Project description:The goal of the experiment was to determine the difference in gene expression between the wild-type strain and a strain lacking rpaA (ΔrpaA). Because gene expression is not at steady-state in the wild-type -- it oscillates with a circadian period -- and we did not know a priori whether it is at steady-state in the ΔrpaA strain, we compared the time-averaged gene expression in the wild-type to the time-averaged gene expression in the ΔrpaA strain. Cultures were grown in a turbidostat as described previously (Vijayan et al, PNAS 2009). Cultures were entrained with two consecutive light/dark cycles and released into continuous light at time T = 0. Cultures were samples every 4 hours for 20 h between T = 24 h and T = 44 h, inclusive (no 4 h sample was acquired because, in a circadianly-oscillating culture, it would be duplicative with the 24 h timepoint). A pool of RNA representing time-averaged wild-type RNA was constructed by pooling equal mass quantities of RNA from each wild-type timepoint. A pool of RNA representing time-averaged RNA for the ΔrpaA strain was constructed by pooling equal mass quantities of RNA from each ΔrpaA timepoint. These two pooled RNA samples were compared by two-color Agilent microarray. To correct for dye biases, two microarrays were performed -- one in which the ΔrpaA pool was labeled with Cy3 and the wild-type pool was labeled with Cy5, and another in which the dyes were swapped. In the manuscript, the average log2 ratio value from these two microarrays was employed (average of log2(ΔrpaA/wild-type) = 0.5 * (log2(ΔrpaA Cy3 / wild-type Cy5) - log2(wild-type Cy3 / ΔrpaA Cy5)), with the minus sign correcting for the sign changed caused by the dye swap). See supplementary file linked at foot of Series record.
Project description:The goal of the experiment was to determine whether gene expression oscillates in the absence of rpaA. It was reported previously (e.g., Takai et al, PNAS 2006) that activity of a handful of individual expression reporters was arrythmic, and we conducted this microarray timecourse to determine whether expression is arrhythmic geneome-wide. Cultures were grown in a turbidostat as described previously (Vijayan et al, PNAS 2009). Cultures were entrained with two consecutive light/dark cycles and released into continuous light at time T = 0. Cultures were samples every 4 hours for 48 h between T = 24 h and T = 72 h, inclusive. Gene expression at each timepoint was compared to the time-averaged gene expression (determined using a pool of equal mass quantities of RNA from all timepoints) using a two-color Agilent microarray.
Project description:The goal of the experiment was to obtain a replicate of the wild-type LL circadian timecourse published in Vijayan et al, PNAS 106: 22564-22568 (2009), in order to identify reproducible circadian genes in LL. Cultures were grown in a turbidostat as described previously (Vijayan et al, PNAS 2009), except that the culture volume was 3 L instead of 4.5 L. Cultures were entrained with two consecutive light/dark cycles and released into continuous light at time T = 0. Cultures were samples every 4 hours from T = 36 h and T = 64 h, inclusive. Gene expression at each timepoint was compared to the time-averaged gene expression (determined using a pool of equal mass quantities of RNA from all timepoints) using a two-color Agilent microarray. Timepoint T = 52 h is omitted due to poor data quality.
Project description:The goal of this experiment was to determine whether global circadian gene expression oscillation depends strictly on the presence of rpaA, even when the KaiABC post-translational oscillator is oscillating with a circadian period. Strains deleted for rpaA lack functional KaiABC post-translational oscillators because their reduced kaiBC expression level leads to a non-permissive Kai protein stoichiometry. We restored KaiABC post-translational oscillator function in a ΔrpaA ΔkaiBC strain by ectopic expression of kaiBC from the Ptrc promoter and used microarrays to measure the timecourse of gene expression globally. As a control, we used microarrays to measure the gene expression timecourse in a ΔkaiBC Ptrc::kaiBC strain, in which gene expression was expected to be rhythmic (Y Murayama et al, J. Bac. 198, 2008), as it is in the pure wild-type strain. Cultures were grown in a flasks bubbled with 1% CO2 in air, initially in the absence of IPTG. Cultures were treated with two consecutive light/dark cycles and released into continuous light at time T = 0, at which time IPTG was added to a final concentration of 6 µM. Cultures were samples every 4 hours for 44 h between T = 24 h and T = 68 h, inclusive. Gene expression at each timepoint was compared to the time-averaged gene expression (determined using a pool of equal mass quantities of RNA from all timepoints) using a two-color Agilent microarray.
Project description:Previous molecular and mechanistic studies have identified several principles of prokaryotic transcription, but less is known about the global transcriptional architecture of bacterial genomes. Here we perform a comprehensive study of a cyanobacterial transcriptome, that of Synechococcus elongatus PCC 7942, generated by combining three high-resolution data sets: RNA sequencing, tiling expression microarrays, and RNA polymerase chromatin immunoprecipitation (ChIP) sequencing. We report absolute transcript levels, operon identification, and high-resolution mapping of 5' and 3' ends of transcripts. We identify several interesting features at promoters, within transcripts and in terminators relating to transcription initiation, elongation, and termination. Furthermore, we identify many putative non-coding transcripts. We provide a global analysis of a cyanobacterial transcriptome. Our results uncover insights that reinforce and extend the current views of bacterial transcription. RNA Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 RNA polymerase ChIP Sequencing of the cyanobacterium Synechococcus elongatus PCC 7942 Tiling Microarray of the cyanobacterium Synechococcus elongatus PCC 7942
Project description:The cyanobacterium Synechococcus elongatus PCC 7942 exhibits oscillations in mRNA transcript abundance with 24-hour periodicity under continuous light conditions. The mechanism underlying these oscillations remains elusive – neither cis nor trans-factors controlling circadian gene expression phase have been identified. Here we show that the topological status of the chromosome is highly correlated with circadian gene expression state. We also demonstrate that DNA sequence characteristics of genes that appear monotonically activated and monotonically repressed by chromosomal relaxation during the circadian cycle are similar to those of supercoiling responsive genes in E. coli. Furthermore, perturbation of superhelical status within the physiological range elicits global changes in gene expression similar to those that occur during the normal circadian cycle. Synechococcus elongatus PCC 7942 was subjected to two consecutive light/dark cycles and released into continuous light (T = 0). Cells were sampled every 4 hours from T = 24 to T = 84 hours for microarray analysis to characterize circadian gene expression. In a separate experiment, to characterize the response of S. elongatus to immediate chromosome relaxation, cells were sampled at T = 56 and T = 64 hours, immediately followed by novobiocin treatment (0.1 ug/ml), and the resulting response was measured by microarray after 5, 10, 30, 90, and 150 minutes. This experiment was designed to test whether chromosomal relaxation is sufficient to induce gene expression changes similar to those observed during the circadian cycle.
