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	Figure 4. Schematic representation of the 3′ part of the gene encoding cyclin B in S. granularis. nt 1 is the first nucleotide of CB5 primer (see Fig. 2). The two splice donor sites and the first acceptor site have been sequenced; the second acceptor site has not been sequenced and is deduced from CBsv cDNA sequence. CBsv mRNA is raised by splicing between donor 2 and acceptor 2, CB by splicing between donor 1 and acceptor 1. The corresponding spliced introns are thus overlapping.
	Figure 1. Alignment of amino acid sequences of cyclin B from the sea urchins Arbacia punctulata (26) and Sphaerechinus granularis (this paper). Residues identical in both sequences are boxed. A high degree of identity is noteworthy between cyclin B of these distant Echinid species.
	Figure 3. PCR characterization of alternative splicing in cyclin B mRNA. (Lanes 1 and 9) Molecular mass markers (φ X 174/HaeIII and λ/EcoRI + HindIII, respectively). (Lanes 2 and 3) RT-PCR was done using poly(A)+ mRNA from unfertilized eggs as a template, and CB5-CB64 and CB5– CB62 primer pairs, respectively (see Fig. 2). Both pairs amplified fragments of the expected sizes. (Lanes 4 and 5) The same primer pairs were used, with genomic DNA as a template. While CB5-CB62 gave rise to the right-sized PCR product (compare lanes 3 and 5), CB5-CB64 amplified a 2.5-kbp PCR fragment (compare lanes 2 and 4). This last product was reamplified with CB5-CB62 and gave rise to a right-sized product (compare lanes 3, 5, and 6), showing part of the clone 2 cDNA sequence comprising primer CB62 was included in the 2.5-kbp genomic CB5-CB64 fragment. (Lanes 7 and 8) PCR amplification of genomic DNA with respectively primers CB8-CB64 and CB10-CB64 (see Fig. 2). Only CB10-CB64 amplified a fragment (1,300 bp, lane 8), whereas amplification with CB8-CB64 was unsuccessful, showing part of the clone 2 cDNA, comprising primer CB8, was not included in the 2.5-kbp genomic CB5-CB64 fragment. All PCR products showed in this figure were confirmed by cloning and partial sequencing (not shown).
	Figure 5. Relative abundance of CB and CBSV mRNAs varies with developmental stage. Northern blots of similar amounts (1 μg as estimated by densitometry) of poly(A)+ mRNAs from unfertilized eggs (lane 1), from 2-cell stage (lane 2), and from swimming late blastulae (lane 3) were probed with a probe specific for CBSV (lanes 1–3). After exposure for 50 h the blot was dehybridized and reprobed with a probe specific for CB and then reexposed for 18 h (lanes 4–6). CBSV mRNAs are essentially distributed in bands between 4 and 5.5 kbp, whereas a single prominent 4.5-kbp CB mRNA is evidenced. As we have used the same blot to visualize first CBSV and then CB mRNAs, we can state that the relative amount of CB mRNA with regard to CBSV mRNA decreases dramatically in swimming larvae, although no rigorous quantitation of poly(A)+ mRNAs was performed.
	Figure 6. Conservation of amino acids at the COOH terminus of A type cyclins (A) and B type cyclins (B). Amino acid 1 is the first of the last translated exon in classical cyclin A and B. 33 cyclin A sequences and 39 cyclin B sequences were visually aligned and compared over the length of the shortest one (25 aa in A, 22 aa in B). The occurrences of each amino acid at a given position were cumulated, and the level of conservation was calculated as the summation of the squares of each score at a given position. The highest possible conservation (the same amino acid at a given position in overall sequences: 332 = 1089 for cyclin A, and 392 = 1521 for cyclin B) was taken as 100%. For example, for three sequences at a given position, the highest conservation will be obtained with three identical aminoacids at this position, thus 32 = 9, 9/9 = 100%; with two identical aminoacids and one divergent, 22 + 1 = 5, 5/9 = 56%; with three different aminoacids, 1 + 1 + 1 = 3, 3/9 = 33%. Given that (a + b)2 > a 2 + b 2, this method is simple and discriminates well the level of conservation. This figure shows that cyclin A COOH terminus is better conserved than cyclin B, and points out the high level of conservation of aminoacids 5 (K) and 6 (Y) in both families of mitotic cyclins.
