* Université Paris VI, Laboratoire Arago, Modèles en Biologie Cellulaire et Evolutive, Banyuls sur Mer, France; Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Ghent, Belgium; and
Institut de Génétique Humaine, Montpellier, France
Correspondence: E-mail: h.moreau{at}obs-banyuls.fr.
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Abstract |
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Key Words: cell division cycle cyclin-dependant kinase cyclin green alga Ostreococcus tauri
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Introduction |
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Cell cycle control genes have been found in the different lineages investigated, including the animal, yeast, and plant lineages. Even though specific regulatory mechanisms are present in all lineages, some have evolved differently, such as the retinoblastoma (Rb/E2F/DP) pathway, which is present in animals and plants but absent in yeast (Rubin et al. 2000). Comparative studies of the cell cycle among model organisms belonging to different eukaryotic lineages can, thus, provide crucial information in distinguishing between the core cell cycle common to all phyla and lineage-specific adaptations. Most of the comparative analysis on the cell cycle regulation in ophistokonts (metazoans and fungi) has already yielded the identification of several evolutionarily conserved cell cycle control genes. Although many of these genes are also known in higher plants, their precise role is hard to grasp because of the high complexity of the plant model genomes; namely, the presence of multiple copies of key genes such as CDKs and cyclins. For example, genome-wide analysis shows that cell division control might involve nine CDKs (one of CDKA, four of CDKB, three of CDKD, and one of CDKF) and 30 cyclins (10 of cyclin A, nine of cyclin B, 10 of cyclin D, and one of cyclin H) in Arabidopsis thaliana (Vandepoele et al. 2002). The function of each copy is very difficult to investigate because their independent roles are blurred: silencing one copy does not necessarily yield the complete phenotype associated with the gene, as part or all of the function of the silenced copy can be rescued by the other copies. Thus, there is a need for a simpler green lineagespecific model organism that can be used to unravel the cell cycle specificities of this phylum. Furthermore, studies in the major "classical" model organisms are not sufficient to account for the common features and the particularities of each model. It is, for instance, difficult to determine whether the presence of only one CDK in yeast is a primeval feature inherited from the ancestral eukaryotic cell or a more recent simplification after the separation between the ophistokonts and the green lineage. These questions can only be answered by the study of new model organisms that occupy key phylogenetic positions. Undoubtedly the genome-wide analysis of their cellular functions, such as the core cell cycle genes, will help in the understanding of the complex green lineagespecific adaptations.
Ostreococcus tauri is a marine unicellular green alga of the Prasinophyceae clade that belongs to the Chlorophyta group of the Planta kingdom (Courties et al. 1998). Because Prasinophyceae have diverged early at the base of the Chlorophyta and consequently of the green lineage (Bhattacharya and Medlin 1998), O. tauri holds a key phylogenetic position in the eukaryotic tree of life. It is, therefore, a potentially powerful model to differentiate between the processes that are common to all eukaryotes (i.e., inherited from the "ancestral eukaryotic cell") and specific adaptations that have occurred after the separation of the different lineages. O. tauri is the smallest free-living eukaryotic cell described to date (Courties et al. 1994), with a diameter of no more than 1 µm. Furthermore, O. tauri has a minimal cellular organization, with a nucleus, a single chloroplast, and only one mitochondrion (Chrétiennot-Dinet et al. 1995). It has a nude plasma membrane without scales or flagella, a reduced cytoplasm (Chrétiennot-Dinet et al. 1995), and a small 12.5-MB to 13-Mb genome, which is currently being sequenced (Derelle et al. 2002). The high-throughput sequencing step of its complete genome is now finished (data not shown), and first analyses indicate that most of the genes have high similarities with genes belonging to the higher plant lineage and can, thus, be easily annotated by sequence similarity. Here, we compare the core cell cycle genes of O. tauri with those of A. thaliana and discuss new features of their evolution.
