Transcriptome profiling of H. polymorpha in response to hydrogen peroxide exposure
ABSTRACT: To gain insight into the basic mechanism of Hydrogen peroxide detoxification in the methylotrophic yeast, H. polymorpha, we analyzed changes in transcriptional profiles in response to hydrogen peroxide exposure. Total RNA samples were collected from H. polymorpha cells after 30 min incubation with 0.5mM hydrogen peroxide. Using the RNA sample obtained prior to hydrogen peroxide addition as a reference, the differential fluorescence intensities of each RNA sample prepared at the indicated time was measured after labeling with Cy3 or Cy5 fluorochromes. For all analyses, we performed dye swapping experiments to avoid dye bias.
Project description:Transcriptome profiling analysis of the Hansenula polymorpha MET4 gene deletion strain have been carried out to obtain comprehensive information on the HpMet4p-mediated regulatory networks in association with the cadmium (Cd) detoxification and sulfur regulation in H. polymorpha. Total RNA samples were collected from H. polymorpha wild type, and HpMET4 deletion strain, under sulfur starvation or Cd (0.6 mM) stress conditions. The differential fluorescence intensities of each RNA sample were measured after labeling with Cy3 or Cy5. For all analyses, we performed dye swapping experiments to avoid dye bias. Thus, four intensity values were generated for each ORF and averaged for analysis.
Project description:Algal photo-bio hydrogen production, a promising method for producing clean and renewable fuel in the form of hydrogen gas, has been studied extensively over the last few decades. In this study, microarray analyses were used to obtain a global expression profile of mRNA abundance in the green alga Chlamydomonas reinhardtii at five different time points before the onset and during the course of sulphur depleted hydrogen production. The present work confirms previous findings on the impacts of sulphur deprivation but also provides new insights into photosynthesis, sulphur assimilation and carbon metabolism under sulphur starvation towards hydrogen production. For instance, while a general trend towards repression of transcripts encoding photosynthetic genes was observed, the abundance of Lhcbm9 (encoding a major light harvesting polypeptide) and LhcSR1 (encoding a chlorophyll binding protein) was strongly elevated throughout the experiment, suggesting remodeling of the photosystem II light harvesting complex as well as an important function of Lhcbm9 under sulphur starvation. This study presents the first global transcriptional analysis of C. reinhardtii during hydrogen production using five major time points at Peak Oxygen, Mid Oxygen, Zero Oxygen, Mid Hydrogen and Peak Hydrogen. Keywords: Time course, sulfur deprivation, hydrogen production. Time course microarray analyses were used to analyze the global gene expression in sulfur depleted hydrogen producing C. reinhardtii. When depleted of sulfur, a sealed an illuminated C. reinhardtii culture slowly becomes anaerobic and produces hydrogen gas. Based on the changes in the dissolved O2 level and H2 production rate, the whole course of H2 production from the starting of S deprivation to the end of H2 production can be divided into five phases including an Aerobic Phase (I) in which the dissolved O2 level goes up (I), an O2 Consumption Phase (II), an Anaerobic Phase (III), a H2 Production Phase (IV) and a Termination phase (V). Samples were taken from three different bioreactors (biological replicates) at the following time points after the start of S depletion: 6 h, 16 h, 21 h, 37 h and 52 h corresponding to Peak O2, Mid O2, Zero O2, Mid H2 and Peak H2.
Project description:Series of DNA microarrays (4) comparing the M. catarrhalis strain 035E response to 50 mM hydrogen peroxide relative to a water-only control. Wild-type cells were exposed to either 50 mM hydrogen peroxide or a water-only control for 15 minutes. RNA was extracted and DNA microarray analysis performed. 4 biologic replicates were studied. One dye swap was included in this series analysis.
