A. nidulans wild type and atmA null mutant strains transcriptome analysis for polar growth upon HU-release

ATM is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease Ataxia Telangiectasia. Previously, we characterized the homologue of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. In order to investigate which pathways are controlled by AtmA during proliferation and polar growth, we determined the transcriptional profile of A. nidulans wild type and delta atmA mutant strains in different growth conditions. Conidia from both wild type and delta atmA mutant were incubated with 50 mM of hydroxyurea to synchronize the germlings in the S-phase of cell cycle. After this time, the cultures were centrifuged, washed and pre-warmed drug-free fresh media was aseptically added to the cultures. They were allowed to grow for additional 60, 90 and 120 minutes after the HU-release. At each time point, the germlings were also harvested by centrifugation and used for competitive microarray hybridizations. Our results indicate several genes that have decreased mRNA expression in the delta atmA mutant which are involved in the formation of a polarized hyphae and control of polar growth; in the synthesis of phosphatidic acid; in the ergosterol biosynthesis; and intracellular trafficking, secretion, and vesicular transport. Keywords: gene expression array-based (log2 ratio) For the time course microarray experiments, 1.0 x 1010 conidia/ml of A. nidulans wild type (GR5) and delta atmA mutant strains were used to inoculate 400 ml of pre-warmed liquid cultures (YG) in 1000-ml erlenmeyer flasks containing 50 mM of hydroxyurea (HU) and allow to grow for 5 hours in a reciprocal shaker (250 rpm) at 37°C to synchronize the germlings in the S-phase of cell cycle. After this time, the cultures were centrifuged, washed and pre-warmed drug-free fresh media was aseptically added to the cultures. They were allowed to grow for additional 60, 90 and 120 minutes after the HU-release. Hybridization experiments were competitive using probes derived from wild type and delta atmA strain grown for zero, 60, 90 and 120 minutes after HU blockage release using timepoint zero as the reference RNA for the hybridizations (wild type or mutant strain). Dye swap hybridizations were performed. Normalized signal intensities were used to generate relative hybridization ratios (query/reference). Following normalization, the values for each gene's in-slide replicates were condensed (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects (see Supplementary files linked below).