TY - JOUR
AU - Peaucellier, Gérard
AB - Abstract Cyclin B3 evolution has the unique peculiarity of an abrupt 3-fold increase of the protein size in the mammalian lineage due to the extension of a single exon. We have analyzed the evolution of the gene to define the modalities of this event and the possible consequences on the function of the protein. Database searches can trace the appearance of the gene to the origin of metazoans. Most introns were already present in early metazoans, and the intron–exon structure as well as the protein size were fairly conserved in invertebrates and nonmammalian vertebrates. Although intron gains are considered as rare events, we identified two cases, one at the prochordate–chordate transition and one in murids, resulting from different mechanisms. At the emergence of mammals, the gene was relocated from chromosome 6 of platypus to the X chromosome in marsupials, but the exon extension occurred only in placental mammals. A repetitive structure of 18 amino acids, of uncertain origin, is detectable in the 3,000-nt mammalian exon-encoded sequence, suggesting an extension by multiple internal duplications, some of which are still detectable in the primate lineage. Structure prediction programs suggest that the repetitive structure has no associated three-dimensional structure but rather a tendency for disorder. Splice variant isoforms were detected in several mammalian species but without conserved pattern, notably excluding the constant coexistence of premammalian-like transcripts, without the extension. The yeast two-hybrid method revealed that, in human, the extension allowed new interactions with ten unrelated proteins, most of them with specific three-dimensional structures involved in protein–protein interactions, and some highly expressed in testis, as is cyclin B3. The interactions with activator of cAMP-responsive element modulator in testis (ACT), germ cell-less homolog 1, and chromosome 1 open reading frame 14 remain to be verified in vivo since they may not be expressed at the same stages of spermatogenesis as cyclin B3. cyclin B3, molecular evolution, intron gain, repeat duplication, alternative splicing, protein–protein interactions Introduction Cyclins are the regulatory subunits of protein kinases, thus called cyclin-dependent kinases (Cdks), which were first discovered as the main regulators of eukaryotic cell division (see Doree and Hunt 2002 for review). Binding of newly synthesized cyclin activates the enzymatic activity of the kinase, to promote entry into M-phase, and then proteolytic degradation of the cyclin shuts down the activity, allowing M-phase exit. A single mitotic Cdk, as Cdk1, can bind different cyclins according to the phase of the cell cycle and thus be involved in different regulations, such as DNA synthesis with cyclin A and mitosis with cyclin B. Cyclins modulate the substrate specificity of the kinases (Miller and Cross 2001) and also their intracellular localization through nuclear and cytoplasmic transfer or retention signals (Pines 1999). Several Cdks and numerous cyclins are now known to regulate all the steps of the cell cycle but also RNA transcription, splicing, and translation (see Satyanarayana and Kaldis 2009 for review). Cyclins have distinctive features, such as a stretch of about 150 amino acids termed “cyclin box” involved in Cdk binding (Kobayashi et al. 1992), which make them easily recognizable in whole-genome sequence data. At least 29 proteins with a cyclin box domain are known in mammals (Malumbres and Barbacid 2005). Although they are referred as cyclins, some of them have no identified Cdk partner and, for most of them, it is not known if they are periodically synthesized and destroyed. Cyclin B3 was initially discovered in a chicken cDNA library (Gallant and Nigg 1994). Although closer to B-type cyclins according to amino acid sequence comparisons, it also displays properties of A-type cyclins, such as a nuclear localization during interphase. It was later detected in Caenorhabditis (Kreutzer et al. 1995), Drosophila (Sigrist et al. 1995), and human (Lozano et al. 2002; Nguyen et al. 2002), and it is now identified in most sequenced metazoan genomes. It is now clear that cyclins B3 form a family distinct from A and B cyclins, since they share more sequence similarity with cyclin B3 of other species than with cyclins B of their own species (Nieduszynski et al. 2002; Gunbin et al. 2011). A first noticeable feature is that no species has more than one cyclin B3 gene, whereas the number of A- or B-type cyclins has been increasing during evolution, with two cyclins A and two to four cyclins B in vertebrates. Since cyclin B3 is present from lowest invertebrates to mammals, it would be expected to have a fixed and essential function, but research in different species has instead shown striking differences. In Drosophila, the first studies demonstrated a mitotic role for cyclin B3, in coordination with cyclins A and B (Sigrist et al. 1995; Sigrist and Lehner 1997). During embryonic mitosis, the three cyclins were found associated with Cdk1, in similar amounts, and their sequential destruction was necessary for mitotic progression and exit. In Caenorhabditis embryo, cyclin B3 was also found to be associated with Cdk1, with associated H1 kinase activity, had a similar temporal and spatial expression as B cyclins, and was found to be involved in mitotic progression (van der Voet et al. 2009; Deyter et al. 2010). In vertebrates, on the contrary, no similar implication of cyclin B3 in embryonic mitosis has been found, with cyclin B3 mRNA levels 10-fold lower than cyclin A and 50-fold lower than cyclin B, in eight-cell human embryo (Kiessling et al. 2009). However, high levels of expression are detected during spermatogenesis in zebrafish (Ozaki et al. 2011), eel (Kajiura-Kobayashi et al. 2004), mouse (Nguyen et al. 2002), and in human testis (Lozano et al. 2002; Nguyen et al. 2002). In these cases, there are no indication of association of cyclin B3 with Cdk1, but, in human, an in vitro association with Cdk2, without associated H1 kinase activity, was reported (Nguyen et al. 2002). Experimental modifications of cyclins expression in invertebrates revealed a redundant function of cyclins B and B3 in mitosis but specific effects during meiosis. In Drosophila, null mutation of the cyclin B3 gene prevents correct achievement of oocyte meiosis, whereas cyclin B mutation also impairs spermatogenesis (Jacobs et al. 1998). In Caenorhabditis also, inhibition of cyclins by RNAi demonstrates that cyclin B3 is necessary for achievement of oocyte meiosis, through its implication in spindle assembly checkpoint (van der Voet et al. 2009; Deyter et al. 2010). A recent study in mouse showed that cyclin B3 is not only involved in male meiosis but is also specifically expressed in female germ cells entering meiosis, at the end of the proliferation phase (Miles et al. 2010). Curiously, there are also reports of high cyclin B3 expression during neurogenesis: in Drosophila neuroblasts (Jacobs et al. 1998) and Xenopus embryo neural plate (Ueno et al. 2008). These exceptionally different uses of cyclin B3 in the various animal lineages raise interest for a careful examination of the evolution of this gene. Of special interest is the 3-fold increase of the size of the protein in mammals, due to the extension of a single exon to a length of about 3,000 nt. This abrupt change is interesting to correlate with the early evolution of mammals, from egg-laying monotremes, to marsupials, and then placental mammals. This period of evolution also coincides with the apparition of the sex chromosomes, and the cyclin B3 gene is located on the X chromosome in higher mammals. This may have consequences on the expression of the gene, due to inactivation of the genes of one X chromosome (reviewed in Payer and Lee 2008), whereas the number of gene copies seems to be important as experimental alteration of the number of copies of the cyclin B3 gene in Caenorhabditis has shown the appearance of toxic effects above two copies (Tarailo-Graovac et al. 2010). Several splice variants of the cyclin B3 gene have been described in humans (Lozano et al. 2002). This process is known to expand the repertoire of transcripts from a single gene (see Nilsen and Graveley 2010 for review). It occurs in the expression of several human cyclins, giving proteins with different properties, and is frequently associated with cancer (Van Dross et al. 2006). Thus, it is interesting to check the existence of alternative transcripts in different species and look at the conservation of the intron–exon structure of the cyclin B3 gene for changes in alternative splicing opportunities. This work was intended to precise cyclin B3 apparition, evolution of intron–exon structure, chromosomal relocation, and exon extension at the emergence of mammals. The possible changes linked to the extension were looked for by structure analysis, checking for retention or deletion in splice variants, and apparition of new interactions by the yeast two-hybrid system. Materials and Methods Biological Materials For brushtail possum (Trichosurus vulpecula), platypus, pig, horse, and mouse, RNA was extracted from testis. Canine RNA was extracted from MDCK, a kidney cell line provided by Dr R. Guyon (IGD, Rennes). Platypus RNA and genomic DNA were provided by Dr J. Deakin (Australian National University, Canberra, Australia). The different cDNAs were synthesized with Powerscript reverse transcriptase (Clontech, Saint Germain-en-Laye, France). cDNA Cloning Based on published sequences, which share high homologies with human cyclin B3: 1 dog EST (gi: 23 706 478), 1 pig EST (gi: 59 828 965), and 1 mouse partial mRNAs (AJ416459) were found on GenBank. All these fragments were amplified by polymerase chain reaction (PCR) and resequenced. These sequences were rapid amplification of cDNA ends (RACE) extended in both directions (Smart RACE cDNA amplification kit, Clontech) to get their full-length cyclin B3 cDNA. For isolation of fragments of cyclin B3 orthologs from other species, we designed degenerate PCR primers corresponding to evolutionally conserved peptides: CPPCVDD and ICEMTLQE (size 215 bp) for horse, EVQENFE and YICDDA (size 200 bp) for possum, and CPPCVDD ET FDINIP for platypus (size 119 pb). PCRs were performed on cDNA and the PCR products were sequenced, and new primers were designed to get the full-length cDNA by RACE extension. To look for cDNA coding for short isoforms of cyclin B3 in mouse, horse, pig, and dog, we used primers designed to encompass the ATG and STOP codons deduced from the sequences of long isoform coding cDNA and performed PCR with the different cDNA. PCR products, which size were lower to known ones, were gel purified, cloned into pGEM-T (vector system 2; Promega), and sequenced. For possum germ cell-less homolog 1 (GMCL1) cloning, two degenerate primers corresponding to evolutionally conserved peptides, EIPDQNID and DQEQVVMN, gave a PCR product, and from its sequence new primers were used to extend in both directions. The sequence is incomplete compared with other known GMCL1 (75 AA N-ter). To clone possum FLH5, we used two primers encompassing the cds of FLH5 of another marsupial, short-tailed opossum (Monodelphis domestica), for which the genome is known. From a tblastn analysis with the human SHCBP1L protein query on platypus WGS databases, we got some sequences with good homology, and primers were designed to perform a PCR. The fragment encodes for a partial SHCBP1L (454AA), which lacks 54AA on C-ter side compared with others and 25–218AA according to the isoform identified in humans (Sood et al. 2001). Partial Platypus Cyclin B3 Gene Cloning The introns of platypus cyclin B3 gene were isolated by PCR using high-molecular-weight genomic DNA. The primers were designed from the cDNA sequence. The PCR products were gel purified and sequenced. Resulting sequences were assembled. Chromosome Walking on Chicken Genomic DNA The same protocol was used for chicken to clone the entire cyclin B3 gene. The primers were designed from the cDNA sequence published (X75757; Gallant and Nigg 1994). After assembling the different parts of cyclin B3 gene and to find DNA sequences adjacent with a potentially known chromosomic location, we used the GenomeWalker universal kit (Clontech). This PCR-based method permits walking on genomic DNA around a known gene. According to the protocol of the manufacturer, separate aliquots of high-molecular-weight genomic DNA (Novagen) were digested with the four restriction enzymes to get the chicken genomic libraries. For the two rounds of PCR on these libraries using Advantage 2 polymerase mix (Clontech), we designed primers chosen upstream of ATG and downstream of STOP sequences: Gal up: 5′-AGC CAG GAG CAC TGC TCA AAG AGG GCT CAG AGC-3′. Gal up nested: 5′-CAA TCC CTC AAT CCA TCC CCT CCT TCC CTC CAA-3′. Gal down: 5′-ATG ATG AAG CTG GAG GAG AAG TTG AAA AGC TAG-3′. Gal down nested: 5′-TAG CCT GTA