1
Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland
2
The Sanger Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA,
UK
3
CREST Research Project of the Japan Science and Technology Corporation, Kansai
Advanced Research Center, Communications Research Laboratory, Kobe 651-2492,
Japan
*Author for correspondence (e-mail: yasushi{at}crl.go.jp )
Accepted April 23, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Meiosis, Fission yeast, Chromosome segregation, Achiasmate segregation
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fission yeast Schizosaccharomyces pombe is a unicellular
eukaryote that undergoes meiotic divisions immediately after the mating of two
haploid cells (Egel, 1989).
Meiosis in fission yeast has some unusual features. Unlike most of the
eukaryotes, S. pombe has no synaptonemal complexes
(Bähler et al.,
1993
) and shows no crossover
interference (Munz, 1994
).
Linear elements, which resemble the axial cores of other eukaryotes, have been
proposed to play a role in meiotic chromosome organization
(Bähler et al.,
1993
; Kohli,
1994
; Kohli and
Bähler,
1994
). The meiotic prophase in
fission yeast is characterized by a strongly elongated nuclear morphology
known as the horse-tail nucleus. (Robinow,
1977
; Robinow and Hyams,
1989
).
Observation of living cells undergoing meiosis has shown that the elongated
prophase nuclei perform striking oscillatory movements between the cell poles.
These movements are led by the spindle pole body (SPB) and the attached
telomere cluster, and this bouquet arrangement of chromosomes is maintained
throughout prophase (Chikashige et al.,
1994; Chikashige et al.,
1997
). The oscillatory nuclear
movements are mediated by the reorganization of astral microtubules
originating from the SPB (Ding et al.,
1998
; Svoboda et al.,
1995
), and require cytoplasmic
dynein as a microtubule motor protein (Yamamoto et al.,
1999
). Pairing of homologous
chromosomes and meiotic recombination occur during prophase nuclear movements.
It has been proposed that telomere clustering and oscillatory nuclear
movements facilitate the encounter of homologous chromosomes, and thus are
required for normal homologous pairing and full level of meiotic recombination
(Chikashige et al., 1997
; Ding
et al., 1998
; Hiraoka,
1998
). Analysis of mutants
that show reduced meiotic recombination, and deficiencies in telomere
clustering (Shimanuki et al.,
1997
; Cooper et al.,
1998
; Nimmo et al.,
1998
) or in nuclear movement
(Yamamoto et al., 1999
)
support this model.
Considering the evidence for the necessity of prophase nuclear movements
for meiotic recombination, we have started live observation of meiosis in
meiotic recombination-deficient mutants (Ponticelli and Smith,
1989; DeVeaux et al.,
1992
). Two of the mutants
(rec8 and rec7) that belong to the genetically best
characterized recombination-deficient mutants (Molnar et al.,
1995
; Krawchuk et al.,
1999
; Parisi et al.,
1999
; Watanabe and Nurse
1999
; Molnar et al.,
2001
) were selected for
detailed study.
Live observation of fission yeast meiosis has so far been concentrated on
the examination of prophase nuclear movements. In the present study we
demonstrate that live observation of meiosis can also reveal details of the
mechanism and accuracy of the meiotic divisions. We have examined the
different meiotic stages in living rec8 and rec7 mutant
cells and have then further analyzed the first meiotic division with the
application of a LacO-LacI-GFP construct that visualizes the centromeric
region of chromosome I (Nabeshima et al.,
1998). We show that the
special features of meiotic divisions detected in the rec7 mutant are
also characteristic of other meiotic mutants and represent a common way of
achiasmate chromosome segregation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains, media and culture conditions
S. pombe strains used in this study are listed in
Table 1. The strains designated
his7+::lacI-GFP lys1+::lacO
carry tandem repeats of LacO DNA sequences at the centromere-linked
lys1 locus on chromosome I, and the integrated GFP-LacI-NLS fusion
construct at the his7 locus (Nabeshima et al.,
1998). Strains were cultivated
on yeast extract agar (YEA) complete medium supplemented with 75 µg/ml
adenine sulfate, at 30°C. Mating and meiosis were induced by transferring
homothallic (h90) or crossing heterothallic
(h+ and h-) strains on malt extract
agar (MEA) sporulation medium (Gutz et al.,
1974
), and incubating the
plates at 26°C. For microscopic observation of meiosis, cells were
resuspended in EMM2-N liquid medium (EMM2 minimal medium without nitrogen
source). Transformants were selected on EMM2 plates supplemented with
appropriate nutrients (Moreno et al.,
1991
).
|
Preparation of specimens for microscopic observation
For live observation of meiosis, cells of control and rec mutant
strains were transferred onto MEA plates and incubated overnight (12-16
hours), at 26°C. Fluorescence staining of nuclei was achieved either by
staining with Hoechst 33342, a DNA-specific fluorescence dye, or with the
application of the jellyfish green fluorescence protein. Hoechst 33342
staining was used to identify and examine the different meiotic stages in
living cells. Samples of mating cells were washed in water, and stained with 1
µg/ml Hoechst 33342 in distilled water for 15 minutes at room temperature.
