A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast.
We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.
Institute of Cell and Molecular Biology, The University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
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CF
Cullen
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May
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Hagan
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Glover
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Ohkura
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins; D005816:Genetic Complementation Test; D005821:Genetic Techniques; D005838:Genotype; D008938:Mitosis / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins; D017386:Sequence Homology, Amino Acid; D013489:Suppression, Genetic; D013696:Temperature; D013997:Time Factors
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C075396:PD1 protein, pig; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C489404:CDC7 protein, S cerevisiae; C489406:cdc7 protein, S pombe; D017346:Protein Serine-Threonine Kinases