H. salinarum NRC-1 vs TFB knockouts and synthetic TFB constructs
Ontology highlight
ABSTRACT:
ORGANISM(S): Halobacterium sp. NRC-1
SUBMITTER: Serdar Turkarslan
PROVIDER: E-GEOD-31308 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Total RNA hybridizations of Halobacterium salinarum NRC-1 total RNA versus a strain overexpressing TFBd were compared to verify the transcriptome landscape changes. Comparison with TFBd binding sites localized using ChIP-chip data and MeDiChI algorithm (Reiss et al, 2008) allowed observation that TF-binding in the middle of coding sequences can also result in transcription initiation or termination internal to a single annotated protein-coding gene. Total RNA sample of plasmid based expression of Cmyc tagged transcription factor TFBd grown in standard growth media with mevinolin.
2010-05-17 | E-GEOD-15788 | biostudies-arrayexpress
Project description:A detailed map of genomic locations where TFs bind DNA and modulate transcription is essential to model mechanisms of gene regulation on a systems-scale. Chromatin immunoprecipitation of transcription complexes coupled to microarray (ChIP-chip (Ren et al, 2000)) or sequencing (ChIP-seq (Robertson et al, 2007)) is a commonly used approach to construct such maps. In ChIP-chip, the resolution to which the protein-DNA binding sites (TFBSs) can be identified is often limited by the genomic spacing of the probes in the array. We utilized the MeDiChI algorithm (Reiss et al, 2008) to estimate precise TFBS locations and their corresponding local false discovery rates (LFDRs) from high-resolution arrays for TFBa, TFBd and TFBf. This regression-based method deconvolves the ChIP-chip enrichment ratios along the genome by fitting them with a 'peak profile' model of binding events, assuming a distribution in enriched DNA fragment lengthsÂ. A comparison of the peak intensities derived from MeDiChI for all three TFs (TFBd, TFBf and TFBa) for which there were biological replicate measurements using 2 different microarray platforms (500 nt resolution spotted arrays, see series GSE7045 vs. 13 nt resolution Nimblegen arrays) provided strong validation (with R2 of 0.66, 0.52, and 0.81, respectively) of most TFBSs. Visualizations comparing the two microarray platforms are available at: http://baliga.systemsbiology.net/regulatory_logic/ IP sample of Cmyc tagged TFBs expressed in Halobacterium NRC-1 cells transformed with pMTFcmyc gene expressing TFBf in standard growth media with 20microgram/ml mevinolin versus a whole cell extract from the same sample. Three biological replicates for TFBd are available.
2010-05-17 | E-GEOD-15786 | biostudies-arrayexpress
Project description:Conditional ChIP-chip for TFBd at different growth stages. cmyc-tagged TFBd was immunoprecipitated at different growth stages (early log, stationary and late stationary phases) and hybridized to microarrays.
2010-05-16 | E-GEOD-13108 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE12923: Halobacterium salinarum NRC-1 growth curve, tiling arrays. GSE12977: Halobacterium salinarum NRC-1 growth curve GSE13108: Halobacterium salinarum NRC-1 conditional ChIP-chip for transcription initiation factor IIB 4 (TFBd) GSE7045: ChIP-Chip of General Transcription factors in Halobacterium NRC-1 GSE15786: Halobacterium sp. NRC-1 ChIP-chip for TFBa, TFBd and TFBf, high resolution array GSE15788: Halobacterium salinarum NRC-1 total RNA hybridization of TFBd overexpression versus Reference sample Despite knowledge of complex prokaryotic transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have played a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ~64% of all genes including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein-DNA interaction datasets revealed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3' ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes - events usually considered spurious or non-functional. With experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements Refer to individual Series
2010-05-17 | E-GEOD-13150 | biostudies-arrayexpress
Project description:Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms. Keywords: stress response, dose response 4 samples were analyzed. Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control.
