Showing papers on "Yeast published in 1979"
TL;DR: A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized in this paper.
Abstract: A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.
1,293 citations
TL;DR: The utility of this cloning system is demonstrated by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.
950 citations
TL;DR: A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described and has recently been certified at the HV2 level by the National Institutes of Health.
819 citations
01 Jan 1979
TL;DR: A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described in this article, where the principle of containment is sterility: the haploid host strains all contain a mating type-non-specific sterile mutation.
Abstract: SUMMARY A system of biological containment for recombinant DNA experiments in Saccharomyces cerevisiae (Brewer's/Baker's yeast) is described. The principle of containment is sterility: the haploid host strains all contain a matingtype-non-specific sterile mutation. The hosts also contain four auxotrophic mutations suitable for selection for the various kinds of vectors used. All vectors are derivatives of pBR322 which can be selected and maintained in both yeast and Escherichia coli. The system has recently been certified at the HV2 level by the National Institutes of Health.
621 citations
TL;DR: The F1-ATPase subunits of yeast were synthesized either in vitro in a reticulocyte lysate programmed with yeast RNA or in vivo (in pulsed and pulsed-chased yeast spheroplasts) to study the mechanism of import.
Abstract: The mitochondrial F1-ATPase consists of five nonidentical subunits that are synthesized outside the mitochondria and imported across both mitochondrial membranes to the matrix side of the inner membrane. In order to study the mechanism of this import, we synthesized the F1-ATPase subunits of yeast either in vitro (in a reticulocyte lysate programmed with yeast RNA) or in vivo (in pulsed and pulsed-chased yeast spheroplasts). Both in vitro and in vivo, each of the three largest ATPase subunits was synthesized as a larger precursor. When the precursors that had been synthesized in vitro were incubated with isolated yeast mitochondria, they were converted to "mature" subunits that were no longer susceptible to externally added proteases. The uptake of the subunit into the mitochondria was thus accompanied by conversion of the precursor. Since uptake of precursors into mitochondria was independent of protein synthesis and since the precursors could also be detected in vivo, the transfer of proteins from the cytosol across both mitochondrial membranes does not occur by vectorial translation. Instead, the proteins destined for import are first made outside the mitochondria as precursors and only subsequently transported into the mitochondria. This step is accompanied by proteolytic conversion of the mature subunit.
396 citations
TL;DR: The production of carotenoid pigments by the yeast Phaffia rhodozyma depended on the culture conditions, and oxygen was an important substrate for optimum yields of yeast and astaxanthin; under microaerophilic growth conditions, astrixanthin production was drastically decreased and P. r Rhodozyma accumulated β-carotene and the monoketone echinenone.
Abstract: Summary: The production of carotenoid pigments by the yeast Phaffia rhodozyma depended on the culture conditions. Astaxanthin, the primary carotenoid in this yeast, was produced mainly during the exponential phase of growth. The concentration of carotenes in P. rhodozyma remained relatively constant [about 5 μg (g yeast)−1] throughout growth in a 1.5% (w/v) glucose medium, but the xanthophyll concentration increased from 90 to 406 μg (g yeast)−1 during fermentation. Active xanthophyll synthesis occurred during the period of accelerating growth and after exhaustion of glucose from the growth medium. In media containing more than 1.5% (w/v) glucose, however, yeast and carotenoid yields were considerably reduced. The pH of the medium affected yeast yields and carotenoid production; the optimum pH was 5.0. At pH 3.5, β-zeacarotene accumulated in P. rhodozyma. β-Carotene was the primary carotene in the yeast under all other conditions tested. The optimum temperature for yeast growth and pigment formation was 20 to 22.C and the best carbon source was d-cellobiose. Oxygen was an important substrate for optimum yields of yeast and astaxanthin; under microaerophilic growth conditions, astaxanthin production was drastically decreased and P. rhodozyma accumulated β-carotene and the monoketone echinenone.
260 citations
TL;DR: The results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.
Abstract: For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.
246 citations
TL;DR: Plasmid pBR313 carrying a 1.4 kb EcoRI fragment from the yeast TRP1 region (designated pLC544) is capable of transforming yeast trp1 mutants to Trp+ at high frequency (10(3)--10(4) transformants/micrograms DNA).
235 citations
TL;DR: Data support the hypothesis that regulation of the ura3 gene in yeast is at the level of transcription, as a coordinate variation in proportion of labeled RNA complementary to the hybrid plasmid was found.