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Materials and Methods |
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Annotation of the Ostreococcus tauri Cell Cycle Genes
All the genes were annotated based on their similarity with other cell cycle genes available in the public databases. These sequences were aligned using Blast (Altschul et al. 1990) against the O. tauri database, and the best hits were further manually annotated using Artemis (Rutherford et al. 2000). The mRNA expression of all the genes described in this study has been confirmed either by their presence among the ESTs sequenced from a cDNA library or from Northern blots or RT-PCR. All the sequences reported in this paper have been submitted to GenBank under the following accession numbers: AY675093 (CDKA), AY675094 (CDKB), AY675095 (CDKC), AY675096 (CDKD/CAK), AY675097 (CycA), AY675098 (CycB), AY675099 (CycD), AY675100 (CycH), AY330645 (Cdc25), AY675101 (Wee1), AY675102 (Rb), AY675103 (E2F), AY675104 (Del), AY675105 (Dp), AY675106 (Cks), AY675107 (Cdc20), AY675108 (CDH1/CCS52), AY675109 (Skp1), AY675110 (Apc1), AY675111 (Apc2), AY675112 (Apc5), AY675113 (Apc6/Cdc16), AY675114 (Apc10), AY675115 (Cdc26p), AY675116 (Apc7), AY675117 (Apc8/Cdc23), AY675118 (Apc11), AY675119 (Apc4), and AY675120 (Apc3).
Phylogenetic Analysis
Sequences were aligned with ClustalW (Thompson, Higgins, and Gibson 1994). The sequence alignments were manually improved using BioEdit (Hall 1999). TreeCon (Van de Peer and De Wachter 1997) was used for constructing the neighbor-joining (Saitou and Nei 1987) trees based on Poisson-corrected distances, only taking into account unambiguously aligned positions. Bootstrap analysis with 500 replicates was performed to test the significance of the nodes.
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Results and Discussion |
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The nuclear genome size of O. tauri has been estimated at approximately 12.6 Mbp by pulsed-field electrophoresis (PFGE), whereas the total size obtained from the contigs is 12.4 Mbp. A total of 1,850 unique O. tauri expressed sequence tags (ESTs) have been sequenced, and around 99% of these sequences mapped with identity greater than 95% onto the genome by using Blast. This is further evidence of the completeness of the genome sequence. and this genome draft has then been used for the complete analysis of the O. tauri core cell cycle genes.
CDK-Cyclin Complexes
CDKs are universally conserved cell cycle regulators. Six classes of CDKs have been described in the plant model A. thaliana (Vandepoele et al. 2002). CDKA has a PSTAIRE cyclin-binding motif and is the plant ortholog of the universal eukaryotic cell cycle regulator CDK1 (Dorée and Hunt 2002). CDKB, whose expression is cell cycle regulated, belongs to a plant-specific CDK clade and plays a role at the G2/M-phase transition. CDKD and CDKF are CDK activating kinases (CAK), which activate the CDK by phosphorylating the threonine residue in the T-loop (Jeffrey et al. 1995). CDKC and CDKE are not directly involved in the cell cycle control. According to this plant nomenclature (Joubès et al. 2000), four CDKs belonging to the A to D classes have been found in the genome of O. tauri (fig.1 and table 1), but only three (CDKA, CDKB, and CDKD) are involved in cell division control.
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Finally, only one PITAIRE motif O. tauri CDKC has been identified, as compared with the two CDKCs described in A. thaliana (fig.1 and table 1). PITAIRE CDKs have been reported to phosphorylate the carboxyl terminal domain (CTD) of the RNA polymerase II, and they do not participate directly in the cell cycle control (Barroco et al. 2003). No E-class or F-class CDKs have been found in the genome of O. tauri.
Cyclins are the regulatory binding partners of the CDKs, which confer the timing and substrate specificity to the activity of the CDK-cyclin complexes (Futcher 1996). O. tauri has the minimum set of cyclins described to date in any organism. More importantly, it has only one copy of each of the A-class, B-class, D-class, and H-class cyclins (Renaudin et al. 1996), thus, presenting even fewer cyclin gene copies than yeasts because S. cerevisiae has three G1 cyclins (CLN) and six S/G2/M cyclins (CLB) (table 2 and fig. 2). The activities of the different cyclins in S. cerevisiae are redundant. In G1-phase, threshold Cln3 kinase activity is necessary for going through the START point, at which time it switches on the other two G1 cyclins, Cln1 and Cln2. The peak activity of these two cyclins induces the activity of the S-phase specific CDK-cyclin complexes by releasing the Clb5-associated and Clb6-associated kinases from the inhibitory CKI Sic1 (Schwob et al. 1994). The Clb5 and Clb6 kinase activities are necessary for progression from the G1 to the end of the S-phases. Finally, the cyclins Clb1-4 are turned on at the G2/M-phase transition, thus, leading the cell into M-phase (Mendenhall and Hodge 1998).
