Copyright  2000 by the Genetics Society of America
The Saccharomyces cerevisiae Centromere Protein Slk19p Is Required
for Two Successive Divisions During Meiosis
Xuemei Zeng and William S. Saunders
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Manuscript received August 5, 1999
Accepted for publication February 18, 2000
ABSTRACT
Meiotic cell division includes two separate and distinct types of chromosome segregation. In the first
segregational event the sister chromatids remain attached at the centromere; in the second the chromatids
are separated. The factors that control the order of chromosome segregation during meiosis have not yet
been identified but are thought to be confined to the centromere region. We showed that the centromere
protein Slk19p is required for the proper execution of meiosis in Saccharomyces cerevisiae. In its absence
diploid cells skip meiosis I and execute meiosis II division. Inhibiting recombination does not correct this
phenotype. Surprisingly, the initiation of recombination is apparently required for meiosis II division.
Thus Slk19p appears to be part of the mechanism by which the centromere controls the order of meiotic
divisions.
M
EIOSIS is a special type of cell division that generates haploid gametes to fulfill the needs of sexual
reproduction. To achieve this end, a single round of
DNA replication is followed by two successive rounds of
chromosome segregation, reductional (meiosis I) and
equational (meiosis II). While meiosis II is mechanically
similar to mitosis, meiosis I is quite distinct from both
mitosis and meiosis II. During anaphase of meiosis I,
sister chromatids do not separate from each other. Instead they segregate as a unit away from the other homologous chromosome.
The two separate meiotic divisions are not interdependent, as demonstrated by single-division meiosis in Saccharomyces cerevisiae mutants, such as cdc5, cdc14, spo12,
spo13, and cdc28 (Klapholz and Esposito 1980b; Shuster and Byers 1989; Sharon and Simchen 1990b).
In these mutants, chromosomes can undergo either a
single reductional division or a single equational division. Although the two nuclear divisions can occur independently, in normal meiosis the progression is regulated so that equational division only happens after
reductional division.
Substantial progress has been made to identify what
determines whether chromosomes will undergo reductional or equational segregation (reviewed in Simchen
and Hugerat 1993; McKim and Hawley 1995). By
fusing two grasshopper spermatocytes at different meiotic stages, Nicklas (1977) found that meiosis I chromosomes still segregated reductionally when micromanipulated into a meiosis II spindle. This result indicates
that it is the chromosome, and not the spindle, or the
Corresponding author: William S. Saunders, Department of Biological
Sciences, 258 Crawford Hall, University of Pittsburgh, Pittsburgh, PA
15260. E-mail: wsaund⫹@pitt.edu
Genetics 155: 577–587 ( June 2000)
general cell environment, which determines the segregational characteristic of the chromosome. The same
conclusion was drawn from studies in S. cerevisiae. Studying mixed segregation in the single-division meiosis mutants cdc5, cdc14, and spo13 revealed that different chromosomes can have different segregational tendencies
even within the same cell. For example, chromosome
IV tends to segregate equationally, while chromosome
XI tends to segregate reductionally in these mutants
(Sharon and Simchen 1990b; Hugerat and Simchen
1993). The chromosomal determinant for segregational
tendencies resides within the centromeric region as
shown by centromere replacement experiments (Sharon
and Simchen 1990a). When the centromere of chromosome XI was replaced with the centromere of chromosome IV, chromosome XI tended to segregate equationally like chromosome IV. However, the molecular
mechanism that the centromere region uses to control
whether chromosomes segregate reductionally or equationally is still unclear.
Centromeres perform multiple functions related to
chromosome segregation during cell division. The most
well characterized is attachment of the chromosome to
the microtubules of the spindle, but centromeres also
play less-well-defined roles in cell cycle control, spindle
structure/assembly, and sister chromatid cohesion (reviewed in Maguire 1990; Bickel and Orr-Weaver
1996; Straight 1997). During meiosis the cohesion
between sister chromatids is lost at two steps. The first
step happens at the metaphase I/anaphase I transition,
during which the cohesion between sister chromosomal
arms disappears but the cohesion at the centromeric
region is maintained and is necessary for the proper
bipolar spindle assembly during meiosis II. The second
step happens at the metaphase II/anaphase II transition.
578
X. Zeng and W. S. Saunders
During this step the cohesion between sister centromeres is also lost in order to separate sister chromatids.
In S. cerevisiae CEN-ARS plasmids missing centromere
DNA element I (CDEI), or chromosomes in cells lacking
CDEI binding protein Cep1p, often missegregate, predominantly due to precocious sister chromatid segregation during meiosis I (Cumberledge and Carbon 1987;
Masison and Baker 1992). Recently, the CDEIII element has also been shown to be important for centromere cohesion in S. cerevisiae (Megee and Koshland
1999). In Drosophila melanogaster, the kinetochore protein MEI-S332 is required to maintain cohesion between
sister centromeres after metaphase I/anaphase I transition. In its absence, a high level of meiosis II nondisjunction was observed (Davis 1971; Kerrebrock et al. 1992,
1995; Moore et al. 1998). These observations indicate
that the centromere and its associated proteins are essential for regulating the stepwise loss of sister chromatid cohesion during meiosis.
