Brendan P. G. Curran

Cellular lipid composition influences stress activation of the yeast general stress response element (STRE).

The heat inducibility of the yeast heat-shock response (HSR) pathway has been shown to be critically dependent on the level of unsaturated fatty acids present in the cell. Here the inducibility by heat or salt of the independently regulated general stress response (GSR) pathway is shown to be affected in the same way. An increase in the percentage of unsaturated fatty acids in heat- or salt-acclimated cells correlated with a decrease in the induction of a general stress-response-promoter-element (STRE)-driven reporter gene by either stress. Despite inducing reporter gene expression, sorbic acid treatment did not confer salt cross-tolerance on the cells. This failure correlated with a failure to increase the percentage of unsaturated fatty acids in the cells, suggesting that GSR pathway induction, in the absence of lipid changes, is insufficient for the induction of cross-tolerance. Cells grown with fatty acid supplements under anaerobic conditions provided further evidence for a potential role for lipids in the acquisition of stress resistance. These cells contained different fatty acid profiles depending on the fatty acid supplement supplied, exhibited differential sensitivity to both heat and salt stress, but had not undergone STRE induction. These results suggest that heat- and salt-stress induction of the GSR are sensitive to the level of unsaturated fatty acids present in the cell and that stress cross-tolerance may be a lipid-mediated phenomenon. Given that an increased level of unsaturated fatty acids also down-regulates heat induction of the HSR pathway, these observations lead to the provocative hypothesis that lipid modifications, rather than HSR or GSR pathway induction, are a major contributor to the induced heat and salt tolerance of yeast cells.

Reactive oxygen species may influence the heat shock response and stress tolerance in the yeast Saccharomyces cerevisiae

Moderate levels of reactive oxygen species (ROS) have been implicated as second messengers in a number of biochemical pathways, and in animal cells have been associated with the activation of the heat shock response (HSR). Here, using an intracellular probe, we demonstrate that differential accumulation of ROS in the yeast Saccharomyces cerevisiae is strongly associated with differential induction of an HS reporter gene over a range of heat shock temperatures. There was a good correlation between cellular ROS levels and the levels of HS‐induced reporter gene expression between 37 °C and 44 °C, both reaching maximal values at 41 °C. Furthermore, the addition of 150 µM H2O2 to the yeast cells during heat treatment resulted in a 3 °C decrease in the temperature required for maximal induction of the HS expression vector—an increased HS sensitivity that corresponded to a concomitant increase in ROS levels at these lower HS temperatures. Conversely, cells treated with 10 mM of the antioxidant ascorbic acid required a temperature that was 2 °C above that required in untreated controls for maximal induction of the HS expression vector. This decreased HS sensitivity corresponded to a decrease in ROS levels at these higher HS temperatures. Finally, cell viability assays reveal that intrinsic thermotolerance remains high in control cells despite concomitant decreases in HS‐reporter gene expression and ROS accumulation between 41 °C and 44 °C. We conclude that the sensitivity of the yeast HSR is strongly associated with ROS accumulation, and suggest that ROS‐mediated signalling ensures cooperation between the HS and the antioxidant responses. Copyright

Alcohols lower the threshold temperature for the maximal activation of a heat shock expression vector in the yeast Saccharomyces cerevisiae

When the yeast Saccharomyces cerevisiae is exposed to elevated growth temperatures, genes containing a heat shock element (HSE) in their promoters are activated. This study demonstrates that alcohols lower the temperature required for the maximal activation of such a promoter and that the concentration of alcohol required decreases as its hydrophobicity increases. A similar correlation has been found between the members of this alcohol series and their effect on a range of membrane functions. Our results therefore indicate that perturbation of the cell membrane may play a role in the heat induced activation of this HSE-containing promoter.

Basic investigations in Saccharomyces cerevisiae.

This chapter aims to provide the reader with a one-stop reference to the basic procedures needed to access, grow, store, mate, and sporulate yeast cells. It starts with an introduction to the Web-based yeast resources, which are becoming increasingly important in the investigation of S. cerevisiae in the post-genomic era. It then goes on to describe recipes for the different types of media and to explain how cells are grown to the appropriate cell numbers at the correct stage in the growth cycle. It also provides a detailed explanation on both short- and long-term storage of yeast cells. It ends by explaining how to set up genetic crosses with special advice on the demanding technique of diploid cell sporulation and spore isolation.

