Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast.
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.
Howard Hughes Medical Institute, University of Colorado School of Medicine, Denver 80262.
J Y
JY
Su
J L
JL
Maller
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005816:Genetic Complementation Test; D016200:Maturation-Promoting Factor / Q000378:metabolism; D008938:Mitosis / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics*; D010641:Phenotype; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D017422:Sequence Analysis, DNA; D013696:Temperature; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000235:genetics*
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D029867:Xenopus Proteins; C530005:WEE1 protein, Xenopus; C068689:wee1 protein, S pombe; D011505:Protein-Tyrosine Kinases; D016200:Maturation-Promoting Factor