As centromeres are a critical part of how the cell
distinguishes between the different meiotic divisions, it
is expected that some centromere proteins will play a
key role in the ordering of meiosis I and II. We find
here that the centromere protein Slk19p is required for
this task. SLK19 was identified previously in a synthetic
lethal screen to identify genes with functions similar to
that of the kinesin motor KAR3. It encodes a potential
coiled-coil protein of ⵑ98 kD and shares no significant
homology to previously characterized proteins. Like
kar3 mutants, slk19 mutants have short spindles and
more cytoplasmic microtubules than normally present
during mitosis (Zeng et al. 1999). Immunofluorescence
studies have shown that slk19 kar3 double mutants are
unable to assemble or maintain spindles, suggesting that
Slk19p and Kar3p work together to stabilize spindle
structure. Here we report that Slk19p is essential during
meiosis. In its absence, a single meiotic division takes
place. Genetic analysis reveals that chromosomes in
slk19 mutants predominantly undergo equational but
not reductional segregation. Recombination is normal
or slightly elevated and may be required for the meiosis
I bypass. If recombination is inhibited, the slk19 mutants
are unable to undergo either meiotic division. Thus
Slk19p appears to be an essential part of the mechanism
at the centromere that helps to ensure the proper ordering of meiosis I and II.
MATERIALS AND METHODS
Yeast strains, plasmids, and media: Strains and plasmids
used in this study are listed in Table 1. All strains are derivatives
of S288C. Media have been described (Sherman 1991). For
sporulation, diploid cells with plasmids were first grown in
selective media. Otherwise they were grown in YEPD. Cells
from fresh overnight cultures (OD600 ⬎ 2) were washed with
sporulation media twice, resuspended in sporulation medium,
and incubated at 26⬚ with shaking for 2 days to allow them
to sporulate.
Nomenclature: The segregation of heterozygous markers
TABLE 1
Yeast strains and plasmids used in this study
Strains/plasmids
WSY993
WSY994
WSY996
WSY990
WSY998
WSY1301
WSY1302
WSY1303
WSY1027
WSY1028
WSY1125
WSY1126
WSY1117
WSY1118
pXZB9
Genotype
MATa/␣ ade2/ADE2 lys2-801/LYS2 his3⌬200/his3⌬200 ura3-52/ura3-52 leu2-3,112/leu2-3,112
MATa/␣ ade2/ADE2 lys2-801/LYS2 his3⌬200/his3⌬200 ura3-52/ura3-52 leu2-3,112/leu2-3,112
trp1⌬1/TRP1 slk19::HIS3/slk19::HIS3
MATa/␣ ade2-101/ade2-101 lys2/LYS2 his3⌬200/his3⌬200 ura3/ura3 leu2-3,112/leu2-3,112
trp1⌬/TRP1 spo13::hisG-URA3-hisG/ spo13::hisG-URA3-hisG
MAT␣ his3⌬200 leu2 ura3-52 trp1⌬63 slk19⌬::SLK19:GFP:URA3
MATa lys2-801 his3⌬200 leu2 ura3-52 slk19⌬::SLK19:GFP:URA3
MATa/␣ ade1/ADE1 lys2-801/LYS2 his3⌬200/his3⌬200 leu2-3,112/leu2-3,112 ura3-52/URA3
trp1⌬1/TRP1 slk19::HIS3/slk19::HIS3
MATa/␣ ade2-101/ADE2 lys2-801/LYS2 his3⌬200/his3⌬200 leu2-3,112/leu2-3,112
trp1-903/TRP1 ura3-52/URA3 tyr1-501/TYR1 can1/CAN1 slk19::HIS3/slk19::HIS3
MATa/␣ lys2-801/LYS2 his3⌬200/his3⌬200 leu2-3,112/leu2-3,112 trp1-903/TRP1 ura3-52/URA3
tyr1-501/TYR1 can1/CAN1
MATa ade2 can1-100r his3-11,15 leu2-3,112 trp1-1 ura3-1 spo11⌬3 atr::HIS3
MAT␣ ade2 can1-100r his3-11,15 leu2-3,112 trp1-1 ura3-1 spo11⌬3 atr::HIS3
MAT␣ ade2 lys2-801his3 leu2-3,112 ura3 slk19::HIS3 spo11⌬3 atr::HIS3
MATa his3 leu2-3,112 ura3 slk19::HIS3 spo11⌬3 atr::HIS3
MATa/␣ ade2-1/ade2-R lys2-801/LYS2 his3⌬200/his3 leu2-3,112/leu2-3,112 trp1/TRP1
ura3-52/ura3-52 slk19::HIS3/slk19::HIS3
MATa/␣ ade2-1/ade2-R lys2-801/LYS2 his3⌬200/his3 leu2-3,112/leu2-3,112 trp1/TRP1
ura3-52/ura3-52
SLK19-TRP1-CEN
Double colon refers to a deletion of the preceding gene and replacement by the marker gene that follows. A single colon
refers to linkage without disruption.
Slk19p Is Required for Meiosis I
in the two-spored asci (dyads) is represented by the format
⫹: ⫺. The ⫹ indicates the number of spores with the phenotype of the dominant allele, while ⫺ indicates the number of
spores with the phenotype of the recessive allele.
Intergenic recombination: Diploid strains WSY1302 (slk19)
and WSY1303 (wild type) were sporulated. The resulting tetrads were dissected as described (Sherman and Hicks 1991).
To unambiguously distinguish dyads from two spores derived
from different asci, dyads were dissected as described (Davidow et al. 1980), except that 0.5 mg/ml zymolyase 20T (U.S.
