Using natural variation Drosophila to discover previously unknown ER stress genes
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ABSTRACT: To measure natural variation in ER stress transcriptional response in a subset of lines from the Drosophila Genetic Reference Panel Transcriptional response to Tunicamycin and control conditions was measured at 8 hours of exposure (20 lines) and 20 hours of exposure (8 lines).
Project description:To measure natural variation in ER stress transcriptional response in a subset of lines from the Drosophila Genetic Reference Panel
Project description:Natural genetic variation is a rich resource for identifying novel elements of cellular pathways such as endoplasmic reticulum (ER) stress. ER stress occurs when misfolded proteins accumulate in the ER and cells respond with the conserved unfolded protein response (UPR), which includes large-scale gene expression changes. Although ER stress can be a cause or a modifying factor of human disease, little is known of the amount of variation in the response to ER stress and the genes contributing to such variation. To study natural variation in ER stress response in a model system, we measured the survival time in response to tunicamycin-induced ER stress in flies from 114 lines from the sequenced Drosophila Genetic Reference Panel of wild-derived inbred strains. These lines showed high heterogeneity in survival time under ER stress conditions. To identify the genes that may be driving this phenotypic variation, we profiled ER stress-induced gene expression and performed an association study. Microarray analysis identified variation in transcript levels of numerous known and previously unknown ER stress-responsive genes. Survival time was significantly associated with polymorphisms in candidate genes with known (i.e., Xbp1) and unknown roles in ER stress. Functional testing found that 17 of 25 tested candidate genes from the association study have putative roles in ER stress. In both approaches, one-third of ER stress genes had human orthologs that contribute to human disease. This study establishes Drosophila as a useful model for studying variation in ER stress and identifying ER stress genes that may contribute to human disease.
Project description:Methicillin resistance in Staphylococcus aureus depends on the production of mecA, which encodes penicillin-binding protein 2A (PBP2A), an acquired peptidoglycan transpeptidase with reduced susceptibility to beta-lactam antibiotics. Here, we show that preventing the expression of wall teichoic acids (WTAs) genetically or with a TarO inhibitor sensitizes MRSA strains to beta-lactams although PBP2A is still expressed. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of S. aureus COL. in order to further understand the sensitization of strain COL to methicillin by tunicamycin we determined the tunicamycin and methicillin transcriptomes alone and in combination. Methicillin treatment of COL at 500 µg/mL had almost no effect on cell growth rate and, remarkably, the only gene in the transcriptome that showed a more than two-fold change in expression was lytM, which was downregulated. The tunicamycin transcriptome of COL, acquired at 0.4 µg/mL, shows modest changes compared to the untreated control both in terms of the total numbers of affected genes and in the degree of up- or downregulation. Several of the genes upregulated upon tunicamycin treatment are part of the cell wall stress stimulon.COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. transcriptome for COL growing in the presence of both agents showed extensive changes in gene expression. , the cell wall stress stimulon, which was not induced by methicillin when tunicamycin was absent, was clearly induced in its presence and the changes were far more dramatic than observed with tunicamycin alone. ). vraS and vraR, which encode a two component signaling system dedicated to the cell wall regulon, were upregulated 3.8 and 3.7 fold, respectively. Other upregulated cell wall stress stimulon genes include pbp2, fmtA, mvaD (mevalonate diphosphate decarboxylase), crtN (dehydrosqualene desaturase) mvak1 (mevalonate kinase), recU, SAV1424 (methionine sulfoxide reductase A), prsA (peptidyl-prolyl cis/trans isomerase), tcaA (Tca protein) and cwrA. A considerable number of genes were also downregulated upon challenge of COL with the combination of tunicamycin and methicillin. These included sspB, lrgA, dltA, capL, SAS0988, sspA, pflB, and spa. Several of these genes have been found to be downregulated in previous studies of cell wall-active antibiotic challenge of S. aureus. Growth conditions for microarray analysis- For transcriptional profiling, an overnight-grown COL culture was diluted (2% vol/vol) to 20 ml TSB medium in a 50-ml Erlenmeyer flask and grown at 30°C with shaking at 200 rpm. For the sensitization experiment, COL was grown with methicillin to an OD600 ~0.4, and challenged with tunicamycin for 2 hrs whereas the control culture contained methicillin alone. Total bacterial RNA was isolated as previously described from S. aureus COL (30°C, 200 rpm). The RNA samples were then converted to fluorescently-labeled cDNA and hybridized to S. aureus microarrays version 7 (NIAID's Pathogen Functional Genomics Resource Center). Hybridization signals were scanned using an Axon4000B scanner (Molecular Devices, Sunnyvale, CA ) with Acuity 4.0 software and scans were saved as TIFF image. Scans were analyzed using TIGR-Spotfinder (www.tigr.org/software/) software and the local background was subsequently subtracted. The data set was normalized by applying the LOWESS algorithm using TIGR-MIDAS (www.tigr.org/software/) software. The normalized log2 ratio of test/reference signal for each spot was recorded. Genes with less than three data points were considered unreliable, and their data points were discarded. The averaged log2 ratio for each remaining gene on the four replicate slides was ultimately calculated. Significant changes of gene expression were identified with SAM (significance analysis of microarrays; www.tat.stanford.edu/~tibs/SAM/index.html. Genes analyzed using these programs were further sorted and grouped based on their function using our in-house software Staphylococcus aureus Gene Sorter (SAGS). Several controls were employed to minimize the technical and biological variations and to ensure that the data obtained were of good quality. First, each ORF was present in triplicate on the array. Second, each RNA preparation was used to make probes for at least two separate arrays for which the incorporated dye was reversed. Contents of raw data files: Channel A = Cy3 dye Channel B = Cy5 dye File names ending with S1,S3 = Cy3 untreated, Cy5 treated File names ending with S2,S4 = Cy5 untreated, Cy3 treated
Project description:Virus infection and over expression of protein in cytosol induce a subset of HSP70s. We named this response the Cytosolic Protein Response (CPR) and have been investigating it in the context of a parallel mechanism in the soluble cytosol with the UPR, and as a subcomponent of the larger HS response. This experiment was carried out to study the transcriptional aspect of CPR. In this analysis, we have triggered CPR by infiltrating proline analogue, L-azetidine-2-carboxylic acid (AZC) into Arabidopsis mature leaves. Since AZC trigger unfolded protein response(UPR) in ER as well as CPR, we have included tunicamycin treatment, which is a specific inducer of UPR to subtract the effect of UPR from the AZC response. Heat shocked samples were included to identify CPR as a subcomponent of larger HS response. We used microarray data to identify the genes upregurated by CPR. These genes were commonly upregulated by AZC and HS but not by tunicamycin treatment. Experiment Overall Design: Arabidopsis mature leaves were infiltrated with AZC or L-Proline (control of AZC) and tunicamycin or DMF(solvent control of tunicamycin). For the heat shock treatment, six mature leaves were detached from plant and incubated at 37 °C or 20°C (as a control) for a period of 1h. The experiment was repeated three times for AZC and HS treatment (3 biological replication). Tunicamycin experiment was repeated five times due to large valiation in the responses (5 biological replication).
