The EMBO Journal vol. 1 0 no. 1 1 pp. 3343 - 3349, 1 991

The role of cyclins in the maturation of Patella vulgata oocytes

Andre E.van Loonl'2,4, Pierre Colas1 2, Hans J.Goedemans2, Isabelle Neant1, Pascal Dalbon3 and Pierre Guerrierl 'Ecole Normale Superieure, Laboratoire de Biologie Cellulaire et Moleculaire, 46 Allee d'Italie, 69364 Lyon Cedex 07, France, 2Department of Experimental Zoology, State University' at Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands and -Biomerieux S.A., 69280 Marcy L'Etoile, France 4Corresponding author (Utrecht) Communicated by T.Hunt We have cloned and sequenced the cDNAs encoding Patella vulgata cyclins A and B. The cDNA clones contain an open reading frame of 426 and 408 amino acids respectively, which present similarity with cyclins from other species. Cyclin A and B RNAs are present as polyadenylated and non-polyadenylated RNA in prophase oocytes and are completely polyadenylated in metaphase I. During the first cleavages after fertilization the level of cyclin A and B mRNAs is high and drops when the free swimming stage is reached. Using pl3sucl-Sepharose bead precipitation we demonstrate that cyclin synthesis is triggered during maturation and that inhibition of protein synthesis makes the cyclins disappear rapidly from the metaphase I oocytes, which shift to interphase condition. By microinjecting antisense oligonucleotides into metaphase I oocytes, we demonstrate that in vivo ablation of cyclin A and B messengers together gives the same result, whereas microinjection of only one oligonucleotide does not show any effect. Key words: antisense oligonucleotides/cyclin A and B/G2-M transition/metaphase arrest/Patella vulgata

Introduction Studies on oocyte maturation of different organisms have identified a maturation promoting factor (MPF) which was shown to be the regulator of this process. It was first described as a factor present in Xenopus mature oocyte cytoplasm that upon microinjection causes G2-arrested oocytes to enter M phase (Masui and Markert, 1971; Smith and Ecker, 1971). MPF also has a regulatory function in the cell cycle of somatic cells. After the identification of the Xenopus homologue of the yeast p34cdc2 kinase, it became clear that oocyte maturation and cell cycle progression may be controlled by the same regulatory factors (Dunphy et al., 1988; Gautier et al., 1988). p34cdc2 was also shown to be a component of the universal histone H 1 kinase (reviewed in Wu et al., 1986) in starfish (Labbe et al., 1989a), sea urchin (Meijer et al., 1989) and Xenopus (Gautier et al., 1990). Thus, MPF and histone H1 kinase constitute two activities of the same entity. (©) Oxford University Press

Among the different regulators involved in the control of

p34cdc2 kinase, cyclin proteins are the best characterized. Cyclins were initially identified in cleaving sea urchin embryos (Evans et al., 1983). They were shown to associate physically with p34cdc2 in a complex and to be phosphorylated by this kinase (Draetta et al., 1989; Labbe et al., 1989b; Meijer et al., 1989; Pines and Hunter, 1989a; Gautier et al., 1990; Pondaven et al., 1990). The key importance of cyclins in MPF regulation is now well established: cyclin synthesis is necessary for entry into mitosis (Minshull et al., 1989; Murray and Kirshner, 1989) whereas cyclin destruction is required for exit from mitosis (Murray et al., 1989). Maturing oocytes are arrested in a, per species, defined stage awaiting fertilization (Guerrier et al., 1990a). If the arrest is at metaphase stage, MPF activity is high and stays high untill the oocyte is fertilized. The mechanism by which this state is maintained could tell us much about the processes which control the formation and breakdown of MPF. In oocytes of Patella vulgata, maturation proceeds in two steps exhibiting a first block at the germinal vesicle stage which can be released in vitro by increasing the intracellular pH. The second block in metaphase I is released upon fertilization (Guerrier et al., 1986). By inhibiting protein synthesis, it has been shown previously that the maintenance of metaphase I conditions requires a continuous supply of new short-lived proteins. Similar results have been obtained with oocytes of the annelid Chaetopterus (Zampetti-Bosseler et al,, 1976) and with the pelecypod mollusc Mytilus edulis (Dube and Dufresne, 1990). In the latter case, it has been shown that release from metaphase block, either by fertilization or emetine, involves a selective and rapid disappearance of a 50 kDa protein, which periodically appeared and disappeared during the meiotic and mitotic cell cycles. Maintenance of the metaphase I condition also requires protein phosphorylation since 6-DMAP, which induces protein dephosphorylation without affecting protein synthesis, leads to chromosome decondensation and the formation of resting nuclei as observed in Patella (Neant and Guerrier, 1988) and Mytilus (Guerrier et al,, 1990b) as well as in the ascidian Phallusia mamillata and the japanese clam Ruditapes philippinarium (P.Guerrier, unpublished). The aim of the present study is to determine whether cyclins are actually involved in maintaining MPF activity during the metaphase block and to follow their expression during early development. Indeed the embryo of Patella, like that of Drosophila, should constitute an excellent system for studying the role of cyclins during early development since its cleavage and differentiation pattern are well known (van den Biggelaar, 1977), can be manipulated experimentally (Kuhtreiber et al., 1988) and seem to correlate with the communication compartments of the embryo (Serras, 1990). We have cloned and sequenced the cyclin A and B cDNAs 3343

A.E.van Loon et al.

of Patella vulgata. The putative encoded proteins resemble cyclins of other species to a high extent. During maturation both cyclin mRNAs are polyadenylated. The amount of cyclin mRNA decreases when the embryo reaches the free swimming stage. Using pl3sucl-Sepharose bead precipitation we examined the synthesis of cyclins during maturation. By ablating the mRNAs encoding these proteins we show that either cyclin A or B is sufficient to stabilize metaphase I conditions.