Biological) in 1 m sorbitol was used to replace glusulase. The
segregation of ura3 and can1 in the resulting colonies was
scored based on their growth on ⫺ura and canavanine plates.
Map distance was determined using Perkins’ (1949) formula
as follows: Map distance ⫽ 50 ⫻ [single crossovers ⫹ 6 (fourstrand double crossovers)]/total. As shown in Figure 4, dyads
resulting from double crossovers and equational division cannot be distinguished from dyads resulting from equational
division without recombination. Therefore, we assumed that
the same frequencies of four-strand double crossovers occur
in reductional division and equational division and calculated
the total number of four-strand double crossovers as follows:
Total four-strand double crossovers ⫽ drd ⫹ drd (ed/rd), where
drd stands for the number of dyads resulting from four-strand
double crossover and reductional division, ed for the number
of dyads resulting from equational division, and rd for the
number of dyads resulting from reductional division (Hugerat and Simchen 1993).
Heteroallelic recombination: To determine the frequencies
of heteroallelic recombination, diploid strains of wild-type
(WSY1118) or slk19⌬ (WSY1117) with ade2 heteroalleles (ade21/ade2-R) were constructed. Individual diploid colonies were
inoculated into YEPD liquid and cultured at 30⬚ with shaking
until OD600 reached 2.0. The cultures were then divided into
two parts. One part was used directly to determine the mitotic
recombination rates by spreading onto YEPD and ⫺ade selective plates and counting the frequency of adenine prototrophy. The other part was washed with sporulation medium
twice and resuspended in sporulation medium to induce meiosis and sporulation. The sporulation culture was vigorously
aerated at 26⬚ for 48 hr, at which time the cells were plated
onto YEPD and ⫺ade plates to determine the meiotic recombination frequencies.
Canavanine reversion: The ploidy of the spores was determined based on the frequencies of canavanine-resistant colonies after UV treatment. Diploid cells homozygous for slk19
and CAN1 were sporulated at 26⬚ and the resulting dyads were
dissected. Resulting individual colonies were serial diluted
onto YEPD plates and incubated at 30⬚ for 2 days. Cells were
then replicated from YEPD plates onto canavanine plates and
treated with UV light immediately after replica plating. The
UV light was delivered from a UV crosslinker (FB-UVXL-1000,
Fisher Scientific) with 9000 ␮J/cm2 energy. The UV-treated
cells were then incubated at 30⬚ for 2 days to allow canavanineresistant colonies to grow.
Immunofluorescence microscopy: To visualize microtubules (MTs) of cells from different meiotic stages, cells were
cultured in the sporulation media for ⵑ20 hr and fixed with
3.7% formaldehyde for 2 hr. Antitubulin indirect immunofluorescence was then performed using monoclonal antibody
YOL 1/34 (Serotec, Oxford, UK) and rhodamine-conjugated
secondary antibody (Jackson Immunoresearch, West Grove,
PA) to visualize MT, and DNA-specific fluorescent dye 4,6diamidino-2-phenylindole (DAPI; Sigma, St. Louis) to identify
the position of the nucleus as described (Pringle et al. 1991;
Hoyt et al. 1992). The slides were examined with an Olympus
BX60 epifluorescence microscope using a 100⫻ oil immersion
objective. Digital images were captured with a Hamamatsu
579
Figure 1.—(A) Sporulation in wild-type and slk19⌬ mutants.
Wild-type and slk19⌬ diploids were sporulated at 26⬚ for 2
days and viewed by DIC microscopy. Sporulation in wild-type
cells usually results in the formation of asci containing four
spores (tetrads). slk19⌬ mutants produced almost exclusively
asci with only two spores (dyads). The sporulation efficiencies
are shown in Table 2. (B) Canavanine resistance test to determine the ploidy of the spores. HP1 and HP2 signify the two
slk19 haploid parents and DP the diploid parent resulting
from the cross of HP1 and HP2. Dyads 1–6 are six dyads
resulting from the sporulation of DP. The left spore of dyad
1 was scored as a haploid and the rest of the spores as diploids
based on this test. All the spores grew well on YEPD plates
made at the same time (not shown).
Argus-20 CCD camera and image processor (Hamamatsu Co.,
Bridgewater, NJ). Figure 2 shows montages made by combining several representative samples using Adobe Photoshop
software (Adobe Systems Inc., Mountain View, CA).
RESULTS
Slk19p is required to obtain two successive divisions
during meiosis: To study the function of Slk19p during
meiosis, a diploid strain homozygous for slk19⌬ (Zeng
et al. 1999) was sporulated. We found that while sporulation of wild-type cells usually led to the formation of
tetrads with four spores enclosed in an ascus, sporulation of slk19⌬ mutants usually led to the formation of
dyads with two spores per ascus (Figure 1A). The sporulation efficiency of slk19⌬ mutants is normal, with ⵑ50%
of the total cells forming asci compared to ⵑ60% in
wild-type cells. The frequency of dyads increased from
ⵑ11% of total asci for wild type to ⵑ80% for slk19⌬
mutants (see Table 4). Tetrads were rarely observed in
slk19⌬ mutants (⬍1%). The spore viability of the slk19⌬
dyads is slightly lower than that of wild-type tetrads.
580
X. Zeng and W. S. Saunders
While wild-type spores maintain ⬎95% viability after
dissection, only 70% of slk19⌬ spores were alive among
239 dyads dissected.
