Showing papers on "Yeast published in 1988"
TL;DR: A simple and convenient phase extraction assay for chloramphenicol (Cm) acetyltransferase (CAT) activity has been developed, based on the enzymatic butyrylation of radiolabelled Cm, which is highly sensitive, and substantially less expensive than all presently available alternatives.
923 citations
TL;DR: Immunofluorescence localization studies using affinity-purified antibody directed against the YPT1 protein are interpreted as suggesting that GTP-binding proteins may act to direct intracellular vesicle traffic.
657 citations
433 citations
TL;DR: An in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS).
Abstract: We have developed an in vitro system in which the posttranslational import of Put2 (delta-pyrroline-5-carboxylate dehydrogenase), into yeast mitochondria is dependent on the addition of yeast postribosomal supernatant (PRS). When mRNA for a nuclear-encoded yeast mitochondrial matrix protein, Put2, was translated in a wheat germ cell-free system, import into posttranslationally added yeast mitochondria was negligible. However, when a yeast PRS was added, significant import was observed. The import stimulating activity of the yeast PRS was shown to consist of at least two distinct factors. One of these is the recently purified 70-kD heat shock-related protein Ssalp/Ssa2p, two proteins that are 98% homologous. The other factor is an N-ethylmaleimide-sensitive protein(s). Both factors act synergistically.
368 citations
TL;DR: An extensive base-pairing interaction with U4 snRNA is described; the destabilization of the U4/U6 complex seen during splicing thus requires a large conformational change.
Abstract: The small nuclear RNA U6 and its gene have been isolated from yeast. In striking contrast to other yeast spliceosomal RNAs, U6 is very similar in size, sequence and structure to its mammalian homologue. The single-copy gene is essential. These properties suggest a central role in pre-mRNA processing. An extensive base-pairing interaction with U4 snRNA is described; the destabilization of the U4/U6 complex seen during splicing thus requires a large conformational change.
356 citations
311 citations
TL;DR: It is shown here that derivatives of the glucocorticoid receptor also enhance transcription when expressed in yeast, indicating that the receptor operates by a common, highly conserved mechanism in yeast and mammalian cells.
Abstract: In mammalian cells, the glucocorticoid receptor binds specifically to glucocorticoid response element (GRE) DNA sequences and enhances transcription from linked promoters. It is shown here that derivatives of the glucocorticoid receptor also enhance transcription when expressed in yeast. Receptor-mediated enhancement in yeast was observed in fusions of GRE sequences to the yeast cytochrome c1 (CYC1) promoter; the CYC1 upstream activator sequences were not essential, since enhancement was observed in fusions of GREs to mutant CYC1 promoters retaining only the TATA region and transcription startpoints. It is concluded that the receptor operates by a common, highly conserved mechanism in yeast and mammalian cells.
292 citations
TL;DR: Less variation in ammonia concentrations and microbial numbers suggest that ruminal fermentation was more stable in cows receiving yeast culture supplement, while rate of disappearance of cellulose in vitro was lower in cows received yeast.
279 citations
TL;DR: Stacked membranes closely associated with the nuclear envelope were observed in strains that over-produced the HMG1 isozyme, one of two isozymes for HMG-CoA reductase in yeast, establishing the existence of cellular mechanisms that monitor the levels of membrane proteins and compensate for changes in these levels by inducing synthesis of particular types of membrane.
Abstract: Overproduction of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in yeast resulted in striking morphological effects on the structure of intracellular membranes. Specifically, stacks of paired membranes closely associated with the nuclear envelope were observed in strains that over-produced the HMG1 isozyme, one of two isozymes for HMG-CoA reductase in yeast. These nuclear-associated, paired membranes have been named "karmellae." In strains that overproduced the HMG1 isozyme, HMG-CoA reductase was present in the karmellar layers. At mitosis, karmellae were asymmetrically segregated: the mother cells inherited all of the karmellae and the daughter cells inherited none. A membranous structure of different morphology was occasionally found in cells that overproduced the HMG2 isozyme. These observations further establish the existence of cellular mechanisms that monitor the levels of membrane proteins and compensate for changes in these levels by inducing synthesis of particular types of membrane.
