Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast.

Abstract

In fission yeast the Wee1 kinase and the functionally redundant Mik1 kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Wee1 and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate wee1 or mik1 homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both wee1 and mik1. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Wee1 kinase function. The expression of this clone in a wee1/mik1-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Wee1 kinase activity.