1 CREST Research Project, Kansai Advanced Research Center, Communications
Research Laboratory, Kobe 651-2492, Japan
2 Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland
* Author for correspondence (e-mail: yasushi{at}crl.go.jp)
Accepted 23 January 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Meiosis, Fission yeast, Linear elements, Sister chromatid cohesion, Homologous chromosome pairing
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The fission yeast Schizosaccharomyces pombe is a haploid,
unicellular eukaryote. Naturally, S. pombe cells undergo meiosis
directly after mating of two cells of opposite matingtype (zygotic meiosis).
However, diploid cells heterozygous for mating-type can be maintained, and
synchronous meiosis can be induced by shifting the culture to nitrogen-free
medium (azygotic meiosis) (Egel,
1973; Egel and Egel-Mitani,
1974
). Meiosis in fission yeast has unusual features. In prophase,
the meiotic nucleus oscillates between the cell poles
(Chikashige et al., 1994
).
These movements confer an elongated shape to the nucleus [horse-tail nucleus
(Robinow, 1977
)], and are led
by the SPB and the attached telomere cluster
(Chikashige et al., 1994
;
Chikashige et al., 1997
). Thus,
the bouquet structure of chromosomes bundled at the telomeres is maintained
during the whole meiotic prophase in fission yeast. Homologous chromosome
pairing and recombination occur during horse-tail movements. Mutants impaired
in telomere clustering or nuclear movement show decreased homologous pairing
and recombination, indicating the importance of these events in homolog
juxtaposition (Shimanuki et al.,
1997
; Cooper et al.,
1998
; Nimmo et al.,
1998
; Yamamoto et al.,
1999
). Fission yeast is highly proficient in meiotic recombination
but shows no crossover interference (Munz,
1994
).
It has been long known that fission yeast does not form synaptonemal
complexes. Instead, filamentous structures (linear elements) appear in meiotic
prophase. They resemble the axial cores of other eukaryotes
(Olson et al., 1978;
Hirata and Tanaka, 1982
). The
adaptation of the nuclear spreading technique to fission yeast made possible a
detailed analysis of linear element formation in meiotic time-course
experiments (Bähler et al.,
1993
). Linear elements do not form continuously along the
chromosomes and undergo morphological changes during meiotic prophase.
Bähler et al. have proposed that the organization of chromatin in the
linear elements may facilitate meiotic chromosome functions, such as sister
chromatid cohesion, chiasma maintenance, homologous pairing and the resolution
of interlocks (Bähler et al.,
1993
). Analysis of the rec8-110 mutant revealed
coincidence of impairment of linear element formation, precocious sister
chromatid separation, and decreased homologous pairing for the first time
(Molnar et al., 1995
).
However, rec8 turned out to be a meiotic cohesin
(Parisi et al., 1999
;
Watanabe and Nurse, 1999
). As
a consequence, direct evidence for the involvement of linear elements in
meiotic chromosome functions is still lacking.
Our preliminary observations have shown that linear elements are altered or
impaired in several meiotic recombination-deficient mutants, indicating a
connection between linear element formation, recombination, and perhaps other
meiotic chromosome functions. To learn more about the functions of linear
elements we studied linear element formation in those rec mutants
that show a strong (rec6, rec12, rec14, rec15, rec16) or intermediate
(rec10, rec11) decrease in meiotic recombination
(Ponticelli and Smith, 1989;
DeVeaux et al., 1992
). Because
of its important role in meiotic chromosome pairing, meu13, homolog
of HOP2 in S. cerevisiae
(Nabeshima et al., 2001
), was
also investigated. In this study we describe several mutants with altered
linear element morphology, and discuss the possible reasons for the
morphological changes. This investigation of linear element formation has
provided a structural explanation for the region-specificity of loss of
recombination observed in the rec8, rec10 and rec11 mutants.
We show that linear elements are dispensable for sister chromatid cohesion,
but contribute to full level homologous pairing of chromosome arms.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
YEA (yeast extract agar) and YEL (yeast extract liquid) complete media, and
MEA (malt extract agar) sporulation medium were as described previously
(Gutz et al., 1974). Diploid
strains JB6 and ED1 to ED9 (Table
1) were constructed through interrupted mating
(Gutz et al., 1974
). Diploid
colonies are prototrophic and white on YEA medium as a result of interallelic
complementation between the ade6-M216 and ade6-M210
mutations (Moreno et al.,
1991
). PM (S. pombe minimal) and PM-N (PM without
NH4Cl) media used for meiotic time-courses were as described
(Beach et al., 1985
;
Watanabe et al., 1988
).