AAT AGC CGG GGG GGA GCA CTG TGT ACA-3′. Vector Constructions Partial or complete coding sequences of the different cyclin B3 orthologs and isoforms were amplified by PCR, with primers containing restriction sites necessary for sub-cloning into pGBKT7 bait vector. The coding sequences of preys we wanted to test were sub-cloned into Pgadt7 prey vector (Cdk5, FLH5, GMCL1, SHCBP1L, and IPO5) using the same method. Human IPO5 coding sequence was sub-cloned into pGEX4T3 (a generous gift of N.R. Yaseen-Fontoura) and human GMCL1 into pGEX4T3. All constructs were confirmed by sequencing. Yeast Two-Hybrid Screening and Tests A commercial human testis MATCHMAKER cDNA library (mRNA from whole testes pooled from 19 Caucasians, aged 17–61 years, died from trauma), cloned into pACT vector (Clontech), was used. It contains 3.5 × 106 independent clones with an average size of 2 kb. The different parts of human cyclin B3 cloned into pGBKT7, in frame with GAL4-binding domain, were used as baits in the yeast two-hybrid system according to the manufacturer’s instructions and as described previously (Lozano et al. 2010). Positive clones were then processed for sequence analysis and identified using the BLAST algorithm. The same two-hybrid system was used to check interactions of the different cyclin B3 orthologs and Ex8ΔCt with full-length prey proteins or nonplacental mammal ortholog preys. In Vitro Binding Assays Human Cdk5 and cyclin B3 were translated in reticulocyte lysates as 35S-labeled, epitope-tagged, proteins with the T7 TNT-coupled transcription–translation system (Promega), from pGADT7-Cdk5 (HA epitope) and pGBKT7-cyclin B3 (c-Myc epitope) expression constructs, following the manufacturer’s instructions. After incubation for 1 h at 30°, followed by 1 h at 0° for cotranslations, the samples were diluted with 400 µl of TBS-T (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.1% Tween-20, and protease inhibitors) and processed for immunoprecipitation with antibodies against c-myc or HA (Clontech). The immunoprecipitated proteins were detected by autoradiography of the dried sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels. Soluble GST, GST-SHCBP1L, and GST-IPO5 fusion proteins expressed in Escherichia coli were purified and immobilized on glutathione-Sepharose beads as instructed by the manufacturer (GE Healthcare). These beads were washed five times with TBS-T. Human cyclin B3 was in vitro–translated and labeled with 35S-Met from the expression plasmid pGBKT7 by using a TNT translation kit (Promega), and 10 µl of the lysate was incubated in phosphate buffered saline buffer with GST alone or GST fusions 1 h at room temperature. Beads were washed four times with the same buffer, and bound proteins were analyzed by SDS–PAGE and processed for autoradiography. Sequences Analysis Protein and genomic sequences were searched on the following servers and web sites (for all: cited 2012 April 30): NCBI (http://blast.ncbi.nlm.nih.gov/Blast), Ensembl (http://www.ensembl.org/), DOE JGI genome (http://genome.jgi.doe.gov/), Sea Urchin Genome Database (http://www.spbase.org/), IMCB elephant shark genome project (http://esharkgenome.imcb.a-star.edu.sg/), and Genoscope’s Oikopleura Genome Browser (http://www.genoscope.cns.fr/externe/GenomeBrowser/Oikopleura/, with the kind assistance of Philippe Dru). Amino acid sequences were aligned with version 2 of Clustal X (Larkin et al. 2007), identical and similar AA were shaded with the BioEdit software (Hall 1999), and phylogenetic analysis and tree drawing were performed with Mega 4 (Tamura et al. 2007). Transposable elements were searched with RepeatMasker (A.F.A. Smit, R. Hubley, and P. Green RepeatMasker at http://repeatmasker.org) using the GIRI Repbase (Kohany et al. 2006; http://www.girinst.org). For analysis of the extension, disordered zones were detected with GlobPlot (Linding et al. 2003; http://globplot.embl.de) or TOP-IDP (Campen et al. 2008; http://www.disprot.org/dev/disindex.php), coiled coils with Coils (Lupas et al. 1991; http://www.ch.embnet.org/software/COILS), and repetitive sequences with Trust (Szklarczyk and Heringa 2004; http://www.ibi.vu.nl/programs/trustwww/). The Gepard software was used for dot matrix analysis (Krumsiek et al. 2007; http://www.helmholtz-muenchen.de/en/mips/services/analysis-tools/gepard/) and WebLogo for calculation and visualization of consensus sequences (Crooks et al. 2004; http://weblogo.berkeley.edu/). Amino acid composition was analyzed with Composition Profiler (Vacic et al. 2007; http://www.cprofiler.org). Results Cyclin B3 Appearance at the Unicellular-Metazoan Transition A single ortholog of the cyclin B3 gene is identified in most metazoan sequenced genomes, including the earliest branching groups as sponges, placozoan, and cnidarians, but not in those of unicellulars and fungi. The single exception is the annotation in the mold Neurospora crassa of a coding sequence “related to cyclin B3.” This led us to check genomic databases for the earliest evidences of the cyclin B3 gene. Different metazoan cyclin B3 amino acid sequences were used to search for similar coding genomic sequences, using NCBI tblastn, in the choanoflagellate Monosiga brevicollis genome, considered as the closest known relative of metazoans (King et al. 2008). Similarities were repeatedly found at three genomic locations on scaffolds 3, 9, and 11, annotated as coding sequences related to cyclins A, F, and B, respectively. Cyclins F have a typical cyclin box sequence but also a specific F box and regulate ubiquitin ligases, involved in protein degradation, instead of protein kinases, and are now designated as SCF (Skp1-Cul1-F-box protein; D'Angiolella et al. 2010). A similar search on the genome of N. crassa revealed similarities at two genomic locations, annotated as coding for proteins related to cyclin B and B3. Comparison of the amino acid sequences, in the highly conserved cyclin box region, of these proteins with their metazoan counterparts (in sponge, placozoan, cnidarian, and sea urchin) confirmed the homologies for the three Monosiga cyclins, but the Neurospora cyclins were found to be close relatives to each other, with similarities with other B type cyclins but not B3 ones (fig. 1). In addition, this comparison of sequences and intron positions supports a closer relationship of cyclin B3 with cyclin B than cyclin A (supplementary material fig. S1, Supplementary Material online). It cannot be excluded that the cyclin B3 gene was missed in Monosiga genome sequencing, but the present data suggest that cyclin B3 appeared, by duplication of the cyclin B gene, in the earliest metazoan ancestor. Fig. 1. View large Download slide Phylogenetic analysis of cyclin B3 apparition at the unicellular-metazoan transition. Phylogenetic tree obtained by the Neighbor-Joining method, with the Mega 4 software, from alignment of amino acid sequences, in the cyclin box region, of cyclins A, B, B3, and F from four metazoans: sea urchin Strongylocentrotus purpuratus, cnidarians Hydra magnipapillata or Nematostella vectensis, placozoan Trichoplax adhaerens, and sponge Amphimedon queenslandica. Also included are three cyclins (A, B, and F) of the unicellular choanoflagellate Monosiga brevicollis and two cyclins (B and B3-related) from the mold Neurospora crassa. The alignment used for the construction of this phylogenetic tree is given in supplementary material fig. S1, Supplementary Material online. References: cyclins B Strongylocentrotus XP_001175791, Hydra XP_002167647.1, Trichoplax jgi|Triad1|3971, Amphimedon jgi|Aqu1|215072, Monosiga jgi|Monbr1|8459, Neurospora XP_963851.1, cyclins B3 Strongylocentrotus XP_795905.1, Hydra XP_002160446.1, Trichoplax jgi|Triad1|56083, Amphimedon XP_003385663.1, Neurospora XP_961608.2, cyclins A Strongylocentrotus NP_999646.1, Hydra XP_002165420.1, Trichoplax jgi|Triad1|24944, Amphimedon jgi|Aqu1|222748, Monosiga jgi|Monbr1|14677, cyclins F Strongylocentrotus XP_001186785.1, Nematostella jgi|Nemve1|124800, Trichoplax jgi|Triad1|53841, Amphimedon XP_003388717.1, and Monosiga jgi|Monbr1|37010. Fig. 1. View large Download slide Phylogenetic analysis of cyclin B3 apparition at the unicellular-metazoan transition. Phylogenetic tree obtained by the Neighbor-Joining method, with the Mega 4 software, from alignment of amino acid sequences, in the cyclin box region, of cyclins A, B, B3, and F from four metazoans: sea urchin Strongylocentrotus purpuratus, cnidarians Hydra magnipapillata or Nematostella vectensis, placozoan Trichoplax adhaerens, and sponge Amphimedon queenslandica. Also included are three cyclins (A, B, and F) of the unicellular choanoflagellate Monosiga brevicollis and two cyclins (B and B3-related) from the mold Neurospora crassa. The alignment used for the construction of this phylogenetic tree is given in supplementary material fig. S1, Supplementary Material online. References: cyclins B Strongylocentrotus XP_001175791, Hydra XP_002167647.1, Trichoplax jgi|Triad1|3971, Amphimedon jgi|Aqu1|215072, Monosiga jgi|Monbr1|8459, Neurospora XP_963851.1, cyclins B3 Strongylocentrotus XP_795905.1, Hydra XP_002160446.1, Trichoplax jgi|Triad1|56083, Amphimedon XP_003385663.1, Neurospora XP_961608.2, cyclins A Strongylocentrotus NP_999646.1, Hydra XP_002165420.1, Trichoplax jgi|Triad1|24944, Amphimedon jgi|Aqu1|222748, Monosiga jgi|Monbr1|14677, cyclins F Strongylocentrotus XP_001186785.1, Nematostella jgi|Nemve1|124800, Trichoplax jgi|Triad1|53841, Amphimedon XP_003388717.1, and Monosiga jgi|Monbr1|37010. Intron–Exon Structure of the Cyclin B3 Gene Genomic databases were searched to check the variability of the cyclin B3 gene, with a particular attention to exon length and thus intron–exon structure. The coding length of the gene is about 1,300 nt, corresponding to proteins of 440 amino acids and 50,000 Da. The cyclin B3 of the nematode worm Caenorhabditis is among the smallest with 382 amino acids for 44,425 Da, whereas that of Drosophila is one of the largest with 497 amino acids for 56,310 Da, thus the size change in mammals has no equivalent in other lineages. There are typically ten coding exons in invertebrates and one more in vertebrates. The 5′UTR of transcripts are also encoded by several exons, four in human, but their exact number is not known in most species. The human gene exon numbering will be used as a reference, with translation beginning in exon 5. As shown in figure 2, there is strong evidence for an ancient origin of most introns, since their number and positions are almost the same in sponges, placozoan, and human. For instance, there is an intron after the destruction box coding sequence, at the end of exon 6, or after conserved amino acid sequences characteristic of the end of exons 9 and 14. Another indication is the good conservation of intron frames, as frame 2 introns found between exons 7 and 8 as well as 14 and 15. There is even evidence of introns predating the divergence of cyclins A and B, such as those surrounding the cyclin box, between exons 9 and 10 and 13 and 14 (supplementary material fig. S1, Supplementary Material online). This panel of species, representative of the mains metazoans phylogenetic lineages, shows numerous probable intron losses and few intron gains. One intron gain is specific to the chordate lineage, as will be analyzed later, the other three are genus specific: one in the leech Helobdella and two in Caenorhabditis. Presumed ancestral introns are missing in 5 of the 11 species of figure 2, up to 7 in Caenorhabditis and complete loss in Drosophila (sequence in Supplementary Data). There is no evidence of gain or loss specific to the protostomian lineage, since the gastropod Lottia gene has the complete set of introns and, in the ecdysozoan lineage, only three are missing in the crustacean Daphnia gene. In the deuterostomian lineage, the ancestral intron pattern is retained, with the exceptions of urochordates, the ascidian Ciona (Supplementary Data), and the appendicularian Oikopleura (not shown), whose cyclin B3 genes have several missing or misplaced introns. All vertebrate cyclin B3 genes have the same intron–exon structure, but in mammals exon 8 is hugely extended and an additional intron is inserted in it in mouse and rat, as will be analyzed later. Fig. 2. View largeDownload slide Early metazoan origin of most introns of the cyclin B3 gene. Schematic representation of cyclin B3 gene intron–exon structure in various metazoans: human (Homo sapiens), chicken (Gallus gallus), sea urchin (Strongylocentrotus purpuratus), leech annelid (Helobdella robusta), gastropod mollusc (Lottia gigantea), honeybee insect (Apis mellifica), crustacean (Daphnia pulex), nematode worm (Caenorhabditis elegans), cnidarian (Hydra magnipapillata), placozoan (Trichoplax adhaerens), and sponge (Amphimedon queenslandica). * indicate species in which intron–exon structure is deduced from a predicted mRNA not, or only partially, validated by ESTs. Exon numbering corresponds to the human gene but only the translated part is