Genome-wide changes in the circadian transcriptional program in Synechococcus elongatus PCC 7942
Ontology highlight
ABSTRACT: Circadian input kinas A (cikA) null cells severly distorts the genome-wide output of the circadian clock Synechococcus cultures (both wildtype and M-NM-^TcikA null cells) were synchronized with two light-dark (12h:12h) cycle before release into continuous light. Samples were collected at 4 h intervals for the next 24 h, total RNA was isolated from samples collected at each time points and genome-wide expression was measured using custom-designed microarrays
Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, most genes show rhythmic expression controlled by the Kai-based clock under continuous light conditions (LL). We found that rpoD6-null mutants impaired expression of clock-controlled genes peaking at hours 8-10 in LL, while sasA-null or rpaA-null mutants each arrested the expression profiles at subjective dawn. Time-course data (0-24 h) of wild type (WT), rpoD6-null, sasA-null and rpaA-null S. elongatus PCC 7942 strains analyzed under continuous light (LL) conditions after two 12h:12h light:dark (LD) cycles using Affymetrix high-density oligonucleotide microarrays (GeneChip CustomExpress Arrays) representing 2,515 predicted protein-coding genes on the genome of Synechococcus elongatus PCC 6301, which can be used also for the almost homologous strain, S. elongatus PCC 7942.
Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, most of genes are downregulated in the dark, while 10% of genes are upregulated. Here, we employed high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild type and kaiABC-null strains under continuous dark (DD) conditions. We found that expression profile of a subset of genes on the genome in DD was dramatically affected by kaiABC-nullification, and the magnitude of dark-induction was dependent on time when cells were transferred from light to DD Keywords: timecourse data (0-12 h) under continuous darkness after dark:light cycles from Synechococcus elongatus PCC 7942 wild type and kaiABC-null strains Wild type (WT) and kaiABC-null (DkaiABC) S. elongatus PCC 7942 strains were analyzed under continuous dark (DD) conditions after two 12h:12h light:dark (LD) cycles using Affymetrix high-density oligonucleotide microarrays (GeneChip CustomExpress Arrays) representing predicted 2,515 protein-coding genes on the genome of Synechococcus elongatus PCC 6301, which can be used also to the almost homologous strain, S. elongatus PCC 7942: Two independent experiments in WT and Dkai strains under DD (hours 0, 0.5, 1, 2, 4, 8 and 12).
Project description:In many organisms, the circadian clock is composed of functionally coupled morning and evening oscillators that regulate the bouts of dawn and dusk activity. In Arabidopsis, oscillator coupling relies on a core loop in which the evening oscillator component TOC1 was proposed to activate a subset of morning-expressed oscillator genes. Our systems-biological approach overturns the current view of the Arabidopsis circadian clock showing that TOC1 does not function as an activator but as a timely-controlled general repressor of morning and evening oscillator components. Repression occurs through rhythmic binding to the promoters of all oscillator genes, suggesting a previously unexpected direct connection between the morning and evening loops. Examination of TOC1 genome-wide binding using TOC1 Minigene (TMG) seedlings expressing the genomic fragment of TOC1 fused to the Yellow Fluorescent Protein in a toc1-2 mutant background (TMG-YFP/toc1-2 seedlings) grown under LD cycles (12h light:12h dark).
Project description:Renal transcriptome analysis of Usp2 knockout mice. Please note 'Zeitgeber time 12' means 12 hours after light onset in 12h: light 12h dark housing conditions. It is of importance in the present design because of the strong circadian oscillation of the interest gene. In the Characteristics[genetic modification] column Usp2-Lac0-SA-IRES-lacZ-WT Neo/Kan is the name of the targeting cassette which was inserted in the gene, in opposition to non for the WT who carry no mutation.
