Figure 5

Microarray analysis of the effects of elevated Tpk3p activity. The effect of high Tpk3p activity on gene transcription was assessed. An affymatrix microarray was performed on RNA isolated from Δpde2 and Δpde2 Δtpk3 strains grown to diaxuic shift in YPD media supplemented with 4 mM cAMP. Confirmation of both the strains genotype and reliability of the micro array data can be seen by comparing expression levels of the effected genes PDE2 and TPK3: TPK1 and TPK2 expression levels are included for information (A). GO-Slim Mapper software was used to identify likely intracellular compartments of genes either up or down regulated by at least 2 fold (B). Genes involved in glycogen biosynthesis were significantly down regulated in Δpde2 cells under these conditions but not in Δpde2 Δtpk3. As further verification of the array data we assayed glycogen levels in wild type, Δpde2 and Δpde2 Δtpk3 cells. In this assay a yellow colour is indicative of glycogen presence (C). Western blotting was used to assess the levels of the mitochondrial Cytochrome c Oxidase protein Cox4p, Cox2p and actin (Act1p) in wild type, Δpde2 and Δpde2 Δtpk3 cells grown in the presence or absence of 4 mM cAMP. Protein equivalent to 2 × 10⁶ cells was run on an SDS-PAGE gel and transferred to PVDF for immunodetection (D).