Abstract: From a large population of strains of Escherichia coli carrying shear fragments of yeast (Saccharomyces cerevisiae) DNA attached by in vitro recombination to the plasmid vector pMB9, two hybrid plasmids were selected that relieve the pyrimidine requirement of nonreverting pyrF mutants of E. coli. An 1100-base-pair DNA fragment common to the two complementing plasmids was recloned into another plasmid vector, pBR322; these new hybrids retained the ability to specify orotidine-5'-phosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase, EC 4.1.1.23) synthesis in E. coli. Evidence is presented that this common fragment is yeast DNA and thus apparently carried the structural information for yeast orotidine-5'-phosphate decarboxylase, the product of yeast gene ura3. A hybrid plasmid containing the 1100-base-pair fragment was used to measure levels of putative ura3 mRNA from yeast cultures labeled with [3H]adenine, ura3 mRNA was unstable with an apparent half-life of 10.5 min. Under different circumstances previously shown to alter the level of orotidine-5'-phosphate decarboxylase in yeast, a coordinate variation in proportion of labeled RNA complementary to the hybrid plasmid was found. These data support the hypothesis that regulation of the ura3 gene in yeast is at the level of transcription.
211 citations
TL;DR: The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis and there were no significant differences between these two yeasts in their control of glucose uptake.
Abstract: Summary: The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40% activation at 1 mM). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor.
194 citations
TL;DR: It is shown that HM, like histones or histone-like proteins, is able to introduce superhelical turns into circular relaxed DNA in the presence of a nicking-closing activity.
Abstract: Analysis of proteins isolated by affinity chromatography on DNA-cellulose from highly purified yeast mitochondria shows that these organelles do not contain histones but have in abundance a DNA-binding protein of 20,000 daltons. The purification yield of this protein, called HM, indicates that mitochondria have at least an equal mass of HM relative to DNA. The amino acid composition and its electrophoretic characterization reveal that HM, rich in lysine, is slightly basic and heat stable. HM appears to be coded by the yeast nucleus, as shown by its presence in several "petite" mutants. We have shown that HM, like histones or histone-like proteins, is able to introduce superhelical turns into circular relaxed DNA in the presence of a nicking-closing activity.
TL;DR: The yeast plasma membrane regulates the movement of compounds into the yeast cell and of yeast metabolites from the cell into the medium, and the rate of penetration of organic acids into the fermentation cell depends on their lipophilic nature, and on their molecular size and degree of branching.
Abstract: The yeast plasma membrane regulates the movement of compounds into the yeast cell and of yeast metabolites from the cell into the medium. The rate of penetration of organic acids into the yeast cell depends on their lipophilic nature, and on their molecular size and degree of branching. During fermentation yeast synthesizes a vast number of aroma compounds. The numerically and quantitatively largest groups of aroma compounds include fusel alcohols, fatty acids and fatty acid esters. The yeast used and the fermentation conditions can influence the formation of aroma compounds. The yeast also has a profound effect on the formation of other aroma compounds, such as sulphur compounds and phenols. In addition to fermentation, the maturing of a beverage can also influence the aroma. During the maturing lactones, phenols and other compounds are extracted from the oak casks in which the beverage is aged. The presence of the so-called “whisky lactone”, β-methyl-γ-octalactone, is characteristic of a beverage that has been matured in oak.
TL;DR: Several additional aspects of yeast transformation are described along with results showing that integrated bacterial plasmid sequences can be retrieved from the yeast genome and reintroduced into bacteria as replicant plasmids.
Abstract: Previous studies showed that the plasmid pYeleu10 could be stably integrated into yeast chromosomes at several locations and, once integrated, the bacterial plasmid sequences (Co1E1) behaved as simple Mendelian elements. In this paper, the authors describe several additional aspects of yeast transformation along with results showing that integrated bacterial plasmid sequences can be retrieved from the yeast genome and reintroduced into bacteria as replicant plasmids; cotransformation of a single cell by two independent plasmids occurs at high frequencies; sequence homology directs the integration of transforming DNA molecules; transformation frequency can be increased dramatically either by converting plasmids from circular plasmids to linear molecules with restriction endonucleases or by creating new recombinant plasmids containing fragments of the indigenous yeast plasmid (ScpI) inserted into the original hybrid plasmids.
TL;DR: The LEU2+ gene, inserted into the rDNA locus, is used as a genetic marker for mapping the r DNA, in a procedure analogous to the use of antibiotic resistance transposons in the mapping of bacterial genes.
TL;DR: Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles, and immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver is not detected.