Two types of dyads have been observed previously in S.
cerevisiae. The first type is due to an ascospore packaging
defect. Under these circumstances, two successive divisions take place but only two of the four nuclei are
enclosed into spores (Davidow et al. 1980). In this case,
the spores are haploid. The second type of dyad is the
result of a single meiotic division. Here two-spored asci
are also seen but the spores are diploid. To investigate
whether dyads of slk19⌬ mutants were the result of an
ascospore packaging defect or a single-division meiosis,
we determined the ploidy of the spores. Typically only
diploid cells can sporulate, and only MATa/MAT␣ and
not MATa/MATa or MAT␣/MAT␣ diploids can sporulate. Dyads of slk19⌬ mutants were dissected (materials
and methods) and the mating capability of viable
spores was tested. Among 42 dyads with two viable
spores, 13 dyads contained two spores capable of mating
and 29 dyads contained two nonmating spores. Dyads
with a mixture of mating and nonmating spores were
not observed. The nonmating spores were able to sporulate and again produced dyads which themselves were
able to sporulate to dyads (six spores from 3 dyads were
tested). These observations indicate that stable diploid
progenies are produced by meiosis in slk19⌬ mutants.
The ploidy of the spores was confirmed by determining
the copy number of chromosome V using a canavanine
mutation test. Haploid CAN1 cells have a much higher
rate of mutation to canavanine resistance than do diploid CAN1/CAN1 cells. Only three spores out of the 42
dyads contained a single copy of CAN1 based on this
analysis. The remaining were concluded to have at least
two copies of CAN1 and thus two copies of chromosome
V (Figure 1B).
The diploid spores are presumably a result of a single
meiotic division. To confirm this, we also examined
slk19⌬ mutants in meiosis with antitubulin indirect immunofluorescence. Only one spindle per cell was observed, consistent with the occurrence of a single meiotic division (Figure 2). In conclusion, slk19⌬ mutants
form dyads after sporulation because there is a single
meiotic division and not because there is an ascospore
packaging defect.
The majority of chromosomes in slk19⌬ mutants undergo an equational segregation: The single meiotic
division could be meiosis I, or meiosis II, or a mixture
of the two. To address which kind of division was taking
place, we studied the segregation pattern of three heterozygous centromere-linked markers. Centromere-linked
markers remain associated with centromeres during
meiosis and thus segregate with the same reductional
and equational properties as chromosomes. For these
experiments the markers chosen were trp1 linked to
CEN4, ade1 linked to CEN1, and ura3 linked to CEN5.
If chromosomes segregate reductionally (meiosis I), we
Figure 2.—Microtubules in slk19⌬ cells during meiosis.
Cells in sporulation medium for 20 hr were fixed and treated
for antitubulin immunofluorescence (materials and methods). Spindles are visible as a bright fluorescent bar. Cytoplasmic MTs are visible as fainter lines at ends of spindles.
Shown are four composite images of representative cells from
the same samples. ben refers to sporulation in the presence
of 5 ␮g/ml benomyl. The level of sporulation in these cultures
is fairly high at ⵑ50% but the synchrony is low and only ⵑ20%
of cells showed spindles at this timepoint.
expect to get 1:1 dyads in which one spore is able to
grow on selective media, but the other spore cannot
(Figure 3). If chromosomes segregate equationally
(meiosis II), we expect to get 2:0 dyads in which both
spores are able to grow on selective media. The majority
of dyads in slk19⌬ mutants were 2:0 dyads for each
chosen marker, suggesting that most chromosomes segregate equationally (Table 2). As shown in Figure 3,
2:0 dyads may result from a single normal equational
division (Figure 3A), or a single equational segregation
accompanied by chromosome loss, or incoordinate segregation (Figure 3B). But only 2:0 dyads resulting from
a normal equational division will have both spores heterozygous for the chosen marker. To address whether
the 2:0 dyads of slk19⌬ mutants were a result of aberrant
chromosome segregation during meiosis, we determined whether these slk19⌬ spores were heterozygous
for the tested marker. We transformed a plasmid containing wild-type SLK19 (pXZB9) into four spores from
two 2:0 dyads for trp1 marker and allowed them to sporu-
Slk19p Is Required for Meiosis I
581
TABLE 3
Coincident segregation of centromere-linked
markers in slk19⌬ dyads
Dyad phenotype (⫹:⫺)
Class
ade1
ura3
trp1
% of dyads
I
II
III
IV
V
VI
VII
VIII
IX
2:0
2:0
2:0
1:1
1:1
1:1
2:0
1:1
0:2
2:0
2:0
1:1
2:0
1:1
2:0
1:1
1:1
0:2
2:0
1:1
2:0
2:0
2:0
1:1
1:1
1:1
1:1
68.4
6.2
4.6
12.3
0
1.5
1.5
4.6
0.8
The same set of dyads from Table 2 was rescored to examine
the segregation of all three centromere-linked markers in the
same cells.
a normal equational division and not from aberrant
chromosome segregation.
While most chromosomes segregate equationally, we
only observed ⵑ68% of the dyads showing 2:0 phenotype for all three markers (Table 3), indicating that at
most 68% of the cells undergo a single equational division of the entire genome. The rest of the dyads showed
a 1:1 phenotype for at least one of the three markers.
