The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells. Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance. Anaerobic conditions caused a 500-to 20,000-fold increase in thermotolerance. The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure. HSP104 is not responsible for the increased resistance of anaerobically grown cells. The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins. By using an oxidation-dependent fluorescent molecular probe a 2-to 3-fold increase in fluorescence was found upon heating. Thus, we conclude that oxidative stress is involved in heat-induced cell death.Most living cells are sensitive to sudden heat exposure. A shift in temperature from a low to an intermediate temperature induces the stress response or heat-shock response (1-3), which is considered to be an evolutionarily conserved genetic system advantageous to living organisms. After a temperature shift from 23 to 37°C in cells of the yeast Saccharomyces cerevisiae, 80 proteins were transiently induced; 20 of these proteins are now classified as major heat-shock proteins (HSPs) (2). Some of these HSPs have been characterized, but the function of many of them is still unclear (4).Initial studies suggested that HSPs play an essential role in the acquisition of stress tolerance. On the other hand, a convincing involvement of HSPs in the development of stress resistance has not been established in yeast. Except for HSP104, none of the other HSP disruption mutants show any block in the acquisition of stress resistance in yeast (5). Furthermore, a yeast strain with a temperature-sensitive mutation in the heat-shock factor (hsfl-m3) that leads to a general block in heat-shock-induced protein synthesis was not affected in the acquisition of thermotolerance (6). Therefore, HSPs may not be important for stress tolerance acquisition but rather for a rapid recovery after heat shock (4).The main factors causing death after heat exposure are still unknown. Thus, the heat-shock response may not elucidate why cells die in response to heat exposure but rather how they repair the damage afterwards. To investigate why cells die in response to heat exposure, we completely avoided the induction of the heat-shock response by exposing cells immediately to lethal heat. In particular, we investigated the possible involvement of oxidative stres...

The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase , f 3 genes (7SBl and 7SB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although 7SBl produces >90% of tryptophan synthase ,f3 mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase B activity. The trp2-1 mutation is complemented by the 7SBl transgene and is linked genetically to a polymorphism in the 7SBl gene, strongly suggesting that trp2-1 is a mutation in 7SB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, 7SB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (7RP1 and 7RP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re‐hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non‐culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat‐killed mixtures. However, for re‐hydrated HADY, Ox stained a significantly (P⩽0·05) higher proportion of cells than did PI. © 1998 John Wiley & Sons, Ltd.

Chromosomal position effects can influence strongly the transcription of foreign genes in transgenic animals. This results in low frequencies and levels of gene expression and, in some cases, in aberrant patterns of expression. Strategies for overcoming these effects are described with particular reference to their application in embryonic stem cells.

The cruciferous plant Arabidopsis thaliana has two closely related, nonallelic tryptophan synthase beta genes (TSB1 and TSB2), each containing four introns and a chloroplast leader sequence. Both genes are transcribed, although TSB1 produces greater than 90% of tryptophan synthase beta mRNA in leaf tissue. A tryptophan-requiring mutant, trp2-1, has been identified that has about 10% of the wild-type tryptophan synthase beta activity. The trp2-1 mutation is complemented by the TSB1 transgene and is linked genetically to a polymorphism in the TSB1 gene, strongly suggesting that trp2-1 is a mutation in TSB1. The trp2-1 mutants are conditional: they require tryptophan for growth under standard illumination but not under very low light conditions. Presumably, under low light the poorly expressed gene, TSB2, is capable of supporting growth. Genetic redundancy may be common to many aromatic amino acid biosynthetic enzymes in plants because mutants defective in two other genes (TRP1 and TRP3) also exhibit a conditional tryptophan auxotrophy. The existence of two tryptophan pathways has important consequences for tissue-specific regulation of amino acid and secondary metabolite biosynthesis.

Strain selection and improvement in the baker’s yeast industry have aimed to increase the speed of maltose fermentation in order to increase the leavening activity of industrial baking yeast. We identified two groups of baker’s strains of Saccharomyces cerevisiae that can be distinguished by the mode of regulation of maltose utilization. One group (nonlagging strains), characterized by rapid maltose fermentation, had at least 12-fold more maltase and 130-fold-higher maltose permease activities than maltose-lagging strains in the absence of inducing sugar (maltose) and repressing sugar (glucose). Increasing the noninduced maltase activity of a lagging strain 13-fold led to an increase in CO2 production in unsugared dough. This increase in CO2 production also was seen when the maltose permease activity was increased 55-fold. Only when maltase and maltose permease activities were increased in concert was CO2 production by a lagging strain similar to that of a nonlagging strain. The noninduced activities of maltase and maltose permease constitute the largest determinant of whether a strain displays a nonlagging or a lagging phenotype and are dependent upon theMALx3 allele. Previous strategies for strain improvement have targeted glucose derepression of maltase and maltose permease expression. Our results suggest that increasing noninduced maltase and maltose permease levels is an important target for improved maltose metabolism in unsugared dough.

We have studied four novel MAL promoters isolated from a single strain of bakers' yeast. Within these promoters we have identified up to five tandem 147 bp repeats located between the MAL UAS region and the MALT TATA box. These repeats strongly reduce MALT (maltose permease) gene expression but only weakly reduce MALS (maltase) gene expression. Insertion of the 147 bp elements into the heterologous CYC1 promoter reduced expression when located between the CYC1 UAS and the TATA box, but not when located upstream of the UAS. We propose that these naturally occurring repeats have evolved as a mechanism to lower the level of MALT expression relative to MALS expression, thus avoiding possible toxic effects associated with over‐expression from multiple copies of the permease gene. Accession numbers are: WIG1, U86359; WIG3, U86360; WIG4, U86361; WIG5, U86362. © 1997 by John Wiley & Sons, Ltd.

Many sets of genes in Saccharomyces cerevisiae are divergently transcribed, but at present there are no vectors generally available for the simultaneous analysis of divergent transcription from these promoters. In the present study MEL1 and lacZ were used to construct a vector capable of measuring the divergent expression initiated by the MAL6T-MAL6S bi-directional promoter. Our observations demonstrate that the expression of both reporter genes was regulated in a similar fashion to the native MAL6T and MAL6S genes, and that induction was dependent upon the presence of a functional MALR activator gene. The results confirmed that the MAL6T-MAL6S promoter was co-ordinately regulated, repressed by glucose, induced by maltose, and that basal expression was more active in the MAL6S direction than in the MAL6T direction.