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  • Saccharomyces cerevisiae  (734)
  • Springer  (734)
  • Nature Publishing Group
  • American Chemical Society (ACS)
  • 1
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    The journal of membrane biology 116 (1990), S. 93-105 
    ISSN: 1432-1424
    Keywords: clathrin ; genetics ; Saccharomyces cerevisiae ; exocytosis ; endocytosis ; prohormone maturation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 2
    ISSN: 1432-1424
    Keywords: vacuole ; lipid bilayer ; K-channel ; single channel ; DIDS ; yeast ; Saccharomyces cerevisiae ; Ca2+ activation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary A voltage-dependent and Ca2+-activated cation channel found in the vacuolar membrane of the yeast,Saccharomyces cerevisiae, was incorporated into planar lipid bilayer and its gating characteristics were studied at the macroscopic and single-channel levels. The open-channel probability at steady state, which was estimated by the macroscopic current measurement, gave a maximum value at −10 mV and decreased in a graded fashion as the voltage became more positive or more negative. The steady-state voltage dependence was explained by assuming two independent gates, which had different rate constants and opposite voltage dependence. The fast-responding gate opened when the voltage of thecis side (the side to which the vesicles were added) was made more negative and the slow-responding gate behaved in the opposite direction. Relatively high concentrations of Ca2+, about 1mm, were required on thecis side for opening the slow gate in a voltage-dependent manner. DIDS increased the open-channel probability of the fast gate when added to thecis side, but was ineffective on the slow gate.
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  • 3
    ISSN: 1432-1424
    Keywords: electron probe X-ray microanalysis ; Saccharomyces cerevisiae ; ethidium ; brontophenol blue ; cationic dye ; cytolysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary K+ efflux provoked by ethidium proceeds partially as an all-or-none effect by which the diffusion barrier for K+ is disrupted and partially from still intact cells, presumably by exchange against ethidium. This is shown by the application of an electron probe microanalysis X-ray technique by which the K+ content of a number of individual cells is analyzed.
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  • 4
    ISSN: 1432-1432
    Keywords: Thiolase ; Peroxisome evolution ; Bootstrap analysis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The thiolase family is a widespread group of proteins present in prokaryotes and three cellular compartments of eukaryotes. This fact makes this family interesting in order to study the evolutionary process of eukaryotes. Using the sequence of peroxisomal thiolase from Saccharomyces cerevisiae recently obtained by us and the other known thiolase sequences, a phylogenetic analysis has been carried out. It shows that all these proteins derived from a primitive enzyme, present in the common ancestor of eubacteria and eukaryotes, which evolved into different specialized thiolases confined to various cell compartments. The evolutionary tree obtained is compatible with the endosymbiotic theory for the origin of peroxisomes.
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  • 5
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    Journal of molecular evolution 38 (1994), S. 363-368 
    ISSN: 1432-1432
    Keywords: Saccharomyces cerevisiae ; 2-μm circle ; DNA sequencing ; Horizontal transmission ; Site-specific recombination ; Selfish DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 μm) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-μm variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-μm plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection.
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  • 6
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    Cellular and molecular life sciences 40 (1984), S. 1159-1161 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; 5-trifluoromethyl-6-àzauracil ; yeast cell cultures ; cell division ; inhibition of
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Cell division, as studied in asynchronous cultures of yeast cells, is sensitive to 5-trifluoromethyl-6-azauracil (F3CAzU). Under defined conditions (10 mmoles l−1 F3CAzU) this compound blocks immediately and completely the process of cell division. Using synchronized cells, the time-point at which division process of yeast cell can be inhibited by F3CAzU has been determined. The inhibitor effect of this compound is completely reversed by thymine, thymidine and uracil.
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  • 7
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    Cellular and molecular life sciences 46 (1990), S. 193-200 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; protein toxin ; yeast toxin precursor ; protease processing ; lectin ; (1→6)-β-D-glucan ; receptor ; resistant mutants ; spheroplasts ; ion-permeable channels ; site-directed mutagenesis ; toxin functional domains
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The K1 killer toxin ofSaccharomyces cerevisiae is a secreted, virally-coded protein lethal to sensitive yeasts. Killer yeasts are immune to the toxin they produce. This killer system has been extensively examined from genetic and molecular perspectives. Here we review the biology of killer yeasts, and examine the synthesis and action of the protein toxin and the immunity component. We summarise the structure of the toxin precursor gene and its protein products, outline the proteolytic processing of the toxin subunits from the precursor, and their passage through the yeast secretory pathway. We then discuss the mode of action of the toxin, its lectin-like interaction with a cell wall glucan, and its probable role in forming channels in the yeast plasma membrane. In addition we describe models of how a toxin precursor species functions as the immunity component, probably by interfering with channel formation. We conclude with a review of the functional domains of the toxin structural gene as determined by site-directed mutagenesis. This work has identified regions associated with glucan binding, toxin activity, and immunity.
