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Vol. 14, Issue 6, 2461-2469, June 2003
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* Department of Biophysics and Biochemistry, Graduate School of Science,
University of Tokyo, Tokyo 113-0033, Japan;
Molecular Genetics Research Laboratory, University of Tokyo, Tokyo 113-0033,
Japan
Submitted November 18, 2002;
Revised January 17, 2003;
Accepted February 11, 2003
Monitoring Editor: Mitsuhiro Yanagida
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Yamamoto et al.,
1997). The mei2 gene is expressed very poorly, if at all,
in cells growing mitotically (Shimoda
et al., 1987;
Watanabe et al.,
1988). In addition, Pat1(Ran1) kinase, which is active during the
mitotic cell cycle, represses the function of Mei2p by phosphorylating it on
two amino-acid residues (Watanabe et
al., 1997). To date, two mechanisms have been elucidated
concerning how the phosphorylation by Pat1 kinase down-regulates Mei2p
activity. 1) Phosphorylated Mei2p is more susceptible to ubiquitin-mediated
proteolysis (Kitamura et al.,
2001). 2) 14-3-3 protein binds preferentially to phosphorylated
Mei2p and thereby decreases the affinity of Mei2p for RNA
(Sato et al.,
2002).
In natural meiosis, the activity of Pat1 kinase is turned off by the
binding of a pseudosubstrate Mei3p, which is expressed only in diploid cells
heterozygous for the mating type genes under starved conditions
(McLeod and Beach, 1988;
Li and McLeod, 1996).
Nutritional starvation also results in enhancement of mei2
transcription (Shimoda et al.,
1987). Thus, diploid cells exposed to starvation accumulate
unphosphorylated Mei2p, which is a critical step to commit the cells to
meiosis (Watanabe et al.,
1997). Mei2p shuttles between the cytoplasm and the nucleus
(Sato et al., 2001),
and the ability of Mei2p to bind to RNA is essential for its activity to
stimulate meiosis (Watanabe and Yamamoto,
1994).
One peculiar property of Mei2p is that it forms a distinct
"dot" in the nucleus at meiotic prophase
(Watanabe et al.,
1997; Yamashita et
al., 1998). The nucleus assumes an elongated shape like a
horse tail and moves back and forth during meiotic prophase, led by the
spindle pole body (SPB) (Chikashige et
al., 1994). The relative position of the Mei2p dot is
apparently fixed in the horse-tail nucleus
(Yamashita et al.,
1998). Formation of the Mei2p dot requires an RNA species called
meiRNA, which specifically binds to Mei2p and colocalizes with it in the dot
(Yamashita et al.,
1998). meiRNA is indispensable for the Mei2p function to stimulate
meiosis I (Watanabe and Yamamoto,
1994). Because the emergence of this dot seems to correlate with
the ability of the cell to perform meiosis I
(Yamashita et al.,
1998; Sato et al.,
2001), we have set out to clarify the identity of the dot, hoping
that the results may provide insight into the molecular function of Mei2p. In
this report, we describe detailed analysis of the location of the dot. The
obtained results lead us to conclude that the dot is closely associated with
the gene for meiRNA, namely, sme2, positioned on the short arm of
chromosome II. Implications of this finding are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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).
Complete medium YE and minimal medium SD
(Sherman et al.,
1986) were used for the routine culture of S. pombe.
Minimal medium MM (Moreno et al.,
1990) was used to induce transcription from the nmt1
promoter. Transformation of S. pombe cells was done by the lithium
acetate method (Okazaki et al.,
1990).
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Microscopy
Fluorescence images were detected by a cooled, charge-coupled device
(Photometrics Peltier-cooled charge-coupled device camera Quantix), which was
attached to a fluorescence microscope Axioplan 2 (Carl Zeiss, Jena, Germany)
and controlled by the Meta-Morph program (Universal Imaging, Downingtown, PA).