	Figure 7. Acquisition of a G2-phase in micromeres is under maternal control and coincides with accumulation of CBsv. (a) BrdU (1 mg/ml) was added during the fourth M-phase to sea urchin embryos. Embryos were directly fixed in 4 N HCl 10 min after the cytodieresis and then processed for BrdU detection, which showed that replication occurred in micromeres (arrowhead) to the same extent as in macromeres (arrow). As cell cycle is much longer in micromeres, this shows that the first G-phase appearing in micromeres is a G2-phase. (b) 200 μg/ml (final concentration) α-amanitin was microinjected in eggs before fertilization. This concentration was far higher than necessary (50 μg/ml) to inhibit hatching and gastrulation (not shown); nevertheless, in microinjected embryos, normal micromeres formed in schedule with control embryos. (c and d) Embryos were fixed after the fifth cell cycle in macromeres and mesomeres and processed for CBsv detection (c) and DNA Hoechst staining (d). While macromeres and mesomeres were in S-phase of the 6th cell cycle, micromeres were in early prophase of the fifth cycle and overexpressed CBsv. Bar, 35 μm.
	Figure 8. CBsv staining along embryogenesis. (a) Mesenchyme–blastula stage (late blastula). On the left a set of cells, representing the bud of archenteron, are heavily stained. Moreover, note that all the cells of this embryo are more or less stained (compare with the following pictures). (b) Young gastrula with a just growing archenteron. CBsv is localized in the cells of the archenteron, and in some secondary mesenchyme cells that have migrated inside the blastocoel. (c) Young gastrula at a later stage. The same categories of cells are stained as in Fig. 9 b. (d) Late gastrula. The tip of the archenteron has reached the apex of the embryo. A few cells (∼50 per embryo at a given time) are heavily stained by CBsv immunofluorescence: at the tip of the archenteron (large arrow), inside the blastocoel (filopodial cells, arrowhead), and at the periphery (presumably pigment cells, tiny arrow). (e) Control staining with anti-CBsv antibody loaded with recombinant CBsv. f is an enlargement of part of c showing that CBsv is concentrated in the cytoplasm as a dot near the nucleus of blastocoelar cells (arrow). The nucleus is seen as a black hole, whereas the surrounding cytoplasm is background stained. Bars: (a–e) 50 μm; (f) 20 μm.
	Figure 9. Anti-CBsv antibodies are specific for the Sphaerechinus granularis splice variant of cyclin B. 20-h-old embryos of S. granularis (a and b) or Paracentrotus lividus, another species of regular sea urchin, were methanol-fixed and processed for cyclin B detection with a polyclonal antibody raised against full-length Arbacia punctulata cyclin B (a and c) or for CBsv detection with the anti-CBsv antibody (b and d). Anti–cyclin B antibody clearly detected the overexpressed CBsv with a similar pattern in both species, even in the presence of 1 mg/ml CBsv peptide (a and c). In contrast, anti-CBsv antibody, which is directed against a specific intronic sequence in S. granularis, did not recognize any epitope in the related species P. lividus (b and d). Bar, 50 μm.
	Figure 10. CBsv distribution in prophase-arrested oocytes (left) and corresponding Hoechst DNA staining (right). (a and b) View at small scale of a field comprising a small oocyte between various embryos, showing CBsv is prominently concentrated in the cortical layer of growing oocytes. (c and d) A near full-grown oocyte that also displays a cortical accumulation of CBsv and two intranuclear dots that do not seem to correspond to any chromatin structure (arrows). Such a nuclear staining in one to three dots was constantly observed in oocytes. Bars: (a and b) 55 μm; (c and d) 20 μm.