shown, thus beginning in exon 5. Exons (boxes) are drawn to scale, but not introns (connecting lines). The extralength of exon 8 corresponding to the mammalian-specific extension is shown on top of the human gene (vertical bar filled box). The presence of recognizable amino acid sequences specific for the end of exon 6 (destruction box), 9, and 14, is indicated by horizontal or oblique bar filling. Frame 1 introns (inserted between the first and second nucleotide of a codon) are indicated by a triangle and frame 2 (between second and third nucleotide) by a star. The position of the cyclin box coding region (Gallant and Nigg 1994) is indicated by a dotted line box. Probable losses of ancestral introns are indicated by arrowheads and acquisition of new introns by filled arrowheads. References: Homo NM_033031.2, Gallus AM691545/NM_205239.1, Strongylocentrotus XM_790812.2, Helobdella jgi|Helro1|186157, Lottia jgi|Lotgi1|236425, Apis XM_397108.3, Daphnia jgi|Dappu1|210441, Caenorhabditis NM_001028941.2, Hydra XM_002160410.1, Trichoplax jgi|Triad1|56083, and Amphimedon XM_003385615.1. Fig. 2. View largeDownload slide Early metazoan origin of most introns of the cyclin B3 gene. Schematic representation of cyclin B3 gene intron–exon structure in various metazoans: human (Homo sapiens), chicken (Gallus gallus), sea urchin (Strongylocentrotus purpuratus), leech annelid (Helobdella robusta), gastropod mollusc (Lottia gigantea), honeybee insect (Apis mellifica), crustacean (Daphnia pulex), nematode worm (Caenorhabditis elegans), cnidarian (Hydra magnipapillata), placozoan (Trichoplax adhaerens), and sponge (Amphimedon queenslandica). * indicate species in which intron–exon structure is deduced from a predicted mRNA not, or only partially, validated by ESTs. Exon numbering corresponds to the human gene but only the translated part is shown, thus beginning in exon 5. Exons (boxes) are drawn to scale, but not introns (connecting lines). The extralength of exon 8 corresponding to the mammalian-specific extension is shown on top of the human gene (vertical bar filled box). The presence of recognizable amino acid sequences specific for the end of exon 6 (destruction box), 9, and 14, is indicated by horizontal or oblique bar filling. Frame 1 introns (inserted between the first and second nucleotide of a codon) are indicated by a triangle and frame 2 (between second and third nucleotide) by a star. The position of the cyclin box coding region (Gallant and Nigg 1994) is indicated by a dotted line box. Probable losses of ancestral introns are indicated by arrowheads and acquisition of new introns by filled arrowheads. References: Homo NM_033031.2, Gallus AM691545/NM_205239.1, Strongylocentrotus XM_790812.2, Helobdella jgi|Helro1|186157, Lottia jgi|Lotgi1|236425, Apis XM_397108.3, Daphnia jgi|Dappu1|210441, Caenorhabditis NM_001028941.2, Hydra XM_002160410.1, Trichoplax jgi|Triad1|56083, and Amphimedon XM_003385615.1. Intron Gain in the Chordate Lineage There is evidence for the acquisition of a new intron, separating exons 10 and 11, at the invertebrate–vertebrate transition. It is inserted in the cyclin box coding region, where amino acid conservation is strong, as shown in figure 3A, in all available vertebrate sequences. Curiously, there is also an intron in this area in the four prochordates for which data are available, but 6 nt before the chordate position. This raises the question of the chronology of apparition of this intron. It could have been acquired independently in prochordates and in chordates, but recent data on deuterostome phylogeny (Putnam et al. 2008) do not support the existence of an ancestor common to amphioxus and tunicates but not to vertebrates. Thus, the intron gained in prochordates should have been lost, and then reacquired at a 6-nt different position in chordates. Examination of the nucleotidic sequences (fig. 3B) suggests a more probable hypothesis. The intron may have been acquired, between E and V codons, in the prochordate–chordate ancestor by tandem genomic duplication using the AGGT site. It would have been displaced later between Q and E codons in the chordate lineage, by 3′-extension, using the Q codon cryptic splice signal, and exonization of the first 6 intron nucleotides under a strong evolutionary constraint to maintain the coding for VQ in transcripts. Remnants of a retrotransposon are detected, by the RepeatMasker software, in the amphioxus intron sequence but not in other prochordates or nonmammalian vertebrates ones, whereas in mammals the size of this intron reaches tens of thousands of nucleotides, with the incorporations of numerous transposable elements and repeats (86% of the 28,252 nt in human). So it is probable that this amphioxus-specific non-LTR retrotransposon, CR1-1_BF (Kapitonov and Jurka 2009), is not the origin of the intron but was inserted later, as in the case of mammals. Fig. 3. View largeDownload slide Gain of introns 10 and 11 in chordates. (A) Alignment of amino acid sequences at the boundary of exons 10 and 11, according to the human sequence (aa 1,160–1,182), showing the conserved position of the phase 0 intron (“/0/”) from fishes to human, its absence in invertebrates and its presence in prochordates, two amino acids before the chordate position. Representative sequences for tetrapods (first block): man Homo sapiens (Homo_s), mouse Mus musculus (Mus_m), horse Equus caballus (Equus_c), dog Canis familiaris (Canis_f), opossum Monodelphis domestica (Mono_d), chicken Gallus gallus (Gallus_g), Xenopus tropicalis (Xenop_t); for invertebrates (upper right block): sea urchin Strongylocentrotus purpuratus (Strong_p), gastropod Lottia gigantea (Lottia_g), insect Apis mellifica (Apis_m), nematode worm Caenorhabditis elegans (Caeno_e), cnidarian Hydra magnipapillata (Hydra_m), placozoan Trichoplax adhaerens (Tricho_a); fishes (lower left block): bony fishes Danio rerio (Danio_r), Oryzias latipes (Oryz_l), Tetraodon nigroviridis (Tetrao_n), and cartilaginous elephantfish Callorhinchus milii (Callor_m); prochordates (lower right block): ascidians Ciona intestinalis (Ciona_i) and savignyi (Ciona_s), appendicularian Oikopleura dioica (Oikopl_d), cephalochordate amphioxus Branchiostoma floridae, (Branch_f). The comparison is based on the chicken sequence for the shading of identical (black) or similar (gray) residues. (B) Comparison of nucleotide sequences at the same intron boundaries in the protochordate amphioxus (Branchiostoma floridae), the sea urchin Strongylocentrotus purpuratus (Strongylo purp), and the zebrafish (Danio rerio). Cryptic splice signals are indicated in bold and underlined characters, intronic sequences are in lower case. References: as in fig. 2 and Mus NM_183015.3, Equus NC_009175.2/AM283100, Canis NM_001005763.1, Monodelphis BN000957, Xenopus ENSXETG00000022597, Danio NM_001076719.1, Oryzias ENSORLT00000010667, Tetraodon ENSTNIT00000022034, Callorhynchus AAVX01309902.1, Ciona i. XM_002126398.1, Ciona s. ENSCSAVT00000005408, Oikopleura GSOIDG00008944001, and Branchiostoma jgi|Brafl1|248144. Fig. 3. View largeDownload slide Gain of introns 10 and 11 in chordates. (A) Alignment of amino acid sequences at the boundary of exons 10 and 11, according to the human sequence (aa 1,160–1,182), showing the conserved position of the phase 0 intron (“/0/”) from fishes to human, its absence in invertebrates and its presence in prochordates, two amino acids before the chordate position. Representative sequences for tetrapods (first block): man Homo sapiens (Homo_s), mouse Mus musculus (Mus_m), horse Equus caballus (Equus_c), dog Canis familiaris (Canis_f), opossum Monodelphis domestica (Mono_d), chicken Gallus gallus (Gallus_g), Xenopus tropicalis (Xenop_t); for invertebrates (upper right block): sea urchin Strongylocentrotus purpuratus (Strong_p), gastropod Lottia gigantea (Lottia_g), insect Apis mellifica (Apis_m), nematode worm Caenorhabditis elegans (Caeno_e), cnidarian Hydra magnipapillata (Hydra_m), placozoan Trichoplax adhaerens (Tricho_a); fishes (lower left block): bony fishes Danio rerio (Danio_r), Oryzias latipes (Oryz_l), Tetraodon nigroviridis (Tetrao_n), and cartilaginous elephantfish Callorhinchus milii (Callor_m); prochordates (lower right block): ascidians Ciona intestinalis (Ciona_i) and savignyi (Ciona_s), appendicularian Oikopleura dioica (Oikopl_d), cephalochordate amphioxus Branchiostoma floridae, (Branch_f). The comparison is based on the chicken sequence for the shading of identical (black) or similar (gray) residues. (B) Comparison of nucleotide sequences at the same intron boundaries in the protochordate amphioxus (Branchiostoma floridae), the sea urchin Strongylocentrotus purpuratus (Strongylo purp), and the zebrafish (Danio rerio). Cryptic splice signals are indicated in bold and underlined characters, intronic sequences are in lower case. References: as in fig. 2 and Mus NM_183015.3, Equus NC_009175.2/AM283100, Canis NM_001005763.1, Monodelphis BN000957, Xenopus ENSXETG00000022597, Danio NM_001076719.1, Oryzias ENSORLT00000010667, Tetraodon ENSTNIT00000022034, Callorhynchus AAVX01309902.1, Ciona i. XM_002126398.1, Ciona s. ENSCSAVT00000005408, Oikopleura GSOIDG00008944001, and Branchiostoma jgi|Brafl1|248144. Intron Gain in Murids The presence of a small intron of about 100 nt, cutting exon 8, only in mouse and rat was intriguing. It was not found in the closer relatives rodents, as squirrel Spermophilus, kangaroo rat Dipodomys, guinea pig Cavia, or rabbits as Ochotona. Thus, it is a recent acquisition, with intriguing features which open the possibility that it results from a newly described mechanism, the intronization of an internal exonic sequence (Irimia et al. 2008). It supposes that point mutations in an exonic sequence can generate functional splice site, recognized by the spliceosome as a new intron. The comparison of amino acid sequences (fig. 4A) is compatible with this hypothesis but not conclusive, since conservation is poor in exon 8–coded sequences, and murid cyclin B3 is highly divergent from those of other mammals. However, Clustal alignment of mouse and human nucleotide sequences favors the intronization hypothesis, with alignment of the intron sequence with an exonic sequence of fairly similar length in human, as shown in figure 4B. The postintron mouse nucleotidic sequence also aligns better with the coding sequence starting at amino acid 266 codon, than with that at amino acid 228 codon as would be expected in an intron insertion hypothesis. The analysis of other mammalian species shows a level of variability in this region which precludes a definitive conclusion, but the postintron mouse and rat nucleotidic sequences were repeatedly found to fit better with the coding sequence starting at the equivalent of amino acid 266 than 228, which supports the intronization hypothesis (results not shown). Fig. 4. View largeDownload slide The intron of muridae is a likely case of intronization of exonic sequence. (A) Alignment of amino acid sequences in the N-terminal part of exon 8 in man (Homo_s), guinea pig Cavia porcellus (Cavia_p), squirrel Spermophilus tridecemlineatus (Spermophilus_t), mouse Mus musculus (Mus_m), and rat Rattus norvegicus (Rattus_n). The comparison is based on the human sequence for amino acid numbering and the shading of identical (black) or similar (gray) residues. The amino acid correspondence to intron nucleotide sequences are indicated in lowercase and stop codons by asterisk. (B) Alignment of nucleotidic sequences corresponding to the muridae intron region (aa 214–279 of the human sequence). Same legends as in A. Additional lines indicate nucleotide identity in mouse and rat intron (*, intron identity) and special intron positions: donor site (D), branching site (B), polypyrimidine track (Y), and acceptor site (A). References: as in fig. 3 and Cavia ENSCPOT00000014349, Spermophilus ENSSTOT00000008336. Fig. 4. View largeDownload slide The intron of muridae is a likely case of intronization of exonic sequence. (A) Alignment of amino acid sequences in the N-terminal part of exon 8 in man (Homo_s), guinea pig Cavia porcellus (Cavia_p), squirrel Spermophilus tridecemlineatus (Spermophilus_t), mouse Mus musculus (Mus_m), and rat Rattus norvegicus (Rattus_n). The comparison is based on the human sequence for amino acid numbering and the shading of identical (black) or similar (gray) residues. The amino acid correspondence to intron nucleotide sequences are indicated in lowercase and stop codons by asterisk. (B) Alignment of nucleotidic sequences corresponding to the muridae intron region (aa 214–279 of the human sequence). Same legends as in A. Additional lines indicate nucleotide identity in mouse and rat intron (*, intron identity) and special intron positions: donor site (D), branching site (B), polypyrimidine track (Y), and acceptor site (A). References: as in fig. 3 and Cavia ENSCPOT00000014349, Spermophilus ENSSTOT00000008336. The Cyclin B3 Gene Was Relocated from an Autosome to X Chromosome During Vertebrate Evolution