Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle. Together with their regulating subunits, cyclins, CDKs regulate progression through the cell cycle by phosphorylating specific targets at distinct points in the cell cycle. We started to characterize CDKs in C. elegans as a model to study cell-cycle regulation during development. Unlike yeast, multiple CDKs are present in C. elegans. We have identified three CDKs (ncc-1, cdk-5 and pct-1) by low stringency hybridization, PCR with degenerate oligonu-cleotide primers and screening of expression libraries with monoclonal anti-bodies (van den Heuvel et al, WBG;12, 5). ncc-1 was previously cloned by Mori and Sternberg and is 64% identical to cdc2 at the amino acid level. cdk-5 and pct-1 are close homologues of the human CDKs cdk5 and PCTAIRE-1, respectively. In addition, the genome project has identified at least 2 other possible CDK family members. To understand the function of these CDKs, we are determining expression patterns and identifying mutant alleles. ncc-1, cdk-5 and pct-1 show different expression patterns during development based on Western and Northern blot analysis. To study expression patterns at a cellular level, GFP-fusion con-structs and HA tagged constructs are being made. Injection of anti-sense ncc-1 RNA into young adults results in up to 100% embryonic lethality. Based on this result and the important roles CDKs play in many organisms, we think that C. elegans CDK mutants could show severe defects during development or gametogenesis. We have determined the genomic location of ncc-1, cdk-5 and pct-1 but were unable to identify any candidate mutations in these regions. We have started a clonal F2 screen to isolate ncc-1 and pct-1 mutants. A strain has been con-structed (N2 heEx1[rol-6, ncc-1, pct-1]) that should rescue mutations in ncc-1 and pct-1. This strain is mutagenized with EMS, single rolling F2 worms are picked and their progeny is examined. Candidate ncc-1 or pct-1 mutant strains segregate both non-rolling mutant progeny and non-mutant rolling progeny. These studies will contribute to our understanding of cell cycle regulation during development and may help us identify targets and regulatory subunits of CDKs.

Affiliation:
- MGH Cancer Center Building 149 13th street, Charlestown, MA 02129