Results The cyclins of Patella vulgata In a first screening with a mixed probe containing cyclin A and B cDNAs from Spisula solidissima, we identified 180 positive clones out of 50 000 clones screened. One clone hybridizing with the Spisula cyclin A cDNA (Swenson et al., 1986) and one hybridizing with cyclin B cDNA (Westendorf et al., 1989) were subcloned in pGEM5 and their nucleotide

A AGACAGAGCGTTTATTTAAAAGCTGGTCTGTTCACTTCAACCTTGACTAGCACACCCGGAACCAGAAAAAGATTTTAGTTTGAAACTTGTTTTATTTATTTGTTTTAGAAAGTTGTGTCG GCTTTTATTAATTTGCTCGTTTGCAAAAATTTATTGCAGCGAGATTCTTACGGAACTGATTTCAAAATGTCTATGGTTCACGGGAGTTCGTTTCAAATTGCACAGGACGGTGAAAATGAG M S M V HG S S F Q I A Q D G E N E AATCAAGGTGTTCAAAGAGTGAAAAAAGCTGGATTGACTGCCCGTGGAAATGTAGCAGTTGCCAAACGATCAGCACTGGGAACAATAACGAACCAAAATATTAGAGTACAACCATCAAGG N Q G V Q R V K K A G L T A R G N V A V A K R S A L G T I T N Q N I R V Q P S R

GCCGCAAAGTCGGGTAATGCAGATTGCCAGGATGAAAATGTTTTTGCAAAACAGAAGTCTTTTGGATCTAGTAACAATGAAAACAAAGGATTTAAAATTCATGTTGATGAACCAACAGTA A

A

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CAGGTACTCACAACTGCTACATTGAAAACTACAAGACAAAGTGAAGACGAAGACATAAAATTAAATGACCAAGTGACATCTTTACCATCTTTACAAGCATTAGAAGACATCCAAGTAGAC Q V L T T A T L K T T R Q S E D E D I K L N D Q V T S L P S L Q A L E D I Q V D AATGAGAATGGCTCGCCCATGGTATTGGATGTTACTATTGAAGATGCAGAAAAGAAACCCATTGATAGAGAAGCTATTATCCTTTCAGTTCCAGAATATGCAGAAGATATTTATAAGCAT N

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CTAAGAGAAGCAGAGTCGAGACACCGTTCTAAGCCCGGCTACATGAAGAAACAACCAGATATAACAAATTCTATGAGAAGTATTTTGGTTGACTGGATGGTTGAAGTTTCGGAAGAATAT L

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AAATTACATCGAGAAACCCTTTTCCTAGCTATAAATTATATTGACAGATTCCTCTCACAAATGTCTGTATTAAGAGGCAAATTACAACTTGTTGGAGCAGCTAGTATGTTTATTGCATCG K

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AAATATGAAGAAATTTACCCACCAGAGGTTTCCGAGTTTGTATACATTACTGATGATACCTATGAACAGAAACAAGTCCTCCGTATGGAACATTTAATATTGAAGGTCTTGTCATTTGAT K Y

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GTAGCTCAACCTACGATAAACTGGTTTACCGATACCTACGCAAAAATGGCTGATACTGATGAAACAACGAAATCTTTATCTATGTATTTATCTGAACTAACATTAGTGGATGCCGACCCT V A Q P

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TATTTAAAATATTTACCCAGTACAATTGCAGCAGCATCTTTATGTTTAGCAAATATCACATTAGGCAGTGAACCATGGCCTTCAAGTTTAGCCAAAGAATCAAAGTATGAAATTAGTGAA Y

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TTTTCTGAATGTTTACAAGAAATGTATCAGACATATTTAAATGCACCAAATCATCCACAACAAGCCATTAGAGAAAAATACAAGTCATCAAAATATCAACAGGTATCATCTATCAGTCCT F S E C L Q E M Y Q T Y L N A P N H P Q Q A I R E K Y K S S K Y Q Q V S S I S P CCCTCCAGTTTGTCCTTCACCATGTAGTCAGCTTCTCTTATCAGTTCTACCACAGTTCTTTCAACAGCCGACCCACCAAATTCAAATCATAGGACACGAAATTTTAATATGCATGGATTC P

