Download Cell Cycle in the Fucus Zygote Parallels a Somatic Cell

The Plant Cell, Vol. 13, 585–598, March 2001, www.plantcell.org © 2001 American Society of Plant Physiologists
Cell Cycle in the Fucus Zygote Parallels a Somatic Cell Cycle
but Displays a Unique Translational Regulation of
Cyclin-Dependent Kinases
Florence Corellou,a Colin Brownlee,b Lenaick Detivaud,c Bernard Kloareg,a and François-Yves Bougeta,1
a Unité
Mixte de Recherche 1931 (Centre National de la Recherche Scientifique and Laboratoires Goëmar), Station
Biologique, 29680 Roscoff, France
b Marine Biological Association, The Laboratory, Citadel Hill, Plymouth PL1 2PB, United Kingdom
c Cell Cycle Group, Station Biologique, 29680 Roscoff, France
In eukaryotic cells, the basic machinery of cell cycle control is highly conserved. In particular, many cellular events during cell cycle progression are controlled by cyclin-dependent kinases (CDKs). The cell cycle in animal early embryos,
however, differs substantially from that of somatic cells or yeasts. For example, cell cycle checkpoints that ensure that
the sequence of cell cycle events is correct have been described in somatic cells and yeasts but are largely absent in
embryonic cells. Furthermore, the regulation of CDKs is substantially different in the embryonic and somatic cells. In
this study, we address the nature of the first cell cycle in the brown alga Fucus, which is evolutionarily distant from the
model systems classically used for cell cycle studies in embryos. This cycle consists of well-defined G1, S, G2, and M
phases. The purine derivative olomoucine inhibited CDKs activity in vivo and in vitro and induced different cell cycle arrests, including at the G1/S transition, suggesting that, as in somatic cells, CDKs tightly control cell cycle progression.
The cell cycle of Fucus zygotes presented the other main features of a somatic cell cycle, such as a functional spindle assembly checkpoint that targets CDKs and the regulation of the early synthesis of two PSTAIRE CDKs, p32 and p34, and
the associated histone H1 kinase activity as well as the regulation of CDKs by tyrosine phosphorylation. Surprisingly, the
synthesis after fertilization of p32 and p34 was translationally regulated, a regulation not described previously for CDKs.
Finally, our results suggest that the activation of mitotic CDKs relies on an autocatalytic amplification mechanism.
INTRODUCTION
During animal early embryogenesis, the fertilized egg undergoes rapid cell cycles without growing. Interphases (S
phase) and mitoses (M phase) alternate in quick succession
without intervening G1 and G2 phases (King et al., 1994).
The oscillation between S and M phases is driven by successive activation and inactivation of a key protein complex
called the M phase–promoting factor (MPF) (Masui and
Markert, 1971; Kishimoto, 1988). MPF is composed of cdc2,
a cyclin-dependent kinase (CDK), and its positively regulating protein, cyclin B. Periodic synthesis and degradation of
cyclin B is the major mechanism of CDK regulation in embryos (Arion et al., 1988; Dunphy and Newport, 1988). In
contrast, additional mechanisms, such as phosphorylation,
regulate CDK activity in somatic cells. In both embryos and
somatic cells, mitotic CDKs operate at the G2/M boundary,
where their activation triggers, through the phosphorylation
1 To whom correspondence should be addressed. E-mail bouget@
sb-roscoff.fr; fax 33-2-98-29-23-24.
of their substrate proteins, several major cellular events, including nuclear envelope breakdown, chromosome condensation, spindle assembly, and cytokinesis (Peter et al., 1990;
Kishimoto, 1994).
In somatic cells and yeasts, however, the cell cycle appears to be more tightly controlled by CDKs than in embryos (Nurse, 1994; Chevalier and Blow, 1996). A restriction
point controls cell cycle progression in G1 in response to
extracellular and/or intracellular cues (Peter and Herskowitz,
1994; Planas-Silva and Weinberg, 1997; Neufeld and Edgar,
1998), and CDK activity promotes the assembly of replication complexes in S phase. In contrast, in the sea urchin zygote, DNA replication can occur while CDKs are maintained
inactive (Moreau et al., 1998), suggesting that CDKs may
not control all aspects of cell cycle progression in animal
embryos.
The differences between cell cycle control in somatic and
embryonic cells are reflected by different mechanisms of
regulation of CDKs. Whereas the reentry of quiescent somatic cells into the cell cycle in G1 is mediated by the successive expression of G1 and mitotic CDKs (Elledge et al.,
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1992), constant levels of CDKs are detected in animal early
embryos. Finally, cell cycle checkpoints of somatic cells differ greatly from those of embryonic cells. Somatic cells display stringent surveillance mechanisms that check the
success of various cell cycle events such as DNA replication
and the integrity of the spindle, preventing mitosis until accurate repairs have been performed (Rudner and Murray,
1996; Paulovich et al., 1997). CDKs are a major target of
these checkpoints, and their regulation after checkpoint activation is usually correlated with changes in their phosphorylation status as well as with associations with regulatory
molecules such as CDK inhibitors (Lew and Kornbluth, 1996;
Rudner and Murray, 1996; Hardwick, 1998). These checkpoints are usually partial in or absent from early embryonic
cells (reviewed in Hartwell and Weinert, 1989; Heichman
and Roberts, 1994; Nurse, 1994; Edgar, 1995).
Although much less is known about the in vivo regulation
of the cell cycle in plants, evidence strongly suggests that
the cell cycle machinery is well conserved between plants
and animals (Mironov et al., 1999). In particular, functional
homologs of the yeast cdc2 gene, which contains the fully
conserved PSTAIRE hallmark, have been identified in several plant species, and these are required for progression
through mitosis (reviewed in Dudits et al., 1998). The lack of
cell cycle mutants in plants has been partially overcome by
using a family of specific inhibitors of CDKs, such as olomoucine, that specifically and reversibly inhibit cdk2 and
cdc2 in many cell types (Vesely et al., 1994; Alessi et al.,
1998). Olomoucine and other purine derivatives were found
to arrest cell cycle progression at both the G1/S and G2/M
transitions in Petunia and Arabidopsis somatic cells (Glab et
al., 1994; Planchais et al., 1997). Finally, tyrosine phosphorylation has been proposed to regulate CDK activity in response
to plant hormones in somatic cells (Bell et al., 1993; Zhang
et al., 1996; McKibbin et al., 1998).