Induction of mating and meiosis was done on MEA plates, and living cells were
observed microscopically in EMM2-N liquid medium [EMM2 minimal medium without
nitrogen source (Moreno et al.,
1991
)].
Time-course experiments
Meiotic cultures of diploid strains were prepared as described
(Bähler et al., 1993).
Shifting a culture to meiotic conditions at highly different cell titers
changes the overall progression of the meiotic time-course [e.g. compare
Fig. 5B and
Fig. 5C in Molnar et al.
(Molnar et al., 1995
)].
Therefore, in order to compare the different mutants, care was taken to grow
each culture to a cell titer of 1x107 to
2x107 in PM medium, before meiosis was induced in PM-N.
Samples were taken hourly for DAPI staining of nuclei
(Bähler et al., 1993
) and
for spreading. Nuclear spreads were prepared and silver-stained as described
(Bähler et al., 1993
),
with one modification. To digest the cell walls 1 mg/ml lysing enzyme L2265
(Sigma) was used instead of Novozyme 234. Silver-stained nuclei were examined
with a Philips EM300 at 60 kV (Philips Eindhoven, The Netherlands).
Approximately 100 silver-stained and 200 DAPI-stained nuclei were evaluated at
each time point. At least two independent time courses were carried out with
each mutant.
|
Examination of sister chromatid cohesion, chromosome segregation and
homologous pairing
Sister chromatid cohesion, chromosome segregation and pairing of homologous
chromosomes were monitored in living fission yeast cells carrying the
lacI/lacO system. Mating and meiosis were induced by transferring homothallic
(h90) or crossing heterothallic (h+
and h-) strains on MEA and incubating the plates overnight
at 26°C. Hoechst 33342, a DNA-specific fluorescence dye, was used to
identify the different meiotic stages in living cells. Cells were stained with
5 µg/ml Hoechst 33342 in distilled water for 15 minutes at room
temperature, resuspended in EMM2-N, and mounted on a coverslip for microscopic
examination. Specimens were observed at 26°C on the CCD microscope system
described previously (Molnar et al.,
2001a). In order to monitor the position and number of GFP
signals, images were taken with an exposure time of 0.5 seconds, in 10 optical
sections covering the whole nucleus. Images were analyzed after deconvolution
using the Delta Vision program (Applied Precision, Seattle, WA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
A comparison of the morphology of elements and the frequency of their
classes in the different meiotic recombination-deficient mutants with the
control revealed that the mutants fall into four groups. (1) Some
recombination-deficient mutants showed regular phenotype qualitatively
(morphology of elements) and quantitatively (frequency of classes). rec6,
rec15, and the previously investigated rec7 mutant
(Molnar et al., 2001b) belong
to this group (data not shown). (2) In the rec12, rec14 and
meu13 mutants both the morphology and frequency of certain classes
were altered, but the observed morphologies still resembled wild-type. (3)
rec11 showed strongly impaired linear element morphology that had
been observed before with the other cohesin mutant rec8
(Molnar et al., 1995
). (4) In
the rec10 and rec16/rep1 mutants no traces of linear
elements were found.
Mutants with altered linear element formation
To investigate the phenotype of linear elements in the rec12
mutant meiotic time courses of strain ED5
(Table 1) were analyzed. In
this mutant two of the observed morphologies of linear elements: class I
(short elements, Fig. 2A) and
class IIa (network, Fig. 2B) strongly resembled those seen in the control strain. However, class IIb and
class III looked different. Instead of having a single, long bundle
(Fig. 1C), class IIb nuclei
contained several, shorter pieces of bundles
(Fig. 2C). The class III nuclei
of rec12 can be characterized by an abundance of single long
elements, some of which were unusually long
(Fig. 2D). The most striking
feature of the rec12 mutant was the high frequency of class III
nuclei throughout the whole time-course
(Fig. 2E). A statistical
analysis of the time course (5x2 table test) clearly indicated that the
distribution of different classes of linear elements in the rec12
mutant is significantly different from that of the wild-type
(P<0.005). Class III was the most frequently observed phenotype in
statistical sense as well, while class I was rare in the rec12
mutant. This suggests that short elements (class I) elongated quickly at the
beginning of the time course, and their disassembling at late phases was also
quicker in this mutant than in the wild-type. Meiotic divisions occurred early
in this mutant (Fig. 2F), and
the intensive sporulation prevented the preparation and evaluation of spreads
at later than 8 hour time points.