Project description:Series of DNA microarrays (4) comparing the M. catarrhalis strain 035E and M. catarrhalis oxyR mutant response to 50 mM hydrogen peroxide. Wild-type cells and oxyR mutant cells were exposed to 50 mM hydrogen peroxide for 15 minutes. RNA was extracted and DNA microarray analysis performed. 4 biologic replicates were studied. One dye swap was included in this series analysis. Control oxyR mutant cells exposed to water
Project description:Background: The mosquito Anopheles gambiae is a major vector of human malaria. Increasing evidence indicates that blood cells (hemocytes) comprise an essential arm of the mosquito innate immune response against both bacteria and malaria parasites. To further characterize the role of hemocytes in mosquito immunity, we undertook the first genome-wide transcriptomic analyses of adult female An. gambiae hemocytes following infection by two species of bacteria and a malaria parasite. Results: We identified 4047 genes expressed in hemocytes, using An. gambiae genome-wide microarrays. While 279 transcripts were significantly enriched in hemocytes relative to whole adult female mosquitoes, 959 transcripts exhibited immune challenge-related regulation. The global transcriptomic responses of hemocytes to challenge with different species of bacteria and/or different stages of malaria parasite infection revealed discrete, minimally overlapping, pathogen-specific signatures of infection-responsive gene expression; 105 of these represented putative immunity-related genes including anti-Plasmodium factors. Of particular interest was the specific co-regulation of various members of the Imd and JNK immune signaling pathways during malaria parasite invasion of the mosquito midgut epithelium. Conclusion: Our genome-wide transcriptomic analysis of adult mosquito hemocytes reveals pathogen-specific signatures of gene regulation and identifies several novel candidate genes for future functional studies. In order to identify hemocyte-specific and immune-responsive transcripts, we first compared transcripts expressed in hemocytes from one day old sugar-fed mosquitoes to transcripts detected in whole mosquitoes of the same age and feeding status. This resulted in identification of the hemocyte-enriched transcriptome. We then compared hemocytes from 1 day old mosquitoes, 1 hour after immune challenge with heat-killed Escherichia coli or Micrococcus luteus, to control female mosquitoes injected with sterile PBS to determine the bacteria challenge responsive transcriptomes. We used heat-killed bacteria in these assays, because our primary interest was in identifying the bacterial responsive transcriptome and to avoid the potentially confounding effects of altered gene expression due to the lethal effects of a systemic infection associated with injection of living bacteria. Lastly, we compared hemocytes from mosquitoes at 24 hours and 19 days after ingestion of a blood meal infected with Plasmodium berghei to mosquitoes of the same age fed a non-infected blood meal to determine the ookinete and sporozoite infection responsive transcriptomes, respectively. This design resulted in a total of five experimental treatments. The following samples are not included in this submission: Hemo E coli vs. hemo unchallenged A Hemo E coli vs. hemo unchallenged B Hemo m luteus vs. hemo unchallenged A Hemo m luteus vs. hemo unchallenged B
Project description:Identify genes with increased transcript abundance after low oxygen stress in Drosophila adults. There are 6 Oregon-R experiments comparing low oxygen exposed to normal oxygen. Samples 1-4 have hypoxic RNA labeled with cy5, and samples 5&6 are dye-swaps with the hypoxic RNA labeled with cy3. Samples 7&8 are Sb/Tm3 flies to control for background effects.
Project description:In this study, DNA microarray technology was used to measure global gene expression in M. catarrhalis cells that had attached to human bronchial epithelial cells in culture. M. catarrhalis ATCC 43617 was allowed to attach (MOI~30) to a monolayer of 16HBE14 human bronchial epithelial cells for 2 hr in OmniTray cell culture plates. Additional portions of these bacteria were incubated in tissue culture medium in 75-cm2 cell culture flasks in the absence of 16HBE14 cells for 2 hr. After this incubation period, the medium in the flask with the 16HBE14 monolayer was decanted and replaced with tissue culture medium containing 25 mM sodium azide (to protect RNA). The 16HBE14 monolayer and attached bacteria were released from the plastic by scraping. After low-speed centrifugation to collect these bronchial cells and attached bacteria, the cell pellet was re-suspended in TE containing 1% saponin (which lyses the human cells). After a 10-min incubation at 37°C, the bacterial cells were collected by centrifugation. The bacteria growing in the flask without human cells were treated identically and then harvested by centrifugation. Total RNA was extracted from both sets of bacterial cells by using the Qiagen RNeasy Midi kit. DNA microarray analysis, slide scanning, and statistical analysis of the data were performed. RNA was isolated four times and used in five DNA microarray hybridizations; “dye swap” experiments were performed twice. Two additional independent experiments were performed to isolate two sets of RNA samples for qRT-PCR analysis to validate DNA microarray data.
Project description:RNA isolated from the 0, 10, 25, 50 and 100 micromolar AFB1 cultures at 120 min treatment was used for cDNA microarray experiments. For each array hybridization experiment, RNAs from the treated sample and its corresponding time-matched control were co-hybridized to arrays and respectively quantified in different channels. A dye swap strategy was used to eliminate the dye bias. Keywords: dose response