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Therefore, O. tauri presents a minimal, yet complete, set of cell division control genes necessary to drive a eukaryotic cell through the complete division cycle: one of CDKA, one of CDKB, one of cyclin A, one of cyclin B, and one of cyclin D. Furthermore, the presence in this organism of two CDKs (the universal regulator CDKA and the green lineagespecific CDKB) supports the hypothesis that having one CDK would be a primeval feature that has been conserved in yeast but not a more recent simplification specifically acquired in this lineage. Furthermore, the simplification to only one copy for each cyclin type, in contrast to the usual high copy number of these genes in the other organisms, makes O. tauri a potentially powerful model for functional plant core cell cycle studies.
CDK subunits (CKS) are proteins that bind to the CDK protein, and their function is important in the transcriptional activation of Cdc20, the activating protein of the APC complex (see below) (Morris et al. 2003). Only one putative Cks has been found in the genome of O. tauri (table 3). This gene has four introns, and the encoded protein has a well-conserved N-terminus sharing an overall 78% and 82% similarity with A. thaliana Cks1 and Cks2, respectively.
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Retinoblastoma (Rb/E2F/DP) Pathway
At the G1/S transition, the Rb pathway is conserved among the animal and plant kingdoms but is absent in yeasts. An Rb homolog has also been described in the unicellular green alga Chlamydomonas reinhardtii (Umen and Goodenough 2001). However, this alga has a peculiar cell division, and the question of whether this Rb pathway is characteristic for multicellular organisms has remained unclear (Cross and Roberts 2001). In algae, plants, and metazoans, the retinoblastoma pathway induces the expression of S-phasespecific genes. The Rb protein sequesters and inactivates the DNA-bound heterodimer transcription factor comprising the E2F protein and its dimerization partner (DP) protein (Weinberg 1995). It also recruits the chromatin remodeling machinery for silencing the target genes (Shen 2002) and is responsible for maintenance of quiescence (Sage et al. 2003). At the G1/S transition, the CDK-cyclin complex phosphorylates Rb. Once phosphorylated, the Rb protein frees the E2F/DP complex, which is able to induce the transcription of its target genes. In contrast to vertebrates, which have three copies of pocket proteins (Rb, p107, and p130), O. tauri, similar to A. thaliana and C. reinhardtii, has only one homolog of Rb gene (fig. 2 and table 3).
The E2F family of transcription factors comprise the subfamilies of activating E2Fs, inhibitory E2Fs, dimerization partner proteins (DPs), and DP-like and E2F-like proteins (DELs) (Shen 2002). The three E2F, two DP, and three DEL genes identified in A. thaliana have approximately 22% overall sequence similarity (Vandepoele et al. 2002). E2FA and E2FB have four binding domains, a DNA-binding, a DP-binding, an Rb-binding, and a transactivation-binging domain. When bound to DPA or DPB proteins, they are transcriptional activators that are repressed by Rb protein. E2FC lacks the transactivation-binding domain and is a homolog of animal inhibitory E2Fs E2F4-6. Also, the three A. thaliana DEL proteins, of which E2F7 is the recently described animal homolog (Di Stefano, Jensen, and Helin 2003), each have two DNA-binding domains but do not have either DP-binding, Rb-binding, or transactivation-binding domains. Hence, DEL proteins contribute to the class of E2F inhibitory subfamilies. Only one E2F, one DP, and one DEL gene have been identified in the genome of O. tauri by alignments of their sequences with their orthologs from A. thaliana (figs. 3 and 4). The phylogenetic analysis of the E2F family confirms the early divergence of O. tauri genes with respect to the higher plants (fig. 4). The O. tauri E2F is a homolog of activating E2Fs and has a conserved binding domain to the DP-binding and Rb-binding domains and DNA-binding and transactivation-binding domains, whereas DEL has only two DNA-binding domains but no other domain (fig. 3).