Immunofluorescence analysis was done essentially as described previously
(Yamashita et al.,
1998). Briefly, S. pombe cells were fixed with 3%
formaldehyde freshly prepared from paraformaldehyde and with 0.2%
glutaraldehyde in PEM buffer (100 mM PIPES, pH 6.9, 5 mM EGTA, 5 mM
MgCl2) for 45 min at room temperature. After fixation, cells were
treated with Zymolyase 100T (0.1 mg/ml; Seikagaku America, Rockville, MD) for
70 min at 37°C. They were suspended in 1% Triton X-100 and washed three
times with 1 mg/ml sodium borohydride. The hemagglutinin (HA)-tagged Mei2
protein was stained with mouse monoclonal anti-HA antibody 16B12 (Babco,
Richmond, CA), followed by Texas Red-conjugated sheep antibodies to mouse IgG
(Amersham Biosciences, Piscataway, NJ). The SPB was visualized with
anti-Sad1p, which recognizes its component
(Hagan and Yanagida, 1995).
DNA was counterstained with Hoechst 33342.
Integration of Epitope-tagged mei2 Alleles into the Chromosomal mei2
Locus
We prepared an EcoRI fragment carrying the mei2 open
reading frame (ORF), which was truncated at the N terminus, tagged with an
epitope at the C terminus, and followed by the nmt1 terminator. The
epitope used was either green fluorescent protein (GFP), or enhanced cyan
fluorescent protein (CFP) (BD Biosciences Clontech, Palo Alto, CA), or three
copies of HA. The EcoRI fragment was cloned into the integration
vector pFA6a-kanMX6 (Bahler et
al., 1998). The resulting plasmid was linearized by cutting
at the KpnI site within the mei2 ORF and transformed into
S. pombe cells. Stable Kanr transformants were isolated,
and they were confirmed by genomic polymerase chain reaction (PCR) to carry
the epitope-tagged mei2 gene generated by integration of the plasmid
at the chromosomal mei2 locus.
Induction of Haploid Meiosis
Two distinctive ways to induce haploid meiosis were used. 1) Cells carrying
both mat1-M and mat1-P were cultured in MM+N medium at
30°C. At the concentration of 
1 x 107 cells/ml, they
were transferred to MM-N medium lacking a nitrogen source to induce meiosis.
2) The pat1-driven meiosis (Iino
and Yamamoto, 1985; Nurse,
1985) was induced in haploid cells, adopting the protocol
described for diploid cells homozygous for the mating-type genes
(Murakami and Nurse, 1999).
Haploid pat1-114 cells were cultured in MM+N medium at 25°C. At
the concentration of 1 x 107 cells/ml, they were
transferred to MM-N medium containing 50 µg/ml leucine, and incubated for
14–16 h at 25°C. NH4Cl and leucine were added to the
cultures at 500 and 50 µg/ml, respectively, immediately before the shift to
34°C. The cells were kept at 34°C to induce meiosis.
Fluorescence Marking of Specific Chromosomal Loci
Marking of chromosomes by the LacI-LacO system was done as described
originally for budding yeast (Straight
et al., 1996). We constructed various derivatives from
the parental S. pombe strains either described previously
(Nabeshima et al.,
1998) or further developed by A. Yamamoto (Kansai Advanced
Research Center, Kobe, Japan). These strains expressed a GFP-LacI-nuclear
localization signal (NLS) fusion protein to recognize the LacO sequence. The
fusion gene for GFP-LacI-NLS was integrated at the his7 locus and
driven by the dis1 promoter. Insertion of the 8-kb-long LacO sequence
to each specific chromosomal locus was carried out using plasmids pCT32-6 and
pCT31-13, provided by A. Yamamoto. pCT32–6 was a variant of
pFA6a-kanMX6, and carried the ura4 gene truncated at its N terminus
(ura4
N) and the kanr gene. The
ura4N and kanr genes were integrated into
a specific chromosomal locus, according to the PCR protocol described for
pFA6a-kanMX6 by Bahler et al.
