Project description:The S. cerevisiae Pop2 protein is an exonuclease in the Ccr4-Not complex that is a conserved regulator of gene expression. Pop2 regulates gene expression post-transcriptionally by shortening the poly(A) tail of mRNA. A previous study has shown that Pop2 is phosphorylated at threonine 97 (T97) by Yak1 protein kinase in response to glucose limitation. However, the physiological importance of Pop2 phosphorylation remains unknown. In this study, we found that Pop2 is phosphorylated at serine 39 (S39) under unstressed conditions. The dephosphorylation of S39 was occurred within 1 min after glucose depletion, and the addition of glucose to the glucose-deprived culture recovered this phosphorylation, suggesting that Pop2 phosphorylation at S39 is regulated by glucose. We previously reported that Pop2 takes a part in the cell wall integrity pathway by regulation of LRG1 mRNA; however, S39 phosphorylation of Pop2 is not involved in LRG1 expression. On the other hand, Pop2 phosphorylation at S39 is involved in the expression of HSP12 and HSP26, encoding small heat shock proteins. In medium supplemented with glucose, Pop2 might be phosphorylated at S39 by Pho85 kinase to repress the expression of HSP12 and HSP26. Glucose starvation inactivated Pho85, which resulted in the derepression of HSP12 and HSP26. Thus, Pop2 phosphorylation at S39 is important for Pop2 to repress the expression of stress response genes, HSP12 and HSP26, in the presence of glucose. Our results suggest that Pop2 phosphorylation by Pho85 kinase is a part of the glucose sensing system in yeast.
Project description:A880 has pop2 deletion and a very slow-growth phenotype with glycerol as carbon source. A880 transformed with 2-micron plasmids encoding STM1 can grow robustly on glycerol plates. The Arg237 on Stm1p can be methylated by Hmt1p. A880 transformed with 2-micron plasmids encoding the Stm1p R237K mutant retain the slow-growth phenotype on glycerol plates. We used microarrays to assess the transcription profiles of A880, and A880 transformed with STM1 or STM1 R237K mutant. The goal is to identify genes that are up- or down-regulated in the presence of STM1 R237K mutant but not in the presence of the wild type STM1. BY4741 (wild type control), A880 (BY4741 with pop2 deletion), A880STM1 (A880 transformed with STM1) and A880STM1K (A880 transformed with STM1 carrying an Arg237 to lysine substitution) cells were grown in SC medium with glucose as carbon source to early log phase. The cells were shifted to SC medium containing 2% glycerol and incubated for 12 hours before harvesting. Total RNAs were extracted for microarray analysis. Two data sets were generated from separate experiments and cell cultures.
Project description:A880 has pop2 deletion and a very slow-growth phenotype with glycerol as carbon source. A880 transformed with 2-micron plasmids encoding STM1 can grow robustly on glycerol plates. The Arg237 on Stm1p can be methylated by Hmt1p. A880 transformed with 2-micron plasmids encoding the Stm1p R237K mutant retain the slow-growth phenotype on glycerol plates. We used microarrays to assess the transcription profiles of A880, and A880 transformed with STM1 or STM1 R237K mutant. The goal is to identify genes that are up- or down-regulated in the presence of STM1 R237K mutant but not in the presence of the wild type STM1.
Project description:Contains a collection of wildtype Saccharomyces cerevisiae strains for estimating the biological variation. Wildtypes are obtained from the yeast wildtypes - wt pool background set HybSet, by randomly taking 100 wt vs. refpool (pooled wts) and 100 refpool vs. wt hybridizations
Project description:To investigate the role of transcriptional factors Gcn4, Leu3, Gat1 and Met31 in the response to 3AT-induced amino acid starvation, we performed time-course microarray studies of wild-type, single deletion and double deletion strains. The analyses provide insight into a complex regulatory response involving at least four transcription factors. Yeast Saccharomyces Cerevisiae BY4741 wild-type and deletion strains were collected at various time points after 3AT induced amino acid starvation.
Project description:Contains a collection of wildtype Saccharomyces cerevisiae strains for estimating the biological variation. Wildtypes are obtained from the yeast wildtypes – platereader, wt pool background set HybSet, by randomly taking 100 wt vs. refpool (pooled wts) and 100 refpool vs. wt hybridizations
Project description:Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. This results in the first network of chromatin interaction pathways. The network is function-based, has a branched, interconnected topology and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are rate-limiting for which genes and predicts new interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin. Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used in one of the channels for each hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Using the Erlenmeyer growth protocol up to five deletion strains were grown on a single day. In the tecan platereader, up to eleven deletion strains could be grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.
Project description:Contains a collection of wildtype Saccharomyces cerevisiae strains for estimating the biological variation. Wildtypes are obtained from the yeast wildtypes - wt pool background set HybSet, by randomly taking 100 wt vs. refpool (pooled wts) and 100 refpool vs. wt hybridizations Two channel microarrays were used. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. Two independent cultures were hybridized on two separate microarrays.
Project description:We created a comprehensive tRNA deletion library in yeast and characterized the phenotypic and further characterized the molecular changes in a subset of deletion strains