BMC Genomics 2004, 5:69 http://www.biomedcentral.com/1471-2164/5/69 interactions among sequences are unknown or poorly understood, it cannot be ruled out as an explanation for our observations. Analyses of additional genomes from diverse eukaryotes are required, both to verify our obser- vations of co-evolution between the CTD and CTD- directed kinases, and to determine its bases. General evolutionary trends in the CDK family Kinases from protistan organisms In an effort to understand the broader evolutionary his- tory of CDKs, three deep-branching protists with com- plete genomes, Plasmodium falciparum, Trypanosoma brucei and Giardia lamblia, were included in our study. Our Blast searches detected 15 putative kinases from these protists; six from Plasmodium falciparum, four from Trypanosoma brucei and five from Giardia lamblia (Table 1). The phylo- genetic positions and orthologous relationships of these kinases generally are not well defined by phylogenetic analyses (Fig. 1). Four of them (GlCdc2L3, GlCAKlike, TbCrk6 and PfCrk4), along with two microsporidian kinases (EcCrkB and EcCrkD) branched close to ScCak1 and SpCsk1, cyclin-activating kinases from yeasts. All of these sequences are highly divergent, and it is difficult to determine, whether their branching positions are due to a phylogenetic artifact or a phylogenetic relationship. As noted above, GlCAKlike kinase has been proposed as a Giardia CDK7 ortholog based on JTT distance data [38], a relationship not supported by our broader phylogenetic analyses. Moreover, there are no experimental data reported on the functions of any of these kinases. Other putative protistan CDKs, GlCdc2L4, PfMrk and PfPk6, scatter among CDKs from other organisms, but with no statistical confidence for any implied relationship. Our most strongly supported results indicate that six of these kinases (TbCdc2L, TbCrk2 and 3, PfPk5, GlCdc2L1 and L2) belong to cell-cycle related kinase families CDK1 and CDK5. In particular, PfPk5 is well-supported as an ortholog of CDK5. In addition, two kinases from Plasmo- dium (PfCrk1 and PfCrk3) appear to be transcription- related kinases, PfCrk1 groups with the CDK10/11 family, and PfCrk3 with CDK9. The phylogenetic distribution of protistan kinases indi- cate that cell-cycle related kinases are present, or at least their functions are more strongly conserved (see discus- sion above regarding CTD/CDK co-evolution), in a more diverse array of eukaryotes than are transcription-related kinases. This pattern also is seen in a more widely-sam- pled analyses including CDKs from a number of organ- isms with incompletely sequenced genomes, including Dictyostelium discoideum that has a canonical RNAP II CTD, and Leishmania major, Cryptosporidium parvum and Entamoeba histolytica, which all lack a CTD (see additional file 1). Thus, the overall results suggest that cell-cycle related kinases are more ancient than transcription- related kinases, and probably ancestral to them, and that their core functions are more similar across the broad diversity of eukaryotic lineages. It will be interesting to see whether these preliminary hypotheses are supported as more genomes are sequenced completely, particularly from diverse protistan organisms. Cell-cycle related kinases Our analyses support well-defined groups for cell-cycle kinases CDK1, CDK4/6 and CDK5. An ortholog of either CDK1 or CDK5 is found in all of the organisms in our study, and these two families appear to be closely related. TbCrk3 was proposed as a functional homolog of CDK1 in Trypanosoma [40]; here it groups among cell-cycle kinases, but is not specifically related to CDK1. CDK4/6 appears to be present only in human, Drosophila and C. elegans. The CDK5 family has undergone expansion in metazoans, including PFTAIRE and PCTAIRE kinases, and putative orthologs of CDK5 are detected in Plasmodium, Trypanosoma and Giardia. Interestingly, no CDK from plants associates strongly with the CDK5 group, while the CdkB-type kinases, which are specific to plants, branch as sister to a broader CDK1/CDK5 clade. Our overall results suggest that cell-cycle kinases have undergone extensive and independent evolutionary diversification in different eukaryotic lineages, and it may be difficult to classify many of them based on orthologous relationships in phy- logenetic analyses. It may be that functional homologies, once established experimentally, will prove to be more consistent criteria for designating CDK groups. The CDK7 family Clear orthologs of CDK7 from animals, plants, yeasts and Microsporidian are strongly supported as a core family, with CDK-activating kinase from Arabidopsis (AtCdkF), and its apparent orthologs from animals, branching as a sister group. In addition to their role as CTD kinases, members of the CDK7 family in plants, animals and fis- sion yeast can function as a CDK-activating kinase (CAK) [41,42]. Unlike animals and yeast, however, four CDK7- like of CAKs were isolated from Arabidopsis [35]. AtCdkF (AtCAK1), which groups with human CCRK and apparent orthologs from Drosophila and Caenorhabiditis, exhibits only CAK activity but no CTD kinase activity. Consistent with the phylogenetic relationships recovered in our anal- ysis, human CCRK and other animal orthologs were recently shown to have CAK activity [43]. In contrast, AtCdkD3 (AtCAK2) and AtCdkD2 (AtCAK4) display both CAK and CTD kinase activity and, along with a single CDK7 from rice, are included in a strongly supported CDK7 clade. Interestingly, and despite its high sequence similarity to AtCdkD3, no kinase activity was reported from AtCdkD1 (AtCAK3) [35]. Apparently CAKs in Arabi- dopsis have diversified substantially, and may be regulated Page 9 of 13 (page number not for citation purposes)