(1998). Stable Kanr
transformants were selected and then transformed with pCT31-13, which carried
the ura4 gene truncated at its C terminus (ura4C) and
256 copies of the lacZ operator sequence. pCT31-13 was linearized by
cutting at the StuI site within the ura4 ORF before
transformation. Thus, the resulting Ura+ strain bore the LacO
sequence at each specific locus on the chromosome. Genomic PCR and Southern
blot hybridization were carried out to confirm the integration of the LacO
sequence at the targeted chromosomal locus. The LacO integration sites A
through F, which are located in noncoding regions, are defined precisely in
Table 2. Expression of
GFP-LacI-NLS visualized the LacO sequence on the chromosome by
fluorescence.
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Reciprocal Chromosome Translocation
We followed the method developed by Virgin et al.
(1995). A 1.8-kb-long
ura4+ fragment was inserted into the HindIII site
on the vector pBluescript (Stratagene, La Jolla, CA). A 2.9-kb-long
PvuII-SpeI fragment carrying the ade6-469 allele
was then inserted into the same plasmid by using the SmaI and
SpeI sites. The resulting plasmid was digested with ClaI and
SpeI, which generated a 4.7-kb-long fragment containing
ura4+ and ade6-469. Plasmids harboring either
site G or site H (Figure 3B)
were constructed by PCR amplification of genomic DNA, and the
ClaI-SpeI fragment was cloned into either the NdeI
site (site G) or the SphI site (site H). The resulting plasmid
carrying site G was digested with SacI and KpnI and that
carrying site H was digested with NotI and KpnI. The
fragments thus obtained were transformed into JW859 (h-
ade6-D19 leu1 ura4-D18), a derivative of the strain provided by G.
Smith (Fred Hutchinson Cancer Research Center, Seattle, WA), and stable
Ura+ transformants were isolated. Genomic PCR was carried out to
confirm that the ura4+ and ade6-469 markers were
properly integrated at either site G or site H. The resultant strains were
crossed with an h90 ade6-M26 strain JW943.
Sporulation was induced and Ade+ spores were selected to isolate
strains with arms of chromosomes II and III translocated reciprocally. Genomic
PCR was carried out to confirm that the chromosomes were crossed over at the
ade6 sequence on chromosome II (site G or H) and the authentic
ade6 locus on chromosome III.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To visualize nucleoli, we expressed a nucleolar protein
Gar2p (Gulli et al.,
1995) conjugated with GFP, from a fusion gene that carried the
authentic gar2 promoter and was integrated at the original
chromosomal locus. Electron microscopic observation has shown previously that
Gar2p spreads throughout the nucleolus in interphase cells
(Sicard et al.,
1998). Consistently, the Gar2p-GFP fusion protein occupied half of
the spherical interphase nucleus, where no staining of DNA was observed
(Figure 1A). This region has
been assigned previously as the nucleolus
(Toda et al.,
1981).
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Using the Gar2p-GFP fusion construct, we visualized the nucleolar region in
the meiotic prophase nucleus. The nucleolus was found to reside in the front
region of the horse-tail nucleus, adjoining the SPB. It was rather elongated
and flattened (Figure 1B).
Similar images were observed when we performed indirect immunostaining by
using the anti-NOP1 (Aris and Blobel,
1988), which recognizes another nucleolar protein fibrillarin
(Girard et al., 1993)
(our unpublished data). Electron microscopic observation has also assigned an
electron-dense region near the SPB as the putative nucleolus
(Bahler et al., 1993).
To determine the position of the Mei2p dot relative to the nucleolus, we fixed
meiotic cells expressing Gar2p-GFP and 3HA-tagged Mei2p. We then immunostained
the cells with anti-HA to detect Mei2p and anti-Sad1p to detect SPB
(Hagan and Yanagida, 1995).
Gar2p-GFP emitted enough fluorescence for detection even after fixation. A
typical result is shown in Figure
1B. In most cells the Mei2p-HA dot was close to the rear edge of
the nucleolus, but it was always outside the nucleolus, indicating that Mei2p
was not associated with part of the nucleolus.