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  • 8
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    Cellular and molecular life sciences 48 (1992), S. 1162-1164 
    ISSN: 1420-9071
    Keywords: Polygodial ; warburganal ; antifungal activity ; Candida albicans ; Saccharomyces cerevisiae ; Pityrosporum ovale ; enhancing effect ; antioxidants ; vitamin C ; BHA ; anethole
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The antifungal activity of two drimane sesquiterpene dialdehydes, polygodial (1) and warburganal (2), alone and in combination with several other substances, was examined against three fungi,Candida albicans, Saccharomyces cerevisiae andPityrosporum ovale employing a broth dilution method. Anethole significantly synergized the activity of the two sesquiterpenoids againstC. albicans andS. cerevisiae however, it had only an, additive effect againstP. ovale. By contrast, two antioxidants, ascorbic acid (vitamin C) and BHA (butylated hydroxyanisole), noticeably enhanced the activity of the sesquiterpenoids againstP. ovale, but had no, effect againstC. albicans andS. cerevisiae.
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  • 9
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    Cellular and molecular life sciences 52 (1996), S. 1111-1116 
    ISSN: 1420-9071
    Keywords: Mitochondria ; mitochondrial inheritance ; cytoskeleton ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; membrane proteins ; organelle movement ; mitochondrial morphology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mechanisms mediating the inheritance of mitochondria are poorly understood, but recent studies with the yeastsSaccharomyces cerevisiae andSchizosaccharomyces pombe have begun to identify components that facilitate this essential process. These components have been identified through the analysis of conditional yeast mutants that display aberrant mitochondrial distribution at restrictive conditions. The analysis of these mutants has uncovered several novel proteins that are localized either to cytoskeletal structures or to the mitochondria themselves. Many mitochondrial inheritance mutants also show altered mitochondrial morphology and defects in maintenance of the mitochondrial genome. Although some inheritance components and mechanisms appear to function specifically in certain types of cells, other conserved proteins are likely to mediate mitochondrial behavior in all eukaryotic cells.
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  • 10
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; mitochondrial ribosomes ; peptidyl transferase ; Varl ribosomal protein ; gene relocation ; posttranscriptional rRNA modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitochondria posses their own ribosomes responsible for the synthesis of a small number of proteins encoded by the mitochondrial genome. In yeast,Saccharomyces cerevisiae, the two ribosomal RNAs and a single ribosomal protein, Varl, are products of mitochondrial genes, and the remaining approximately 80 ribosomal proteins are encoded in the nucleus. The mitochondrial translation system is dispensable in yeast, providing an excellent experimental model for the molecular genetic analysis of the fundamental properties of ribosomes in general as well as adaptations required for the specialized role of ribosomes in mitochondria. Recent studies of the peptidyl transferase center, one of the most highly conserved functional centers of the ribosome, and the Varl protein, an unusual yet essential protein in the small ribosomal subunit, have provided new insight into conserved and divergent features of the mitochondrial ribosome.
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  • 11
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    Cellular and molecular life sciences 43 (1987), S. 888-890 
    ISSN: 1420-9071
    Keywords: Thiaminase ; thiamine ; thiamine antagonist ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary It was found that cell-free extracts ofSaccharomyces cerevisiae contain thiaminase II which hydrolyzes thiamine and thiamine analogs. The possible involvement of this enzyme and thiamine-synthesizing enzymes in thiamine production from thiamine antagonists is discussed.
