Influence of the composition of the fusion medium on the yield of electrofused yeast hybrids

Abstract Electrofusion between cells of yeast strains with different genetic markers in isotonic sorbitol solutions leads to high yields of hybrids when 0.1 mM Ca²⁺ and 0.5 mM Mg²⁺ salts are aded. On average, 1000–2000 hybrids are obtained when electrofusion is performed (in a helical chamber) compared to a yield of about 40–120 in the absence of these bivalent cations. A further increase in yield can be achieved by the addition of 1 mg/ml albumin, which results in up to 4000 hybrids per experimental run. The entire fusion process leads to very reproducible results in the presence of these substances.     

Use of live Saccharomyces cerevisiae cells as a biological response modifier in experimental infections

Abstract A short-term oral administration of live Saccharomyces cerevisiae cells, strain Sillix Hansen DSM 1883, resulted in enhanced resistance of mice toward infections with K. pneumoniae, S. pneumoniae and S. pyogenes A produced by intranasal inoculation. Yeast pre-treatment also increased the efficacy of antibiotic therapy in bacterial infections and of antiviral drugs in viral infections. Yeast treatment of animals stimulated phagocytosis, activated the complement system and induced interferon which are likely to represent the main mechanisms of action whereby pretreatment of mice with live S. cerevisiae cells increases resistance to infection. It is concluded that preventive administration of live Saccharomyces cerevisiae cells should be used for increasing resistance to bacterial infections, in particular of the respiratory tract, or to viral infections, as well as an adjunct to antibiotic and antiviral drug therapy.     

Effects of glucose, tetrapyrroles and protein kinase C activators on cell proliferation in cultures of Saccharomyces cerevisiae

Abstract Saccharomyces cerevisiae was inoculated into a yeast nitrogen base with either glycerol or glucose as carbon source. Cell proliferation was followed by colony counts on agar medium. Cells in the glycerol-supplemented medium divided less than once in 10 days. When glucose, 6-deoxy-glucose or protoporphyrin IX was added, the cells had doubling times of about 24 h and increased in number to about 0.5 × 10⁶ cells ml⁻¹ Addition of either of the protein kinase C activators oleoyl-acetylglycerol or phorbol-12-myristate-13-acetate did not activate cell proliferation in the glycerol medium. However, when (i) glucose was combined with either protoporphyrin IX or chlorophyllin, or (ii) either protoporphyrin IX or chlorophyllin was combined with either of the protein kinase C activators, the cells had doubling times of about 12 h. Hence, (i) glucose can act as both a carbon source and a signalling molecule for proliferation, and (ii) two systems are involved in activating cell proliferation in S. cerevisiae : one operating through a protein kinase C system and another through a guanylate cyclase system.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Enantioselective hydrolysis of esters of secondary alcohols using lyophilized Bakers'' yeast

A number of acetoxycarboxylic acid esters were hydrolysed enantioselectively by Saccharomyces cerevisiae Hansen leading to chiral hydroxycarboxylic acid esters of high optical purity. The scope and limitations of this method with respect to the substitutional pattern of substrates were investigated. Subcellular localization of the hydrolytic activity on the plasma membrane led to the assumption that unspecific carboxyl esterases are responsible for hydrolysis of this type of substrate. Comparative experiments using viable cells and lyophilized cells as source of enzyme revealed the latter to be superior with respect to enantioselection and ease of handling.     

The pentafunctional FAS1 genes of Saccharomyces cerevisiae and Yarrowia lipolytica are co-linear and considerably longer than previously estimated

Summary The fatty acid synthetase (FAS) gene FAS1 of the alkane-utilizing yeast Yarrowia lipolytica was cloned and sequenced. The gene is represented by an intron-free reading frame of 6228 by encoding a protein of 2076 amino acids and 229980 Da molecular weight. This protein exhibits a 58% sequence similarity to the corresponding Saccharomyces cerevisiae FAS -subunit. The sequential order of the five FAS1-encoded enzyme domains, acetyl transferase, enoyl reductase, dehydratase and malonyl/palmityl-transferase, is co-linear in both organisms. This finding agrees with available evidence that the functional organization of FAS genes is similar in related organisms but differs considerably between unrelated species. In addition, previously reported conflicting data concerning the 3 end of S. cerevisiae FAS1 were re-examined by genomic and cDNA sequencing of the relevant portion of the gene. Thereby, the translational stop codon was shown to lie considerably downstream of both published termination sites. The S. cerevisiae FAS1 gene thus has a corrected length of 6153 by and encodes a protein of 2051 amino acids and 228667 Da molecular weight.     

Influence of the curing of the killer phenotype inSaccharomyces cerevisiae wine strains on their fermentative behaviour

Fermentative behaviour and cell growth have been studied in grape juice inoculated either with two killerSaccharomyces cerevisiae wild strains or with their Acridine Orange-cured isogenic counterparts. The number of viable cells/ml at the beginning of the fermentation, as well as during exponential growth, were higher in grape juices inoculated with the cured strains. The CO₂ production, fermentative rate and ethanol and acetic acid production were also higher in the cured strains, particularly during the stage of active fermentation. These differences, however, were minimal at the end of the fermentations.     

cAMP-dependent phosphoprotein changes in the yeast Saccharomyces cerevisiae

Abstract cAMP-dependent phosphoprotein changes were determined using 1-dimensional SDS-gel electrophoresis in a cAMP-requiring yeast mutant ( Saccharomyces cerevisiae AM18). During cAMP starvation, the yeast cells accumulated 3 ³²P-labeled bands with M _r/ 72000, 54000, and 37000. The M _r/ 72000 protein was the most prominent phosphorylated protein. After the readdition of cAMP, these phosphoproteins lost their ³²P-label while phosphoproteins with M _r/ 76000, 65000, 56000 and 30000 were accumulated. Similar phosphoprotein changes were also detected in cdc35 at the nonpermissive temperature, but not in wildtype (A363A) or cdc7 strains of S. cerevisiae .