The cyclin B3 gene is located on the short arm of the X chromosome in man (Xp 11.2; Lozano et al. 2002) and in other placental mammals. At the beginning of this work, the chromosomal location of cyclin B3 was unknown in nonplacental mammals, as well as in chicken. The interest raised by the origin of mammals’ sex chromosomes led us to give it further investigation. In chicken we looked for cyclin B3 gene on genomic DNA, by PCR with primers deduced from the cDNA sequence. The complete sequence of cyclin B3 gene was obtained, and then extended in both directions, by chromosome walking, until a sequence with a known chromosomal location was found (last exon of diacylglycerol kinase kappa). This showed us that cyclin B3 gene was located on chicken chromosome 4 (accession number AM691545). In marsupials, we cloned a complete cyclin B3 cDNA from T. vulpecula (silver-gray brushtail possum) testis (AM262813) and intron–exon boundaries on genomic DNA, but attempts of chromosomal location were unsuccessful. In M. domestica (short-tailed opossum), we identified most of the cyclin B3 gene from unannotated genomic DNA sequences and reconstituted the cDNA sequence from contig AAFR03062587 (BN00957 and BN00958). This allowed mapping by fluorescent in situ hybridization (FISH) to proximal Xq with two separate bacterial artificial chromosomes (BACs; results not shown). This localization was confirmed in the second version of the genome assembly (Mikkelsen et al. 2007). In the monotreme platypus (Ornithorhynchus anatinus), we cloned the complete cyclin B3 mRNA (AM262748) and most of the genomic sequence (exons 3–15, AM262812). Part of this sequence was missed during platypus genome sequencing published later (Warren et al. 2008), and the chromosomal location of ultracontig 315, containing the cyclin B3 gene, was initially not known. Further BACs FISH mapping revealed that this ultracontig is located on chromosome 6q (Veyrunes et al. 2008). These results support the recent view that therian X originates from chicken chromosome 4 p and corresponds to platypus chromosome 6 (Veyrunes et al. 2008). Database analysis also showed that the synteny of genes surrounding CB3 are informative on the chromosomal rearrangements, which occurred during these crucial steps of mammal X genesis (supplementary fig. S3, Supplementary Material online). This synteny study brought the accessory discovery of the peculiar evolution of the two closer genes to cyclin B3. The gene adjacent to the 3′-end of cyclin B3, in chicken, zebra finch, and lizard, codes for a protein characterized by two galactose-binding domains (here called GBP), which is affiliated to, but distinct of the C21orf63 gene, an heparin-binding protein expressed in epithelia (Mitsunaga et al. 2009), which is present on another chromosome in all species. Since no trace of an homolog of GBP could be found in marsupial and placental mammals, this isozyme was probably lost at the beginning of mammalian evolution. The gene adjacent to the 5′-end of cyclin B3 is the kappa isozyme of diacylglycerol kinase (DGKK), which, in human, has a specific enlargement of the N-terminal exon, of intriguing structure and unknown role (Imai et al. 2005). These features are absent from nonmammalian DGKK, where the first exon is very similar to that of the eta isozyme (DGKH), located on a different chromosome. Comparison of amino acid sequences of DGKK and DGKH first four exons, in fish, amphibian, lizard, and birds, reveals a close similarity, suggesting an origin by duplication of an ancestral gene, with little later evolution (supplementary fig. S3B and Supplementary Data, Supplementary Material online). This is also true in platypus and opossum, whereas all available data on placental mammals yield indications of enlargement of the first exon by repetitions, as in human. This is a striking parallel with the evolution of exon 8 in the neighbor cyclin B3 gene. The Large Exon 8 of Placental Mammals Has Unusual Features Exon 8 is characterized by the ancestral frame 2 intron, which separates it from exon 7 in all vertebrates and several invertebrates. However, its amino acid–encoded sequence is variable from eight amino acids in leech to 39 in Xenopus and shows no conserved amino acid motif (supplementary fig. S2, Supplementary Material online). An abrupt change must have occurred during the emergence of mammals, where the size of this exon is always between 2,400 and 3,000 nt. Since this change must have significant functional changes, we investigated the apparition and characteristics of this extension. Our cloning of cyclin B3 cDNA in platypus, brushtail possum, and genomic sequences of the short-tailed opossum confirmed the absence of the extension in nonplacental mammals exon 8 (which encodes for 44 amino acids in platypus, 52 in possum, and 64 in opossum; supplementary fig. S2, Supplementary Material online), whereas it is present in the early divergent groups of xenarthrans, with more than 855 amino acids encoded in exon 8 of the armadillo (Dasypus novemcinctus) (ENSDNOT00000013199 and our annotation BN00965) and afrotherians, with about 800 amino acids encoded in elephant exon 8 (ENSLAFT00000028769). Thus, the extension appeared after the divergence of placental from nonplacental mammals. To get unambiguous sequence information, we cloned cyclin B3 messenger RNAs in mouse (AJ555464), dog (AJ833648), horse (AM283100), and pig (AM261212). The comparison of these sequences shows that exon 8 has, by far, the lowest sequence conservation (fig. 5). The analysis of exon 8–encoded sequences with structure prediction programs revealed no clear features, beside small patches of disordered structure or coiled coil, at different positions in the five species (see supplementary fig. S4A, Supplementary Material online). Actually, disorder analysis methods yield highly variables predictions for human exon 8, ranging from 3% of disordered zones with GlobProt (Linding et al. 2003) to 94% with TOP-IDP (Campen et al. 2008). However, it has a characteristic highly repetitive organization. This is apparent in dot matrix analysis, as shown in the graphical dotplot in figure 6A. It reveals a frequent periodicity of 18 amino acids. These repetitions are also recognized by internal repeat detection software as Trust (Szklarczyk and Heringa 2004). In the human sequence, 24 repetitions of 36 amino acids were detected, 21 in pig, and 19 in dog. The consensus of these repetitions was highly similar, differing mostly by a shift of two or three amino acids. To get an average, the repeats from human and pig, which had the same alignment, were pooled. The resulting consensus is shown in figure 6B. It is clear that these 36 amino acid repeats are in fact doublets of 18 amino acid repeats, with an overall consensus of EESLFKEPLALQEKPTTE. This repeat is detected 48 times in the human exon 8–encoded sequence, whose length is 997 amino acids, which implies that it accounts for more than 80% of the sequence. Protein database searches revealed no other proteins with significantly similar repeats and not even a single occurrence of similarity superior at 61% with the 18-amino-acid sequence in any animal protein. Thus, the origin of this sequence is unclear, but there are hints that it may have appeared in noneutherian mammals’ exon 8–encoded sequence, by unconstrained mutations, as it is there that the best alignment is found by ClustalX, in the three available cyclin B3 noneutherian mammals’ whole sequences but not in other vertebrate sequences (supplementary fig. S2, Supplementary Material online). The levels of similarity and identity are, respectively, 72% and 39% in opossum, 61% and 28% in possum and platypus, which do not allow for a definitive conclusion but still support the possibility. Curiously, in mouse and rat, exon 8 sequences show very few similarities with those of other mammals, including other rodents, such as squirrel and guinea pig, and repetitive structure is barely detectable in amino acid sequences as well as nucleotidic ones. This suggests the absence of strong evolutionary constraint on the amino acid sequence and three-dimensional structure of the protein product of exon 8. However, comparison of amino acid content, with the Composition Profiler software (Vacic et al. 2007), reveals that murid exon 8 has a fairly similar bias as that of other mammals (supplementary fig. S4B, Supplementary Material online), with an enrichment in disorder promoting residues (E, K, and S) and a depletion in order promoting residues (VWY; Dunker et al. 2001), by comparison with the average of all proteins in the SwissProt database. Thus, the absence of structure and tendency to disorder seems the most specific feature of this extended exon 8. The repetition of ancestral amino acid sequence is probably at the origin of the unusually large size of the exon, through high-frequency duplications. Two examples of recent duplications are shown in figure 7. The first occurred during early primate evolution in the simiiforme lineage, after the divergence of the new world monkeys (Platyrrhini) since the marmoset Callithrix jacchus does not have it, as well as earlier primate divergent groups, as the tarsiiforme Tarsius syrichta and the lorisiforme Otolemur garnettii. It is present in Catarrhini from the rhesus monkey Macaca mulatta, an old world monkey (Cercopithecoidea), to man (fig. 7A) and all hominoidea for which sequences are available: gibbon (Nomascus leucogenys), orangutan (Pongo pygmaeus), gorilla (Gorilla gorilla), and chimpanzee (Pan troglodytes; results not shown). Just before this duplication site is another example of duplication (fig. 7B). There is an N-ter duplication of 28 amino acids, which occurred recently, in the hominoidea lineage, since it is present from the gibbon N. leucogenys to man, but not in the Cercopithecoidea rhesus monkey (M. mulatta). The C-ter duplication of 30–36 amino acids occurred earlier since it is present in Macaca as well as earlier primates (Callithrix, Tarsius, and Otolemur, results not shown). Fig. 5. View large Download slide The amino acid sequence encoded by extended mammalian exon 8 has the lowest conservation of the cyclin B3 protein. The amino acid sequences of cyclin B3 exons of five placental mammals (human, mouse, dog, horse, and pig) were aligned with clustalX 2.0 (see supplementary material fig. S4A, Supplementary Material online). The conservation scores for each exon are reported on the graph. Fig. 5. View large Download slide The amino acid sequence encoded by extended mammalian exon 8 has the lowest conservation of the cyclin B3 protein. The amino acid sequences of cyclin B3 exons of five placental mammals (human, mouse, dog, horse, and pig) were aligned with clustalX 2.0 (see supplementary material fig. S4A, Supplementary Material online). The conservation scores for each exon are reported on the graph. Fig. 6. View largeDownload slide Exon 8 is mostly coding for multiple repetitions of an 18 amino acid sequence. (A) Visualization by dot matrix analysis of repetitions common to human and pig cyclin B3 complete amino acid sequences. Graph from the Gepard software, with a window size of 18. The limits of the N-Terminal part (N_Ter), exon 8 and cyclin box are indicated. (B) Consensus repetitive sequences detected in human and pig exon 8 by the Trust software, visualized by WebLogo. Trust detected 36 amino acid–long repeats, 24 in human and 21 in pig, which were pooled for obtaining the consensus sequence by Weblogo. A gap is inserted after the 18th amino acid to visualize the similarity between both halves. Fig. 6. View largeDownload slide Exon 8 is mostly coding for multiple repetitions of an 18 amino acid sequence. (A) Visualization by dot matrix analysis of repetitions common to human and pig cyclin B3 complete amino acid sequences. Graph from the Gepard software, with a window size of 18. The limits of the N-Terminal part (N_Ter), exon 8 and cyclin box are indicated. (B) Consensus repetitive sequences detected in human and pig exon 8 by the Trust software, visualized by WebLogo. Trust detected 36 amino acid–long repeats, 24 in human and 21 in pig, which were pooled for obtaining the consensus sequence by Weblogo. A gap is inserted after the 18th amino acid to visualize the similarity between both halves. Fig. 7. View largeDownload slide Exon 8 sequences show evidence of multiple cases of sequence duplication. (A) Indication of the duplication of a sequence coding for 83 amino acids in early primate evolution. The duplication is present in man (Homo_s) and rhesus monkey Macaca mulatta (Maca_m) but not in marmoset Callithrix jacchus (Call_j), tarsier Tarsius syrichta (Tars_s) and the galago Otolemur garnettii (Otol_g). (B) Indication of the duplication of a sequence coding for 28 amino acids during later primate evolution and an earlier 30–36 amino acid duplication. The N-ter duplication is present in hominoidea from man (Homo_s) to gibbon Nomascus leucogenys (Noma_l) but not present in the