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: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: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:NIH 3T3 cells were challenged with tunicamycin for 2, 5 or 10h. MICRO-RNAs that are upregulated or down regulated were identified by micro-ARRAY. NIH 3T3 cell were challenged with 2ug/ml tunicamycin for 2, 5 or 10 hrs. Total RNA was purified using miRNeasy Mini Kit (QIAGEN). Microarray was carried out using GeneChip® miRNA Arrays (Affymetrix, CA) and results were analyzed using GENESPRING software. ANOVA and T-tests were used to calculate fold change and p-values. Spotfire (Somerville, MA) software was used to generate heat maps.
Project description:Investigation of whole genome gene expression level changes in response to tunicamycin, an inhibitor of protein N-glycosylation, in Candida glabrata CBS138 delta-cnb1, delta-crz1, delta-slt2, and delta-ire1 mutants, compared to the wild-type strain. The mutations engineered into these strains render them hypersusceptible to tunicamycin. The mutants analyzed in this study are further described in Miyazaki, T. et al. (2010) Antimicrob Agents Chemother. 54(4):1639-1643 (PMID: 20100876) and Miyazaki, T. et al. (2010) FEMS Yeast Res 10(3):343-352, 2010 (PMID: 20214686). A fifteen chip study using total RNA isolated from Candida glabrata wild-type control and four mutant strains, in which the entire ORFs of CNB1 (XP_448800), CRZ1 (XP_449644), SLT2 (XP_447735), or IRE1 (XP_446111) are deleted. Each chip measures the expression level of 5,217 genes from Candida glabrata CBS138 with six 60-mer-probe pairs per gene, with two-fold technical redundancy.