Article |
Address correspondence to Marie-Hélène Verlhac, Biologie Cellulaire et Moleculaire du Developpement, UMR 7622, Centre National de la Recherche Scientifique/Université Pierre et Marie Curie, 9 quai Saint Bernard-Bat. C-5, 75252 Paris, cedex 05, France. Tel.: 33-14-427-3401. Fax: 33-14-427-3498. E-mail: marie-helene.verlhac{at}snv.jussieu.fr
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Abstract |
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Key Words: MISS; MAP kinase; spindle stability; morpholino; mouse
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Introduction |
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The cascade initiated by c-mos, which activates mitogen-activated protein kinases (MAPK) (the Mos/.../MAPK pathway), controls many important events of meiotic maturation (for an extensive review see Abrieu et al., 2001). This pathway controls the migration of the germinal vesicle (GV) to the cortex in Xenopus oocytes and the migration of the first meiotic spindle to the cortex in mouse oocytes (Choi et al., 1996; Gavin et al., 1999; Verlhac et al., 2000a). It is therefore essential for the establishment of asymmetric divisions during meiosis. This pathway also controls microtubule organization in mouse oocytes (Verlhac et al., 1994, 1996). Moreover, the Mos/.../MAPK pathway promotes the cytostatic factor (CSF; Masui and Markert, 1971) arrest of vertebrate oocytes (Haccard et al., 1993; Colledge et al., 1994; Hashimoto et al., 1994; Verlhac et al., 1996). Indeed, vertebrate oocytes are arrested at metaphase of the second meiosis by an egg cytoplasmic substance called CSF. Then fertilization will release the egg from the arrest and allow it to complete meiosis and proceed to mitosis. The CSF arrest of the cell cycle is affected by the Mos/.../MAPK cascade, which results in the activation of the ribosomal S6 kinase p90rsk (Bhatt and Ferrell, 1999; Gross et al., 1999, 2000). However, the mechanism of the cell cycle arrest in MII has not been fully clarified. Particularly puzzling is the fact that the cell cycle is not arrested at metaphase I (MI), whereas all the kinases mentioned above have already been activated before MI. So molecules activated only in MII and targets of the Mos/.../MAPK pathway could also be involved in the CSF arrest.
To look for physiological effectors of the Mos/.../MAPK pathway in meiosis, we generated a mouse immature oocyte cDNA library that we screened by two hybrid using MAPK as a bait. We isolated a new protein, MAP kinaseinteracting and spindle-stabilizing protein (MISS), which is rich in proline residues and has four potential MAPK phosphorylation sites, a MAPK docking site, a PEST sequence, and a bipartite nuclear localization signal (NLS). Apart from a potential human homologue, we did not find any homologies with other proteins in the databases. In mouse oocytes, MISS is a potential in vivo substrate for the Mos/.../MAPK pathway. The endogenous protein accumulates during mouse meiotic maturation and localizes as discrete dots on the MII spindle. Also, a MISSGFP construct localizes to kinetochore microtubules in MII oocytes. The stability of MISS is regulated during mouse meiotic maturation. It is unstable in MI, where its degradation is microtubule dependent, and becomes stable in MII. MISS is again unstable after parthenogenetic activation and the endogenous protein is no longer detected in early embryos. By microinjecting both antisense (as) RNA, double-stranded (ds) RNA, and morpholino oligonucleotides (Summerton and Weller, 1997) directed against MISS endogenous mRNA, we show that it is involved in maintaining MII spindle integrity. Mouse oocytes microinjected with any of these three different molecules, and not control molecules, display disorganized MII spindles and numerous asters in their cytoplasm. Moreover, the spindle defects induced after microinjection of morpholino oligonucleotides directed against 25 bp upstream of the ATG can be rescued by the overexpression of MycMISS from RNA corresponding to the full-length ORF devoid of MISS 5' UTR. The injection of dsRNA against MISS has yet no effect on the first two mitotic divisions, suggesting a specific role for MISS in meiosis. MISS is the first example of a physiological MAPK substrate that is stabilized in MII and specifically regulates MII spindle integrity during the CSF arrest.
