EFFECTS OF THE MITOTIC CELL-CYCLE MUTATION
cdc4 ON YEAST MEIOSIS
G. SIMCHEN
AND
J. HIRSCHBERG
Department of Genetics, The Hebrew University,
Jerusalem, Israel
Manuscript received May 25, 1976
Revised copy received December 20, 1976
ABSTRACT
The mitotic cell-cycle mutation cdc4 has been reported to block the initiation of nuclear DNA replication and the separation of spindle plaques after
their replication. Meiosis in cdc4/cdc4 diploids is normal at the permissive temperature (25") and is arrested at the first division (one-nucleus stage) at the
restrictive temperature (34" or 36"). Arrested cells at 34" show a high degree
of commitment to recombination (at least 50% of the controls) but no haploidization, while cells arrested at 36" are not committed to recombination.
Meiotic cells arrested at 34" show a delayed and reduced qynthesis of DNA
(at most 40% of the control), at least half of which is probably mitochondrial.
It is suggested that recombination commitment does not depend on the completion of nuclear premeiotic DNA replication in sporulation medium.-Transfer of cdc4/cdc4 cells to the restrictive temperature at the onset of sporulation
produces a uniform phenotype of arrest a t a I-nucleus morphology. On the
other hand, shifts of the meiotic cells to the restrictive temperature at later
times produce two additional phenotypes of arrest, thus suggesting that the
function of cdc4 is required at several points in meiosis (at least at three different times).
MITOTIC functions are required in yeast meiosis, as is evident from the inability of temperature-sensitive cdc mutants that are defective in nuclear
mitotic division and/or DNA synthesis to complete normal sporulation ( SIMCHEN 1974). In the present paper, we develop the methodology for an analysis
CULOTTIand REID 1970) in
of the behavior of the cdc mutants (HARTWELL,
meiosis.
Our first line of inquiry relates to the dissection of meiosis. Given that the cdc
lesions serve as landmarks in the cell-cycle (HARTWELL
1974) and that the
mitotic functions of the nuclear cdc genes are also executed in meiosis, we ask
which meiotic characteristics are independent of the cdc lesions.
The second line of inquiry relates to the primary effects of the cdc mutations
in mitosis (and also in meiosis). Each cdc mutant, when arrested in the mitotic
cell cycle at the restrictive temperature, exhibits a multitude of defects, resulting
from the interdependence of functions. The distinction between the primary
lesion of a mutant and its secondary effects can be guided by the meiotic behavior
of the mutant, because meiosis includes two divisions that share certain features
Genetlcs 86: 57-72 May, 1977
58
G . SIMCHEN A N D J. H I R S C H B E R G
but differ in others. The terminal phenotypes of the meiotic cdc lesion might lead
us to the recognition of the primary effect, or at least to the dismissal of certain
effects as secondary ones. For example, the spindle and its components are
formed in both meiotic divisions and therefore a cdc lesion affecting primarily
the spindle should bring about arrest at either division, depending upon the time
of transfer to the restrictive temperature. On the other hand, centromere division takes place only in the second division and DNA synthesis only in (or prior
to) the first meiotic division. A mutation affecting primarily DNA synthesis is
thus expected to result in arrest at the first meiotic division, while a mutation
that affects a product that is primarily required for the second division of meiosis
will cause an arrest at that division.
The analysis of meiosis in the cdc mutants may therefore enlighten us with
respect to the genetic control of both meiosis and mitosis, as illustrated in the
present analysis of cdc4. I n vegetative cells under restrictive conditions, this
mutation has been reported to block the initiation of nuclear DNA replication
(HARTWELL
1971; PETESand NEWLON1974) and the separation of spindle
plaques after their replication (BYERSand GOETSCH
1973).
MATERIALS A N D METHODS
Strains: The diploid 212 was obtained from the mating of 135.1.1, which is a derivative of
et al. 1973) and of the genotype a adel ade2 ural tyrl his7 lys2 cdc4-3, with
458 (HARTWELL
our standard strain CY ade2-Rd met canl-11. The two diploids 212-1 and 212-10, were obtained
as temperature-sensitive colonies following UV irradiation of cells of 212 (see SIMCHEN1974
for details) and were shown to he homozygous for cdc4-3 by tetrad analysis in which all the
haploid progeny were of the typical cdc4 phenotype (when replicated colonies were incubated
at 36"). 212 is heterozygous for cdc4-3 and does not differ phenotypically from our standard
diploid 419. The latter does not carry temperature-sensitive cdc mutations and results from the
et al. 1973) with our above mentioned CY strain.
mating of A364A (HARTWELL
Media: YEP and PSP2 are glucose and acetate vegetative growth media, respectively, and
PIAON and SALTS1972). M I N is a minimal
SPM is sporulation medium (for details see SIMCHEN,
medium containing 6.7 g yeast nitrogen base (without amino acids) and 20.0 g glucose in one
liter water. -AD serves to detect adenine prototrophs and consists of MIN to which the following
amino acids are added: lysine, methionine, tyrosine (40 mg each), histidine, uracil ( I O mg each).
