The functional analysis of
cki-1, the C.elegans
p27KIP cyclin-dependent kinase inhibitor homologue, has demonstrated a major role for this cell cycle regulator in the developmental regulation of the G1/S progression throughout postembryonic development. The removal of
cki-1 function through dsRNA-mediated interference causes extra cell divisions in multiple postembryonic lineages. In order to further understand how cell division is coordinated with developmental processes, and to identify upstream regulators of
cki-1 , we have been performing screens to identify mutants which phenocopy
cki-1(RNAi) animals in specific lineages. Joel Rothman’s group has previously shown that deficiencies that remove
cki-1 result in significantly more intestinal cells in embryos. Similarly, following
cki-1(RNAi) the average number of intestinal nuclei in
cki-1(RNAi) animals is 50 (+/-3), as compared to 32-34 in wildtype animals. The extra nuclei mostly arise from an additional cell division during embryogenesis. In a screen for mutants which phenocopy the extra intestinal cell proliferation, we have found a mutant,
rr31 , with an average number of 57(+/-4) intestinal nuclei. As in
cki-1(RNAi) animals, the additional cells arise during embryogenesis, although at an earlier stage. The loss of
cki-1 activity in
rr31 mutants does not enhance the total number of intestinal nuclei suggesting that these two proteins might cooperate to regulate divisions of the E lineage. Interestingly, the
rr31 mutation was shown to be a fully dominant, maternal-effect, gain-of-function allele. The
rr31 mutation was mapped using a combination of STS-polymorphism, snip-SNP mapping and three-factor crosses to a region between 0.97 and 1.23 on LGI. This region was analysed for the existence of known cell cycle regulators and candidates were tested using PCR amplification of the corresponding genes from mutant animals followed by injection into wild type animals. A 7.3kb PCR product amplified from the
rr31 mutant that corresponded to the
cdc25.1 cell-cycle phosphatase (corresponding cosmid region K06A5.7a) was injected into N2. This resulted in transgenic animals with extra intestinal nuclei, albeit at low frequency, consistent with maternal expression of transgenes. Furthermore,
cdc25.1(RNAi) in
rr31 animals fully suppressed the extra intestinal cell defect. Finally, a GC to AT transition was found at the splice site between the first and second exon in the mutant
cdc25.1 (
rr31) . We are currently investigating the effect of the gain-of-function allele in postembyonic lineages through ectopic expression studies. We are also trying to understand the basis of the intestinal specificity of this mutant cell cycle regulator through the characterization of extragenic suppressors and two-hybrid interactions. Biochemical and structural methods will be undertaken to compare the mutant and wild type forms of CDC25.1.