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Results |
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MISS mRNA is present in immature oocytes but MISS protein accumulates after meiosis resumption and localizes to the MII spindle
To check if the full-length RNA encoding MISS was present in mouse oocytes, we performed RT-PCR using primers specific for the MISS mRNA. By RT-PCR, we could amplify a specific band of 800 bp corresponding to the full-length MISS ORF (Fig. 1 C). As shown in Fig. 1 C, MISS mRNA is expressed in mouse ovaries, mouse immature oocytes (at the GV stage), and two cell stage mouse embryos. However, both by immunoblotting (Fig. 1 D, lane 1) and immunofluorescence (Fig. 3 A) using an affinity-purified anti-MISS peptide antibody, we could not detect the presence of the endogenous protein in immature oocytes. MISS was present in mature oocytes (Fig. 1 D, lane 2). The apparent molecular weight being around 45 kD suggests that MISS undergoes posttranslational modifications. By immunofluorescence, MISS was undetectable in GV oocytes (Fig. 3 A), was hardly detectable in the vicinity of the chromosomes in late MI oocytes (Fig. 3 B), and accumulated strongly and became localized in MII-arrested oocytes (Fig. 3, C and D). Both the cytoplasmic and the MII spindle staining are specific because they are no longer present when the purified antibody is preincubated with the immunogenic peptide (Fig. 3 F). Interestingly, we could not detect any staining in early mouse embryos (Fig. 3 E), suggesting that MISS accumulates specifically in meiosis. A MISSGFP construct also behaves like the endogenous protein during meiotic maturation. Indeed, the overexpressed MISSGFP protein is detected by immunoblotting in MII oocytes (Fig. 4 F), and no localization of the MISSGFP protein is detected in MI oocytes (Fig. 4 A). In MII oocytes, the MISSGFP localizes in patches along fibers that evoke kinetochore fibers emanating from the chromosomes (Fig. 4, B, F, and G). The MISSGFP protein also localizes along the spindle in mitotic dividing embryos (Fig. 4 D). After activation and in interphase, the MISSGFP protein is completely excluded from the cytoplasm and present only in the nucleus, where small dots are observed (Fig. 4, C and E). This result suggests that the NLS present in the protein is functional and involved in a tight regulation of MISS localization.
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First we checked that the MycMISS behaves like the endogenous MISS. Indeed, the exogenous protein could not be detected in immature oocytes, whereas it was present in mature oocytes (Fig. 5 A, lanes 1 and 2). We microinjected chimeric RNA that contained the MISS ORF flanked by the 5' and 3' UTR of the Xenopus ß-globin gene, thus the translation of the MycMISS encoding RNA was not submitted to translational regulations by MISS endogenous 5' and 3' UTR. Using the same construct, we typically observed similar amounts of translation of the exogenous RNA both in immature and mature oocytes when allowed to overexpress for the same amount of time (Fig. 5 A; the levels of expression of MycERK2 are similar in immature and mature oocytes).
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Because the kinetics of MycMISS accumulation are similar in wild-type compared with mos-/- oocytes (Fig. 5 C), which do not have MAPK activity (Verlhac et al., 1996), the Mos/.../MAPK pathway does not regulate MISS stability.
To demonstrate that MycMISS is indeed unstable in MI due to a high turnover of the protein, we examined its stability in oocytes incubated in nocodazole. In mouse oocytes, cyclin B1 is stabilized by nocodazole treatment, suggesting that its degradation requires intact microtubules (Kubiak et al., 1993). We treated MycMISS-injected oocytes with nocodazole for 3 h. As shown in Fig. 5 D, this treatment induces MycMISS and cyclin B1 accumulation before polar body extrusion. This shows that MISS is unstable in MI and is degraded by a microtubule-dependent mechanism.
To analyze MISS behavior after parthenogenetic activation, we microinjected MycMISS RNA into MII-arrested oocytes. Half of the injected oocytes were activated by ethanol treatment, whereas the other half was not treated. As shown in Fig. 5 E, MycMISS accumulates during the MII arrest (lanes 3 and 4), whereas it is unstable after activation (lanes 1 and 2).
As we observed by immunofluorescence on the endogenous protein, MycMISS protein accumulated late in MI. Based on the nocodazole experiment and the behavior of other Myc-tagged proteins in mouse oocytes (i.e., MycERK2; Fig. 5 A; Verlhac et al., 2000b), we conclude that MISS is stabilized only during the MII stage.
