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Over-expression of CTR1 delta-300 alters element and transcription profiles in yeast

ABSTRACT: In an approach to generate Saccharomyces cerevisiae strains with increased intracellular copper amounts for technical applications we over-expressed the copper transporter CTR1 and a variant of CTR1 with a truncation in the C-terminus after the 300 amino acids (CTR1 delta-300). We determined the copper sensitivity of the generated strains and used inductively coupled plasma spectrometry (ICP-OES and ICP-MS) analysis to investigate the effects of over-expression of both constructs under excess copper on the cellular content of different elements in S. cerevisiae. In addition, we ran DNA microarray analysis to obtain the gene expression profile under the changed element contents. Over-expression of CTR1 increased the copper content in the cells up to 160% and 78 genes were differentially regulated. Over-expression of the truncated CTR1 delta-300 resulted in an increased copper, iron and zinc content of more than 200% and 980 genes showed differential expression. We found that transition metal ion homeostasis was disrupted in CTR1 delta-300 over-expressing strains under excess copper and that this was combined with a transcriptional remodelling of cellular processes

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE44043 | GEO | 2014/01/28

SECONDARY ACCESSION(S): PRJNA188412

REPOSITORIES: GEO

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Project description:Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes including frozen dough baking. The cell viability and fermentation activity after freeze-thaw were dramatically decreased due to freeze-thaw injury. Because freeze-thaw injury involves complex phenomena, the mechanisms of it are not fully understood. We attempted to analyze the mechanisms of freeze-thaw injury by indirect gene expression analysis during post-thaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that a high frequency of the genes involved in the homeostasis of metal ions were up-regulated depending on the freezing period. The phenotype of the deletion mutants of the up-regulated genes extracted by indirect gene expression analysis was assessed. The deletion strains of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. Supplementation with copper ions during post-thaw incubation increased intracellular superoxide dismutase activity. Inverse correlated with intracellular superoxide dismutase activity, intracellular levels of reactive oxygen species were decreased. Moreover, cell viability increased by supplementation with copper ions under specific assessment conditions. This study suggested that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury. Total RNA was extracted from the stress-treated yeast cells by using a hot phenol method. Poly(A)+ RNA was enriched from total RNA by using an Oligotex dT30 (Super) mRNA purification kit (Takara Bio, Ohtsu, Japan). cDNA synthesis, cRNA synthesis, and labeling were performed according to the Affymetrix user’s manual (Affymetrix, Santa Clara, USA). Biotinyated cRNA was fragmented and then used as a probe.Affimetrix Yeast Genome 2.0 arrays (Affymetrix) were used as DNA microarrays. All experiments were done in triplicate independently.

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Over-expression of CTR1 delta-300 alters element and transcription profiles in yeast

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