|
In the rec14 mutant (strain ED6, Table 1) linear element formation began with regular class I morphology (Fig. 3A), but nuclei of this type remained scarce throughout prophase (Fig. 3E). The single short elements developed into networks (class IIa, Fig. 3B), which is the most frequently seen morphology in rec14 (Fig. 3E). Many of these networks contained loosely connected elements and showed a sort of `moth-eaten' appearance (Fig. 3B, right). These nuclei may represent impaired network morphology. Alternatively, since the high frequency of networks suggests that rec14 nuclei spend longer time at this stage, the moth-eaten morphology may represent processing or disassembly of networks. Class IIb nuclei were rare, and contained 2-4 short pieces of bundles (Fig. 3C). The processing of linear elements seems to pass through the single long element stage (class III, Fig. 3D), but the quick progression to meiosis I and the heavy sporulation prevented an evaluation of the latest stages (Fig. 3E,F). The difference in the distribution of linear element classes between the rec14 and wild-type strains is statistically significant (P<0.005). Classes I and IIa show the major differences.
|
Time-course experiments with strain ED8 (Table 1) revealed that linear element formation begins in an unusual way in the meu13 mutant. Instead of class I, small, compact nuclei with 1-2 pieces of bundle-like structures started the process (class IIb early, Fig. 4A). These nuclei were distinguishable from those appearing later (class IIb late, Fig. 4C) by their smaller size. Because the same specimens contained nuclei of different sizes and linear element morphologies (Fig. 4E, see time points 5 to 7 hours), these compact nuclei are not likely to be spreading artifacts. Nuclei designated class IIb late (Fig. 4C) were larger and contained 2 to 4 short bundle pieces. Networks (class IIa) were also altered in the meu13 mutant. They frequently appeared as a kind of combination of the network and regular bundle morphologies (Fig. 4B). Single long elements was the morphology observed latest (Fig. 4D). Divisions in meu13 occurred with a timing similar to the control (Fig. 4F). Because the meu13 mutant showed some obvious differences in LE organization compared to the wild-type (lack of class I nuclei and two types of bundles), we did not carry out a statistical analysis on the distribution of different LE classes. The same applies for the cohesin mutants (see below).
|
Linear elements in cohesin mutants
Experiments with strain ED4 (Table
1) revealed that the morphologies of linear elements in the
rec11 mutant are virtually indistinguishable from those seen in
rec8. Therefore, the same designation of classes was used to describe
rec11 as that previously used for the rec8-110 mutant
(Molnar et al., 1995). Class A
stands for nuclei with a few, very short elements
(Fig. 5A). In class B nuclei
usually 2 or 3 short and thick elements were visible
(Fig. 5B). Class C nuclei
appeared last, and contained a single long element
(Fig. 5C,D). The above
morphologies suggest a severe defect in linear element formation. It is a
distinct phenotype, observed so far only in the meiotic cohesin mutants
rec8 and rec11 [(Molnar
et al., 1995
) and this study]. Thus we call it the cohesion-defect
phenotype of linear elements. The same morphologies were detected in strain
ED2 (Table 1) where both
meiotic cohesins are deleted (compare the nuclei in
Fig. 5B and C, and data not
shown).
Mutants that do not form linear elements
Two of the meiotic recombination-deficient mutants, rec16/rep1 and
rec10, did not contain any element-like structures. The majority of
cells in the rec16/rep1 strain ED9
(Table 1) did not undergo
meiosis. In two time-course experiments, 18% and 25% final sporulation was
measured. Although only a small fraction of cells underwent meiosis, nuclei in
meiotic prophase were still detectable in the electron microscope. Before
meiotic prophase, the spindle pole body (SPB) locates far from the nucleolus,
and consists of a single body or two bodies of equal sizes
(Bähler et al., 1993).
Normally, meiotic prophase nuclei contain linear elements and have their
spindle pole body (SPB) located close to the nucleolus. The SPB consists of a
large and two adjacent smaller bodies in meiotic prophase [see Figs
1,2,3,4,5
(Bähler et al., 1993
)]. At
time points 3 to 9 hours, up to 20% of nuclei showed this morphology in the
rec16 mutant, but these meiotic nuclei never contained linear
elements (Fig. 6A). Sugiyama et
al. have shown that rec16/rep1 is deficient in premeiotic DNA
synthesis (Sugiyama et al.,
1994
). This explains the described severe defects.
|
Meiotic time-courses with strain ED3 (Table 1) gave an unexpected result. In the rec10 mutant no trace of linear elements could be observed. The rec10 mutant progressed through meiosis similarly to the JB6 control strain (Fig. 6B). In rec10, empty nuclei with meiotic prophase SPB configuration were observed with a frequency similar to that seen in linear-element-containing nuclei in the control. Horse-tail nuclei appeared with similar dynamics in the two strains. In contrast to rec16/rep1, the rec10 mutant underwent meiosis efficiently (Fig. 6B). Thus rec10 can be a useful tool to evaluate the consequences of the lack of linear elements in fission yeast.