	Figure 11. CBsv microinjection delays activation and reduces activity of the mitotic H1 kinase during hormone-stimulated meiosis reinitiation of starfish oocytes. A. aranciacus oocytes were microinjected with 1 μM (final concentration) CB or CBsv and then allowed to mature. Individual oocytes of each batch, comprising one batch of uninjected control oocytes, were periodically sampled and frozen in 2 μl of distilled water. H1 kinase assay was performed by addition of 8 μl of the assay buffer containing 5 mM MgCl2, 50 μM ATP, 0.5 mg/ml histone H1, 5 mM Hepes, pH 7.0, and 50 μCi γ-[32P]ATP, for 10 min. After SDS-PAGE, the band containing histone H1 in each sample was excised and scintillation counted. This experiment was partially reproduced three times more with essentially the same results. In controls (solid line) H1 kinase reached maximal activity at 40 min, dropped almost completely at 100 min, and rose again for the second meiotic cell cycle. CB microinjection (dotted line) resulted in a significant advance in activation of the kinase, since the maximal activity was reached at 20 min, in an increase of the maximal activity, and a delay for mitosis exit. In contrast, CBsv microinjection (broken line) resulted in a large delay for activation of the kinase (maximal value at 55 min) and a lowering of its activity.
	Figure 12. CBsv microinjection induces first cell cycle lengthening and developmental disorders in sea urchin embryos. CBsv (1 μM final concentration) was microinjected before fertilization in sea urchin eggs. Pictures were then taken during cleavage of microinjected (a, c, and e) or control uninjected (b, d, and f) embryos. (a and b) At 140 min controls were in metaphase of the third cycle, whereas CBsv microinjected just initiated anaphase of the first cycle. (c and d) At 170 min, controls were at the 8-cell stage, and microinjected eggs at the 2-cell stage. (e) At 240 min, injected embryos displayed various numbers of blastomeres irregular in size, here six blastomeres, one large and five small. (f) At 205 min, control embryos formed micromeres. a, c, and e are successive pictures of the same microinjected embryo. Bar, 50 μm.
	Figure 13. CBsv binds to cdc2 with a reduced affinity and is less efficient than CB for rescuing a CLN1-3 triple deletion in budding yeast. (a) Western blot showing binding of CBsv (lanes 1–3) and CB (lanes 4–6) after microinjection of the recombinant proteins in Xenopus laevis oocytes: (input) total amount of microinjected material; (cyclin) amount of cyclin (CB or CBsv) recovered on p13suc1 beads after a 2-h incubation in the cells: CB (right) was better recovered than CBsv (left), whereas the amount of cdc2 in the purified material was constant (cdc2). (b) The triple CLN deleted strain 589-5 was transformed with either the vector pADNS alone (lanes 1 and 4), pADNS containing CBsv ORF (lanes 2 and 5), or pADNS containing CB (lanes 3 and 6). Homogenates of the three transformed strains grown on galactose were subjected to detection with CBsv antibody (lanes 1–3, lower arrowhead), or with CB antibody (lanes 4–6, upper arrowhead). This panel shows that similar amounts of both proteins were efficiently produced, together with homologous degradation products. (c) Supernatants of the same homogenates were subjected to p13suc1 affinity purification, then the purified material detected with antibodies specific for CB (lane 2, upper arrowhead), or CBsv (lane 1, lower arrowhead), showing that the binding of CBsv to endogenous CDC28p (in similar amounts on p13 beads, Cdc28p) was reduced compared with the binding of CB. (d) Accordingly, when grown on dextrose, CB-transformed cells (arrowheads) were efficiently rescued, whereas CBsv-transformed cells grew only hardly (arrow). Cells transformed with vector alone (tiny arrow) did not grow at all.

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