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  • 12
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; growth conditions ; kinaseless mutant ; plasma membrane vesicles ; glucose transport ; kinetics and computer simulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In this study experimental data on the kinetic parameters investigated by other authors1–5, 11 together with own data on plasma membrane vesicles, have been subjected to a computer simulation based on the equations describing facilitated diffusion. The simulation led to an ideal fit describing the above data. From this it can be concluded that glucose is transported by facilitated diffusion, and not by active transport as was postulated by Van Steveninck14, 15. The simulation method also demonstrates that the fast sampling technique used by these authors1–5,11 underestimates the fluxes. Thus, the parameters given do not contribute to the understanding of glucose transport under different metabolic conditions. The K value of plasma membrane vesicles prepared from glucose-repressed cells is around 7 mM. Derepression, particularly by galactose, causes a highly significant increase in affinity as shown by a decrease in the K value to 2 mM. The highest affinity was measured in a triple kinaseless mutant grown on glycerol with a K value of 1 mM. If seems, therefore, that the kinetic parameters derived from initial uptake rates of glucose in intact cells1–5,11 using single flux analysis, such as Eadie-Hofstee- or Lineweaver-Burk-plots, are in error.
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  • 13
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    Cellular and molecular life sciences 52 (1996), S. 1033-1041 
    ISSN: 1420-9071
    Keywords: Ubiquitin ; yeast ; Saccharomyces cerevisiae ; Dictyostelium discoideum ; cytoskeleton ; mutants ; endocytosis ; actin ; myosin ; calmodulin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Endocytosis is a general term that is used to describe the internalization of external and plasma membrane molecules into the cell interior. In fact, several different mechanisms exist for the internalization step of this process. In this review we emphasize the work on the actin-dependent pathways, in particular in the yeastSaccharomyces cerevisiae, because several components of the molecular machinery are identified. In this yeast, the analysis of endocytosis in various mutants reveals a requirement for actin, calmodulin, a type I myosin, as well as a number of other proteins that affect actin dynamics. Some of these proteins have homology to proteins in animal cells that are believed to be involved in endocytosis. In addition, the demonstration that ubiquitination of some cell surface molecules is required for their efficient internalization is described. We compare the actin, myosin and ubiquitin requirements for endocytosis with recent results found studying these processes usingDictyostelium discoideum and animal cells.
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  • 14
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    Cellular and molecular life sciences 52 (1996), S. 1130-1135 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; mitochondria ; mRNA-specific translational activation ; synthetic genes ; gene regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Mitochondrial gene expression in yeast,Saccharomyces cerevisiae, depends on translational activation of individual mRNAs by distinct proteins encoded in the nucleus. These nuclearly coded mRNA-specific translational activators are bound to the inner membrane and function to mediate the interaction between mRNAs and mitochondrial ribosomes. This complex system, found to date only in organelles, appears to be an adaptation for targeting the synthesis of mitochondrially coded integral membrane proteins to the membrane. In addition, mRNA-specific translational activation is a rate-limiting step used to modulate expression of at least one mitochondrial gene in response to environmental conditions. Direct study of mitochondrial gene regulation and the targeting of mitochondrially coded proteins in vivo will now be possible using synthetic genes inserted into mtDNA that encode soluble reporter/passenger proteins.
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  • 15
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    Cellular and molecular life sciences 43 (1987), S. 886-888 
    ISSN: 1420-9071
    Keywords: Saccharomyces cerevisiae ; trichothecenes ; mycotoxins ; vitamins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Several trichothecene mycotoxins were shown to inhibit the growth ofSaccharomyces cerevisiae. This effect was most pronounced with the macrocyclic trichothecenes, especially verrucarin A. Much less growth inhibition was observed with T-2 toxin. Verrucarol, diacetoxyscirpenol, acetyl T-2 toxin, HT-2 toxin, T-2 tetraol and neosolaniol were inactive at a concentration of 75 μg of toxin per disc. Incubation ofS. cerevisiae with verrucarin A together with vitamins resulted in a decrease in toxicity. Pyridoxine-HCl, Ca-pantothenate, thiamine-HCl and α-tocopheryl acetate were amongst the most potent of the vitamins tested which reversed growth inhibition, overcoming the inhibitory potential of the toxins.
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  • 16
    ISSN: 1423-0127
    Keywords: Acquired immunodeficiency syndrome ; Human immunodeficiency virus ; Nef protein ; Myristylation ; Membrane permeabilisation ; Saccharomyces cerevisiae ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.
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  • 17
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    Archives of microbiology 110 (1976), S. 313-318 
    ISSN: 1432-072X
    Keywords: Yeast protoplasts ; Saccharomyces cerevisiae ; Conjugation ; Cell wall ; Morphogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.