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ATAATCTCATTTTTGTTTTTTTAAATCGTTCCAATCATTTTAAATCATTTTTTTAATCAACAGAATTTGTTTTTAATTAAAACTATGGTATTGTTTTTATAATAGTGCAATATAAAGATG GTCATTGTAATAAATGATCTAATCATGTTAATAGTGTGAATGTATTTTGAATGTATTTTTTGATTGTATTTTTTGCATGGAGTGAGTGAATGGGACCAAGACATTTATAAATCAGTGAAT TAAATCCAGAGTTAAACCATAATTTTTAATCACTGTAAATAAAATTGCACTATTAATTTTAAAGATTAAGTTTTAATTTAATTTGTTTTTAAAATGGATAAATTATCGGTGCAAATTTTG

TAAGTCGGATAGTTTATTTATTTTCAGAGTTTTCCCATTTTTACACACTAAACTGTTAAAATTTCTGTTTCTTCTATTTTCTTAGTTTTCTCATCTTTTGTTTCTGTCTTCCTTCGGCTG TAACTGTGCTACTCTACAGCGTTCTGTGTAAACCTGGGAATTCTCTTTAAACTAGTATTGTGTAGATTTTATGACAAGCTTTTGTAAATGGCATTCCTTTAAT 2143

B AAACTAATGCTTATATTCCAGTCTCTGTTGTAAATCCATGTTTGAAAATGACTACAGTTACACGCTCTTCATCGGCTAATCTTGGTGCTAGTCAAAAGTTGGCTGTGAAGAAAGGTGATG_ M

T T V

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CAATGATGTCGAGTAAAGGTATTTCCAACCGAAGCAATCGGTCTGCTTTAGGAGACATAGGAAATAAAGTTTCTAATATGACTATTGACCCAACGAAAAAAGCATTAGGCCTACCTGTAA A M M S

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TCAAAAAGGAGATAATTCAGAAATCCAAGTTTACAAGAAGTAAGACTACCGTTTCGGAATCGGATATCTTGTTACAGGAGAAAGAGTCTGCCTGTTGTAGTAGAGCTTATACCATCTTTA I

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AAGATGCCATCGAACCAATTGTGTCGACTGTTGATCTTATGGACATTAGTGAAGACAAACCAGACGCCTTCTCCAAAGTTTTATTAACAGTTGAAGATATTGACGCCAACGACAAAGATA K D A I

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ACCCCCAACTTGTCAGTGACTATGTCAATGATATTTACCATTACATGAGACATTTAGAGGAAACGTTTGCCGTAAAAGCTAACTTTCTTGAAGGACAAGAAGTAACTGGTAAAATGAGAT N

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CAATCTTGATTGATTGGTTATGCCAAGTTCATCACAGATTCCATCTATTACAAGAAACTTTATATTTAACGGTTTCTATAATAGACAGGTTTTTGCAGGTTCATCCGATTTCTAGAAATA S

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AGTTGCAGTTAGTTGGGGTTACCTCAATGCTGCTTGCTTCAAMAATATGAAGAAATGTACGCCCCCGAAGTGGCGGATTTTGTGTACATAACGGACAATGCTTATACCAAAGCCGATATTAA K L Q L V G V

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GGACTATGGAACAAACAATATTAAAAACACTAGATTTTAGTTTTGGAAAGCCATTGTGTTTACATTTTTTACGAAGGAATTCTAAAGCTGGTCAGGTTGATGCAACCAAACATACACTTG R T M E Q T

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CCAAGTATTTAATGGAACTTACCATTATAGAGTATGATATGGTGCATTGTAATCCATCTATTATAGCTGCTGCAGCTTTATGTTTATCTATGAAGGTGCTAGATGATTCACAATGGTCTG

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AAACCTTAGCTCATTACAGTAACTACTCGGAAAAAGAGATTTATCCAGTAATGCAGAAATTAGCTCAGTTGGTGGTTAAAGCAGAAACTAGTAAATTAACGGCTGTAAAGATAAAATATT E

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CCAGTTCAAGGTTTATGAAAATCAGTTCCATTCCAGAATTGAAATCTAATGCTATAACGGATCTCGTATTATAGTGAATGTAGTGTAATTGTTTATTGACAATTTTATATAGTTGAGTGT S S S R F M K

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TGGAAGAATGCCAGAATTCATATTTGTATCAAGTGTCAGAATGCAACATGTCTTTCATATGTATTTTTATATTGTATTGTGTTTATTTGTTGGTATATGTGAATGAAAACAAGCACGATT TTTATTTGTATGAATGTTTTAATCAATGCCTTTCCAAATGTTTTTTATAGTGAAATACTGTACTAATTATGTAAGATTGTTTTAAAATGGCGTTTTTGTTTTTAAATGTCGACGGACTTG TATAAATAACACTGTGGATGTTTTTCGAAGTCCAGGAACATACAGAATTGCAATGAATGACGAATCAAGTCCTGTCACATGACCTTATCACTGGGGTCTTGTCTCATTCGTGGGATTGGA AATCAATTCTGAAACATTTCAATGAAGTATCCCCTAGGTGTTAGGGAAATTGTAAGCACTGCAGAATCATGTACAGTTTTAATAATACTAATTGACATGTACAGGGTTAAAACAGACCAA AAGAAATGACTGTGATTTTTAGCTTATTCCAT 1832

Fig. 1. Sequence of Patella vulgata cyclins A and B. The coding strand of the cloned cyclin A (A) and cyclin B (B) cDNAs are shown with the encoded amino acids in one letter code. The T-rich region in cyclin A (---), polyadenylation signal (-) and oligonuclotide specific for cyclin A or B (- -) are indicated. -

