Project description:The synthesis of starch-like glucans can be reconstituted in S. cerevisiae using the genes from Arabidopsis thaliana: heterologous expression of SS1 to SS4, BE2, BE3, ISA1, and ISA2, together with a bacterial ADPglucose pyrophosphorylase gene for substrate provision in yeast strain 29, yielded insoluble granules with a semi-crystalline structure similar to that of plant amylopectin (Pfister et al., 2016). Here we conducted a label-free proteomics experiment to compare yeast strains with and without glucan production. This experiment was performed on strains 29, 48A, 362.1, 363.1 and WT (n = 4 replicates each, arising from independent replicate cultures). Strains including genotypes are described in the associated publication. Reference: Pfister, B., Sánchez-Ferrer, A., Diaz, A., Lu, K., Otto, C., Holler, M., Shaik, F.R., Meier, F., Mezzenga, R., and Zeeman, S.C. (2016). Recreating the synthesis of starch granules in yeast. Elife 5: 1–29.
Project description:The mitochondrial oxidative phosphorylation system (OXPHOS) contains subunits of dual genetic origin, namely the nuclear and the mitochondrial genome. Mounting the different subunits into functional OXPHOS complexes is aided by dedicated chaperones termed assembly factors. How unfunctional or superfluous proteins are recognized during assembly and directed to degradation is currently not understood. Here, we reveal that protein quality control is an integral part of assembly, which is organized in dedicated hubs gathering mitochondrial translation, protein import, assembly, and protein turnover. Fate-decision of newly synthesized proteins is achieved by molecular triaging engaging the evolutionary conserved prohibitin/m-AAA protease supercomplex and assembly chaperones. The localization of these competing activities in vicinity to the mitoribosomal tunnel exit allows for a rapid decision whether newly synthesized proteins are fed into OXPHOS assembly or to degradation. In total, three projects are hosted in this repository. 1) Phb1-Alfa DSG cross-linked immunoprecipitation 2) Metabolic switch from Glycerol to Glucose followed by Phb1-ALFA DSG cross-linked immunoprecipitation. 3) Phb2-ALFA DSG cross-linked immunoprecipitation.
Project description:Cohesin acetylation by Eco1 during DNA replication establishes sister chromatid cohesion. We show that acetylation makes cohesin resistant to Wapl activity from S-phase until mitosis. Wapl turns out to be a key regulator of cohesin dynamics on chromosomes by controling cohesin maintenance following its establishment in S-phase and its role in chromosome condensation. The Affymetrix Saccharomyces cerevisiae Chip Tiling 1.0R Arrays were used to analyze the binding pattern of Scc1 along the genome of Saccharomyces cerevisiae in late G1 and metaphase arrested cells.
Project description:Long read SMRT cDNA sequencing of nascent RNA from exponentially growing S. cerevisiae and S. pombe cells was employed to obtain transcription elongation and splicing information from single transcripts. Nascent RNA was prepared from the yeast chromatin fraction (Carrillo Oesterreich, Preibisch, Neugebauer, Mol Cell 2010). The nascent 3â?? end was labeled with a 3â?? DNA adaptor through ligation. The adaptor sequence served as template for full-length reverse transcription and double-stranded cDNA was obtained in a PCR (gene-specific or transcriptome-wide). SMRT DNA sequencing libraries were prepared subsequently. Nascent RNA profiles for mainly intron-containing genes were generated with long-read SMRT cDNA sequencing.
Project description:This experiment was performed in order to assess the specificity of Rad9 binding to S. cerevisiae genome. In another ChIP-chip experiment in SC BCS BPS growth conditions we have found Rad9 present to a significant number of genomic loci with a bias to transcriptionally active regions. In order to see if that pattern was random and depended or not on the activity state of the genes, we conducted a Rad9 ChIP on chip experiment with the strain grown in medium with galactose instead of glucose, where it is known that particular gene groups are transcriptionally activated. We then compared to the results of our previous experiment where the strain was grown in glucose.
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:26972c yeast cells were transformed with either empty vector (pYES3) or pYES3:Gm:bHLHm1. Cells were grown on low ammonium concentrations to observe transcriptional changes in the yeast genome in response to the soybean bHLHm1 transcription factor. The ammonium transport mutant (26972c) was transformed with either a empty vector control (pYES3) or pYES3 containing GmbHLHm1.Independent replicates (3) for each treatment were grown for 12 hours in the presence of 1 mM ammonium and 2 % w/vol galactose to activate the Gal promoter located on pYES3. Cells were harvested and total RNA extracted. Quantified RNA was analaysed using microarray analysis using the [Yeast_2] Affymetrix Yeast Genome 2.0 Array.