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  • 18
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    Archives of microbiology 114 (1977), S. 91-92 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Cell wall ; Glucan ; Mannan ; Synchronous culture
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The mode of increase in cell wall polysaccharides of yeast (glucan and mannan) during cell cycle was analyzed using cell wall samples obtained from a synchronous culture. The increase in mannan and total glucan proceeded almost linearly throughout the cell cycle except for a short period of their leveling off at the time of cell division. However, the constituents of glucan behaved characteristically: Alkalisoluble glucan and insoluble glucan increased mainly in the former and the latter half of the cell cycle, respectively.
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  • 19
    ISSN: 1432-072X
    Keywords: Ethanol inhibition ; Solute accumulation ; Saccharomyces cerevisiae ; Plasma membrane ; Lipids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Incorporation of ethanol (1.0 or 1.25 M) into exponential-phase cultures of Saccharomyces cerevisiae NCYC 366 growing anaerobically in a medium supplemented with ergosterol and an unsaturated fatty acid caused a retardation in growth rate, which was greater when the medium contained oleic rather than linoleic acid. Ethanol incorporation led to an immediate drop in growth rate, and ethanol-containing cultures grew at the slower rate for at least 10 h. Incorporation of ethanol (0.5 M) into buffered (pH 4.5) cell suspensions containing d-[6-3H] glucose, d-[1-14C] glucosamine, l-[U-14C] lysine or arginine, or KH2 32PO4 lowered the rate of solute accumulation by cells. Rates of accumulation of glucose, lysine and arginine were retarded to a greater extent when cells had been grown in the presence of oleic rather than linoleic acid. This difference was not observed with accumulation of phosphate. Ethanol was extracted from exponential-phase cells by four different methods. Cells grown in the presence of linoleic acid contained a slightly, but consistently, lower concentration of ethanol than cells grown in oleic acid-containing medium. The ethanol concentration in cells was 5–7 times greater than that in the cell-free medium.
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  • 20
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Catabolite repression and inactivation ; Inhibition of protease B
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Catabolite inactivation of fructose-1,6-bisphosphatase, isocitrate lyase, phosphoenolpruvate carboxykinase and malate dehydrogenase in intact cells could be prevented by phenylmethylsulfonyl fluoride added 40 min prior to the addition of glucose. Protein synthesis, fermentative and respiratory activity and catabolite repression were not affected. Elimination of catabolite inactivation by the addition of PMSF revealed that catabolite repression started at different times for different enzyme.
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  • 21
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    Archives of microbiology 128 (1980), S. 157-161 
    ISSN: 1432-072X
    Keywords: Lactobacillus brevis ; Streptococcus lactis ; Saccharomyces cerevisiae ; Concanavalin A ; Symbiosis ; Tibi
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tibi grains consist of a unique and very stable symbiotic association of Lactobacillus brevis, Streptococcus lactis and Saccharomyces cerevisiae embedded in a dextran matrix. The structural organization of the grain was examined by light, scanning and transmission electron microscopy. The grain was constituted of an outer compact layer and an inner spongy structure. The outer layer was more densely populated by the microorganisms than the inner layer but dextran, stained on frozen thin sections with fluorescein-conjugated concanavalin A, was more abundant in the inner layer.
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  • 22
    ISSN: 1432-072X
    Keywords: a Pheromone ; α Pheromone ; Hansenula wingei ; Inducible mutant ; Saccharomyces cerevisiae ; Saccharomyces kluyveri ; Sexual agglutinability ; Shmoo ; Synthetic analogues
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three analogues of the peptidyl pheromone, α pheromone of Saccharomyces kluyveri, synthesized based on the amino acid sequence proposed by Sato et al. (Agric Biol Chem 45:1531–1533, 1981) were tested for both shmoo-inducing and agglutinability-inducing actions. Purified natural α pheromone of the yeast showed the highest activity among the peptides tested. When methionine in the peptides was oxidized, the activity decreased significatly. α Pheromone of S. kluyveri induced sexual agglutinability in a cells of Saccharomyces cerevisiae, and shmoo in a cells of S. cerevisiae and S. kluyveri. a Pheromone of S. kluyveri had no agglutinability-inducing action on α cells of S. cerevisiae. a Cells of S. kluyveri inactivated only α pheromone of the same species, but a cells of S. cerevisiae inactivated α pheromones of both S. cerevisiae and S. kluyveri.