Regular sister chromatid cohesion in rec10
It has been proposed that linear elements have a role in meiotic chromatin
organization and that they may be necessary for the proper completion of
meiotic chromosome functions (Bähler
et al., 1993). In order to test the involvement of linear elements
in sister chromatid cohesion, rec10 and control heterothallic strains
bearing the lacI-GFP/lacO recognition system were crossed with strains lacking
GFP labeling (heterozygous cross for GFP). Deletion strains of the meiotic
cohesins rec8 and rec11
(Table 1) were also
investigated. Sister chromatid cohesion was checked at the centromere and
three different loci along the right arm of chromosome II
(Fig. 7F). Cells in meiotic
prophase (horse-tail nuclei) were identified after Hoechst 33342 staining, and
the number of GFP signals was determined in living cells (see Materials and
Methods). In a heterozygous cross a single GFP signal indicates regular sister
chromatid cohesion. Appearance of two separated GFP signals is an obvious sign
of the loss of sister chromatid cohesion. Sometimes nuclei with two closely
associated but still unseparated signals were detected. Because it was
frequently seen in the cohesin mutants, this doubling of the GFP signal
probably indicates a loosening of sister chromatid association. Nuclei with
separated and doubled GFP signals were scored separately
(Fig. 7).
Sister chromatids were rarely separated at the centromere in prophase in the rec8 and rec11 mutants (Fig. 7A). In contrast, an increased impairment of sister chromatid cohesion was detected along the chromosome arm in both mutants (Fig. 7B-D). The degree of impairment was fairly constant at all loci examined in rec8. In rec11, a slight increase was observed towards the telomere. This is in contrast to the rec10 mutant where only slight aberrancies were detected at each locus (Fig. 7A-D).
2 test showed no statistical difference in sister chromatid
cohesion between the wild-type and the rec10 mutant at any of the
chromosomal loci. A comparison of rec8 and rec11 showed that
there clearly was no difference between them either. To get an idea about the
difference between the wild-type and the cohesin mutants, we compared the
combined data of the wild-type strains (wt and rec10) to the combined
data of the cohesin mutant strains (rec8 and rec11). The
reduction in sister chromatid cohesion in the cohesin mutants was
statistically not significant at the centromere (0.05<P<0.1).
In contrast, cohesion is significantly reduced at all the other loci
(his2, ade1, ade8) examined in the cohesin mutants
(P=0.005). We conclude that the cohesion of sister chromatids is
basically intact despite the lack of linear elements in meiotic prophase of
rec10.
Precocious separation of sister chromatids may occur at the first meiotic
division. It is conceivable that cytologically invisible pieces of linear
elements remain at the chiasmata and support the proper segregation at meiosis
I. Therefore, we examined chromosome segregation at the first meiotic division
in each mutant. Cells having two nuclei were identified after Hoechst 33342
staining and the number of GFP signals was determined. When both nuclei
carried a GFP signal in a heterozygous cross, sister chromatids segregated
prematurely. To examine segregation of homologous chromosomes, homothallic
strains (homozygous cross for GFP, Table
1) were also analyzed. In a homozygous cross both homologous
chromosomes have GFP labeling. If one of the sister nuclei after meiosis I had
no GFP signal, nondisjunction of homologous chromosomes had occurred. In
accordance with previous studies (Watanabe
and Nurse, 1999; Molnar et
al., 2001a
), a high level of precocious sister chromatid
separation was detected in rec8 (PSSC;
Fig. 7E). Precocious sister
chromatid separation was rare in rec10 and rec11, but both
mutants showed high level of chromosomal nondisjunction at the first division
(NDJI; Fig. 7E). A comparison
of the combined data of the rec10 and rec11 strains to the
combined data of the wt and rec10 strains showed a statistically
highly significant difference in NDJI (P=0.005). In summary, our
observations with GFP-labeled chromosomes in living cells suggest that linear
elements are dispensable for sister chromatid cohesion in fission yeast.
Homologous chromosome pairing is decreased in rec10 in a
region-specific manner
Next we asked whether linear elements are necessary for homologous
chromosome pairing. Homothallic rec10 (strains 119, 148, 149 and 166)
and control (CT2111-2, JW555, JW558 and 159) strains
(Table 1) bearing GFP labeling
at different chromosomal loci (Fig.