The 1:1 phenotype may be a result of a single reductional segregation, or an aberrant segregation, or a rare
meiotic recombination between centromere and linked
marker followed by a single equational segregation (Figure 3). The expected number of 1:1 dyads resulting
from meiotic recombination followed by a single equational division was calculated based on the distance between the marker locus and the centromere (Table 2).
The observed frequency was much higher than expected based on recombination alone, suggesting that
Figure 3.—Schematic diagram showing the expected segregation patterns of heterozygous centromere-linked markers
in dyads resulting from abnormal meiotic events. The meiotic
products are shown in the squares with each oval representing
one spore. The markers are indicated on the chromosome.
⫹ indicates the dominant allele and ⫺ indicates the recessive
allele. No recombination events are shown. (A) Normal segregation in wild-type or single-division meiosis. (B) Two aberrant
segregational events that could also result in 2:0 dyads or 1:1
dyads.
late. Seven resulting tetrads for each spore were dissected (see materials and methods) and the trp1
marker segregated 2:2 for each, indicating that those
spores were heterozygous for trp1 and thus resulted from
TABLE 2
Segregation of heterozygous centromere-linked markers in slk19⌬ dyads
Dyad phenotye (⫹:⫺)
Chromosome
I
V
VI
Marker
2:0
1:1
0:2
Total
Gene-centromere
distance (cM)a
Expected 1:1 dyadsb
ade1
ura3
trp1
104
111
117
25
18
13
1
1
0
130
130
130
4
7
1
2.6
4.6
0.7
Strain WSY1301 was sporulated and resulting dyads were dissected (see materials and methods). Dyads
with two viable spores were tested for growth on ⫺ade, ⫺trp, and ⫺ura plates.
a
From Saccharomyces Genome Database.
b
Refers to the expected number of 1:1 dyads resulting from recombination between marker and centromere
followed by equational division. The number is calculated using the formula E ⫽ (D/100) ⫻ T ⫻ (1/2), where
E is the expected number of 1:1 dyads; D is the distance between marker and centromere; and T is the total
number of dyads. All recombination events are assumed to be single crossing over for ease of calculation. Only
half of those dyads with recombination followed by equational division can be distinguished phenotypically
(see Figure 4).
582
X. Zeng and W. S. Saunders
TABLE 4
TABLE 5
Effect of benomyl on sporulation
Intergenic recombination is elevated in slk19⌬ strains
Number of spores (%)
Strain
Wild type
Wild type ⫹ ben
slk19⌬
slk19⌬ ⫹ ben
slk19⌬/slk19⌬
0
1
2
3
4
41.2
45.3
51.7
65.5
1.2
0.7
7.0
10.7
6.5
5.0
39.0
23.0
12.8
12.2
1.5
0.3
38.3
36.8
0.8
0.5
Strains WSY993 (wild type) and WSY994 (slk19⌬) were sporulated at 26⬚ for 2 days. A total of 600 cells were scored for
the number of enclosed spores. ben refers to sporulation in
the presence of 5 ␮g/ml benomyl.
Dyad phenotype
Phenotypic
class
A
B
C
D
E
Spore
A
Spore
B
P
D
P
D
No. of dyads
⫹
⫹
⫺
⫺
⫹
⫹
⫺
⫹
⫺
⫺
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫹
⫹
⫹
62
38
6
1
4
Distance
(cM)
64.2
SLK19/SLK19
most of these 1:1 dyads were not due to recombination,
but more likely from a single reductional segregation
or an aberrant segregation event. The cells rarely go
through reductional division for the entire genome, as
we observed only 4.6% of dyads showing 1:1 for all three
markers (Table 3). The rest of the dyads showing 1:1
phenotypes for at least one of the markers were probably
from a mixed division with some chromosomes segregating equationally and some chromosomes segregating
reductionally. The chromosomes did not behave equivalently. For example, chromosome I has a higher tendency than chromosome V to segregate reductionally
as evidenced by the observation that more 1:1 dyads
were observed for the ade1 marker than for the ura3
marker, even though ade1 is closer to the centromere
than ura3.
In conclusion, chromosomes predominantly segregate equationally during meiosis upon loss of Slk19p.
Consistent with the previous finding that different chromosomes may have different segregational properties
within the same cell, we have also observed different
segregational tendencies for different chromosomes.
An excess of cytoplasmic microtubules is not the direct cause of the meiosis I bypass: Immunofluorescence
with antitubulin antibodies revealed that like vegetatively growing slk19⌬ cells, meiotic slk19⌬ cells have an
increased amount of cytoplasmic microtubules (Figure
2). These results indicate that Slk19p also influences
microtubule numbers and distribution in meiosis. This
excess is not an inevitable result of a single equational
division, since we did not observe this phenotype in
spo13⌬ mutants, which are known to have a single
equational division (Figure 2; Klapholz and Esposito
1980b). Among 200 spindles examined, the average
number of cytoplasmic microtubules was three per spindle for slk19⌬ mutants and one per spindle for spo13⌬
mutants. Moens and Rapport (1971) have shown using
electron microscopy that yeast meiotic cells lose most
of their cytoplasmic microtubules when they enter meiosis I. The functional significance of this loss is not clear,
but it may be an important part of meiotic progression.
