Isolation and characterization of the new conditional-lethal mutations in byr4 of Schizosaccharomyces pombe by in vitro mutagenesis

Coordination of nuclear division, cytokinesis, and septation is essential for maintaining the genomic stability during the cell division cycle. byr4 in fission yeast Schizosaccharomyces pombe encodes an essential gene that regulates the timing of cytokinesis and septation in a dosage-dependent manner (Song et al., 1996). The knock-out of byr4 causes cell cycle arrest in late mitosis with multiple cytokinesis and septation, while byr4 overexpression inhibits cytokinesis and produces multinuclear cells. Since byr4 is an essential gene, characterization of the byr4 null phenotypes and investigation of its genetic interactions with other mutants entail technical limitations. To better characterize the functional mechanisms of byr4 through phenotypic and genetic analyses, we generated five temperature-sensitive byr4 mutant alleles. A truncated byr4 with a deletion corresponding to the N-terminal 29 amino acids was randomly mutagenized by hydroxylamine in vitro. The mutagenized byr4 with an N-terminal truncation was integrated into the byr4 locus of S. pombe genome. Cells that formed colonies at the permissive temperature, 25°C,but could not grow at the restrictive temperatures, 18°C or 35°C, were isolated. We successfully isolated five temperature-sensitive byr4 alleles (KSY1-5) that could not grow at 35°C. In the restrictive temperature, KSY1, KSY3, and KSY5 alleles arrested cells with multiple septation while chromosome segregation was normal in these alleles. KSY2 and KSY4 alleles exhibited two phenotypes at the restrictive temperature: cells were arrested with multiple nuclei due to the inhibition of cytokinesis or with multiple nuclei that were separated by septum. These newly isolated byr4 conditional alleles will be useful for the deduction of cellular processes where byr4 functions. Genetic studies and suppressor screens of the conditional alleles can provide useful tools for the isolation of interacting proteins with Byr4p.