Dynamic transcriptional response of Saccharomyces cerevisiae cells to copper.

Copper is a crucial trace element for all living systems and any deficiency in copper homeostasis leads to the development of severe diseases in humans. The observation of extensive evolutionary conservation in copper homeostatic systems between human and Saccharomyces cerevisiae made this organism a suitable model organism for elucidating molecular mechanisms of copper transport and homeostasis. In this study, the dynamic transcriptional response of both the reference strain and homozygous deletion mutant strain of CCC2, which encodes a Cu2+-transporting P-type ATPase, were investigated following the introduction of copper impulse to reach a copper concentration which was shown to improve the respiration capacity of CCC2 deletion mutants. The analysis of data by using different clustering algorithms revealed significantly affected processes and pathways in response to a switch from copper deficient environment to elevated copper levels. Sulfur compound, methionine and cysteine biosynthetic processes were identified as significantly affected processes for the first time in this study. Stress response, cellular response to DNA damage, iron ion homeostasis, ubiquitin dependent proteolysis, autophagy and regulation of macroautophagy, DNA repair and replication, as well as organization of mitochondrial respiratory chain complex IV, mitochondrial organization and translation were identified as significantly affected processes in only CCC2 deleted strain. The integration of the transcriptomic data with regulome revealed the differences in the extensive re-wiring of dynamic transcriptional organization and regulation in these strains

Transcriptomic response of yeast cells to ATX1 deletion under different copper levels.

BACKGROUND: Iron and copper homeostatic pathways are tightly linked since copper is required as a cofactor for high affinity iron transport. Atx1p plays an important role in the intracellular copper transport as a copper chaperone transferring copper from the transporters to Ccc2p for its subsequent insertion into Fet3p, which is required for high affinity iron transport. RESULTS: In this study, genome-wide transcriptional landscape of ATX1 deletants grown in media either lacking copper or having excess copper was investigated. ATX1 deletants were allowed to recover full respiratory capacity in the presence of excess copper in growth environment. The present study revealed that iron ion homeostasis was not significantly affected by the absence of ATX1 either at the transcriptional or metabolic levels, suggesting other possible roles for Atx1p in addition to its function as a chaperone in copper-dependent iron absorption. The analysis of the transcriptomic response of atx1∆/atx1∆ and its integration with the genetic interaction network highlighted for the first time, the possible role of ATX1 in cell cycle regulation, likewise its mammalian counterpart ATOX1, which was reported to play an important role in the copper-stimulated proliferation of non-small lung cancer cells. CONCLUSIONS: The present finding revealed the dispensability of Atx1p for the transfer of copper ions to Ccc2p and highlighted its possible role in the cell cycle regulation. The results also showed the potential of Saccharomyces cerevisiae as a model organism in studying the capacity of ATOX1 as a therapeutic target for lung cancer therapy.The authors greatly acknowledge the Turkish State Planning Organization DPT09K120520, Bogazici University Research Fund through Project No 5562 and TUBITAK through Project No 110 M692 for the financial support provided for this research.This is the final version of the article. It first appeared from BioMed Central via http://dx.doi.org/10.1186/s12864-016-2771-

A Novel Strategy for Selection and Validation of Reference Genes in Dynamic Multidimensional Experimental Design in Yeast