279 citations
TL;DR: This chapter focuses on the purification and reconstitution of H + -ATPase from plasma membranes of Saccharomyces cerevisiae and arena sativa roots, which represents a novel type of proton pump.
Abstract: Publisher Summary This chapter focuses on the purification and reconstitution of H + -ATPase from plasma membranes of Saccharomyces cerevisiae and arena sativa roots The plasma membranes of fungi and plants contain a similar ATPase, which represents a novel type of proton pump Two convenient sources for these enzymes are bakers' yeast ( Saccharomyces cerevisiae ) and oat (Arena sativa) roots The purified plasma membrane ATPases from these organisms can be reconstituted into proteoliposomes that catalyze ATPdriven proton transport A summary of the purification of the yeast and oat root plasma membrane ATPases is shown in the chapter The evaluation of the purifications is complicated by the activating and inactivating effects of detergents Detergent activation results from unmasking latent enzyme molecules present in closed vesicles, while detergent inactivation may be caused by delipidation of the enzymes The purified plasma membrane ATPase from yeast and oat roots contain a major polypeptide of about 105 kDa, which represents more than 70–80% of the protein in the preparations Some minor polypeptides of lower molecular weight are usually present in variable and less than stoichiometric amounts
257 citations
TL;DR: Examination of the linking number distributions of plasmids bearing the inducible promoters of GAL1 and GAL10 genes indicates that the generation of supercoiled domains of opposite signs is related to transcription.
TL;DR: Results suggest that the metabolic pathways of furfural and 5-HMF are different depending on their entry in the fermentative route or in the oxidative one in the strains of yeasts studied.
TL;DR: Yeast strains that secrete functional mouse-human chimeric antibody and its Fab fragment into the culture medium are constructed and chimeric whole antibody exhibited antibody-dependent cellular cytot toxicity activity but not complement-dependent cytotoxicity activity.
Abstract: We have constructed yeast strains that secrete functional mouse-human chimeric antibody and its Fab fragment into the culture medium For chimeric whole antibody, cDNA copies of the chimeric light-chain and heavy-chain genes of an anti-tumor antibody were inserted into vectors containing the yeast phosphoglycerate kinase promoter, invertase signal sequence, and phosphoglycerate kinase polyadenylylation signal Simultaneous expression of these genes in yeast resulted in secretion of properly folded and assembled chimeric antibody that bound to target cancer cells Yeast chimeric antibody exhibited antibody-dependent cellular cytotoxicity activity but not complement-dependent cytotoxicity activity For production of Fab fragments, a truncated heavy-chain (Fd) gene was created by introducing a stop codon near the codon for the amino acid at which papain digestion occurs Simultaneous expression of the resulting chimeric Fd and light-chain genes in yeast resulted in secretion of properly folded and assembled Fab fragment that bound to target cancer cells
TL;DR: The DPM1 gene is essential for viability in yeast since disruption of the gene is lethal, and it is suspected Dol-P-Man synthase is not an essential protein due to its role in N-glycosylation since mutations in other genes that affect the late steps in lipid-linked oligosaccharide synthesis do not affect cell growth.
TL;DR: It is shown that the AP-1 recognition element (ARE) found in the SV40 enhancer can activate transcription from a heterologous promoter in S. cerevisiae, suggesting that the yeast and mammalianAP-1 are evolutionarily conserved and perhaps functionally related.
TL;DR: Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose, and both of the two recombinant cellulases were able to degrade amorphous cellulose.
TL;DR: A crucial role of intracellular pH in the growth control of animal cells is supported and a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase7 in fibroblasts is introduced, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells.
