Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Measuring the effects of hypoxia on transcription in Caulobacter crescentus

ABSTRACT: To test the effects of hypoxia on transcription in Caulobacter crescentus, we cultured cells in a New Brunswick bioreactor under controlled conditions. Prior to innoculation, the medium was bubbled with laboratory air at maximum flow and stirred at 300 rpm for 2 hours. After this period, the medium was considered saturated with air and the oxygen probe was set to 100%. Untreated cultures were grown in air-saturated complex medium at 30 degrees C to OD660=0.5 at pH=7 (continuous air-bubbling; 300 rpm stirring). At cell harvest in aerated culture, the dissolved oxygen probe remained above 98%. To subject cells to hypoxia, culture at OD660=0.5, pH=7 was sparged continuously with nitrogen gas; the dissolved oxygen level as measured by the gas probe dropped from 100% to 0% over the course of 5 minutes under this condition. Hypoxic cultures were continually stirred and bubbled with nitrogen for another 20 minutes after the dissolved gas probe read 0%. Hypoxic cells were then harvested for RNA isolation. Four independent biological samples are included in this study. Two batches of cells were subjected to 20 minutes of hypoxia in a bioreactor; two cell batches were highly aerated.

ORGANISM(S): Caulobacter vibrioides

SUBMITTER: Sean Crosson

PROVIDER: E-GEOD-21127 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Interaction specificity, toxicity and regulation of a paralogous set of ParE/RelE-family toxin-antitoxin systems.

Fiebig Aretha A Castro Rojas Cyd Marie CM Siegal-Gaskins Dan D Crosson Sean S

Molecular microbiology 20100512 1

Toxin-antitoxin (TA) gene cassettes are widely distributed across bacteria, archaea and bacteriophage. The chromosome of the alpha-proteobacterium, Caulobacter crescentus, encodes eight ParE/RelE-superfamily toxins that are organized into operons with their cognate antitoxins. A systematic genetic analysis of these parDE and relBE TA operons demonstrates that seven encode functional toxins. The one exception highlights an example of a non-functional toxin pseudogene. Chromosomally encoded ParD a ...[more]

PMID: 20487277

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Project description:Adult zebrafish (TÃ¼bingen strain, sex not specified) at approximately 1 year of age were analysed. For experiments conducted under low oxygen conditions, nitrogen gas was bubbled through water to deplete oxygen before exposure of individual fish to the medium. Oxygen concentrations were measured using a dissolved oxygen meter (DO 6+, EUTECH instruments, Singapore). The dissolved oxygen level for hypoxia treatment was measured to be 1.20 Â± 0.6 mg/l, whereas normal ambient oxygen levels were 6.6 Â± 0.45 mg/l. Zebrafish were exposed to the hypoxic medium for 3 hours. Briefly, after each hypoxia trial, the animals were euthanized by hypothermic shock and then decapitated to remove the brain. Total RNA was extracted from samples mentioned above using the QIAGEN RNeasy mini kit (QIAGEN, GmbH, Hilden, Germany) and stored at â??80Â°C before further analysis. RNA concentration was determined with a NanoVueâ?¢ UVâ??vis spectrophotometer (GE Healthcare Life Sciences, Fairfield, USA). RNA integrity and quality were then estimated using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and the RNA integrity number (RIN) index was calculated for each sample. Only RNAs with a RIN number >7.0 were processed further. Microarray analysis of gene expression was performed using the Zebrafish Gene 1.0 ST Array (Affymetrix Inc. Santa Clara, CA). Briefly, 300ng of total RNA derived from a single adult brain was converted to amplified sense strand cDNA using the Ambion WT Expression Kit (Life Technologies, Carlsbad, CA). The resulting sense cDNA was fragmented and Biotin end-labelled using the Affymetrix Genechip WT Terminal Labeling Kit prior to hybridisation to the array at 45 Â°C for 16 hours. Two treatments including hypoxia and normoxia were studied. Each treatment had three biological replicates (i.e. three fish exposed to hypoxia and three fish exposed to normoxia). Six samples were analysed. Microarray analysis of gene expression was performed using the Zebrafish Gene 1.0 ST Array. The signal intensity of the chip was scanned using a GeneChipR Scanner 3000TG and analysed using Expression Console software (www. Affymetrix.com). CEL files were imported and intensities adjusted by RMA background correction and quantile normalization.

Dataset Information

Measuring the effects of hypoxia on transcription in Caulobacter crescentus

Publications

Interaction specificity, toxicity and regulation of a paralogous set of ParE/RelE-family toxin-antitoxin systems.

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