Worm Breeder's Gazette 12(5): 85 (February 1, 1993)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

The cdc2 Gene & Family in C. elegans

Sander van den Heuvel, Li-Huei Tsai, Ed Harlow.

MGH Cancer Center, Building 149, 13th Street; Charlestown, MA 02129

The cdc2 protein kinase is conserved as a key regulator of the cell cycle from yeast to humans. In yeast only a single cdc2 kinase has been discovered. However, in vertebrates many different cdc2 -likekinases have been identified. Based on sequence homology, our lab has recently cloned seven novel members of this family from human cells. Although not yet established for every family member, where known the activation of these kinases requires the association with a regulatory subunit known as a cyclin. Hence, this class of kinases is now known as the "cyclin-dependent kinases" (cdk's). Where no cyclin partner has been identified, the kinases are named by the single amino acid code in a well conserved region, the so-called PSTAIRE-motif in cdc2 .All eukaryotes express a series of different cyclins, some of which are now known to be implicated in the regulation of specific transitions in the cell cycle.

To establish a genetic system in which the function of cdk's can be studied, we have cloned a number of these genes from C. elegans. In a first approach, we used an antibody raised against the PSTAIRE-region of cdc2 .Screening an expression library revealed several independent cDNA clones all arising from a single gene. The protein predicted to be encoded by this gene shares 66% identity with human cdc2 ,and 62% with both cdk2 and cdk3 .Despite the high sequence identity, it has two characteristics that distinguish it from cdc2 proteins in other species. One is the change of valine for isoleucine in the PSTAIRE region, giving the sequence PSTAVRE. The other is a small N-terminal extension of 19 amino acids. The gene maps on chromosome IIl to the same region as ncc- 1 and therefore is likely to be the cdc2 homolog cloned previously by H. Mori and Paul Sternberg. Moreover, it was also identified in the sorted cDNA library from Waterston, Martin, and Sulston; clones cm04 g2 , cm04 g9 and cm13 c2 from this library contain sequences derived from this cdc2 -likegene.

In the second approach, degenerate oligonucleotides corresponding to highly conserved regions of cdc2 were used for PCR reactions. Two other cdc2 -relatedgenes were isolated using this strategy. One was a homolog of the cdk5 gene, and its predicted ORF shares 74% amino-acid identity with the human gene. There are several indications that cdk5 may be an important kinase in neuronal cells. In rat and man, the mRNA is highly expressed in neuronal tissues, and the protein has been purified as the major histone H1 kinase from bovine brain. Therefore, C. elegans promises to provide a particularly attractive system to study cdk5 .Our preliminary mapping suggests that this gene is located on the right arm of chromosome III, and we are currently identifying cosmids which harbor its sequences.

The third clone was highly homologous to the PCTAIRE kinases. In human cells, three different PCTAIRE kinases have been identified, and they share a high sequence identity in the catalytic domain and contain N-terminal and C-terminal extensions. These characteristics are conserved in the worm kinase. Within a 220 amino-acid stretch in the catalytic core, the C. elegans PCTAIRE kinase was 80% identical to the human PCTAIRES's. The gene was mapped near to unc-24 on chromosome IV and has been found on two overlapping cosmids from this region ( C27B3 and C07G1 ).In collaboration with Michael Hengartner and Bob Horwitz, we are currently trying to rescue mutants that map to this region.

Using these and other methods, we have not identified any other cdc2 -relatedgenes, and our cloning efforts are now shifting to the cyclins.