Showing papers on "Yeast published in 1984"

Transformation of intact yeast cells treated with alkali cations or thiol compounds

Abstract: When intact cells of Saccharomyces cerevisiae were treated with alkali cations or thiol compounds, the cells gained the ability to take up plasmid DNAs. The transformation efficiencies of yeast cells treated with alkali cations was greatly influenced by both the kind and concentration of cation used. The transformation efficiency also varied depending on the yeast strain. Polyethylene glycol was indispensable for the transformation. The uptake of plasmid DNAs into the yeast cells was found only in the presence of this polymer. Based on these results, the properties of transformation of intact yeast cells treated with alkali cations or thiol compounds were discussed.

A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance.

Abstract: Mutations at the URA3 locus of Saccharomyces cerevisiae can be obtained by a positive selection. Wild-type strains of yeast (or ura3 mutant strains containing a plasmid-borne URA3+ gene) are unable to grow on medium containing the pyrimidine analog 5-fluoro-orotic acid, whereas ura3- mutants grow normally. This selection, based on the loss of orotidine-5'-phosphate decarboxylase activity seems applicable to a variety of eucaryotic and procaryotic cells.

The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein.

Abstract: Ubiquitin1, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species2,3, from chromosomal histones4–6 to cytoplasmic proteins. Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes7,8. We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction. We have isolated the S. cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement. This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly–Met peptide bonds between the last and first residues of mature ubiquitin, respectively. Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences. As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.

NADH-linked aldose reductase: The key to anaerobic alcoholic fermentation of xylose by yeasts

Abstract: The kinetics and enzymology of o-xylose utilization were studied in aerobic and anaerobic batch cultures of the facultatively fermentative yeasts Candida utilis, Pachysolen tannophilus, and Pichia stipitis. These yeasts did not produce ethanol under aerobic conditions. When shifted to anaerobiosis cultures of C. utilis did not show fermentation of xylose; in Pa. tannophilus a very low rate of ethanol formation was apparent, whereas with Pi. stipitis rapid fermentation of xylose occurred. The different behaviour of these yeasts ist most probably explained by differences in the nature of the initial steps of xylose metabolism: in C. utilis xylose is metabolized via an NADPH-dependent xylose reductase and an NAD+-linked xylitol dehydrogenase. As a consequence, conversion of xylose to ethanol by C. utilis leads to an overproduction of NADH which blocks metabolic activity in the absence of oxygen. In Pa. tannophilus and Pi. stipitis, however, apart from an NADPH-linked xylose reductase also an NADH-linked xylose reductase was present. Apparently xylose metabolism via the NADH-dependent reductase circumvents the imbalance of the NAD+/NADH redox system, thus allowing fermentation of xylose to ethanol under anaerobic conditions. The finding that the rate of xylose fermentation in Pa. tannophilus and Pi. stipitis corresponds with the activity of the NADH-linked xylose reductase activity is in line with this hypothesis. Furthermore, a comparative study with various xylose-assimilating yeasts showed that significant alcoholic fermentation of xylose only occurred in those organisms which possessed NADH-linked aldose reductase

Evolution of yeasts and lactic Acid bacteria during fermentation and storage of bordeaux wines.

Abstract: The levels of yeasts and lactic acid bacteria that naturally developed during the vinification of two red and two white Bordeaux wines were quantitatively examined. Yeasts of the genera Rhodotorula, Pichia, Candida, and Metschnikowia occurred at low levels in freshly extracted grape musts but died off as soon as fermentation commenced. Kloeckera apiculata (Hanseniaspora uvarum), Torulopsis stellata, and Saccharomyces cerevisiae, the dominant yeasts in musts, proliferated to conduct alcoholic fermentation. K. apiculata and eventually T. stellata died off as fermentation progressed, leaving S. cerevisiae as the dominant yeast until the termination of fermentation by the addition of sulfur dioxide. At least two different strains of S. cerevisiae were involved in the fermentation of one of the red wines. Low levels of lactic acid bacteria (Pediococcus cerevisiae, Leuconostoc mesenteroides, and Lactobacillus spp.) were present in grape musts but died off during alcoholic fermentation. The malolactic fermentation developed in both red wines soon after alcoholic fermentation and correlated with the vigorous growth of at least three different strains of Leuconostoc oenos.

