Glucose-depletion time-course experiment in Saccharomyces cerevisiae wild-type cells.
ABSTRACT: To investigate the glucose regulatory system in Saccharomyces cerevisiae, we conducted a time-course in which glucose-depleted wildtype (WT) cells were inoculated into fresh media (SC, 2% glucose). Their subsequent transcriptional output was monitored over a period of five hours by DNA microarrays: samples for gene expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium. Transcripts upregulated are involved in translational processes such as the GO biological processes “ribosome biogenesis” and “ribosome localization”. Transcripts downregulated are enriched for the GO biological processes “cellular respiration” and various metabolism related processes. The time-course was used to verify the physiological relevance of gene expression profiles determined for individual deletions of glucose regulatory system components. Importantly, transcripts up- or downregulated in WT cells upon the addition of glucose are similarly up- or downregulated in deletion mutants that each lack a component of the glucose regulatory system. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Two overnight WT cultures were used to inoculate 50 ml cultures at an OD600 of 0.15. These were depleted of glucose by growing for 24 hours and were used the next day to inoculate 500 ml cultures in fresh medium to an OD600 of 0.15. Samples for expression profiling were taken immediately after, as well as 3, 7.5, 15, 30, 60, 110, 150, and 300 minutes after inoculation into fresh medium.