Microspectrophotometric analysis of the DNA content in
sperm nuclei has suggested specificities of the cell cycle in
higher plant gametes, such as the initiation of DNA replication before gametic fusion (Friedman, 1999). Furthermore, in
in vitro–fertilized zygotes from maize, the transcripts of three
cyclin genes are detected only after fertilization, suggesting
that there may be different patterns of cell cycle activity in
higher plant embryos (Sauter et al., 1998). However, higher
plant embryos are embedded deeply in maternal tissue and
are largely inaccessible to experimentation, and, to our
knowledge, in vitro fertilization systems do no provide
enough material for biochemical approaches. Consequently,
little is known about the molecular mechanisms of cell cycle
regulation in these embryos.
In fucoid algae, including the genera Fucus and Pelvetia,
fertilization is external, and large populations of synchronously developing zygotes can be obtained (Brownlee and
Bouget, 1998). This characteristic, coupled with the length
of the first cell cycle (24 hr), makes fucoid algae powerful
experimental systems for cellular and biochemical investigations of cell cycle regulation. In a previous study, we
demonstrated the presence in fucoid zygotes of a DNA replication checkpoint, a common feature of somatic cells but
also of a few animal embryos (Corellou et al., 2000a). To determine more precisely the nature of the first cell cycle in Fucus (embryonic versus somatic), we investigated the
regulation of CDKs and their involvement in controlling the
first embryonic cell cycle of Fucus zygotes during normal
development and after various cell cycle arrests. Our results
indicate that in Fucus zygotes, many events of the cell cycle
and cell cycle arrest are tightly controlled by CDKs, which
themselves appear to be regulated, as in somatic cells, both
at the level of synthesis and by reversible phosphorylation.
The presence of a functional spindle assembly checkpoint
further confirmed that the cell cycle in Fucus is somatic in
nature. Interestingly, CDKs appear to be synthesized from
maternal mRNAs, suggesting a unique regulation of CDKs
at the translational level. Our results also suggest the presence of a mechanism of autocatalytic amplification of
mitotic cyclin/CDK activity similar to those described previously in animals and yeasts. We propose a model of the
possible roles and regulations of CDKs throughout the first
cell cycle of fucoid algae.
RESULTS
We studied three different aspects of the Fucus first cell cycle to determine more precisely its nature (embryonic versus
somatic): (1) the requirement for CDKs for cell cycle progression, particularly at the G1/S transition; (2) the regulation of CDKs; and (3) the presence of a spindle assembly
checkpoint.
Olomoucine Induces Different Cell Cycle Arrests in
Fucus Zygotes
Previous results have shown that in Fucus zygotes, the purine derivative olomoucine specifically inhibits mitotic CDKs
in vitro and blocks cell cycle progression from G2 to mitosis
(Corellou et al., 2000a). Zygotes treated with olomoucine
from 6 hr after fertilization (AF) display a decondensed nucleus and a spindle with an aligned centrosomal axis that is
typical of zygotes in premitosis (Corellou et al., 2000a). Taking advantage of this specific inhibitor of CDKs, we further
investigated the involvement of CDKs in the control of the
Fucus first cell cycle. Zygotes were treated with various
concentrations of olomoucine from 2 hr until 36 hr AF. Concentrations of ⵑ35 ␮M were required to block cytokinesis in
most embryos (data not shown). At these concentrations,
zygotes exhibited condensed chromosomes that were often
scattered in the cytoplasm (Figure 1B). Between 50 and 100
␮M olomoucine was required to arrest and maintain all zygotes with a decondensed nucleus. A concentration of 100
␮M olomoucine therefore was used to block chromatin con-
Cell Cycle Control in Fucus Zygotes
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Figure 1. Effect of Olomoucine on Cell Cycle Progression in Zygotes.
Olomoucine (100 ␮M) was added at various times until 36 hr AF. Cells were fixed and subsequently stained with mithramycin A ([A] to [D]).
(A) to (D) Zygotes treated with 100 ␮M olomoucine from 12 (A), 16 (B), 20 (C), and 24 (D) hr until 36 hr AF. Bars ⫽ 25 ␮m.
(E) Zygotes treated with 100 ␮M olomoucine at various times AF (x axis) displayed either one or two sets of dispersed chromosomes (white dots
and black dots, respectively) or one or two decondensed nuclei (black bars and white bars, respectively). The data shown are representative of
the results of two independent experiments.
(F) RNA gel blot analysis with a Fucus histone H3 DNA probe (left). Equal amounts of RNA (10 ␮g), extracted at the times indicated, were loaded
in each lane, and RNA integrity was checked by staining with ethidium bromide (data not shown). Corresponding quantifications (right) were performed with a phosphorimager in control zygotes (open circles) as well as in zygotes treated from 3 hr AF with either 20 ␮M aphidicolin (closed
squares) or 100 ␮M olomoucine (closed triangles) until the times indicated on the x axis. The data shown are representative of the results of two
independent experiments. AF, after fertilization.
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densation (data not shown) and was added at various times
AF. When added in early G2, that is, when in most cells division was no longer sensitive to the DNA replication inhibitor
aphidicolin (Corellou et al., 2000a), 100 ␮M olomoucine
blocked nuclei before mitosis, that is, at the G2/M transition
(Figures 1A and 1E). In contrast, olomoucine treatments
started just before mitosis, at 16 hr AF, allowed ⵑ40% of
the chromosomes to condense (Figures 1B and 1E), like the
arrest induced by continuous treatment with 35 ␮M olomoucine. Treatment of zygotes with olomoucine at 18 hr AF had
little effect on nuclear events of the first cell cycle (Figures
1C to 1E), even though cytokinesis was still inhibited (data
not shown). Olomoucine (100 ␮M) induced the same succession of arrests during the second cell cycle (Figures 1C
to 1E) but more rapidly, consistent with the faster pace of
this second cell cycle. These findings indicate that the CDK
inhibitor olomoucine induces various cell cycle arrests, including one at the G2/M transition and one in mitosis, and
that the targets of olomoucine display different degrees of in
vivo sensitivity to this drug.
It proved impossible to investigate directly the effect of olomoucine on the G1/S transition by quantifying DNA levels
in zygotes stained with mithramycin A or with any other DNA
dye (Corellou et al., 2000a). In the one-celled zygote the nucleus is decondensed and occupies a central position, and
the cell is large and filled with organelles with high autofluorescence, such as plastids and polyphenol-containing physodes. These features make it difficult to directly measure the
nuclear DNA levels in zygotes; therefore, we used an indirect method to monitor the G1/S progression and the effect
of cell cycle inhibitors. In most eukaryotic cells, transcription
of certain histone genes, including histone H3 , begins at
Figure 2. Regulation of the Expression and Activity of CDKs during Early Development.