CAN medium supports the growth of canavanine-resistant colonies only, and is similar to -AD
hut contains also adenine (40 mg) and canavanine sulfate (40 mg). Media are solidified with
1.5% Difco Bacto-agar.
Vegetatiue growth and sporulation: Details of growth conditions are given in previous publications (SIMCHEN,PIFJONand SAcrs 1972; SIMCHEN1974). In order to examine sporulation at
the permissive and restrictive temperatures, cells were grown in PSP2 liquid medium supplemented with adenine (40 pg/ml), at the permissive temperature of 25", to a titer of IO' cells/ml.
The cells were washed in water, resuspended in liquid SPM, and incubated for 1-1.5 hrs in a
shaker at 25". At this point, one o r more subcultures were transferred to the restrictive temperature and some subcultures remained at 25" as controls. The initial short incubation of sporulating cultures at the permissive temperature was adopted in order to enable temperaturesmsitive mutants to complete the mitotic cell cycles in which they were engaged without being
subjected to the temperature block. The restrictive temperature in sporulation experiments was
usually 33.5"-34" (SIMCHEN1974) and not 36", which was the restrictive temperature for
et al. 1973). Certain sporulation experiments, however,
growth for most cdc mutants (HARTWELL
were carried out at 36", hut at that temperature most of our nonmutant strains already showed
]-educed sporulation. For instance, the cdc4 heterozygote 212 produced after 24 hours in SPM
MITOTIC DEFECT I N YEAST MEIOSIS
59
61% asci when incubated at 25", 65% asci at 33" and 16% asci at 36". Similar results were
obtained with the strain 419.
Fization and staining: Cells were fixed in formaldehyde and stained with Giemsa (ROBINOW
and MARAK
1966; HARTWELL,
CULOTTI
and REID1970).
DNA measurements: The fluorescence assay for DNA was developed by C. P. MILNEand
L. H. HARTWELL
and is described by SIMCHEN,PIGON and SALTS(1972). A second assay is based
upon the increase in radioactive label in DNA, in sporulating cells (in SPM) that were prelabelled in the vegetative medium (PSP2). The label used was 14C-2-uracil (A.E.C., Nuclear
Research Center, Negev). Further details are given elsewhere (SIMCHEN,PIGON and SALTS
1972).
Equilibrium sedimentation of D N A : Cells in PSP2 were prelabelled with 14C-2-uracil and
were labelled again in SPM with 3H-2-adenine (same source as 14C label). Spheroplast preparation, lysis, and preparation of CsCl gradients were done following PIGON, SALTSand SIMCHEN
(1974). DNA sedimentation was carried out for 60 hours at 35000 rpm i n the Beckman Type 50
rotor.
RESULTS
The results reported here concern three diploid strains, 212, heterozygous for
cdc4-3, and 212-1 and 212-10, both of the homozygous constitution cdc4-3/cdc4-3.
The three strains sporulate normally in sporulation medium (SPM) at the permissive temperature of 25" (50-75% asci by 24 hrs) and so does 212 at the restrictive temperature of 34". The two homozygotes, however, do not sporulate
at 34" o r show at most 0.5% aberrant, 2-spored asci.
Similar behavior was observed for homozygotes obtained in the same way
from the other alleles of cdc4.
Timing of the lesion
Cultures of the homozygotes 212-1 and 212-10, grown at the permissive conditions and transferred to SPM at 25", were split into subcultures, each being
transferred to 34" at a different time. Sporulation was assessed 24 hours after
the cultures were exposed to SPM. Subcultures that were transferred to 34" at
early times did not sporulate. while late transfers did, thus reflecting on the last
point, with reference to the course of meiotic events at 25", at which the function
is executed. Execution here refers to either functioning of the thermosensitive
gene product at the permissive temperature, o r alternatively to the thermosensitive formation of the gene product. In any case, since there may be more than
one execution point f o r a given lesion during meiosis, only the last one can be
located by this experimental procedure. Similar consideration was given to meiotic lesions by ESPOSITO
et al. (1970).
