cul-4 is a member of the cullin family. Cullins are components of ubiquitin-ligase assemblies that promote ubiquitin mediated degradation of specific target proteins. The functions of three cullins, CUL-1, CUL-2 and CDC53, are known and all are cell cycle regulators. Little is known about CUL-4. It has been reported that the human homologue of the
cul-4 gene is amplified and overexpressed in primary breast cancers (Chen, L. C. et al. Cancer research, 38: 3677-3683, 1998), suggesting a role of CUL-4 in cancer progression. We used dsRNA microinjection to get information about the mutant phenotypes. With
cul-4 dsRNAi, we observed that the majority of larvae arrested in the L2 stage. In these larvae, we saw huge hypodermal nuclei (up to 9 times bigger than normal nuclei) while other cells appeared to have normal sized nuclei. The huge hypodermal nuclei may result either from endoreplication, in which cells undergo extra rounds of S phase while bypassing mitosis, from fusion of synctial nuclei, or from G1 arrest. In certain L2 arrested animals, we observed that some germ cells had differentiated into sperm, suggesting that certain developmental programs, including germ cell differentiation, continued in
cul-4 RNAi animals although cell division was arrested. Alternatively,
cul-4 may normally function to inhibit sperm differentiation. The
cul-4 RNAi results indicate that loss of
cul-4 function is lethal. We have found that there are several lethal genes in the vicinity of
cul-4 . Among them,
let-263 and
let-236 are unidentified genes. We have started to test whether these lethals are
cul-4 . One strain,
let-263 has a phenotype of L2 arrest and large hypodermal cells, similar to the
cul-4 RNAi phenotype. We are currently determining whether
let-263 strain has DNA mutations in the
cul-4 gene with the chemical-cleavage-mismatch method developed by Cotton et al,1988. We are also in the process of generating deletion mutants by PCR screening animals mutated with EMS. Finally, we are in the process of making a GFP-tagged
cul-4 to determine its expression profile.