Total proteins were extracted from Fucus zygotes at the times indicated, and CDKs were purified by affinity on p9CKShs1–Sepharose beads. The
results shown are representative of the results of three independent experiments. Purified proteins were assayed for their ability to phosphorylate histone H1 in vitro ([A] and [C]). Eluted proteins were immunoblotted with an anti-PSTAIRE antibody ([B] and [D]).
(A) Histone H1 kinase activity during normal development.
(B) Synthesis of PSTAIRE CDKs during normal development.
(C) Histone H1 kinase activity in extracts from zygotes treated from 1 hr AF with either 16 ␮M actinomycin D (gray bars) or 0.2 ␮M cycloheximide
(black bars), compared with control cells (white bars).
(D) Expression of PSTAIRE CDKs in extracts from zygotes treated from 1 hr AF with either 16 ␮M actinomycin D or 0.2 ␮M cycloheximide.
Cell Cycle Control in Fucus Zygotes
the onset of S phase (Kapros et al., 1992; Chaubet and
Gigot, 1998). We monitored histone H3 transcription as an S
phase–specific marker using RNA gel blot analysis with a
fragment of the coding region from a Fucus histone H3
gene. In Fucus zygotes, histone H3 transcripts were clearly
detected at 6 hr AF, and they accumulated progressively
until 12 hr AF (Figure 1F). This is unlikely to be due to a general increase of transcription, because the transcript of actin, a constitutively expressed gene, displays constant levels
of transcript throughout the first cell cycle (Bouget et al.,
1995). Although the transcription of histone H3 still occurred
in zygotes in which DNA replication was blocked by aphidicolin (Figure 1F), no histone H3 transcripts were detected in
zygotes incubated with 100 ␮M olomoucine from 3 hr AF.
These results suggest that in Fucus zygotes, S phase begins at 4 to 6 hr AF, and 100 ␮M olomoucine induced a cell
cycle arrest before the arrest induced in early S phase by
aphidicolin, that is, in G1 or at the G1/S transition.
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vision was fully inhibited for at least 26 hr, incubations with
actinomycin D from 1 hr AF did not affect the synthesis of
PSTAIRE CDKs (Figure 2D), suggesting that the synthesis of
CDKs is translationally regulated. In these conditions, however, histone H1 kinase activities remained at levels lower
than those of 14-hr-old controls (Figure 2C), suggesting that
the synthesis of mitotic CDK-activating proteins is transcriptionally regulated.
When added at 2 hr AF, the microtubule depolymerizing
agent nocodazole prevented the fusion of pronuclei but had
no effect on early morphogenesis (i.e., germination; Figure
3A). The male pronucleus remained condensed and always
entered mitosis after the female pronucleus, as described
previously in other fucoid algae (Brawley and Quatrano,
1979; Swope and Kropf, 1993; Motomura, 1995). Similarly,
when nocodazole was added after pronuclei fusion, zygotes
were arrested in mitosis with clustered, heavily condensed
Histone H1 Kinase Activity and the Synthesis of
PSTAIRE CDKs Increase Dramatically AF and Are
Regulated at the Translational Level, but They Do Not
Depend on Karyogamy
CDKs were purified using their affinity for the human suc1
homolog p9CKShs1, and their histone H1 kinase activity was
measured in vitro. The levels of two PSTAIRE CDKs bound
to p9CKShs1, p32 and p34, were monitored using a monoclonal anti-PSTAIRE antibody (Corellou et al., 2000a). Extracts from unfertilized eggs exhibited very low levels of
histone H1 kinase activity (Figure 2A), and both p32 and p34
were not detectable or only barely detectable before fertilization (Figure 2B). Histone H1 kinase activity increased
ⵑ10-fold between 0 and 2 hr AF (Figure 2A), which correlated with a significant increase in the synthesis of both p34
and p32 (Figure 2B). This initial increase in histone H1 kinase activity was followed by a steady and continuous increase (Figure 2A; see also controls in Figures 2C and 6A),
and the synthesis of PSTAIRE CDKs increased in a similar
manner (Figure 2B; see also control in Figure 2D). The specific activity of histone H1 kinase relative to total protein in
crude zygote extracts gave a similar profile (data not
shown), suggesting that these postfertilization changes in
histone H1 kinase activity did not result from a general increase in protein synthesis.
The requirements for the synthesis of PSTAIRE CDKs and
for the associated histone H1 kinase activity were investigated using actinomycin D and cycloheximide, two inhibitors of transcription and translation, respectively, in Fucus
zygotes (Quatrano, 1968). When added before 12 hr AF, actinomycin D (16 ␮M) and cycloheximide (0.2 ␮M) inhibited
cell division (data not shown). When cells were treated with
cycloheximide from 1 hr AF, the levels of both histone H1 kinase activity and PSTAIRE CDKs remained low for at least
26 hr (Figures 2C and 2D). In contrast, and although cell di-
Figure 3. Effect of the Inhibition of Pronuclei Fusion by Nocodazole
on the Activity of CDKs.
(A) A zygote treated with 0.33 ␮M nocodazole from 2 to 36 hr AF
and then stained with mithramycin A.
(B) A zygote treated with 0.33 ␮M nocodazole from 12 to 36 hr AF
and then stained with mithramycin A.
(C) Histone H1 kinase activity in extracts from cells treated with 0.33
␮M nocodazole from either 2 hr AF (closed circles) or 12 hr AF
(closed triangles, arrow) until the times indicated on the x axis. Histone H1 kinase activity in extracts from control zygotes is represented by open squares. The data shown are representative of the
results of two independent experiments.
Bar in (A) ⫽ 20 ␮m for (A) and (B).
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chromosomes (Figure 3B). The developmental course of histone H1 kinase activity was almost identical in zygotes
treated with nocodazole before or after pronuclei fusion,
and the mitotic activity was much higher than in control zygotes (Figure 3C). These results suggest that the synthesis
of CDKs and the activation of their kinase activity is initially
triggered by fertilization independently of karyogamy.
