Comment[ArrayExpressAccession] E-GEOD-46013 MAGE-TAB Version 1.1 Public Release Date 2013-10-12 Investigation Title How does orotic acid affect the growth of R.etli CE3? Comment[Submitted Name] How does orotic acid affect the growth of R.etli CE3? Experiment Description Growth inhibition of Rhizobium etli and other organisms by exogenous OA has not been previously reported. We conducted genome-wide transcriptomic analysis to determine the possible mechanism by which OA exerts its growth-inhibitory effect on R. etli CE3. The observed changes fall into several broad categories. First, the decreased expression of genes involved in protein synthesis (including ribosomal genes), replication and energy production indicates that the cells arrest or at least slow their growth, which agrees with the observed growth arrest induced by OA. The upregulation of a key enzyme (PrsAch) for 5'-phosphoribosyl-1'-pyrophosphate (PRPP) synthesis suggests that cells exposed to OA suffer PRPP deprivation. We hypothesized that the PRPP pools were depleted as a result of an increase in the synthesis of OMP stimulated by exogenous OA. PRPP depletion would result in a marked decrease in purine synthesis, which would lead to an imbalance between purine and pyrimidine nucleotides, causing arrest of DNA synthesis and a lack of energetic nucleotides (ADP, ATP). Three independent biological materials with one dyeswap were performed. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Encarnación Andrade Salazar Encarnación Person First Name Sergio Andres Emmanuel Sergio Person Mid Initials Manuel Person Email encarnac@ccg.unam.mx Person Affiliation Universidad Nacional Autonoma de México Person Phone 777 3291899 Person Fax 777 3175094 Person Address Centro de Ciencias Genomicas, Universidad Nacional Autonoma de México, Av. Universidad S/N, Cuernavaca, Morelos, Mexico Person Roles submitter Protocol Name P-GSE46013-1 P-GSE46013-5 P-GSE46013-6 P-GSE46013-2 P-GSE46013-3 P-GSE46013-4 P-GSE46013-7 Protocol Description Background correction, lowest normalization, intensity filter, variance analysis of replicates and selection of differentially expressed genes were performed with GenArise software (http://www.ifc.unam.mx/genarise/). The software identifies differential expressed genes by calculating an intensity-dependent z-score that measures the number of standard deviations (SD) a data point is from the mean [zi=(Ri-mean(R))/sd(R)]. One biological replicate was washed the slide twice, because at the first time several grids was detected dirty, and in the second one this area was observed in perfect conditions. For that reasons I analyzed both images and then we cut and pasted the grids 16 and 20 in data table obtained for best image (second washing). ID_REF = VALUE = Lowess normalized log2 ratio test/reference 10 μg of total RNA were reversed transcribed into cDNA incorporating dUTP-Cy3 or dUTP-Cy5 and employing the CyScribe First-Strand cDNA labeling kit (Amersham Biosciences, New Jersey, USA). A dye-swap experiment was performed. Incorporation of fluorophore was analyzed by using the absorbance at 555 nm for Cy3 and 655 nm for Cy5. Both Cy3 and Cy5 labeled cDNAs to be compared were mixed, dried to completion, and then resuspended in 4 ul of water, then were incubated at 94 C and added 45 ul of hybridization buffer Nexterion Hyb (SCOTT nexterion). The mixture was hybridized at 50 C for 14 to 16 h. Hybridized arrays were washed with 2X SSC, 0.1% SDS at 42 C for 2 min, and, subsequently, with 0.1X SSC at room temperature for 2 min, and twice with 0.1X SSC at room temperature for 2 min. After 4 h incubation at 30 ºC with constant shaking (200 rpm), 100 ml culture was supplemented with 100 µg/ml orotic acid (treatment) and control cultures were supplemented with water. R. etli CE3 was grown overnight in PY medium. Cells were collected by centrifugation and washed twice with water. These cells were used to inoculate minimal medium ((1.6 mM KH2PO4, 0.83 mM MgSO4, 10 mM NH4Cl, 5 mM dextrose, 5 mM CaCl2, 18.49 µM FeCl3.6H2O, 2 µg/L biotin, 178 µM uracil) at an OD600 of 0.2. After 30 min incubation at 30 ºC with constant shaking (200 rpm), cells were collected by centrifugation at 5520 g for 5 min, flash-frozen, and stored in liquid nitrogen. Total RNA was extracted using RNeasy minikit (Qiagen, Hilden, Germany) following the manufacturer's instructions. The array was scanned using a pixel size of 10 um with a Scan Array Lite microarray scanner (Perkin-Elmer, Boston, MA). For each spot the Cy3 and Cy5 density mean value and the Cy3 and Cy5 background mean value were calculated with software ArrayPro® Analyzer from Media Cybernetics. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Comment[SecondaryAccession] GSE46013 Comment[GEOReleaseDate] 2013-10-12 Comment[ArrayExpressSubmissionDate] 2013-04-12 Comment[GEOLastUpdateDate] 2013-10-12 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-46013.sdrf.txt