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determined. The nucleotide sequence of the coding strands with the predicted amino acid sequence is shown in Figure 1. The cyclin A clone has a total length of 2143 nucleotides (nt) and an open reading frame of 426 amino acids (aa) starting with the ATG at nt 187 with an A at the -3 position conforming to the Kozak consensus sequence for the initiation codon (Kozak, 1986). No ATGs are present in the thus defined 5' untranslated region (UTR). The 3' UTR is 678 nt long. The cyclin B cDNA has a total length of 1832 nt with a 408 aa open reading frame starting with the ATG at nt 48. This ATG matches the Kozak consensus sequence for an initiation codon (Kozak, 1986). Two out of frame ATGs are found upstream of this initiation codon which do not have an A in the -3 position. There are two in frame ATGs starting at nt 126 and 198 with an A at the -3 position. The stop codon at nt 1273 results in a 3' UTR of 560 nt. A number of structural features from the cDNAs can be distinguished. Both cyclin A and B from Patella contain a number of ATTTA motifs in the 3' UTR (11 and two copies respectively) which were shown to be associated with the susceptibility of mRNA to degradation in human (Shaw and Kamen, 1986). The presence of these sequence motifs in cyclins was first noticed in starfish cyclin B mRNA by Labbe et al. (1989b). In cyclin B mRNAs from other species a varying number of this sequence element was reported [between nine in Spisula (Westendorf et al., 1989) and none in human (Pines and Hunter, 1989)]. Cyclin A mRNAs from other species also contain this element in the 3' UTR: twice in the cloned 3' UTR of both Spisula (Swenson et al., 1986), Drosophila (Lehner and O'Farrell, 1989) and Xenopus (Minshull et al., 1990) and several times in the cyclin A from human (Pines and Hunter, 1990). In human cells both cyclin A and B mRNA levels drop rapidly after mitosis. In Drosophila, cyclin A mRNA is degraded following mitosis 13 (Lehner and O'Farrell, 1989). Whether the ATTTA motif is required for these degradations is still not known. Two related structural features of the 3' UTR should be mentioned: the presence of the polyadenylation signal (AATAAA) which is present only in cyclin A cDNA beginning at nt 1838, and upstream of this signal, the T5A2T site which is probably required for the polyadenylation of maternal mRNAs (McGrew et al., 1989). The T5A2T signal in Patella is located 7 nt upstream of the poly(A) signal, a position similar to that found in Spisula. In Patella the cyclin A mRNA is polyadenylated at maturation (see below). Both in Patella and Spisula cyclin A, the polyadenylation site is located upstream of the putative poly(A) tail much further away than the proposed optimal distance in vertebrate cells (Birnstiel et al., 1985). In other cyclin A mRNAs (Drosphila and human) the distance between the poly(A) tail and the AATAAA site is much shorter and these mRNAs do not contain the T5A2T sequence

sequence.

The calculated molecular weight of the predicted cyclin A and cyclin B proteins are 48 kDa and 46 kDa respectively. This differs from the measured molecular weight (see Figure 4) as was found in other species. Table I compares the derived amino acid sequences encoded by cyclin cDNA clones of different species. The amino acid identity of both Patella cyclins is highest with the corresponding Spisula cyclins, which is not surprising since they belong to the same phylum. The central region of different cyclins is even more conserved, while the replacements are conservative.

Furthermore, cyclin A from Patella is

more

homologous

to cyclin A from other species than to Patella cyclin B, which supports the already suggested independent evolutionary

conservation of cyclins A and B. A previously unnoticed difference between cyclin A and cyclin B proteins is their calculated pI. For cyclin A, this tends to be more acidic (between 4.9 and 6.1) than for cyclin B (7.4 for human cyclin B and between 8.0 and 9.6 for the other cyclin Bs shown in Table I). Cyclin messengers during development In order to establish the mRNA levels during maturation and early development of Patella vulgata we isolated RNA from different stages of development and performed Northern blots (Figure 2). The mRNA length is 2.9 and 2.5 kb for cyclin A and cyclin B respectively. For cyclin A this compares closely to the 2950 nt found for Spisula cyclin A mRNA (Swenson et al., 1986). Maturation of Patella oocytes has a dramatic effect on the polyadenylation of cyclin RNA (both A and B). At metaphase all cyclin A and B mRNAs appear in the poly(A)+ fraction (Figure 2A). Polyadenylation upon maturation of cyclin A and B mRNAs was also reported for starfish (Standart et al., 1987; Tachibana et al., 1990). After fertilization, the level of RNA encoding both cyclins increases until the 32-cell stage (1 and 3.5 h after fertilization, Figure 2B). It then stays at the same level until the early trochophore stage (12 h after fertilization), at which time the level of cyclin mRNA falls to a barely detectable level. Thus high levels of cyclin mRNA are correlated with early cell divisions. Cyclins and MPF activation Neant and Guerrier (1988) have previously shown that meiosis reinitiation in Patella is accompanied by a quantitative increase in protein synthesis and by the de novo Table I. Comparison between Patella vulgata cyclins and cyclins of other species Patella vulgata cyclin A Patella vulgata cyclin B Total protein aa 180-360 Total protein aa 161 -341 Patella A'