Olomoucine Inhibits Both in Vivo and in Vitro Histone H1
Kinase Activity and Induces an Accumulation of
Tyrosine-Phosphorylated CDKs
The role of tyrosine phosphorylation in CDK regulation was
investigated after various cell cycle arrests. Histone H1 kinase activities were monitored after treatment of 3-hr-old
zygotes with either 100 or 35 ␮M olomoucine for 36 hr in zygotes that were arrested before S phase and in mitosis, respectively. In control zygotes, histone H1 kinase activity
doubled from 4 to 14 hr AF, and the maximal value of the
mitotic peak of activity corresponded to an eightfold increase compared with the activity detected at 4 hr AF. In
contrast, no peak of activity was observed when zygotes
were treated with 100 ␮M olomoucine, and the level of H1
kinase remained similar to that of 6- to 8-hr-old control zygotes even at 36 hr AF (Figure 4A). The histone H1 kinase
activity from zygotes incubated with 35 ␮M olomoucine increased rapidly, reaching a level similar to that of control zygotes after 14 to 16 hr. This kinase activity remained stable
until 24 hr AF, increased again between 24 and 36 hr AF (a
four- to 20-fold increase depending on the experiment), and
finally remained stable for at least another 24 hr (Figure 4A).
Staining with mithramycin A revealed that, after treatment
Figure 4. Effect of Olomoucine on Histone H1 Kinase Activity and
Tyrosine Phosphorylation of CDKs.
(A) Histone H1 kinase activities in extracts from control zygotes
(open squares) and zygotes treated from 3 hr AF with either 100 ␮M
olomoucine (closed circles) or 35 ␮M olomoucine (open circles) until
the times indicated on the x axis. The data shown are representative
of the results of three independent experiments.
(B) Dose-dependent in vitro inhibition by olomoucine of histone H1
kinase activity in extracts from cells treated from 3 to 36 hr AF with
either 35 ␮M olomoucine (black bars) or 100 ␮M olomoucine (white
bars). For each treatment, the kinase activity is reported as a percentage of control activity (no olomoucine). The data shown are representative of the results of two independent experiments.
(C) Protein gel blot analysis of the expression and phosphorylation
of PSTAIRE CDKs after treatment with olomoucine. Immunoblotting
was performed with anti-PSTAIRE (PSTAIRE) or anti-phosphotyrosine (PY) antibodies. Top two panels, zygotes were treated from 3
hr AF with either 100 or 35 ␮M olomoucine until the times indicated
(data shown are from the same experiment represented in [A]). Bottom panel, zygotes were treated from 4 or 10 hr AF with 100 ␮M olomoucine or with 0.33 ␮M nocodazole (Noco) until 36 hr AF. The data
shown are representative of the results of three independent experiments.
Cell Cycle Control in Fucus Zygotes
with 35 ␮M olomoucine, the nuclei were decondensed at 24
hr AF, whereas condensed chromosomes were observed at
36 hr AF (data not shown).
Because olomoucine is a reversible inhibitor of CDKs, it
was possible to investigate the in vitro effect of olomoucine
on histone H1 kinase in cell extracts from zygotes treated
previously with olomoucine (Corellou et al., 2000a). In vitro
treatments with olomoucine inhibited histone H1 kinase in
extracts from 36-hr-old zygotes arrested before S phase
with 100 ␮M olomoucine or in mitosis with 35 ␮M olomoucine (Figure 4B). Even though the initial levels of kinase activities were dramatically different (Figure 4A), the patterns of
in vitro dose-dependent inhibition were similar in both types
of cell cycle arrests, suggesting that similar CDK/cyclins
were targeted in vivo by olomoucine. The higher kinase activities observed after mitotic arrest with lower concentrations of olomoucine (Figure 4A) therefore may arise from a
partial activation of mitotic CDKs (see Discussion).
Immunological detection of PSTAIRE CDKs in olomoucine-treated zygotes showed that until 12 to 16 hr AF, levels of both p34 and p32 increased normally as in controls
(data not shown), suggesting that the synthesis of PSTAIRE
CDKs was not inhibited by olomoucine regardless of the
concentration of the drug (Figure 4C, which corresponds to
the kinase assay shown in Figure 4A). In contrast, the immunological detection of phosphotyrosine residues revealed
that tyrosine phosphorylation increased progressively on
both proteins from 8 to 36 hr AF. In control zygotes, tyrosine
phosphorylation of these two proteins has been shown to
be cell cycle regulated, being maximal in G2 phase and absent at the time of mitosis (Corellou et al., 2000a). Zygotes
treated with 100 ␮M olomoucine at 10 hr AF, that is, at the
S/G2 transition, were arrested with a decondensed nucleus
at the G2/M transition (Figure 1). In these cells, the expression patterns of both PSTAIRE and phosphotyrosine epitopes were similar to those observed in zygotes treated with
100 ␮M olomoucine from 3 hr AF, and the 32- and 34-kD
proteins remained phosphorylated on tyrosine until at least
36 hr AF (Figure 4C). In contrast, no phosphorylation was detectable in zygotes arrested in mitosis by nocodazole (Figure 4C, bottom).
591
from zygotes arrested in mitosis by 35 ␮M olomoucine than
in extracts from zygotes arrested in mitosis by nocodazole
(Figure 5). However, treatments with the human phosphatase glutathione S-transferase (GST)–cdc25A led to a
dramatic increase of histone H1 kinase activities from cells
arrested by olomoucine in their cell cycle progression but
not in cells arrested by nocodazole. The relative activation
was higher when the zygotes were arrested early in the cell
cycle by 100 ␮M olomoucine, that is, when the kinase activities were initially low. Although the factors of activation
were variable (from 5.0 to 15.5), the final levels of kinase activity after phosphatase treatments were broadly similar to
the mitotic level of histone H1 kinase observed in extracts
from cells arrested by nocodazole. Together, Figures 4 and
5 demonstrate that, as in animal somatic cells, tyrosine
phosphorylation is a major mechanism involved in CDK regulation in Fucus zygotes.
A Spindle Assembly Checkpoint Targets Mitotic CDKs in
Fucus Zygotes
Nocodazole was used to inhibit mitotic spindle formation.
Zygotes that were treated continuously with 0.33 ␮M nocodazole displayed heavily condensed chromosomes for at
least 36 hr and never proceeded through mitosis (Figure
6C), suggesting the presence of a spindle assembly checkpoint. In zygotes treated with 0.33 ␮M nocodazole from 12 hr
Treatments with cdc25A Phosphatase Restore Mitotic
Levels of Histone H1 Kinase in Extracts from Cells
Arrested by Olomoucine
Various arrests were induced during cell cycle progression
by olomoucine and nocodazole (Figure 5). The earlier the
cells were arrested by olomoucine during cell cycle progression, the lower their levels of histone H1 kinase activities
(Figure 5; see also Figure 4A). Histone H1 kinase activities
from cells arrested by 100 ␮M olomoucine added at 3 hr AF
were similar to those obtained from zygotes blocked in early
S phase by aphidicolin (Corellou et al., 2000a; data not
shown). Histone H1 kinase activities were lower in extracts
Figure 5. In Vitro Activation of Histone H1 Kinase by the Human
Phosphatase GST-cdc25A.
