Large-scale identification of genes important for apical growth in Saccharomyces cerevisiae by directed allele replacement technology (DART) screening

Abstract.

In Saccharomyces cerevisiae, apical bud growth occurs for a brief period in G1 when the deposition of membrane and cell wall is restricted to the tip of the growing bud. To identify genes important for apical bud growth, we have utilized a novel transposon-based mutagenesis system termed DART (Directed Allele Replacement Technology) that allows the rapid transfer of defined insertion alleles into any strain background. A total of 4,810 insertion alleles affecting 1,392 different yeast genes were transferred into a cdc34-2 mutant strain that arrests in the apical growth phase when grown at the restrictive temperature of 37 °C. We identified 29 insertion alleles, containing mutations in 17 different genes (SMY1, SPA2, PAN1, SLA1, SLA2, CBK1, SEC22, FAB1, VPS36, VID22, RAS2, ECM33, OPI3, API1/YDR372c, API2/YDR525w, API3/YKR020w, and API4/YNL051w), which alter the elongated bud morphology of cdc34-2 cells arrested in the apical growth phase. Upon treatment with mating pheromone at 25°C, cells containing insertion alleles affecting ten of these genes (SMY1, SPA2, PAN1, SLA1, SLA2, CBK1, FAB1, VPS36, VID22, and API2/YDR525w) form abnormal mating projections. Additionally, cells containing insertion alleles affecting SEC22, RAS2, API1/YDR372c, API3/YKR020w, and API4/YNL051 display severe mating projection formation defects at the elevated temperature of 37°C. DART mutagenesis has many advantages over traditional mutagenesis methods and will be a useful tool for dissecting gene networks important for biological processes.