Project description:Analysis of transcriptional response to UV irradiation in two related crenarchaea, Sulfolobus solfataricus and Sulfolobus acidocaldarius.
Project description:Genome-wide occupancy of the basal transcription machinery in Saccharolobus solfataricus strain P2 (Sulfolobus solfataricus) after hydrogen peroxide treatment Recruitment of RNA polymerase and initiation factors to the promoter is the only known mechanisms for transcription activation and repression in archaea. Whether any of the subsequent steps towards productive transcription elongation is involved in regulation is not known. We characterised how the basal transcription machinery is distributed along genes in the archaeon Sulfolobus solfataricus. We discovered a distinct early elongation phase where RNA polymerases sequentially recruit the elongation factors Spt4/5 and Elf1 to form the transcription elongation complex (TEC) before the TEC escapes into productive transcription. TEC escape is rate-limiting for transcription output during exponential growth. Oxidative stress causes changes in TEC escape that correlate with changes in the transcriptome. Our results thus establish that TEC escape contributes to the basal promoter strength and facilitates transcription regulation. Impaired TEC escape coincides with the accumulation of initiation factors at the promoter and recruitment of termination factor aCPSF1 to the early TEC. This suggests two possible mechanisms for how TEC escape limits transcription, physically blocking upstream RNA polymerases during transcription initiation and premature termination of early TECs.
Project description:Experimentally mapped transcriptome structure of Sulfolobus solfataricus P2 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 25 nt).
Project description:Experimentally mapped transcriptome structure of Sulfolobus solfataricus P2 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 25 nt). Sulfolobus solfataricus P2 growth curve experiments were conducted in batch culture. Reference samples were cultured at mid-log phase (OD600 = 0.312). Eight samples were collected that spanned the key phases of the growth curve. Total RNA from samples of growth curve and reference were directly labeled with Cy3 or Cy5, and were hybridized to the tiling array. Dye-flip experiments were done for each sample. Log ratios were calculated for each probe (growth curve sample/reference). Transcriptome browser is available at http://baliga.systemsbiology.net/enigma/.
Project description:Time series of infection of Sulfolobus solfataricus strain 2-2-12 cells infected with STIV are compared with uninfected cells over 32 h.
Project description:To investigate the efficacy of CRISPR-Csm complexes for RNA- knockdown in eukaryotes, we quantified transcript abundance in HEK293T cells after targeting several nuclear or cytoplasmic RNAs. RNA-seq demonstrates efficient and specific knockdown of CRISPR-Csm compared to Cas13 and shRNA knockdown.
Project description:Genome reorganization by large scale indels, gene displacements, and horizontal gene transfers allow an organism to re-organize genes into operons (“operonization”) and explore novel strategies for adapting to its changing environment. We have characterized the process of operonization by mapping and comparing transcriptome structures (TSs) of four phylogenetically diverse exptremophilic archaea: a hydrogenotrophic methanogen (Methanococcus maripaludis S2), an anaerobic thermophile (Pyrococcus furiosis DSM 3638), an acidophilic and aerobic thermophile (Sulfolobus solfataricus P2), and a photoheterotrophic halophile (Halobacterium salinarum NRC-1). We demonstrate how the evolution of new transcriptional elements (promoters and terminators) is utilized as a mechanism to incorporate translocated, inverted, and newly acquired genes into existing gene regulatory programs. This SuperSeries is composed of the following subset Series: GSE26777: Methanococcus maripaludis S2 growth curve, tiling arrays GSE26778: Pyrococcus furiosus DSM 3638 growth curve, tiling arrays GSE26779: Sulfolobus solfataricus P2 growth curve, tiling arrays Refer to individual Series
Project description:RNA interference (RNAi) is a powerful experimental tool for RNA knockdown, but not all organisms are amenable. Here, we provide a “proof of principle” demonstration that CRISPR endoribonuclease can be used for programmable mRNA transcript degradation in eukaryotes. Using zebrafish as the animal model and Csm complex as the CRISPR endoribonuclease, we targeted EGFP transcript expressed from a variety of promoters. A drastic decrease of fluorescence was achieved in germ cells of the vasa:EGFP line. Weaker effects were also seen in fish lines that express EGFP zygotically. Knockdown was statistically significant in cmcl2:EGFP and fli1:EGFP zebrafish lines at 1 day post fertilization (dpf), but reduced to background levels at 2 dpf. The nkx2.5:EGFP fish line was least susceptible to Csm mediated EGFP knockdown. We also tested Csm mediated knockdown on the endogenous tdgf1 (oep) transcript. At optimal Csm dose, we observed a penetrance of the characteristic one-eyed phenotype at greater than 50% penetrance, and hence with similar efficiency to morpholino-mediated knockdown. We conclude that Csm mediated knockdown is very efficient for maternal transcripts and can also be used for mixed maternal/early zygotic and early zygotic transcripts, in some cases reaching comparable efficiency to morpholino-based knockdown with no significant off-target effects in our model.
Project description:Time series of infection of Sulfolobus solfataricus strain 2-2-12 cells infected with STIV are compared with uninfected cells over 32 h. Two-condition experiment, Control vs. Infected cells sampled at 5 time points (0, 8, 16, 24, 32 h post infection). Biological replicates: 3 control, 3 infected, paired samples independently grown. One replicate per array. Dye swaps.
Project description:Sulfolobus solfataricus P2 was grown aerobically, with O2 concentrations ranging from 1.5 to 26 % (v/v; gas phase). To gain some insight in control of the respiratory system, transcriptomes of the strain cultivated in different O2 concentrations (1.5 % vs 21 %, 1.5 % vs 26 %) were compared using a DNA microarray approach.