Department of Genetics, University of Bayreuth, 95440 Bayreuth, Germany
* Author for correspondence (e-mail: chle{at}uni-bayreuth.de)
Accepted 5 February 2003
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Summary |
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Key words: Pimples, Sister chromatid separation, Separase, Cyclin A, Polo, APC/C
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Introduction |
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Mitotic securin degradation involves polyubiquitinylation by a special
ubiquitin ligase known as anaphase-promoting complex/cyclosome (APC/C) (for a
review, see Peters, 2002).
During mitosis, APC/C activity is controlled by the mitotic spindle checkpoint
pathway. This checkpoint assures that securin protein remains stable until all
chromosomes have reached the correct bi-orientation within the mitotic
spindle. The fact that securin stabilization by the mitotic spindle checkpoint
prevents premature sister chromatid separation has been clearly shown in
budding yeast (Ciosk et al.,
1998
; Yamamoto et al.,
1996
). By contrast, premature sister chromatid separation has not
been observed in human cells lacking securin when arrested during mitosis
(Jallepalli et al., 2001
),
suggesting that sister chromatid separation in these cells involves additional
securin-independent pathways.
In addition to mitotic securin degradation, other levels of regulation have
been implicated in the temporal control of separase activity and of sister
chromatid separation. In budding yeast, phosphorylation of the Scc1 cohesin
subunit by the Cdc5/Polo-kinase provides regulation at the substrate level
(Alexandru et al., 2001). After
phosphorylation, Scc1 is a better substrate for the yeast separase Esp1.
Regulated Scc1 phosphorylation therefore provides sufficient temporal control
of sister chromatid separation when the securin Pds1 is absent and Esp1 is
constitutively active throughout the cell cycle. Cohesin phosphorylation by
Polo-like kinase also controls the separase-independent dissociation of
cohesin complexes from higher eukaryotic chromosomes during mitotic prophase
(Losada et al., 2002
;
Sumara et al., 2002
).
In higher eukaryotes, where separase is required for the removal of those
cohesin complexes that remain on chromosomes until the metaphase-to-anaphase
transition (Hauf et al.,
2001), cyclin-dependent kinase 1 (Cdk1) has been proposed to
inhibit separase activity in parallel to securin
(Stemmann et al., 2001
).
Separase is phosphorylated by cyclin-Cdk1 complexes and thereby inhibited in
cells arrested by the mitotic spindle checkpoint. High levels of
non-degradable Cyclin B have been shown to inhibit sister chromatid separation
in Xenopus egg extracts and in PtK1 cells
(Stemmann et al., 2001
;
Hagting et al., 2002
). In
Drosophila embryos, expression of non-degradable Cyclin A, which is
found exclusively in Cdk1(Cdc2) and not in Cdk2(Cdc2c) complexes, delays
sister chromatid separation significantly
(Jacobs et al., 2001
;
Kaspar et al., 2001
;
Parry and O'Farrell, 2001
;
Sigrist et al., 1995
). Cdk1
inactivation resulting from the APC/C-dependent proteolysis of the mitotic
cyclins at the metaphase-to-anaphase transition therefore presumably leads to
separase activation, similar to securin degradation. Securin- and
Cdk1-dependent separase regulation might be largely redundant, explaining why
human cells display only very subtle defects in the absence of securin
function.
In securin-expressing cells, mitotic sister chromatid separation is
generally assumed to be strictly dependent on mitotic securin degradation.
However, the corresponding evidence is from experiments involving
overexpression of securin variants with mutant degradation signals. By
contrast, our experiments in Drosophila embryos involving expression
at physiological levels raised the possibility that sister chromatid
separation might not depend on PIM degradation
(Leismann et al., 2000).
Therefore, we have further analyzed the role of PIM degradation.
We have previously shown that PIM contains a novel D-box variant that
functions as a mitotic degradation signal
(Leismann et al., 2000).
D-boxes that form an RxxLxxxxN consensus sequence
(Peters, 1999
) were initially
identified in B-type cyclins where they are required for mitotic destruction.