The results of such an experimcnt are given in Figure 1, which also contains
data concerning the timing o€ meiotic events at the permissive temperature (Figure lb, see also Table 1 ) . It was suggested by HARTWELL
(1971) that cdc4 mutants are defective in a DNA initiation function. Therefore, it was expected that,
with respect to meiosis, the function will be required only in the first division
and that the thermosensitivity of the meiotic cells will be over when all the cells
have initiated premeiotic DNA synthesis. From the kinetics of the various meiotic
parameters at 30" (SIMCHEN,
PISONand SALTS1972) and at 25" (HIRSCHBERG,
60
G . SIMCHEN A N D J. HIRSCHBERG
10
HOURS
20
IN SPM
FIGURE
1.-a. Sporulation of 212-10 (cdc4/cdc4) upon transfer to restrictive temperature
(34") at various times. Sporulation was estimated at 24 hrs. The abscissa marks the time of
transfer. The points represent the means of five experiments. b. Meiotic events at the permissive
temperature (25") at the time oI transfer. The curve represents DNA content and each point
is the mean of three replicates. Arrow I indicates point at which half the sporulating cells have
gone into or passed through the 1st meiotic division. Arrow I1 indicates a corresponding point
for the 2nd meiotic division.
unpublished data), we estimate that the premeiotic replication in a single cell
lasts approximately 2 hrs. Based on the completion of the premeiotic replication
at 12 hrs (Figure lb), it is assumed that at 10 hrs most of the cells have initiated
DNA synthesis. Yet only half of these cells could sporulate upon transfer to the
restrictive temperature (Figure l a ) . Similar considerations show that the thermosensitivity curve in Figure l a is approximately 2 hrs later than expected
from the DNA synthesis data (Figure Ib). This result means that the last execution point of cdc4 in meiotic cells occurs approximately 2 hrs after the initiation of the premeiotic replication.
For further clarification of the thermosensitive lesion, the terminal phenotypes
of cells in stained preparation were examined in the subcultures that were transferred at various times to the restrictive conditions.
Terminal phenotypes
Vegctative cdc4 cells arrested at the restrictive temperature show three distinct features. The cells arrest prior to the initiation of the nuclear DNA synthesis (HARTWELL
1971; PETES
and NEWLON1974), they have undergone rep-
61
MITOTIC DEFECT I N YEAST MEIOSIS
TABLE 1
Nuclear phenotypes of cells of 212-10 fixed at various times during sporulation at 25°C
Hrs in
SPM
I-nucleus
cells
1st division
cells
Percent
2-nuclei
2nd division
cells
cells
4-nuclei
cells
asci
~~
(a) 7
8
9
10
12
11
12.5
13
14
15.5
25
100
0
0
99
0
1
96
83
68
2
6
2
6
9
87
76
57
51
2
5
5
4
10
3
6
10
8
5
4
41
1
27
22
0
0
2
0
0
0
4
5
3
4
16
16
24
5
0
0
0
0
0
1
16
0
0
0
6
0
14
19
23
51
4.
0
0
0
0
0
0
14
72
Notes: (a) and (b) represent two different experiments; a t each of the given times, a sample
of 200 cells and asci were scored following fixation and staining.
lication of the spindle plaques but not their separation (BYERSand GOETSCH
1973) and they show rhythmical budding corresponding to several cell cycles
(HARTWELL
1971). The uncontrolled budding cannot be the primary lesion of
cdc4 since budding is absent in meiosis and the homozygotes are defective in
meiosis (at 34") in a manner totally unrelated to this feature. This leaves US
with the following possibilities: (i) DNA initiation is the primary function of
cdc4 and plaque separation is dependent on DNA initiation. (ii) Plaque separation is the primary function of cdc4 and DNA initiation is dependent on it.
(iii) Both functions, DNA initiation and plaque separation, are secondary and
are dependent on an unknown primary function of cdc4. In order to distinguish
between these possibilities, experiments were performed in which cdc4 homozygotes were transferred from the permissive to the restrictive temperatures (25'
to 34" or to 36") at various times and the terminal phenotypes were examined
in Giemsa stained preparations 24-30 hrs following the onset of sporulation
(Table 2).
Following this staining technique, one can observe in meiotic yeast cells sevand ROBINOW
1969, and our Figure 2), includeral phenotypes ( MATILE,MOOR
ing cells with single nuclei, cells with single stretched-out nuclei that are
regarded as first-division nuclei, cells with two separate nuclei each, cells with
two stretched-out nuclei that are interpreted to be second-division cells, and asci
containing four o r sometimes two spores. Electron microscopy has shown that the
nuclear membrane remains intact until spore formation (MOENS
and RAPPORT
1971), suggesting that the Giemsa stained "nuclei" are in fact chromatin structures that segregate from each other, but are not bona fide nuclei with separate
nuclear membranes.
