Project description:The goal was to use microarray to compare changes in the global transcription profiles between an engineered bacterial strain and one of its descendants subject to 600 generations of experimental evolution in batch culture. This comparison allowed us to identify a beneficial mutation that substantially increased expression of a novel cobalt transporter cassette in this descendant. The total RNA of the an engineered strain of Methylobacterium extorquens AM1 and an descendant derived from this engineered strain was extracted. RNA extractions were performed 3 times using 3 independent growth cultures of a given strain. The final RNA samples for the following microarray experiments consisted of an equal proportion of 3 independent extractions for each strain. The cDNA of the descendant was labeled with Alexa_555 while the engineered strain was labeled with Alexa_647 in the array dataset GSM371896.
Project description:Organisms cope with physiological stressors through acclimatizing mechanisms in the short-term, and through adaptive mechanisms over evolutionary timescales. Whereas the former offer a consistent and largely predictable buffer against stressors, myriad paths of adaptation are often possible. Our work examined whether knowledge of acclimatizing responses could be informative ofM-BM- aspects of future adaptation, using as a model system a strain of Methylobacterium extorquens AM1 that was experimentally engineered and then evolved with a novel central metabolism. The engineered strain is markedly slower and less fit than wild-type, which is reflected in microarray analyses by hundreds of genes with differential expression, and also altered levels NAD(P)(H) metabolites. Yet after 600 generations of evolution in the lab, eight replicate populations founded from an engineered ancestor showed substantial but variable improvements in growth using the engineered metabolic pathway. Using information from wild-type, engineered, and adapted physiological states, we determined the extent to which physiological processes were restored, unrestored, reinforced, or novel after experimental evolution. Overall, we found that the vast majority of gene expression perturbations from the engineered strain are restored to wild-type like conditions after experimental evolution but were accompanied by a modest number of unrestored processes, varying instances of novel expression, and a few rare instances where expression changes from acclimation were reinforced through adaptive evolution. One such example was in the reinforced up-regulation of pntAB transhydrogenase, whose increased expression and activity correlated with the restoration of NAD(P)(H) metabolism towards wild-type levels and with increased growth rate in the evolved lineages. Thus, while trajectories of physiological adaptation may still be difficult to predict a priori, our results demonstrate that information from acclimatizing responses can provide a M-bM-^@M-^\directionM-bM-^@M-^] to hypothesize which changes in physiology arose as a consequence of adaptation versus those that may have caused itM-BM- . Single-color microarray hydridization of total RNA isolated from mid-exponentially grown cultures of wildtype, engineered, and eight experimentally evolved populations of Methylobacterium extorquens AM1, each represented by three biological replicates
Project description:Inferring the heritability of gene expression is one of the main areas of the field of genetical genomics. With the possibility to treat the abundances of gene transcripts as a suite of quantitative traits, genetical genomics can make an extensive use of the microarray technology. Here we extended a major method for estimating the heritability of a quantitative trait, single parent-offspring regression, to assess the heritability of the expression of genes with two-channel microarrays. In a series of maternal parent-offspring pairs of Interior spruce (Picea glauca x engelmannii, our focus in the outer stem tissues is the expression of defense-related genes, the heritability of which can affect fitness and necessary for evolution by natural selection. Parent-offspring pairs of Interior spruce planted as a part of Tree Breeding and Improvement Program of the Forest Genetics Section, British Columbia Ministry of Forests and Range, Prince George, BC, Canada (http://www.for.gov.bc.ca/hre/forgen/interior/spruce.htm#1) were used. A total of 30 trees, comprising 15 parent-offpsring pairs were sampled. These include15 offsprings and 15 maternal parents (grafts), which were planted within ~ 1Km range west (122º 42’ 43’’W, 53º 45’41’’N) of the offsprings, hence grown in a relatively similar environment. The bark and the attached phloem were separated from inner layers in the mid-morning hours of June 25, 2008, flash-frozen in liquid nitrogen, and transferred into separate containers. A chain design was decided for gene expression profiling (a total of 45 slides) using the Treenomix third generation cDNA microarray platform (GEO accession #: GPL5423). Array350 kit (Genisphere, Hatfield, USA) was chosen for the microarray hybridizations. The slides were scanned with a ProScanArray scanner (PerkinElmer, Downers Grove, IL, USA), and the scanned TIF images were processed by ImaGene software (BioDiscovery, Inc., El Segundo, CA, USA) to quantify spot signals. Normalization was done between arrays using variance stabilizing (VSN) method for ratio based analysis of the expression data.
Project description:Transcriptional profiling comparing Escherichia coli simultaneously exposed to tellurite and CTX with untreated control cells; Tellurite with control; CTX with control Three-condition experiment, antibacterial (tellurite; CTX or tellurite/CTX) vs. Untreated control cells. Biological replicates: 3 control, 3 toxicants exposed cells, independently grown and harvested. One replicate per array.