The D-box variant identified in PIM starts with a K instead of an R. Apart
from the D-box, a different destruction signal, the KEN-box, can also mediate
APC/C-dependent degradation of various proteins
(Pfleger and Kirschner, 2000
;
Peters, 2002
). A KEN-box has
recently been shown to contribute to the mitotic degradation of human securin
(Hagting et al., 2002
;
Zur and Brandeis, 2001
). Here,
we show that PIM contains a functional KEN-box as well. Moreover, we show that
physiological levels of PIM with mutations in both the D- and the KEN-box do
not support sister chromatid separation, in contrast to our previous findings
with D-box mutants. Sister chromatid separation in Drosophila,
therefore, might well be strictly dependent on securin degradation. However,
we also show genetic interactions arguing for the presence of additional,
securin-independent regulation of sister chromatid separation.
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Materials and Methods |
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UAS-pimkena-myc lines were generated with a pUAST
construct (Brand and Perrimon,
1993). Its construction involved the introduction of the desired
KEN-box mutations by enzymatic amplification using the plasmid
pKS+gpim-myc as a template. pKS+gpim-myc was constructed by
first ligating into the NotI and KpnI sites of pKS+ the
regulatory, the coding and the 3' region of pim+
enzymatically amplified from the pKS+S2P4B1 plasmid containing a genomic
pim+ fragment
(Stratmann and Lehner, 1996
)
using the primer pairs RS37
(5'-ATAAGAATGCGGCCGCAACAGCTCGCGCAGAAGAGG-3') and RS61
(5'-CGGGGATCCTTTTTGTTAGCATTTATTGG-3'), RS34
(5'-GGATGGATCCGATTTTAAACAAGGAAA-3') and RS35
(5'-GAAGATCTATGCCTTCCAAGTTAGGCAATG-3'), or RS38
(5'-GTAGATCTTCTATTTTAGATCCTTGTTTAAAA-3') and RS39
(5'-GGGGTACCGGGCGATCTTGGCCGAAC-3'), respectively, to yield
pKS+gpim. These primers introduced a BamHI site immediately
after the start codon, a BglII site just before the stop codon, and
mutations (Q3P, V197L) that do not affect PIM function
(Stratmann and Lehner, 1996
).
The construction of pKS+gpim-myc was then completed by amplifying a
fragment encoding six copies of the myc epitope with the primers RS68
(5'-GGATCCCATCGATTGGATCCCATGGAGCAAAA-3') and RS51
(5'-GAGAGGCTCGAGAAGATCTGAATTCAAG-3') from pCS+MT
(Rupp et al., 1994
) and
inserting it into the BglII site of pKS+gpim. Primers OL33
(5'-GGAAAAAGGCGGCCGCGTTTAAAATCGGATCCAT-3') and OL35
(5'-GGAGGTACCAAGTGCCGCTCGTTTTCAG-3') were then used for
amplification of a first fragment from pKS+gpim-myc and OL32
(5'-GGAAAAAGCGGCCGCCACCGGCATTAATTGTAAG-3') and OL2
(5'-GCATCTAGAAGTTTTTATAGTTGCTTTAATTC-3') for a second fragment.
Fragment 1 was digested with Asp718 and NotI, fragment 2
with NotI and XbaI. Both fragments were ligated into the
Asp718 and XbaI sites of pUAST.
The transgenes gpim-myc and gpimdba-myc, in
which expression is directed by the pim+ regulatory
region, have been described previously
(Leismann et al., 2000;
Stratmann and Lehner, 1996
).
For the generation of gpimdba lines, we first
enzymatically amplified a fragment from pKS+gpimdba-myc
with the primers RS33 (5'-TTCAATACGTAGGCGCC-3') and OL59
(5'-CAAGGAAAACACCGGCATTAATTG-3'). The resulting 300 bp
amplification product with the D-box mutations was used as a primer, which was
extended after hybridization to the plasmid pKS+S2P4B1. The newly synthesized
strands were annealed and ligated, yielding pKS+S2P4B1dba.
Its insert was excised by BamHI and transferred into the germ line
transformation vector pCaSpeR 4 (Pirrotta,
1988
).