Tetrad phenotype
Distance (cM)
PD
NPD
T
Total
37
4
26
67
This study
37.3
SGD
43
WSY1302 (slk19) and WSY1303 (wild type) were sporulated
and dyads or tetrads, respectively, were dissected. In both
strains, one copy of chromosome V contains can1 and URA3,
and the other contains CAN1 and ura3. A total of 111 dyads
with two viable spores for slk19 mutants and 67 tetrads with
four viable spores for wild type were scored based on their
growth on ⫺ura and canavanine plates. The recombination
and segregational events generating each class of dyads are
shown in Figure 4. P refers to the proximal marker ura3 and
D refers to the distal marker can1. ⫹ refers to the phenotype
of the wild-type allele; ⫺ refers to the phenotype of the mutant
allele. The distance between ura3 and can1 was determined
as described (see materials and methods).
To test whether the excess of cytoplasmic microtubules
is the direct cause of the meiosis I bypass, we let slk19⌬
mutants sporulate in the presence of benomyl, a microtubule depolymerization drug (Davidse 1986). The
amount of cytoplasmic microtubules was substantially
reduced, as shown by immunofluorescence with antitubulin antibody (Figure 2, a montage of representative
cells from the same sample is shown), but we found no
increase in the frequency of tetrads (Table 4). Cytoplasmic microtubules can also be reduced by genetic
deletion of the nonessential ␣-tubulin TUB3 gene
(Schatz et al. 1986) or the kinesin-related KIP2 gene
(Huyett et al. 1998). slk19⌬tub3⌬ and slk19⌬kip2⌬ mutants also showed no increase in the frequency of tetrad
formation compared to slk19⌬ alone (not shown).
These results indicate that the excess of cytoplasmic
MTs is not likely to be the direct cause of the meiosis
I bypass in slk19⌬ mutants.
slk19 mutants exhibit normal or slightly elevated meiotic recombination rates: Proper meiosis I segregation
depends on a stable bipolar spindle, which in turn relies
on a stable attachment between homologous chromosomes. Recombination is responsible for most of the
association between homologues. In the absence of re-
Slk19p Is Required for Meiosis I
583
TABLE 6
Mitotic and meiotic heteroallelic recombination
in wild-type and slk19⌬ mutants
Division
Genotype
Recombination
frequency
Mitosis
Mitosis
Meiosis
Meiosis
Wild type
slk19⌬
Wild type
slk19⌬
(6.8 ⫾ 8.5) ⫻ 10⫺6
(9.0 ⫾ 10.4) ⫻ 10⫺6
(2.6 ⫾ 0.6) ⫻ 10⫺4
(3.1 ⫾ 0.2) ⫻ 10⫺4
A total of five individual cultures for wild type and four
for slk19⌬ mutants were tested. Results shown were mean of
different cultures ⫾ standard deviation. More than 600 Ade⫹
colonies (on seven plates) for mitotic recombination and more
than 1000 (on nine plates) for meiotic recombination were
counted for each individual culture.
Figure 4.—Recombination and segregational events that
produce the various types of dyads shown in Table 5. ⫹ represents the dominant allele while ⫺ represents the recessive
allele. SCO stands for single crossing over, DCO for double
crossing over, equational for equational segregation, and reductional for reductional segregation. (*)These dyads are phenotypically indistinguishable.
combination, a high level of meiosis I nondisjunction
is observed (John 1990). To test whether slk19⌬ mutants
have normal levels of meiotic recombination, we examined two types of recombination, intergenic recombination and heteroallelic recombination. For intergenic
recombination, wild type and slk19⌬ diploid strains heterozygous for ura3 and can1 were sporulated and the
resulting tetrads (wild type) or dyads (slk19⌬) were dissected as described (materials and methods) and
scored for the segregation of markers (Table 5; the
meiotic events producing various types of dyads are
shown in Figure 4). The genetic distance between can1
and ura3 in the slk19⌬ background was calculated to be
64.2 cM, which is higher than the value of 37.3 cM
observed in our wild-type background, or the 43 cM
registered with the Saccharomyces Genome Database,
suggesting that slk19⌬ diploids might have an increased
frequency of recombination (see materials and methods for a description of the calculation of map distance). To further examine recombination frequencies,
we measured the intragenic recombination between two
ade2 alleles, ade2-1 and ade2-R. Recombination between
these heteroalleles is mostly due to gene conversion
(Lichten et al. 1987) and will generate a wild-type ADE2
allele, resulting in adenine prototrophy. For this analysis
we measured both mitotic and meiotic recombination
by determining the frequency of Ade⫹ colonies follow-
ing growth on YEPD and sporulation media. The number of Ade⫹ colonies varied widely for different cultures
following vegetative growth, most likely due to differences in the time of occurrence of the Ade⫹ recombinants (Table 6). However, in all cases the frequency of
mitotic recombination was ⬍3% of the meiotic frequency. The frequency of meiotic recombination was
similar in wild-type and slk19 diploids, with slightly elevated levels observed for the slk19 diploids (Table 6).
Loss of SPO11 cannot rescue the meiotic bypass of
slk19 mutants: If the recombination attachment between
homologues cannot be resolved at the metaphase I/
anaphase I transition, the intact bivalent (the attached
meiotic homologues) could segregate to one pole during meiosis I. This may result in disomic spores and
genetically appear as a meiosis I bypass. Since recombinant dyads were observed, the recombination attachment must eventually be resolved, but this may not occur
until the second meiotic division, with loss of the cohesion between the arms of sister chromatids, which has
been proposed as a mechanism to maintain chiasmata
(Moore and Orr-Weaver 1998). To examine the influence of recombination on the slk19 meiotic phenotype, we measured sporulation in slk19⌬spo11⌬ double
mutants. Spo11p is the catalytic subunit of the doublestrand break-associated protein (Keeney et al. 1997).
spo11 mutants have been shown previously to be able
to bypass the requirement of recombination for meiotic
nuclear division (Klapholz et al. 1985; Bishop et al.