Copper metallothionein of yeast, structure of the gene, and regulation of expression

Abstract: Addition of copper to yeast cells leads to the induction of a low molecular weight, cysteine-rich protein that binds copper. This protein, termed copper chelatin or thionein, is related to the metallothionein family of proteins that are induced in response to cadmium and zinc in vertebrate cells. We have determined the structure of the yeast copper-binding protein by DNA sequence analysis of the gene. Although the 6573-dalton yeast protein is substantially divergent from vertebrate metallothioneins, the arrangement of 12 cysteine residues, which is a hallmark of metal-binding proteins, is partially conserved. We analyzed the regulatory DNA sequence of the gene by fusing it with the Escherichia coli galactokinase gene and assaying the levels of enzyme activity in yeast in response to copper. The transcriptional activation has a specific requirement for copper. Zinc, cadmium, and gold were unable to regulate the galactokinase activity. The yeast copper metallothionein regulatory sequences represent a previously unreported class of yeast promoter that is regulated by copper.

Primary structure and transcription of an amplified genetic locus: the CUP1 locus of yeast.

Abstract: Copper resistance in yeast is controlled by the CUP1 locus. The level of resistance is proportional to the copy number of this locus, which can be found in up to 15 tandemly iterated copies. To elucidate the molecular mechanisms controlling the amplification and expression of the CUP1, locus, we determined its full nucleotide sequence. We have also identified and mapped two transcription units within the basic amplification unit of CUP1 in laboratory yeast strains. One of those transcription units is inducible by copper and encodes a low molecular weight copper binding protein--copper chelatin. The increased production of chelatin, due to both gene amplification and induction of transcription, leads to increased resistance of yeast cells to copper ions.

High-gravity brewing: effects of nutrition on yeast composition, fermentative ability, and alcohol production.

Abstract: A number of economic and product quality advantages exist in brewing when high-gravity worts of 16 to 18% dissolved solids are fermented Above this level, production problems such as slow or stuck fermentations and poor yeast viability occur Ethanol toxicity has been cited as the main cause, as brewers' yeasts are reported to tolerate only 7 to 9% (vol/vol) ethanol The inhibitory effect of high osmotic pressure has also been implicated In this report, it is demonstrated that the factor limiting the production of high levels of ethanol by brewing yeasts is actually a nutritional deficiency When a nitrogen source, ergosterol, and oleic acid are added to worts up to 31% dissolved solids, it is possible to produce beers up to 162% (vol/vol) ethanol Yeast viability remains high, and the yeasts can be repitched at least five times Supplementation does not increase the fermentative tolerance of the yeasts to ethanol but increases the length and level of new yeast cell mass synthesis over that seen in unsupplemented wort (and therefore the period of more rapid wort attenuation) Glycogen, protein, and sterol levels in yeasts were examined, as was the importance of pitching rate, temperature, and degree of anaerobiosis The ethanol tolerance of brewers' yeast is suggested to be no different than that of sake or distillers' yeast

Alcoholic Fermentation of d-Xylose by Yeasts

Abstract: Type strains of 200 species of yeasts able to ferment glucose and grow on xylose were screened for fermentation of d-xylose. In most of the strains tested, ethanol production was negligible. Nineteen were found to produce between 0.1 and 1.0 g of ethanol per liter. Strains of the following species produce more than 1 g of ethanol per liter in the fermentation test with 2% xylose: Brettanomyces naardenensis, Candida shehatae, Candida tenuis, Pachysolen tannophilus, Pichia segobiensis, and Pichia stipitis. Subsequent screening of these yeasts for their capacity to ferment d-cellobiose revealed that only Candida tenuis CBS 4435 was a good fermenter of both xylose and cellobiose under the test conditions used.