Stained cells were resuspended in EMM2-N and mounted either on a coverslip or
in a glass-bottom culture-dish (MatTek, Ashland, MA) coated with concanavalin
A (1 mg/ml).
To observe the duration of prophase nuclear movements, nuclei were stained
with the use of jellyfish GFP protein. Control, rec7 and
rec8 homothallic strains were transformed with the plasmid pYC551
according to the LiCl method (Moreno et al.,
1991). This pREP1 expression
vector-based plasmid (Maundrell,
1993
) carries the sequences
for the NLS-GFP protein and the Saccharomyces cerevisiae LEU2 gene as
selection marker. Transformants grew on appropriately supplemented EMM2-leu
plates containing 2 µM thiamine, and prepared for microscopic observation
of meiosis as described above.
To examine the final meiotic phenotypes of rec mutants, the MEA plates were incubated for 3 days at 26°C. In each mutant, approx. 200 mature asci were classified in phase-contrast microscope according to their spore numbers. From the same crosses, samples were taken for fluorescence staining of nuclei in the asci. Samples were treated with 70% ethanol for 5 minutes, resuspended in 1 µg/ml DAPI (4',6-diamidino-2-phenylindol) and examined in fluorescence microscope.
Fluorescence imaging of living fission yeast cells
Specimens of living fission yeast cells were observed at 26°C on the
CCD microscope system using an Olympus oil immersion objective lens (Plan Apo
60/NA=1.4). Images were obtained on the cooled CCD with an exposure time of
0.2-0.5 seconds under the illumination of a mercury arc lamp. An excitation
filter with a narrow peak at 380 nm for Hoechst 33342 and a high-selectivity
fluorescein excitation filter for GFP in combination with high-selectivity
barrier filters for DAPI and fluorescein (Chroma Technology, Brattleboro, VA),
respectively, were used. A single dichroic mirror with quadruple-bandpass
properties (Chroma Technology) was used. During the observation of different
meiotic stages images were taken in a single optical section. In order to
monitor the position and number of GFP signals visualizing the
centromere-proximal region of chromosome I, images were taken in ten optical
sections covering the whole nucleus at each time-point. In the presented
figures, the images are projected into two dimensions after deconvolution.
Image processing, analysis and display were carried out using the DeltaVision
program (Applied Precision, Seattle, WA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We were interested in whether these aberrations influence the length of
prophase nuclear movements. The duration of prophase nuclear movements was
determined after fluorescence staining of the nuclei with GFP (see Materials
and Methods). In the wild-type strain, in two experiments time lengths of 140
and 160 minutes were measured. These numbers are in good agreement with the
previously presented data (146±14 minutes, in Hiraoka et al.,
2000). Prophase nuclear
movements lasted 147±25 minutes in rec8, and 153±6
minutes in rec7 mutant strains (averages of three measurements).
Because the prophase nuclei did perform oscillatory movements in both mutants,
and these movements were of similar duration as those in wild-type nuclei, we
conclude that the rec8 and rec7 mutations have an effect on
the nuclear shape and chromosome organization rather than on the movements per
se.
Analysis of meiotic divisions in rec8 and rec7
mutants
Live observation of fission yeast meiosis has been centered on the
understanding of the most striking meiotic phenomenon, the prophase nuclear
movements (see Introduction). Live observation of meiotic divisions in mutants
that are strongly impaired in meiotic recombination may contribute to a better
understanding of meiotic chromosome segregation. rec8 and
rec7 are particularly interesting in this respect, as they are the
meiotic recombination-deficient mutants, in which the two missegregation types
(precocious separation of sister chromatids in rec8, Molnar et al.,
1995; nondisjunction I in
rec7, Molnar et al.,
2001
) have been detected
genetically.
To examine the first division, zygotes with a single almost round nucleus
were selected after staining the cells with Hoechst 33342 dye. These nuclei
had completed the prophase movements and were in the nuclear condensation
stage, which precedes the first division (Chikashige et al.,
1994; Hiraoka et al.,
2000
). To see the detailed
dynamics of nuclear divisions, images were taken at 2 minute intervals.
Apparently equal amounts of nuclear material moved to the two cell poles in
the first division, both in the wild-type and the rec8 mutant cell.
Fig. 2A shows an example of the
dividing nucleus of a rec8 zygote (for wild type, data not shown). In
rec8, regular distribution of the nuclear material was observed in
five cases studied. According to previous studies, which detected precocious
separation of sister chromatids on spreads of rec8 prophase nuclei
(Molnar et al., 1995
), a more
irregular first division was expected in this mutant. However, a complete
change of the reductional segregation pattern to equational (Watanabe and
Nurse, 1999
) could account for
the observed regular phenotype, if sister chromatids are held together at
least at the centromeric region until anaphase I.
|
In sharp contrast to the rec8 mutant, rec7 exhibited a highly irregular first division. In six divisions out of nine observed in rec7, unequal amounts of nuclear masses were distributed to the cell poles. A striking feature of the rec7 mutant was that chromosomes moved back and forth between the cell poles during the first division. Seven out of the nine divisions exhibited this phenotype. Selected frames of two examples are shown in Fig. 2B,C. Individual chromosomes were clearly discernable in some images. In Fig. 2C, for example, six chromosomes can be seen at 48 minutes and the arrow indicates a chromosome wandering between the cell poles. Chromosome movements ended in an apparently regular segregation in two cases (Fig. 2C), while in five divisions the final chromosome distribution was seemingly irregular (Fig. 2B).
