ABSTRACT: An intergenic region found to be enriched from a genomic library under butyrate stress was overexpressed and challenged with butyrate (0.6%). The overexpression strain was compared to the plasmid control to determine the transcriptional changes due to overexpression and butyrate stress. RNA samples were taken from both the overexpression strain (pRDNA7) and the plasmid control strain (pSOS95del) at 0, 15, 40, 120, 240, and 360 min post a 0.6% butyrate (pH 6.7) stress. Two slides per timepoint were hybridized on a dye swap configuration.
Project description:S288C was transformed with plasmids expressing the GCN5 F221A mutant at varying levels. We sought to examine the global impact on gene expression using the Affymetrix yeast 2.0 arrays. TEF promoter strength varied from 32, 68 and 95% of the wild-type expression. Controls included wild-type with empty & GCN5 null strain with empty plasmid. Strains were grown for approximately 7 dblings in triplicate, followed by a mid-log phase RNA extraction. Whole cell RNA was processed for Affymetrix yeast 2.0 arrays.
Project description:Cupriavidus metallidurans CH34 is a metal resistant beta-proteobacterium. The genome of this bacterium contain many genes involved in heavy metal resistance. Gene expression of C. metallidurans was studied after the addition of of Zn(II), Cd(II), Cu(II), Ni(II), Pb(II), Hg(II) or Co(II). Keywords: Heavy metal stress response Cultures of C. metallidurans CH34 were grown at 30°C until OD reached 0.6 (mid- exponential phase cultures). Heavy metals (0.8 mM of Zn(II), 0.5 mM of Cd(II), 0.1 mM of Cu(II), 0.6 mM of Ni(II), 0.4 mM of Pb(II), 5 uM of Hg(II) and 0.5 mM of Co(II)) were added to the culture for 30 minutes induction time. Total RNA was extracted, reverse-transcribed and labeled with Cy3-dCTP for the control (without metal) and with Cy5-dCTP for each conditions (challenged with one metal). Labeled cDNA were (control and one condition) added to a spotted slide for overnight hybridization at 42°C. Slides were scanned with a laser at 532 and 635 nm.
Project description:Volatile fatty acids found in effluents of the dark fermentation of biowastes can be used for mixotrophic growth of microalgae, improving productivity and reducing the cost of the feedstock. Microalgae can use the acetate in the effluents very well, but butyrate is poorly assimilated and can inhibit growth above 1 gC.L-1. The non-photosynthetic chlorophyte alga Polytomella sp. SAG 198.80 was found to be able to assimilate butyrate fast. To decipher the metabolic pathways implicated in butyrate assimilation, a large-scale differential proteomics study was developed comparing Polytomella sp. cells grown on acetate and butyrate at 1 gC.L-1.
Project description:Pseudomonas aeruginosa is an opportunistic human pathogen, infecting immuno-compromised patients and causing persistent respiratory infections in people affected from cystic fibrosis. Pseudomonas strain Pseudomonas aeruginosa PA14 shows higher virulence than Pseudomonas aeruginosa PAO1 in a wide range of hosts including insects, nematodes and plants but the precise cause of this difference is not fully understood. Little is known about the host response upon infection with Pseudomonas and whether or not transcription is being affected as a host defense mechanism or altered in the benefit of the pathogen. In this context the social amoeba Dictyostelium discoideum has been described as a suitable host to study virulence of Pseudomonas and other opportunistic pathogens.
Project description:OLE RNA (Ornate, Large, Extremophilic RNA) is a large, noncoding RNA from extremophilic and/or anaerobic Firmicutes that binds to an integral membrane protein (OAP, OLE-Associated Protein) and localizes to the cell membrane. It is highly abundant in cells, and knockouts show increased sensitivity to short-chain alcohols, especially ethanol. We treated Bacillus halodurans C-125 cells (both wild-type and OLE-OAP knockouts) with 5% (v/v) ethanol for 3 hours to determine differences in their response to this stress. There are almost no significant differences between cells before ethanol stress, while after ethanol stress a small number (~40) genes show differences in expression between the two strains. These genes show little connection between their function, however, leading us to conclude that the majority of differential adaptation in kncokouts may occur at an earlier time or at the post-trancriptional level. These data also serve as a reference for a Gram-positive alkaliphile's response to acute ethanol stress. Examination of wild-type and OLE-OAP knockout cells before and after ethanol stress, in duplicate (8 samples total)
Project description:Integrating omics data with quantification of biological traits provides unparalleled opportunities for discovery of genetic regulators by in silico inference. However, current approaches to analyze genetic-perturbation screens are limited by their reliance on annotation libraries for prioritization of hits and subsequent targeted experimentation. Here, we present iTARGEX (identification of Trait-Associated Regulatory Genes via mixture regression using EXpectation maximization), an association framework with no requirement of a priori knowledge of gene function. After creating this tool, we used it to test associations between gene expression profiles and two biological traits in single-gene-deleted budding yeast mutants, including transcription homeostasis during S phase and global protein turnover. For each trait, we discovered novel regulators without prior functional annotations. The functional effects of the novel candidates were then validated experimentally, providing solid evidence for their roles in the respective traits. Hence, we conclude that iTARGEX can reliably identify novel factors involved in given biological traits. As such, it is capable of converting genome-wide observations into causal gene function predictions. Further application of iTARGEX in other contexts is expected to facilitate the discovery of new regulators and provide observations for novel mechanistic hypotheses regarding different biological traits and phenotypes.