Abstract: A common early response of eukaryotic cells to stimuli which activate their proliferation is an increase in intracellular pH (ref. 1). In animal cells this is caused by the activation of an Na+/H+ exchange system2–5; in fungi and plants an H+-pumping ATPase6 is involved. The critical question is whether this intracellular alkalinization is merely coincident with the activation of cell proliferation or whether it is a regulatory signal2. To increase intracellular pH bypassing the usual physiological stimuli (growth factors, hormones etc.) alkaline media or ammonia have been used in the past2. Both approaches suffer from long-term toxicity effects and cannot be used in tumorigenic assays with whole organisms. We introduce here a more specific approach which involves expressing the gene for the yeast plasma membrane H+-ATPase7 in fibroblasts. The resulting cells have an elevated intracellular pH and acquire tumorigenic properties, suggesting that the yeast ATPase gene behaves as an oncogene in mammalian cells. These experiments support a crucial role of intracellular pH in the growth control of animal cells.
TL;DR: Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier, including 13 species of genera other than Saccharomyces.
Abstract: The mannoprotein which is a major component of the cell wall of Saccharomyces cerevisiae is an effective bioemulsifier. Mannoprotein emulsifier was extracted in a high yield from whole cells of fresh bakers' yeast by two methods, by autoclaving in neutral citrate buffer and by digestion with Zymolase (Miles Laboratories; Toronto, Ontario, Canada), a beta-1,3-glucanase. Heat-extracted emulsifier was purified by ultrafiltration and contained approximately 44% carbohydrate (mannose) and 17% protein. Treatment of the emulsifier with protease eliminated emulsification. Kerosene-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 5% sodium chloride or up to 50% ethanol in the aqueous phase. In the presence of a low concentration of various solutes, emulsions were stable to three cycles of freezing and thawing. An emulsifying agent was extracted from each species or strain of yeast tested, including 13 species of genera other than Saccharomyces. Spent yeast from the manufacture of beer and wine was demonstrated to be a possible source for the large-scale production of this bioemulsifier.
TL;DR: Examination of structure/activity relationships in the COOH-terminal regions of octapeptide substrates of yeast N-myristoyltransferase (NMT) suggests that either multiple NMT activities exist in rat liver or the yeast and rodent enzymes have similar but distinct peptide substrate specificities.
TL;DR: The effects of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Kloeckera apiculata, Candida stellata,candida krusei,Candida pulcherrima and Hansenula anomala were examined during mixed culture in grape juice.
Abstract: The effects of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Kloeckera apiculata, Candida stellata, Candida krusei, Candida pulcherrima and Hansenula anomala were examined during mixed culture in grape juice. At 25°C, pH 3.0 and pH 3.5, S. cerevisiae dominated the fermentation and the other species died off before fermentation was completed. Saccharomyces cerevisiae also dominated the fermentation at 20°C but there was increased growth and survival of the other species. At 10°C the fermentation was dominated by the growth of both S. cerevisiae and K. apiculata and there was extended growth and survival of C. stellata and C. krusei. Juices fermented at 10°C exhibited ethanol concentrations between 7.4 and 13.4% and populations of K. apiculata, C. stellata and C. krusei in the range 106-108 cells/ml. However, these species produced maximum ethanol concentrations in the range 2.7–6.6% when grown as single cultures in grape juice.
TL;DR: Using either permeabilized cells or microsomes the authors have reconstituted the early events of the yeast secretory pathway in vitro, and determined the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo.
Abstract: Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.
Book•
01 Jan 1988
TL;DR: Culture, storage, isolation and identification of yeasts Cytological methods Synchronous cultures and age fractionation Yeast genetics, molecular aspects and isolation of yeast nuclei and chromatin.
Abstract: Culture, storage, isolation and identification of yeasts Cytological methods Synchronous cultures and age fractionation Yeast genetics Yeast genetics, molecular aspects Isolation of yeast nuclei and chromatin for studies of transcription-related processes Isolation of yeast mRNA and in vitro translation in a yeast cell-free system Isolation and analysis of cell walls Yeast mitochondria Membranes and lipids of yeasts.
TL;DR: M mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo and may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.
Abstract: The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.
TL;DR: There is a remarkable conservation of many genes involved in eukaryotic cell cycle control and controlling elements in mammalian cells, and the power of yeast genetics for investigation of mammalian gene function is highlighted.
TL;DR: The gene for copper, zinc-superoxide dismutase from the yeast Saccharomyces cerevisiae has been cloned, sequenced, and shown to have physiological activity, and the role of Cu,Zn-SOD in defense against dioxygen toxicity is discussed in the light of these findings.