Measurement of duration of the first division further emphasized the difference between the two mutants. The first meiotic division took 22±3 minutes in the wild type and 21±3 minutes in rec8 mutant cells (averages of three and five measurements, respectively). In rec7, an average of 57±26 minutes was measured in five observations. In the rec7 mutant, the observed divisions were widely different in duration. Notably, the shortest one (20 minutes) resulted in an apparently equal distribution of the nuclear mass and was followed by a reasonably regular second division (data not shown).
To observe the second meiotic division, zygotes with two nuclei were chosen after Hoechst 33342 staining. Generally, irregular distribution of the nuclear masses was characteristic for the second division in both mutants (Fig. 3B,C; for wild-type, see Fig. 3A). In addition, in rec7 the second division did not always take place or only one of the daughter nuclei divided. Because the first meiotic division was irregular in both mutants, it was difficult to decide whether the second meiotic divisions themselves were impaired as well, or whether they simply were a consequences of impairment during the first divisions. Therefore, the second meiotic division was not analyzed further.
|
Tracing individual chromosomes through the first meiotic
division
Live observation of the first meiotic division in the rec7 mutant
resulted in a puzzling observation. It gave the impression that chromosomes
moved back and forth between the cell poles several times, until finally they
stopped at either pole. To understand the chromosome movements better in the
rec7 and rec8 mutants, we traced individual chromosomes
through the first meiotic division.
For visualization of individual chromosomes, a GFP-LacI-NLS fusion protein
was used that binds to a LacO array integrated at the centromere-proximal
region of chromosome I (Nabeshima et al.,
1998). This construct was
introduced into homothallic and heterothallic, wild-type and mutant strains
(Table 1), and was observed as
described in Materials and Methods. Results are summarized in
Table 2 (see the next section
for details); some examples of images are shown in Figs
4,5,6
below.
|
|
|
|
To examine chromosome segregation in rec7, a homothallic strain
was applied because, according to genetic studies (Molnar et al.,
2001), nondisjunction of
homologs at the first division was expected. In homothallic crosses, sister
chromatids of the homologs are labeled with GFP fluorescence. In the
homothallic wild-type strain (control), segregation of homologous chromosomes
occurred in two phases (Fig.
4). After separation the centromere signals maintained their
position at a short distance (from 15 to 30 minutes,
Fig. 4), then moved to the cell
poles. In some images (30, 35 and 45 minutes in
Fig. 4), one of the chromosomes
showed two very closely spaced signals, suggesting that during this division
in one of the chromosomes a loosening of sister chromatid attachment occurred.
Fig. 5 shows examples of
rec7 homothallic cross: chromosome segregation occurred in one phase
without having a period of short-distance separation. After an initial regular
separation (10 minutes to 20 minutes in
Fig. 5A; 5 minutes to 15
minutes in Fig. 5B), one of the
chromosomes lost orientation, and started to move to the same pole as its
homolog (see 35 minutes in Fig.
5A, and 20 minutes in Fig.
5B). This loss-of-orientation resulted in nondisjunction of the
homologs in Fig. 5A. In another
example, however, a successful re-orientation took place, ending with normal
disjunction of the homologs (Fig.
5B).
To observe chromosome segregation in rec8, an h- strain carrying the GFP construct was crossed with an h+ strain lacking the construct. In such a heterothallic cross, sister chromatids of only one of the homologs are labeled; thus, separation of sister chromatids can be distinguished from separation of homologs. In accordance with this, the signals in Fig. 6 are weaker than in the images, where they represent homologous chromosomes (homothallic crosses, Figs 4 and 5). The separating GFP signals in Fig. 6 indicate that sister chromatids moved to opposite poles precociously at the first division. The segregation of sister chromatids also occurred in one phase.
Fidelity of chromosome segregation at meiosis I in rec8 and
rec7 mutants
Next we tried to answer two questions: how frequently the separation of
sister chromatids in rec8 occurs and how efficiently the chromosome
re-orientation works in the rec7 mutant. To test the accuracy of the
first meiotic division, we first stained the cells with Hoechst 33342 dye in
order to identify zygotes having two nuclei. Then, the number of GFP signals
in the daughter nuclei was determined as described in Materials and
Methods.
To examine the separation of sister chromatids, crosses heterozygous for the GFP construct were applied. Crossing a h- rec8 strain carrying the GFP construct with a h+ strain that lacked the construct revealed that precocious separation of sister chromatids at meiosis I occurred fairly regularly in this mutant (crosses heterozygous for GFP in Table 2). In Table 2, class 1 represents precocious separation of sister chromatids at the first meiotic division. Classes 2 to 4 represent regular segregation; in these divisions the labeled chromosome moved to one pole (classes 2 and 3) and started the second meiotic division (class 4). Precocious separation of sister chromatids was detected in 90% of the divisions observed in rec8 heterothallic cross (class 1 in Table 2), whereas it was observed infrequently in the control cross (one out of 38 zygotes) and in rec7 heterothallic cross (one out of 40 zygotes).
