Project description:Genome-wide expression profiling was used to identify genes that showed altered expression in response to treatment with the fungistatic saponin tomatidine. Overnight batch cultures of strain S288C were diluted to an optical density at 600nm of 0.1. After 1 hour of growth at 30°C, the compounds were added and the cultures were incubated for an additional 5 hours prior to harvesting. Control cultures were treated with the solvent dimethylformamide (DMF). Tomatidine treated cultures were grown in the presence of tomatidine (dissolved in DMF) at concentations of 7.5 uM and 15 uM. Growth inhibition by the compounds was monitored by measuring the OD600nm. Cells were pelleted and frozen in liquid nitrogen. Acid-washed glass beads (0.5 mm diameter; Sigma) were added and the cells disrupted using two 20 s cycles at speed setting 6 in the Savant Bio 101 Fast Prep FP120. Total RNA was isolated using the Qiagen RNeasy kit (Qiagen, Inc., Valencia, CA, USA). Microarray hybridisation was performed using the Affymetrix GeneChip® Yeast genome S98 array using protocols described by Affymetrix, Inc. (Santa Clara, CA, USA) (as previously described (Zhu et al., 2001). Data were analyzed using Affymetrix® Microarray Suite version 5.0 software
Project description:Genome-wide expression profiling was used to identify genes that showed altered expression in response to treatment with the saponins tomatidine, tomatine and the sterol biosynthesis inhibitory compound fenpropimorph; Growth inhibition by the compounds was monitored by measuring the OD600nm. Cells were pelleted and frozen in liquid nitrogen. Acid-washed glass beads (0.5 mm diameter; Sigma) were added and the cells disrupted using two 20 s cycles at speed setting 6 in the Savant Bio 101 Fast Prep FP120. Total RNA was isolated using the Qiagen RNeasy kit (Qiagen, Inc., Valencia, CA, USA). Microarray hybridisation was performed using the Affymetrix GeneChip® Yeast genome S98 array using protocols described by Affymetrix, Inc. (Santa Clara, CA, USA) (as previously described (Zhu et al., 2001). Data were analyzed using the RMA algorithm using Expressionist Pro (GeneData Inc., Switzerland). Experiment Overall Design: Overnight batch cultures of strain S288C were diluted to an optical density at 600nm of 0.1. After 1 hour of growth at 30°C, the compounds were added and the cultures were incubated for an additional 5 hours prior to harvesting. Control cultures were treated with the solvent dimethylformamide (DMF). Tomatidine treated cultures were grown in the presence of 0.625 ppm tomatidine, 13.9 ppm Tomatine and 0.313 ppm Fenpropimorph, all dissolved in DMF. Each of the three replicates of the cultures produced on 3 independent days.