Spisula A2 Drosophila A3 Human A4 Xenopus A5 Patella B' .Spisula B6 Drosophila B7 Arbacia B5 Marthasterias B9 Xenopus B"'° Xenopus B210 Human All

100/100 45/61 32/51 38/57 38/55 25/45 25/46 16/38 21/41 24/42 20/39 20/40 20/43

100/100 73/86 60/83 62/78 64/82 38/59 40/60 28/51 40/61 39/60 35/59 39/61 34/59

25/45 21/40 19/41 17/40 21/43 100/100 54/70 25/46 33/50 43/60 38/58 40/57 39/60

38/58 38/56 34/57 33/51 36/56

100/100 80/89 39/63 64/78 70/86 63/83 66/82 62/82

The percentage of identical or similar amino acids are given, before and after the slash (/) respectively. The region of aa 180-360 in cyclin A of Patella and 161 -341 in cyclin B, show the highest similarity. The aa sequences of cyclin A (A) or B (B) from different species were published in the following papers: 'this study, 2Swenson et al. (1986), 3Lehner and O'Farrell (1989), 4Wang et al. (1990), 5Minshull et al. (1990), 6Westendorf et al. (1989), 7Lehner and O'Farrell (1990), 8Pines and Hunt (1987), 9Labbe et al. (1989b), 0Minshull et al. (1989), "Pines and Hunter (1989).

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A.E.van Loon et al.

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Fig. 4. RNase H-mediated destruction of cyclin A and B mRNAs in vitro. Total RNA was incubated with anti-cyclin A or anti-cyclin B oligonucleotide indicated in Figure 1. The RNA was treated with 41

F-

Fig. 2. (A) Polyadenylation of cyclin mRNAs during maturation. 0.2 ,tg poly(A)+ RNA from prophase and metaphase I oocytes (lanes 1 and 3 respectively) and 20 Ag poly(A)- RNA from the same stages (lanes 2 and 4) were separated on a formamide containing agarose gel, blotted onto nitrocellulose and probed with cyclin A or B cDNA. The molecular weight markers are in kb (1.4 and 2.4) are shown on the right side. The length of the cyclin A and cyclin B mRNAs are indicated. (B) mRNA steady state levels of cyclins A and B during development. At the indicated time after fertilization RNA was isolated and a Northern blot was perfomed.

RNase H and translated in vitro. Lane 1: without oligo, lane 2: oligo anti-A, lane 3: oligo anti-B, lanes 4, 5 and 6: immunoprecipitation with an antibody directed agains a cyclin A peptide (see Materials and methods) of in vitro translated products shown in lanes 1, 2, 3, 7, 8 and 9: immunoprecipitation with an antibody directed against a cyclin B peptide (see Materials and methods) of in vitro translated products shown in lanes 1, 2 and 3. Molecular weight markers are shown on the left in kDa. Cyclin A and B are indicated by arrows.

performed on lysates of oocytes labelled in vivo with [35S]methionine. During prophase stage (lane 1), cyclins are almost undetectable whereas, at the metaphase stage, they appear strongly labelled (lane 2), thus demonstrating that a dramatic increase in cyclin synthesis occurs during maturation. The increase in protein synthesis does not affect all proteins, since less p34cdc2 seems to be synthesized in metaphase than during prophase. More than two bands are obtained in the region of the molecular weights of cyclins. They probably result from different states of phosphorylation of the cyclins, as treatment with alkaline phosphatase reduces the number of bands (not shown).

Fig. 3. pl3sucl-Sepharose beads precipitation from prophase and metaphase oocytes. Prophase and metaphase oocytes were labelled in vivo with [35S]methionine as described in Materials and methods and pI3Suc1 -Sepharose beads precipitation was performed. Lane 1: prophase oocytes, lane 2: metaphase oocytes, lane 3: emetine treated oocytes. Cyclins ( [ ) are indicated. Molecular weight markers are shown on the left in kDa.

Cyclins and maintenance of metaphase I conditions Inhibition of protein synthesis by emetine drives metaphase I arrested Patella oocytes towards interphase within 0.5 h (Neant and Guerrier, 1988). In order to determine whether cyclins are among the first proteins that disappear in emetine treated oocytes, we performed an affinity precipitation on a lysate prepared 30 min after emetine addition and checked for the appearance of resting nuclei in the same batch. Lane 3 (Figure 3) shows that cyclins are no longer present in the treated oocytes while a p34cdc2 signal is still present. It can thus be concluded that cyclins are among the first proteins to disappear in emetine-treated oocytes, suggesting a short half-life of 15 min. Because of this short half-life, it is likely that cyclins belong to that group of proteins whose constant synthesis is required for the maintenance of metaphase I conditions. To address this question directly, we designed two antisense oligonucleotides (oligos) from the cyclin A and B cDNA sequences (indicated in Figure 1), to inhibit specifically cyclin protein synthesis in vivo. First, we checked the -

synthesis of a few proteins. As cyclins play a major role in MPF activation, we determined whether they belong to those newly synthesized proteins. To visualize cyclins, we used the pl3sucl yeast gene product, which, coupled to Sepharose beads, was shown to bind the p34cdc2 -cyclin complex from all species tested so far. Figure 3 shows the result of such affinity precipitations 3346