Project description:Transcriptome responsiveness was further tested by attempts to invoke the unfolded protein repsonse (UPR), a classic ER-based pathway stimulated by the presence of increased levels of unfolded polypeptides. The UPR is mediated via transcriptional responses in both yeast and metazoan cells, and can be reliably activated by addition of dithiothreitol (DTT). Using DTT at concentrations that invoke a UPR in mammalian cells, Arabidopsis, yeast and other systems, we found that, in T. brucei, DTT exposure led to rapid cell death. We analysed the transcriptome at 1 mM DTT, under conditions where cells remained viable, as assessed by motility. <br><br>part 1: 3 biological replicates of SMB cells grown under normal conditions, and 3 replicates of SMB cells treated with 1mM DTT for 1hr, as well as dye swaps were used. <br><br>part 2: 3 biological replicates of SMB cells <br>grown under normal conditions, and 3 replicates of SMB cells treated <br>with 1mM DTT for 4hr, as well as dye swaps were used.<br><br>The UPR can also be activated by addition of tunicamycin. Using tunicamycin at concentrations that invoke a UPR in mammalian cells, Arabidopsis, yeast and other systems, we found that, in T. brucei, tunicamycin exposure efficiently inhibits trypanosome N-glycosylation and that it resulted in growth arrest over a period of up to 24 hours. We analysed the transcriptome at 5 ?g/ml tunicamycin under conditions where cells remained viable, as assessed by motility.<br><br>part 3: 3 biological replicates of SMB cells grown under normal conditions, and 3 replicates of SMB cells treated with 5 ?g/ml tunicamycin for 4hr, as well as dye swaps were used.<br><br>part 4: 3 biological replicates of SMB cells grown under normal conditions, and 3 replicates of SMB cells treated with 5 ?g/ml tunicamycin for 24hr, as well as dye swaps were used.
Project description:Polysomes of untreated (NT) or tunicamycin-treated (TM) human cardiomyocyte AC16 cells were immunoprecipitated (IP MRPS15) with anti-MRPS15 antibody, followed by RNA sequencing. As a control, RNAseq was performed on polysome-associated RNAs of untreated or tunicamycin-treated human cardiomyocyte AC16 cells before immunoprecipitation (input).
Project description:AtbZIP60 is one of the transcription factors involved in the endoplasmic reticulum (ER) stress response in Arabidopsis. To identify genes under the control of AtbZIP60 during ER stress, we compared the genome-wide expression profiles of wild-type and atbzip60 mutant plants in response to the ER stress inducer tunicamycin.
Project description:We used microarrays to examine gene expression levels from 131 unrelated CEPH-Utah grandparents with either DMSO or tunicamycin. We measured gene expression levels in immortalized B cells from 131 unrelated CEPH-Utah grandparents. Each individual was treated for 8 hours with either DMSO or with 4 ug/ml of tunicamycin. Gene expression was measured.
Project description:Diatoms are a major phytoplankton group that play important roles in maintaining oxygen levels in the atmosphere and sustaining the primary nutritional production of the aquatic environment. Among diatoms, the genus Chaetoceros is one of the most abundant and widespread. Temperature, climate, salinity, nutrients, and predators were regarded as important factors controlling the abundance and population dynamics of diatoms. Here we show that a viral infection can occur in the genus Chaetoceros and should therefore be considered as a potential mortality source. Chaetoceros salsugineum nuclear inclusion virus (CsNIV) is a 38-nm icosahedral virus that replicates within the nucleus of C. salsugineum. The latent period was estimated to be between 12 and 24 h, with a burst size of 325 infectious units per host cell. CsNIV has a genome structure unlike that of other viruses that have been described. It consists of a single molecule of covalently closed circular single-stranded DNA (ssDNA; 6,005 nucleotides), as well as a segment of linear ssDNA (997 nucleotides). The linear segment is complementary to a portion of the closed circle creating a partially double-stranded genome. Sequence analysis reveals a low but significant similarity to the replicase of circoviruses that have a covalently closed circular ssDNA genome. This new host-virus system will be useful for investigating the ecological relationships between bloom-forming diatoms and other viruses in the marine system. Our study supports the view that, given the diversity and abundance of plankton, the ocean is a treasury of undiscovered viruses.