1992). In its absence, cells skip recombination and undergo two successive divisions with low spore viability.
Interestingly, we found that slk19⌬spo11⌬ double mutants failed to perform either meiotic division. No nuclear division was visible by DAPI staining and no spores
were observed by differential interference contrast
(DIC) microscopy. Therefore, lack of recombination is
apparently unable to rescue the meiosis I bypass of
slk19⌬ mutants. Furthermore, this result implies that at
least the beginning step of recombination is actually
584
X. Zeng and W. S. Saunders
TABLE 7
Effect of hydroxyurea on the sporulation of slk19⌬
and spo13⌬ mutants
Genotype
slk19⌬
slk19⌬
spo13⌬
spo13⌬
HU
(mm)
Asci/cells
(%)
Triads ⫹ tetrads/asci (%)
0
5
0
5
61.3
53.8
33.7
38.2
4.9
5.2
0
9.4
Diploid strains WSY994 (slk19⌬) and WSY996 (spo13⌬) were
sporulated at 26⬚ in the presence of the indicated concentrations of hydroxyurea. A total of 600 cells were scored. The
presence of either triads or tetrads indicates entry into the
second meiotic division.
required for slk19 mutants to skip meiosis I or to undergo meiosis II.
slk19 mutants utilize a different mechanism from
spo13 mutants to bypass meiosis I: The meiotic phenotype of slk19 mutants is similar to that of spo13 mutants.
Both are competent for meiotic recombination but skip
meiosis I (Klapholz and Esposito 1980b). But unlike
slk19 spo11 double mutants, spo11 spo13 double mutants
are able to sporulate and produce dyads with viable
spores (Klapholz et al. 1985). Spo13p has been proposed to act as a regulatory protein to slow down progression into meiosis I in order to allow sufficient time
to assemble the meiosis I spindle apparatus (McCarroll and Esposito 1994). This conclusion is supported
by the observation that the meiosis I bypass in spo13⌬
mutants can be partially rescued by delaying meiotic
progression, for example, by sporulating at low temperature or in the presence of sublethal concentration of
hydroxyurea (HU). HU is a DNA synthesis inhibitor
(Timson 1975) and has been shown to be able to delay
the cell cycle through the RAD9-dependent pathway
(Weinert and Hartwell 1988). The different meiotic
phenotypes of slk19 spo11 and spo11 spo13 diploids suggest that slk19 mutants utilize a different mechanism
from spo13 to bypass meiosis I. To confirm this, we
examined sporulation of slk19⌬ mutants at 20⬚ or in the
presence of 5 mm HU. The meiotic defect of slk19⌬
mutants could not be improved by these treatments
(Table 7 and data not shown). This result further suggests that slk19⌬ mutants bypass meiosis I under different circumstances than spo13 mutants.
slk19 mutant diploids begin the first meiotic division
and spore morphogenesis at the same time as the wildtype cells: Since the slk19 mutant diploids undergo only
a single meiotic division, they might be expected to
sporulate more rapidly than wild-type cells, which must
divide twice prior to spore wall formation. Alternatively,
if slk19 mutants attempt both meiotic divisions or if the
timing of sporulation is independent of meiotic division,
the rates of sporulation may be similar. To see which
Figure 5.—Timing of the sporulation events in wild-type
and slk19 mutant diploids. Isogenic wild-type and slk19 mutant
diploids were sporulated at 26⬚. Samples were taken throughout sporulation and the percentage of bi- and tetranucleate
cells (by DAPI staining) and asci was determined. A total of
400 cells were counted for each sample. (A) Percentage of
binucleate and tetranucleate cells vs. time in sporulation. (B)
Percentage of asci vs. time in sporulation.
case is true for slk19 mutants, isogenic slk19 and wildtype diploid cells were sporulated and samples at various
timepoints were stained with DAPI to visualize the DNA.
The number of binucleate and tetranucleate cells and
asci for each sample were counted. Interestingly, we
found that slk19 mutants started their meiotic division
at nearly the exact time as the first meiotic division of
wild-type cells (Figure 5A). The second meiotic division
Slk19p Is Required for Meiosis I
in wild-type cells occurred ⵑ3 hr later than the first
meiotic division. Surprisingly, we also found that although only a single meiotic division occurred in slk19
mutants, both wild-type and slk19 mutant cells form asci
at the same rate (Figure 5B). This can be explained by
two possible mechanisms. First there is a delay between
the completion of meiotic division and the spore wall
formation in slk19 mutants. Alternatively, there may be
an internal clock that regulates the timing of spore wall
formation that is independent of meiotic division.