Protein extracts from zygotes treated with olomoucine or nocodazole from the times indicated until 36 hr AF were assayed for histone
H1 kinase activity after treatment with 62 units of GST-cdc25A for
20 min (black bars) or after incubation in the dephosphorylation
buffer (gray bars). The relative degrees of activation by GST-cdc25A
are indicated above the black bars. The data shown are representative of the results of two independent experiments.
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AF, histone H1 kinase activity increased progressively from
4 to 16 hr AF. This was followed, at the time of entry into mitosis at 16 to 18 hr AF, by a sharp increase in activity until
22 hr AF (Figure 6A), and high levels of kinase activity were
observed for at least another 12 hr (data not shown). This
was in contrast to the levels of histone H1 kinase activity
during a normal cell cycle, which decreased after the mitotic
peak at 18 hr AF (Figure 6A). The abrupt increase of histone
H1 kinase activity preceded chromosome condensation in
both controls (38 and 80% of cells in mitosis at 18 and 20
hr, respectively) and zygotes treated with nocodazole (22
and 68% of cells in mitosis at 18 and 20 hr, respectively), indicating that the peak or high levels of H1 kinase corresponded to kinase activities of mitotic CDKs. No significant
Figure 6. A Spindle Assembly Checkpoint Targets Mitotic CDKs.
(A) Histone H1 kinase activity in extracts from either control zygotes (open circles) or cells treated with 0.33 ␮M nocodazole from 4 hr AF (closed
circles) until the times indicated on the x axis. The proportion of cells in mitosis was determined by staining DNA with mithramycin A.
(B) Protein gel blot analysis of PSTAIRE CDKs in extracts from either control zygotes or zygotes treated with 0.33 ␮M nocodazole from 4 hr AF
until the times indicated.
(C) A zygote arrested in mitosis with 0.33 ␮M nocodazole from 6 to 36 hr AF, fixed, and stained with mithramycin A. Note the highly condensed
and clustered chromosomes (arrowhead).
(D) A zygote arrested in mitosis with 0.33 ␮M nocodazole from 6 to 36 hr AF (e.g., as in [C]), subsequently treated with both 100 ␮M olomoucine
and 0.33 ␮M nocodazole for 6 hr, and finally fixed and stained with mithramycin A. Note the decondensed nucleus at 42 hr AF (arrowhead).
(E) Dose-dependent in vitro inhibition by olomoucine of histone H1 kinase activity in extracts from cells treated with 0.33 ␮M nocodazole from 4
to 36 hr AF. Kinase activity is represented as a percentage of control activity (no olomoucine). The data shown are representative of the results
of three independent experiments.
Bar in (C) ⫽ 10 ␮m for (C) and (D).
Cell Cycle Control in Fucus Zygotes
differences in the levels of the two PSTAIRE CDKs, p32 and
p34, were observed between control zygotes and zygotes
arrested in mitosis by nocodazole (Corellou et al., 2000a),
suggesting that the mitotic kinase activity of PSTAIRE CDKs
is not regulated by the synthesis and/or degradation of
these proteins (Figure 6B).
Zygotes arrested in mitosis by 0.33 ␮M nocodazole (Figure 6C) were then treated with 100 ␮M olomoucine for 6 hr.
This allowed the chromatin to decondense in ⬎90% of the
cells (Figure 6D). Furthermore, olomoucine inhibited, in a
dose-dependent manner, the high histone H1 kinase activity
found in extracts from cells incubated with 0.33 ␮M nocodazole from 6 to 36 hr AF (Figure 6E), suggesting that
CDKs present in these extracts are a target of olomoucine in
vivo. Together, these results suggest strongly that in Fucus
zygotes, the spindle assembly checkpoint operates by
maintaining high levels of activity of mitotic CDKs. These
data also suggest indirectly that the inactivation of CDKs is
required for chromatin decondensation at the end of mitosis.
DISCUSSION
We have shown previously that the G2 phase in Fucus zygotes starts at 10 to 12 hr AF and ends at 16 to 18 hr AF,
whereas cytokinesis is completed by 22 to 24 hr AF (Corellou
et al., 2000a). Based on the increase of histone H3 transcripts, which are typically detected at the onset of S phase
(Kapros et al., 1992), the S phase begins between 4 and 6 hr
AF in Fucus zygotes, that is, after pronuclei fusion (3 to 4 hr AF),
an event that is required for full DNA replication (Motomura,
1995). Therefore, like the cell cycles of dividing somatic
cells, the first cell cycle in Fucus encompasses four welldefined phases: G1, S, G2, and M (Figure 7).
CDK Activity Is Required for S Phase Entry in
Fucus Zygotes
Although in mammalian somatic cells and yeasts the onset of
S phase is controlled by G1/S CDKs (reviewed in Heichman
and Roberts, 1994), in animal embryos the role of G1/S
CDKs in promoting DNA replication is unclear. In the sea urchin zygote, cdk2 activity is not required for DNA replication
(Moreau et al., 1998). Furthermore, treatments with olomoucine do not impede DNA replication (Moreau et al., 1998). In
contrast, immunodepletion of cdk2 from Xenopus egg extracts prevents DNA replication, and a peak of cyclin E/cdk2
activity correlates with the onset of S phase in frog early embryos (Fang and Newport, 1991). In Fucus zygotes, histone
H3 mRNA was detected even upon inhibition of DNA replication by aphidicolin, but the transcription of histone H3 was
fully inhibited by olomoucine, suggesting that this drug induces a cell cycle arrest before S phase. This cell cycle arrest
was directly demonstrated in multicellular embryos by quan-
593
tifying the relative DNA levels in nuclei stained with mithramycin
A (data not shown). Therefore, in contrast to the observations
in sea urchin zygote, CDK activity appears to be required to
trigger S phase entry in the Fucus zygote.