Abstract: The gene for copper, zinc-superoxide dismutase (Cu,Zn-SOD; EC 1.15.1.1) from the yeast Saccharomyces cerevisiae has been cloned, sequenced, and shown to have physiological activity. The gene was isolated from a lambda gt11 library by using a long, unique deoxyoligonucleotide probe. The probe sequence was deduced from the known amino acid sequence by using a computer-generated yeast codon preference table. The sequence of the coding and flanking regions is reported. The cloned gene was expressed and shown to be active in vivo. A 3.2-kilobase fragment containing the coding region and 160 upstream bases, subcloned in a yeast/Escherichia coli shuttle vector, was used to transform a yeast strain lacking Cu,Zn-SOD activity. The presence of the Cu,Zn-SOD gene-containing plasmid corrected the characteristic dioxygen sensitivity of this strain. Electrophoretic transfer blots with antibody to yeast Cu,Zn-SOD showed the presence of the protein in transformants and wild-type yeast but not in the mutant. The role of Cu,Zn-SOD in defense against dioxygen toxicity is discussed in the light of these findings.
TL;DR: Three approaches suggest that AGE exerts its effect by the oxidation of thiol groups present in the essential proteins, causing inactivation of enzymes and subsequent microbial growth inhibition.
Abstract: Summary: The mode of action of aqueous garlic extract (AGE) was studied in Candida albicans. The minimum inhibitory concentration (MIC) of AGE against six clinical yeast isolates ranged between 0.8 and 1.6 mg ml−1. Scanning electron microscopy and cell leakage studies showed that garlic treatment affected the structure and integrity of the outer surface of the yeast cells. Growth of C. albicans in the presence of AGE affected the yeast lipid in a number of ways: the total lipid content was decreased; garlic-grown yeasts had a higher level of phosphatidylserines and a lower level of phosphatidylcholines; in addition to free sterols and sterol esters, C. albicans accumulated esterified steryl glycosides; the concentration of palmitic acid (16:0) and oleic acid (18:1) increased and that of linoleic acid (18:2) and linolenic acid (18:3) decreased. Oxygen consumption of AGE-treated C. albicans was also reduced. The anticandidal activity of AGE was antagonized by thiols such as L-cysteine, glutathione and 2-mercaptoethanol. Interaction studies between AGE and thiols included growth antagonism, enzymic inhibition and interference of two linear zones of inhibition. All three approaches suggest that AGE exerts its effect by the oxidation of thiol groups present in the essential proteins, causing inactivation of enzymes and subsequent microbial growth inhibition.
TL;DR: Mouse liver cytochrome P-450 P1 was produced in the yeast Saccharomyces cerevisiae transformed by various expression vectors and found to be integrated into the microsomal membrane in a fully functional form, as judged by Western blotting, optical spectra and enzymatic activities.
TL;DR: Synthesis of polyols such as xylitol, arabitol, glycerol and ribitol reduced the ethanol yield in some instances and was related to the yeast strain, carbon source and oxygen availability, and in general, these yeasts fermented d-glucose more rapidly than d-xylose.
Abstract: The effect of oxygen availability on d-xylose and D-glucose metabolism by Pichia stipitis, Candida shehatae and Pachysolen tannophilus was investigated. Oxygen was not required for fermentation of d-xylose or d-glucose, but stimulated the ethanol production rate from both sugars. Under oxygen-limited conditions, the highest ethanol yield coefficient (Ye/s) of 0.47 was obtained on d-xylose with. P. stipitis, while under similar conditions C. shehatae fermented d-xylose most rapidly with a specific productivity (qpmax) of 0.32 h-1. Both of these yeasts fermented d-xylose better and produced less xylitol than. P. tannophilus. Synthesis of polyols such as xylitol, arabitol, glycerol and ribitol reduced the ethanol yield in some instances and was related to the yeast strain, carbon source and oxygen availability. In general, these yeasts fermented d-glucose more rapidly than d-xylose. By contrast Saccharomyces cerevisiae fermented d-glucose at least three-fold faster under similar conditions.
TL;DR: Experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.
Abstract: Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.