7F) were examined. Horse-tail stage cells were identified after
Hoechst 33342 staining and the GFP signals were analyzed as described in
Materials and Methods. Homologous chromosomes were scored as paired when their
GFP signals touched each other or only a single signal was visible. The
results are summarized in Table
2.
|
The highest level of chromosome `pairing' was measured at the centromere in
the control strain. [Actually, in fission yeast a clustering of all the
centromeres occurs in prophase (Scherthan
et al., 1994).] Along the chromosome arm, an increase of pairing
was detectable towards the telomere. The rec10 mutant showed
wild-type level of clustering of centromeres but decreased pairing at all
other loci examined. A comparison of the homologous pairing in rec10
to the control (Fig. 8)
revealed that in rec10 the impairment was slight (statistically not
significant) at the his2 and ade8 loci. These loci are
located towards the centromere and telomere ends of the chromosome arm,
respectively (Fig. 7F). At the
ade1 locus, which is situated in the middle of the right arm of
chromosome II, significantly decreased pairing (0.01<P<0.025)
was detected. These results suggest that linear elements contribute to regular
homologous chromosome pairing and their role is especially important in the
interstitial regions of chromosome arms.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conclusions from the analysis of mutants with altered linear element
formation and processing
Three of the recombination deficient mutants, rec12, rec14 and
meu13 showed altered linear element formation, but the morphology
patterns were close to those seen in wild-type. Because the prophase stages in
other organisms are defined by the development of the synaptonemal complex, a
similar subdivision of meiotic prophase in fission yeast is not possible. A
correlation of recombination events other than DSBs, as detected by physical
analysis, with the cytological changes is still missing in fission yeast.
Nevertheless, it is noteworthy that the three mutations caused different
changes of morphologies and their frequencies, at different stages of meiotic
prophase. This argues for a biological significance of the different linear
element morphologies.
Rec12 is a homolog of Spo11, a protein that forms recombination-initiating
double-strand breaks in S. cerevisiae
(Cervantes et al., 2000;
Davis and Smith, 2001
;
Keeney et al., 1997
). The
conservation of the catalytic site suggests an identical function in S.
pombe (Keeney et al.,
1997
; Cervantes et al.,
2000
). In the rec12 mutant of fission yeast no detectable
meiosis-specific breakage of chromosomes occurs
(Cervantes et al., 2000
;
Young et al., 2002
).
Therefore, the rec12 mutant is an appropriate tool to address a
possible interdependence of formation of double-strand breaks and linear
elements. Linear elements, although with altered morphology, were observed in
the rec12 mutant (Fig.
2). Consequently, the formation of linear elements is not
dependent on the occurrence of double-strand breaks. Analysis of the rec6,
rec7 (Molnar et al.,
2001b
) and rec15 mutants confirmed this conclusion. They
are defective in DSB formation (Cervantes
et al., 2000
; Davis and Smith,
2001
), but show normal linear element morphology. In addition,
meiotic breakage of chromosomes does not occur in strains lacking the
rec14 gene product (Cervantes et
al., 2000
; Davis and Smith,
2001
), but it showed altered linear elements
(Fig. 3) that differed from
those in rec12 (Fig.
2).
Conversely, although LE formation does not depend on DSB formation, some
early prophase proteins influence both processes and recombination. The
rec8 and rec10 mutants were shown to form meiotic breaks
with reduced efficiency (Cervantes et al.,
2000). In the cohesin mutants rec8
(Molnar et al., 1995
) and
rec11, as well as in the double mutant rec8rec11
(Fig. 5), identical and, from
wild-type, strongly deviating LE structures were observed. In rec10
the linear elements were missing completely
(Fig. 5). Obviously cohesins
and rec10 are involved in LE and DSB formation (see below).
rec12 and rec14 are also involved in both processes. The
most striking feature of the rec12 mutant was the abundant occurrence
of nuclei with single long elements at the expense of networks and bundles
(Fig. 2E). Networks and bundles
are the most complicated linear element morphologies. Their rarity indicates
that linear element processing is altered in the rec12 mutant. Thus
fission yeast Rec12 is similar to its budding yeast homolog in the sense that
both proteins are involved in the initiation of homologous recombination and
also in proper chromosome organization. However, their role in chromosome
organization might be somewhat different: spo11 null mutants are
capable of forming axial elements, but exhibit severe homolog synapsis defects
(Loidl et al., 1994
).
The most striking feature of the rec14 mutant was the high
frequency of networks (Fig. 3).
rec14 is a functional homolog of REC103 of S. cerevisiae
(Evans et al., 1997). Both
genes were shown to be involved in early stages of meiotic recombination
(Evans et al., 1997
;
Gardiner et al., 1997
;
Cervantes et al., 2000
), but
the known phenotypes of their mutants do not allow a clear conclusion about
the function of rec14 in linear element formation. However, it should
be noted that the expression pattern of rec14 is different from that
of other recombination genes in fission yeast: its transcript is present both
in meiotic and mitotically dividing cells, and the mutant shows a slow mitotic
growth phenotype (Evans et al.,
1997
). Thus it is conceivable that the observed alteration in
linear element formation is a consequence of a disturbance of basic chromosome
structure not directly related to the initiation of recombination.
meu13 participates in homologous chromosome pairing in fission
yeast in a recombination-independent mechanism and shows significant sequence
homology to Hop2 (Nabeshima et al.,
2001), a protein that ensures synapsis between homologous
chromosomes in S. cerevisiae (Leu
et al., 1998
). Double-strand breaks form in meu13
deletion strains and their repair is retarded
(Shimada et al., 2002
). The
typical morphological change in this mutant was the frequent occurrence of
network-and bundle-like structures (Fig.