To examine the fidelity of chromosome disjunction (regular segregation versus nondisjunction I), meioses in homothallic (h90) strains were analyzed (crosses homozygous for GFP in Table 2). In a homothallic strain, segregation of sister chromatids to opposite poles is not distinguishable from segregation of homologs, therefore results obtained from rec8 are not informative because of the high frequency of separation of sister chromatids. However, precocious separation of sister chromatids is rare in rec7, results obtained from rec7 well reflect disjunction of homologs. In this mutant, classes 5, 6 and 8 are typical for a regular first meiotic division. In class 6, one of the chromosomes was represented by tightly associated signals of sister chromatids. Class 8 represented zygotes, which have already started the second meiotic division. Class 7 indicated that in rec7 the coordination of second meiotic division between the daughter nuclei was impaired: only one of the daughter nuclei entered the second division. In wild-type strains this class was not observed. The segregation patterns of classes 5 to 8 arose from regular disjunction of homologs, while classes 9 and 10 showed nondisjunction of homologs at the first meiotic division. In rec7, nondisjunction I was observed in 36% of divisions analyzed. Precocious separation of sister chromatids occurred in the mutant with low frequency (class 11). This is consistent with results obtained from crossing heterothallic strains, where class 1 gave direct evidence for precocious separation of sister chromatids.
Sister chromatid cohesion was also examined during meiotic prophase nuclear movements. In a rec8 heterothallic cross (same cross as in Table 2), horse-tail nuclei were selected after Hoechst 33342 staining and the number of signals determined in 40 nuclei. Two separated, but closely spaced signals were observed in three horse-tail nuclei (7.5%). Sister chromatids were represented by tightly associated signals in 11 cases (27.5%). A single signal was observed in the remaining 26 nuclei (65.0%). In the control cross (rec+ heterothallic cross, same as in Table 2) either a single signal was observed (in 36 out of 40 nuclei) or signals of tightly associated sister chromatids were seen (in four nuclei).
Observation of GFP signals in a rec7 homothallic strain (same as
in Table 2) during horse-tail
nuclear movements confirmed that pairing of centromere proximal regions takes
place regularly in the mutant, and precocious separation of sister chromatids
does not occur more frequently than in the wild-type strain (data not shown;
see Molnar et al., 2001).
Common features of meiosis in rec7, rec14 and rec15
mutants
Studying additional meiotic recombination deficient mutants revealed that
the special features of meiotic divisions observed in the rec7 mutant
are typical not only for this mutant. The phenotype of the first meiotic
division in the rec14 and rec15 mutants is virtually
indistinguishable from that observed in rec7
(Fig. 2B and data not shown).
These mutants were chosen for study because they showed aberrations in meiotic
prophase nuclear movements. The observed common features of the three mutants
can be described as follows. Irregular distribution of the nuclear mass in the
first meiotic division occurred frequently. This was detected in six divisions
out of 10 observed in the rec14 mutant, and in six cases out of 12
observations in the rec15 mutant. During the first meiotic division
the chromosomes moved back and forth between the cell poles. Eight divisions
in the rec14 mutant and 9 divisions in the rec15 mutant
showed this phenotype. Chromosome movements sometimes resulted in seemingly
regular segregation (three and four cases in rec14 and
rec15, respectively), other times in apparently irregular
distribution (five observations in both mutants). The first division lasted
significantly longer than in wild-type cells (see Results above and data not
shown). Interruption of meiosis after the first division was detected during
live observation of all three mutants (data not shown).
In rec7, it was demonstrated genetically that interruption of
meiosis led to the formation of two-spored asci (Molnar et al.,
2001). To analyze sporulation
in the mutants, zygotes were allowed to complete meiosis and the number of
spores in mature asci was determined (see Materials and Methods).
Fig. 7 shows that frequent
occurrence of two-spored asci was typical for rec7, rec14 and
rec15, and that this phenotype was most strongly expressed in
rec15.