Subtle alterations in growth medium composition can dramatically alter the percentage of unsaturated fatty acids in the yeast Saccharomyces cerevisiae

The essence of the scientific method is the production of reproducible results in repeated experiments. Cells of the yeast strain DBY747 normally contain 36% unsaturated fatty acids but suddenly, and initially inexplicably, lipid analysis revealed 72% unsaturated fatty acids in the same strain at the same growth temperature. A comparative lipid analysis of DBY747 grown in YEPD and in a number of different types and batches of Yeast Nitrogen Base media revealed two heretofore unreported phenomena. We provide mass spectroscopy and yeast bioassay evidence suggesting that the increase in lipid unsaturation can be attributed to the presence of the plasticizing agent dioctylphthalate in YNB and bactopeptone packaged in ‘new’ plastic containers first introduced by Difco some 3–4 years ago. We also demonstrate that L‐methionine plays an important role in determining the percentage of unsaturated fatty acids in cells grown in laboratory‐produced YNB. The results illustrate a novel aspect of methionine metabolism while at the same time highlighting the need for more stringent control to be exercised by the companies that formulate and package defined media. Copyright

Can the different heat shock response thresholds found in fermenting and respiring yeast cells be attributed to their differential redox states

In this study we used a heat‐shock (HS) reporter gene to demonstrate that respiring cells are intrinsically less sensitive (by 5 °C) than their fermenting counterparts to a sublethal heat shock. We also used an oxidant‐sensitive fluorescent probe to demonstrate that this correlates with lower levels of sublethal reactive oxygen species (ROS) accumulation in heat‐stressed respiring cells. Moreover, this relationship between HS induction of the reporter gene and ROS accumulation extends to respiring cells that have had their ROS levels modified by treatment with the anti‐oxidant ascorbic acid and the pro‐oxidant H2O2. Thus, by demonstrating that the ROS/HSR correlation previously demonstrated in fermenting cells also holds for respiring cells (despite their greater HS insensitivity and higher level of intrinsic thermotolerance), we provide evidence that the intracellular redox state may influence both the sensitivity of the heat‐shock response (HSR) and stress tolerance in the yeast Saccharomyces cerevisiae. Copyright

Alpha-factor enhancement of hybrid formation by protoplast fusion in the yeast Saccharomyces cerevisiae.

Two a strains of Saccharomyces cerevisiae, carrying complementary genetic markers, were arrested for 3 h with alpha-factor. These were then protoplasted, prior to fusion using polyethylene glycol. The number of viable fusion products was enhanced by a factor of 20 as compared with unarrested controls.

Is mRNA sequestration involved in the regulation of progesterone 14α-hydroxylase cytochrome P-450 expression in Mucor hiemalis ?

During a 6 h incubation at 26 °C, Mucor hiemalis hydroxylated progesterone predominantly at the 14α site. Further incubation resulted in the production of several 14α-dihydroxyprogesterones and a novel microbial transformation product, 8(9), 14(15)-didehydroprogesterone (pregna-4,8[9], 14[15]-triene-3,20-dione). Azole inhibition indicates that the hydroxylases are cytochromes P-450. Mycelia transformed in the presence of the translation inhibitor cycloheximide failed to hydroxylate progesterone, whereas identical treatment with the transcription inhibitor actinomycin D, or heat-shock for 1 h at 37 °C prior to progesterone transformation, stimulated hydroxylation. 14α-Hydroxylation was stimulated if mycelia were pre-incubated with progesterone, transcription or translation inhibitors. These data are consistent with a model in which cytochrome P-450 steroid hydroxylase mRNA is stored in cells in a dormant form by a labile sequestering protein.

Differential effects of hydrogen peroxide and ascorbic acid on the aerobic thermosensitivity of yeast cells grown under aerobic and anoxic conditions.

We have previously demonstrated that in aerobically‐grown cells of the yeast Saccharomyces cerevisiae, hydrogen peroxide (H2O2) increases and ascorbic acid decreases cellular thermosensitivity, as determined by the inducibility of a heat shock (HS)‐reporter gene. In this work, we reveal that the aerobic thermosensitivity of anaerobically‐grown yeast cells also increases in the presence of H2O2, albeit differentially between cells with two different lipid profiles. In comparison to aerobically‐grown fermenting cells treated with the same H2O2 concentration, both these types of anaerobically‐grown cells were found to be considerably less sensitive to aerobic heat shock and considerably more thermotolerant. Paradoxically, and in contrast to ascorbate‐pretreated aerobically‐grown yeast cells, when anaerobically‐grown cells were heat‐shocked aerobically in the presence of the same ascorbic acid concentration, they exhibited increased thermosensitivity and decreased intrinsic thermotolerance with respect to their untreated counterparts. These findings are discussed with respect to what is currently known about the redox and physiological status of yeast cells grown aerobically and cells reoxygenated following anoxic growth. Copyright

A Terrible Beauty is Born: Clones, Genes and the Future of Mankind

Uniquely Similar! Incredible Journeys. What is it about the Nucleus? The Health of the Nation. Dealing with the Invisible. Awesome Analysis. Knowledge is Power. Molecular Magic. Miracle Cures. Beyond Belief. All is Changed, Utterly Changed.