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.
Su
J Y
JY
Howard Hughes Medical Institute, University of Colorado School of Medicine, Denver 80262.
Maller
J L
JL
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