|
DAPI staining of nuclear material in the asci (Materials and Methods)
revealed the final phenotypes of meiotic divisions in the mutants. Our
observations on the ascus morphology and chromosome distribution are in
agreement with those of Krawchuk et al. (Krawchuk et al.,
1999): in rec8,
regular four-spored asci were the predominant class
(Fig. 7) but the spores
frequently contained unequal amounts of nuclear material
(Fig. 8B). We explain this
terminal phenotype by the occurrence of a fairly regular, but equational first
division followed by a random second division. Two-spored asci, which
contained two large nuclei, were found in all the other mutants
(Fig. 8C,D and E), in agreement
with the observed interruption of meiosis. Interestingly, rec7, rec14
and rec15 also formed large spores, in which more than one
DAPI-stainable body was enclosed. The distribution of nuclear material was
rather unequal in all the three mutants.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rec8 is required for proper nuclear movement during meiotic
prophase
Live observation of different meiotic stages in rec8 revealed that
prophase nuclear movements are impaired in this mutant. Only the leading end
of the horse-tail nucleus moved, whereas the bulk nuclear mass remained in the
cell center (Fig. 1B). However,
both the dynamics and the duration of these movements were normal, suggesting
that the observed phenotype is caused by a deficiency in the nuclear
organization. A similar phenotype was observed in taz1, a mutant
deficient in telomere clustering (Cooper et al.,
1998; Hiraoka et al.,
2000
). Telomere clustering
occurs regularly in rec8 (Molnar et al.,
1995
), indicating that here
another deficiency of the nuclear structure has led to the same phenotype. In
rec8, the formation of linear elements is impaired (Molnar et al.,
1995
). An important role for
these structures in regular meiotic chromosome organization has been proposed
(Bähler et al.,
1993
; Kohli,
1994
; Kohli and
Bähler,
1994
). We infer that the
observed unusual shape of the prophase nucleus is due to the lack of regular
linear elements. Linear elements provide scaffolding structures for the
chromosomes, which may be necessary for driving the entire chromosomes during
prophase nuclear movements.
Meiotic chromosome segregation in rec8
The first meiotic division showed basic differences in the rec8
and rec7 mutants. In rec8 the division was regular
(Fig. 2A), but mainly
equational (Table 2).
rec8 encodes a meiotic cohesin which localizes to chromosomes in
premeiotic S phase (Watanabe and Nurse,
1999) and completely
dissociates only at anaphase II (Parisi et al.,
1999
; Watanabe and Nurse,
1999
). In rec8
mutants, impaired linear element formation, reduced meiotic chromosome pairing
and, on prophase nuclear spreads, precocious separation of sister chromatids
have been detected (Molnar et al.,
1995
). Watanabe and Nurse
(Watanabe and Nurse, 1999
)
have shown that Rec8p has a role in the establishment of meiosis specific
centromere structure; thus, it is required for reductional chromosome
segregation at meiosis I. Data obtained from live monitoring of meiosis in the
mutant are fully compatible with such a function.
Examination of cen1 GFP signals in horse-tail nuclei showed that the sister
centromeres remain together in rec8 during prophase, although their
association is weaker than in wild-type strains (see Results). This may
suggest the involvement of additional proteins in sister chromatid cohesion at
the centromeres throughout prophase. Alternatively, the association of
centromeres might be due to the regular centromere clustering in rec8
(Molnar et al., 1995). Rec8p
fulfills several functions during meiosis: (1) it establishes a
meiosis-specific centromere structure, and (2) it contributes to sister
chromatid cohesion at the centromeres and centromere-proximal regions of
chromosomes. In the rec8 mutant, the chromosomes are predisposed to
undergo an equational division: their sister centromeres face to opposite
poles, and they lack the cohesive function of Rec8p in the centromeric region.
We detected equational chromosome segregation in 90% of divisions
examined.
In rec8 mutant strains, sister chromatid cohesion at the telomeric
regions of chromosomes is likely to be maintained by the Rad21p mitotic
cohesin (Watanabe and Nurse,
1999). Meiotic double strand
breaks occur in rec8, although at strongly reduced levels (Cervantes
et al., 2000
). Meiotic
recombination is practically abolished at the central regions of chromosomes,
but a moderate recombination is still detectable at the telomere-proximal
regions (Krawchuk et al.,
1999
; Parisi et al.,
1999
). These findings suggest
that incidental meiotic recombination interfered with a perfect equational
division in the rec8 mutant, and resulted in regular reductional
disjunction of chromosomes observed in the minority of divisions
(Table 2).
An explanation for the common features of rec7, rec14 and
rec15: chromosomes segregate in an achiasmate first meiotic
division
Live observation of meiosis I detected a lengthened first meiotic division
in rec7, during which individual chromosomes seemed to change their
position several times between the cell poles (Figs
2 and
5). rec7 is an early
meiotic recombination gene (Fox and Smith,
1998; Molnar et al.,
2001
). In rec7,
meiotic double strand breaks have not been detected (Cervantes et al.,
2000
). Rec7p was localized in
horse-tail nuclei in live observation, and on meiotic prophase nuclear spreads
to about 50 foci per nucleus (Molnar et al.,
2001
). All these findings are
consistent with a role for Rec7p in the initiation of meiotic recombination. A
failure of initiation of meiotic recombination obviously leads to lack of
crossover formation, a severe overall reduction in meiotic recombination
(DeVeaux and Smith, 1994
;
Molnar et al., 2001
) and the
lack of functional chiasmata.
We propose that the observed oscillation of chromosomes between the cell
poles is a consequence of the lack of chiasma formation. Studies in higher
eukaryotes have shown that mechanical tension stabilizes the proper chromosome
configuration on the metaphase plate, and controls the cell cycle checkpoint
(Nicklas et al., 1995;
Nicklas, 1997
). In meiosis, a
stable configuration is achieved if chiasmata between the homologous
chromosomes balance the pulling forces exerted on the kinetochores that are
attached to opposite poles. The short-distance separation of homologous
centromeres observed in the wild-type strain probably reflects this stable
configuration (Fig. 4).