Cyclins of Patella vulgata

specificity of these oligonucleotides by incubating each of them with total RNA extracted from metaphase I-arrested oocytes in the presence of RNase H (Standart et al., 1987). Figure 4 demonstrates that each oligo leads to the disappearance of one band which is precipitated, in the absence of the oligos, with antibodies directed against oligopeptides which are part of the cyclin proteins. We conclude that the oligos are specific for Patella cyclin A and B messengers and can be used to try to inhibit in vivo cyclin protein synthesis. The oligonucleotides were thus microinjected into metaphase I-arrested oocytes. After 2.5 h, oocytes were fixed and stained with Hoechst in order to visualize their chromosomes. In two independent experiments a total of eight oocytes were injected with cyclin A together with cyclin B antisense oligo. Each of these oocytes exhibited resting nuclei, whereas uninjected control oocytes remained arrested in metaphase I (Figure 5). When antisense oligonucleotides were injected separately (28 injections performed with the cyclin A antisense oligo and 19 with the cyclin B oligonucleotide), no effect was observed (Figure 5). In each injection 1 ng of oligonucleotide(s) was injected and no attempts have been made to determine the threshold concentration of the oligos required to obtain metaphase disruption. A good internal control, however, arguing for the innocuity and specificity of these oligonucleotide injections is provided by the fact that no effect could be observed when cyclin A or cyclin B antisense oligos were microinjected separately at the same concentration as used for the injections of both antisense oligos together. From these data we conclude that a constant synthesis of cyclin A or B is required to maintain the metaphase I state in the oocytes of Patella vulgata.

Discussion cDNA sequence and gene expression We have cloned and sequenced Patella vulgata cyclins A and B and determined the abundance of their respective mRNAs during early development. The putative proteins encoded by these cloned cDNAs resemble Spisula cyclins more than cyclins from other species. Furthermore, Patella cyclin A resembles cyclin A from other species more than cyclin B from Patella and vice versa, confirming the already suggested independent evolutionary conservation. The resemblance is even higher when one considers only the middle part of the proteins. After maturation the total amount of cyclin messenger increases up to the fifth cleavage (3.5 h after fertilization). As the number of cells continues to increase, the number of cyclin mRNAs per cell decreases after this stage. This decrease is even more prominent in the trochophore larva where the level of cyclin mRNA is very low compared with that in the cleavage stages. Whether a direct correlation exists between the level of cyclin RNA and the division rate of individial cells has to be established.

Cyclin synthesis and MPF activity The use of p1 3suc I-Sepharose beads enabled us to show that the onset of cyclin synthesis coincides with meiosis reinitiation and therefore with the activation of MPF. The onset of cyclin synthesis is correlated with the polyadenylation of the mRNAs which was shown to be

Fig. 5. Effects of emetine treatment and of the microinjection of cyclin antisense oligonucleotides on metaphase I-arrested Patella vulgata oocytes. (A) Hoechst 33342 staining of a control uninjected oocyte. (B) Hoechst 33342 staining 90 min after treating the metaphase I oocyte with 90 mM emetine. (C) Polynuclei observed in vivo in a similarly treated oocyte. Nucleoli are visible. Nomarski optics. (D) Hoechst 33258 staining of a metaphase I oocyte which was injected 150 min earlier with both anticyclin A and B oligonucleotides. The bars represent 25 Am.

required for protein synthesis from mRNAs (McGrew et al., 1989). The coincidence of cyclin synthesis and meiosis reinitiation is in agreement with the large body of recent data which describes cyclins as MPF activator proteins (see Introduction). However, Neant and Guerrier (1988) showed that meiosis reinitiation in Patella does not require protein synthesis since germinal vesicle breakdown can occur normally in the presence of emetine. It is thus likely that the prophase I-arrested oocyte contains stockpiles of cyclin A and/or cyclin B proteins that could be unmasked by the increase of intracellular pH which triggers maturation (Westendorf et al., 1989). The pl3sucl affinity precipitations also allowed us to determine that cyclins disappear rapidly in metaphase Iarrested oocytes treated with emetine. This result indicates that cyclins exhibit a short half-life (- 15 min). It also suggests that the effect of emetine can be accounted for by the disappearance of cyclins. To verify this assumption we performed an in vivo antisense oligonucleotide-mediated destruction of cyclin messengers. Antisense oligonucleotides have been used in Xenopus oocytes to destroy either exogenous (Jessus et al., 1988; Cazenave et al., 1989) or endogenous messengers (Shuttleworth et al., 1988). In these cells, the mechanism of destruction involves a strong RNase H activity (Cazenave et al., 1987). In Patella oocytes the biological effect exerted by these oligonucleotides could be due either to a similar activity or to an inhibition of the initiation of translation due to the presence of a RNA -DNA duplex near the initiation codon (Figure 1). In order to check the specificity of the oligos we designed