4), some of which rather resembled an unspecific deposition of
linear element material than a functional structure
(Fig. 4A). Meu13p is localized
to meiotic chromatin during the horse-tail nuclear movement stage
(Nabeshima et al., 2001
).
Because linear elements were most aberrant at early prophase stages in the
meu13 mutant (Fig.
4A), it is suggested that Meu13 might serve as a loading site for
linear element proteins. In turn, linear elements are needed to achieve the
full level of meiotic chromosome pairing
(Table 2; Fig. 8). Thus the decreased
meiotic pairing and recombination observed in the meu13 mutant might
be a consequence of imperfect formation of linear elements.
A possible explanation for the regional specificity of recombination
loss in rec8, rec10 and rec11 mutants
DeVeaux and Smith have observed first a regional specificity of loss of
meiotic recombination in the rec8, rec10 and rec11 mutants
(DeVeaux and Smith, 1994).
They have found that meiotic recombination was impaired most severely in a
2 Mb region surrounding the ade6 locus of chromosome III, while
other loci examined were less severely affected. Parisi et al. and Krawchuck
et al. extended their study and showed that meiotic recombination is decreased
severely in the centromeric region of each chromosome in these mutants
(Parisi et al., 1999
;
Krawchuck et al., 1999). Based on epistasis analysis and classical chromosome
segregation studies, Krawchuk et al. proposed that Rec8, Rec10 and Rec11 are
involved in a `meiotic sister chromatid cohesion pathway' and promote
homologous chromosome pairing in the centromer proximal regions of chromosomes
(Krawchuk et al., 1999
). Our
results largely confirm this hypothesis and suggest that the underlying
structural reason for the regional specificity is a defect in the formation of
linear elements in the rec8, rec10 and rec11 mutants.
Rec8 and Rec11 are meiotic cohesins
(Watanabe and Nurse, 1999;
Parisi et al., 1999
;
Davis and Smith, 2001
). Rec8
has two different functions correlating with distinguishable localization. It
was found to locate to the centromeres of chromosomes
(Watanabe and Nurse, 1999
) and
to ensure reductional segregation at the first meiotic division
(Watanabe and Nurse, 1999
;
Molnar et al., 2001a
). In
addition, Rec8 as well as Rec11 are involved in sister chromatid cohesion
along the chromosome arms (Fig.
6). We have found a severe defect in linear element formation in
rec8 and rec11 mutants
(Molnar et al., 1995
)
(Fig. 5). Deletion of either of
the meiotic cohesins resulted in a typical morphological change observed so
far only in these mutants. Moreover, the double mutant showed the same
phenotype (Fig. 5). The most
straightforward interpretation of these observations is that Rec8 and Rec11
work in a complex, and that functional cohesin complexes are indispensable for
proper linear element formation. Cohesin complexes may serve as loading sites
for linear element polymerization. A similar function of S.
cerevisiae Rec8 for axial element formation has been suggested
(Klein et al., 1999
). The
rec10 mutant does not form linear element material at all, but it
undergoes meiosis with similar timing and efficiency to that of a wild-type
strain (Fig. 6). Direct
investigation of the sister chromatid cohesion along chromosome II in the
rec10 mutant has shown that linear elements are basically dispensable
for sister chromatid cohesion in fission yeast
(Fig. 7).
The role of linear elements in chromosome pairing
Cells employ several mechanisms to effect chromosome pairing. The
contribution of horse-tail movements and telomere clustering to homologous
chromosome pairing is well-demonstrated in fission yeast (for a review, see
Yamamoto and Hiraoka, 2001).
The rec10 mutant shows regular horse-tail movements (M.M.,
unpublished). Because mutants impaired in telomere clustering perform aberrant
nuclear movement (Cooper et al.,
1998
; Nimmo et al.,
1998
; Hiraoka et al.,
2000
), the above observation indirectly indicates that telomere
clustering is regular in rec10. Therefore, the decrease in chromosome
pairing in the rec10 mutant is likely to be attributable to the lack
of linear elements.