To generate the g>stop>pim,
g>stop>pimkenadba, and
g>stop>pimkenadba-myc lines, we first constructed
pKS+5'FRTgpim3' by inserting a fragment with a
FLP recombinase target sequence (FRT) amplified from the plasmid pKB345
(kindly provided by K. Basler, University of Zurich) with the primers RS62
(5'-GCGAGATCTACCGGGGGATCTTGAAGTTC-3') and RS63
(5'-CGCGGATCCATTTTTGTACCCAGCTTCAAAAGCGC-3') after digestion with
BglII and BamHI into the BamHI site of
pKS+gpim. pKB345 contains a 2.4 kb Asp718 fragment with
3'UTR and transcriptional terminator sequences from the heat-shock
protein gene, hsp70 (stop cassette) flanked by two FRT sites
(Struhl and Basler, 1993). For
the introduction of the stop cassette along with the second FRT site into
pKS+5'FRTgpim3', we first transferred the
Asp718 fragment from pKB345 into pKS+, in which the BamHI
site had been destroyed by religation after filling the restricted site. The
BamHI site within the insert fragment of the resulting plasmid was
also eliminated. The plasmid was then used as a template for enzymatic
amplification with the primers OL83
(5'-GCCGGTGTGCTGACGCATGTGAAG-3') and RS63
(5'-CGCGGATCCATTTTTGTACCCAGCTTCAAAAGCGC-3'). The amplification
product containing the 1500 bp stop cassette followed by the downstream FRT
site was digested with BglII and BamHI and ligated into the
BamHI site of pKS+5'FRTgpim3'. Ligation in the
correct orientation resulted in pKS+>stop>gpim, a
plasmid with an intact stop cassette flanked by FRT sites in front of the
start codon. Its Asp718-NotI insert fragment was transferred
into the corresponding sites of pCaSpeR 4. To introduce the KEN-box mutations,
the corresponding pim region was amplified using the primers OL13
(5'-CCATCTCTAGAAAAGTGCCGC-3') and OL84
(5-GCCGGTGGCAGCCGCGTTTAAAATCGGATC-3') from the template
pUASTpimkena-myc. To add the D-box mutations, the
resulting product was used as a primer for an additional polymerase chain
reaction in combination with a second primer OL8
(5'-ATTAGTAGTACAAAGATACCTAGC-3') and the template pKS+
gpimdba-myc. The fragment with the kena and
dba mutations was used to replace the wild-type sequence in
pKS+>stop>gpim either by using BamHI and
SnaBI (in the case of g>stop>pimkenadba) or
BamHI and BglII (in the case of
g>stop>pimkenadbamyc). The final pCaSpeR 4
constructs were again obtained by transposing Asp718-NotI
fragments. All constructs were verified by DNA sequencing and used for
Drosophila germ line transformation according to standard
procedures.
Antibodies, immunoprecipitation and immunolabeling
Mouse monoclonal antibodies against a myc epitope (9E10), against
Drosophila Cyclin B (F2) and against -tubulin (Neomarkers,
Fremont, CA) were used. Rabbit polyclonal antibodies against
Drosophila Cyclin B, Three rows (THR), PIM and Separase (SSE) have
been described previously (Jäger et
al., 2001
). Double labeling with mouse (Promega, Madison,WI) or
rabbit antibodies against ß-galactosidase (Cappel, Aurora, OH) in
combination with blue balancers was used for the identification of embryo
genotypes.
For co-immunprecipitation experiments, extracts were prepared with eggs
collected from either gpim-myc III.1, or UAS-Cdk1-myc II.2;
armGAL4 flies, or from a cross of
pim1/CyO, P{w+,
ftz-lacZ} females with pim1,
g>stop>pimkenadba-myc II.1/CyO,
P{w+, ftz-lacZ}; P{bTub85D-FLP}/+ males
during 2 hours on apple-juice agar plates followed by aging for 6 hours at
25°C. Immunoprecipitations were performed as described previously
(Leismann et al., 2000).
Fixation of embryos and immunolabeling was performed essentially as
described previously (Leismann et al.,
2000). Eggs were collected for 2 hours and aged at 25°C from
the following crosses:
To analyze a potential synergism between gpimdba and reduced polo+ function, all embryos with a strong abnormal central nervous system (CNS) phenotype were first identified on the basis of the DNA labeling before genotypes were assigned on the basis of the anti-ß-galactosidase labeling. Thereby 80% of the embryos with a strong abnormal CNS phenotype were found to be polo10 homozygotes with gpimdba II.1. An additional 10% were polo10 homozygotes without gpimdba II.1, and 10% were polo+ siblings with gpimdba II.1.
