BMC Genomics 2004, 5:69 http://www.biomedcentral.com/1471-2164/5/69 in different ways from those in yeast, animals, and even rice. ScCAK1 and SpCSK1 from yeasts also have CAK activity; however, despite their functional similarity to kinases in the CCRK group, they do not group with animal or plant CAKs (Fig. 1). Interestingly, in the single most likely tree recovered in our expanded Bayesian analysis of 133 sequences, ScCAK1 and SpCsk1 group with other CAKs in the sister clade to CDK7 (additional file 1); however, there is no support for this placement in the Bayesian probabil- ity distribution. ScCAK1 and SpCSK1 sequences are highly divergent from all CDKs, and the regulation of CAK activ- ity in yeast is very different from that of animals and plants [42,44]. Thus, alternative lines of evidence may be required to determine whether there is any specific evolu- tionary relationship among all CAKs. The CDK 8 family CDK 8 (SRB10 in yeast) is a component of the multi-sub- unit Mediator complex, which transduces signals from cis regulatory elements to RNAP II; it is proposed to inhibit transcription initiation by phosphorylation of the CTD. CDK8/SRB10 and its partner cyclin C/SRB11, together with SRB8 and SRB9, form a specific sub-module that is variably associated with the RNAP II holoenzyme, and potentially with the free mediator complex [45]. Apparent orthologs of CDK8 form a well-defined group, including sequences from plants, animals and yeasts. Interestingly, although a member of the CTD clade (Fig. 2 and note that all microsporidian RPB1 genes isolated to date encode a CTD), no ortholog of CDK8 was identified from Encepha- litozoon. Our further blast results (unpublished data) failed to identify any of the units of the CDK8/SRB10 (SRBs8-11) sub-module in the Microsporidia suggesting a loss of CDK8/SRB10 unit from these highly reduced parasites. Although the CDK8/SRB10 sub-module has been impli- cated in negative regulation of transcription by phospho- rylation of TFIIH, leading to the inhibition of the TFIIH CTD kinase and transcription [46], the exact mechanism still is unclear. Recent research shows that the Mediator containing this sub-module is isolated only in free form, not associated with RNAP II. In contrast, Mediator lacking this sub-module associates with the polymerase [47]. There also is experimental evidence that negative Media- tor-RNAP II regulation by the SRB8-11 sub-module is evo- lutionarily conserved from yeast to humans [47]. Therefore, the absence of identifiable components of the SRB8-11 sub-module in Encephalitozoon suggests CDK8/ SRB10 function is absent from the Microsporidia. The loss of CDK8 from Microsporidia, along with absolute conser- vation of CDKs7 and 9 in all members of the "CTD-clade" (Figs. 1 and 2) implies that interactions between the CTD and Mediator complex are less strongly entrained into essential RNAP II function, than are those regulated by TFIIH and P-TEFB kinase activity. The CDK 9 family CDK9 is a component of the P-TEFb complex, which is a positive-acting RNAP II transcription elongation factor [48,49]. Research has focused on P-TEFb from animals and budding yeast. A definitive yeast homolog of animal P-TEFb has not yet been determined from functional stud- ies, but two candidates have emerged: the BUR1 complex and the CTDK-I complex [26]. Based on our blast and phylogenetic analyses, BUR1 and CTK1 (subunit of CTDK-I complex) are found in two distinct but related kinase groups, each with orthologs from other eukaryotes. BUR1 is identified as the specific ortholog of CDK9 from metazoans, budding yeast and probably the Microsporidia. Unexpectedly, the CDC2-like5 kinases and CrkRS from animals are highly supported as orthologs of CTK1 from yeasts. Although their functions are not yet clear [50], our results suggest that human CDC2-like5 kinases and CrkRS have CDK9 function. Recent analyses of CrkRS (CDC2- related kinase with an RS-rich domain) suggest that it has CTD kinase activity and helps to link transcription directly to intron splicing [51]. This CTK1 clade also contains putative CDK9 (CdkC) kinases from plants and as well as a CDC2-like kinase from Plasmodium (PfCRK3). The latter is the only apparent ortholog of a CTD-directed kinase (CDKs 7, 8 or 9) identified in our analyses from any organism outside the "CTD-clade." It remains to be determined whether PfCRK3 possesses the P-TEFb func- tion of CTK1, since it is the only protistan sequence present in either CDK9 sub-group, and the RNAP II CTD has not been conserved in apicomplexans or their closest relatives (Fig. 2). In addition to the two previously identified copies of CDK9 (CdkC1 and CdkC2) from Arabidopsis, and one from Oryza (CdkC1) [33,34], our Blast searches also retrieved a large group of CDK9-like sequences (14 from Arabidopsis and 8 from Oryza) (Table 1). These kinases are annotated as "Cdc2-like" in databases and some of them also were identified in previous analyses of CDK evolu- tion [38]. With one exception (Os1562.H01.5), all of these kinases group in a single cluster, with 100% support, and as sister to previously identified CDK9s of Arabidopsis and Oryza (additional file 1). Os1562.H01.5 (Gi: 38424086) from Oryza is extremely similar to OsCdkC1 and very likely a second copy of CdkC (CDK9) from Oryza. There is no evidence of biological functions for these kinases as yet, but our results indicate that they are part of a large CDK9 complex specific to plants. Page 10 of 13 (page number not for citation purposes)