The Mei2p Dot Segregates Like a Chromosome in Haploid Meiosis
We next examined the possibility that the Mei2p dot might be associated
with chromosomes. As a characteristic of the meiotic nuclei in fission yeast,
telomeres cluster near the SPB during prophase (Chikashige et al.,
1994,
1997). Centromeres are away
from the SPB and each part of the chromosomes apparently occupies a specific
position relative to the SPB (Niwa et
al., 2000). However, the telomere clustering is disrupted and
the prophase-specific chromosome arrangement is distorted in the
taz1 mutant, which lacks a telomere-binding protein
(Cooper et al., 1998).
We visualized the Mei2p dot by immunostaining in taz1 cells,
and stained the cells also with anti-Sad1p, which marked the SPB at the
leading edge of the horse-tail nucleus. Compared with wild-type cells
(Figure 1B), the Mei2p dot was
far distant from the SPB in taz1 cells
(Figure 1C). These results
suggested that the location of the Mei2p dot was unlikely to be directed by
the SPB. Rather, the Mei2p dot seemed to have been displaced together with the
bulk of chromosomes disconnected from the SPB in taz1
cells.
To investigate the possibility that the Mei2p dot is attached to one of the
three chromosomes, we used a combination of two haploid-meiosis systems in
fission yeast. The first system was a strain that carried the mat1-M
gene in addition to its endogenous mat1-P gene, which could perform
haploid meiosis under nutrient starvation
(Kelly et al., 1988).
In this system, meiosis is activated through mating pheromone signaling, and
the chromosome segregation in meiosis I is reductional
(Yamamoto and Hiraoka, 2003).
In other words, the two sister chromatids generated by replication do not
separate in the first division but move together into one daughter nucleus
(Figure 2A). In the second
system, haploid meiosis was induced by inactivation of thermolabile Pat1p
kinase (Iino and Yamamoto,
1985; Nurse,
1985). The first division in pat1-driven haploid meiosis,
which proceeds in the absence of mating pheromone signaling, is mostly
equational, i.e., the sister chromatids tend to segregate into two daughter
nuclei at meiosis I (Yamamoto and Hiraoka,
2003) (Figure
2A).
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In natural meiosis, the Mei2p dot persists until telophase of meiosis I and
segregates into two daughter nuclei
(Yamashita et al.,
1998). Thus, we expected that, if the Mei2p dot was linked to a
chromosome, the dot would be seen in only one of the two split daughter nuclei
in the first haploid meiosis system, whereas in both of them in the second
system. The experimental results indicated that the expectation was correct.
In the first system, the Mei2p dot was observed in only one daughter nucleus
at telophase of meiosis I (Figure
2B). In contrast, we could observe the dot in each daughter
nucleus in the second system (Figure
2C). Thus, the Mei2p dot was suspected to be associated with one
of the three chromosomes.
Association of the Mei2p Dot with Chromosome II
We used the first haploid-meiosis system further to identify the chromosome
with which the Mei2p dot cosegregated. We marked each of the three chromosomes
by using the lac repressor/operator system
(Nabeshima et al.,
1998). The lac operator sequence LacO, which could be
visualized by the binding of the GFP-conjugated lac repressor protein
LacI, was integrated into specific sites on chromosomes. Two sites were chosen
for each chromosome: chromosome I, lys1 and "site A"
(between SPAC521.01 and SPAC521.02); chromosome II, cut3 and
"site B" (between SPBC31F10.05 and SPBC31F10.06); and chromosome
III, ade6 and "site C" (between SPCC663.02 and
SPCC663.03). These sites are schematically shown in
Figure 3A. A haploid strain
carrying both the mat1-P and mat1-M mating-type genes, which
was marked with LacO at one of these sites and was expressing CFP-conjugated
Mei2p, was subjected to nutrient starvation to induce meiosis. Because the
first division was reductional in this system, the Mei2p dot segregated into
one nucleus at telophase of meiosis I, and so did each chromosome marker. We
then calculated the frequency of cosegregation of the Mei2p dot with each
chromosome marker. As summarized in Table
3, the LacO marker integrated in chromosome II frequently
cosegregated with the Mei2p dot, whereas that in either chromosome I or
chromosome III showed no positive correlation. These results suggested that
the Mei2p dot was coupled with chromosome II. Also, the data in
Table 3 seem to suggest that
chromosomes I and II might preferentially segregate from each other in this
haploid meiosis system, whereas chromosomes II and III behaved randomly with
respect to each other.