The highest level of homologous chromosome contact was detected at the
centromere of chromosome II, both in the control and the rec10 mutant
strains (Table 2). A similar
result was obtained in a wild-type strain by fluorescence in situ
hybridization experiments performed on nuclear spreads: Scherthan et al. has
shown that all the centromeres form a cluster and maintain this state
throughout meiotic prophase in fission yeast
(Scherthan et al., 1994). This
observation suggests that cells have a mechanism to accomplish the clustering
of centromeres independently from linear element formation or rec10
function. In the control strain, a gradual increase in chromosome pairing was
observed towards the telomere (Table
2). This can be explained by the effect of telomere clustering,
which increases the possibility of chance contacts primarily at the telomere
proximal regions of chromosomes.
In the rec10 mutant a decrease in homologous chromosome pairing was measured at each locus along the chromosome arm (Table 2). The reduction was slight at the his2 and ade8 loci (Fig. 8). Among the examined loci, ade8 is closest to the telomere and his2 is to the centromere. Telomere clustering may promote pairing of homologous chromosomes most efficiently in the ade8 region in absence of linear elements. The efficient clustering of centromeres in rec10 might exert a similar supporting effect for pairing of centromere proximal loci. An alternative explanation is based on the fact that the his2 locus is near the mating-type locus, which contains heterochromatin. Thus, fission yeast may use the heterochromatin of the mating-type and centromeres to achieve linear element independent pairing of the centromere-proximal region in this chromosome arm. The ade1 locus is situated in the middle of the chromosome arm, and the most severe defect in chromosome pairing was detected at this locus (Fig. 8). Linear elements are thus likely to be most important for chromosome pairing at interstitial arm regions. Clustering of telomeres and centromeres provide for alignment of the telomere and centromere proximal regions, respectively. This probably ensures frequent contact of the corresponding regions, and homologous sequences may have a better chance for pairing despite the lack of chromatin organization by linear elements.
Concluding remarks
rec10-155::LEU2 is a partial deletion lacking residues 683 to 791
(Lin and Smith, 1995). This
mutant undergoes meiosis similarly to wild-type, but lacks linear elements
completely. This provided a good opportunity to analyze the role of linear
elements in meiosis. We have found that linear elements are basically
dispensable for sister chromatid cohesion, but contribute to homologous paring
of chromosomes. Although we cannot rule out the possibility that
rec10 promotes homologous chromosome pairing independently from its
function in linear element formation, the most straightforward interpretation
of our data is that Rec10 exerts its effect on homologous chromosome pairing
through its function in linear element formation. What is the role of
rec10 in linear element formation? rec10 might encode a
structural protein of linear elements. However, a regulatory role is also
plausible. rec10 may regulate linear element formation directly or
through more general processes, for example, through the regulation of
chromatin structure.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bähler, J., Wyler, T., Loidl, J. and Kohli, J. (1993). Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis. J. Cell Biol. 121,241 -256.[Abstract]
Bähler, J., Wu, J.-Q., Longtine, M. S., Shah, N. G., McKenzie, A., III, Steever, A. B., Wach, A., Philippsen, P. and Pringle, J. R. (1998). Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe.Yeast 14,943 -951.[CrossRef][Medline]
Beach, D., Rodgers, L. and Gould, J. (1985). RAN1+ controls the transition from mitotic division to meiosis in fission yeast. Curr. Genet. 10,297 -311.[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.[CrossRef][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.
Davis, L. and Smith, G. R. (2001). Meiotic
recombination and chromosome segregation in Schizosaccharomyces pombe.Proc. Natl. Acad. Sci. USA
98,8395
-8402.
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, R. and Smith, G. R. (1998). Global control of meiotic recombination genes by Schizosaccharomyces pombe rec16 (repl). Mol. Gen. Genet. 258,663 -670.[CrossRef][Medline]
Egel, R. (1973). Commitment to meiosis in fission yeast. Mol. Gen. Genet. 121,277 -284.
Egel, R. and Egel-Mitani, M. (1974). Premeiotic DNA synthesis in fission yeast. Exp. Cell. Res. 88,127 -134.[Medline]
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.
Gardiner, J. M., Bullard, S. A., Chrome, C. and Malone, R.
E. (1997). Molecular and genetic analysis of REC103, an early
meiotic recombination gene in yeast. Genetics
146,1265
-1274.
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.
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 104,203 -214.[CrossRef]
Hirata, A. and Tanaka, K. (1982). Nuclear behavior during conjugation and meiosis in the fission yeast Schizosaccharomyces pombe. J. Gen. Appl. Microbiol. 28,263 -274.
