Nucleosome organization plays a key role in the regulation of gene expression. However, despite the striking advances in the accuracy of nucleosome maps, there are still severe discrepancies on individual nucleosome positioning and how this influences gene regulation. The variability among nucleosome maps, which precludes the fine analysis of nucleosome positioning, might emerge from diverse sources. We have carefully inspected the extrinsic factors that may induce diversity by the comparison of ...[more]
Project description:Yeast strain BY4741 was grown overnight at 30C in YPD rich media. The yeast culture was diluted to an OD600 of 0.1 using fresh YPD media and further grown until an OD600 of 0.2. Then, alpha -factor mating pheromone (GenScript) was added to a final concentration of 10 uM to allow cell synchronization at G1 phase. After 2.5 h, the alpha-factor was removed by harvesting the cells for 10 min at 6000 rpm and decanting supernatant. The arrested cells were inoculated in fresh YPD rich medium to be released from G1-arrest. Samples were collected at 0, 30, 40, 50, and 70 mins, 7.5 ml samples were collected for RNA extraction while 40 ml samples were taken for nucleosomal DNA preparation and for flow cytometry (FACS). Samples for RNA isolation were collected by pipetting the culture directly into 15-ml Falcon tubes containing 7.5 ml of icy-water to quickly chill the cells. Cells were harvested by spinning for 3-4 min at 6000 rpm, frozen in liquid N2 and stored at - 80C. Total cellular RNA was extracted using the RNeasy kit (Qiagen), following the manufacturers instructions with the spheroplasting protocol. Purified RNA samples were quantified by Qubit fluorometer (Invitrogen, Inc.) and Nanodrop spectrophotometer (Thermo Scientific, Inc.). The total RNA was hybridized to Affymetrix GeneChip Yeast Genome 2.0 arrays for gene expression analysis.
Project description:Nucleosome organization and dynamics play a central role in controlling the DNA accessibility to regulatory factors of many critical cellular functions, especially gene regulation. However, despite extensive studies, the main factors determining nucleosome positioning and its fluctuation during cell cycle still remain elusive. Here, we present a large-scale study of nucleosome plasticity throughout the cell cycle and its interplay with gene expression based on genome-wide nucleosome positioning and mRNA abundance. We have clusterized distinct nucleosome architectures around transcription start sites and replication origins and studied their dynamics during the cell cycle progression. The most significant cell cycle-dependent changes occur at G1-S and G2-M transitions due to a large changes in gene expression in cell cycle regulatory genes. Taken together, our accurate study provides a dynamic picture of chromatin organization along cell cycle and its interplay with gene expression.
Project description:Seven temperature sensitive Saccharomyces cerevisiae BY4741 mutants (rsc3-1, abf1-101, reb1-212, rap1-1, mcm1, tbf1 and cep3-1) were grown at restrictive temperatures until a difference in OD600 was observed relative to a wild-type control. Nucleosomal DNA, whole genomic DNA and total RNA were isolated and hybridized onto yeast whole-genome tiling arrays.<br>
Project description:The Low MP laboratory strain BY4741 was subjected to a stepwise selection procedure during 120-150 generations, enabling enrichment for beneficial mutations that allow growth at increasing pH. In order to identify the mutations responsible for the ability to grow at high pH, we used tiling arrays to genotype the Low MP ancestor and three evolved strains: two clones from two independent lines that had acquired the ability to grow at pH 8.5 and pH 8.6 (A8.5 and C8.6) and one clone from an intermediate stage of line C (C8.0). The identity and nature of the mutated alleles was further confirmed by direct DNA sequencing. <br>
Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip. Time Course ChIP-ChIP experiment, Rap1-Flag IP and Rap1-Myc IP. 10 Time Points (0,10,20,30,40,50,60,90,120,150 Minutes) 2 Biological Replicates. Total Rap1 Occupancy at times 0 and 60 minutes in the time course; 2 Biological Replicates. mRNA expression levels at times 0 and 60 minutes in the time course; 2 biological Replicates.
Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip. Time Course ChIP-ChIP experiment, Rap1-Flag IP and Rap1-Myc IP. 13 Time Points (0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240 Minutes) 2 Biological Replicates. Total Rap1 Occupancy at times 0 and 60 minutes in the time course; 2 Biological Replicates. mRNA expression levels at times 0 and 60 minutes in the time course; 2 biological Replicates. ChIPs comparing the occupancy of Rap1 in a strain containing two copies of Rap1, one with a Flag tag and one with a Myc tag, and expressed from an identical promoter.
Project description:Transcripitonal profiling of Saccharomyces cerevisiae treated with 2% n-alkanes vs untreatment Multiple condition experiment, S. cerevisiae treated with n-alkanes (n-nonane, n-decane, n-undecane, n-dodecane, respectively) for 24hrs or 48hrs, compared to control (those without treatment)
Project description:Influence of a C-terminal deletion of the RNA polymerase II elongation factor Spt6 on the transcription of the yeast genome. In parallel the influence of a Spt6 overexpression on gene expression is analyzed.
Project description:Total RNA from three replicate cultures of wild-type and mutant strains was isolated and the expression profiles were determined using Affymetrix arrays. Comparisons between the sample groups allow the identification of genes regulated by histone H2B K111A mutant. Comparison of genes whose mRNA levels are affected in a histone H2B K111A mutant yeast strain compared to wild-type yeast strain.