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Localization of the Mei2p Dot at the sme2 Locus on Chromosome II
To pinpoint the location of the Mei2p dot, we marked a number of sites on
chromosome II by integrating the LacO sequence, as indicated in
Figure 3A, in homothallic
h90 strains carrying
mei2+-CFP. Meiosis after self-conjugation was
induced in each strain, and the distance between the Mei2p dot and the
respective marked site in a horse-tail nucleus was measured. In an initial
attempt, we examined four loci, namely, cut3, his2, ade1, and
ade8. Each of these loci (Figure
4, green dot) occupied a position in the horse-tail nucleus that
was colinear with its map distance from the nearest telomere. The Mei2p dot
(Figure 4, red dot) seemed to
localize very close to the ade8 marker, which is 300 kb away
from the long-arm telomere, and moderately close to the cut3 marker,
which is 1 Mb away from the short-arm telomere
(Figure 3A).
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Based on these observations, we examined two additional loci, namely, site
D between SPBC428.16 and SPBC428.17, and site E between SPBC 56F2.05 and
SPBC56F2.06 (Figure 3A). These
two loci are 400 kb away from the short-arm and long-arm telomeres,
respectively. The average distance observed between the Mei2p dot and each of
these sites was <1 µm in either case
(Figure 4), but the SD of the
distance was significantly smaller between the Mei2p dot and site D than
between the Mei2p dot and site E (our unpublished data). We suspected from
these results that the dot might be associated with the short arm. This was
subsequently confirmed by using the taz1 mutant, in which the
prophase chromosomal arrangement is distorted
(Cooper et al., 1998).
The distance between the Mei2p dot and either site D or site E was measured in
this mutant at meiotic prophase (Figure
4), and the average distance ± SD was calculated. Site D
was always close to the Mei2p dot (0.58 ± 0.23 µm, n = 22), whereas
the distance of site E from the dot was more variable (1.2 ± 0.75
µm, n = 25). These observations indicated that the location of the Mei2p
dot was near to site D on the short arm of chromosome II. It is reported that
pairing of homologous chromosomes is affected in the taz1 mutant
(Cooper et al., 1998).
We observed two Mei2p dots sitting near the split LacO/LacI dots at site D in
taz1 mutant cells, although not frequently, reinforcing that the
Mei2p dot was linked to site D (our unpublished data). Given these results, an
idea occurred to us that the Mei2p dot might be associated with sme2,
the gene for meiRNA, which is located 250 kb away from the end of the
short arm of chromosome II. Therefore, we integrated the LacO sequence at site
F between SPBC1271.13 and SPBC1271.14, which is only 2kb away from the
sme2 gene (Figure 3B).
The Mei2p dot and the LacO/LacI dot overlapped closely in this strain
(Figure 4).
We then set out to examine strains in which the telomeric regions were
translocated reciprocally between chromosomes II and III. Strain construction
was carried out essentially as described previously
(Virgin et al.,
1995). Briefly, a DNA fragment containing the ade6-469
allele and the ura4+ marker was integrated at either site
G (between SPBC106.02 and SPBC 106.03) or site H (between SPBC1271.14 and SPBC
1271.15, 3 kb apart from sme2) on chromosome II
(Figure 3B). Site G was on the
centromere-proximal side of sme2, whereas site H was on the
telomere-proximal side. An ade6-deletion (ade6-D19) strain
with the ade6-469 allele integrated at either site G or site H was
crossed with an ade6-M26 strain. Recombination between the
ade6-469 allele on chromosome II and the ade6-M26 allele on
chromosome III resulted in the generation of an ade6+
strain with chromosome arms translocated
(Figure 5). When arms were
exchanged at site G (Figure
5A), the resultant strain exhibited the Mei2p dot at nearly the
same position as the wild-type cells
(Figure 6A). In contrast, when
chromosome translocation was induced at site H and hence the sme2
locus was expected to be 1.8 Mb away from the telomere
(Figure 5B), the position of
the Mei2p dot shifted toward the rear of the nucleus and became much distant
from the leading edge (Figure
6A). These results indicate that the Mei2p dot resides between
site G and site H, where sme2 is located.