Early transfers to the restrictive temperature (at 0, 1.5 and 2 hrs) gave a uni-
62
G. S I M C H E N A N D J. H I R S C H B E R G
TABLE 2
Terminal phenotypes at 24 hrs of cells transferred to the restrictive temperature at various times
Time of shift
1-nucleus
cells
2-nucleus
cells
Percent'
4-nucleus
cells
%spored
asci
4-spoyed
asci
STRAIN 212-10 (cdc4/cdc4)
No shift
control at 25°C
Shifts 25"C-t34"C
0 hrs
2 hrs
4 hrs
6 hrs
8 hrs
10 hrs
12 hrs
14 hrs
Shifts 25"C+36"C
0 hrs
2 hrs
4 hrs
7 hrs
9 hrs
11 hrs
12 hrs
control a t 25°C
control at 34°C
32
2
99
99
90
1
1
74
58
39
33
33
Icw)
100
98
97
89
61
52
35
26
2
7
10
15
12
5
3
2
62
0
0
1
5
7
12
9
0
0
0
4
0
0
0
0
4
4
1
10
10
4
5
12
3
STRAIN 212 (CDC4/cdc4)
1
3
0
1
4
4
0
0
2
3
0
0
0
0
6
12
34
51
58
0
0
0
0
2
15
28
61
65
* Each sample contained 200 stained cells and asci.
form phenotype of arrest of one-nucleus cells. Later transfers, however, showed
additional phenotypes of arrest, namely %nuclei and +nuclei cells (Table 2 and
Figure 2a). In none of these experiments were cells observed to be arrested in
the middle of either the first o r the second nuclear divisions of meiosis (stretchedout nuclei), although these configurations are quite frequent when a sporulating
culture is stained following fixation at the appropriate times (see Table 1 and
Figure 2b). In other transfer experiments (to 34") where cell samples were
stained at 48-50 hrs, terminal phenotypes were obtained in proportions similar
to those given in Table 2. These data show that the 2-nuclei and 4-nuclei phenotypes are not transient and that the effect of cdc4 on nuclear morphology does
not show leakiness at 34".
Possibility (i) above could be distinguished from the other two because it predicted that the only terminal phenotype to be found in arrested meiotic cells
would be of arrest at a one-nucleus stage, and that cells that were not arrested
at that point would be able to complete meiosis and form asci. As the additional
phenotypes were observed (Table 2), the former possibility may be dismissed
and the defect in DNA initiation in meiotic cells can be regarded as a secondary
effect resulting from the cdc4 lesion. This conclusion was reached independently
63
MITOTIC DEFECT IN YEAST MEIOSIS
W
' c,
Y
FIGURE
2.-a. Terminal phenotypes of meiotic cells arrested at the restrictive temperature.
These cells of strain 212-10 were incubated in SPM at 25" for 10 hrs, transferred to 34" and
then fixed and stained at 24 hrs. b. Intermediate phcnotypes in a sporulating culture of 21210
at 12 hrs (at 25'). Note cells during the 1st or 2nd meiotic divisions. Bar represents 10 pm.
(HARTWELL
et al. 1974) following the observation that another mutant, cdc7,
goes through plaque separation in mitosis at 36" (BYERS
and GOETSCH
1973) but
does not initiate DNA synthesis.
The finding of three terminal phenotypes (with regard to nuclear morphology) suggests that the primary function of cdc4 is required on at least three occasions during meiosis, the first leading to the first division, the second leading
to the second division and the third prior to spore formation. It should be noted
that in the control culture (Table 2) and in comparable temperature shifts of
strains that are not temperature-sensitive (including the heterozygote 212), the
frequencies of 2-nuclei and 4-nuclei cells at 24 hrs in SPM are usually not higher
than 3% and 4%, respectively. The three terminal phenotypes, as well as other
meiotic phenotypes such as dividing nuclei, that could only be observed during
meiosis but not as terminal phenotypes of cdc4 homozygotes, are shown in Figure 2.
Meiotic events at the restrictive temperature
In the previous section, the terminal phenotype of meiotic cells in the restrictive conditions was assessed by light microscopy of Giemsa-stained preparations.
The meiotic parameters used were those of nuclear morphology. In order to further understand the cdc4 lesion, we have examined some additional meiotic
parameters following the arrest at the restrictive temperature.
Two meiotic events, recombination and haploidization, may be signalled by
64
G. SIMCHEN A N D J. HIRSCHBERG
the ability of sporulating cells to form colonies on selective media. The strains
we used in this study could not form colonies on medium lacking adenine (-AD),
unless they haw e undergone intragenic recombination in ade2 and produced
ADE prototrophs. The detection of such prototrophs depends also on the presence of the wild-type allele at the adel locus, which has to be taken into account
when recombination frequencies are calculated for haploid segregants. Haploidization is signaled by the ability of cells to form colonies on canavanine containing
medium (CAN), on which the heterozygous diploid C A N l l c a n l cannot grow,
because the mutant allele for resistance ( c a n l ) is recessive. The haploid can1
cells, which segregate in meiosis, form colonies on CAN medium (recombination
between can1 and the centromere may also result in resistant cells, but their frequency is much lower than that obtained as a result of haploidization). The
plating of meiotic cells on selective media was followed by 3-5 days incubation
at 25" and only then were colonies scored. This delay in scoring meant that the
ability of the plated cells to form recombinant or haploid colonies was assessed,
rather than the presence of recombinant or haploid cells at the time of plating.