Notably, this phase was not detectable in either rec8 or in
rec7 (Figs 5 and
6). In the lack of stabilizing
tension, chromosomes might detach from the spindle microtubules in
rec7, and change their position until a new capture by microtubules
or final migration to either cell pole occurs.
Studying meiosis I in rec14 and rec15 mutants has
provided further support for this hypothesis. The phenotypes of first meiotic
division in these mutants were virtually indistinguishable from that of
rec7. rec14 is a homolog of REC103, an early meiotic
recombination gene in budding yeast (Evans et al.,
1997; Fox and Smith,
1998
). Meiotic double strand
breaks have not been detected in rec14 (Cervantes et al.,
2000
), and a severe reduction
of meiotic recombination was measured in all the chromosomal intervals tested
(Evans et al., 1997
). In
rec15, occurrence of meiosis initiator breaks have not been tested,
and rec15 shows no sequence homology to any other reported
polypeptides (Lin and Smith,
1995
). However, its transient
induction at early meiotic stages, the observed severe reduction in meiotic
recombination in the rec15 deletion strain (Lin and Smith,
1995
) and its additional
common phenotypes with early genes (see Results) suggest that rec15
is also an early meiotic recombination gene. We suggest that the common
features of meiosis I in rec7, rec14 and rec15 can be
attributed to the achiasmate segregation of homologs.
All achiasmate mutants showed two additional features: the frequent
omission of meiosis II and enclosure of nuclear masses into huge spores.
Persistence of Rec7p in the meiotic nuclei after meiosis I has been
demonstrated (Molnar et al.,
2001); thus, a direct role for
Rec7p in the initiation of meiosis II is possible. Surprisingly, interruption
of meiosis after the first division was detected in live observation of
rec14 and rec15 as well; therefore we now propose an
alternative explanation for the omission of meiosis II in the early
recombination deficient mutants. A failure of initiation of meiotic
recombination may trigger a block after meiosis I. This contributes to the
improvement of spore viability by the omission of a frequently irregular
second meiotic division. The block after meiosis I in recombination-deficient
mutants is not complete. Finally, all achiasmate mutants formed two-spored
asci, and occasionally enclosed several DAPI-stainable bodies into huge
spores. Spindle pole body modification (a differentiation of the SPB into
multiplaque structure which is necessary for the assembly of forespore
membranes) usually occurs during meiosis II (Tanaka and Hirata,
1982
). The two-spored asci and
huge spores in the early rec mutants suggest that in these mutants a
modification in spore formation might have occurred, in order to ensure a
better enclosure of the irregularly distributed chromosomes.
Fidelity of achiasmate chromosome segregation
In achiasmate meiosis, random segregation of homologous chromosomes at the
first division is expected. Random segregation results in regular disjunction
of homologs (50%) or in nondisjunction I (50%). For three pairs of
chromosomes, a completely random distribution results in proper segregation
only in 12.5% of divisions. Some of our observations indicate that the
fidelity of achiasmate chromosome segregation in the recombination deficient
mutants may exceed randomness. In rec7, nondisjunction of chromosome
I was detected in only 36% of divisions examined
(Table 2). Live observation of
the first meiotic division detected apparently regular distribution of the
nuclear mass in each mutant more frequently than it would be expected on
random basis (see Results).
We consider two possible mechanisms that may contribute to the improvement
of achiasmate chromosome segregation. First, occasional crossovers arising
from the repair of spontaneously occurring double strand breaks or from
meiotic double strand breaks may lead to a regular first division. Although
meiosis initiator breaks have not been detected in these mutants (Cervantes et
al., 2000) rare occurrence of
DNA breaks that escaped the current detection level can not be excluded.
Considering that severe reduction in meiotic recombination was measured in
each mutant, this mechanism cannot be frequent. Second, a backup system may
exist in fission yeast that improves the fidelity of segregation of achiasmate
chromosomes. This may be achieved by triggering a checkpoint response that
gives time for missegregating chromosomes to correct their error. In
Fig. 5B we show that S.
pombe is able to re-orient a chromosome if it moves to the same pole as
its homolog. Chromosome wandering between the cell poles during the first
division was common in each mutant. These divisions usually lasted longer than
those where segregation happened to be regular. Although they resulted in
apparently regular distribution more frequently than 12.5% of the divisions,
it has to be noted that chromosome movements did not always lead to an
improvement of segregation. Thus, the achiasmate backup system of fission
yeast may work with low efficiency.
Meiosis is not an error free process. The presented data
(4,
Table 2) show that both slight
irregularities and obvious missegregation occur even in
recombination-proficient, wild-type strains. Considering that mutation in a
single gene may lead to achiasmate meiosis and that the consequences of
aneuploidy is frequently death, to assume the operation of an achiasmate
backup system in fission yeast is an appealing hypothesis. Distributive
disjunction has been detected also in S. cerevisiae, another organism
highly proficient in meiotic recombination (Dawson et al.,
1986; Guacci and Kaback,
1991
; Loidl et al.,
1994
).
