Genetic Suppression of Stress Sensitivity Following Loss of SSPI (CAMKK) in Schizosaccharomyces Pombe

Abstract

Loss of the ssp1 protein kinase (CAMKK) gene results in stress sensitivity, cell elongation, slow growth and in some cases cell cycle arrest. In order to identify new components of the ssp1 stress response pathway, a transposon mediated suppressor screen was used to identify loss of function suppressors of a Schizosaccharomyces pombe ssp1 gene disruption. The Musca domestica Hermes transposon was used to randomly insert the KanMx6 selectable marker in the genome. The selection was for Hermes insertions which rescued the G2 cell cycle arrest phenotype of ssp1- when grown at pH 3.5 and 36 C. Second site mutations that rescued the cell cycle arrest and allowed for colony formation were identified. In total 121 mutant strains with elongated morphology but capable of colony formation at pH 3.5 and 36 C were isolated and 22 insertion sites were identified by inverse PCR and sequencing. Genes for a transcriptional suppressor, scr1 (SPBC1D7.02c ), a spermidine transporter (SPCC569.05c ), cyp9 cyclophilin 9 (SPCC553.04), complexed with cdc5 (cwf4) (SPBC31F10.11c), ptr8 (SPAC17A5.06), (SPBC1921.07c), and set7 (SPCC297.04c) were identified as second site loss of function suppressors of the ssp1 deletion. Identifying these genes and their phenotype in conjunction with loss of ssp1, substantially improves our understanding of the Ssp1 molecular pathway in cell cycle control and cell stress response