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Cdc25/Wee1 Control of CDK-Cyclin Activity
The kinase Wee1 and the phosphatase Cdc25 regulate the G2/M transition in metazoans and yeasts by posttranslational regulation of the CDK-cyclin complex. In plants, an ortholog of the wee1 gene has been found, but the cdc25 gene has never been identified either in the fully sequenced genomes of higher plants such as A. thaliana and rice or in that of the unicellular green alga C. reinhardtii. The absence of the M-phase inducer Cdc25 phosphatase in plants is puzzling because the other actors of this regulation pathway, namely the CDK-cyclin B complex and the Wee1 kinase, are evolutionarily conserved. Furthermore, the activating dephosphorylation at the M-phase entry is conserved in plants (Zhang, Letham, and John 1996; McKibbin, Halford, and Franci 1998).
An intronless putative wee1 gene has been identified in the genome of O. tauri (table 3). It is similar to the Wee1 kinase of A. thaliana, maize, and C. reinhardtii and potentially inhibits the activity of the CDK-cyclin complex by its inhibitory phosphorylation. Surprisingly, the ortholog of the activating phosphatase cdc25 gene has been identified in O. tauri (table 3). It is the first time that cdc25 is described in the green lineage, and it shows that the cdc25 gene is present at the base of this lineage. The O. tauri cdc25 gene codes for a protein that is able to rescue the S. pombe cdc25-22 conditional mutant. Furthermore, microinjected O. tauri Cdc25 specifically activates starfish oocytes arrested in the prophase of the first meiotic division, thus, causing germinal vesicle breakdown. In vitro phosphatase assays, namely antiphosphotyrosine Western blotting and the histone H1 kinase assay confirmed the in vivo activity of O. tauri Cdc25 (Khadaroo et al. 2004).
The presence of the first functional green-lineage Cdc25 dual-specificity phosphatase discovered in O. tauri indicates that this gene was probably present in the ancestor of the eukaryotic cell. In this respect, it should be noted that a putative Cdc25-like gene has also been identified in the complete genome of C. reinhardtii. Furthermore, because its activity is necessary in higher plants, the most parsimonious hypothesis is that the sequence of Cdc25 in higher plants has diverged so much that it can no longer be recognized by sequence homology analysis (Khadaroo et al. 2004). This has been very recently confirmed by the identification in A. thaliana of a poorly conserved Cdc25-related protein having a tyrosine-phosphatase activity stimulating the kinase activity of A. thaliana CDKs (Landrieu et al. 2004).
Ubiquitin Ligases APC and SCF
Both the anaphase promoting complex (APC) and Skp1/Cdc53/F box protein (SCF) complex, which are the key enzymes for tagging proteins in the ubiquitin pathway, are the E3 ligases responsible for the specificity of protein degradation by the 26S proteasome (Irniger 2002; Cope and Deshaies 2003). The two evolutionarily conserved functions of the APC is the cell cyclespecific targeting of securin and the mitotic cyclin B for proteasome-mediated destruction. The APC is composed of at least 13 protein subunits initially identified in yeast and animals (Schwickart et al. 2004). All vertebrate APC subunits have their homologs in plants (Capron, Okresz, and Genschik 2003; Tarayre et al. 2004). Interestingly, although many genes (and notably cell cycle genes [see above]) are present as multiple copies in A. thaliana, its APC genes are present as single copies, except for APC3, which is represented by two slightly different genes (table 4, modified from Capron, Okresz, and Genschik [2003]). In O. tauri, putative orthologs of most of these genes have also been found as single copies, including only one copy of APC3. They show very high similarity scores with the A. thaliana APC genes. Furthermore, putative orthologs of the two activators (Cdc20 and Cdh1) regulating the activity of the APC complex have also been found in the O. tauri genome. In contrast with the many putative orthologs of Cdc20 and Cdh1 in A. thaliana (Capron, Okresz, and Genschik 2003), there seems to be only one Cdc20 and one Cdh1 gene in O. tauri (table 4). Thus, O. tauri has a complete set of APC genes, with a minimal number of activators.
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Once more, O. tauri presents a conserved set of APC and SCF genes with a lower copy number of genes than in A. thaliana.
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Conclusion |
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Acknowledgements |
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Footnotes |
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1 Present address: Molecular Biology Group, Trieste, Italy.
Geoffrey McFadden, Associate Editor
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References |
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