Project description:Wild-type cells and cells with abundant high-affinity transporters (PHO84C) were transferred from rich medium (SD) to medium containing 0.5mM phosphate
Project description:Investigation of Saccharomyces cerevisiae phosphate metabolism. Cells starved for phosphate, cells grown with intermediate and high phosphate concentrations, and PHO4 mutant cells examined. Keywords: other
Project description:Arsenic is known as a human carcinogen that easily be exposed by the living organisms through environment and food consumption. The arsenic is transport into the cells via phosphate transporters due to its structural similarity with phosphate in both prokaryotes and eukaryotes. We here evaluated and analyzed the toxicogenomic impacts of arsenate and the role of different phosphate concentrations on arsenic toxicity. Our results showed that arsenic uncoupled phosphate levels which eventually affected the growth rate of yeast cells. Analysis of arsenate levels in the medium over 4 to 10 h of its exposure clearly showed that arsenate was easily taken up by the cells in phosphate limited condition.
Project description:Wild-type cells and cells with abundant high-affinity transporters (PHO84C) were transferred from rich medium (SD) to medium containing 0.5mM phosphate Cells were grown to logarithmic phase in SC medium (OD=0.5), washed and then incubated in SC medium with intermediate phosphate level (0.5mM, initial OD=0.05). During the experiment (0, 1, 5, 8 and 23 hours) cells were harvested, pelleted and frozen for further analysis. Total RNA was extracted using MasterPure™fication Kit (Epicentre). The samples were amplified, labeled, hybridized to yeast dual color expression microarrays and scanned, all using standard Agilent protocols, reagents, and instruments. The scanned images were analyzed using SpotReader software (Niles Scientific).
Project description:Investigation of Saccharomyces cerevisiae phosphate metabolism. Cells starved for phosphate, cells grown with intermediate and high phosphate concentrations, and PHO4 mutant cells examined.
Project description:Inorganic phosphate (Pi) is a critical building block for key biomolecules including ATP, DNA, RNA, and phospholipids. Consequently, cells monitor phosphate levels and acquire phosphate when intracellular levels become low. Here we demonstrate an unanticipated connection between the enzymatic activity of the S. cerevisiae RNase T2 ortholog, Rny1, and inorganic phosphate availability. Rny1 has been studied for its role in autophagy-linked RNA degradation under starvation conditions. Here we find that in nutrient-rich conditions, cells lacking Rny1 function exhibit phosphate starvation phenotypes and aberrantly activate the PHO signaling pathway despite the presence of high levels of inorganic phosphate in the growth media.
Project description:The Mycobacterium tuberculosis genome encodes two complete high-affinity Pst phosphate-specific transporters. We previously demonstrated that a membrane-spanning component of one Pst system, PstA1, was essential both for M. tuberculosis virulence and for regulation of gene expression in response to external phosphate availability. To determine if the alternative Pst system is similarly required for virulence or gene regulation, we constructed a deletion of pstA2. Transcriptome analysis revealed that PstA2 is not required for regulation of gene expression in phosphate-replete growth conditions. PstA2 was also dispensable for replication and virulence of M. tuberculosis in a mouse aerosol infection model. However, a ∆pstA1∆pstA2 double mutant was attenuated in mice lacking the cytokine interferon-gamma, suggesting that M. tuberculosis requires high-affinity phosphate transport to survive phosphate limitation encountered in the host. Surprisingly, ∆pstA2 bacteria were more resistant to acid stress in vitro. This phenotype is intrinsic to the alternative Pst transporter since a ∆pstS1 mutant exhibited similar acid resistance. Our data indicate that the two M. tuberculosis Pst transporters have distinct physiological functions, with the PstA1 transporter being specifically involved in phosphate sensing and gene regulation while the PstA2 transporter influences survival in acidic conditions.