The ability of cells to form recombinant colonies is called recombination commitment and it precedes the actual act of meiotic recombination (ESPOSITO
and &P O S I T ~ 1974; SILVA-LOPEZ,
ZAMBand ROTE 1975; SALTS,
SIMCHEN
and PIGON
1976). Recombination commitment can be defined as a reversible stage of meiosis
at which the conditions for recombination have been established, enabling the
cell to recombine even if it continues to develop as a nonmeiotic cell. Table 3
TABLE 3
Recombination (ADE prototrophs/IOfi survivors) and sporulation at 24 hrs
Strain
25oc
Recombination Sporulation
34oc
Recombination Sporulation
Exp. 1 212
212-1
212-10
180
200
180
60%
73%
75%
120
40
40
65%
0
Exp.2 212
212-1
212-20
150
170
165
51 %
65%
56%
120
60
95
62%
0
0
Exp.3 212
212-1
212-10
175
155
110
55%
64%
74%
100
50
80
61%
0
0
Exp.4 212-1
160
71%
120
0
115
54
61 %
0
Exp.5 212
212-1
36'C
Recombination Sporulation
0
Exp. 6 212
212-1
212-10
131
152
156
52%
M%
57%
100
97
94
70%
0
0
Exp. 7 212
212-1
212-10
237
206
22 1
61 %
67 %
75%
166
129
201
65 %
0
0
208
11
8
16%
0
0
65
MITOTIC DEFECT I N YEAST MEIOSIS
contains data of seven experiments in which cells were plated on -AD medium
after 24 hours in the sporulation conditions (liquid SPM) . In six of the experiments only the lower restrictive temperature for sporulation, 34", was used,
while in the seventh experiment an additional restrictive temperature of 36"
was employed. In all cases, the titers of the cultures and cell viability were assessed by dilution platings on YEP medium, and the recombination estimates
given in Table 3 are based on these estimates and on the number of colonies
fomed on -AD plates. All plates were incubated at 25". Recombination kinetics
of another experiment are shown iii Figure 3. Cell viability in these experiments
was between 50% and 80%. With very long incubations (30-50 hrs) of meiotic
cells at the restrictive temperatures, viability commonly went down: 40-80%
viability values were observed upon such incubations at 34" and 30-60% were
2.0- 212
A
A
-A
w
A
- 12
0
-8
1.0A
-4:
0
1
0
3
m
z
8
I-
z 2.0 w
+
z
s
z
212-1
m
1.0 4
al
-4
z
0
-L
0,
LLI
->
k
4
w
a
2.0212 - 10
-8
A
A
FIGURE
3.-Recombination kinetics of 212 (CDC4/cdc4) and of 212-1 and 212-10 (both
34". The cells were grown in PSP2 f adenine (40 pg/ml) at 25" to a titer of IO7
cells/ml, washed in water, resuspended in SPM and incubated in 25" shaker for 1.5 hrs. At this
cdcl/cdc.l) at
stage the cultures were transferred to a 34" shaker, and at given times samples were plated on
5 -AD and on 5 YEP plates, which were incubated at 25", to estimate the frequency of ADE
prototrophs ( 0 ) .DNA content was measured by the fluorescence assay, on 3 one-ml samples
from each culture at the given times (A-means
of triplicate measurements.)
66
G.
SIMCHEN A N D J. HIRSCHBERG
obtained following long incubations at 36". In all cases, however, recombination
frequencies were calculated per survivor.
It is evident that following incubation in sporulation medium at 34", ADE
prototrophs are produced by the homozygous cdc4 strains at a frequency of at
least 50% of that found in the heterozygote at the same conditions. The frequency of prototrophs produced by the homozygotes following similar incubation at 36", however, is not significantly higher than in mitotic cells; thus apparently no recombination events took place in these mutant strains at 36". The
heterozygote 212 behaves in a similar way to the wild-type homozygote 419
( SIMCHEN1974). The homozygous mutant strains do not differ from each other
and at the permissive temperature they are very similar to the heterozygote.
One further point that should be noted is that in the heterozygote the level of
recombination following sporulation at 25" is consistently higher than at 34".
This phenomenon of higher recombination frequency at 25" compared to 34" is
common to many strains that are not temperature-sensitive, and has been discussed elsewhere ( SIMCHEN,
IDAR
and KASSIR1976).
Haploidization was normal at 25 ",ranging between 50-75 %, and corresponding to the sporulation frequencies observed. This correspondence was also held
at 34": the homozygotes gave 2% or less can1 colonies (per survivor), and these
were diploid resulting from recombination between can1 and the centromere,
while the heterozygote reached thi. noma1 level. Thus at the restrictive temperature of 34", meiotic cells of the homozygotes were not committed to haploidization, but were committed to recombination. To test whether the ADE recombinant colonies of 212-1 were haploid, we replica-mated 200 such colonies on
YEP to Zysl testers that were either a or cy, and after an overnight incubation
replicated the plates onto MIN plates, and incubated them at 35-36". Only 6%
of the recombinant colonies from 34" were maters (i.e. haploids), while 90%
were maters in a similar sample of colonies from the same strain at 25". Recombinant colonies obtained from the heterozygote 212 at both temperatures were
mostly maters (75%-85%, but thz test was only made on approximately half of
the recombinants, those that were temperature-sensitive) .