Synthesis of PSTAIRE CDKs and Early Increase in
Histone H1 Kinase Activity Are Triggered by Fertilization
but Do Not Rely on Karyogamy
As illustrated in mitogen-activated quiescent human cells,
which reenter the cell cycle by promoting the successive
transcription of cdk2 and cdc2 genes (Pagano et al., 1993),
the sequential expression of CDK genes plays an important
role in regulating CDK activity and ordering cell cycle progression in animal somatic cells. In rice, the phytohormone
gibberellin was reported to promote the expression of cdc2
and associated H1 kinase activity, and in both alfalfa and Arabidopsis, the expression of several cdc2-related genes is cell
cycle regulated and correlates with their specific activation
(Sauter et al., 1995; Magyar et al., 1997; Mironov et al., 1997).
In contrast, in animal early embryos, regulation of MPF activity relies primarily on the periodic synthesis of cyclin B. Cdc2
is already present in the oocyte, and many rounds of cell division can occur when transcription is inhibited (Gerhart et al.,
1984; Arion and Meijer, 1989). In Fucus eggs, the levels of
both CDK activity and PSTAIRE CDKs are very low, and the
postfertilization synthesis of PSTAIRE CDKs correlates with a
dramatic increase of CDK activities. Although the inhibition of
transcription had no effect on either the synthesis of PSTAIRE
CDKs or histone H1 kinase activity until early G2 phase, the
inhibition of translation completely prevented both events,
suggesting an early regulation of CDK activity at the translational level. To complete the first cell cycle, however, the Fucus zygote also requires transcription for ⵑ10 hr AF, or until
early G2 phase. Furthermore, the mitotic peak of histone H1
kinase is dependent on transcription, even though upon inhibition of transcription, the level of PSTAIRE CDKs is similar to
that of mitotic controls. Therefore, it is likely that the synthesis
of fully active G1/S CDK relies on maternal transcripts,
whereas the transcription of proteins such as cyclin B is required to activate mitotic CDKs. Such a regulation of CDK activities represents an intermediate situation between the cell
cycle control of animal early embryos and cell cycle reentry in
somatic cells.
Interestingly, karyogamy is dispensable for the synthesis
and activation of CDKs during the first cell cycle of Fucus
zygotes, suggesting that, as in the sea urchin egg, plasmogamy is sufficient to commit the egg to enter the first
cell cycle (Shatten et al., 1989). When pronuclei fusion is inhibited, DNA replication occurs in the egg pronucleus but
not in the male pronucleus, which remains condensed
throughout the first cell cycle of Fucus zygotes (Motomura,
1995). Furthermore, the female pronucleus always enters
mitosis before the male pronucleus (Motomura, 1995). We
propose that the transcription of cell cycle genes occurs
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The Plant Cell
Figure 7. The First Cell Cycle and the Possible Roles and Regulations of CDKs in Fucoid Algae.
This diagram is based on the results of this study and on those reported previously (Corellou et al., 2000a). Activating and inhibitory mechanisms
are shown in red and green, respectively. Active and inactive CDKs are shown in orange and yellow, respectively. The first cell cycle comprises
well-defined G1, S, G2, and M phases. (1) PSTAIRE CDKs are synthesized from maternal mRNAs after fertilization. (2) The CDK activity that is required for the G1/S transition is inhibited by olomoucine. The transcription of histone H3 in S phase is inhibited by olomoucine but not by aphidicolin. (3) Transcription, before 10 hr AF, of the genes encoding activating proteins is required for mitotic activity of CDKs. (4) CDKs are
maintained inactive in G2 by inhibitory phosphorylation (P) on tyrosine residues (Y) and are activated in mitosis by a cdc25-like phosphatase. (5)
Olomoucine, by inhibiting CDKs, prevents entry into and progression through mitosis. (6) A DNA replication checkpoint prevents mitosis, including chromatin condensation and spindle formation, through inactivation of mitotic CDKs by inhibitory phosphorylation. (7) The autocatalytic amplification of mitotic CDK activity (⫹), which may rely on the activation of a cdc25-like protein by CDKs, is inhibited by olomoucine (5). (8) A
spindle assembly checkpoint prevents progression through mitosis, including chromatin decondensation, by inhibiting the inactivation of CDKs
by an unknown mechanism (?). F, fertilization.
only in the decondensed female pronucleus, because the
transcription-dependent histone H1 kinase is still observed
when pronuclei fusion is inhibited. The male pronucleus,
which remains condensed at all times, would not be able to
either replicate DNA or transcribe the cell cycle genes required for entry into mitosis; thus, its entry into mitosis
would depend on the female pronucleus.
Tyrosine Phosphorylation Is a Major Mechanism
Involved in Regulating the Mitotic Activity of CDKs in
Fucus Zygotes
The regulation of cdc2 during early animal embryo cell cycles usually relies on the periodic synthesis and degradation
of cyclin B. In addition to this regulatory mechanism, in fission yeast and mammalian somatic cells the activation of
mitotic cdc2 operates by tyrosine dephosphorylation of
cdc2 by the phosphatase cdc25 (Lew and Kornbluth, 1996).
In various plant taxa, several cdc2 homologs contain a conserved tyrosine residue, most often at position 15 (Zhang et
al., 1996), and in tobacco cells, tyrosine phosphorylation
was shown to regulate cdc2-like kinase activity upon stimulation with cytokinin (Zhang et al., 1996). Tyrosine phosphorylation of the PSTAIRE CDKs p32 and p34 was shown to
be cell cycle regulated in Fucus zygotes, and dephosphorylation of p34 and p32 correlates with entry into mitosis
(Corellou et al., 2000a). Furthermore, the S/M DNA checkpoint was shown to operate by maintaining mitotic CDKs inactive by tyrosine phosphorylation (Corellou et al., 2000a),
Cell Cycle Control in Fucus Zygotes
whereas after activation of the spindle assembly checkpoint, highly active mitotic CDKs were not phosphorylated
on tyrosine residues. Our results indicate that the various
cell cycle arrests induced by olomoucine also trigger tyrosine phosphorylation of both p32 and p34 proteins. Olomoucine treatments did not impede the synthesis of
PSTAIRE CDKs, and the timing of tyrosine phosphorylation
during the first cell cycle was similar in zygotes arrested in
G1, at the G2/M transition, or in mitosis. Furthermore, treatment with cdc25A led to an activation of CDK activity, confirming that phosphorylation on tyrosine residues negatively
regulates the activity of CDKs.
In Fucus zygotes, all of the samples treated with olomoucine (i.e., arrested in G1/S, G2/M, or mitosis) displayed, after
dephosphorylation by cdc25A, kinase levels similar to the high
mitotic levels observed after nocodazole treatment. Therefore, the inhibition of G1/S CDKs by olomoucine does not appear to prevent the formation of potentially active mitotic
cyclin/CDK complexes, but it likely inhibits their activation by
preventing their dephosphorylation on tyrosine residues.
Such a downregulation of mitotic CDKs also occurs when
DNA replication is inhibited (Corellou et al., 2000a). How the
inhibition of G1/S CDKs leads to a downregulation of mitotic
CDKs remains unclear in Fucus zygotes. As in Xenopus egg
extracts, G1/S CDKs may directly and positively regulate mitotic CDKs through the reversible phosphorylation and activation of cdc25-like proteins (Guadano and Newport, 1996).
Alternately, the inhibition of G1/S CDKs, by preventing DNA
replication, may activate the DNA replication checkpoint,
which in turn would lead to an inactivation of CDKs by tyrosine phosphorylation (Corellou et al., 2000a).
Inhibition of mitosis by nocodazole yielded maximal CDK
activities associated with dephosphorylated p34 and p32,
whereas zygotes arrested in mitosis by low doses of olomoucine displayed higher CDK activities than did zygotes arrested at the G2/M transition by high doses of olomoucine.
Conversely, the activation by cdc25A was much higher in zygotes arrested by olomoucine at the G2/M transition than in
cells arrested by olomoucine in mitosis. Together, our data
suggest that a gradual dephosphorylation, or activation, occurs between G2 phase and mitosis, as reported in starfish
oocytes (Borgne and Meijer, 1999). This activation seems to
require CDK activity, because it is much lower after a cell cycle arrest at the G2/M transition than after an arrest in mitosis.
These results suggest a possible inhibition by olomoucine of
an autocatalytic amplification of mitotic cyclin/CDK activity
similar to those described previously in animals and yeasts
(reviewed in Lew and Kornbluth, 1996).
Fucus Zygotes Display a Functional Spindle
Assembly Checkpoint
Mitotic cyclin B/cdc2 is known to promote the nucleation of
the mitotic spindle (Verde et al., 1990; Kishimoto, 1994), and
inactivation of the mitotic complex cyclin B/cdc2 at meta-
595
phase is required for a cell to exit from mitosis and reenter the
next cell cycle (King et al., 1994). Inactivation of mitotic CDKs
is first achieved by the degradation of cyclin B (Hershko,
1997). In animal somatic cells and in Schizosaccharomyces
pombe, the lack of a functional spindle prevents the exit
from mitosis by inhibiting the degradation of key proteins
implicated in chromosome cohesion as well as that of cyclin
B, thus maintaining high levels of mitotic CDK activity
(Nishimoto et al., 1992). However, the early embryos of several animal species, such as Xenopus, lack a fully active
spindle assembly checkpoint and undergo abnormal mitosis
in the absence of a fully functional spindle (Clute and Masui,
1992; Murray, 1992; Sluder et al., 1994). Fucus zygotes
lacking a functional spindle after treatment with nocodazole
feature high levels of mitotic CDK. These zygotes did not divide or rereplicate DNA, indicating the presence of a fully
active spindle assembly checkpoint. The fact that the inactivation of CDK activity by olomoucine triggers a decondensation of chromatin in zygotes continuously treated with
nocodazole strongly suggests that inactivation of CDKs is
required for the exit from mitosis and that, as in animal somatic cells, the spindle assembly checkpoint operates at
least in part by hampering the inactivation of mitotic CDKs.
These findings are summarized in Figure 7, which describes the major steps of the Fucus first cell cycle together
with the possible roles and regulations of CDKs. Cell cycle
progression, including S phase entry, is tightly regulated by
CDKs. Two functional DNA replication and spindle assembly
checkpoints block cell cycle progression by altering CDK
activities. CDKs are regulated at both the transcriptional and
transductional levels and by phosphorylation. Together, our
results indicate that in Fucus zygotes, the zygotic cell cycle
resembles a somatic cell cycle more than the typical cell cycles of animal embryos in which controls are reduced.
METHODS
Culture and Inhibitors
Sexually mature receptacles of Fucus spiralis were collected at Le
Dossen (Brittany, France) and stored at 4⬚C for up to 14 days. Gametes
were released by standard osmotic shock procedures in filtered seawater for 1 hr (Corellou et al., 2000b). The time of fertilization (0 hr)
was taken to be 30 min after the first eggs were released. Zygotes
and embryos were grown at 14⬚C. Unfertilized eggs were obtained
by inducing receptacles to release in high-potassium artificial seawater, according to Brawley (1987). Aphidicolin (20 ␮M; Sigma) was
used to inhibit DNA replication and subsequent inhibition of cell division. Aphidicolin was added at various times after fertilization to determine experimentally the end of S phase, that is, the beginning of
G2 phase (Corellou et al., 2000a). Olomoucine was used to inhibit cyclin-dependent kinases (CDKs) in vivo and in vitro. The structural analog iso-olomoucine had no effect on cell division at concentrations as
high as 500 ␮M. Nocodazole (0.33 ␮M; Sigma) was used to prevent
spindle formation, and it arrested the cells in mitosis. Actinomycin D
596
The Plant Cell
(16 ␮M) and cycloheximide (0.2 ␮M) were used to prevent transcription and translation (Quatrano, 1968; Bouget et al., 1996). Aphidicolin, olomoucine, nocodazole, actinomycin D, and cycloheximide
were stored in DMSO at 10, 300, 1, 10, and 0.5 mg/mL, respectively,
and further diluted in artificial seawater before use. Control experiments were performed in filtered seawater containing the same final
concentrations of DMSO.
cence (ECL) or ECL⫹ detection. Protein gel blotting was performed
with either a monoclonal antiphosphotyrosine antibody (PY20; Santa
Cruz Biotechnology, Santa Cruz, CA) at a 1:20,000 dilution or a monoclonal anti-PSTAIRE antibody (Sigma) at a 1:3000 dilution. The antiphosphotyrosine antibody could not be detected using the classic
ECL detection system. Therefore, the blots were probed first with this
antibody, revealed in ECL⫹, and reprobed with the anti-PSTAIRE antibody, which required short times of exposure in ECL detection.
DNA Staining and Quantification
Zygotes and embryos were fixed for 12 hr in 0.2 M citric acid and
0.2% Triton X-100 and kept in 100% methanol for long-term storage.
Fixed cells were attached to poly-L-lysine–coated cover slips, processed and stained with 50 ␮g/mL mithramycin A, as described
previously (Corellou et al., 2000a), and then observed by epifluorescence microscopy (540- to 590-nm excitation filter, 585-nm band
pass, a 32-nm band pass emission filter, centered at 585 nm). As reported earlier (Corellou et al., 2000a), one-celled zygotes did not
yield reproducible results. DNA was therefore quantified by measuring mithramycin A fluorescence on two-celled and older embryos.
Mithramycin A fluorescence was measured either with a confocal microscope (model 1024; Bio-Rad, Hemel Hempstead, UK) or with a
CCD camera (Spot-RT Slider Eurocam model 2.3.0; Diagnostic Instruments, Inc., Sterling Heights, MI). The camera was standardized
with Fluoresbrite fluorescent beads (Polyscience Inc., Warrington,
PA), and the images were analyzed with the Image Tool software (a
freeware from UCSB computer, science supported software;
www.cs.ucsb.edu). For each sample, an average of 50 to 75 nuclei
were scanned. In the first experiment, the highest fluorescence value
obtained in nocodazole-treated embryos was given the arbitrary
value of 8.0 relative fluorescence units (RFU), and the average fluorescence value of nocodazole-treated cells was determined to be 5.7
RFU. In the following experiment, this value was used as a standard
to normalize the values of both experiments. The relative fluorescence of nuclei was represented using a scale of 0 to 8.0 RFU
divided into eight equal 1-unit classes. Histograms with the percentage of nuclei in each class were established. For each sample, the
average relative fluorescence were also determined. The average relative fluorescence of nuclei in nocodazole-treated embryos (5.7
RFU) was close to the average value of mitotic figures in metaphase
(5.3 RFU), corresponding to a 4C DNA content. The average relative
fluorescence corresponding to a 2C DNA content, which was determined in derivative nuclei in anaphase (2.7 RFU), was close to the
average fluorescence value of nuclei treated with aphidicolin from
mitosis (2.6 RFU).
Protein Extraction, Purification of CDKs, and Protein Gel
Blot Analysis
Protocols for protein extraction, CDK purification, and protein gel
blot analysis have been described in detail by Corellou et al. (2000a,
2000b). Briefly, embryos were harvested, frozen in liquid nitrogen,
and stored at ⫺80⬚C until extraction. Frozen samples were ground in
liquid nitrogen. Purification of CDKs was performed on p9CKShs1–
Sepharose beads. Eluted proteins were resolved on a 10 or 12% (w/v)
SDS–polyacrylamide denaturing gel and electrotransferred onto a nitrocellulose or a polyvinylidene difluoride membrane (Amersham
Pharmacia Biotech, Little Chalfont, UK) for enhanced chemilumines-
Histone H1 Kinase Activity and in Vitro Dephosphorylation of
Proteins Bound to p9CKShs1
The histone H1 kinase activity of proteins bound to p9CKShs1–
Sepharose beads was measured at 30⬚C for 30 min using ␥-32P-ATP,
as reported previously (Corellou et al., 2000a). Quantification of radioactive histone H1 was performed using a STORM phosphorimager
and Image QuanT software (Molecular Dynamics, Sunnyvale, CA).
For each lane, a protein-free region of the area under quantification
was used to determine the background according to the object average method. Kinase activities are represented either as relative
STORM units or as a percentage of control activity for in vitro inhibition experiments. The glutathione S-transferase (GST)–cdc25A fusion protein was overproduced in Escherichia coli and purified by
affinity on glutathione–agarose beads, as described previously
(Corellou et al., 2000a). Dephosphorylations were initiated by adding
to the proteins bound to the p9CKShs1 beads 100 ␮L (62 units) of the
purified GST-cdc25A in buffer B (50 mM Tris-HCl, pH 8.0, 50 mM
NaCl, 1 mM EDTA) containing 20 mM DTT. Dephosphorylation reactions were performed at 30⬚C for 1 hr. Control samples were treated
identically but without GST-cdc25A.
Cloning of the histone H3 Gene and RNA Gel Blot Analysis
DNA was extracted from Fucus sperm using a standard protocol for
brown algae (Apt and Grossman, 1993). A representative genomic library (5 ⫻ 105 clones) was established in Lambda DASH II, as recommended by the manufacturer (Stratagene, La Jolla, CA). This library
was screened under low-stringency conditions (Bouget et al., 1995)
with a Laminaria digitata histone H3 cDNA probe (kindly provided by
Dr. Florent Crépineau, Centre National de la Recherche Scientifique
Roscoff; GenBank accession number AW400773). A 300-bp exon
fragment encoding a putative histone H3 (GenBank accession number
AJ276797) ⬎95% identical to the L. digitata deduced amino acid sequence of histone H3 was subcloned and used as a probe for RNA gel
blot analysis as follows. RNA was extracted, electrophoresed through
a 1.2% formaldehyde denaturing gel, and transferred to a Hybond N⫹
membrane (Bouget et al., 1996). The Fucus histone H3 DNA probe
was labeled with radioactive ␣-32P-dCTP using a random priming labeling kit (Megaprime, Amersham Life Science). High-stringency hybridization was performed overnight at 42⬚C in 50% deionized
formamide, 7% (w/v) SDS, 250 mM NaCl, and 120 mM Na(PO4)2, pH
7.2. Hybridization washes were performed twice at 42⬚C in 2 ⫻ SSC
(1⫻ SSC is 0.15 M NaCl and 0.015 M sodium citrate) containing 0.1%
SDS and once in 1 ⫻ SSC containing 0.1% SDS. Membranes were exposed to a PhosphorScreen (Molecular Dynamics, Amersham Pharmacia Biotech) overnight. Quantification of bound radioactive probe
was performed using a STORM phosphorimager and Image QuanT
software.
Cell Cycle Control in Fucus Zygotes
ACKNOWLEDGMENTS
We thank Blandine Baratte and Sophie Leclerc for producing GSTcdc25A and Florent Crépineau for providing us with the L. digitata histone H3 cDNA. Thanks also to Laurent Meijer for helpful criticism of
the manuscript. F.C. was a recipient of a doctoral fellowship from the
Conseil Régional de la Région Bretagne, whose help is gratefully
acknowledged.
597
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Cell Cycle in the Fucus Zygote Parallels a Somatic Cell Cycle but Displays a Unique Translational
Regulation of Cyclin-Dependent Kinases
Florence Corellou, Colin Brownlee, Lenaick Detivaud, Bernard Kloareg and François-Yves Bouget
Plant Cell 2001;13;585-598
DOI 10.1105/tpc.13.3.585
This information is current as of August 9, 2017
References
This article cites 57 articles, 14 of which can be accessed free at:
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