A bacterial repressor protein or a yeast transcriptional terminator can block upstream activation of a yeast gene

Abstract: A bacterial repressor protein blocks transcription of a gene in yeast when its operator is placed in the promoter between the upstream activator sequence and the transcription start point. Putative transcription terminators from yeast installed in the same region of the promoter have a similar effect.

Economic evaluation of alternative ethanol fermentation processes

Abstract: Eleven alternative fermentation schemes for ethanol production are compared. Conventional batch, continuous, cell recycle, and immobilized cell processes, as well as membrane, extraction, and vacuum processes which remove ethanol from the broth selectively as it is produced, are considered. The processes are compared on identical bases using a consistent model for the yeast metabolism. Both molasses and cellulose hydrolyzate are considered as feeds. Optimized ethanol plants, including feed preparation, fermentation, and product recovery sections are designed and total costs are projected.

Xylose fermentation by yeasts

Abstract: Utilization and fermentation of xylose by the yeasts Pachysolen tannophilus I fGB 0101 and Pichia stipitis 5773 to 5776 under aerobic and anaerobic conditions are investigated. Pa. tannophilus requires biotin and thiamine for growth, whereas Pi. stipitis does not, and growth of both yeasts is stimulated by yeast extract. Pi. stipitis converts xylose (30 g/l) to ethanol under anaerobic conditions with high yields of 0,40 and it produces only low amounts of xylitol. The yield coefficient is further increased at lower xylose concentrations.

Requirement of either of a pair of ras-related genes of Saccharomyces cerevisiae for spore viability

Abstract: Cells of the yeast, Saccharomyces cerevisiae, containing disruptions of either of two genes that are members of the ras oncogene family are viable, but haploid yeast spores carrying disruptions of both genes fail to grow.

Inhibition of alcoholic fermentation of grape must by Fatty acids produced by yeasts and their elimination by yeast ghosts.

Abstract: In a complete nutritive medium rich in sugar, such as grape must, the inhibition of alcoholic fermentation is caused by substances produced by the yeast which, acting synergistically with ethanol, are toxic to the yeasts themselves. Among these are decanoic and octanoic acids and their corresponding ethyl esters. Their adsorption by yeast ghosts permits the prevention and treatment of fermentation stoppages.

Glucan compositions and process for preparation thereof

Abstract: Three dimensional glucan matrix compositions are prepared by separating growing Saccharomyces cerevisiae yeast from its growth medium, subjecting the yeast with cell walls intact to aqueous hydroxide and treating the insoluble glucan with acetic acid to alter the β(1-6) linkages. The glucans have viscosity characteristics dependent upon the strain of Saccharomyces cerevisiae utilized and are useful as stabilizer or thickeners.

In vitro binding of Candida albicans yeast cells to human fibronectin

Abstract: The binding of Candida albicans yeast cells to human fibronectin (Fn), a major glycoprotein of mammalian cells, was studied using an in vitro assay. Adherence was quantitated in microtiter dishes coated with Fn to which radiolabeled yeast cells were added. Under optimum conditions of the assay, i.e., 1 mM CaCl2 and 70 μg Fn protein, approximately 40% of the radiolabeled yeast cells adhered to the Fn. Adherence to Fn was greater at 30 °C than at 4 °C and was greater with viable yeast cells than with heat-killed cells. Candida albicans (two strains) and C. tropicalis adhered to Fn to a greater extent than C. pseudotropicalis, C. krusei, or Saccharomyces cerevisiae. Pretreatment of C. albicans with chymotrypsin, pronase, or papain, but not pepsin, decreased adherence to Fn. Blocking experiments using mannan, sugars, or amino sugars were carried out by preabsorbing the Fn with each of the above-mentioned compounds. Candida mannan blocked adherence of C. albicans to Fn. The mannan effect was dose dependent. Ho...