2010-06-11 | E-GEOD-7613 | biostudies-arrayexpress
Project description:Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms. Keywords: stress response, dose responseH 4 samples were analyzed. Each sample was dye-swapped (2 replicates per condition) and hybridized against a standard control
2010-06-11 | E-GEOD-7612 | biostudies-arrayexpress
Project description:100mM EMS was added to mid-log phase Halobacterium NRC-1 cultures. After constant stress of EMS for 30 minutes, cultures were spun down and pellets were re-suspended in same volume of GN101 media. Cultures were then harvested by centrifugation and pellets were snap frozen on a dry-ice ethanol bath. Samples for RNA preparation were collected during recovery time points at 0, 10, 20, 30, 40, 60 and 120 minutes. 16 samples (8 samples from EMS perturbed and 8 non-perturbed controls) were analyzed on replicate arrays (dye-flips) all against the same standard reference sample.
2010-05-27 | E-GEOD-7609 | biostudies-arrayexpress
Project description:50mM H2O2 was added to mid-log phase Halobacterium NRC-1 cultures. After constant stress of H2O2 for 30 minutes, cultures were spun down and pellets were resuspended in same volume of GN101 media. Samples for RNA preparation were collected during recovery time points at 0, 10, 20, 30, 40, 60 and 120 minutes. 16 samples (8 exposed to H2O2 and 8 non-exposed controls) were each hybridized on duplicate arrays (as dye-flips) against the same standard control sample.
2010-05-27 | E-GEOD-7610 | biostudies-arrayexpress
Project description:Halobacterium cells, specified general transcription factor knock-out strains or plasmid expressed 6-His tagged general transcription factor strains, were grown to mid-log phase. RNA extractions were performed using the Stratagene Absolutely RNA Miniprep Kit and RNA quality checked with the Agilent Bioanalyzer and with Oligo Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1 Labeling, hybridization and washing have been previously described (Baliga et al. 2002) with 10 μg of RNA from the sample and reference. RNA from the final time point of a replicate experiment was used as the reference. . Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda (λ) values determined by a maximum likelihood method. Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) Proc Natl Acad Sci U S A 99:14913-84. each knockout or perturbed expression strain was analyzed at a single point during growth. This sample was analyzed on duplicate microarrays as a dye flip (2 microarrays per sample).
2010-05-27 | E-GEOD-7713 | biostudies-arrayexpress
Project description:We report detailed characterization of physiological changes encoded by 63% of all genes within the archaeon Halobacterium salinarum subsp. NRC-1 during routine laboratory growth. While the majority of these changes occur during the transition from rapid exponential growth to the stationary phase, we were also able to detect transient changes in gene expression. This demonstrated the presence of several additional albeit subtle physiological states that exist beyond what might be anticipated from a direct interpretation of the growth profile. Changes in the abundance of several key intracellular metabolites over the growth curve corroborated observations of changes in gene expression of enzymes that catalyze their synthesis. Next, we investigated the roles of general transcription factors (GTFs) in mediating these global growth-associated gene expression changes. This revealed numerous phenotypic perturbations including slowed growth, gas vesicle biogenesis deficiency and morphologic abnormalities upon the overexpression of a single GTF - TBPd. These phenotypes were quantified and were attributed to a perturbation in the regulation of aconitase. Importantly, our results demonstrate why an experiment design as simple as a simple batch culture can be enormously informative of activities and interrelationships of a large fraction of all genes in a microbe. Halobacterium NRC-1 was grown in shaken (220RPM) complete medium liquid culture at 37°C in duplicate flasks. Samples were selected at 7 timepoints during the growth of this organism representing different phases of growth (e.g. early exponential, logrithmic, stationary etc.). RNA extractions were performed using the Stratagene Absolutely RNA Miniprep Kit and RNA quality checked with the Agilent Bioanalyzer and with Oligo Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1 Labeling, hybridization and washing have been previously described (Baliga et al. 2002) with 10 μg of RNA from the sample and reference. RNA from the final time point of a replicate experiment was used as the reference. Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda (λ) values determined by a maximum likelihood method.Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) Proc Natl Acad Sci U S A 99:14913-84.
2010-05-14 | E-GEOD-14832 | biostudies-arrayexpress