To analyze a potential synergy between gpimdba and
expression of mitotically stabilized Cyclin A, embryos with
UAS-CycA1-53 III.2 and prd-GAL4 at the stage
of mitosis 16 were scored for the presence of gpimdba II.1
and for a strong enrichment of metaphase plates in
prd-GAL4-expressing epidermal segments compared with the intervening
segments. Although 82% of the embryos with gpimdba II.1
displayed a strong metaphase enrichment, only 25% of the embryos without
gpimdba II.1 were comparably affected.
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Results |
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Instead of being required during each mitosis, PIM degradation might be important to keep protein levels below a critical threshold. We have previously shown that already moderate overexpression of wild-type pim (about fivefold) is sufficient to block sister chromatid separation. Moreover, although gpimdba rescued sister chromatid separation during mitosis 15 and 16 in pim mutants, it did not allow later divisions (data not shown), perhaps because the levels of stabilized PIMdba had built up beyond the critical threshold.
If degradation of the securin PIM was not an obligatory process required during each mitosis, separase bound to securin would be expected to have sufficient basal activity to allow sister chromatid separation. In this case, premature sister chromatid separation during interphase and early mitosis would have to be prevented by securin-independent regulation. As securin-independent regulation at the level of Scc1 phosphorylation by Cdc5/Polo kinase has been described in yeast, we analyzed whether a reduction in polo function enhances the effects of stabilized PIMdba. Within the CNS of polo-mutant embryos, we observed many abnormal cells with very large polyploid nuclei, when these embryos also carried gpimdba (Fig. 1E,F). Similar abnormal cells were almost never observed in either polo+ sibling embryos with gpimdba (Fig. 1A,B) or in polo- sibling embryos without gpimdba (Fig. 1C,D). In the presence of stabilized PIMdba, therefore, the remaining level of maternal polo+ contribution is no longer sufficient to mask phenotypic abnormalities in polo-mutant embryos. Moreover, reduced polo+ function enhances the effects of stabilized PIMdba.
|
In addition to Scc1 regulation by Cdc5/Polo kinase, vertebrate Cdk1 has
been shown to regulate separase independently of securin
(Stemmann et al., 2001). The
effects of stabilized Cyclin A in Drosophila embryos
(Sigrist et al., 1995
;
Jacobs et al., 2001
) are
consistent with the finding that vertebrate Cdk1 phosphorylates and thereby
inhibits separase. Mutant Cyclin A versions that cannot be degraded during
mitosis delay progression through the embryonic cell divisions during
metaphase before sister chromatid separation. Therefore, Drosophila
Cyclin A-Cdk1 complexes might inhibit separase activity. Accordingly, the
effects of stabilized Cyclin A
1-53 are expected to be enhanced by
expression of stabilized PIMdba. Labeling with antibodies against
tubulin (data not shown) and a DNA stain clearly revealed an increased number
of metaphase figures in epidermal regions of embryos expressing both Cyclin
A
1-53 and PIMdba (Fig.
1I,J), compared with embryos expressing only Cyclin A
1-53
(Fig. 1G,H). The stabilized
Cyclin A
1-53 therefore results in a more pronounced metaphase delay in
the presence of the stabilized PIMdba.
In principle, stabilized Cyclin A might delay cells in metaphase because it
results in an inhibition of PIM degradation during mitosis. However, cells
delayed in metaphase by stabilized Cyclin A1-170 no longer contained
PIM-myc according to immunolabeling experiments, whereas metaphase cells that
do not express Cyclin A
1-170 were always positive for PIM-myc
(Fig. 2). We conclude,
therefore, that the metaphase delay induced by stabilized Cyclin A does not
result from delayed PIM degradation.
|
The phenotypic interactions between stabilized PIMdba and Polo or Cyclin A are consistent with the notion that separase complexed with non-degradable securin might have sufficient activity to allow sister chromatid separation and that the timing of this process is controlled by pathways other than securin degradation. observed However, the sister chromatid separation in PIMdba-expressing cells might also be supported by residual mitotic PIMdba degradation. A KEN motif, which is found close to the N-terminus in all of the securins (Fig. 3A), might allow some limited mitotic PIMdba degradation, escaping detection by confocal microscopy as applied in our previous experiments.
|
To determine whether the KEN motif of PIM functions as a degradation
signal, we analyzed the mitotic stability of a myc-tagged PIM version with a
mutant KEN-box (PIMkena-myc with AAA instead of KEN).