DISCUSSION
We have identified a gene in S. cerevisiae that is required if the diploid cell is to complete two successive
meiotic divisions. When mutants homozygous for slk19
sporulate, they produce mostly two-spore dyads. The
dyads are a result of a single equational division as demonstrated by the following observations: (1) Nonmating
spores of the dyads can sporulate, indicating that they
are diploid. (2) Mutational frequencies indicate spores
of the dyads contain at least two copies of chromosome
V on which the CAN1 gene is located, consistent with
being diploid. (3) Antitubulin indirect immunofluorescence revealed only a single spindle per cell during
meiosis. (4) The segregation pattern of centromerelinked markers indicates that most chromosomes segregate equationally but not reductionally. Therefore
Slk19p is essential for the reductional division from a
diploid to a haploid genome. Recombination in slk19
mutants was normal or slightly elevated over wild-type
cells, consistent with previous observations that meiotic
recombination is not necessarily associated with reductional division and that equational division does not rely
on prior reductional division (Klapholz and Esposito
1980b). These observations confirm the independent
and nonlinear organization of the meiotic pathway.
The meiotic phenotype of slk19 mutants is similar to
that of the previously described spo12 and spo13 mutants
(Klapholz and Esposito 1980b). Each has a single
equational division meiosis and normal or elevated levels of meiotic recombination. Spo13p has been proposed to act as a timing regulatory factor to slow down
the meiotic progression to allow sufficient time for assembly of the meiosis I spindle apparatus (McCarroll
and Esposito 1994). But unlike spo13 mutation, slk19
mutation cannot allow spo11 mutants to form viable
spores, and the meiotic defect of slk19 mutants cannot
be improved by the addition of HU or by lowering the
sporulating temperature. These results suggest that the
meiosis I bypass in slk19 mutants is fundamentally different from that of spo13 mutants. slk19 mutants are more
similar to spo12 mutants in that both require recombination for their single meiotic division. Both slk19 spo11
double mutants and spo12 spo11 double mutants are
unable to sporulate (Esposito and Klapholz 1981).
585
But the underlying mechanism for the meiotic defect
in spo12 mutants is also unclear.
We propose two models to explain the slk19 meiotic
phenotype. The first is that Slk19p may be uniquely
required for spindle stability in meiosis I. Thus Slk19p
has a similar role in meiosis I as mitosis, but becomes
essential in meiosis I. Slk19p could become essential
due to a difference in the nature of the kinetochoremicrotubule attachment. In meiosis I each sister kinetochore pair is attached to only a single spindle pole.
Bipolar attachment is important to generate tension
and stabilize the microtubule-kinetochore association
(reviewed in Nicklas 1997). Slk19p has also been proposed to stabilize kinetochore microtubules in mitosis
(Zeng et al. 1999) and in meiosis I, without the benefit
of the tension associated with bipolar attachment, this
activity may become critical. However, the apparent low
level of chromosome loss in viable slk19 spores may
argue against this model. Slk19p could also become
essential due to a change in activity of Kar3p. Previous
work has shown that Slk19p and Kar3p work together
during mitosis to assemble and maintain spindles (Zeng
et al. 1999). During meiosis I Kar3p appears to be required for homologous pairing (Bascom-Slack and
Dawson 1997), an event not observed in mitotic division
or meiosis II. If Kar3p becomes diverted to other tasks
during meiosis I, Slk19p could be expected to become
essential for spindle stability at this division.
A second model proposes that what appears to be a
single meiosis II division in slk19 mutants is in fact an
aberrant meiosis I. This model can explain why slk19⌬
mutants start their single meiotic division at the same
time as the first meiotic division of wild-type cells. The
delay between the completion of meiotic division and
the spore wall formation in slk19 mutants could result
from the failed attempt at a second meiotic division in
the slk19 mutants. Chromosomes may segregate equationally during meiosis I if for example, Slk19p is required to hold the sister centromeres together in meiosis I. In slk19 mutants the sister centromeres may
separate prematurely in meiosis I, giving the appearance
of an equational division. The sister chromatids may
also missegregate equationally during meiosis I if Slk19p
is required to regulate the timing of sister kinetochore
differentiation. Substantial experimental evidence
suggests that sister kinetochores undergo functional
(Goldstein 1981; Rufas et al. 1989) and spatial differentiation during meiosis I (reviewed in Nicklas 1997),
which limits their ability to bind to both poles. If the
timing of sister kinetochore differentiation depends on
Slk19p then mutational loss could lead to precocious
sister kinetochore differentiation. As a result, sister chromatids may bind microtubules from opposite poles and
segregate equationally during meiosis I.
We may be able to distinguish between these two
models by electron microscopy. The spindle pole bodies
during meiosis I and mitosis have a similar structure,
586
X. Zeng and W. S. Saunders
while the meiosis II spindle pole body has an outer
plaque that is larger and denser (Moens and Rapport
1971; Peterson et al. 1972). This observation was used
by Moens and co-workers to determine that ATCC4117
yeast cells, from which the original spo12-1 and spo13-1
mutations were isolated (Klapholz and Esposito
1980a), initiated some aspect of meiosis I even though
only equational division is observed genetically (Moens
1974; Moens et al. 1977). Similarly, the recent advent
of green fluorescent protein-labeled centromeres may
allow us to determine if centromeres divide prematurely
in slk19 mutants (Klein et al. 1999).
Although the underlying mechanism for the single
division meiosis of slk19 mutants is not clear, the observation that loss of SLK19 leads to a single equational
division is the first identification of a centromere protein that influences the choice between equational and
reductional division in meiosis.
The authors thank Drs. Giora Simchen of the Hebrew University
of Jerusalem, Israel, for the spo13 mutants and ade2 heteroalleles and
Rochelle Esposito of the University of Chicago for the spo11 mutants.
The work was funded through American Cancer Society award CB171 to W.S.S.
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Communicating editor: M. D. Rose