The finding that cdc4 homozygotes could go through with recombination commitment at the restrictive temperature of 34" contradicted the supposition from
mitotic cells (HARTWELL
1971), that the mutation prevented initiation of DNA
synthesis and agreed with our previous conclusion that the latter effect of cdc4
was a secondary one. Generally, it is reasonable to relate recombination events
to the G3 stage or to S rather than to GI. I n yeast sporulation especially, it has
been common experience that recombination commitment occurs concomitantly
with the premeiotic DNA replication (SHERMAN
and ROMAN1963; ESPOSITO
1971; SIMCHEN1974; SILVA-LOPEZ,
ZAMB
and ESPOSITO
1974; ROTHand FOGEL
IDAR
and KASSIR
1976). We therefore tried to analyze
and ROTH1975; SIMCHEN,
further the DNA synthesis of 212-1 and 212-10 (cdc4/cdc4) in sporulation
conditions at the permissive and restrictive conditions. Figures 3 and 4 describe
DNA synthesis measured by two different methods, namely, radioactive label
incorporation and spectrophotofluorometric measurements. I n both experiments
67
M I T O T I C DEFECT IN YEAST MEIOSIS
1
I
I
10
20
HOURS IN SPM
I
30
FIGURE
4.-DNA synthesis of 212-1 (cdc4/cdc4) at the permissive temperature, 25" (A)
and at the restrictive temperature of 34" ( a ) .The culture was grown overnight in PSP2 -k
adenine (40 /.tg/ml) f2 pCi/ml 14C-2-uracil (specific activity 60.4 pCi/p mole), to a titer of
1.3 x l o 7 cells/ml. Cells were washed twice and resuspended in SPM, and incubated in a 25"
shaker. The culture was split and one subculture was transferred to 34" after 1.5 hrs. Triplicate
0.3 ml samples were taken a t the given times and label in DNA was counted following overnight
incubation in NaOH and TCA precipitation. Sporulation a t 26 hrs was 68% a t the permissive
temperature and zero at the 34" (at 26 hrs and later, at 50 hrs).
the increase in DNA in the homozygotes at 34" is very slow, particularly if
compared to the heterozygote 212 in the same conditions. But a certain increase
in DNA does exist, and it varies from 20% up to almost 40% by 24 hours in
some experiments. By contrast, no more than a 10% increase in DNA content
was observed when 212-1 was incubated in SPM at 36".
One possible explanation for the synthesis observed in the homozygotes at 34"
was that it was of mitochondrial DNA. We examined this possibility by prelabelling DNA in vegetative medium with 14C-uraciland then labeled the DNA
in sporulation medium with 3H-adenine.Cells were grown at both temperatures,
25" and 34", and were harvested at different times. DNA was extracted, and
analyzed by equilibrium sedimentation in CsCl gradients. Nuclear and mitochondrial DNA synthesis could be followed separately by incorporation of 3H
label into the respective fractions. An example of such an experiment is presented in Figure 5. This series of experiments did not provide us with quantitative estimates of the synthesis of nuclear and of mitochondrial DNA, or of their
relative amounts. However, the comparison of the gradients of DNA from cultures that were incubated in SPM at 25" and at 34" indicates that at the latter
temperature label is preferentially incorporated into mitochondrial DNA and
that nuclear DNA replication at 34" starts only at 12 hours. We suggest that
probably half of the total increase in DNA observed at 34" is mitochondrial.
DISCUSSION
We report two main findings concerning the effect of cdc4 mutation on mei-
68
G . S I M C H E N A N D J . HIRSCHBERG
!
340 c
25OC
4 hrs
8 hrs
34OC
12 hrs
34OC
3H 14c
--
16 h r s
n,
10
20
30
10
20
3c
FRACTION
FIGURE
5.-Preparative CsCl gradients of DNA extracts from cells of 212-1 (cdc4/cdc4)
incubated in SPM at 25" and at 34". Cells were grown as in Figure 4, but the vegetative culture
contained also "cold" uracil 40 pg/ml. In SPM 10 yCi/ml sH-%adenine was added (specific
activity 8.37 mCi/p mole). 3 ml samples were taken for each DNA preparation, and treated as
described by PICON, SALTSand SIMCHEN
(1974). Sporulation at 26 hrs was 55% in the 25"
culture and zero in the 34" culture. Nuclear DNA peak is in fractions 8-10.
osis. The first is the occurrence of at least three terminal phenotypes at the restrictive temperature, the relative frequency of which depends on the time of
transfer from the permissive to the restrictive conditions. The second finding is
that at 34", cdc4/cdc4 diploids show a high level of recombination commitment,
though only a limited portion of the nuclear DNA is replicated.