What mechanism re-orients the chromosomes in meiosis I? Does the meiotic
spindle checkpoint play as crucial a role in meiotic chromosome segregation in
fission yeast as it has been demonstrated in budding yeast (Shonn et al.,
2000)? The meiotic spindle
checkpoint in fission yeast has not been characterized. Is the second meiotic
division really coupled to the initiation of meiotic recombination?
Experiments to clarify the answers to these questions are in progress.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baker, B. S., Carpenter, A. T. C., Esposito, M. S., Esposito, R. E. and Sandler, L. (1976). The genetic control of meiosis. Annu. Rev. Genet. 10,53 -134.[Medline]
Bähler, J., Wyler, T., Loidl, J. and Kohli, J. (1993). Unusual nuclear structures of meiotic prophase of fission yeast: a cytological analysis. J. Cell Biol. 121,241 -256.[Abstract]
Carpenter, A. T. (1994). Chiasma function. Cell 77,957 -962.[Medline]
Cervantes, M. D., Farah, J. A. and Smith, G. R. (2000). Meiotic DNA breaks associated with recombination in S. pombe. Mol. Cell 5,883 -888.[Medline]
Chikashige, Y., Ding, D.-Q., Funabiki, H., Haraguchi, T., Mashiko, S., Yanagida, M. and Hiraoka, Y. (1994). Telomere-led premeiotic chromosome movement in fission yeast. Science 264,270 -273.[Medline]
Chikashige, Y., Ding, D.-Q., Imai, Y., Yamamoto., M., Haraguchi,
T. and Hiraoka, Y. (1997). Meiotic nuclear reorganization:
switching the position of centromeres and telomeres in fission yeast
Schizosaccharomyces pombe. EMBO J.
16,193
-202.
Cooper, J. P., Watanabe, Y. and Nurse, P. (1998). Fission yeast Taz1 protein is required for meiotic telomere clustering and recombination. Nature 23,828 -831.
Dawson, D. S., Murray, A. W. and Szostak, J. W. (1986). An alternative pathway for meiotic chromosome segregation in yeast. Science 234,713 -717.[Medline]
DeVeaux, L. C., Hoagland, N. A. and Smith, G.R.
(1992). Seventeen complementation groups of mutations decreasing
meiotic recombination in Schizosaccharomyces pombe.Genetics 130,251
-262.
DeVeaux, L. C. and Smith, G. R. (1994). Region-specific activators of meiotic recombination in Schizosaccharomyces pombe. Genes Dev. 8,203 -210.[Abstract]
Ding, D-Q., Chikashige, Y., Haraguchi, T. and Hiraoka, Y. (1998). Oscillatory nuclear movement in fission yeast meiotic prophase is driven by astral microtubules as revealed by continuous observation of chromosomes and microtubules in living cells. J. Cell Sci. 111,7001 -7012.
Egel, R. (1989). Mating-type genes, meiosis, and sporulation. In Molecular Biology of the Fission Yeast (ed. A. Nasim, P. Young, and B. F. Johnson), pp.31 -73. Academic Press, San Diego.
Evans, D. H., Li, Y. F., Fox, M. E. and Smith, G. R.
(1997). A WD repeat protein, Rec14, essential for meiotic
recombination in Schizosaccharomyces pombe. Genetics
146,1253
-1264.
Fox, M. E. and Smith, G. R. (1998). Control of meiotic recombination in Schizosaccharomyces pombe. Prog. Nucleic Acid Res. Mol. Biol. 61,345 -378.[Medline]
Guacci, V. and Kaback, D. B. (1991).
Distributive disjunction of authentic chromosomes in Saccharomyces
cerevisiae. Genetics 127,475
-488.
Gutz, H., Heslot, H., Leupold, U. and Loprieno, N. (1974). Schizosaccharomyces pombe. In Handbook of Genetics. Vol. 1 (ed. R. C. King), pp. 395-446. New York: Plenum Press.
Haraguchi, T., Kaneda, T. and Hiraoka, Y.
(1997). Dynamics of chromosomes and microtubules visualized by
multiple-wavelength fluorescence imaging in living mammalian cells: effects of
mitotic inhibitors on cell cycle progression. Genes
Cells 2,369
-380.
Haraguchi, T., Ding, D.-Q., Yamamoto, A., Kaneda, T., Koujin, T. and Hiraoka, Y. (1999). Multiple-color fluorescence imaging of chromosomes and microtubules in living cells. Cell Struct. Funct. 24,291 -298.[Medline]
Hiraoka, Y. (1998). Meiotic telomeres: a
matchmaker for homologous chromosomes. Genes Cells
3, 405-413.
Hiraoka, Y., Ding, D.-Q., Yamamoto, A., Tsutsumi, C. and Chikashige, Y. (2000). Characterization of fission yeast meiotic mutants based on live observation of meiotic prophase nuclear movement. Chromosoma 109,103 -109.[Medline]
Kohli, J. (1994). Telomeres lead chromosome movement. Curr. Biol. 4,724 -727.[Medline]
Kohli, J. and Bähler, J. (1994). Homologous recombination in fission yeast: absence of crossover interference and synaptonemal complex. Experientia 50,296 -306.