Cercopithecoidea Macaca mulatta (Maca_m). The earlier C-ter duplication is present in all three species. Identical amino acids are shaded black, similar are shaded gray. Amino acid numbering is indicated for species whose complete cyclin B3 sequence is available. References: as in previous figures and Macaca XM_002808531.1, Callithrix XM_002763798.1, Tarsius ENSTSYT00000002261, Otolemur ENSOGAT00000003115, and Nomascus XM_003276862.1. Fig. 7. View largeDownload slide Exon 8 sequences show evidence of multiple cases of sequence duplication. (A) Indication of the duplication of a sequence coding for 83 amino acids in early primate evolution. The duplication is present in man (Homo_s) and rhesus monkey Macaca mulatta (Maca_m) but not in marmoset Callithrix jacchus (Call_j), tarsier Tarsius syrichta (Tars_s) and the galago Otolemur garnettii (Otol_g). (B) Indication of the duplication of a sequence coding for 28 amino acids during later primate evolution and an earlier 30–36 amino acid duplication. The N-ter duplication is present in hominoidea from man (Homo_s) to gibbon Nomascus leucogenys (Noma_l) but not present in the Cercopithecoidea Macaca mulatta (Maca_m). The earlier C-ter duplication is present in all three species. Identical amino acids are shaded black, similar are shaded gray. Amino acid numbering is indicated for species whose complete cyclin B3 sequence is available. References: as in previous figures and Macaca XM_002808531.1, Callithrix XM_002763798.1, Tarsius ENSTSYT00000002261, Otolemur ENSOGAT00000003115, and Nomascus XM_003276862.1. Splice Variant Transcripts, Coding for Shorter Cyclin B3 Proteins, Are Found in Several Mammals The existence of several splice variants isoforms has been reported for human cyclin B3 mRNA, three modifying the transcript 5′UTR part and two the coding sequence (Lozano et al. 2002). These variants may be of important functional significance, especially isoform 1, which lacks exons 7–10, whose translation yields a protein without the insertion but with most of the cyclin box, thus having both extremities of nonmammalian cyclin B3 (fig. 8). We looked for such variant transcripts in other species and found cases in mouse, dog, horse, and pig, as summarized in figure 8. Curiously, in each case the modification of the splicing pattern is different. In mouse, the transcript is depleted of most of exon 8, through the use of the murid-specific intron, without modification of the reading frame, allowing normal translation of exons 9–15. Thus, this protein may function like a nonmammalian cyclin B3. In dog, a similar protein may be produced by the skipping of exon 8 and the use of an alternative 5′ splice site at the end of exon 7 maintaining the right reading frame. However, in horse and pig, alternative splicing also skipped exon 8, totally in pig or partially in horse through the use of an alternative 5′ splice site at the beginning of exon 8, but this resulted in a shift of the reading frame, interrupting translation at the beginning of exon 9, as in human isoform 2. Such transcripts with a premature termination codon may be targeted for nonsense-mediated decay (Chang et al. 2007), making them less easily detectable. Thus, there seems to be no conservation of an alternative splicing pattern providing, in each species, cyclin proteins without extended exon 8, but with a functional cyclin box, as in mouse. Fig. 8. View largeDownload slide Splice variants transcripts, coding for shorter cyclin B3 proteins, are found in several mammals. Schematic representation of transcripts in human (Homo), mouse (Mus), dog (Canis), horse (Equus), and pig (Sus). Exons are indicated by boxes (with black filling for the coding parts and dotted lines if not retained in the transcript), introns by connecting lines. Exon numbering corresponds to the human gene but only the translated part is shown, thus beginning in exon 5. Frame 2 introns (inserted between the first and second nucleotide of a codon) are indicated by a star. The position of the cyclin box coding region is indicated by a dotted line box. References: Homo sapiens isoform 3 AJ314764/NM_033031 (4524 nt, 1395 aa), isoform 1 AJ314765/NM_033670 (1201 nt, 291 aa), isoform 2 AJ314766 (1521 nt, 111 aa); Mus musculus splice variant 2 AJ555465 (1766 nt, 493 aa); Canis familiaris splice variant 2 AM261211 (1149 nt, 382 aa); Equus caballus splice variant 2 AM283101 (1824 nt, 167 aa); and Sus scrofa splice variant 3 AM292028 (1142 nt, 102 aa). Fig. 8. View largeDownload slide Splice variants transcripts, coding for shorter cyclin B3 proteins, are found in several mammals. Schematic representation of transcripts in human (Homo), mouse (Mus), dog (Canis), horse (Equus), and pig (Sus). Exons are indicated by boxes (with black filling for the coding parts and dotted lines if not retained in the transcript), introns by connecting lines. Exon numbering corresponds to the human gene but only the translated part is shown, thus beginning in exon 5. Frame 2 introns (inserted between the first and second nucleotide of a codon) are indicated by a star. The position of the cyclin box coding region is indicated by a dotted line box. References: Homo sapiens isoform 3 AJ314764/NM_033031 (4524 nt, 1395 aa), isoform 1 AJ314765/NM_033670 (1201 nt, 291 aa), isoform 2 AJ314766 (1521 nt, 111 aa); Mus musculus splice variant 2 AJ555465 (1766 nt, 493 aa); Canis familiaris splice variant 2 AM261211 (1149 nt, 382 aa); Equus caballus splice variant 2 AM283101 (1824 nt, 167 aa); and Sus scrofa splice variant 3 AM292028 (1142 nt, 102 aa). The Mammalian-Specific Extension of Cyclin B3 Exon 8 Allows New Protein Interactions The yeast two-hybrid system was used to search for proteins interacting with the part of the cyclin B3 protein encoded by the extended exon 8. A human testis cDNA library was screened with complete or truncated human cyclin B3 as baits. The “Ex5-8ΔCt” bait coded for amino acids 1–801, corresponding to exons 5–7 and 70% of exon 8, and “Ex8ΔCt” to 70% of exon 8 only (amino acids 112–801). The deletion of the C-terminal part of exon 8, which contains the two large repeated sequences, was required since baits truncated at the end of exon 8 were found to be transactivators in our screening conditions. As shown in table 1, 11 interactions were identified: 3 with the complete cyclin B3, 5 with the cyclin box-deleted bait, and 3 with exon 8 alone. Only one was a Cdk, Cdk5, which is not the regular cyclin B3 partner but has already been found to interact with it in an echinoderm two-hybrid screen (Lozano et al. 2010). Only two of the other preys were related (PPP2R5A and PPP2R5E), these protein phosphatase 2 A regulatory subunit isoforms sharing 84% identity and 98% similarity. Four preys are known to be highly expressed in testis: activator of cAMP-responsive element modulator (CREM) in testis (ACT), GMCL1, Ran-binding protein 5 (RanBP5), and Chromosome 1 open reading frame 14 (C1orf14). Six are not specially expressed in testis. They belong to unrelated functional families but are known to associate in multiprotein complexes through special structures, as alpha-alpha superhelix ARM repeats, in phospholipase-activating protein (PLAA) and the two protein phosphatase regulatory subunits (PP2R5A and E), or seven-bladed beta propeller fold WD40 in RACK1. EIF3D is a subunit of a large multiprotein scaffold, binding to the 40 S ribosomal subunit but the cyclin B3 interacting clone corresponds only to the 12% C-terminal part, with a peculiar Glu-rich coiled coil structure. Table 1. Yeast Two-Hybrid Screen of Human Cyclin B3 Interacting Proteins. Prey  Bait   Amino Acids in Prey   Prey Clone Coded Domains    Cyc B3 tot aa 1–1,395  Ex5-8ΔCt aa 1–801  Ex8ΔCt aa 112–801  Clone coded aa  Percentage of Total    CDK5  +      1–1,395  100  Ser-Thr protein kinase-like domain  EIF3D      +  485–548  12  Coiled coil, Glu-rich  FHL5/ACT    +    33–284  90  LIM (4,5×)  GMCL1      +  84–515  86  BTP/POZ  GNB2L1/RACK1    +    197–317  40  WD40 (3×)  IPO5/RanBP5  +      1,055–1,115  5    KAT5/HTATIP/Tip60    +    1–513  100  Chromo/Tudor-knot, MOZ/SAS  PLAA      +  251–795  74  PFU, PUL, ARM repeat  PPP2R5A    +    130–486  94  PP2A-B56, ARM repeat  PPP2R5E  +      1–467  100  PP2A-B56, ARM repeat  SHCBP1L/C1orf14    +    1–653  100  PbH1 (4×), CASH, Pectin lyase-like  Prey  Bait   Amino Acids in Prey   Prey Clone Coded Domains    Cyc B3 tot aa 1–1,395  Ex5-8ΔCt aa 1–801  Ex8ΔCt aa 112–801  Clone coded aa  Percentage of Total    CDK5  +      1–1,395  100  Ser-Thr protein kinase-like domain  EIF3D      +  485–548  12  Coiled coil, Glu-rich  FHL5/ACT    +    33–284  90  LIM (4,5×)  GMCL1      +  84–515  86  BTP/POZ  GNB2L1/RACK1    +    197–317  40  WD40 (3×)  IPO5/RanBP5  +      1,055–1,115  5    KAT5/HTATIP/Tip60    +    1–513  100  Chromo/Tudor-knot, MOZ/SAS  PLAA      +  251–795  74  PFU, PUL, ARM repeat  PPP2R5A    +    130–486  94  PP2A-B56, ARM repeat  PPP2R5E  +      1–467  100  PP2A-B56, ARM repeat  SHCBP1L/C1orf14    +    1–653  100  PbH1 (4×), CASH, Pectin lyase-like  Note.—Prey abbreviations and mRNA references: CDK5, cyclin-dependent kinase 5 (NM_004935); EIF3D, eukaryotic translation initiation factor 3, subunit D (NM_003753); FHL5/ACT, four-and-a-half LIM domain 5/activator of CREM in testis (NM_020482); GMCL1, germ cell-less homolog 1 (NM_178439); GNB2L1/RACK1, guanine nucleotide-binding protein, beta polypeptide 2-like/receptor for activated C kinase 1 (NM_006098); IPO5/RanBP5, Importin 5/Importin beta3/Ran-binding protein 5 (NM_002271); KAT5/HTATIP, lysine acetyl transferase 5/HIV-1 TAT-interactive protein (NM_006388); PLAA, phospholipase A2-activating protein (NM_001031689); PPP2R5A, protein phosphatase 2, regulatory subunit B' (B56), alpha (NM_006243); PPP2R5E, protein phosphatase 2, regulatory subunit B' (B56), epsilon (NM_006246); SHCBP1L/C1orf14, SHC SH2-domain binding protein 1-like/chromosome 1 open reading frame 14 (NM_030933). Domain references: Ser-Thr protein kinase-like domain (Pfam00069), glutamic acid-rich region (Glu-rich, Prosite 50313), LIM domain (Pfam 00412), BTP/POZ domain (Pfam 00651), WD40 repeat (Pfam 00400), Chromatin organization modifier (Chromo, Smart00298), RNA binding activity-knot of a chromodomain (Tudor-knot, Pfam 11717), MOZ/SAS family (Pfam 01853), PLAA ubiquitin-binding domain (PFU, Pfam 09070), PUL domain (Pfam 08324), Armadillo repeat (ARM repeat, Superfamily 48371), PP2A regulatory subunit B56 (PP2A-B56, Pfam 01603), Parallel beta-helix repeats (PbH1, Smart00710), carbohydrate-binding proteins and sugar hydrolases domain (CASH, Smart00722), Pectin lyase-like (Superfamily 51126). View Large The ability of the extended exon 8–encoded polypeptide to interact with full open reading frame (ORF) prey proteins was further checked in the yeast two-hybrid system for Cdk5 and the four testis-specific proteins (Supplementary Data). Six baits were used: the human complete sequence or only exon 8 or splice variant isoform 1 (lacking exons 7–9), the mouse splice variant (with most of exon 8 skipped), and the complete cyclin B3 from two nonplacental mammals, possum, and platypus, which have a short exon 8. As expected, the interaction with human Cdk5 occurred only with baits containing the C-ter cyclin box, whatever the species. All other tested preys showed interaction only with baits containing the extended exon 8, alone or in complete cyclin B3 sequence. To further illustrate that extension of exon 8 in placental mammals brought new interaction abilities, three prey orthologs were sequenced in nonplacental mammals: FHL5 and GMCL1 in possum and SHCBP1L in platypus. These proteins were not able to interact with cyclin B3 of the same species, but did interact with extended exon 8 containing human baits. This heterologous interaction is not surprising since these proteins showed good sequence conservation with their human homologs (66% identity and 85% similarity for FHL5, 93% and 97% for GMCL1, and 83% and 95% for SHCBPL1). Some of these interactions were also verified in vitro by pull-down experiments with mRNA of human cyclin B3, Cdk5, IPO3, and SHCBP1L, translated in reticulocyte lysates (Supplementary Data). Discussion Our detailed search of early-branching metazoan genomic databases indicates that cyclin B3 was already present in sponges, placozoan, and cnidarians but not in their supposed closest ancestors, the unicellular choanoflagellate, Monosiga, and the mold, Neurospora, whereas cyclins A and B are of ancient eukaryotic origin (Srivastava et al. 2010). Thus, cyclin B3 probably appeared by duplication of the cyclin B gene and may be added to the list of cell cycle genes, such as Cdk2 and cyclin E, which appeared in metazoans and allow new regulations (Srivastava et al. 2010). Genomic studies have revealed a significant conservation of intron positions from early-branching metazoan groups to man. This is clearly the case for the cyclin B3 gene whose intron positions and phases found in sponge and placozoan are still present in man. The significant loss