Project description:FLOWERING LOCUS C (FLC) is a MADS box transcription factor that plays a well characterised role in repressing the vegetative to floral transition of Arabidopsis thaliana. FLC has also been shown to affect the Arabidopsis circadian clock, with mutant seedlings showing short circadian periods. In a previous study, we identified the temperature-dependent circadian period QTL PerCv5b near the FLC locus on the top arm of Chromosome 5. PerCv5b caused a significant period effect at 27oC but not at 12oC or 22oC. Temperature-dependent circadian period phenotypes and a known polymorphism in the Ler allele made FLC a strong candidate gene for PerCv5b. The period effect of FLC was enhanced by combination with alleles of FRIGIDA (FRI), a gene shown to up-regulate FLC's expression. We were interested in identifying how FLC affects the circadian clock, so we decided to identify its target genes. Greatest phenotypic difference was observed between fri; flc and FRI; FLC genotype seedlings at 27oC, so expression was compared between these lines (previously described in Michaels and Amasino1999 and 2001) on the Affymetrix ATH1 microarray. Seedlings were grown on media (MS 1.5% Agar containing 3% Sucrose) for 6 days under constant cool white fluorescent light (55-60 micro EINSTEINS) at 22oC then entrained for 4 days under (12h , 12h) light , dark cycles at 22oC. At dawn on the fourth day of entrainment they were transferred to constant light (25-30 micro EINSTEINS) at 27oC. Four samples were taken at 6 hour intervals starting 24h after the transfer to continuous conditions at the times 24h, 30h, 36h and 42h. Equal amounts of total RNA were pooled from the three time points to produce one sample per genotype. The pooling strategy was employed to reduce the effect of circadian regulation on genes expression. This was particularly important in our case as some interesting genes would likely be regulated by the circadian clock and may only show expression differences at particular phases that could easily be missed if using just one time point. Experimenter name = Kieron Edwards; Experimenter phone = 0131 651 3326; Experimenter fax = 0131 650 5392; Experimenter department = Institute of Molecular Plant Sciences; Experimenter institute = University of Edinbugh; Experimenter address = Kings Buildings; Experimenter address = Mayfield Road; Experimenter address = Edinburgh; Experimenter zip/postal_code = EH9 3JH; Experimenter country = UK Experiment Overall Design: 4 samples were used in this experiment
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 â?? 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:Inducible overexpression of Arabidopsis meristem regulators by AlcR / AlcA system. Plants harboring 35S::AlcR/AlcA::GOI (GUS control, LEAFY, SHOOTMERSTEMLESS, WUSCHEL)constructs were grown in continous light for 12 days and induced with 1% Ethanol. After 12h of EtOH treatment, seedlings were dissected and RNA was processed from the shoot apex and young leaves. Affymetrix Ath1 arrays were hybridized in duplicates from each experiment.
Project description:In the unicellular cyanobacterium, Synechococcus elongatus PCC 7942, essentially all promoter activities are under the control of the circadian clock in continuous light (LL) conditions. Here, we employed high-density oligonucleotide arrays to explore comprehensive profiles of genome-wide Synechococcus gene expression in wild type, kaiABC-null and kaiC-overexpressor strains under LL and continuous dark (DD) conditions. In the wild type strain more than 30% of transcripts significantly oscillated in a circadian fashion, peaking at subjective dawn and dusk. Such circadian control was nullified in kaiABC-null strains. Although KaiC has been proposed to globally repress gene expression, our analysis revealed that dawn expressing genes were upregulated by kaiC-overexpression, such that the clock was arrested at subjective dawn. Transfer of cells to continuous dark (DD) conditions from LL immediately suppressed expression of most of genes, while the clock keeps time even in the absence of transcriptional feedback. Thus, the Synechococcus genome seems primarily regulated by the light/dark cycles and dramatically modified by the protein-based circadian oscillator. Keywords: timecourse data (~48 hours under continuous light or darkness) from Synechococcus elongatus PCC 7942 (wild type, kaiABC-null, and inducible kaiC-overexpressor) strains Wild type (WT), kaiABC-null (Dkai), and inducible kaiC-overexpressor (oxC) S. elongatus PCC 7942 strains were analyzed under continuous light (LL), continuous dark (DD) using Affymetrix high-density oligonucleotide microarrays (GeneChip CustomExpress Arrays) representing predicted 2,515 protein-coding genes on the genome of Synechococcus elongatus PCC 6301, which can be used also to the almost homologous strain, S. elongatus PCC 7942: Two independent experiments in WT under LL (hours 0 to 52) and DD (hours 0 to 48); Two independent experiments in Dkai under LL (hours 0 to 48); Two independent experiments in oxC under LL in the presence or absence of an inducer, IPTG, from hour 25 to 33 in LL; a single experiment in WT under DD in the presence of an transcriptional inhibitor, rifampicin.
Project description:Quantification of circadian gene expression in WT Synechocystis sp. PCC 6803 cells We quantified circadian gene expression of the wild type Synechocystis PCC6083 strain. Over a 24 h time course, 6 samples for RNA isolation were taken at the following time points: 30 minutes before and after light is switched off (sample 1 - CT 11.5 and sample 2 - CT 12.5), 30 minutes before midnight (sample 3 - CT 17.5), 348 30 minutes before and after light onset (sample 4 - CT 23.5 and sample 5 - CT 0.5) and 30 minutes before noon (sample 6 - CT 5.5).