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  • 23
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    Archives of microbiology 132 (1982), S. 236-240 
    ISSN: 1432-072X
    Keywords: α Pheromone ; Cycloheximide ; Inducible a strain ; Phenylmethylsulfonyl fluoride ; Saccharomyces cerevisiae ; Sexual agglutinability ; Temperature-sensitive
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When α pheromone-pretreated cells of an inducible a strain of Saccharomyces cerevisiae carrying the inducible gene saa1 were incubated in a growth medium at 28°C, induction of sexual agglutinability began after a 10 min lag period. If the cells were incubated at 38°C during the lag period, no induction occurred even after incubation at 28°C. Contrary to this, if the cells were incubated at 28°C during the lag period, almost complete induction occurred, even after transfer to 38°C. Temperature shift experiments revealed that 5 min incubation at 28°C was necessary for the initiation of the temperature-sensitive period and further 5 min incubation for the completion of the period. The temperature-sensitive period was sensitive to phenylmethylsulfonyl fluoride.
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  • 24
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    Archives of microbiology 134 (1983), S. 171-174 
    ISSN: 1432-072X
    Keywords: Acetate growth medium ; Anti-microtubule agent ; Bud initiation ; Ethyl N-phenylcarbamate ; Meiosis ; Mitotic cell cycle ; Saccharomyces cerevisiae ; Sporulation induction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract When diploid cells of Saccharomyces cerevisiae were incubated in acetate growth media containing 2.5 mM ethyl N-phenylcarbamate (EPC), bud initiation was inhibited preferentially, and eventually overgrown, unbudded cells accumulated. During subsequent incubation, meiosis and ascospore formation occurred at high frequencies. The behavior of EPC-treated cells was essentially the same as that of cells transferred to a starvation sporulation medium. EPC thus has a pronounced effect on the mitotic growth of yeast cells, which leads to meiotic development. Our observations indicate that EPC has a decisive function in the initiation of meiosis in rich growth media.
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  • 25
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    Archives of microbiology 137 (1984), S. 357-361 
    ISSN: 1432-072X
    Keywords: Yeast ; Saccharomyces cerevisiae ; Killer toxin ; Extracellular glycoprotein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of Mr=16,000. The amino acid composition of the toxin type K2 of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.
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  • 26
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    Archives of microbiology 143 (1985), S. 88-93 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Potassium transport mutant ; Rubidium transport ; Sodium transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A mutant in Saccharomyces cerevisiae required one hundred times more K+ than wild type for the same half maximal growth rate. Mutant cells and wild type cells grown at millimolar K+ did not show significant differences in Rb+ transport. In the mutant, a rapid K+ loss induced by azide or incubation (4 h) in K+-free medium decreased the Rb+ transport K m by one half; in the wild type, those treatments decreased the Rb+ K m twenty and one hundred times, respectively. Mutant and wild type did not show significant differences in Na+ transport and in the Na+ inhibition of Rb+ transport, either in normal-K+ cells or in K+-starved cells. The results suggest that either two systems or one system with two interacting sites mediate K+ transport in S. cerevisiae.
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  • 27
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    Archives of microbiology 143 (1985), S. 220-224 
    ISSN: 1432-072X
    Keywords: Peroxy benzoic acid ; Saccharomyces cerevisiae ; ATP ; Glycolysis ; Glyceraldehyde-3-phosphate dehydrogenase ; Colony forming capacity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Concentrations of m-Cl-peroxy benzoic acid (CPBA) higher than 0.1 mM decrease the ATP-content of Saccharomyces cerevisiae in the presence of glucose in 1 min to less than 10% of the initial value. In the absence of glucose, 1.0 mM CPBA is necessary for a similar effect. After the rapid loss of ATP in the first min in the presence of glucose caused by 0.2 mM CPBA, the ATP-content recovers to nearly the initial value after 10 min. Aerobic glucose consumption and ethanol formation from glucose are both completely inhibited by 1.0 mM CPBA. Assays of the activities of nine different enzymes of the glycolytic pathway as well as analysis of steady state concentrations of metabolites suggest that glyceraldehyde-3-phosphate dehydrogenase is the most sensitive enzyme of glucose fermentation. Phosphofructokinase and alcohol dehydrogenase are slightly less sensitive. Incubation for 1 or 10 min with concentrations of 0.05 to 0.5 mM CPBA causes a) inhibition of glyceraldehyde-3-phosphate dehydrogenase, b) decrease of the ATP-content and c) a decrease of the colony forming capacity. From these findings it is concluded that the disturbance of the ATP-producing glycolytic metabolism by inactivation of glyceraldehyde-3-phosphate dehydrogenase may be an explanation for cell death caused by CPBA.