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Finally, we removed a 3-kb-long DNA segment, which carried the entire sme2 but no other probable gene, from the original sme2 locus on chromosome II, and inserted it into the lys1 locus near the centromere of chromosome I. In the resulting strain, the Mei2p dot was seen to be far away from the leading edge and overlapped with the LacO marker integrated at the lys1 locus (Figure 6B). Together, we conclude that the sme2 gene directs assembly of Mei2p into a dot structure around it.
Transcription of the sme2 Gene Is Essential for the Dot Assembly
around It
The question we addressed next was whether the DNA sequence of
sme2 directs the position of the dot or its transcripts do so. To
answer this question, we newly constructed a mutant allele of sme2
(sme2-m), which carried a number of substitutions in the TATA
sequence (Figure 7A) and was
much reduced in the transcription activity
(Figure 7C). We inserted this
mutant allele into the lys1 locus on chromosome I. The resultant
strain (JW941), which carried the authentic sme2+ allele
at its original locus, was compared with a control strain (JW937), which was
isogenic to JW941 except that it had the sme2+ allele
integrated at the lys1 locus instead of sme2-m. When the
Mei2p dot was visualized in these two strains, the former showed only one
nuclear dot at the original sme2 locus
(Figure 7B, left). In contrast,
the latter strain harbored two nuclear dots, which corresponded to the
original sme2 locus and the lys1 locus
(Figure 7B, right). These
observations suggest that the sme2 allele must be transcriptionally
active to generate the Mei2p dot around it and that meiRNA may play a major
role in positioning of the dot. It seems that the DNA sequence of the gene is
not a key determinant of the dot position, although we cannot deny the
possibility that the DNA sequence may also participate in the positioning.
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The Dot Is Unlikely to Be a Simple Reflection of sme2
Transcription
The above-mentioned results suggested that the dot was likely to be
assembled on nascent meiRNA, which might not have detached from the
sme2 gene. At the same time, however, we suspected that the dot was
unlikely to be a simple visualization of sme2 transcription by
binding of Mei2p, because our previous observations had indicated that nascent
meiRNA transcripts alone would not be detectable as a dot-like structure. The
rationale was as follows. In situ hybridization analysis has shown that meiRNA
can be observed as a dot overlapping the Mei2p dot in meiotic prophase nuclei,
but no meiRNA dot is detectable in a mutant strain that either lacks Mei2p
(mei2) or carries Mei2p defective in binding to RNA
(mei2–644A) (Yamashita
et al., 1998). It has been also shown that transcription
of meiRNA is inducible by nutrient starvation irrespective of the presence or
absence of Mei2p (Watanabe and Yamamoto, unpublished data; see below). These
observations together lead to a conclusion that nascent meiRNA transcripts are
not detectable as a dot unless they are bound by Mei2p.
To confirm the above-mentioned inference, we reexamined the previous
observations more quantitatively. Northern blotting indicated that the amount
of meiRNA in mei2 cells subjected to starvation was equal to,
or slightly more abundant than that in wild-type cells (our unpublished data).
This suggested that transcription of sme2 must be ongoing in
mei2 cells under meiotic conditions. We then examined the
mei2 strain carefully for generation of a dot-like structure
by meiRNA. In situ hybridization performed as described previously
(Yamashita et al.,
1998) revealed no nuclear meiRNA dot in any of 10
mei2 cells that evidently bore meiRNA in the cytoplasm. In
contrast, similar analysis visualized a nuclear meiRNA dot in 17 of 24
mei2+ cells. Therefore, we conclude that the Mei2p dot is
unlikely to reflect simple association of Mei2p to nascent meiRNA being
transcribed from the sme2 gene, but that it is probably a more
sophisticated and organized structure.
