the
dark for 1 hour at 30 degrees C until greater than 95% of the cells were budded
at which time α-factor was added at 1 µM. The cultures were treated as
described above so that the mothers and daughters from the culture of mother
cells (MM and MD) were isolated and the mothers and daughters from the culture
of daughter cells (DM and DD) were isolated. DNA was extracted and subjected
to electrophoresis using a Bio-Rad CHEF-mapper XA system according to
manufacturers instructions. The gel was subjected to Sothern blot following
standard procedures and the BrdU was detected by immunoblotting using mouse
anti-BrdU (G3/G4). In the second protocol, DNA was purified from the BrdU-
labeled mothers and daughters and heated to 95 degrees C for 5 min in 360 µl
distilled water and then placed on ice. Forty µl of 10X PBS was added and 20 µl
of 1 mg/ml mouse anti-BrdU (G3/G4) and the mixture was incubated for an hour.
The immuno-complexes were recovered by adding 50 µl of sheep anti-mouse
dyanbeads. The unbound fraction was analyzed by slot blot and greater than
95% of the BrdU was bound to the beads. The BrdU containing DNA was
recovered by resuspending the beads in 100 µl of 1.7 mM BrdU. The recovered
DNA was extracted with phenol chloroform isoamyl alcohol and concentrated by
ethanol precipitation. DNA was labeled with random priming (In Vitrogen) with
using biotin-dCTP according to manufacturers instructions and used for
hybridization to Affymetrix arrays following manufacturers instructions.
B.?? E.?? Keyes?? et?? al.?? 10?? SI??
Table?? S1?? ?? ?? Wilcoxon?? ranked?? sign?? test??
Null?? hypothesis:?? that?? the?? mean?? of?? the?? distribution?? of?? the?? mother-‐daughter?? ratios?? for?? probes?? to?? the?? Watson?? and?? Crick??
strands?? for?? each?? chromosome?? is?? equal?? to?? zero.??
Chromosome?? P?? Value??