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against cyclin mRNAs, in vitro translation products of total oocyte RNA were subjected to immunoprecipitation. We were able to precipitate the in vitro translation product of cyclin mRNA. Microinjection of anti-cyclin A and cyclin B oligos together led to the formation of resting nuclei in all the injected oocytes. This demonstrates that the specific inhibition of the protein synthesis of both cyclins is sufficient to reproduce the effects brought about by the general protein synthesis inhibitor emetine. The failure of the microinjections of only one oligo (anti-A or anti-B) to produce resting nuclei shows that this effect cannot be accounted for by the microinjection itself or by the simple introduction of an oligonucleotide into the oocyte. However, the synthesis of cyclins is not the only requirement for maintaining metaphase I conditions. Indeed, the in vivo application of 6-DMAP, which inhibits protein phosphorylation, also triggers the transition from metaphase I to interphase without affecting cyclin synthesis or the association with p34cdc2 (our unpublished data). The regulation of MPF through the synthesis of cyclin proteins has been hinted at in other molluscs (Dub6 and Dufresne, 1990). However, in this case the inhibition of protein synthesis leads to the formation of a polar body, which indicates that the nuclei have gone on to the interphase between the two meiotic divisions. The regulation of MPF in molluscs through the synthesis of cyclin is different from that found in Xenopus where the activity of MPF is under the control of active cytostatic factor, which is the equivalent of mosXE kinase (Sagata et al., 1989). As the mos proto-oncogene is probably not present in invertebrates (unpublished results cited in Roy et al., 1991), MPF activity is regulated in these species via synthesis of cyclin protein. Inhibition of translation of either cyclin A or cyclin B does not induce the shift from metaphase I to interphase. This result suggests that cyclin A and B have the same function in the maintenance of MPF activity. The ability of clam cyclin A alone to cause a metaphase arrest when added to Murray extracts, supports this conclusion (Roy et al., 1991). Although other data argue also in favour of a similar role of both cyclins in MPF activation (Westendorf et al. 1989; Murray and Kirshner 1989), the independent evolutionary conservation of cyclins A and B and several recent observations in Drosophila (Lehner and O'Farrell, 1990; Whitfield et al. 1990) and in Xenopus (Minshull et al., 1989, 1990) indicate that cyclin A and B fulfil distinct roles in the control of mitosis, at least in some species and/or at some stages of development. We think that Patella should offer a very attractive model system for further distinguishing the respective roles of cyclin A and B during maturation and early development.

Materials and methods cDNA library construction, clone isolation and sequence analysis The cDNA library used for the isolation of the Patella vulgata cDNAs encoding cyclin A and B was made from mRNA (see Northern blots) isolated from oocytes treated for 7 min with Millipore-filtered sea water of pH 8.8. This treatment induces germinal vesicle breakdown after which oocytes arrest in metaphase I stage until fertilization (Guerrier et al., 1986). Synthesis of double stranded cDNA and addition of linkers were performed with the Pharmacia cDNA synthesis kit. The double stranded cDNA was ligated into EcoRI digested XgtlO arms (obtained from Promega) and packaged with Promega packaging extracts. The primary library was amplified from 0.8 x 106 to > 1010. 50 000 plaques were screened with a mixed probe of

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Spisula solidissima cyclin A and B cDNA kindly provided by Dr J.Ruderman (Swenson et al., 1986; Westendorf et al., 1989). Hybridization was carried out in 5 x SSPE, 0.1 % SDS, 5 x Denhardt's solution, 10 Agg/ml Escherichia coli DNA at 65°C for 16 h (Sambrook et al., 1989). After hybridization the filters were washed twice in 6 x SSPE, 0.1 % SDS for 15 min at 20°C, 2x in 4x SSPE, 0.1% SDS and once in 3x SSPE, 0.1% SDS at 37°C. Ten clones were rescreened in the same way and DNA was isolated from five clones. The identity of the different clones was established by a Southern blot of NotI and EcoRI digested DNA. Two clones hybridized with the Spisula cyclin A and three hybridized with the Spisula cyclin B cDNA. Cyclin A hybridizing clones had identical insert lengths and EcoRl restriction patterns. The same was observed for clones hybridizing with clam cyclin B. The inserts of one cyclin A and one cyclin B clone were subcloned into the pGEM5 (Promega) NotI site. Subsequent deletions from both sides of the clones were constructed with the Promega Erase-a-base kit. The nucleotide sequence from these deletions was determined with the dideoxy chain termination reaction using the Pharmacia T7 sequencing kit (Sanger et al., 1980). Sequence data were analysed with MacVector and PC Gene.

Northern blots and DNA manipulations RNA from different developmental stages were isolated as described in Rosenthal and Wilt (1986). Northern blots were performed using standard methods (Sambrook et al., 1989). DNA modification enzymes and restriction enzymes were purchased from Boehringer Mannheim or Pharmacia and used according to the manufacturer.