Effects of the Translocation of the sme2 Gene on Meiosis
We finally investigated whether translocation of the sme2 gene
might affect the proficiency of the host cell to perform meiosis. JW938, in
which sme2 was moved to the lys1 locus, formed spores as
efficiently as the wild type. JW936, in which the 1.8-Mb telomeric region of
chromosome III was exchanged with the telomeric region of chromosome II distal
to sme2, could also sporulate as efficiently as the wild type. In the
case of JW935, in which sme2 was moved to chromosome III together
with the telomeric region of chromosome II distal to it, we noticed a
sporulation deficiency. However, this deficiency was observed also in the
strain JW968, which carried an insertion of ade6-469 at site G on
untranslocated chromosome II. Thus, the insertion at site G apparently
affected function of a certain gene required for sporulation.
4,6-Diamidino-2-phenylindole staining of JW935 cells shifted to the
sporulation medium revealed that many of them carried four nuclei, indicating
that the meiotic divisions could proceed normally. Together, we conclude that
relocation of the sme2 gene from its original chromosomal locus does
not significantly hamper the progression of meiosis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), because chromosomes are thought to align linearly
in the prophase nucleus, with their telomeres held at the SPB (Chikashige
et al., 1994,
1997;
Niwa et al.,
2000). Our results have indicated unambiguously where the Mei2p dot is located. However, the question what function this dot performs is still left open. Roughly speaking, we can imagine two types of possibilities about its function. One possibility is that the dot represents an assembly farm for Mei2p and meiRNA and does not have an intrinsic function to stimulate meiosis. The emergence of the dot will be an indication of active transcription of meiRNA and vigorous assembly of it with Mei2p in this case. Mei2p coupled with meiRNA may then fulfill a function critical for meiosis I, either within the nucleus or after migrating to the cytoplasm. Even if this is the case, we like to emphasize that the dot is unlikely to be a simple reflection of the attachment of Mei2p to meiRNA undergoing transcription, as substantiated in the RESULTS. The dot may be regarded as a specialized structure for the assembly of Mei2p and meiRNA, just like the nucleolus for the assembly of ribosomal proteins and RNAs.
The other possibility is that the dot structure itself performs some
function essential for meiosis I. So far, however, we have no concrete
evidence to support this idea. Rather, the results we have obtained seem to
deteriorate this possibility. Only one of the three chromosomes bears the
Mei2p dot, and translocation of the dot to another chromosome does not
significantly affect the ability of the cell to undergo meiosis. These suggest
that the dot is unlikely to be involved in the meiosis-specific modification
of the chromosome structure. Nevertheless, we cannot abandon this possibility
completely. We have previously observed that Mei2p-NLS, which can suppress
sme2, forms a nuclear dot in the absence of meiRNA, probably
attached by another RNA species (Yamashita
et al., 1998). The rad24 mutant, missing
the major 14-3-3 isoform (Ford et
al., 1994), allows formation of a Mei2p dot under the
conditions where sme2 is unlikely to be actively transcribed
(Sato et al., 2002).
These observations suggest that Mei2p, bound with RNA, has an intrinsic
tendency to coagulate and form a complex, which may be vital for its
function.
Our preliminary analysis of the Mei2p dot by electron microscopy has shown
that the dot is likely to be electron dense (Yoneda, Kamasawa, Osumi,
Yamashita, and Yamamoto, unpublished data), suggesting that it may be
identical to the "black bodies," an entity found electron
microscopically in the meiotic prophase nucleoplasm
(Bahler et al., 1993).
Together, whatever its function is, the Mei2p dot seems to possess a
specialized structure relevant to the progression of meiosis, significance of
which deserves further investigation.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Corresponding author. E-mail address:
myamamot{at}ims.u-tokyo.ac.jp.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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