MycMISS is a substrate of the Mos/.../MAPK pathway
The protein sequence of MISS displays four potential ERK phosphorylation sites and a MAPK docking site. We have shown that active MAPK can phosphorylate MISS in vitro (unpublished data). To determine whether the protein is an in vivo substrate of MAPK, we checked for its electrophoretic mobility in oocytes from mos-/- oocytes. In these oocytes, the migration of MycMISS is faster compared with its electrophoretic mobility in wild-type oocytes, reflecting a posttranslational modification of MycMISS in wild-type oocytes (Fig. 6 A). Among the posttranslational modifications, we show that at least some of them are associated with phosphorylation. Indeed, treatment of the protein isolated from MII-arrested oocytes with -phosphatase induced a downshift of its electrophoretic mobility (Fig. 6 B, compare lanes 1 and 2).
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Altogether our results show that MycMISS is an in vivo substrate of the Mos/.../MAPK pathway.
Interfering with endogenous MISS mRNA induces severe spindle defects in MII-arrested oocytes
To determine the potential role of MISS during mouse meiotic maturation, we decided to interfere with endogenous mRNA expression. Three different types of molecules were used. First, asRNA (asMISS) or dsRNA (dsMISS) directed against the full-length ORF were microinjected into immature oocytes. The microinjection of either asRNA or dsRNA directed against endogenous MISS mRNA had no effect on GVBD or first polar body extrusion (data not shown). However, both injections induced severe spindle disorganization in MII-arrested oocytes. As shown in Fig. 7, B and D, the MII spindle loses its bipolarity and numerous asters appear in the cytoplasm. This phenotype is visible right after the MII spindle is formed (13 h after GVBD). It is never observed in noninjected oocytes or in oocytes injected with an irrelevant dsRNA at the same concentration and directed against Xenopus frizzled 7 (Fig. 7, A and C). As expected, the injection of dsMISS induced a disappearance of MISS endogenous protein in MII oocytes (Fig. 7, compare E and F). Moreover, this phenotype was highly reproducible, as it was observed in >90% of the 96 asMISS- or dsMISS-injected oocytes (Fig. 7 E). Interestingly, this phenotype is similar to the one observed in mos-/- oocytes, which arrest in metaphase III (MIII) with monopolar spindles after second polar body extrusion (Fig. 8 F; Verlhac et al., 1996).
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The fact that we observed the same phenotype using three different types of molecules directed against endogenous MISS mRNA, and not with control molecules, is a strong argument in favor of the specificity of the observed spindle defects. This is further demonstrated by the rescue experiment using MycMISS RNA in morpholino-injected oocytes.
Altogether our results using three different approaches demonstrate that MISS maintains a bipolar MII spindle during the CSF arrest of mouse oocytes.
MISS does not induce mitotic spindle destabilization in early embryos
Considering the severe phenotypes observed on MII spindles, we can assume that if such spindle defects are observed after injection of dsMISS into early embryos, they should block cell cycle progression. Indeed, similar defects are observed in mos-/- oocytes (Fig. 8 F) and induce an MIII arrest. To test this hypothesis and therefore see whether MISS also stabilizes mitotic spindles, we microinjected dsMISS into either zygotes or two cell embryos and checked whether cell cycle progression was inhibited. The injection of dsMISS into zygotes does not block the division from the one to two cell stage: 99% (n = 38) of dsMISS-injected zygotes undergo the one to two cell division like the control (n = 36) dsFz-injected ones. Also, dsMISS injection does not block the two to four cell division: 79% (n = 68) of dsMISS-injected two cell blastomeres divided to four cell blastomeres, comparable to 81% (n = 70) of dsFz-injected blastomeres. We checked that our dsMISS were indeed efficient by coinjecting them together with MISSGFP RNA (Fig. 9). As for zygotes in interphase (Fig. 4 C), the MISSGFP protein accumulated in the nuclei of the injected blastomeres (Fig. 9 B). However, the MISSGFP accumulation was very weak when coinjected with dsMISS, compared with its coinjection with dsFz (Fig. 9, B and D). We confirmed this by immunoblotting with an anti-GFP antibody (Fig. 9 E). The MISSGFP protein accumulated in dsFz- but not in dsMISS-injected blastomeres.