Oocyte labelling and p 13"c1 - Sepharose beads affinity precipitations Oocytes were obtained at different stages as described previously (Guerrier et al., 1986). To label the proteins synthesized in the oocytes, a 20% cell suspension at the desired stage was incubated for 2 h with 75 yCi [35S]methionine (Amersham, SJ1515). For each sample, 0.5 ml was collected, pelleted, frozen in liquid nitrogen and stored at -70°C until use. Each frozen pellet was thawed in 0.5 ml of cold bead buffer and homogenized. After a 14 000 r.p.m. centrifugation, the supernatant was collected and incubated in a total volume of 1 ml with 10 IL pI3SuC -Sepharose beads. Affinity precipitations were performed essentially as described in Meijer et al. (1989). Proteins were analysed on

10% SDS-polyacrylamide gels. Preparation of the oligopeptides and antibodies Oligopeptides were selected by using several computer programs to predict major antigenic sequences (Hopp and Woods, 1981; Karplus and Schulz, 1985; Kyte and Doolittle, 1982; Parker et al., 1986). Oligopeptides corresponding to aa 74-88 of cyclin A and aa 68-86 of cyclin B proteins were synthesized on a Model 430A Peptide Synthesizer (Applied Biosystems) using t-boc protected amino acids. The resin-bound protected peptides were dried and then treated for 45 min. at -2°C with 10 ml of hydrogen fluoride, 1 ml anisole and 1 ml dimethylsulphide per gram of resin. Purification of the peptides was carried out by RP-HPLC on a Vydac C18 column (The Separations Group) (2 x25 cm) in 0.1% TFA using a linear gradient of acetonitrile. The peptides were checked for homogeneity by analytical HPLC. Their structures were confirmed by amino acid analysis and mass spectrometry. Conjugates were prepared using glutaraldehyde to couple peptides via their NH2 groups to the tetanus toxoid. The amounts of peptide and carrier were calculated to give - 1.2 peptide NH2 equivalents for each amino group on the carrier. The coupling reaction was carried out at 200C in 0.1 M sodium bicarbonate, pH 8, with glutaraldehyde at 2.63 mM. The reaction was allowed to proceed for 6 days with constant stirring, followed by dialysis against PBS. Antisera were obtained by immunization of Dunkin Hartley guinea pigs with the peptide-tetanus toxoid conjugates emulsified in complete Freund's adjuvant. After the first injection with 0.1 mg of the conjugates in the footpads, the animals were boosted subcutaneously 4 weeks later. Blood was drawn 6 weeks after initial immunization. The presence of antipeptide antibodies in the immune sera was determined by ELISA assays.

Immunoprecipitation An aliquot of each in vitro translation mixture was taken and prepared for protein electrophoresis. The rest underwent immunoprecipitation as described in Westendorf et al. (1989), using 1/100 diluted antibodies. Samples were loaded on a 10% SDS-polyacrylamide gel. In vitro oligonucleotide-mediated destruction of cyclin A and B mRNAs The oligonucleotides specific for cyclin A and B mRNAs whose sequences are indicated on Figure 1 were synthesized by G.Lombard-Platet using an Applied Biosystems DNA synthesizer. The RNase H digestion step was

Cyclins of Patella vulgata performed as described by Standart et al. (1987). RNAse H treated RNA were translated in vitro with an Amersham kit according to the instructions of the manufacturer. Proteins were analysed on 15 % SDS-polyacrylamide gels.

Microinjections Antisense oligonucleotides were dissolved in 10 mM HEPES pH 7.0 and microinjected according to the method of Hiramoto (1974) as precisely described in Meijer et al. (1984) and Kishimoto (1986). Briefly, the metaphase I arrested oocytes were maintained in artificial sea water (ASW) between two pieces of coverslip which formed a sharp acute angle and were supported by a U-shaped piece glued to a slide. Using a Narishige IM200 microinjection device, each sample (- 100 pl) was aspirated into an oil-filled micropipette (tip diameter: 10 tim) followed by a small volume of silicone oil (DC200, Serva, Heidelberg). The samples were thus pressureinjected between two drops of silicone oil. When a complete set of injected oocytes was obtained (i.e. 12-15 oocytes within 1 h), all the oocytes of the preparation were flushed out into a small finger bowl containing ASW. About 150 min after injection of the last oocyte, both injected and control companion uninjected oocytes were fixed for 1 h in 2% formaldehyde in glucamine acetate buffer, washed and stained with 0.5 tg/ml of the DNA specific fluorescent probe Hoechst 33258 (Dufresne et al., 1988). Observations were carried out with an Olympus epifluorescence microscope.

Acknowleaements The order of the first two authors is arbitrary; they both contributed to the same extent to this paper. We wish to thank Dr Joan Ruderman for her kind gift of Spisula cyclin A and B cDNA clones. We thank Dr M.Jolivet for helpful discussions on the oligopeptide synthesis and antibody preparation. Many thanks are also due to Drs Tim Hunt, Roy Golsteyn and Marcel Dor6e for providing p13suc -Sepharose beads and to Dr J.A.M.van den Biggelaar for critical reading of the manuscript. We acknowledge the exellent technical asssistance of Luc Vauzelas at Biomerieux. We are grateful to Dr Brichon and the fishermen of the Station Biologique de Roscoff for collecting and sending Patella vulgata. We wish to thank sincerely Mr Maxims and Mr Taitinger for their continuous and stimulating support. This work was supported in part by a grant from the Association pour la Recherche sur le Cancer (ARC6462) and by the Fondation pour la Recherche Medicale. A.E.v.L. was supported in part by CNRS and P.C. was supported by a short term fellowship from EMBO.

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Note added in proof The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers X58357 (cyclin A) and X58358 (cyclin B).

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