Our lab is interested in the regulation of cell division during development. Cyclin depen-dent kinases (cdks) are known as key regulators of the cell cycle. In yeast, a single cdk,
p34Cdc2/CDC28, regulates progression through multiple transitions in the cell cycle, whereas several cdks are used in higher eukaryotes. To determine which cdks are required for cell division in C. elegans, we identified C. elegans cdk homologs by low-stringency hybridization, PCR with degenerate oligos, ex-pression-library screening with anti-Cdc2 antibodies and BLAST homology searches. Only one of the 7 cdks identified is likely to be a general cell cycle regulator. This kinase,
ncc-1, was previously identified in Paul Sternberg's lab (1). NCC-1 is 64% identical at the amino acid level to human Cdc2, and nearly as closely related to Cdk2 and Cdk3. NCC-1 differs from these kinases in that it has an amino acid change in the conserved sequence PSTAIRE and has an 19 amino-acid extension at the N-terminus. We set out to determine whether
ncc-1 is the universal cdk in C. elegans, analogous to
cdc2/CDC28 in yeast. As a first approach, we studied the effects of RNAi by injecting N2 worms with
ncc-1 RNA and analyzing the F1 progeny. Most progeny arrested at the one cell stage. We fol-lowed early events after fertilization in vivo and used DNA and immunostaining to further characterize the defects. The maternal chromosomes appeared to remain arrested in a con-densed state after fertilization, and meiosis I and II are not completed. Migration of the ma-ternal and paternal pronuclei is delayed and pseudocleavage does not occur. The zygote ar-rests with the maternal pronucleus enlarged in size while the paternal pronucleus is small and not very well defined. A bipolar spindle is established at the maternal pronucleus in these mutant embryos. These results indicate an essential role for
ncc-1 in meiosis. Interestingly, some of the first mutant progeny developed normally through embryo-genesis and arrested as sterile and uncoordinated larvae with no apparent cell divisions after hatching. We assume that these larval arrested animals lack zygotic expression but did re-ceive maternal product, whereas the one cell arrested progeny may lack both. The pheno-type of the larval arrested animals was highly reminiscent to that of animals homozygous for
n3064, an allele isolated previously in a screen for thin, sterile, and uncoordinated mu-tants (van den Heuvel and Horvitz). We mapped
n3064 in close proximity to
ncc-1 and identified a G-to-A nucleotide transition in the
ncc-1 gene from
n3064 mutant animals. This mutation results in an E-to-K change of one of the most highly conserved amino acids in protein kinases. Consistent with such a mutation, animals homozygous for
n3064 have an equally strong phenotype as animals carrying
n3064 in trans of a deficiency. We conclude that
n3064 is a strong loss-of-function or null allele of
ncc-1. If
ncc-1 is indeed the universal cdk in C. elegans, cells should arrest early in the cell cy-cle with a 2N DNA content. To characterize the cell-cycle arrest, we are addressing the de-fects caused by
ncc-1 mutations in a number of genetic backgrounds. In
lin-5 animals, DNA replication continues after abortive mitosis, which results in polyploid cells. In
lin-6 animals, mitosis continues in the absence of DNA replication. If
ncc-1 animals arrest early in the cell cycle,
ncc-1 mutations should be epistatic to both
lin-5 and
lin-6 mutations. In addition, we will analyze expression of a fusion construct between ribonucleotide reductase and GFP (rnr::gfp, kindly provided by Richard Roy and Victor Ambros) in homozygous
ncc-1(
n3064) animals. Expression of ribonucleotide reductase correlates with DNA repli-cation, therefore, GFP expression should not be detected in these animals if
ncc-1 is re-quired early in the cell cycle. We are currently screening for conditional alleles of
ncc-1 which will be used to further define its role in various cell-cycle transitions. Developmental cues are likely to influence entry into the cell cycle. Therefore, we hope to be able to use a conditional allele of
ncc-1 in modifier screens, with the ultimate goal to identify novel genes involved in cell-cycle regu-lation during development. 1. Mori H, Palmer RE, Sternberg PW. Mol Gen Genet 1994 Dec 15;245(6):781-786