PIMkena-myc, and PIM-myc for control, were expressed in the
anterior region of embryos during cycle 14, as described previously
(Leismann et al., 2000).
Immunolabeling at the stage of mitosis 14 indicated that
PIMkena-myc is largely stable throughout mitosis
(Fig. 3F-I), in contrast to
PIM-myc, which was detected before but not after the metaphase-to-anaphase
transition (Fig. 3B-E).
Progression beyond the metaphase-to-anaphase transition was monitored by the
labeling of DNA and Cyclin B, which is rapidly degraded when cells enter
anaphase. Our results show that the KEN-box is required and that the variant
D-box (KKPLGNLDN), which is still present in PIMkena-myc, is not
sufficient for normal mitotic PIM degradation.
Overexpression of PIMkena-myc resulted in mitotic defects.
Normal anaphase and telophase figures were not observed in
PIMkena-myc-positive cells that had progressed beyond the
metaphase-to-anaphase transition according to the absence of anti-Cyclin-B
labeling. Instead of pairs of well-separated telophase daughter nuclei, which
were readily observed in Cyclin-B-negative regions in the PIM-myc control
experiments (Fig. 3D,E,
arrowheads), Cyclin-B-negative regions of PIMkena-myc-expressing
embryos displayed decondensing metaphase plates or chromatin bridges between
partially separated nuclei (Fig.
3H,I arrows). These abnormalities caused by PIMkena-myc
were indistinguishable from those previously observed with
PIMdba-myc which has been shown to inhibit sister chromatid
separation (Leismann et al.,
2000).
Sister chromatid separation is also inhibited by strong overexpression of
wild-type PIM-myc (Leismann et al.,
2000). By contrast, at low physiological expression levels,
PIM-myc and, remarkably, also the stabilized versions PIMdba-myc
(Leismann et al., 2000
) and
PIMkena-myc (Fig.
3J-L), can promote sister chromatid separation in pim
mutants.
To analyze the function of PIM with mutations in both D- and KEN-box, we
constructed additional transgenes (g>stop>pimkenadba
and g>stop>pimkenadba-myc), allowing the expression
of PIMkenadba or PIMkenadba-myc under the control of the
normal pim regulatory region. To establish chromosomal insertions of
these potentially detrimental transgenes, we inserted a stop cassette flanked
by FLP recombinase target sites (>stop>) into the 5'
untranslated region. This stop cassette was eventually excised by transmitting
the established insertions via males expressing FLP recombinase specifically
in spermatocytes. Expression of the paternally recombined transgenes
(g>pimkenadba and
g>pimkenadba-myc) started at the onset of zygotic
expression during cycle 14 of embryogenesis. Expression of
g>pimkenadba and
g>pimkenadba-myc in pim-mutant embryos did not
allow sister chromatid separation during mitosis 15
(Fig. 4M-O and data not shown).
Instead of normal mitotic figures, which were readily apparent in
pim+ sibling embryos
(Fig. 4A, arrows), only
decondensing metaphase plates were observed during exit from mitosis
(Fig. 4M, arrowheads). Thus,
pim-mutant embryos expressing g>pimkenadba and
g>pimkenadba-myc displayed the same phenotype as
pim mutants without transgene
(Leismann et al., 2000) (and
data not shown) or with the non-recombined
g>stop>pimkenadba transgene
(Fig. 4I-K).
|
Control experiments with g>stop>pim transgenes encoding
wild-type PIM showed that expression after stop-cassette removal was
sufficient to promote normal sister chromatid separation in pim
mutants (Fig. 4E-G). Moreover,
additional control experiments showed that the recombined
g>pimkenadba-myc transgene was expressed as expected.
