ABSTRACT: Microarrays analysis was undertaken to compare mRNA levels in exponentially growing wild-type and sub1Δ cells, or after 1 h treatment with 4NQO. After spot quantification and normalization, only few genes were found to be differentially expressed when the two strains were exponentially grown in rich medium. In contrast, the distribution of gene expression ratios was clearly different upon 4NQO treatment showing that the transcriptional response to DNA damages was affected in the absence of Sub1. Most of the differentially expressed genes were up-regulated in sub1Δ cells, suggesting a global repression effect of Sub1. About half of them were bound by Sub1 demonstrating that Sub1 occupancy correlates with gene regulation. A GoTermFinder analysis revealed an enrichment in genes implicated in cell cycle process for the down-regulated genes. The up-regulated genes were enriched in ribosome biogenesis and cell wall structure GO categories, confirming that most of the genes bound by Sub1 were up-regulated in sub1Δ cells upon 4NQO treatment. A third GO category corresponded to genes encoding proteins involved in amino acid metabolism whose expression is under the control of the Gcn4 transcriptional activator. This GO category could reflect an indirect effect since the GCN4 gene is bound by Sub1, up-regulated in sub1Δ cells and induced at the translational level in response to DNA-damaging agents. Keywords: Transcriptomic Genomic Comparative Cell culture and RNA extraction. Wild-type (yPH500) or sub1Δ cells were grown to exponential phase (OD600 = 1) in YPD medium at 30°C and an aliquot of cells was collected to serve as the time-zero reference. After the addition of 4NQO (1 µg/µl), the cells were further incubated for 1h. The cells were harvested by centrifugation and resuspended in 50 mM Na acetate, pH 5.3, 10 mM EDTA. Total RNA was isolated by heating and freezing the cells in the presence of SDS and phenol as described (Schmitt et al., 1990). Preparation of labeled cDNAs Labeled cDNAs were synthesized from 20 to 30μg total RNA using an indirect labeling protocol adapted from P.Brown (http://cmgm.stanford.edu/pbrown/protocols/aadUTPCouplingProcedure.htm) with the Superscript II reverse transcription kit (Gibco-BRL) and amino-allyl dUTP. lAmino-allyl modified DNA was purified on Microcon filters (Amicon) recovered with 20 µL of H2O and the DNA was lyophilized. The DNA pellet was resuspended in 9 µL of 100 mM sodium bicarbonate, pH 9.0. This sample DNA was used to dissolve a dry pellet of monofunctional NHS-ester Cy3 or Cy5 (1/16e of the quantity delivered from the mono-reactive dye pack PA23001 or PA25001 respectively from Amersham) and the mixture was incubated for 1 hour at room temperature in the dark. The coupling reaction of the Cy dyes to the amino-allyl dUTP was stopped by quenching with the addition of 4.5 µL of 4M hydroxylamine (Sigma) and the solution was further incubated for 15 min in the dark. Cy3-immunoprecipitated DNA and Cy5-WCE control DNA were mixed and unincorporated/quenched Cy dyes were removed using a Microcon YM-30 filter (Amicon/Millipore). DNA was recovered with 60 µL of TE and ethanol precipitated. Construction of ORF-microarrays S. cerevisiae open reading frames (ORF), reamplified using a pair of universal primers from 6144 full-length ORFs (1 ng) amplified from genomic DNA by Research Genetics. The PCR products were purified by ethanol precipitation, and their size and concentration were measured by agarose gel electrophoresis. 92.4% of the ORFs were correctly amplified and purified. The purified DNAs were spotted onto amino-silane coated glass slides (GAPS II, Corning) using an automated arrayer (MicroGrid II, BioRobotics) with a spotting success percentage higher than 96 %.. Prehybridization and hybridization. Prehybridization and hybridization conditions were those described on P. Brown's web site (http://brownlab.stanford.edu/protocols.html) with few minor modifications. The labeled precipitated DNA samples were recovered in hybridization buffer (50% formamide, 5X Denhardt’s, 0.5% SDS, 7X SSPE, 10 µg herring sperm DNA) and hybridized to the DNA microarray in a Corning slide chamber at 42°C overnight. Arrays were washed at room temperature once with 0.1X SSC + 0.1% SDS, twice with 0.1X SSC, then dried by centrifugation. Data analysis Hybridized arrays were scanned using a GenePix 4000A scanner (Axon Instruments, Inc.) and fluorescence ratio measurements were determined with the GenePix Pro 6.0 software (Axon Instruments, Inc.). Array analyses were undertaken using the Limma package (Smyth, 2005) from the R/Bioconductor software (R-Development-Core-Team, 2007). Microarray spot intensities have been normalized by subtracting the background and using the LOWESS method (Cleveland, 1979) with the smooth parameter set to 0.33 as recommended (Quackenbush, 2002). Data from four technical replicates corresponding to two independent experiments were compiled. Normalized measures served to compute the ratios of Cy3/Cy5 intensity and the associated log2-transform (denoted log2-ratios) for each gene. To identify genes with a log2-ratio significantly different between the mutant and wild-type strains, p-values were calculated for each gene using a moderated t-test. The moderated t test applied here was based on an empirical Bayes analysis and was equivalent to shrinkage (or expansion) of the estimated sample variances towards a pooled estimate, resulting in a more stable inference.