of introns already noticed in Drosophila and Caenorhabditis (Putnam et al. 2007) is also visible in the cyclin B3 gene, with the loss of two-thirds of them in the nematode and total loss in the fly. The genomes of these species, as well as those of urochordates (Ciona and Oikopleura), are known to have underwent extensive rearrangements, for still unclear reasons, but among which was pressure for genome compaction (Edvardsen et al. 2004). The presence of unmodified ancestral intron pattern in the gastropod Lottia or in the cephalochordate amphioxus indicates that gene structures in these divergent lineages are clearly not representative of basic phylogenetic evolution (Putnam et al. 2008). However, available data are still too fragmental to exclude that the three losses of ancestral introns in the cyclin B3 gene of the crustacean Daphnia are not representative of evolution in the ecdysozoan lineage. In the extreme case of Drosophila, the total loss of introns, in all species of this genus, fits with the best supported hypothesis that intron losses result from recombination with an intronless cDNA of the gene (Coulombe-Huntington and Majewski 2007). An incidental discovery of this study is the apparition of two new introns, one in chordates and the other in mouse and rat. This is interesting since intron gain is reported to be extremely rare in vertebrate evolution (Loh et al. 2008), and the underlaying mechanisms are still debated (Roy and Gilbert 2006). The first case is the intron separating exons 10 and 11, which is present in all known cyclin B3 genes from a cartilaginous fish to human, and in a nearby position in prochordates but in no invertebrate sequence. The most likely hypothesis for the origin of this intron is by tandem genomic duplication and use of cryptic splice signals (Zhuo et al. 2007), but this event is too ancient to be further supported by remaining sequence similarity in the intron and flanking exons. The most favorable splice site is that of the prochordates’ intron. So if this intron was only acquired once, it may have been at this site, and lately slid to the chordate position, by alternative splicing using the Q codon cryptic splice site. Nevertheless, the gain of this intron may have relied on very low probability circumstances, such as genome duplications, since the VEVQ coding sequence, with its associated cryptic splice signals, is frequent in invertebrates but there is no indication of sporadic intron occurrence. It is also surprising that the intron position was kept unchanged in all vertebrates for which data are available, despite the presence of cryptic splice signals, associated with several valine codons occurrences on the 5′-side and glutamic acid codons on the 3′-side. Intronization of exonic sequences has been recently described in Caenorhabditis (Irimia et al. 2008) and primates (Szczesniak et al. 2010). This new mechanism of intron formation may be at the origin of the small intron inserted in mouse and rat exon 8, by deletion, in murid transcripts, of a previously coding sequence. The cryptic splice sites are not apparent in the other rodent sequences, but the intron creation is more ancient than in the case of Caenorhabditis, where it concerns species of the same genus. Another difference is that the murid intron has a stop codon and thus unspliced transcripts could not be translated, whereas both kinds of transcripts have been found in Caenorhabditis elegans. Thus, the deletion of the intronized exonic sequence is compulsory, but the removal of about 38 amino acids has probably no significant functional consequence, since the placental mammalian extended exon 8 has the lowest constraint for amino acid sequence conservation. This intron may rather be advantageous since, being of frame 0 instead of frame 2 for the previous intron, it allows the generation of a short alternative transcript, lacking most of extended exon 8, but allowing in frame translation of subsequent exons. The study of cyclin B3 gene location at the transition between sauropsids and mammals revealed that it belongs to the autosomal group of genes at the origin of marsupial X chromosome. From the short arm of chicken chromosome 4, then platypus chromosome 6, they constitute the whole X chromosome in marsupials and finally the X conserved region of human X (Veyrunes et al. 2008). Although not in close synteny with the cyclin B3 gene, this group contains the SOX3 gene, probable ancestor of the testis-determining gene SRY (Foster and Graves 1994). Of all known human cyclin genes, only the cyclin B3 one is on the X chromosome, but in nontherian vertebrates, as well as invertebrates, in which chromosomal location is known, it is never located on a sex chromosome. So if its function in gametogenesis fits well with the observed excess of reproduction-related genes on human chromosome X (Saifi and Chandra 1999), this specific localization was not favored in nonmammalian species. The localization on the X chromosome should, however, have had consequences on the evolution of the gene, such as more rapid selection in the hemizygous male (Graves 2006; Vicoso and Charlesworth 2006). This may have favored the abrupt extension of cyclin B3 exon 8. It also implied new constraints on gene expression, notably a reduction due to absence of a second gene copy in males or its silencing in females. This may be especially important for cyclin B3, whose critical gene dosage has been demonstrated in Caenorhabditis (Tarailo-Graovac et al. 2010). Escape from X-inactivation is not rare, but the cyclin B3 gene was found to be mostly subjected to inactivation in L. Carrel experiments (expression in two of nine Xi hybrids; Carrel and Willard 2005). Although the dosage compensation, by overexpression of X-linked genes, is currently debated (Xiong et al. 2010), levels of gene expression are still reported to be higher in testis and brain than in other organs. So the change in localization must have resulted in a modest reduction of transcription, almost similar in both sexes. Another peculiarity of X-linked genes is their inactivation during male meiosis, called meiotic sex chromosome inactivation (MSCI; see Turner 2007 for review). In this process, transcription is inactivated through post-translational modifications of core histones and replacement with other highly modified histone variants, in unsynapsed chromatin, at the pachytene stage of meiosis. The X and Y chromosomes are further compartmentalized into a peripheral subdomain, the sex or X-Y body, until the end of the diplotene stage and, even after meiotic divisions, they remain mostly repressed as postmeiotic chromatin in spermatids. This is in accordance with the reported downregulation of cyclin B3 at the pachytene stage of mouse spermatogenesis (Nguyen et al. 2002), whereas other A, B, and D type cyclin expressions continue until the spermatid stage (Wolgemuth and Roberts 2010). In addition, experimental upregulation of cyclin B3 expression in transgenic mice leads to abnormal spermatogenesis (Refik-Rogers et al. 2006). A significant number of X-linked genes, involved in male germline function, are reported to be at the origin of functional retrogenes on autosomes, thus insuring continuation of transcription during MSCI (Emerson et al. 2004). However, this is not the case for cyclin B3, since the only retrogene found in human (LOC100131678) is largely incomplete, covering only 36% of C-ter ORF, and without continuous coding frame. The most intriguing feature of cyclin B3 is the abrupt change in exon 8 size, at the divergence of placental from nonplacental mammals. The high conservation of the intron–exon structure leaves no doubt that this change did not result from intron loss but from internal extension of the exon. The presence of a detectable repetitive amino acid sequence of 18 amino acids indicates a probable origin through multiple internal duplications. The part of cyclin B3 proteins encoded by exon 8 has the lowest amino acid conservation between species, which is indicative of a zone submitted to a very low pressure selection and thus allowing for duplications without functional consequences. There are no indications in databases that the basic amino acid motif is present in other proteins, which make improbable the transfer of a large exogenous sequence. The amplification may have rather started in the therian mammal ancestor gene, probably rapidly since the exon size is fairly similar in all actual species. There are good indications that accumulation of tandem repeats in a protein is frequently associated with acquisition of new functions (De Grassi and Ciccarelli 2009), but it is generally assumed to occur after gene duplication, which is improbable for cyclin B3 gene, which is always found as a single copy. There is no indication of repetitive tertiary structure in the exon 8–encoded amino acid sequence, in contrast with many well-studied protein repeats, such as zinc fingers, armadillo, and HEAT motifs (Andrade et al. 2001). Thus, this part of the protein is probably intrinsically unstructured, which would fit with its fast evolution (Brown et al. 2011), and accumulation of repeats (Simon and Hancock 2009; Tompa 2003). It would also allow for new interactions since there are reports that the lack of rigid structure is important for protein–protein interaction (Tompa and Fuxreiter 2008), notably for cell cycle proteins such as the p27 Cdk inhibitor (Galea et al. 2008). In the hypothesis of unconstrained repeat expansion, it is curious that the size of exon 8 is not very different in all mammalian cyclin B3 genes, between 2,400 and 3,000 nt, whereas there are recognizable duplications of several hundreds of nucleotides just in the primate lineage. This suggests the existence of a constraint preventing a size increase above 3,000 nt. The mechanisms allowing efficient splicing of very large exons are unknown, since most exons do not exceed 300 nt (Gudlaugsdottir et al. 2007) and this seems to be the upper limit for proper function of the regular splicing machinery when exon size was experimentally increased (Berget 1995). However, databases indicate the existence of exons up to 10,000 nt in mammals (Gudlaugsdottir et al. 2007) and some well-studied proteins, as apolipoprotein B and Ki-67 antigen (Schluter et al. 1993), have exons of 7,572 and 6,845 nt, respectively, which implies that splicing is still possible above 3,000 nt. Thus, the 3,000 nt limit for cyclin B3 exon 8 is probably due to a constraint on the function of the protein. Alternative splicing is common, especially in genes with large exons, which are frequently skipped in mRNA variants. This is confirmed in this study for the cyclin B3 gene where short transcripts, lacking the totality or most of exon 8, were found in the four species investigated, but with different splicing patterns, producing or not premature translation termination. The alternative transcripts of horse and pig code for proteins without functional cyclin box, suggesting that there was no selection pressure to maintain the coexistence of protein products with and without the exon 8 extension in mammals. So the enlargement of exon 8 probably did not prevent the cyclin B3 protein functioning as in nonmammalians and, in contrast to what is observed in many other genes (Nilsen and Graveley 2010), alternative transcripts may not even yield functionally significant proteins. The search for new interaction brought to the cyclin B3 protein by the extension of exon 8, with the yeast two-hybrid system, identified different interacting proteins. One was the expected interaction of a Cdk with cyclin B3 cyclin box, but curiously it is not one of the physiological Cdk partners, Cdk1 or Cdk2, but the neuronal-specific Cdk5, not involved in cell cycle control (see Dhariwala and Rajadhyaksha 2008 for review). Its physiological activators are proteins structurally similar to cyclins but without amino acid sequence homology, p35 and p39, or a typical cyclin, cyclin I (Brinkkoetter et al. 2009). It can also bind cyclins D and E, but without stimulation of its kinase activity, and, in a previous experiment, sea urchin Cdk5 was found to interact with starfish cyclin B3, in the two-hybrid system but not in vivo (Lozano et al. 2010). Six interacting proteins do not have a high probability to be cyclin B3 partners under physiological conditions, since they are not known to be specially expressed in testis, and are rather indicative of the new ability of the extended exon 8 to bind specialized structures. One is a small fragment of a 40 S ribosomal subunit (see Hinnebusch 2006 for review), probably interacting through its Glu-rich coiled coil. RACK1 is a scaffold protein undergoing protein–protein interaction through its WD40 beta propeller fold (see Sklan et al. 2006 for review). KAT5/Tip60 is mostly incorporated in a stable nuclear complex of 20 proteins and can bind transiently many other proteins, but interaction with cyclin B3 has never been reported (see