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  • 28
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    Archives of microbiology 100 (1974), S. 243-252 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Carbon Monoxide Trace-Measurement ; 14C-Glucose ; CO Production ; Atmospheric Cycle of Trace Gases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Description / Table of Contents: Zusammenfassung 1. Mit einer empfindlichen Analysenmethode, die auf die Reaktion CO+HgO→CO2+Hg basiert und den CO-Gehalt auf Grund der Absorption des freigesetzten Hg bei 2537 Å ermittelt, wurden im Gasraum über wachsenden Kulturen von Saccharomyces cerevisiae, S. oviformis, Escherichia coli, Aerobacter aerogenes, Pseudomonas spec. und Lactobacillus brevis 0.4–2.6 ppm CO nachgewiesen. Bei Lactobacillus arabinosus, Bacillus cereus var. mycoides und Aspergillus niger war eine CO-Bildung nicht meßbar. 2. Bei S. cerevisiae war die CO-Bildung bei Konzentrationen von 10–50 g Glucose pro Liter Medium am größten. Außerdem wurde die CO-Bildung proportional zum anfänglichen Sauerstoffgehalt im Gasraum über den Kulturen gefördert. 3. Mit 14C-markierter Glucose wurde nachgewiesen, daß CO aus Glucose entsteht. 4. Die CO-Bildung der untersuchten Mikroorganismen ist so gering, daß sie keine Bedeutung für den Kreislauf dieses Spurengases in der Atmosphäre hat.
    Notes: Summary 1. Growing cultures of Saccharomyces cerevisiae, S. oviformis, Escherichia coli, Aerobacter aerogenes, Pseudomonas spec. and Lactobacillus brevis produce trace amounts of CO (0.4–2.6 ppm) that can be detected in the gas space above the cultures using a sensitive analytical method based on the reaction CO+HgO→CO2+Hg. The liberated Hg is quantitatively measured by atomic absorption at 2537 Å. No CO could be detected above cultures of Lactobacillus arabinosus, Bacillus cereus var. mycoides and Aspergillus niger. 2. The maximum CO production by Saccharomyces was obtained with concentrations of 10–50 g glucose per liter medium. The amount of CO formed increased with the oxygen concentration in the gas space above the cultures. 3. Using 14C-glucose it was shown that S. cerevisiae forms CO from glucose. 4. The formation of CO by the microorganisms investigated is very small. The ratio of CO/CO2 produced is much smaller than in environmental air. Therefore it can be concluded that the production of CO by these microorganisms has probably no significance for the atmospheric cycle of this trace gas.
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    Archives of microbiology 104 (1975), S. 23-28 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Yeast ; Chemostat ; Nutrient Concentration ; Thermal Death ; Thermal Association ; Optimum Temperature for Growth ; Maximum Temperature for Growth ; Microbial Ecology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Saccharomyces cerevisiae was grown in a chemostat under glucose limitation at three superoptimal temperatures. In each steady state the specific growth rate was the sum of the dilution rate and the specific death rate, exponential death concurring with exponential growth. The specific death rate was a function of the temperature while the specific growth rate was a function of both the temperature and the concentration of the limiting nutrient. Each superoptimal temperature was characterized by a critical glucose concentration below which net growth was not possible. The critical glucose concentration increased with the temperature. Consequently the maximum temperature for growth was a function of the concentration of the limiting nutrient and approached the optimum temperature for growth with decreasing glucose concentrations.
