Project description:To identify substrates of the ubiquitinating E3 enzyme Rsp5 we applied purified Rsp5 to duplicate protein arrays. The Rsp proteins were expressed as fusion proteins to GST. We used as a control Ubr1, a RING domain containing E3 ligase We analyzed Rsp5 from S.cerevisiae on duplicate arrays, with four control chips, two without Rsp5 and two with Ubr1.
Project description:The Myc-Max heterodimer is a DNA binding protein that regulates expression of a large number of genes. Genome occupancy of Myc-Max is thought to be driven by E-boxes (CACGTG or variants) to which the heterodimer binds in vitro. By analyzing ChIP-Seq datasets, we demonstrated that the positions occupied by Myc-Max across the human genome correlate with the RNA polymerase II (Pol II) transcription machinery better than with E-boxes. Metagene analyses showed that in promoter regions, Myc was uniformly positioned about 100 bp upstream of essentially all promoter proximal paused polymerases with Max about 10 bp upstream of Myc. We re-evaluated the DNA binding properties of full length Myc-Max proteins using electrophoretic mobility shift assays (EMSA) and protein-binding microarrays (PBM). EMSA results demonstrated Myc-Max heterodimers have high affinity for both E-box containing and non-specific DNA. Quantification of the relative affinities of Myc-Max for all possible 8- mers using PBM assays showed that sequences surrounding core 6-mers significantly affect binding. Comparing to the in vitro sequence preferences, Myc-Max genomic occupancy measured by ChIP-Seq was largely, although not completely, independent of sequence specificity. Our results suggest that the transcription machinery and associated promoter accessibility play an important role in genomic occupancy of Myc. Two protein binding microarray (PBM) experiments were performed: one for the heterodimer of the human transcription factors c-Myc and Max, and one for the Max-Max homodimer. Briefly, 4x44K arrays (Agilent Technologies; AmadID 015681) containing the M-bM-^@M-^Xall 10-merM-bM-^@M-^Y universal PBM design were used. Arrays were incubated with a PBS buffer based protein mixture of wither 10nM His-tagged Myc-Max heterodimer or 10nM His-tagged Max-Max homodimer, 2% milk, 200ng/M-BM-5L BSA, 50ng/M-BM-5L Salmon Testes DNA, and 0.02% TX-100. Bound protein was tagged with 10ng/M-BM-5L anti-His antibody conjugated to Alexa 488 (Qiagen; 35310) in PBS with 2% milk. Data were analyzed to obtain fluorescence intensities for all 8mers. The PBM protocol is described in Berger et al., Nature Biotechnology 2006 (PMID 16998473).
Project description:This SuperSeries is composed of the following subset Series: GSE24037: Salivary cytokine alterations in HIV infection part 1 GSE24064: Salivary cytokine alterations in HIV infection part 2 Refer to individual Series
Project description:To study their metabolic potential in natural ecosystems, we developed a species-independent LAB microarray, containing 2,269 30-mer oligonucleotides, and targeting 406 genes that play a key role in the production of sugar catabolites, bacteriocins, exopolysaccharides, and aromas, in probiotic and biosafety characteristics, and in stress response. Also, genes linked to negative traits such as antibiotic resistance and virulence are represented. This experiment is a validation experiment, where we hybridized labelled RNA from 20 LAB strains, covering 86% of all oligos. Keywords: Platform validation experiment Loop design, hence all RNA samples were hybridized once in each colour (Cy3 and Cy5).
Project description:Malaria parasites dramatically alter the rheological properties of infected red blood cells. In the case of Plasmodium vivax, the parasite rapidly decreases the shear elastic modulus of the invaded RBC, enabling it to avoid splenic clearance. This study highlights correlation between rosette formation and altered membrane deformability of P. vivax-infected erythrocytes, where the rosette-forming infected erythrocytes are significantly more rigid than their non-rosetting counterparts. The adhesion of normocytes to the PvIRBC is strong (mean binding force of 440pN) resulting in stable rosette formation even under high physiological shear flow stress. Rosetting may contribute to the sequestration of PvIRBC schizonts in the host microvasculature or spleen.