Keeney, S., Giroux, C. N. and Kleckner, N. (1997). Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88,375 -384.[Medline]
Kleckner, N. (1996). Meiosis: how could it
work? Proc. Natl. Acad. Sci. USA
93,8167
-8174.
Klein, F., Mahr, P., Galova, M., Buonomo, S. B. C., Michaelis, C., Nairz, K. and Nasmyth, K. (1999). A central role for cohesins in sister chromatid cohesion, formation of axial elements, and recombination during yeast meiosis. Cell 98, 91-103.[Medline]
Kohli, J. (1994). Telomeres lead chromosome movement. Curr. Biol. 4,724 -727.[CrossRef][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.
Leu, J.-Y., Chua, P. R. and Roeder, G. S. (1998). The meiosis-specific Hop2 protein of S. cerevisiae ensures synapsis between homologous chromosomes. Cell 94,375 -386.[Medline]
Li, Y. F., Numata, M., Wahls, W. P. and Smith, G. R. (1997). Region-specific meiotic recombination in Schizosaccharomyces pombe: the rec11 gene. Mol. Microbiol. 23,869 -878.[CrossRef][Medline]
Lin, Y. and Smith, G. R. (1994). Transient,
meiosis-induced expression of the rec6 and rec12 genes of
Schizosaccharomyces pombe. Genetics
136,769
-779.
Lin, Y. and Smith, G. R. (1995). Molecular cloning of the meiosis-induced rec10 gene of Schizosaccharomyces pombe. Curr. Genet. 27,440 -446.[Medline]
Loidl, J., Klein, F. and Scherthan, H. (1994). Homologous pairing is reduced but not abolished in asynaptic mutants of yeast. J. Cell Biol. 125,1191 -1200.[Abstract]
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., Bähler, J., Kohli, J. and Hiraoka, H. (2001a). Live observation of fission yeast meiosis in recombination-deficient mutants: a study on achiasmate chromosome segregation. J. Cell Sci. 114,2843 -2853.[Medline]
Molnar, M., Parisi, S., Kakihara, Y., Nojima, H., Yamamoto, A.,
Hiraoka, Y., Bozsik, A., Sipiczki, M. and Kohli, J. (2001b).
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 of fission yeast: Dis1 is implicated in force balance in metaphase
bipolar spindle. Mol. Biol. Cell
9,3211
-3225.
Nabeshima, K., Kakihara, Y., Hiraoka, Y. and Nojima, H.
(2001). A novel meiosis- specific protein of fission yeast,
Meu13p, promotes homologous pairing independently of homologous recombination.
EMBO J. 20,3871
-3881.
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.
Olson, L. W., Eden, U., Egel-Mitani, M. and Egel, R. (1978). Asynaptic meiosis in fission yeast? Hereditas 89,189 -199.
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 sistercohesion phosphoprotein of the Rad21p family conserved
from fission yeast to human. Mol. Cell. 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.
Roeder, G. S. (1997). Meiotic chromosomes: it
takes two to tango. Genes Dev.
11,2600
-2621.
Scherthan, H., Bähler, J. and Kohli, J. (1994). Dynamics of chromosome organization and pairing during meiotic prophase of fission yeast. J. Cell. Biol. 127,273 -285.[Abstract]
Shimada, M., Nabeshima, K., Tougan, T. and Nojima, H.
(2002). The meiotic recombination checkpoint is regulated by
checkpoint rad+ genes in fission yeast. EMBO
J. 21,2807
-2818.
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 meioticprophase-specific nuclear architecture. Mol. Gen. Genet 254,238 -249.[CrossRef][Medline]
Sugiyama, A., Tanaka, K., Okazaki, K., Nojima, H. and Okayama, H. (1994). A zinc finger protein controls the onset of premeiotic DNA synthesis of fission yeast in a Mei2-independent cascade. EMBO J. 13,1881 -1887.[Abstract]
Watanabe, Y. and Nurse, P. (1999). Cohesin Rec8 is required for reductional chromosome segregation at meiosis. Nature 400,461 -464.[CrossRef][Medline]
Watanabe, Y., Iino, Y., Furuhata, K., Shimoda, C. and Yamamoto, M. (1988). The S. pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under the regulation of cAMP. EMBO J. 7,761 -767.[Abstract]
Yamamoto, A. and Hiraoka, Y. (2001). How do meiotic chromosomes meet their homologous partner? Lessons from fission yeast. BioEssays 23,526 -533.[CrossRef][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.
Young, J. A., Schreckhise, R. W., Steiner, W. W. and Smith, G. R. (2002). Meiotic recombination remote from prominent break sites in S. pombe. Mol. Cell 9, 253-263.[Medline]
Zickler, D. and Kleckner, N. (1999). Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33,603 -754.[CrossRef][Medline]
Related articles in JCS: