Download Cyclin synthesis and degradation and the embryonic cell cycle

y . Cell Sci. Suppl. 12, 6 5 -7 6 (1989)
P rin ted in G reat B ritain (E) The Company o f Biologists L im ited 1989
65
Cyclin synthesis and degradation and the embryonic cell cycle
A N D R E W W. M U R R A Y
Department of Biochemistry and Biophysics, University of California at San Francisco,
San Francisco, CA 94143-0448, USA
Summary
I discuss recent advances in the study of somatic and embryonic cell cycles. In the frog embryonic
cell cycle, cyclin is the only newly synthesized protein required to activate maturation-promoting
factor and induce mitosis. Diminishing the rate of cyclin synthesis increases the length of
interphase. Cyclin degradation is required for the progression from mitosis to interphase.
Comparison of the frog embryonic cell cycle to other cell cycles suggests that all cell cycles will rely
on the same closely conserved set of components. However, the component that is rate-limiting for
any step in the cell cycle will vary in different cell cycles.
Introduction
For many years the investigation of embryonic and somatic cell cycles had proceeded
independently. The identification of p34cdc2, the protein product of a major somatic
cell cycle control gene, as a subunit of maturation-promoting factor (M PF), the
protein complex that induces mitosis in embryonic cell cycles (Dunphy et al. 1988;
Gautier et al. 1988; Labbe et al. 1988), has linked thee two previously separate areas
of cell cycle research. In this article I will briefly discuss the genesis of the
conventional views of the embryonic and somatic cell cycles, describe how the
synthesis and degradation of a single protein, cyclin, can drive the embryonic cell
cycle, and finally discuss ways of rationalizing the differences between the embryonic
and somatic cell cycles.
The somatic cell cycle
The key advances in our understanding of the somatic cell cycle have come from
genetic studies on two yeasts: the budding yeast, Saccharomyces cerevisiae and the
fission yeast, Schizosaccharomycespombe. This approach was pioneered by Hartwell
(reviewed by Hartwell, 1978), who argued that the underlying logic of the cell cycle
could be revealed by isolating mutants that arrested the cell cycle at specific stages,
studying their phenotypes and examining the interaction between different cell cycle
I mutants. He and his colleagues found that the cell cycle could be largely described as
a series of steps where the initiation of each step required the completion of the
v preceding step in the cell cycle. Thus cells which have not completed D N A synthesis
fail to enter mitosis, and cells which have failed to correctly assemble a mitotic
spindle do not make the transition between metaphase and anaphase (Hartwell,
Key words: cell cycle, MPF, proteolysis, cdc2.
66
A. W. Murray
Go
Spindle correctly assembled?
<
3
0
Fig. 1. The somatic cell cycle in buddingyeast. The cell cycle is divided into four stages:
G j, S, G 2 and M. The main points in the cell cycle where progress is dependent on earlier
events in the cell cycle are shown as STOP signs labelled with the conditions that must be
met in order for the cell cycle to continue. START is shown as a single point in Gj.
Passage through START requires adequate nutrients and a attainment of a critical size.
At the edge of the circle the appearance of budding yeast cells at different points in the cell
cycle is shown (the nucleus is shown solid).
1978). (I believe that it makes good sense to regard this transition as marking the end
of mitosis and the beginning of the subsequent interphase.) This type of cell cycle
has been described as a dependent cell cycle and this view is supported by
experiments in which tissue culture cells in different stages of the cell cycle were
fused to each other (Rao and Johnson, 1970). The dependent cycle can be viewed
either as a traditional biochemical pathway where the product of each step acts as a
substrate for the next, or as a series of essentially independent events that are linked
to each other by feedback controls. The latter view is illustrated in Fig. 1, where the
feedback controls have been illustrated as points where the cell cycle stops until
certain conditions have been met: the boundary between G 2 and mitosis, which
cannot be crossed until D N A synthesis is complete; the boundary between
metaphase and anaphase, whose passage requires the correct assembly of the mitotic
spindle, and a special point called Start where cells must have attained a certain
m inimum size and have sufficient nutrients available in order to commit themselves
to passage through the next cell cycle. Between the end of mitosis and Start, cells can
enter a specialized resting phase called Gq; they cannot then re-enter the mitotic cell
cycle without passing through Start. The strongest evidence for feedback controls is
the existence of mutants and drugs that destroy the normal dependency relations in
the cell cycle: for instance the racL9 mutant of budding yeast allows cells that have
failed to repair D N A damage to pass through mitosis (and in doing so kill
themselves; Weinert and Hartwell, 1988), while tissue culture cells that are arrested
in S phase can be induced to enter mitosis by treatment with caffeine (Schlegel and
Pardee, 1986).
Cyclin and the embryonic cell cycle
67
Nurse and his colleagues have performed a genetic analysis of the cell cycle of the
fission yeast (reviewed by Lee and Nurse, 1988). Their main interest has been the
v induction qf mitosis. The key player in this transition is the product of the cdc2 gene
known as p34cdc2, whose ability to induce mitosis is increased by the activity of the
, cdc25 gene and diminished by the activity of the weel gene (Fantes, 1979). Altering
the relative dosage of the weel and cdc25 genes affects the m inim um cell size
required to enter mitosis, and extreme alterations lead either to arrest in G 2 as very
large cells (no cdc25 function), or entry into mitosis at a cell size that is so small that it
kills the cells (excess of cdc25 function in the absence of weel function; Russell and
Nurse, 1986). Since the cell cycle length of fission yeast is mostly regulated by
controlling the length of G 2, cell size and nutrient availability must interact in some
way with the gene products that control the activity of cdc2.
Intriguingly, the activity of cdc2 is also required for passage through Start (Nurse
and Bissett, 1981), although the cdc25 and weel genes do not appear to influence the
rate of this transition. The fission yeast cdc2 is functionally interchangeable and
homologous with the budding yeast CDC28 gene (Beach et al. 1982). In budding
yeast, the activity of CDC28 is clearly required for passage through Start but has not
been shown to be required for the induction of mitosis, although it can clearly act in
the induction of mitosis when it is expressed in the fission yeast (Beach et al. 1982).
The embryonic cell cycle
Studies on the cell cycle of early embryos began with the cytologists of the late 19th
and early 20th century (reviewed by Wilson, 1928) and received their modern
stimulus from the discovery of a protein complex which could induce meiosis when
injected into immature oocytes (Masui and Markert, 1971; Reynhout and Smith,
1974) and was therefore named maturation-promoting factor (M PF). M PF is
conventionally assayed by its ability to induce maturation in frog oocytes and has
been detected by this assay in extracts from cells in either mitosis or meiosis from a
wide variety of eukaryotes, demonstrating both its ubiquity and its functional
conservation during evolution (Kishimoto and Kanatani, 1977).
M PF has recently been purified from frog eggs by Lohka and Mailer and shown to
be a protein kinase with strong activity towards histone H I (Lohka et al. 1988). A
number of criteria suggest that M PF is identical to the growth-associated H I kinase
previously identified in extracts of mitotic tissue culture cells (J. Mailer, personal
communication). Immunological tests demonstrate that one of the subunits of M PF
purified from frog or starfish eggs is the product of the homologue of the cdc2 gene
(Dunphy et al. 1988; Gautier et al. 1988; Labbe et al. 1988) and a similar
conclusion has been reached for the mitotic H I kinase of HeLa cells studied by
Draetta and Beach (1988).
The work of Kirschner, Gerhart, Newport and their colleagues has shown that the
embryonic frog cell cycle is driven by fluctuations in the activity of M PF (reviewed
■by Kirschner et al. 1985). High levels of M PF activity induce entry into mitosis
1 (Newport and Kirschner, 1984), while M PF activity decreases as the cells leave
A. W. Murray
68
Protein
synthesis
Fig. 2. The embryonic cell cycle. The oscillation between interphase and mitosis of the
embryonic cell cycle. Note that protein synthesis is required for the transition between
interphase (I) and mitosis (M) but not for the transition from mitosis to interphase.
mitosis and enter interphase (Gerhart et al. 1984). Unlike the somatic cell cycle there
are no detectable feedback controls in this embryonic cell cycle: inhibiting D N A
synthesis, microtubule assembly, or even removing the egg nucleus all fail to prevent
the regular oscillation of interphase and mitotic states (Hara et a l. 1980). This cell
cycle has therefore been called an autonomous oscillator, to indicate that there is
some biochemical machinery that oscillates in a way that drives nuclear events but is
not dependent upon them (Fig. 2).
Protein synthesis is required during interphase of each embryonic cell cycle for the
occurrence of the subsequent mitosis (Wagenaar, 1983), suggesting that at least one
of the components required for the induction of mitosis must be synthesized de novo
in each cell cycle. Since the protein synthesis requirement can also be met by the
injection of M PF (Newport and Kirschner, 1984) it appears that the newly
synthesized proteins must act somewhere on the pathway of events that leads to the
activation of M PF. Hunt and his colleagues discovered cyclin, a prominent candidate
for a newly synthesized inducer of M PF activity, when they examined the pattern of
protein synthesis in early sea urchin embryos (Evans et al. 1983). The abundance of
cyclin increases during each interphase only to decline precipitously at the end of
each mitosis. The level of cyclin is regulated entirely by controlling the half-life of the
newly synthesized protein: it is long during interphase and becomes extremely short
at the end of mitosis (Evans et al. 1983).
Cyclin induces m itosis
The kinetics of cyclin appearance and disappearance suggest an attractive model for
Cyclin and the embryonic cell cycle
I
MPF
activity
M
I
M
69
_n
I
M
High
Low
Hypothetical
MPF
inactivase
High
Cyclin
Low
Nuclear breakdown
Chromosome condensation
Spindle assembly
CYCLIN DEGRADATION
Fig. 3. A model for a cyclin-based cell cycle oscillator. (A) From top to bottom are
shown the alternation of interphase (I) and mitosis (M), the fluctuations of MPF activity
and the levels of cyclin and a hypothetical MPF inactivase during the cell cycle. (B) A
representation of the reactions that control the cell cycle.
the embryonic cell cycle (Murray, 1987; Murray and Kirschner, 1989). Fig. 3 shows
this model, where we suggest that there are two competing activities: cyclin, which
stimulates the conversion of an inactive form of M PF into an active one, and a so far
hypothetical M PF inactivase which converts the active form of M PF into the inactive
one (Fig. 3). While cyclin abundance fluctuates during the cell cycle, the activity of
the M PF inactivase remains constant. The last postulate is that M PF induces its own
degradation as well as inducing nuclear envelope breakdown, chromosome conden­
sation and spindle assembly. These components create a version of the autonomous
oscillator. At the beginning of each interphase the level of cyclin is low, so that the
activity of the M PF inactivase is dominant and M PF is kept in its inactive form. As
interphase proceeds the level of cyclin increases until the rate of M PF activation
exceeds that of M PF inactivation and M PF activity increases, leading to the
induction of mitosis. However, the appearance of M PF activity leads ultimately to
the destruction of cyclin, the subsequent inactivation of MPF and exit from mitosis.
A number of lines of evidence support this scheme. Clam cyclin A (Swenson et al.
1986) or sea urchin cyclin B (Pines and Hunt, 1987) mRNAs can induce meiotic
maturation when injected into immature frog oocytes. As far as the exit from mitosis
is concerned, in both clams (T. Hunt and J. Ruderman, personal communication)
and flies (Lehner and O ’Farrell, 1989) cyclin is destroyed immediately before the
onset of anaphase, and in starfish, protease inhibitors can block the exit from meiosis
70
A. W. Murray
I (Picard et al. 1985). Suggestive as these lines of evidence are, none of them directly
demonstrates that cyclin accumulation induces mitosis, or that cyclin degradation is
required for the exit from meiosis.
To address these issues, we turned to in vitro cell cycle extracts of frog eggs that
were prepared by an adaptation of the method pioneered by Lohka and Masui
(1983). These cell cycle extracts are capable of several cell cycles that proceed at rates
similar to those of the in vivo cell cycle (Hutchison et al . 1987; Murray and
Kirschner, 1989) and closely mimic the properties of the in vivo cell cycle: in
interphase the nuclei are intact, D N A synthesis occurs (Blow and Watson, 1987;
Hutchison et al. 1987) and the activity of M PF and H I kinase are low (Murray and
Kirschner, 1989); as mitosis is initiated the activity of M PF and H I kinase increases,
the chromosomes condense, nuclear envelope breakdown occurs and at the end of
mitosis cyclin is degraded (Hutchison et al. 1988; Murray and Kirschner, 1989).
The cell cycle extracts can be treated with pancreatic RNase followed by RNase
inhibitor, so that their m R N A is destroyed but they retain the ability to synthesize
proteins in response to exogenous m R N A added after the RNase treatment. These
mRNA-dependent extracts are arrested in interphase but can be induced to start
cycling by adding pure sea urchin or frog cyclin B m R N A . In each cycle, cyclin
accumulates during interphase and is then destroyed at the end of mitosis,
demonstrating that cyclin synthesis, in the absence of other protein synthesis, is
sufficient to induce the entry into mitosis (Murray and Kirschner, 1989). By
specifically destroying cyclin m R N A , Minshull et al. (1989) have shown that cyclin
B synthesis is necessary for the induction of mitosis in frog cell cycle extracts. The
finding that cyclin synthesis is both necessary and sufficient for the induction of
mitosis in frog egg extracts makes it likely that in vivo, cyclin is the only newly
Tsynthesized protein required for the induction of mitosis.
Is cyclin accumulation the rate-limiting step, or trigger, of mitosis in frog
embryos? In the mRNA-dependent extracts, the length of the cell cycle increases as
the amount of added cyclin m R N A decreases, suggesting that cyclin accumulation
can be rate-limiting for the induction of mitosis (Murray and Kirschner, 1989). On
the other hand, experiments in vivo show that inhibiting protein synthesis during the
second half of interphase does not prevent or delay the next mitosis (Karsenti et al.
1987; Wagenaar, 1983). The combination of these observations suggest that
interphase of the frog embryonic cell cycle may be divided into two stages: an initial
stage during which cyclin must be accumulated to some critical level in order to
ensure the subsequent entry into mitosis, and a later stage during which posttranslational events lead to the activation of M PF. Since the rate of cyclin synthesis
will affect the duration of the initial stage but will not grossly affect the rate of the
post-translational modification step, increasing the rate of cyclin synthesis will not be
able to diminish the length of interphase indefinitely. I will address the identity of the
triggers of mitosis in other cell cycles below.
The role of cyclin destruction in the exit from mitosis was investigated by
constructing a truncated cyclin that was capable of inducing mitosis but not of being
degraded in mitotic cytoplasm. Introduction of the m R N A for this protein either
Cyclin and the embryonic cell cycle
71
into frog cell cycle extracts or intact frog eggs arrested the cell cycle in metaphase but
did not prevent the destruction of the endogenous full length cyclin (Murray et al.
1989). This experiment demonstrates that the truncated cyclin is a dominant
inhibitor of the exit from mitosis and therefore that cyclin degradation is required for
exit from mitosis.
Structure of cell cycles
How can we relate the autonomous oscillations of the embryonic cell cycle to the
dependent pathways of the somatic one? The cyclin-based cell cycle described above
appears to match the properties required of the autonomous oscillator. In this
scheme mitosis and interphase alternate because both cell cycle states are unstable:
interphase because the accumulation of cyclin leads to the induction of mitosis, and
mitosis because the activity of M PF leads ultimately to the destruction of cyclin. We
speak of the trigger of mitosis as the step in the activation of M PF that is rate-limiting
for the induction of mitosis. Even if the set of reactions required for the activation of
M PF is evolutionarily conserved, the step that is rate-limiting may be different in
different organisms and in different cell cycles in the same organism. For instance,
although the rate of cyclin accumulation appears to be rate-limiting for at least one
part of interphase in the early embryonic frog cell cycle, it is clearly not rate-limiting
for the post-cellularization cell divisions of the Drosophila embryo (Edgar and
O ’Farrell, 1989; Lehner and O ’Farrell, 1989). In this case the rate-limiting step
appears to be the synthesis of the homologue of the cdc25 gene that is probably
required for the slow post-translational steps after cyclin accumulation has been
completed. If this step is very slow relative to the rate of cyclin accumulation, then
the rate of cyclin synthesis would have to be very drastically reduced before it became
rate-limiting. Because there is no requirement for cdc25 synthesis in the early frog
cell cycle, this function must either not be required, or more likely supplied by
maternally inherited stores of the cdc25 protein.
In the frog embryonic cell cycle, the cyclin-based oscillator runs without any
feedback controls to entrain it to events of the nuclear cycle. However, in some
embryonic cell cycles feedback controls do exist, and it is easy to imagine that these
act through cyclin (Fig. 4). Thus in sea urchin embryos inhibition of D N A synthesis
blocks the phosphorylation of cyclin as well as entry into mitosis (N. Standart and
T. Hunt, personal communication). It is tempting to speculate that cyclin
phosphorylation is one of the post-translational steps required for the activation of
M PF and that the protein kinase that performs this modification can only be
activated by some signal that is generated when D N A replication is completed. In
contrast, this kinase would be constitutively active in frog embryos.
In both clams and sea urchins the depolymerization of microtubules increases the
length of mitosis (Sluder et al. 1986) and stabilizes cyclin during mitosis (Evans
et al. 1983), suggesting the existence of a feedback system that can detect incorrectly
assembled spindles and as a result inhibit cyclin degradation. Although frog embryos
lack this control, unfertilized frog eggs provide a clue as to what one of its
A. W. Murray
72
Embryonic cell cycle
Without feedback
With feedback
Failure to
Failure to
finish DNA
\assemble
replication
/ v i ’ sP 'ndle
/
Somatic cell cycle
Fig. 4. Models of the embryonic and somatic cell cycles. The levels of cyclin (--- ),
MPF ( • --- • ) and a putative G j cdc'2-associated protein kinase activity (---) are
shown throughout an embryonic cell cycle without feedback controls, an embryonic cell
cycle with feedback control, and a somatic cell cycle. Feedback controls (-||-) are
indicated. In the somatic cell cycle G i is divided into two portions: G[u, the period before
START and G ic, the period after START. During G iu cells can leave the cell cycle to
enter a specialized resting stage named G q. (From Murray and Kirschner, 1989).
components might be. When frog oocytes are induced to mature they pass rapidly
through meiosis I and then arrest in metaphase of meiosis I I. This arrest is mediated
by an activity that has been named cytostatic factor (CSF), and is preserved as the
mature oocyte passes down the oviduct and is laid as an unfertilized egg (Masui,
1974; Masui and Markert, 1971). The activity of CSF is calcium-sensitive (Masui,
1974) so that the rise in intracellular calcium concentration that is induced upon
fertilization leads to the inactivation of CSF, allowing M PF levels to decline and the
cell cycle to progress into interphase. One of the effects of CSF activity is to stabilize
cyclin in metaphase cytoplasm (Murray et al. 1989), suggesting that CSF may arrest
the cell cycle by preventing the degradation of cyclin. In frogs, CSF activity does not
appear during the early embryonic cell cycles, which may account for the lack of
feedback controls seen after fertilization. In embryos that do have feedback controls
it is possible that CSF, or some related activity, mediates the mitotic feedback
control by requiring some aspect of mitotic spindle assembly, rather than fertiliz­
ation, to generate the signal required for CSF inactivation.
Proper coordination of the cell cycle requires that the chromosomal D N A is
replicated once and only once before the initiation of mitosis. The studies of Rao and
Johnson (1970) demonstrate that over-replication is prevented by some mechanism
that modifies the chromosomes during the course of replication so that they cannot be
re-replicated within the same cell cycle even if the D N A replication machinery
Cyclin and the embryonic cell cycle
73
remains active within the same cell. In early embryonic cell cycles this re-replication
block is removed during passage through mitosis (Newport and Kirschner, 1984) so
that D N A replication begins as soon as chromosome decondensation and nuclear
reformation begins. Blow and Laskey (1988) suggest that the re-replication block is
removed as a consequence of the physical act of nuclear envelope breakdown,
although the alternative possibility that nuclear envelope breakdown and the removal
of the re-replication block are independent events that both require the activation of
M PF has not been rigorously excluded.
In somatic cells D N A replication requires passage through Start, suggesting that
the block to re-replication might be removed as a consequence of passage through
Start, rather than through mitosis. If this possibility is true, then in some sense the
mitotic and D N A replication cycles are under separate controls and therefore require
coordination. This coordination may be facilitated by the fact that both Start and
mitosis require the activity of the cdc2 gene product (Nurse and Bisset, 1981),
especially if the two different activities of this protein are induced by different
accessory proteins at the two cell cycle transitions. At mitosis the activation of cdc2
requires the activity of both cdc25 and the fission yeast cyclin B homologue, cdcl3
(Booher and Beach, 1988; Hagan et al. 1988; Solomon et al. 1988). The finding that
cdcl3 in fission yeast and cyclin A and B in clams are associated with p34cflfc2 (Draetta
et al. 1989) suggests that the association of cyclin with p 3 ¥ dc2 may both activate the
protein kinase activity of p34crfc2 and determine its substrate specificity. Since neither
,cdcl3 nor cdc25 activity are required for passage through Start, it is tempting to
suggest that either cyclin A or some undiscovered member of the cyclin family might
act at Start to activate p34crfc2 to a pattern of substrate specificity to produce the
biochemical changes that correspond to passage through Start (Fig. 4). In the
budding yeast one such cyclin has been identified as the product of the gene variously
referred to as whil and DAF1. Mutants in this gene which truncate the protein
(possibly making it resistant to degradation) allow cells to pass Start at much smaller
sizes than normal (Cross, 1988; Nash et al. 1988). If there are different cyclins
responsible for the Start and mitosis-inducing activities of p34cafc2 in the somatic cell
cycle, then the orderly progress of the cell cycle requires that the accumulation
and/or activation of these different cyclins alternate during the cell cycle. This could
be achieved by making the accumulation of the mitosis-specific cyclin dependent on
the occurrence of Start and the accumulation of the Start-specific cyclin dependent
on the occurrence of mitosis. In the embryonic cell cycle, Start and mitosis may
occur as a single event, either by making the kinetics of the Start and mitosis-specific
cyclins identical, or by making both Start (the removal of the block to re-replication
of D N A ?) and mitosis dependent on the same cyclin.
How many clocks run the cell cycle? If there are separate but intertwined clocks for
mitosis and D N A replication may there not also be other clocks for other cell cycle
. events? This question has been clearly answered in the affirmative by Mitchison and
his colleagues who have discovered two clocks which continue to run when the other
cell cycle clocks have been stopped by temperature-sensitive mutations in cdc2
(Creanor and Mitchison, 1986; Novak and Mitchison, 1986). Under these conditions
74
A. W. Murray
,v' these two clocks, whose molecular mechanisms are currently mysterious, run with
slightly different periods. It seems likely that we still have a great deal to learn about
timekeeping and coordination in the cell cycle.
I am extremely grateful to Marc Kirschner for his support, hospitality and for many stimulating
discussions, and to my colleagues in the cell cycle field for many valuable discussions that have
shaped my thinking. I am a Lucille P. Markey Scholar and am supported by a grant from the
Lucille P. Markey Charitable Trust.
References
P. (1982). Functionally homologous cell cycle control genes
in budding and fission yeast. Nature, Land. 300, 706-709.
B l o w , J. a n d W a t s o n , J. (1987). Nuclei act as independent and integrated units of replication in a
Xenopus cell-free D N A replication system. EMBO J. 6, 1997-2002.
B l o w , J. J. a n d L a s k e y , R . A. (1988). A role fo r the n u c le a r envelope in c o n tro llin g D N A
re p lic a tio n w ith in th e cell cycle. Nature, Lond. 332, 546-548.
B o o h e r , R . a n d B e a c h , D . (1988). Involvement of cdcl3+ in mitotic control of Schizosaccharomyhces pombe: possible interaction of the gene product with microtubules. EMBO J. 7,
2321-2327.
■
n | C r e a n o r , J. a n d M i t c h is o n , J. M . (1986). Nucleoside diphosphokinase, an enzyme with step
. changes in activity during the cell cycle of the fission yeast Schizosaccharomyces pombe. J. Cell,
Be a c h , D ., D u r k a c z , B. a n d N u r se ,
"
Sci. 86,
207-215.
F. (1988). DAF1 , a mutant gene affecting size control, pheromone arrest, and cell cycle
kinetics of Saccharomyces cerevisiae. Molec. cell Biol. 8, 4675-4684.
D r a e t t a , G. a n d B e a c h , D . (1988). Activation of cdc2 protein kinase during mitosisin human
cells: cell cycle-dependent phosphorylation and subunit rearrangement. Cell 54, 17-26.
D r a e t t a , G ., L u c c a , F., W e s t e n d o r f , J., B r i z u e l a , L . , R u d e r m a n , J. a n d B e a c h , D . (1989).
cdc2 protein kinase is complexed with both cyclin A and B : evidence for proteolytic inactivation
of MPF. Cell 56, 829-838.
D u n p h y , W. G., B r i z u e l a , L., B e a c h , D . a n d N e w p o r t , J. (1988). The Xenopus cdc2 protein is
a component of MPF, a cytoplasmic regulator of mitosis. Cell 54, 423-431.