Anti-myc immunoblotting clearly showed expression (data not shown), and
co-immunoprecipitation experiments (Fig.
4U) indicated that the PIMkenadba-myc protein
associates efficiently with Separase (SSE) and Three rows (THR), a
Drosophila protein known to form trimeric complexes with SSE and PIM
(Jäger et al., 2001). In
addition, although g>pimkenadba-myc expression in
pim+ sibling embryos had little effect during the initial
embryonic cell divisions (mitosis 14-16), it resulted in a severe mutant
phenotype in the CNS where additional cell divisions occur
(Fig. 4T). Wild-type PIM
therefore appears to protect cells from the effects of
PIMkenadba-myc but only as long as the latter has not yet
accumulated to high levels.
In summary, our experiments with g>pimkenadba and g>pimkenadba-myc in pim mutants show that sister chromatid separation does not occur in the presence of physiological levels of the double mutants PIMkenadba and PIMkenadba-myc, in contrast to our findings with the single mutants PIMdba, PIMdba-myc and PIMkena-myc.
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Discussion |
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Mutations in either the D- or the KEN-box result in significant
stabilization of PIM protein during mitosis. Neither the D- nor the KEN-box,
therefore, are sufficient for normal degradation during the embryonic cell
divisions in Drosophila. Similar observations have been described for
human securin (Hagting et al.,
2002; Zur and Brandeis,
2001
). However, in contrast to Drosophila, mitotic
degradation of human securin still occurs quite effectively when either only
the D- or the KEN-box is intact. The D- and KEN-boxes of Drosophila
PIM, therefore, might function less independently than the corresponding
motifs in human securin. Eventually, the understanding of D- and KEN-box
function will require structural analyses of their interactions with
Fizzy/Cdc20 and Fizzy-related/Cdh1, which recruit proteins with these
degradation signals to the APC/C (Burton
and Solomon, 2001
; Pfleger et
al., 2001
). Fizzy and Fizzy-related are clearly both involved in
PIM degradation, at least indirectly, as PIM is stabilized in both
fizzy and fizzy-related mutants
(Leismann et al., 2000
) (data
not shown).
Under the assumption that PIMkenadba and
PIMkenadba-myc are still capable of providing the positive PIM
function, our results with these stabilized mutants suggest that PIM must be
degraded during each and every mitosis to allow sister chromatid separation.
Although not detectable by confocal microscopy, the single mutants
PIMdba and PIMkena might not be completely stable in
mitosis. After low-level expression in pim-mutant embryos, residual
mitotic degradation of single-mutant proteins might free some separase
activity sufficient for sister chromatid separation. Similar results have been
observed with the fission yeast securin Cut2, which is completely stabilized
in a Xenopus extract destruction assay by mutations in either of the
two D-boxes, and yet, low-level expression of single-but not double-mutant
proteins is able to complement growth of cut2-ts strains at the
restrictive temperature (Funabiki et al.,
1997). We emphasize that even in wild-type cells, mitotic PIM
degradation appears to be far from complete, and it can be speculated that it
is the PIM protein of a special pool of separase complexes that is more
efficiently degraded, perhaps on kinetochores or during transport on spindles
towards kinetochores. At high expression levels of PIM with or without single
mutations, free excess of this securin might rapidly re-associate and inhibit
the activated separase, resulting in the observed block of sister chromatid
separation.
Our results also point to alternative pathways that might regulate separase
activity and sister chromatid separation independently of PIM degradation. As
in yeast, the success of mitosis in cells with reduced separase function is
dependent on Polo kinase in Drosophila embryos. Moreover, as
expression of mitotically stabilized Cyclin A versions result in a metaphase
delay without inhibiting PIM degradation, Cyclin A appears to contribute
independently of PIM to the inhibition of premature sister chromatid
separation. Even though it remains to be analyzed whether Polo kinase and
Cyclin A-Cdk1 act during Drosophila divisions as proposed for Polo
homologs (Alexandru et al.,
2001) and vertebrate Cyclin B-Cdk1
(Stemmann et al., 2001
), our
results indicate that separase and sister chromatid separation are unlikely to
be regulated exclusively by securin degradation.
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Acknowledgments |
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