Continuous measurement of ethanol production by aerobic yeast suspensions with an enzyme electrode

Abstract: An alcohol electrode was constructed which consisted of an oxygen probe onto which alcohol oxidase was immobilized. This enzyme electrode was used, in combination with a reference oxygen electrode, to study the short-term kinetics of alcoholic fermentation by aerobic yeast suspensions after pulsing with glucose. The results demonstrate that this device is an excellent tool in obtaining quantitative data on the short-term expression of the Crabtree effect in yeasts. Samples from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae not producing ethanol, immediately (within 2 min) exhibited aerobic alcoholic fermentation after being pulsed with excess glucose. With chemostat-grown Candida utilis, however, ethanol production was not detectable even at high sugar concentrations. The Crabtree effect in S. cerevisiae was studied in more detail with commercial baker's yeast. Ethanol formation occurred only at initial glucose concentrations exceeding 150 mg·l-1, and the rate of alcoholic fermentation increased with increasing glucose concentrations up to 1,000 mg·l-1 glucose. Similar experiments with batch cultures of certain ‘non-fermentative’ yeasts revealed that these organisms are capable of alcoholic fermentation. Thus, even under fully aerobic conditions, Hansenula nonfermentans and Candida buffonii produced ethanol after being pulsed with glucose. In C. buffonii ethanol formation was already apparent at very low glucose concentrations (10 mg·l-1) and alcoholic fermentation even proceeded at a higher rate than in S. cerevisiae. With Rhodotorula rubra, however, the rate of ethanol formation was below the detection limit, i.e., less than 0.1 mmol·g cells-1·h-1.

Testing of chemicals for genetic activity with Saccharomyces cerevisiae: a report of the U.S. Environmental Protection Agency Gene-Tox Program.

Abstract: The yeast Saccharomyces cerevisiae is a unicellular fungus that can be cultured as a stable haploid or a stable diploid . Diploid cultures can be induced to undergo meiosis in a synchronous fashion under well-defined conditions. Consequently, yeasts can be used to study genetic effects both in mitotic and in meiotic cells. Haploid strains have been used to study the induction of point mutations. In addition to point mutation induction, diploid strains have been used for studying mitotic recombination, which is the expression of the cellular repair activities induced by inflicted damage. Chromosomal malsegregation in mitotic and meiotic cells can also be studied in appropriately marked strains. Yeast has a considerable potential for endogenous activation, provided the tests are performed with appropriate cells. Exogenous activation has been achieved with S9 rodent liver in test tubes as well as in the host-mediated assay, where cells are injected into rodents. Yeast cells can be recovered from various organs and tested for induced genetic effects. The most commonly used genetic end point has been mitotic recombination either as mitotic crossing-over or mitotic gene conversion. A number of different strains are used by different authors. This also applies to haploid strains used for monitoring induction of point mutations. Mitotic chromosome malsegregation has been studied mainly with strain D6 and meiotic malsegregation with strain DIS13 . Data were available on tests with 492 chemicals, of which 249 were positive, as reported in 173 articles or reports. The genetic test/carcinogenicity accuracy was 0.74, based on the carcinogen listing established in the Gene-Tox Program. The yeast tests supplement the bacterial tests for detecting agents that act via radical formation, antibacterial drugs, and other chemicals interfering with chromosome segregation and recombination processes.

Isolation of the nuclear yeast genes for citrate synthase and fifteen other mitochondrial proteins by a new screening method.

Abstract: To isolate nuclear genes specifying imported mitochondrial proteins, a yeast genomic clone bank was screened by an RNA hybridization-competition assay. This assay exploited the fact that mRNAs for imported mitochondrial proteins are enriched in polysomes which are bound to the mitochondrial surface in cycloheximide-inhibited yeast cells. Clones selectively hybridizing to these enriched mRNAs were further screened by hybrid-selected translation and immunoprecipitation with monospecific antisera against individual mitochondrial proteins. Thirty-six clones were isolated which contained complete or partial copies of 16 different genes for imported mitochondrial proteins. Several of these clones caused expression of the corresponding precursor polypeptide in Escherichia coli or over-expression of the corresponding mature protein in yeast. The gene for the matrix enzyme citrate synthase was sequenced; the derived amino acid sequence of the precursor polypeptide revealed an amino-terminal extension containing basic but no acidic residues.