E d g a r , B. A. a n d O ’F a r r e l l , P. H. (1989). Genetic control of cell division patterns in the
Drosophila embryo. Cell 57, 177-187.
E v a n s , T . , R o s e n t h a l , E . T . , Y o u n g b l o m , J . , D is t e l , D . a n d H u n t , T . (1983). C y c lin : a
C ross,
p ro te in specified b y m a te rn a l m R N A in sea u r c h in eggs th a t is d e stroyed at each cleavage
d iv is io n . Cell 33, 389-396.
F a n t e s , P. (1979). Epistatic interactions in the control of division in fission yeast. Nature, Land.
279, 428-430.
G a u t ie r , J., N o r b u r y , C., L o h k a , M . , N u r s e , P. a n d M a l l e r , J. (1988). Purified Maturationpromoting factor contains the product of aXenopus homolog of the fission yeast cell cycle control
gene cdc2+. Cell 54, 433-439.
G e r h a r t , J., Wu, M. a n d K i r s c h n e r , M. W. (1984). Cell cycle dynamics of an M-phase specific
cytoplasmic factor in Xenopus laevis oocytes and eggs. J. Cell Biol. 98, 1247-1255.
H a g a n , I., H a y l e s , J. a n d N u r s e , P. (1988). Cloning and sequencing of the cyclin-related
cdcl3+ gene and a cytological study of its role in fission yeast mitosis. J. Cell Sci. 91, 587-595.
H a r a , K . , T y d e m a n , P. a n d K i r s c h n e r , M. W. (1980). A cytoplasmic clock with the same period
as the division cycle in Xenopus eggs. Proc. natn. Acad. Sci. U.S.A. 77, 462-466.
H a r t w e l l , L. H . (1978). Cell division from a genetic perspective. J 1
’. Cell Biol. 77, 627-637.
H u t c h is o n , C. J., Cox, R., D r e p a u l , R. S., G o m p e r t s , M . a n d F o r d , C. C. (1987). Periodic
D N A synthesis in cell-free extracts of Xenopus eggs. EMBO J. 6, 2003-2010.
H u t c h is o n , C. J., Cox, R. a n d F o r d , C. C. (1988). The control of D N A replication in a cell-free
extract that recapitulates a basic cell cycle in vitro. Development 103, 553-566.
K a r s e n t i , E., B r a v o , R. a n d K i r s c h n e r , M . W . (1987). Phosphorylation changes associated with
the early cell cycle in Xenopus eggs. Devi Biol. 119, 442-453.
Cyclin and the embryonic cell cycle
75
M ., N e w p o r t , J. a n d G e r h a r t , J. (1985). The timing of early developmental events
in Xenopus. Trends Genet. 1, 41-47.
K is h im o t o , T. a n d K a n a t a n i , H. (1977). Lack of species specificity of starfish maturationpromoting factor. Gen. comp. Endocrinol. 33, 41-44.
L a b b é , J. C., L e e , M. G ., N u r s e , P ., P i c a r d , A. a n d D o r é e , M. (1988). Activation at M-phase
of a protein kinase encoded by a starfish homologue of the cell cycle control gene cdc2+. Nature,
Lond. 355, 251-254.
L e e , M. a n d N u r s e , P. (1988). Cell cycle control genes in fission yeast and mammalian cells.
Trends Genet. 4, 287-290.
L e h n e r , C. a n d O ’F a r r e l l , P. (1989). Expression and function of Drosophila cyclin A during
embryonic cell cycle progression. Cell 56, 957-968.
L o h k a , M . J. a n d M a s u i , Y . (1983). F o r m a tio n in vitro of s p e rm p ro n u c le i a n d m ito tic
ch ro m o so m e s in d u c e d b y a m p h ib ia n o o p la s m ic c o m p o n e n ts . Science 220, 719-721.
L o h k a , M . J., H a y e s , M . K . a n d M a l l e r , J. L . (19 88 ). P u rific a tio n o f m a tu ra tio n - p r o m o tin g
K ir s c h n e r ,
facto r, a n in tr a c e llu la r re g u la to r o f early m ito tic events.
Proc. natn. Acad. Sci. U.S.A.
85,
3009-3013.
M a s u i, Y .
a tio n .
