cAMP mediated gene expression in S.Cerevisae

cAMP is a central signaling molecule involved in a plethora of cellular regulation processes, e.g. stress response, nutrient response and pseudohyphal growth. In order to identify transcriptional changes in cells that are due to changing cAMP levels we constructed a strain in which internal cAMP levels can be regulated by adding cAMP externally. A strain that is deficient in RAS1, RAS2 and PDE2 fullfills this criteria. Since it can not produce cAMP by itself and is responsive to external cAMP, it's internal cAMP levels can be determined by adding cAMP to the media. Transcriptional profiles of this strain, exposed to different cAMP concentrations, were analyzed using Affymetrix microarrays. Four different concentrations of cAMP (no cAMP, 0.5, 1.0 and 2.0 mM cAMP) in SC-media were used. The resulting transcritional profiles were compared to each other and to the transcriptional profile of a WT strain grown w/o cAMP. cAMP has a dramatic effect on gene expression in S. cerevisiae. We could observe a greater than 2 fold change in expression levels for about 20 per cent of the genome (about 1300 genes) as we compared the transcriptional profiles of cells grown without to cells grown with 2 mM cAMP. This result supports the fundamental role of cAMP in cellular processes. To validate our results we looked at genes that are known to be regulated by cAMP, e.g. CTT1 and FLO11. We could verify that low cAMP levels induce CTT1 strongly (up to - 80 fold) and high levels of cAMP induce FLO 11 (about 10 fold). Furthermore genes involved in stress response, like HSP12 and GRE1 are among the most strongly cAMP regulated genes. The induction of several of these genes can be attributed to STRE-elements. High cAMP levels strongly induce transcription of ribosomal protein subunits and ribosomal RNA. Another aspect of our analysis is the clustering of sets of genes required for specific cellular processes. We could find similar induction of genes required for respiration (COX-genes, QCR-g enes, F(O)-ATPase) in response to low cAMP levels. This indicates a direct link between the cAMP levels in the cell and its means to generate energy. These profiles also serve as a basis to analyze cellular processes of RAS and IRA, distinct from their role in cAMP regulation. Transcriptional profiles of strains carrying dominant allels of RAS2 or loss of function allels of IRA 112 have been gathered and are compared to the transcriptional profiles described above.