Abstract: Precipitation of Saccharomyces cerevisiae ribosomes by ethanol under experimental conditions that do not release the ribosomal proteins can affect the activity of the particles. In the presence of 0.4 M NH4Cl and 50% ethanol only the most acidic proteins from yeast and rat liver ribosomes are released. At 1 M NH4Cl two more non-acidic proteins are lost from the ribosomes. The release of the acidic proteins causes a small inactivation of the polymerizing activity of the particles, additional to that caused by the precipitation itself. The elongation-factor-2-dependent GTP hydrolysis of the ribosomes is, however, more affected by the loss of acidic proteins. These proteins can stimulate the GTPase but not the polymerising activity when added back to the treated particles. Eukaryotic proteins cannot be sustituted for bacterial acidic proteins L7 and L12. We have not detected immunological cross-reaction between acidic proteins from Escherichia coli and those from yeast, Artemia salina and rat liver or between acidic proteins from these eukaryotic ribosomes among themselves.
TL;DR: Two chimeric plasmids containing yeast ura3 gene and 2-μm yeast DNA linked to the bacterial plasmid pCR1 are used to transform Escherichia coli and the specific activity of orotidine 5′-monophosphate decarboxylase in yeast transformants was 10- to 30-fold higher than in the wild type.
TL;DR: The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme, which indicate that the microbial and the mammalian enzymes may have separate evolutionary origins.
Abstract: Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione–Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4°C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide `maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The kcat. and Km values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a kcat./Km value near 107s−1·m−1, which is close to the theoretical diffusion-controlled rate of enzyme–substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.
TL;DR: Four yeast strains, causally associated with infection in a patient with acute myelogenous leukemia, were identified by standard methods currently used in yeast taxonomy as representatives of Candida lusitania van Uden et do Carmo-Sousa, thus confirming their classification by conventional taxonomy.
Abstract: Four yeast strains, causally associated with infection in a patient with acute myelogenous leukemia, were identified by standard methods currently used in yeast taxonomy as representatives of Candida lusitania van Uden et do Carmo-Sousa. Because this species has not been recognized previously as an opportunistic yeast in humans, molecular taxonomic methods were applied to confirm its identity. The nuclear deoxyribonucleic acid (DNA) base composition of two clinical isolates was shown to be 45.1 mol% guanine plus cytosine as compared to 44.7 mol% guanine plus cytosine for the type strain of this species. DNA/DNA reassociation experiments revealed more than 95% complementarity between the DNAs from the clinical isolates and that of the type strain of C. lusitaniae, thus confirming their classification by conventional taxonomy. A key is provided to differentiate C. lusitaniae from two phenotypically similar Candida species.
TL;DR: The effect of temperature on the fatty acyl transfer reaction of the fatty acid synthetase complex showed that the activation energies of the palmityl transfer and stearyl transfer activities vary with temperature and may account, in part, for the increased production of fatty acids of CIs chain length over CIs.
TL;DR: This paper showed that stationary phase cells are not endowed with a specific "competence" to germinate, however, cells at any stage of growth are able to form germ-tubes giving true hyphae when incubated at 37 °C in the presence of inducers such as serum or Nacetyl-d-glucosamine.
Abstract: Summary: Yeast cells of Candida albicans growing at 25 °C in glucose/yeast extract medium cannot germinate by dilution into fresh medium at 37 °C before reaching the stationary phase of growth However, cells at any stage of growth are able to form germ-tubes giving true hyphae when incubated at 37 °C in the presence of inducers such as serum or N-acetyl-d-glucosamine Thus, stationary phase cells are not endowed with a specific ‘competence' to germinate
TL;DR: Spheroplast fusion thus affords a means to genetically analyse brewing yeast strains, such an analysis having been difficult if not impossible by conventional hybridization techniques.
Abstract: Spheroplasts of brewing polyploid yeast strains have been successfully fused with spheroplasts of haploid yeast strains. After regeneration of the cell wall, stable fusion recombinants were isolated. Genetic analysis of these recombinants revealed that they contained the genotype of both parents, sporulated well with each ascus containing four spores and were indeed diploid. Spheroplast fusion thus affords a means to genetically analyse brewing yeast strains, such an analysis having been difficult if not impossible by conventional hybridization techniques.
TL;DR: Inclusion of magnetic material apparently had no adverse effect on the yeast cells as judged from their fermentation capacity, their operational stability as well as their ability to propagate in situ in the presence of nutrients.
Abstract: Saccharomyces cerevisiae was immobilized in calcium alginate gel together with varying concentrations of iron oxide, in the form of magnetite or a colloidal ferrite suspension, Ferrofluid™. Inclusion of magnetic material apparently had no adverse effect on the yeast cells as judged from their fermentation capacity, their operational stability as well as their ability to propagatein situ in the presence of nutrients. The usefulness of magnetic preparations in viscous or particle containing media is discussed.