(1974). A cy tostatic factor in a m p h ib ia n oocytes: its e x tra ctio n a n d p a rtia l ch a rac te riz­
J. exp. Zool. 187, 141-147.
Y. a n d M a r k e r t , C. (1971). Cytoplasmic control of nuclear behavior during meiotic
maturation of frog oocytes. J. exp. Zool. 177, 129-146.
M i n s h u l l , J., B l o w , J. a n d H u n t , T. (1989). Translation of endogenous B-type cyclin m RN A is
necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 56, 947-956.
M u r r a y , A. W. (1987). Cyclins in meiosis and mitosis. Nature. Lond. 326, 542-543.
M u r r a y , A. W. a n d K i r s c h n e r , M . W. (1989). Cyclin synthesis drives the early embryonic cell
cycle. Nature, Lond. 339, 275-280.
M u r r a y , A. W . , S o l o m o n , M . J. a n d K i r s c h n e r , M . K . (1989). The role of cyclin synthesis and
degradation in the control of M P F activity. Nature, Lond. 339, 280-286.
N a s h , R., T o k i w a , G ., A n a n d , S ., E r i c k s o n , K. a n d F u t c h e r , A . B. (1988). The W HI1+ gene
of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBOJ. 7,
4335-4346.
N e w p o r t , J. W. a n d K i r s c h n e r , M. W. (1984). Regulation of the cell cycle during early Xenopus
development. Cell 37, 731-742.
"■^N o v a k , B. a n d M it c h is o n , J. M . (1986). Changes in the rate of C 0 2 production in synchronous
1
cultures of the fission yeast Schizosaccharomyces pombe\ a periodic cell cycle event which
persists after the DNA-division cycle has been blocked. .7- Cell Sci. 86, 191-206.
N u r s e , P . a n d B is s e t t , Y. (1981). Gene required in G i for commitment to cell cycle and in G 2 for
control of mitosis in fission yeast. Nature, Lond. 292, 558-560.
P i c a r d , A., P e a u c e l l i e r , G ., L e B o u f f a n t , F., L e P e u c h , C. a n d D o r e e , M. (1985). Role of
protein synthesis and proteases in production and inactivation of maturation-promoting activity
during meiotic maturation of starfish oocytes. Devi Biol. 109, 311-320.
P i n e s , J. a n d H u n t , T. (1987). Molecular cloning and characterization of the m RN A for cyclin
from sea urchin eggs. EM BOJ. 6, 2987—2995.
R a o , P. N. a n d J o h n s o n , R . T. (1970). Mammalian cell fusion studies on the regulation of D N A
synthesis and mitosis. Nature, Lond. 225, 159-164.
R e y n h o u t , J. K. a n d S m it h , L. D . (1974). Studies on the appearance and nature of a maturationinducing factor in the cytoplasm of amphibian oocytes exposed to progesterone. Devi Biol. 38,
394-400.
R u s s e l l , P. & N u r s e , P. (1966). Negative regulation of mitosis by w eel , a gene encoding a protein
kinase homolog. Cell 49 , 559-567.
S c h l e g e l , R. a n d P a r d e e , A. B. (1986). Caffeine-induced uncoupling of mitosis from the
completion of D N A replication in mammalian cells. Science 232, 1264-1266.
S o l o m o n , M ., B o o h e r , R., K ir s c h n e r , M. a n d B e a c h , D . (1988). Cyclin in Fission Yeast. Cell
54, 738-739.
S l u d e r , G ., M i l l e r , F. J. a n d S p a n j ia n , K. (1986). The role of spindle microtubules in the
timing of the cell cycle in echinoderm eggs. J'. exp. Zool. 238, 325-336.
S w e n s o n , K. I., F a r r e l l , K. M. a n d R u d e r m a n , J. V. (1986). The clam embryo protein cyclin
M a s u i,
76
A. W. Murray
A induces entry into M phase and the resumption of meiosis in Xenopus oocytes. Cell 47,
861-870.
W a g e n a a r , E. B. (1983). The timing of synthesis of proteins required for mitosis in the cell cycle
of the sea urchin embryo. Expl Cell Res. 144, 393-403.
W e in e r t , T. A. a n d H a r t w e l l , L. H . (1988). The RAD9 gene controls the cell cycle response to
D N A damage in Saccharomyces cerevisiae. Science 241, 317-322.
W i l s o n , E . B . (1928). The Cell in Development and Heredity , 3rd edition. New York: Macmillan.