Project description:Sourdough is a very competitive and challenging environment for microorganisms. Usually, a stable microbiota composed of lactic acid bacteria (LAB) and yeasts comes to dominate this ecosystem. Although rich in carbohydrates, thus providing an ideal environment to grow, the low pH presents a particular challenge. The nature of the adaptation to this low pH was investigated for Lactobacillus plantarum IMDO 130201, an isolate from a laboratory wheat sourdough fermentation. Batch fermentations were carried out in wheat sourdough simulation medium, and total RNA was isolated from mid-exponential growth phase cultures, followed by differential gene expression analysis using a LAB functional gene microarray. At low pH values, an increased expression of genes involved in peptide and amino acid metabolism was observed as well as of genes involved in plantaricin production and lipoteichoic acid synthesis. The results highlight cellular mechanisms that allow L. plantarum to function at a low environmental pH. The labeled aRNA samples were hybridized using a loop design, i.e. two consecutive samples (e.g., pH 3.5 and pH 4.0, pH 4.0 and pH 4.5, etc.) were hybridized on the same microarray slide, each labeled with another fluorescent dye (Cy3 or Cy5), and the loop was closed by hybridizing sample pH 5.5 together with sample pH 3.5.
Project description:Until now, it has been reasonably assumed that specific base-pair recognition is the only mechanism controlling the specificity of transcription factor (TF)M-bM-^HM-^RDNA binding. Contrary to this assumption, here we show that nonspecific DNA sequences possessing certain repeat symmetries, when present outside of specific TF binding sites (TFBSs), statistically control TFM-bM-^HM-^RDNA binding preferences. We used high-throughput proteinM-bM-^HM-^RDNA binding assays to measure the binding levels and free energies of binding for several human TFs to tens of thousands of short DNA sequences with varying re- peat symmetries. Based on statistical mechanics modeling, we iden- tify a new proteinM-bM-^HM-^RDNA binding mechanism induced by DNA se- quence symmetry in the absence of specific base-pair recognition, and experimentally demonstrate that this mechanism indeed gov- erns proteinM-bM-^HM-^RDNA binding preferences. Four custom protein binding microarray (PBM) experiments of human transcription factors were performed. Briefly, the PBMs involved binding his-tagged transcription factors c-Myc, Max, and Mad1(Mxd1) to double-stranded 180K Agilent microarrays in order to determine their binding specificity for GTCACGTGAC DNA binding sites flanked by repetitive DNA elements with different symmetries and correlation length scales. Briefly, we represent three categories of 36-bp sequences: 1) 28800 probes centered at a GTCACGTGAC site and flanked by repetitive elements (probe names starting with Ariel_); 2) Unbound probes (or negative controls); and 3) Bound probes, which correspond to randomly selected genomic regions bound in vivo by c-Myc, Max, or Mad2 (ChIP-seq P < 10^(-10) in HeLaS3 or K562 celld (ENCODE)), which contain at least two consecutive 8-mers with universal PBM E-score > 0.4 (Munteanu and Gordan, LNCS 2013). Each DNA sequence represented on the array is present in 6 replicate spots. We report the gcPBM signal intensity for each spot (raw files) as well as the median intensity over the 6 replicate spots (normalized data). The PBM protocol is described in Berger et al., Nature Biotechnology 2006 (PMID 16998473).
Project description:rs10-05_tcv - gene profiling of turnip crinkle virus (tcv) sirna - 1. What are the genes (including miRNA precursors) that are differentially regulated in a set of viral siRNA in A.thaliana? 2. There are also differentially regulated during an evolution and a fitness process? - This is a plant evolution project on TCV in which, the biological questions are: 1. What are the genes (including miRNA precursors) that are differentially regulated in Col0 and dcl234 mutant in wt conditions? 2. What are the genes (including miRNA precursors) that are differentially regulated in a set of viral siRNA in Col0 and dcl234 mutant? 3. There are also differentially regulated between the plant generations 1 (G1) and 11 (G11)? 4. What are the genes (including miRNA precursors) that are differentially regulated in a set of viral siRNA after fitness experiment? 24 dye-swap - gene knock out,treated vs untreated comparison
Project description: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:Measure the relative changes of gene expression upon GIS2 overexpression; 100 ml of BY4741 cells bearing plasmid pBG1805-Gis2 (GIS2 under the control of the galactose promotor) or the empty plasmid pBG1805 (=control) were grown in synthetic medium lacking uracil (SC-Ura supplemented with 2% raffinose) at 30 degrees to an OD600 of 0.45- 0.5. Expression of GIS2 was induced with 2% galactose for 1.5 h and cells were harvested by centrifugation, washed twice with 800 ul of ice-cold sterile water. 100 ul (1/8) of cells were removed and RNA was isolated by hot phenol extraction for microarray analysis. 6.7 ug of total RNA derived from cells expressing the empty vector (pBG1805) or Gis2p (pBG1805-Gis) were reverse transcribed in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs with a mixture of N9 and dT20V primers, and cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences Cat# RPN5661), respectively, and competitively hybridized on yeast oligo arrays at 42 degrees celsius for 14 hours in formamide-based hybridization buffer. Microarrays were scanned with an Axonscanner 4200 (Molecular Devices) and analyzed with GenePix Pro 5.1 (Molecular devices). Biological Replicate