Project description:E. coli cultures were exposed to tellurite 0.5 µg/ml during 15 min. Total RNA was extracted and cDNA labeled probes were generated by reverse transcription using Alexa 555 and Alexa 647 fluorophores. These probes were used to hybridize genomic slides containing genomic arrays to determine global transcriptional changes. Two-conditions experiment, antibacterial vs. Untreated control cells. Biological replicates: 2 control, 2 toxicants exposed cells, independently grown and harvested. One replicate per array. Dye swap conditions.
Project description:The angio-suppressive effect of 20(R)-ginsenoside Rg3 (Rg3-R) has been previously demonstrated, and microRNAs (miRNAs) are a vital group of small non-coding RNAs that function as post-transcriptional modulator of gene expression. Thus, using human umbilical vein endothelial cells (HUVEC) as model, we compared the microRNA (miRNA) expression profile of vascular endothelial growth factor (VEGF)-induced cells with the profile of the cell co-treated with VEGF and Rg3-R. Among the screened 553 human miRNAs, 6 up-regulated (miR-520h, miR-487b, miR-197, miR-524*, miR-342 and miR-219) and 3 down-regulated (miR-23a, miR-489 and miR-377) miRNAs were detected in Rg3-R treated vascular endothelial growth factor (VEGF)-induced HUVECs compared to VEGF alone. Real time RT-PCR was subsequently performed to verify the miRNA microarray result. Two condition experiment: VEGF-induced HUVEC and VEGF-induced HUVEC treated with Rg3-R. Three independent microarray experiments, with triplicate per microarray.
Project description:Extending lifespan from yeast to mammals, calorie restriction (CR) is the most conserved longevity intervention. Numerous conserved pathways regulating aging and mediating CR have been identified; however, the overall proteomic changes during these conditions remain largely unexplored. We compared proteomes between young and replicatively aged yeast cells under normal and CR conditions using SILAC quantitative proteomics and discovered distinct signatures in the aging proteome. We found remarkable similarities between aged and CR cells, including induction of stress response pathways, providing evidence that CR pathways are engaged in aged cells. These observations also uncovered aberrant changes in mitochondria membrane proteins as well as a proteolytic cellular state in old cells. These proteomics analyses also help identify potential genes and pathways that have causal effects on longevity.
Project description:The goal of this study was to use microarrays to identify genes differentially regulated under conditions of formaldehyde stress relative to two other stress conditions (oxidative, osmotic) in an effort to identify genes that might be involved in a formaldehyde-specific stress response, rather than a general stress response, in the model methylotroph Methylobacterium extorquens AM1. Two color experiment, three treatments, three biological replicates per treatment, and two technical (dye swap) replicates per biological replicate: formaldehyde-stressed vs. unstressed cells; oxidative-stressed vs. unstressed cells; and osmotic-stressed vs. unstressed cells.
Project description:Brucellosis is still a widespread zoonotic disease. Very little is known about the interaction between B. abortus and trophoblastic cells, which is essential for better understanding the pathogenesis of the Brucella-induced placentitis and abortion, a key event for transmission of the disease. The goal of this study was to evaluate the profile of gene expression by bovine trophoblastic cells during infection with B abortus. Explants of chorioallantoic membranes were inoculated with B. abortus strain 2308. Microarray analysis was performed at 4 h after infection, and expression of cytokines and chemokines by trophoblastic cells was assessed by real time RT-PCR at 6 and 12 h after inoculation. In addition, cytokine and chemokine expression was evaluated in placentomes from experimentally infected cows. Expression of pro-inflammatory genes by trophoblastic cells was suppressed at 4 h after inoculation, whereas a significant up-regulation of CXC chemokines, namely CXCL6 (GCP-2) and CXCL8 (IL-8), was observed at 12, but not at 6 h after inoculation. Placentomes of experimentally infected cows had a similar profile of chemokine expression, with upregulation of CXCL6 and CXCL8. Our data indicate that B. abortus modulates the innate immune response by trophoblastic cells, suppressing expression of pro-inflammatory mediators during the early stages of infection that is followed by a delayed and mild expression of pro-inflammatory chemokines, which is similar to the profile of chemokine expression in the placentomes of experimentally infected cows. This trophoblastic response is likely to contribute to the pathogenesis of B. abortus-induced placentitis. Keywords: trophoblast response to Brucella Total RNA used for microarray analysis was obtained from 6 placentas with 12 infected and 12 control explants from each placenta. At 4 h after inoculation, total RNA was isolated from the trophoblastic surface of the explants. Three RNA pools from two placentas each were generated prior to cDNA synthesis.