The occurrence of three recognizable terminal phenotypes of cdc4 in meiosis
suggests that the function of this gene is required at least at three different points
in the process. There are at least two additional known phenotypes of meiotic
nuclei that were not observed in these experiments as terminal phenotypes,
namely cells with dividing nuclei during the first or the second meiotic divisions
(compare Tables 1 and 2), thus suggesting that once a meiotic nuclear division
starts it is completed (at least as far as light microscopy is concerned). As mentioned earlier, the primary defect cannot be the failure to initiate DNA replica-
MITOTIC DEFECT IN YEAST MEIOSIS
69
tion because there is only one round of replication in meiotic cells, which occurs
prior to the first nuclear division (meiosis I). A primary defect in initiation of
DNA replication could not explain the 2-nuclei and 4-nuclei phenotypes, unless
these phenotypes resulted from the failure to initiate a late meiotic DNA replication such as the zygotene replication or the pachytene repair synthesis (STERN
1973). Moreover, the nuclear phenotype of cells arrested during the
and HOTTA
first two hours of sporulation was the same (100% l-nucleus cells) when the
temperature of arrest was 34" or 36", in spite of the fact that nuclear DNA replication was initiated in the former but not in the latter temperature. Transfers
of cdc4/cdc4 cells to 36" at later times during sporulation produced the same
terminal phenotypes as transfers to 34", though the higher temperature of incubation reduces sporulation frequency even in nonmutant yeast strains. It appears, therefore, that the leakiness of cdc4 with respect to DNA replication at
34" does not affect the stages of arrest with respect to nuclear morphology, thus
supporting the notion that the DNA replication defect, whether in meiotic or
vegetative cells is olnly a secondary result of the cdc4 defect. Another possibility
for the primary role of cdc4 is that of providing for the separation of the spindle
plaques (BYERSand GOETSCH
1973). This would imply that meiotic cells may
be arrested at either the first or the second division, depending on the time of
the temperature shift. However, the finding of a third terminal phenotype (4nuclei cells) shows that the function of cdc4 may be required for spore formation, and is therefore unlikely to be primarily plaque separation. Thus, both
initiation of DNA replication and spindle-plaque separation are dependent on
a third, yet undefined function, which is temperature-sensitive in cdc4 cells. We
suggest, tentatively, that this primary function of the normal CDC4 allele may
be related to chromosome decondensation. The latter may in turn be required
for DNA initiation prior to the first meiotic division, for plaque separation before
both the first and second division, and for the packaging of nuclei into spores
after the second division of meiosis, at the 4-nuclei stage. It should be noted in
this context that the frequency of the second and third terminal phenotypes
(cells with 2 and 4 nuclei, respectively) is never very high (Table 2). The explanation for these low frequencies may be that the three occasions on which
cdc4 functions are required in meiosis are close to each other, in comparison to
the interval between the beginning of sporulation and the first of these three
occasions, and that due to the high degree of asynchrony ocf sporulation (3-4
hours at 25") only a small fraction of the cells at any given time is found bebetween the first and the third occasions.
Recombinant colonies are produced by ascospores following meiosis, but may
also be formed by meiotic cells that are plated on the selective medium while
still undergoing meiotic processes, from the time of the premeiotic DNA replica1971; ESPOSITO
and
tion onwards (SHERMAN
and ROMAN1963; ROTHand FOGEL
ESPOSITO
1974). Thus it may be argued that meiotic recombination actually takes
place during the premeiotic replication. On the other hand, sensitivity to UV
irradiation and specific DNA repair activities in meiotic yeast cells reach a peak
after replication, at a time comparable to the meiotic prophase (SIMCHEN,
SALTS
70
G.
SIMCHEN A N D J. HIRSCHBERG
and PIGON 1973). Moreover, at that time recombination frequency can still be
reduced by chemical treatment (SILVA-LOPEZ,
ZAMBand ROTH 1975) or UV
irradiation (SALTS,SIMCHENand P I ~ O1976)
N thus suggesting that recombination events are initiated during the premeiotic replication, but are consummated
only during the meiotic prophase. According to this interpretation, cells that are
plated during the premeiotic DNA replication complete the process of recombination (and the replication) while on the vegetative selective medium. I n fact,
these cells are committed only to recombination, but not to other meiotic events
such as haploidization and ascus formation (ESPOSITO
and ESPOSITO
1974), and
the recombinant colonies are mostly diploid. The behavior of cdc4/cdc4 diploids
in meiosis is consistent with our understanding of recombination commitment as
an went that is related to the premeiotic DNA replication, but is separable from
later meiotic events such as haploidization and spore formation. At 36", the
meiotic cdc4 cells arrest at a l-nucleus phenotype, show a negligible amount
(510%) of DNA synthesis, which is most likely mitochondrial (cdc4 block
arrests nuclear but not mitochondrial DNA synthesis; NEWLONand FANGMAN
1975), and show no recombination commitment. This finding is in accordance
with the behavior of other mutants that block the premeiotic DNA replication
(ROTH 1973) and with the behavior of meiotic cells in which replication is
inhibited by hydroxyurea ( SILVA-LOPEZ,
ZAMBand ROTH1975; SIMCHEN,IDAR
and KASSIR1976). However, when the slightly lower restrictive temperature
of 34" was employed, cells were also arrested at the l-nucleus morphology, but
a high level of recombination commitment was attained. At the same time a
small, but noticeable, amount o€ nuclear DNA synthesis was observed at 34".