Secretion of a wheat α-amylase expressed in yeast

Abstract: The translocation of secretory proteins across the endoplasmic reticulum involves the recognition and cleavage of an amino-terminal extension called the signal sequence1. The structure of signal peptides appears to be ubiquitous in having a very hydrophobic central core2, so that the signal sequence in secretory proteins from one organism could possibly be recognized by the processing and transport apparatus of another. We therefore wished to investigate whether a protein, α-amylase, one of several hydrolytic enzymes secreted from the aleurone of wheat into the endosperm during germination, could be processed and secreted in an active form from the yeast Saccharomyces cerevisiae, secretion being dependent upon the plant signal sequence. Here, synthesis of α-amylase was by inserting a cDNA clone coding for the entire α-amylase structural gene3 into a yeast expression vector4. The α-amylase protein coded for by this gene fusion has the signal sequence located internally, not at the N-terminal end of the polypeptide. Nevertheless, it is processed and the processed form is secreted into the medium in an active form. There are potential industrial applications for yeast that secrete a functional α-amylase.

Yeast expression systems with vectors having a gapdh promoter, and synthesis of foreign proteins

Abstract: Yeast cells containing DNA plasmids having foreign DNA, wherein foreign DNA is expressed, are described. For example, DNA coding for hepatitis B and its virus surface antigen (HBsAg) is ligated to a yeast plasmid to yield a product that is used to transform yeast cells. the plasmids of this invention have either GAPDH or PyK promoters, and are capable of replicating in either a yeast cell or a bacterial cell.

Method for expressing foreign genes in schizosaccharomyces pombe and the use in therapeutic formulations of the products, DNA constructs and transformant strains of schizosaccharomyces pombe usable in such method and their preparation

Abstract: A method is provided for producing heterologous polypeptides in yeast Schizosaccharomyces pombe. DNA constructs are provided for use in this method comprising a segment coding for an S. pombe promoter. A particular construct is provided comprising the S. pombe alcohol dehydrogenase promoter and human alpha-1-antitrypsin gene, which is used to transform S. pombe to produce a substantially unglycosylated protein having activity of human alpha-1-antitrypsin.

Nucleotide sequence of two rasH related-genes isolated from the yeast Saccharomyces cerevisiae

Abstract: A complete nucleotide sequence of two ras-related yeast genes (c- rassc -1 and c- rassc -2) isolated from the yeast strain Saccharomyces cerevisiae is reported. They encode predicted polypeptides of 40,000 and 41,000 daltons, respectively. The N-terminal 170 amino acids from both genes show extensive amino acid homology to other ras genes from vertebrates, whereas their C-termini have diverged. These genes should be useful in the elucidation of a normal biological function of ras-related genes in a simple system like yeast.

Hygromycin B resistance as dominant selectable marker in yeast.

Abstract: Saccharomyces cerevisiae is normally sensitive to the drug hygromycin B; a hygromycin B concentration of 200 µg/ml in agar plates is sufficient to completely inhibit growth. We constructed yeast-E. coli bifunctional plasmids which confer hygromycin B resistance to Saccharomyces cerevisiae. Promoters and amino terminal coding regions of a heat shock gene, a heat shock cognate gene, and the phosphoglycerate kinase gene from yeast were fused to a bacterial hygromycin B resistance gene. In all three cases, yeast cells containing plasmids with the hybrid hygromycin B resistance gene were resistant to high levels of the drug. Yeast cells containing these plasmids can also be directly selected after transformation by using hygromycin B. The intact bacterial hygromycin B resistance gene and the kanamycin resistance gene from Tn903 were also tested in yeast for their ability to confer resistance to hygromycin B and G418. The intact bacterial genes were not effective in conferring drug resistance to yeast cells.