In Saccharomyces cerevisiae , several genes, which encode components of the mitotic checkpoint, such as MAD1, 2, 3 or BUB1, 2, 3 , are required for cell cycle arrest in response to defects in the chromosome segregation machinery. We investigated the function of mitotic checkpoint genes in multicellular organisms. We have identified a gene encoding 203 amino acids, having 37% and 50% sequence identity with budding yeast Mad2p and hMAD2, respectively, and named the gene
mdf-2 after mitosis arrest deficiency. The
mdf-2 gene product is capable of rescuing the benomyl sensitive phenotype of S. cerevisiae
mad2 mutants. Using a yeast two-hybrid system, we identified a gene encoding an MDF-2 interacting protein, designated
mdf-1 which is predicted to encode a coiled-coil protein sharing weak sequence similarity with the S. cerevisiae gene, MAD1 . Antibody to MDF-2 localized to the centrosomes and the central spindle region in the early cleaving embryonic cells in a cell cycle dependent manner. In addition, we found the prominent staining of MDF-2 in the nuclei of gut cells destined for endreduplication, and in the germ cells at prophase of meiosis I. Silencing of
mdf-1 and
mdf-2 genes by RNAi resulted in larval arrest, adult sterility, or mature adults with incorrect chromosome number at meiotic prophase, giving few viable offspring and a high frequency of XO males. In addition, we obtained a deletion mutant of the
mdf-1 locus (isolated by the C. elegans gene knockout facility, University of British Columbia). The deletion,
gk2 , removed 2724 bases of DNA in the
mdf-1 locus, including nucleotides that encode amino acids 3-679 of MDF-1. The
mdf-1 deletion homozygotes look normal, but produced a range of mutant phenotypes including unhatched embryos, arrested L1 larvae, fertile hermaphrodites that give few progeny and a high incidence of males, as well as sterile adults. Mutant phenotype and RNAi phenocopy results are identical. The strain cannot be maintained as a homozygote beyond the F2 generation, making it an unconditional maternal effect sterile. These results revealed the essential function of
mdf-1 and
mdf-2 for normal development and fertility as well as the maintenance of proper chromosome number.
mdf-1 and
mdf-2 for normal development and fertility as well as the maintenance of proper chromosome number.