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

Abstract

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.

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