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Discussion |
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From the screen performed using ERK2 as bait, we isolated a novel protein, MISS, to which, except for a potential human homologue, we cannot find any obvious homologues in the databases. MISS is very rich in proline residues in its NH2-terminal end and has four potential ERK2 phosphorylation sites, a perfect PEST sequence, and a bipartite NLS that contains a potential MAPK docking site. We confirmed biochemically the two-hybrid interaction by showing that MISS coimmunoprecipitates with endogenous MAPK.
We show by RT-PCR that the mRNA is present in immature oocytes as well as early mouse embryos. We also show by immunoblotting and immunofluorescence using an affinity-purified antipeptide antibody that MISS protein is present in mouse mature oocytes.
MISS is stabilized only in MII and is phosphorylated by the Mos/.../MAPK pathway
Both by immunoblotting and immunofluorescence, we show that although the mRNAs are present in immature oocytes, MISS protein is absent from these oocytes. The endogenous protein starts to accumulate late in MI, around the first polar body extrusion. RNA encoding MISS, fused either to a Myc epitope in an NH2-terminal position or to a GFP epitope in a COOH-terminal position, cannot induce MISS accumulation in GV or early MI oocytes, whereas RNA encoding MycERK2 is typically associated with similar accumulation of MycERK2 in GV and MII oocytes. Moreover, treatment of injected oocytes with nocodazole induces accumulation of the protein in MI, where it normally does not accumulate. Altogether, these data show that MISS is unstable in MI. The stabilization of the protein occurs in MII, after the first polar body extrusion. After parthenogenetic activation, the MycMISS protein does not accumulate and is, therefore, probably degraded. This observation is consistent with the fact that we cannot detect endogenous MISS in early embryos by immunofluorescence. The regulation of MISS stability during meiosis is in agreement with the presence of a true PEST sequence in the protein.
The activity that is involved in MISS degradation during MI and disappears in MII does not depend on the Mos/.../MAPK pathway because MycMISS also accumulates in mos-/- oocytes.
From studies in Xenopus oocytes, we know that MISS is phosphorylated by active MAPK in vitro (unpublished data), and we show that in vivo, MISS is partly phosphorylated by the Mos/.../MAPK pathway. First, we show that MISS is hyperphosphorylated in MII oocytes. Indeed, both endogenous and Myc-tagged MISS migrate at an apparent molecular weight much higher than the calculated one (45 instead of 29 kD), suggesting the presence of posttranslational modifications. Second, MycMISS protein migrates as discrete spots in 2D gel electrophoresis, which suggests different phosphorylation states. Third, treatment of MycMISS from MII oocytes with -phosphatase induces a severe downshift of the protein. Eventually, we show by 1D and 2D electrophoresis that MycMISS is in part phosphorylated by the Mos/.../MAPK pathway.
As MISS is stable only in MII, it is reasonable to assume that the protein is regulated in two steps: (1) stabilization, which is independent of the Mos/.../MAPK pathway but might depend on phosphorylation events by other kinases, and, most likely, (2) full activation through phosphorylation by the Mos/.../MAPK pathway. This implies that the protein is mainly active in MII, when CSF activity is present (Masui and Markert, 1971).
MISS is localized in dots on the MII spindles and disappears after fertilization
The endogenous MISS accumulates in dots on the MII spindles. Also, a MISSGFP fusion protein shows a strong localization of the protein as bigger patches along fibers that evoke kinetochore microtubules of the MII spindles. In interphase, although we cannot detect the endogenous MISS protein, the MISSGFP protein concentrates into dots inside the nucleus and is excluded from the cytoplasm. This suggests that the NLS present in the protein is functional.
We cannot explain why the MISSGFP protein accumulates in early embryos when the endogenous protein does not. One explanation could be that the activity that degrades MISS in GV, early MI, and activated oocytes is no longer present in early embryos, and therefore allows accumulation of an exogenous MISSGFP protein, whereas it does not allow its accumulation in GV or early MI oocytes. This would imply that a mechanism specific to meiotic maturation controls MISS stability, and this mechanism disappears in early embryos. Alternatively, MISS could undergo other posttranslational modifications during early development that prevent its recognition by the antipeptide antibody.