Krawchuk, M. D., DeVeaux, L. C. and Wahls, W. P.
(1999). Meiotic chromosome dynamics dependent upon the
rec8+, rec10+ and
rec11+ genes of the fission yeast Schizosaccharomyces
pombe. Genetics 153,57
-68.
Lin, Y. and Smith, G. R. (1995) An intron-containing meiosis-induced recombination gene, rec15, of Schizosaccharomyces pombe. Mol. Microbiol. 17,439 -448.[Medline]
Loidl, J., Scherthan, H. and Kaback, D. B. (1994). Physical association between nonhomologous chromosomes precedes distributive disjunction in yeast. Proc. Natl. Acad. Sci. USA 91,331 -334.[Abstract]
Maguire, M. P. (1974). Letter: the need for a chiasma binder. J. Theor. Biol. 48,485 -487.[Medline]
Maundrell, K. (1993). Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene 123,127 -130.[Medline]
Molnar, M., Bähler, J.,
Sipiczki, M. and Kohli, J. (1995). The rec8 gene of
Schizosaccharomyces pombe is involved in linear element formation,
chromosome pairing and sister-chromatid cohesion during meiosis.
Genetics 141,61
-73.
Molnar M., Parisi, S., Kakihara, Y., Nojima, H., Yamamoto, A.,
Hiraoka, Y., Bozsik, A., Sipiczki, M. and Kohli, J. (2001).
Characterization of rec7, an early meiotic recombination gene in
Schizosaccharomyces pombe. Genetics
157,519
-532.
Moreno, S., Klar, A. and Nurse, P. (1991). Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.Methods Enzymol. 194,795 -823.[Medline]
Munz, P. (1994). An analysis of interference in
the fission yeast Schizosaccharomyces pombe. Genetics
137,701
-707.
Nabeshima, K., Nakagawa, T., Straight, A. F., Murray, A.,
Chikashige, Y., Yamashita, Y. M., Hiraoka, Y. and Yanagida, M.
(1998). Dynamics of centromeres during metaphase-anaphase
transition in fission yeast: Dis1 is implicated in force balance in metaphase
bipolar spindle. Mol. Biol. Cell
9,3211
-3225.
Nicklas, R. B. (1997). How cells get the right
chromosomes. Science
275,632
-637.
Nicklas, R. B., Ward, S. C. and Gorbsky, G. J. (1995). Kinetochore chemistry is sensitive to tension and may link mitotic forces to a cell cycle checkpoint. J. Cell Sci. 130,929 -939.
Nimmo, E. R., Pidoux, A. L., Perry, P.E. and Allshire, R. C. (1998). Defective meiosis in telomere-silencing mutants of Schizosaccharomycs pombe. Nature 23,825 -828.
Parisi, S., McKay, M. J., Molnar, M., Thompson, M. A., van der
Speck, P. J., van Drunnen-Schoenmaker, E., Kanaar, R., Lehmann, E.,
Hoeijmakers, J. H. J. and Kohli, J. (1999). Rec8p, a meiotic
recombination and sister chromatid cohesion phosphoprotein of the Rad21p
family conserved from fission yeast to humans. Mol. Cel.
Biol. 19,3515
-3528.
Ponticelli, A. S. and Smith, G. R. (1989).
Meiotic recombination-deficient mutants of Schizosaccharomyces pombe.Genetics 123,45
-54.
Robinow, C. F. (1977). The number of
chromosomes in S. pombe: light microscopy of stained preparations.
Genetics 87,491
-497.
Robinow, C. F. and Hyams, J. S. (1989). General cytology of fission yeasts. In Molecular Biology of the Fission Yeast (ed. A. Nasim, P. Young, and B. F. Johnson), pp.31 -73. Academic Press, San Diego.
Shimanuki, M., Miki, F., Ding, D-Q., Chikashige, Y., Hiraoka, Y., Horio, T. and Niwa, O. (1997). A novel fission yeast gene, kms1+, is required for the formation of meiotic prophase-specific nuclear architecture. Mol. Gen. Genet. 254,238 -249.[Medline]
Shonn, M. A., McCarroll, R. and Murray, A. W.
(2000). Requirement of the spindle checkpoint for proper
chromosome segregation in budding yeast meiosis.
Science 289,300
-303.
Svoboda, A., Bähler, J. and Kohli, J. (1995). Microtubule-driven nuclear movements and linear elements as meiosis-specific characteristics of the fission yeasts S. versatilis and S. pombe. Chromosoma 104,203 -214.[Medline]
Tanaka, K. and Hirata, A. (1982). Ascospore development in the fission yeasts Schizosaccharomyces pombe and S. japonicus. J. Cell Sci. 56,263 -279.[Abstract]
Watanabe, Y. and Nurse, P. (1999). Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature 400,461 -464.[Medline]
Yamamoto, A., West, R. R., McIntosh, J. R. and Hiraoka, Y.
(1999). A cytoplasmic dynein heavy chain is required for
oscillatory nuclear movement of meiotic prophase and efficient meiotic
recombination in fission yeast. J. Cell Biol.
145,1233
-1249.