Project description:The cyanobacterium Synechococcus elongatus contains a circadian clock which coordinates circadian changes in gene expression of a large percentage of its genes. The response regulator RpaA has been implicated as an important regulator of many circadian genes, but the role of this protein in regulating changes in gene expression genome-wide is not known. We show that deletion of rpaA abrogates circadian gene expression genome-wide and arrests cells in a gene expression state highly similar to that of wildtype cells in the morning. Furthermore, we show that RpaA binds DNA in an circadian manner that is dependent on phosphorylation of the protein. To demonstrate the sufficiency of phosphorylated RpaA in driving global changes in gene expression, we used RNA sequencing to measure changes in gene expression elicited by a phosphomimetic of RpaA (RpaA D53E) and compared these changes to those that occur during a circadian cycle in wildtype cells. This analysis reveals that induction of RpaA D53E is sufficient to drive all circadian gene expression changes that happen from dawn to dusk in wildtype cells. Interestingly, the dynamics of gene expression elicited by RpaA D53E induction mirror those observed during a circadian cycle in wildtype cells, suggesting that the dynamics of circadian gene expression and hard-wired in the regulon downstream of RpaA. Enriched mRNA was prepared from synchronized wildtype S. elongatus cells every four hours over a circadian cycle and sequenced using the Illumina TruSeq Stranded mRNA Sample Prep Kit and Illumina HiSeq technology. To test the role of phosphorylated RpaA in driving circadian gene expression, we generated a strain which we refer to as OX-D53E that lacks core clock components (ΔrpaA, ΔkaiBC) with an RpaA phosphomimetic (RpaA D53E) under the control of an IPTG-inducible promoter (Ptrc::rpaAD53E). We used the same methodology to measure gene expression in OX-D53E before and after induction of RpaA D53E. As a control, we also measured gene expression in the OX-D53E strain over time in the absence of IPTG. Also, we generated a strain similar to OX-D53E in which the only difference was that no gene was inserted downstream of the IPTG inducible promoter (OX-Empty). We measured gene expressio in OX-Empty before and after IPTG addition to test for off-target effects of IPTG.
Project description:In the present study, the Agilent-016251 Sparus aurata oligo microarray platform (GPL6467) was used to compare expression profiles of mineralization-induced VSa13 cell cultures against untreated ones. ECM mineralization was induced for 4 weeks by supplementing medium with 50 µg/ml of L-ascorbic acid, 10 mM β glycerophosphate and 4 mM CaCl2. For each group, total RNA was extracted from three (3) independent biological replicates, each consisting of pools of cells. Data analysis demonstrated that expression profiles were strongly affected by ECM mineralization with hundreds of genes differentially expressed with relevant fold-change. In this study, we analyzed six (6) cell samples, three (3) collected from untreated VSa13 cell cultures and three (3) collected from mineralization-induced VSa13 cell cultures. Gene expression profiling was performed using Agilent-016251 Sparus aurata oligo microarray platform (GPL6467) (6 arrays, no replicate) based on single-colour detection (Cyanine-3 only). Microarrays were scanned with Agilent scanner G2565BA (barcode on the left, DNA on the back surface, scanned through the glass) at a resolution of 5 microns; all slides were scanned twice at two different sensitivity settings (XDRHi 100% and XDRLo 10%); the scanner software created a unique ID for each pair of XDR scans and saved it to both scan image files. Feature Extraction (FE) 9.5 used XDR ID to link the pairs of scans together automatically when extracting data. The signal that was left after all the FE processing steps was ProcessedSignal that contains the Multiplicatively Detrended, Background-Subtracted Signal.
Project description:Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway leading to the expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive (pho7+) and negative (csk1+) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response for the ScPHO5 (pho1+), ScPHO84 (spbc8e4.01c+), and ScGIT1 (spbc1271.09+) orthologs. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. We provide a global analysis of the PHO pathway in S. pombe. Our results elucidate the conserved core regulon required for responding to phosphate starvation between distantly related ascomycetes and a better understanding of flexibility in environmental stress response networks. Schizosaccharomyces pombe 972 h- cells were starved for inorganic phosphate for 0, 30, 60, 120, or 240 minutes pior to microarray preparation to determine the extent and temporal resolution of the phosphate starvation response. At 120 minutes post-starvation we define a set of genes that are directly and specifically induced by phosphate starvation, providing a time-point at which all other experiments were performed. We characterize the pho7+- and csk1+- dependency of this PHO response at 120 minutes post-starvation in pho7+csk1+, pho7Δ, csk1Δ, and pho7Δcsk1Δ cells. In a seperate set of experiments we characterized the S. pombe stress response to copper limitation, iron limitation, and carbon switching at 120 minutes post-stress and osmotic shift at 20 minutes post-stress in both pho7+ and pho7Δ cells.