Nonetheless, we believe that MISS is no longer synthesized during early development. First, we showed that MycMISS, which behaves like endogenous MISS and can rescue the depletion of the endogenous protein, is no longer accumulated after parthenogenetic activation. Second, we showed that dsMISS has no effect on the first two mitotic divisions, although we demonstrated its activity on abundant exogenous MISSGFP RNA.
MISS is involved in MII spindle stability during the CSF arrest of mouse oocytes
The localization of MISS and MISSGFP to MII spindles is consistent with the phenotypes obtained after asRNA, dsRNA, and morpholino injection. Indeed, interfering with the endogenous mRNA by three different means induces severe spindle defects only in MII oocytes, where the protein is present, stable, and probably active. Moreover, we show that the phenotype is associated with a lack of accumulation of endogenous MISS protein in MII. Oocytes that do not possess MISS protein show disorganized spindles often lacking one pole, or have long microtubules emanating from both poles and numerous cytoplasmic asters, which are likely nucleated by cytoplasmic microtubule organizing centers (Maro et al., 1985). The phenotype is specific, because it is rescued by reintroducing MycMISS-encoding RNA into anti-MISS morpholinoinjected oocytes. This phenotype is also very consistent with the microtubule defects observed in mos-/- oocytes and with the localization of MAPK at spindle poles of mouse oocytes (Verlhac et al., 1994, 1996). The fact that mos-/- oocytes present some spindle defects could be due to MISS, which is not fully phosphorylated in this strain and probably not active.
Our results suggest that MISS is a spindle-associated protein that is involved in the stabilization of the MII spindle during the CSF arrest. As mentioned in the introduction, it is puzzling that although the Mos/.../MAPK cascade is active in MI, it does not induce CSF arrest in MI. MISS could be an important target of the MAPK cascade that is only stable in MII (Fig. 10). Because we do not observe a significant drop in histone H1 kinase activity as well as a degradation of cyclin B1 in oocytes treated with dsMISS, we think that MISS plays a role in controlling microtubule dynamics, through an interaction with a microtubule associated protein, rather than a direct role in CSF arrest (Fig. 10).
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Materials and methods |
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Preparation of the mouse oocyte cDNA expression library
Immature oocytes were removed as previously described (Verlhac et al., 1996), collected in sterile PBS, frozen immediately in dry ice, and stored at -80°C. Samples corresponding to 1,200 oocytes were lysed by freezing and thawing. RNA was prepared using the Rneasy Mini Kit (QIAGEN). The genomic DNA was digested by adding 2 U of RQ1 Dnase (Promega) and treating with 40 U of RNase inhibitor (Promega). The RNA was precipitated with ethanol and resuspended in water treated with 0.1% diethyl-pyrocarbonate (H2O-DEPC). Double-stranded cDNA was synthesized from RNA using a commercial cDNA synthesis kit (SMART PCR cDNA Synthesis Kit; CLONTECH Laboratories, Inc.), as recommended by the manufacturer, and using 5'-AAGCAGTGGTAACAACGCAGAGTGAATTCGCGGG and 5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAG(T)18 primers for the first strand cDNA synthesis. PCR amplification was performed as described in the cDNA synthesis kit using 5'-AAGCAGTGGTAACAACGCAGAGTGAATTCGCGGG and 5'-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTT primers. The cDNAs between 500 pb and 1 kb were gel purified and cloned into pYESTrp (Invitrogen) at EcoRI and XhoI sites. We estimated that the cDNA library consisted of 3 x 106 transformants.
Two-hybrid screen
A two-hybrid screen was performed using the Hybrid Hunter Kit (Invitrogen). The bait constructs were generated by PCR amplification from plexA-ERK2WT and plexA-ERK2KD (Waskiewicz et al., 1997) using 5'-TGACTAGGATCCGTATGGCGGCGGCGGCG and 5'-TGCATCGAGTTAAGATCTGTATCCTGG primers and cloned as a BamH1/XhoI fragment into pEG202. plexA-Su(Fu) (mouse suppressor of fused) was a gift of A. Plessis (Institut Jacques Monod, Paris, France). We thank J. Cooper for the gift of the pLexA-ERKWT and plexA-ERKKD plasmids.