Sapountzi et al. 2006 for review). PLAA is a phospholipase A2-activating protein, which modulates either response to inflammation (Zhang et al. 2008) or ubiquitin-mediated protein degradation through its ubiquitin-binding domain PUL (Fu et al. 2009). The serine/threonine protein phosphatase 2 A is a very abundant enzyme, with conserved catalytic and scaffold subunits able to associate with highly variable regulatory subunits (see Shi 2009 for review), but no interaction with cyclin B3 has been reported. Thus, the two-hybrid interaction is more probably indicative of an ability of extended exon 8 to bind the super helical structures common to PLAA and the B56 PP2A regulatory subunit. For the four testis-specific proteins, there is also no conclusive indication of physiological interaction. RanBP5 is not known to mediate nuclear import of cyclins but rather histones and ribosomal proteins (Chou et al. 2010), and additional two-hybrid checking do not show interaction with platypus and possum cyclin B3, despite the probable presence of functional nuclear localization signals. The interaction is limited to the exon 8 extension, which contains some unconventional nuclear localization signals (Tschop et al. 2006), but RanBP5 has HEAT and ARM-type folds which may mediate an interaction similar to that of the phosphatases and phospholipase preys. FHL5/ACT was discovered as an activator of the sperm-specific transcription factor CREM which regulates the expression of various postmeiotic genes (Fimia et al. 1999). It is nuclear but expressed in late spermatogenesis, in round and elongated spermatids, whereas cyclin B3 is not expressed after meiosis. The two-hybrid interaction may result from a protein–protein interaction with the four and a half repetitions of LIM domains, characterized by two adjacent zinc fingers, which were first identified in homeodomain transcription factor, and can mediate the formation of homo and heterocomplexes. Germ cell-less was discovered in Drosophila, as a gene required for germ line specification, before a close ortholog was found in mouse, with its highest expression during spermatogenesis. In both cases, the protein localizes to the nuclear envelope and deletion of the gene impairs fertility in male mice (Kimura et al. 2003). It contains a BTZ/POZ domain, promoting protein–protein interactions, and may be a physiological partner of cyclin B3, although their maximal expressions during mouse spermatogenesis, at pachytene and diplotene, respectively, are not fully synchronous. Less is known on the last interacting protein, C1orf14, which has no known function but is strongly expressed in testis (Sood et al. 2001) and breast cancer (WO/2008/086799). It has 30% identity to mouse SHC-binding protein, which belongs to an evolutionary conserved family, recently found associated to central spindle in Drosophila during spermatocytes cytokinesis, where it can bind kinesin-like proteins, Rho family GTPase-activating proteins and carbohydrates, via its pectin lyase domain (Montembault et al. 2010). Further work will be needed to determine the physiological relevance of the new interactions detected, but it seems already clear that the expansion of exon 8 in placental mammals brought new possibilities of interaction to cyclin B3. A last point of interest is a probable oncogenic ability, since in a very recently published work, cyclin B3 abnormal expression was found to be associated with a new subtype of human bone sarcoma. In a recurrent paracentric inversion, the complete corepressor BCOR gene is fused to the C-terminal part of cyclin B3, starting with complete in frame exon 8 (thus lacking exon 6 destruction box), leading to a high level of expression of the chimeric protein (Pierron et al. 2012). This is of special interest since only cyclin D and E gene amplification (Stamatakos et al. 2010) and virally modified cyclin A (Berasain et al. 1998) have been reported to be oncogenic triggers. Acknowledgments This publication is dedicated to the memory of André Picard, who initiated this work and gave it enthusiastic and unfailing support. We thank Dr Jennifer A. Marshall Graves, La Trobe University, Melbourne, Australia, and Dr Jeanine Deakin, Australian National University, Canberra, Australia, for the generous gift of platypus biological material, Dr Nadine Gerard, UMR 6175 INRA-CNRS U. de Tours, France, for the generous gift of pig and horse mRNA, and Dr Fabrice Senger and Dr Richard Guyon, Institute of Genetics and Development of Rennes, France, for the generous gift of dog biological material. This work was supported by the Ligue Nationale contre le Cancer. References Andrade MA,  Perez-Iratxeta C,  Ponting CP.  Protein repeats: structures, functions, and evolution,  J Struct Biol. ,  2001, vol.  134  2–3(pg.  117- 131) Google Scholar CrossRef Search ADS PubMed  Berasain C,  Patil D,  Perara E,  Huang SM,  Mouly H,  Brechot C.  Oncogenic activation of a human cyclin A2 targeted to the endoplasmic reticulum upon hepatitis B virus genome insertion,  Oncogene ,  1998, vol.  16  10(pg.  1277- 1288) Google Scholar CrossRef Search ADS PubMed  Berget SM.  Exon recognition in vertebrate splicing,  J Biol Chem. ,  1995, vol.  270  6(pg.  2411- 2414) Google Scholar CrossRef Search ADS PubMed  Brinkkoetter PT,  Olivier P,  Wu JS,  Henderson S,  Krofft RD,  Pippin JW,  Hockenbery D,  Roberts JM,  Shankland SJ.  Cyclin I activates Cdk5 and regulates expression of Bcl-2 and Bcl-XL in postmitotic mouse cells,  J Clin Invest. ,  2009, vol.  119 (pg.  3089- 3101) Google Scholar CrossRef Search ADS PubMed  Brown CJ,  Johnson AK,  Dunker AK,  Daughdrill GW.  Evolution and disorder,  Curr Opin Struct Biol. ,  2011, vol.  21  3(pg.  441- 446) Google Scholar CrossRef Search ADS PubMed  Campen A,  Williams RM,  Brown CJ,  Meng J,  Uversky VN,  Dunker AK.  TOP-IDP-scale: a new amino acid scale measuring propensity for intrinsic disorder,  Protein Pept Lett. ,  2008, vol.  15  9(pg.  956- 963) Google Scholar CrossRef Search ADS PubMed  Carrel L,  Willard HF.  X-inactivation profile reveals extensive variability in X-linked gene expression in females,  Nature ,  2005, vol.  434  7031(pg.  400- 404) Google Scholar CrossRef Search ADS PubMed  Chang YF,  Imam JS,  Wilkinson MF.  The nonsense-mediated decay RNA surveillance pathway,  Annu Rev Biochem. ,  2007, vol.  76 (pg.  51- 74) Google Scholar CrossRef Search ADS PubMed  Chou CW,  Tai LR,  Kirby R,  Lee IF,  Lin A.  Importin beta3 mediates the nuclear import of human ribosomal protein L7 through its interaction with the multifaceted basic clusters of L7,  FEBS Lett. ,  2010, vol.  584  19(pg.  4151- 4156) Google Scholar CrossRef Search ADS PubMed  Coulombe-Huntington J,  Majewski J.  Intron loss and gain in Drosophila,  Mol Biol Evol. ,  2007, vol.  24  12(pg.  2842- 2850) Google Scholar CrossRef Search ADS PubMed  Crooks GE,  Hon G,  Chandonia JM,  Brenner SE.  WebLogo: a sequence logo generator,  Genome Res. ,  2004, vol.  14  6(pg.  1188- 1190) Google Scholar CrossRef Search ADS PubMed  D'Angiolella V,  Donato V,  Vijayakumar S,  Saraf A,  Florens L,  Washburn MP,  Dynlacht B,  Pagano M.  SCF(Cyclin F) controls centrosome homeostasis and mitotic fidelity through CP110 degradation,  Nature ,  2010, vol.  466  7302(pg.  138- 142) Google Scholar CrossRef Search ADS PubMed  De Grassi A,  Ciccarelli FD.  Tandem repeats modify the structure of human genes hosted in segmental duplications,  Genome Biol. ,  2009, vol.  10  12pg.  R137  Google Scholar CrossRef Search ADS PubMed  Deyter GM,  Furuta T,  Kurasawa Y,  Schumacher JM.  Caenorhabditis elegans cyclin B3 is required for multiple mitotic processes including alleviation of a spindle checkpoint-dependent block in anaphase chromosome segregation,  PLoS Genet. ,  2010, vol.  6  11pg.  e1001218  Google Scholar CrossRef Search ADS PubMed  Dhariwala FA,  Rajadhyaksha MS.  An unusual member of the Cdk family: Cdk5,  Cell Mol Neurobiol. ,  2008, vol.  28  3(pg.  351- 369) Google Scholar CrossRef Search ADS PubMed  Doree M,  Hunt T.  From Cdc2 to Cdk1: when did the cell cycle kinase join its cyclin partner?,  J Cell Sci. ,  2002, vol.  115  Pt 12(pg.  2461- 2464) Google Scholar PubMed  Dunker AK,  Lawson JD,  Brown CJ, et al.  ,  (20 co-authors).  Intrinsically disordered protein,  J Mol Graph Model ,  2001, vol.  19  1(pg.  26- 59) Google Scholar CrossRef Search ADS PubMed  Edvardsen RB,  Lerat E,  Maeland AD,  Flat M,  Tewari R,  Jensen MF,  Lehrach H,  Reinhardt R,  Seo HC,  Chourrout D.  Hypervariable and highly divergent intron-exon organizations in the chordate Oikopleura dioica,  J Mol Evol. ,  2004, vol.  59  4(pg.  448- 457) Google Scholar CrossRef Search ADS PubMed  Emerson JJ,  Kaessmann H,  Betran E,  Long M.  Extensive gene traffic on the mammalian X chromosome,  Science ,  2004, vol.  303  5657(pg.  537- 540) Google Scholar CrossRef Search ADS PubMed  Fimia GM,  De Cesare D,  Sassone-Corsi P.  CBP-independent activation of CREM and CREB by the LIM-only protein ACT,  Nature ,  1999, vol.  398  6723(pg.  165- 169) Google Scholar CrossRef Search ADS PubMed  Foster JW,  Graves JA.  An SRY-related sequence on the marsupial X chromosome: implications for the evolution of the mammalian testis-determining gene,  Proc Natl Acad Sci U S A. ,  1994, vol.  91  5(pg.  1927- 1931) Google Scholar CrossRef Search ADS PubMed  Fu QS,  Zhou CJ,  Gao HC,  Jiang YJ,  Zhou ZR,  Hong J,  Yao WM,  Song AX,  Lin DH,  Hu HY.  Structural basis for ubiquitin recognition by a novel domain from human phospholipase A2-activating protein,  J Biol Chem. ,  2009, vol.  284  28(pg.  19043- 19052) Google Scholar CrossRef Search ADS PubMed  Galea CA,  Wang Y,  Sivakolundu SG,  Kriwacki RW.  Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits,  Biochemistry ,  2008, vol.  47  29(pg.  7598- 7609) Google Scholar CrossRef Search ADS PubMed  Gallant P,  Nigg EA.  Identification of a novel vertebrate cyclin: cyclin B3 shares properties with both A- and B-type cyclins,  Embo J. ,  1994, vol.  13  3(pg.  595- 605) Google Scholar PubMed  Graves JA.  Sex chromosome specialization and degeneration in mammals,  Cell ,  2006, vol.  124  5(pg.  901- 914) Google Scholar CrossRef Search ADS PubMed  Gudlaugsdottir S,  Boswell DR,  Wood GR,  Ma J.  Exon size distribution and the origin of introns,  Genetica ,  2007, vol.  131  3(pg.  299- 306) Google Scholar CrossRef Search ADS PubMed  Gunbin KV,  Suslov VV,  Turnaev II,  Afonnikov DA,  Kolchanov NA.  Molecular evolution of cyclin proteins in animals and fungi,  BMC Evol Biol. ,  2011, vol.  11 pg.  224  Google Scholar CrossRef Search ADS PubMed  Hall TA.  BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT,  Nucleic Acids Symp Ser. ,  1999, vol.  41 (pg.  95- 98) Hinnebusch AG.  eIF3: a versatile scaffold for translation initiation complexes,  Trends Biochem Sci. ,  2006, vol.  31  10(pg.  553- 562) Google Scholar CrossRef Search ADS PubMed  Imai S,  Kai M,  Yasuda S,  Kanoh H,  Sakane F.  Identification and characterization of a novel human type II diacylglycerol kinase, DGK kappa,  J Biol Chem. ,  2005, vol.  280  48(pg.  39870- 39881) Google Scholar CrossRef Search ADS PubMed  Irimia M,  Rukov JL,  Penny D,  Vinther J,  Garcia-Fernandez J,  Roy SW.  Origin of introns by “intronization” of exonic sequences,  Trends Genet. ,  2008, vol.  24  8(pg.  378- 381) Google Scholar CrossRef Search ADS PubMed  Jacobs HW,  Knoblich JA,  Lehner CF.  Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like Cyclin B,  Genes Dev. ,  1998, vol.  12  23(pg.  3741- 3751) Google Scholar CrossRef Search ADS PubMed  Kajiura-Kobayashi H,  Kobayashi T,  Nagahama Y.  The cloning of cyclin B3 and its gene expression during hormonally induced spermatogenesis in the teleost, Anguilla japonica,  Biochem Biophys Res Commun. ,  2004, vol.  323  1(pg.  288- 292) Google Scholar CrossRef Search ADS PubMed  Kapitonov VV,  Jurka J.  Young families of CR1 non-LTR retrotransposons from the amphioxus genome,  Repbase Reports ,  2009, vol.  9  7pg.  1628  Kiessling AA,  Bletsa R,  Desmarais B,  Mara C,  Kallianidis K,  Loutradis D.  Evidence that human blastomere cleavage is under unique cell cycle control,  J Assist Reprod Genet. ,  2009, vol.  26  4(pg.  187- 195) Google Scholar CrossRef Search ADS PubMed  Kimura T,  Ito C,  Watanabe S, et al.  ,  (14 co-authors).  Mouse germ cell-less as an essential component for nuclear integrity,  Mol Cell Biol. ,  2003, vol.  23  4(pg.  1304- 1315) Google Scholar CrossRef Search ADS PubMed  King N,  Westbrook MJ,  Young SL, et al.  ,  (36 co-authors).  The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans,  Nature ,  2008, vol.  451  7180(pg.  783- 788) Google Scholar CrossRef Search ADS PubMed  Kobayashi H,  Stewart E,  Poon R,  Adamczewski JP,  Gannon J,  Hunt T.  Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits,  Mol Biol Cell ,  1992, vol.  3  11(pg.  1279- 1294) Google Scholar CrossRef Search ADS PubMed  Kohany O,  Gentles AJ,  Hankus L,  Jurka J.  