Project description:A comparative genomic hybridisation experiment using Affymetrix YG-S98 arrays to study the genetic background of S. Boulardii compared to S. Cerevisiae strain BY4743. Background: Saccharomyces boulardii, a yeast that was isolated from fruit in Indochina has been used as a remedy for diarrhoea since 1950, and is now a commercially available treatment throughout Europe, Africa and South America. Though initially classified as a separate species of Saccharomyces, recent publications have shown that the genome of S. boulardii is so similar to Saccharomyces cerevisiae that the two should be classified as conspecific. This raises the question of the distinguishing molecular and phenotypic characteristics present in S. boulardii that make it perform more effectively as a probiotic organism compared to other strains of S. cerevisiae. This investigation reports some of these characteristics including enhanced ability for pseudohyphal switching upon nitrogen limitation and increased resistance to acidic pH. However, these differences did not correlate with increased adherence to epithelial cells or transit through mouse gut. Pertinent characteristics of the S. boulardii genome such as trisomy of chromosome IX, altered copy number of a number of individual genes and sporulation deficiency have been revealed by comparative genome hybridisation using oligonucleotide-based microarrays coupled with a rigorous statistical analysis. The contributions of the different genomic and phenotypic features of S. boulardii to its probiotic nature are discussed.
Project description:Gene expression profiles of human cell (THP-1) lines exposed to a novel Daboiatoxin (DbTx) isolated from Daboia russelli russelli, and specific cytokines and inflammatory pathways involved in acute infection caused by Burkholderia pseudomallei. Experiment Overall Design: 1. Group I:- Human monocytic macrophage (THP-1) cell lines grown in the culture medium without any bacterial infection served as untreated control group (Three Biological Replicates). Experiment Overall Design: 2. Group II:- THP-1 cells were infected with Burkholderia pseudomallei (A600 nm = OD 0.6, ~5 x 107 cfu/ml) for 24h served as a disease control group (Three Biological Replicates). Experiment Overall Design: 3. Group III:- THP-1 cells were infected with B. pseudomallei and treated with Daboiatoxin (0.5 mM) isolated from Daboia russelli russelli venom served as a treatment group (Three Biological Replicates). Experiment Overall Design: 4. Group IV:- THP-1 cells were infected with B. pseudomallei (A600 nm = OD 0.6, ~5 x 107 cfu/ml) treated with standard antimicrobial drug ceftazidime (10mg/ml) served as a drug control (Three Biological Replicates). Experiment Overall Design: 5. Group V:- THP-1 cells were exposed to Daboiatoxin (0.5 mM) without bacterial infection (Three Biological Replicates).
Project description:Chromatin insulators are DNA-protein complexes situated throughout the genome capable of demarcating independent transcriptional domains. Previous studies point to an important role for RNA in gypsy chromatin insulator function in Drosophila; however, the identity of these putative insulator-associated RNAs is not currently known. Here we utilize RNA-immunoprecipitation and high throughput sequencing (RIP-seq) to isolate RNAs stably associated with gypsy insulator complexes. Strikingly, these RNAs correspond to specific sense-strand, spliced, and polyadenylated mRNAs, including two insulator protein transcripts. In order to assess the functional significance of these associated mRNAs independent of their coding function, we expressed untranslatable versions of these transcripts in developing flies and observed both alteration of insulator complex nuclear localization as well as improvement of enhancer-blocking activity. Together these data suggest a novel, noncoding mechanism by which certain mRNAs contribute to chromatin insulator function. RIP-seq of insulator proteins with different library preparations and multiple biological replicates