When the cells were plated on the selective medium (-AD), they were incubated
at 25" (i.e.,the cdc4 block was lifted). The cells could now complete the recombination process as well as the DNA replication which they have started at 34".
Recombination was consummated at the permissive temperature during, or following, this round of replication. The decision t o recombine (i.e.,recombination
commitment), however, was undertaken in the sporulation medium, at the
restrictive temperature of 34", which allowed only the early steps of premeiotic
replication. The absence of recombination commitment at 36" may be due to the
fact that the premeiotic replication could not even start at this temperature, or
to the fact that the cdcl block at 36" was not reversible when the cells were
returned to 25"; the Mock at 34", on the other hand, could readily be reversed
and the cells could continue with the recombination process at 25" (on vegetative
medium).
We interpret the limited nuclear DNA replication in cdc4 diploids under
sporulation conditions at 34" as homogenous throughout the cell population, with
leakiness with respect to the early steps of replication. These early steps are
required for recombination commitment, and their absence at 36" does not enable
the mutant cells to become committed to recombination at the higher temperature. An alternative interpretation of the data might be that the limited amount
of nuclear DNA synthesis observed was due to full rounds of replication in
10-20% of the cells that leaked through the cdc4 block under the restrictive
MITOTIC DEFECT IN YEAST MEIOSIS
71
conditions, and that recombination commitment took place in many more cells,
including those that were not synthesizing DNA.We reject this interpretation
f o r the following reasons: (a) When the homozygous diploids were sporulated
at 34", almost all the cells arrested at the one-nucleus stage (Table 2 and other
experiments). Had a noticeable fraction of the cells gone through a full round
of DNA replication, it is likely that they would have been arrested at a subsequent terminal phenotype or even would have completed sporulation. (b) Recombination commitment does not occur when the initiation of premeiotic DNA
replication is inhibited by hydroxyurea (SILVA-LOPEZ,
ZAMBand ROTH1975;
SIMCHEN,
IDAR
and KASSIR1976) or by the cdc4 block at 36" (Table 3, experiment 7 ) . A variation on the above interpretation might be that recombination
commitment occurred only in the fraction of cells (10-20%) in which full
rounds of nuclear DNA replication took place. This possibility is rejected because
of the relative high frequency of prototrophs attained, when compared to the
small increase in DNA content (see, for instance, Figure 3). Thus we conclude
that the early steps of the premeiotic replication took place in most, or even all,
cdc4 arrested cells at 34", and that these steps enabled recombination commitment to occur at 34" at an almost normal level. The completion of nuclear DNA
replication occurred only later, on the vegetative medium at 25". In addition to
the separation between recombination commitment and the completion of the
premeiotic DNA replication, cdc4 diploids that are arrested at 34" also manifest
the separation between recombination commitment and haploidization, which
may be attained transiently during the premeiotic DNA replication of nonmutant strains of yeast (SHERMAN
and ROMAN1963; ESPOSITO
and ESPOSITO
1974).
Further insight into recombination commitment of cdc4-arrested cells can be
derived from the finding of BYERSand GOETSCH
(1975 and personal communication) that our strains 212-1 and 212-10 accumulate synaptonemal complexes
in SPM at 34", and that at 24 hrs the majority of cells contains these meiosisspecific structures. Thus the ability to complete the recombination process (i.e.,
recombination commitment) may be correlated with the presence in the cells
of synaptonemal complexes, as well as with the initiation of nuclear DNA replication. For such a correlation to be meaningful, no synaptonemal complexes
should be found in meiotic cdc4 cells at 36". On the other hand, it is also possible
to argue that the accumulation of synaptonemal complexes in cdc4-arrested
meiotic cells is related to, or even caused by, the failure to go through meiotic
segregation (haploidization).
This research was supported by a grant from the United States-Israel Binational Science
Foundation (BSF), Jerusalem, Israel. We thank Ms. YONAKISSIRand DRS.R. FALK
and L. H.
HARTWELL
for their valuable comments on the manuscript.
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Corresponding editor: R. E. ESPOSITO