Annotation, submission and screening of repetitive elements in Repbase: RepbaseSubmitter and Censor,  BMC Bioinformatics ,  2006, vol.  7 pg.  474  Google Scholar CrossRef Search ADS PubMed  Kreutzer MA,  Richards JP,  De Silva-Udawatta MN,  Temenak JJ,  Knoblich JA,  Lehner CF,  Bennett KL.  Caenorhabditis elegans cyclin A- and B-type genes: a cyclin A multigene family, an ancestral cyclin B3 and differential germline expression,  J Cell Sci. ,  1995, vol.  108   Pt 6(pg.  2415- 2424) Google Scholar PubMed  Krumsiek J,  Arnold R,  Rattei T.  Gepard: a rapid and sensitive tool for creating dotplots on genome scale,  Bioinformatics ,  2007, vol.  23  8(pg.  1026- 1028) Google Scholar CrossRef Search ADS PubMed  Larkin MA,  Blackshields G,  Brown NP, et al.  ,  (13 co-authors).  Clustal W and Clustal X version 2.0,  Bioinformatics ,  2007, vol.  23  21(pg.  2947- 2948) Google Scholar CrossRef Search ADS PubMed  Linding R,  Russell RB,  Neduva V,  Gibson TJ.  GlobPlot: exploring protein sequences for globularity and disorder,  Nucleic Acids Res. ,  2003, vol.  31  13(pg.  3701- 3708) Google Scholar CrossRef Search ADS PubMed  Loh YH,  Brenner S,  Venkatesh B.  Investigation of loss and gain of introns in the compact genomes of pufferfishes (Fugu and Tetraodon),  Mol Biol Evol. ,  2008, vol.  25  3(pg.  526- 535) Google Scholar CrossRef Search ADS PubMed  Lozano JC,  Perret E,  Schatt P,  Arnould C,  Peaucellier G,  Picard A.  Molecular cloning, gene localization, and structure of human cyclin B3,  Biochem Biophys Res Commun. ,  2002, vol.  291  2(pg.  406- 413) Google Scholar CrossRef Search ADS PubMed  Lozano JC,  Schatt P,  Verge V,  Gobinet J,  Villey V,  Peaucellier G.  CDK5 is present in sea urchin and starfish eggs and embryos and can interact with p35, cyclin E and cyclin B3,  Mol Reprod Dev. ,  2010, vol.  77  5(pg.  449- 461) Google Scholar CrossRef Search ADS PubMed  Lupas A,  Van Dyke M,  Stock J.  Predicting coiled coils from protein sequences,  Science ,  1991, vol.  252  5010(pg.  1162- 1164) Google Scholar CrossRef Search ADS PubMed  Malumbres M,  Barbacid M.  Mammalian cyclin-dependent kinases,  Trends Biochem Sci. ,  2005, vol.  30  11(pg.  630- 641) Google Scholar CrossRef Search ADS PubMed  Mikkelsen TS,  Wakefield MJ,  Aken B, et al.  ,  (64 co-authors).  Genome of the marsupial Monodelphis domestica reveals innovation in non-coding sequences,  Nature ,  2007, vol.  447  7141(pg.  167- 177) Google Scholar CrossRef Search ADS PubMed  Miles DC,  van den Bergen JA,  Sinclair AH,  Western PS.  Regulation of the female mouse germ cell cycle during entry into meiosis,  Cell Cycle ,  2010, vol.  9  2(pg.  408- 418) Google Scholar CrossRef Search ADS PubMed  Miller ME,  Cross FR.  Cyclin specificity: how many wheels do you need on a unicycle?,  J Cell Sci. ,  2001, vol.  114  Pt 10(pg.  1811- 1820) Google Scholar PubMed  Mitsunaga K,  Harada-Itadani J,  Shikanai T,  Tateno H,  Ikehara Y,  Hirabayashi J,  Narimatsu H,  Angata T.  Human C21orf63 is a heparin-binding protein,  J Biochem. ,  2009, vol.  146  3(pg.  369- 373) Google Scholar CrossRef Search ADS PubMed  Montembault E,  Zhang W,  Przewloka MR,  Archambault V,  Sevin EW,  Laue ED,  Glover DM,  D'Avino PP.  Nessun Dorma, a novel centralspindlin partner, is required for cytokinesis in Drosophila spermatocytes,  J Cell Biol. ,  2010, vol.  191  7(pg.  1351- 1365) Google Scholar CrossRef Search ADS PubMed  Nguyen TB,  Manova K,  Capodieci P,  Lindon C,  Bottega S,  Wang XY,  Refik-Rogers J,  Pines J,  Wolgemuth DJ,  Koff A.  Characterization and expression of mammalian cyclin B3, a prepachytene meiotic cyclin,  J Biol Chem. ,  2002, vol.  277  44(pg.  41960- 41969) Google Scholar CrossRef Search ADS PubMed  Nieduszynski CA,  Murray J,  Carrington M.  Whole-genome analycsis of animal A- and B-type cyclins,  Genome Biol. ,  2002, vol.  3  12(pg.  701- 708) Google Scholar CrossRef Search ADS   Nilsen TW,  Graveley BR.  Expansion of the eukaryotic proteome by alternative splicing,  Nature ,  2010, vol.  463  7280(pg.  457- 463) Google Scholar CrossRef Search ADS PubMed  Ozaki Y,  Saito K,  Shinya M,  Kawasaki T,  Sakai N.  Evaluation of Sycp3, Plzf and Cyclin B3 expression and suitability as spermatogonia and spermatocyte markers in zebrafish,  Gene Expr Patterns. ,  2011, vol.  11  5–6(pg.  309- 315) Google Scholar CrossRef Search ADS PubMed  Payer B,  Lee JT.  X chromosome dosage compensation: how mammals keep the balance,  Annu Rev Genet. ,  2008, vol.  42 (pg.  733- 772) Google Scholar CrossRef Search ADS PubMed  Pierron G,  Tirode F,  Lucchesi C,  Reynaud S,  Ballet S,  Cohen-Gogo S,  Perrin V,  Coindre JM,  Delattre O.  A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion,  Nat Genet. ,  2012, vol.  44 (pg.  461- 466) Google Scholar CrossRef Search ADS PubMed  Pines J.  Four-dimensional control of the cell cycle,  Nat Cell Biol. ,  1999, vol.  1  3(pg.  E73- E79) Google Scholar CrossRef Search ADS PubMed  Putnam NH,  Butts T,  Ferrier DE, et al.  ,  (37 co-authors).  The amphioxus genome and the evolution of the chordate karyotype,  Nature ,  2008, vol.  453  7198(pg.  1064- 1071) Google Scholar CrossRef Search ADS PubMed  Putnam NH,  Srivastava M,  Hellsten U, et al.  ,  (37 co-authors).  Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization,  Science ,  2007, vol.  317  5834(pg.  86- 94) Google Scholar CrossRef Search ADS PubMed  Refik-Rogers J,  Manova K,  Koff A.  Misexpression of cyclin B3 leads to aberrant spermatogenesis,  Cell Cycle ,  2006, vol.  5  17(pg.  1966- 1973) Google Scholar CrossRef Search ADS PubMed  Roy SW,  Gilbert W.  The evolution of spliceosomal introns: patterns, puzzles and progress,  Nat Rev Genet. ,  2006, vol.  7  3(pg.  211- 221) Google Scholar PubMed  Saifi GM,  Chandra HS.  An apparent excess of sex- and reproduction-related genes on the human X chromosome,  Proc Biol Sci. ,  1999, vol.  266  1415(pg.  203- 209) Google Scholar CrossRef Search ADS PubMed  Sapountzi V,  Logan IR,  Robson CN.  Cellular functions of TIP60,  Int J Biochem Cell Biol. ,  2006, vol.  38  9(pg.  1496- 1509) Google Scholar CrossRef Search ADS PubMed  Satyanarayana A,  Kaldis P.  Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms,  Oncogene ,  2009, vol.  28  33(pg.  2925- 2939) Google Scholar CrossRef Search ADS PubMed  Schluter C,  Duchrow M,  Wohlenberg C,  Becker MH,  Key G,  Flad HD,  Gerdes J.  The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins,  J Cell Biol. ,  1993, vol.  123  3(pg.  513- 522) Google Scholar CrossRef Search ADS PubMed  Shi Y.  Serine/threonine phosphatases: mechanism through structure,  Cell ,  2009, vol.  139  3(pg.  468- 484) Google Scholar CrossRef Search ADS PubMed  Sigrist S,  Jacobs H,  Stratmann R,  Lehner CF.  Exit from mitosis is regulated by Drosophila fizzy and the sequential destruction of cyclins A, B and B3,  Embo J. ,  1995, vol.  14  19(pg.  4827- 4838) Google Scholar PubMed  Sigrist SJ,  Lehner CF.  Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles,  Cell ,  1997, vol.  90  4(pg.  671- 681) Google Scholar CrossRef Search ADS PubMed  Simon M,  Hancock JM.  Tandem and cryptic amino acid repeats accumulate in disordered regions of proteins,  Genome Biol. ,  2009, vol.  10  6pg.  R59  Google Scholar CrossRef Search ADS PubMed  Sklan EH,  Podoly E,  Soreq H.  RACK1 has the nerve to act: structure meets function in the nervous system,  Prog Neurobiol. ,  2006, vol.  78  2(pg.  117- 134) Google Scholar CrossRef Search ADS PubMed  Sood R,  Bonner TI,  Makalowska I, et al.  ,  (16 co-authors).  Cloning and characterization of 13 novel transcripts and the human RGS8 gene from the 1q25 region encompassing the hereditary prostate cancer (HPC1) locus,  Genomics ,  2001, vol.  73  2(pg.  211- 222) Google Scholar CrossRef Search ADS PubMed  Srivastava M,  Simakov O,  Chapman J, et al.  ,  (33 co-authors).  The Amphimedon queenslandica genome and the evolution of animal complexity,  Nature ,  2010, vol.  466  7307(pg.  720- 726) Google Scholar CrossRef Search ADS PubMed  Stamatakos M,  Palla V,  Karaiskos I,  Xiromeritis K,  Alexiou I,  Pateras I,  Kontzoglou K.  Cell cyclins: triggering elements of cancer or not?,  World J Surg Oncol. ,  2010, vol.  8 pg.  111  Google Scholar CrossRef Search ADS PubMed  Szczesniak MW,  Ciomborowska J,  Nowak W,  Rogozin IB,  Makalowska I.  Primate and rodent specific intron gains and the origin of retrogenes with splice variants,  Mol Biol Evol. ,  2010, vol.  28  1(pg.  33- 37) Google Scholar CrossRef Search ADS PubMed  Szklarczyk R,  Heringa J.  Tracking repeats using significance and transitivity,  Bioinformatics ,  2004, vol.  20  Suppl 1(pg.  i311- i317) Google Scholar CrossRef Search ADS PubMed  Tamura K,  Dudley J,  Nei M,  Kumar S.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0,  Mol Biol Evol. ,  2007, vol.  24  8(pg.  1596- 1599) Google Scholar CrossRef Search ADS PubMed  Tarailo-Graovac M,  Wang J,  Tu D,  Baillie DL,  Rose AM,  Chen N.  Duplication of cyb-3 (cyclin B3) suppresses sterility in the absence of mdf-1/MAD1 spindle assembly checkpoint component in Caenorhabditis elegans,  Cell Cycle ,  2010, vol.  9  24(pg.  4858- 4865) Google Scholar CrossRef Search ADS PubMed  Tompa P.  Intrinsically unstructured proteins evolve by repeat expansion,  Bioessays ,  2003, vol.  25  9(pg.  847- 855) Google Scholar CrossRef Search ADS PubMed  Tompa P,  Fuxreiter M.  Fuzzy complexes: polymorphism and structural disorder in protein-protein interactions,  Trends Biochem Sci. ,  2008, vol.  33  1(pg.  2- 8) Google Scholar CrossRef Search ADS PubMed  Tschop K,  Muller GA,  Grosche J,  Engeland K.  Human cyclin B3. mRNA expression during the cell cycle and identification of three novel nonclassical nuclear localization signals,  Febs J. ,  2006, vol.  273  8(pg.  1681- 1695) Google Scholar CrossRef Search ADS PubMed  Turner JM.  Meiotic sex chromosome inactivation,  Development ,  2007, vol.  134  10(pg.  1823- 1831) Google Scholar CrossRef Search ADS PubMed  Ueno H,  Nakajo N,  Watanabe M,  Isoda M,  Sagata N.  FoxM1-driven cell division is required for neuronal differentiation in early Xenopus embryos,  Development ,  2008, vol.  135  11(pg.  2023- 2030) Google Scholar CrossRef Search ADS PubMed  Vacic V,  Uversky VN,  Dunker AK,  Lonardi S.  Composition Profiler: a tool for discovery and visualization of amino acid composition differences,  BMC Bioinformatics ,  2007, vol.  8 pg.  211  Google Scholar CrossRef Search ADS PubMed  van der Voet M,  Lorson MA,  Srinivasan DG,  Bennett KL,  van den Heuvel S.  C. elegans mitotic cyclins have distinct as well as overlapping functions in chromosome segregation,  Cell Cycle ,  2009, vol.  8  24(pg.  4091- 4102) Google Scholar CrossRef Search ADS PubMed  Van Dross R,  Browning PJ,  Pelling JC.  Do truncated cyclins contribute to aberrant cyclin expression in cancer?,  Cell Cycle ,  2006, vol.  5  5(pg.  472- 477) Google Scholar CrossRef Search ADS PubMed  Veyrunes F,  Waters PD,  Miethke P, et al.  ,  (16 co-authors).  Bird-like sex chromosomes of platypus imply recent origin of mammal sex chromosomes,  Genome Res. ,  2008, vol.  18  6(pg.  965- 973) Google Scholar CrossRef Search ADS PubMed  Vicoso B,  Charlesworth B.  Evolution on the X chromosome: unusual patterns and processes,  Nat Rev Genet. ,  2006, vol.  7  8(pg.  645- 653) Google Scholar CrossRef Search ADS PubMed  Warren WC,  Hillier LW,  Marshall Graves JA, et al.  ,  (103 co-authors).  Genome analysis of the platypus reveals unique signatures of evolution,  Nature ,  2008, vol.  453  7192(pg.  175- 183) Google Scholar CrossRef Search ADS PubMed  Wolgemuth DJ,  Roberts SS.  Regulating mitosis and meiosis in the male germ line: critical functions for cyclins,  Philos Trans R Soc Lond B Biol Sci. ,  2010, vol.  365  1546(pg.  1653- 1662) Google Scholar CrossRef Search ADS PubMed  Xiong Y,  Chen X,  Chen Z,  Wang X,  Shi S,  Wang X,  Zhang J,  He X.  RNA sequencing shows no dosage compensation of the active X-chromosome,  Nat Genet. ,  2010, vol.  42  12(pg.  1043- 1047) Google Scholar CrossRef Search ADS PubMed  Zhang F,  Sha J,  Wood TG, et al.  ,  (11 co-authors).  Alteration in the activation state of new inflammation-associated targets by phospholipase A2-activating protein (PLAA),  Cell Signal ,  2008, vol.  20  5(pg.  844- 861) Google Scholar CrossRef Search ADS PubMed  Zhuo D,  Madden R,  Elela SA,  Chabot B.  Modern origin of numerous alternatively spliced human introns from tandem arrays,  Proc Natl Acad Sci U S A. ,  2007, vol.  104  3(pg.  882- 886) Google Scholar CrossRef Search ADS PubMed  © The Author 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
TI - Evolution of Cyclin B3 Shows an Abrupt Three-Fold Size Increase, due to the Extension of a Single Exon in Placental Mammals, Allowing for New Protein–Protein Interactions
JO - Molecular Biology and Evolution
DO - 10.1093/molbev/mss189
DA - 2012-07-23
UR - https://www.deepdyve.com/lp/oxford-university-press/evolution-of-cyclin-b3-shows-an-abrupt-three-fold-size-increase-due-to-l6bHLUs1WS
SP - 3855
EP - 3871
VL - 29
IS - 12
DP - DeepDyve
ER -