In a screen for embryonic lethal mutants with aberrant expression of a
pes-10 :GFP transgene, we isolated a number of mutants that fail to activate mRNA transcription in early embryos. Most of these mutants arrest with a terminal phenotype similar to that observed when RNA polymerase II function is removed - 100-cell stage arrest with no apparent differentiation (Powell-Coffman et al., 1996). In contrast, one mutant,
ax224 , showed a more severe terminal phenotype - mutant embryos arrest at about the 50 cell-stage, and DAPI staining reveals defects in chromatin segregation and DNA replication. We have mapped and cloned this mutant, and have shown that it is an allele of the C. elegans homologue of Cdk7 (Thanks to Amy Walker for first pointing out the map position of C. elegans
cdk-7 ). Cdk7 is the catalytic subunit of TFIIH and is thought to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II during transcription. Consistent with this role, we find that
ax224 embryos have reduced levels of phosphorylated CTD. Cdk7 has also been proposed to be the Cdk activating kinase (CAK) that activates cell cycle Cdks. CAK activity of Cdk7 has been observed in vitro in several systems, but in vivo evidence for an essential role in cell cycle progression has been difficult to obtain due to Cdk7's essential role in transcription. Evidence from yeast actually argues against Cdk7 acting as a CAK since the budding yeast homologue of Cdk7, Kin28, does not possess CAK activity in vivo or in vitro . Instead a distantly related gene Cak1 functions as the sole essential CAK in S. cerevisiae . Cak1 is not conserved in higher eukaryotes (Liu and Kipreos, 2000), leaving open the question as to which Cdk functions as the CAK outside of yeast. Consistent with
cdk-7 acting as the CAK in C. elegans , we find that
ax224 embryos have cell cycle defects beyond what is expected from a block in transcription. These defects include lengthened interphase and M phase, and inappropriate DNA replication. In addition, we find that phosphorylation of NCC-1 ( C. elegans
cdc2, Boxem et al., 1999) is reduced in
ax224 mutants (Thanks to Andy Golden for the NCC-1 antibody). Finally, the
cdk-7(RNAi);
cdk-7(
ax224) "double mutant", which presumably lacks most
cdk-7 activity, arrests in the one-cell stage with a phenotype similar to that observed in
ncc-1(RNAi) embryos. Taken together, our data demonstrates that
cdk-7 has independent roles in transcriptional activation and cell cycle control, and suggests that
cdk-7 may be the sole essential CAK in C. elegans .