The pEG202-lexA-ERK2WT construct was introduced into the yeast EGY48 (MAT ura3 trp1 his3 6lexAop-LEU2) strain transformed with pSH18-34. pEG202-lexA-ERK2WT was tested for spontaneous activation of the LEU2 and lacZ reporter genes. The EGY48 strain transformed with pEG202-lexA-ERK2WT and pSH18-34 was further transformed with the cDNA library cloned into pYESTrp. We screened 4 x 106 transformants.
The two-hybrid tests of specificity were performed using different baits in the strain RFY206 mated with the strain EGY48 containing the potential positive clones.
RT-PCR assay
RT-PCR was processed as previously described (Ledan et al., 2001). PCR amplification was performed using 5'-ATGTATCCCTTCATCCCTCCA and 5'-TCATTTAGACCCTTCACTTTC primers.
Plasmid construction and in vitro synthesis of RNA
pRN3MycMISS was constructed by RT-PCR amplification from mouse ovaries. Total RNA was extracted using the Rneasy Mini Kit (QIAGEN). The reverse transcription was performed on 500 ng of RNA. The PCR amplification was done on 50 ng of RNA/DNA using 5'-ATGCGAATTCAGAATGTATCCCTTCATCCCT and 5'-ATGCGCGGCCGCTTAAATTAAACTGTTTATTA primers. The 814-bp PCR product was then cloned into pRN3Myc2. pRN3MISSGFP was obtained by PCR subcloning at XhoI/EcoRI sites using 5'-ATGCCTCGAGATGTATCCCTTCATCCCTCC and 5'-ATGCGAATTCCTTTAGACCCTTCACTTTCA primers. The in vitro synthesis of capped RNA was performed as previously described (Verlhac et al., 2000b).
Microinjection
Microinjection of in vitrotranscribed RNA was performed as previously described (Verlhac et al., 2000b). The sequence of the control morpholino oligonucleotide is 5'-CATACTTCTTGCTGCTGCCCCGTAG and the sequence of the anti-Miss morpholino oligonucleotide is 5'-CATTCTTGTTGGTGCTGCTCCGAAG.
Coimmunoprecipitation in Xenopus oocyte extracts
Xenopus oocyte microinjection as well as extraction were performed as previously described (Gavin et al., 1999). For the immunoprecipitation of xp42mpk1, we used an anti-ERK2 antibody conjugated to agarose (Santa Cruz Biotechnology, Inc.). Xenopus oocyte extracts (300 µl) were precleared with 20 µl of protein A coupled to agarose beads for 30 min at 4°C. The cleared extracts were then incubated for 2 h at 4°C with the antibody coupled to agarose beads. The beads were washed four times in lysis buffer supplemented with 150 mM NaCl and then processed for immunoblotting.
Dephosphorylation assay
Just after collection and lysis, oocytes were incubated with or without 400 U of -phosphatase (New England BioLabs, Inc.) in
-phosphatase buffer at 37°C for 1 h and then processed for immunoblotting.
2D . gel electrophoresis
Oocytes were stored at -80°C in water containing 1 µM okadaic acid (LC Laboratories) and 80 µM ß-glycerophosphate. They were then processed as previously described (Louvet-Vallee et al., 2001).
Immunoblotting
Oocytes at the appropriate stage of maturation were collected in sample buffer (Laemmli, 1970) and heated for 3 min at 100°C. We used the following antibodies: an affinity-purified anti-MISS antibody directed against the peptide TMLHVNSQHESEGSK; the 9E10 monoclonal antibody (sc-40; Santa Cruz Biotechnology, Inc.) for Myc-tagged MISS; the monoclonal antibody (no. 1814460, Boehringer) against GFP-tagged MISS; the monoclonal antibody (no. M3530; Dako) against cyclin B1; and the anti-ERK antibody (sc-94; Santa Cruz Biotechnology, Inc.).
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Footnotes |
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Acknowledgments |
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This work was supported by grants from the Association pour la Recherche sur le Cancer (9913 and 5792 to M.H. Verlhac). C. Lefebvre and M.E. Terret are recipients of fellowships from the Ministère de l'Education et de la Recherche Technologique.
Submitted: 12 February 2002
Revised: 3 April 2002
Accepted: 5 April 2002
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