TL;DR: It was interesting to develop a rapid test for the detection of this character on agar plates because yeast strains carrying an Am$ gene on a plasmid express this gene and produce penicillinase (personal communication).
TL;DR: The addition to wort of lipids derived from malt spent grains had a pronounced effect on yeast metabolism and the effect of the lipids on the synthesis of esters, fusel alcohols and medium chain fatty acids could be explained solely by their content of unsaturated fatty acids.
Abstract: The addition to wort of lipids derived from malt spent grains had a pronounced effect on yeast metabolism. The lipids allowed the fermentation of de-oxygenated wort and also stimulated yeast growth and the corresponding rate and extent of fermentation of air-saturated wort by yeast strains having a high oxygen requirement. The lipids increased the fusel alcohols content of beer and decreased the content of esters and medium chain-length fatty acids. The yeast incorporated sitosterol and unsaturated fatty acids from the spent grain lipids and the unsaturated fatty acids changed the pattern of fatty acids and sterols synthesized by the yeast. The fatty acids were present in the spent grain lipids mainly as triglycerides, free fatty acids and phospholipids. Using pure lipid compounds it was shown that the triglycerides were inactive and that the spent grain lipids exerted their effect on fermentation through the synergistic action of free unsaturated fatty acids, sitosterol and phospholipid. Phospholipid could be replaced by the detergent, Triton X-100. The effect of the lipids on the synthesis of esters, fusel alcohols and medium chain fatty acids could be explained solely by their content of unsaturated fatty acids.
TL;DR: In this article, the authors evaluated one of the most promising of yeast that has flocculent characteristics and that is capable of fermentation at high sugar concentrations - Saccharomyces carlsbergensis (uvarum).
Abstract: The fermentation of alcohol from renewable substrates is attracting attention as a promising energy resource. Brazil has started to develop its fermentation industries for producing fuel ethanol from crops such as sugar-cane and cassava. However, before alcohol production costs become competitive, significant improvements in technology will have to be made. This paper evaluates one of the most promising of yeasts that has flocculent characteristics and that is capable of fermentation at high sugar concentrations - Saccharomyces carlsbergensis (uvarum). A maximum specific ethanol production rate of 0.58 g/g/hour was found in batch culture at high yeast concentrations and a value of 0.75 g/g/hour in continuous culture with recycle. It is suggested that where flocculation properties, as well as fermentation properties are required, the use of this yeast could well be justified.
TL;DR: Glucose repressed the synthesis of 1,3-β-glucanase in Neurospora crassa, but if deprived of carbon source the fungus actively synthesized high levels of enzyme when growth ceased.
Abstract: Summary: Glucose repressed the synthesis of 1,3-β-glucanase in Neurospora crassa. The production of enzyme during growth in glucose-supplemented medium was negligible, but if deprived of carbon source the fungus actively synthesized high levels of enzyme when growth ceased. Similar results were obtained for 1,6-β-glucanase but less enzyme was produced. A different pattern of production of these enzymes was found in Trichoderma viride and Saccharomyces cerevisiae. The enzymes were produced in glucose-supplemented medium with increasing specific activity during growth. Resting cells deprived of glucose either failed to produce β-glucanases or produced them in smaller quantities.
TL;DR: The DNase I (EC 3.1.21.1) sensitivity of transcribed yeast chromatin has been examined and it is found that the entire yeast genome exists in a state that represents a restricted proportion of total chromatin in higher eukaryotes.
Abstract: The DNase I (EC 3.1.21.1) sensitivity of transcribed yeast chromatin has been examined. We find that, in contrast to chromatin from higher eukaryotes, transcribed yeast chromatin and total yeast chromatin are equally sensitive to DNase I digestion. We interpret these results to mean that the entire yeast genome exists in a state that represents a restricted proportion of total chromatin in higher eukaryotes.
TL;DR: The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain.
Abstract: The retardation and arrest of fermentation, observed before the complete sugar consumption of high-sugar grape must, come from an inhibition of the yeast metabolism during its decline phase and are variable with the strain. The addition of nutritional growth factors stimulates the initial growth of the yeast but is ineffective in the decline phase. Some substances, known previously as yeast anaerobic growth factors (sterols, oleanolic acid, oxytocin), in some conditions (initially aerated grape must and aerobically cultivated yeast) act by increasing the viability of the resting cells and prolonging their fermentation activity. These substances have been named “survival factors.”