Project description:The differentially expressed genes in mutation strain and control strain were analyzed using RNA-Seq DGE method to reveal the mechanism of the oxidation stress tolerance in yeast.
Project description:Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BP and 5'SS of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We used RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings reveal BP-based regulation and demonstrate unanticipated complexity of splicing in yeast.
Project description:The modification of the The modification of the tolerance of xylose-fermenting yeast is an urgent issue for improving ethanol production. In this study, multiple genes involving in superoxide dismutase, glutathione biosynthesis, NADPH regeneration and acetic acid degradation were overexpressed using stress-induced promoters, which is selected from the transcriptome data. Stress-induced promoters were used to realize the feedback control of the tolerant genes, which can ultimately improve the tolerance and ethanol production. We reported the stress-induced promoters for overexpressing tolerant genes and increasing yeast tolerance in a feedback manner
Project description:We performed massive screening of the genes in yeast that were involved in the tolerance to isopropanol using the non-essential genes deleted yeast collection, and identified sixty-five disruptants that grew slower than the wild type strain in the presence of isopropanol. The isopropanol sensitive mutants were tested to know their behaviour under other alcohol stresses. Besides, we conducted microarray analysis to reveal the transcriptional response to isopropanol stress in yeast. Our results certainly provide new insights into yeast response to C3 alcohol isopropanol.
Project description:Yeast enolase (Eno2p) conjugated with EGFP and Flag-tag (Eno2p-EGFP-FLAG) and Eno2p with V22A substitution (Eno2V22Ap) conjugated with EGFP and Flag-tag (Eno2V22Ap-EGFP-FLAG) were produced in baker's yeast S. cerevisiae. After semi-anaerobic culture at 30 ˚C for 12h, cells producing Eno2p-EGFP-FLAG formed fluorescent foci, while cells producing Eno2V22Ap-EGFP-FLAG did not. The cells were collected and lysed, and proteins Eno2p-EGFP-FLAG or Eno2V22Ap-EGFP-FLAG and the associated proteins were coimmunoprecipitated using ANTI-FLAG M2 affinity gel and analyzed. Data contain two biological replicates and two technical replicates (n = 4). As the results, 96 proteins were detected with both recombinant Eno2p-EGFP-FLAG and Eno2V22Ap-EGFP-FLAG protein, 29 proteins were detected only with recombinant Eno2p-EGFP-FLAG protein, and 16 proteins were detected only with recombinant Eno2V22Ap-EGFP-FLAG protein. Data Processing/Data Analysis: The separated analytes were detected on an LTQ Velos linear ion trap mass spectrometer (Thermo Scientific). For data-dependent acquisition, the method was set to automatically analyze the five most intense ions observed in the MS scan. The mass spectrometry data were used for protein identification by the Mascot search engine on Protein Discoverer software (ver. 1.2, Thermo Scientific) against the information in the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org). Search parameters for peptide identification included a precursor mass tolerance of 1.2 Da, a fragment mass tolerance of 0.8 Da, a minimum of one tryptic terminus, and a maximum of one internal trypsin cleavage site. Cysteine carbamidomethylation (+57.021 Da) and methionine oxidation (+15.995 Da) were set as a differential amino acid modification. The data were then filtered at a q value ≤ 0.01 corresponding to 1% FDR at the spectral level.