<div><h3>Background</h3><p>Understanding the dynamic mechanism behind the transcriptional organization of genes in response to varying environmental conditions requires time-dependent data. The dynamic transcriptional response obtained by real-time RT-qPCR experiments could only be correctly interpreted if suitable reference genes are used in the analysis. The lack of available studies on the identification of candidate reference genes in dynamic gene expression studies necessitates the identification and the verification of a suitable gene set for the analysis of transient gene expression response.</p> <h3>Principal Findings</h3><p>In this study, a candidate reference gene set for RT-qPCR analysis of dynamic transcriptional changes in <em>Saccharomyces cerevisiae</em> was determined using 31 different publicly available time series transcriptome datasets. Ten of the twelve candidates (<em>TPI1</em>, <em>FBA1</em>, <em>CCW12</em>, <em>CDC19</em>, <em>ADH1</em>, <em>PGK1</em>, <em>GCN4</em>, <em>PDC1</em>, <em>RPS26A</em> and <em>ARF1</em>) we identified were not previously reported as potential reference genes. Our method also identified the commonly used reference genes <em>ACT1</em> and <em>TDH3</em>. The most stable reference genes from this pool were determined as <em>TPI1</em>, <em>FBA1</em>, <em>CDC19</em> and <em>ACT1</em> in response to a perturbation in the amount of available glucose and as <em>FBA1</em>, <em>TDH3</em>, <em>CCW12</em> and <em>ACT1</em> in response to a perturbation in the amount of available ammonium. The use of these newly proposed gene sets outperformed the use of common reference genes in the determination of dynamic transcriptional response of the target genes, <em>HAP4</em> and <em>MEP2</em>, in response to relaxation from glucose and ammonium limitations, respectively.</p> <h3>Conclusions</h3><p>A candidate reference gene set to be used in dynamic real-time RT-qPCR expression profiling in yeast was proposed for the first time in the present study. Suitable pools of stable reference genes to be used under different experimental conditions could be selected from this candidate set in order to successfully determine the expression profiles for the genes of interest.</p> </div

Identification of point mutations in Turkish DMD/BMD families using multiplex-single stranded conformation analysis (SSCA)

Small mutations are the cause of the disease in one third of cases of Duchenne and Becker muscular dystrophy (DMD/BMD), The identification of point mutations in the dystrophin gene is considered to be very important, because it may provide new insights into the function of dystrophin and direct information for genetic counselling, In this study, we have screened 18 deletion-prone exons (25.5% of the coding region) of the dystrophin gene by using a modified non-isotopic multiplex single-stranded conformation analysis (SSCA). Mutations responsible for the disease phenotype could be identified in five out of 56 unrelated DMD/BMD patients without detectable deletions, Two of these mutations, 980-981delCC and 719G > C, are novel mutations which have not been described previously. Four of the five mutations, including 980-981delCC detected in this study are found to be nonsense or frameshift mutations leading to the synthesis of a truncated dystrophin protein. The missense mutation, 719G > C, causing the substitution of highly conserved alanine residue at 171 with proline in the actin binding domain of the dystrophin, is associated with a BMD phenotype. This study also revealed the presence of six polymorphisms in Turkish DMD/BMD patients

Additional file 6: of Transcriptomic response of yeast cells to ATX1 deletion under different copper levels

Intracellular and extracellular iron levels. This figure represents the intracellular (blue) and extracellular (red) iron levels in the reference and ATX1 deleted cells under copper deficient and high copper conditions. Error bars show the standard deviation. (TIF 59 kb

Saccharomyces cerevisiae adapted to grow in the presence of low-dose rapamycin exhibit altered amino acid metabolism

Abstract Background Rapamycin is a potent inhibitor of the highly conserved TOR kinase, the nutrient-sensitive controller of growth and aging. It has been utilised as a chemotherapeutic agent due to its anti-proliferative properties and as an immunosuppressive drug, and is also known to extend lifespan in a range of eukaryotes from yeast to mammals. However, the mechanisms through which eukaryotic cells adapt to sustained exposure to rapamycin have not yet been thoroughly investigated. Methods Here, S. cerevisiae response to long-term rapamycin exposure was investigated by identifying the physiological, transcriptomic and metabolic differences observed for yeast populations inoculated into low-dose rapamycin-containing environment. The effect of oxygen availability and acidity of extracellular environment on this response was further deliberated by controlling or monitoring the dissolved oxygen level and pH of the culture. Results Yeast populations grown in the presence of rapamycin reached higher cell densities complemented by an increase in their chronological lifespan, and these physiological adaptations were associated with a rewiring of the amino acid metabolism, particularly that of arginine. The ability to synthesise amino acids emerges as the key factor leading to the major mechanistic differences between mammalian and microbial TOR signalling pathways in relation to nutrient recognition. Conclusion Oxygen levels and extracellular acidity of the culture were observed to conjointly affect yeast populations, virtually acting as coupled physiological effectors; cells were best adapted when maximal oxygenation of the culture was maintained in slightly acidic pH, any deviation necessitated more extensive readjustment to additional stress factors