Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Expression analysis of fast-fermenting high-gravity maltose Saccharomyces cerevisiae Y55 mutants

ABSTRACT: Investigation of whole genome gene expression level changes in three S. cerevisiae Y55 mutants, compared to the wild-type strain. The UV-induced mutations enable the mutant strains to ferment high-gravity maltose faster than the WT. The mutants analyzed in this study are further described in Baerends, R.J.S., J.L. Qiu, L. Gautier, and A. Brandt. A high-throughput system for screening of fast-fermenting Saccharomyces cerevisiae strains. Manuscript in preparation. A single-dye 12-plex array chip study using double-stranded DNA prepared from messenger RNA purified from total RNA recovered from three separate Saccharomyces cerevisiae Y55 wild-type cultures and 3x three separate cultures each corresponding to a fast-fermenting UV-induced mutant (mutant 1, 2 and 3), during fermentation of high-gravity maltose at day 2. Each array on the 12-plex chip measures the expression level of 5,777 genes from Saccharomyces cerevisiae S288C with eight 60-mer probes per gene, with three-fold technical redundancy.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Richard Baerends

PROVIDER: E-GEOD-25015 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The combination of genetic analysis and genome-wide expression profiles make Saccharomyces cerevisiae the cradle of Systems Biology. The way S. cerevisiae uses carbon sources has long been a landmark for studies concerning cellular responses to environmental conditions. S. cerevisiae conserves the entire machinery for utilization of fatty acids, yet with a different compartmentalization compared to higher eukaryotes. We propose S. cerevisiae as a model to understand how perixosomal compartmentalization of oleate metabolism co-evolved with the transcriptional regulatory networks of FA metabolism. With this aim, we performed growth competition experiments in oleate on the pooled collection of all viable yeast null mutants. We integrated results of competitive fitness with transcriptional profiles in oleate, identifying two genes previously not directly associated to FA metabolism: OAR1 and FET3. For the first time we show the importance of iron metabolism not only in maintaining functional mitochondria, but also in influencing peroxisome wellness. An aliquot of the Yeast Homozygous Diploid Deletion Pool (95401.H1Pool, Invitrogen), containing equal amounts of each barcoded viable knock-out mutant from the Saccharomyces Genome Deletion project, was grown in the presence of G418 in YPD medium for 24h, then aliquots of 5x10^7 cells were harvested and stored at -80M-BM-0C. From the same pooled culture, equal amounts of cells were washed and transferred to YP medium added with oleic acid 0.1% and 5%. We collected samples of cells 24h and 72h after carbon source metabolic shift, and genomic DNA was extracted. Each experiment was performed in biological duplicate. The upTAG and downTAG regions were amplified separately in PCR reactions, one for each experimental condition, using specific primers. Amplified upTAG and downTAG sequences were combined in equal amounts and used to probe high-density oligonucleotide arrays (Affymetrix GeneChip_DNA_Tag3). Data analysis was performed as previously described (Pierce et al., 2007). In order to adapt this data analysis protocol to our experimental design, and to the use of GeneChip_DNA_Tag3 arrays, some changes to the procedure have been improved.

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Expression analysis of fast-fermenting high-gravity maltose Saccharomyces cerevisiae Y55 mutants

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