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  • 30
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    Archives of microbiology 105 (1975), S. 187-192 
    ISSN: 1432-072X
    Keywords: Peroxisomes (microbodies) ; Saccharomyces cerevisiae ; Catalase ; Urate oxidase ; Glyoxylate cycle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Peroxisomes were isolated from derepressed (lactose grown) Saccharomyces cerevisiae cells following homogenization with a “Merkenschlager” cell mill (at 0°C using glass beads). Catalase and urate oxidase, along with low activities of d-amino acid oxidase and l-α-hydroxyacid oxidase (glycollate oxidase), were associated with the peroxisomes. No catalase activity was present in glucose repressed cells. When protoplasts prepared from derepressed cells were used for peroxisome isolation, catalase activity was not sedimentable through gradients. Apparently peroxisomes were destroyed as the cells became fermentative during protoplast preparation. The distribution of glyoxylate cycle enzymes was examined. Isocitrate lyase was not sedimentable, suggesting that, if the enzyme is peroxisome-associated, it is either readily released or present in a labile second class of peroxisomes. Low activities of malate dehydrogenase and citrate synthetase were found in peroxisome fractions from gradients, but may represent mitochondrial contamination. Citrate synthetase was not found associated with a low-density particle as had been previously reported.
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  • 31
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    Archives of microbiology 146 (1986), S. 221-226 
    ISSN: 1432-072X
    Keywords: Exoglucanase ; Saccharomyces cerevisiae ; Secretory mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37°C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (〈65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t 1/2=150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.
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  • 32
    ISSN: 1432-072X
    Keywords: Yeast ; Saccharomyces cerevisiae ; Mating reaction ; Zygote formation ; Mating pheromone ; Fatty acid ; Arachidonic acid
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    Topics: Biology
    Notes: Abstract Effect of exogenous fatty acids on zygote formation in Saccharomyces cerevisiae was studied. Arachidonic and oleic acids considerably stimulated zygote formation, but other fatty acids tested, linoleic, linolenic, stearic and palmitic acids, did not. Pretreatment experiments with arachidonic acid showed that the stimulation of zygote formation by the fatty acid required the presence of mating pheromone.
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  • 33
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    Archives of microbiology 151 (1988), S. 20-25 
    ISSN: 1432-072X
    Keywords: Yeast ; Saccharomyces cerevisiae ; Mating ; Zygote formation ; Chloroquine ; Lysosomotropic agent ; Plasma membrane ; Cell fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Haploid cells of opposite mating type of Saccharomyces cerevisiae conjugate to form zygote. During the conjugation process, the degradation or reorganization of the cell wall and the fusion of the two plasma membranes take place. Since chloroquine inhibits cellular events associated with the reorganization of the plasma membrane, the effect of the drug on conjugation was studied. Chloroquine at a concentration, at which cell growth was not retarded, inhibited zygote formation, while it did not affect other mating functions, such as sexual agglutination, production of and response to mating pheromone. Cells in a mating culture containing chloroquine formed no “prezygote” suggesting that they were not prepared for entering into fusion process. The inhibitory effect of chloroquine was reversible as cells formed zygote when they were washed after treatment with chloroquine. Zygote formation was unaffected in cells possessing chlorquine within vacuoles after incubation with the drug in complete medium (YPD) at pH 7.5, followed by washing. This suggests that chloroquine inhibits zygote formaton by adsorbing to the plasma membrane of S. cerevisiae.
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  • 34
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Yeast ; Phospholipase B ; Lysophospholipase ; Enzyme inhibition ; AMP ; Unesterified fatty acids
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    Notes: Abstract Divalent cations activate the lysophospholipase and transacylase reactions catalyzed by the same enzymes in the yeast Saccharomyces cerevisiae. The activation was observed at neutral pH, but not at the pH optimum of lysophospholipase/transacylase, near 3.5. Adenine nucleotides, especially AMP and ADP, are strong inhibitors of the same group of enzymes. Half maximal inhibition by AMP was found at a concentration of about 20 μM. The inhibition by nucleotides in low concentrations is enhanced by divalent cations.
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  • 35
    ISSN: 1432-072X
    Keywords: cAMP ; Cat mutants ; Glucose repression ; Glucose-induced ; Intracellular pH ; Ras ; Saccharomyces cerevisiae ; Signal transduction ; Trehalase ; Yeast
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    Topics: Biology
    Notes: Abstract Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells. The glucose-induced artefactual increase occasionally observed in repressed cells is probably caused by the fact that their low intracellular pH is only restored after the ATP level has increased to such an extent that it is no longer limiting for cAMP synthesis. It is unclear why the artefactual increases are not always observed. Measurement of glucose- and DNP-induced activation of trehalase confirmed the physiological validity of the changes observed in the cAMP level. Our results are consistent with the idea that the glucose-induced signaling pathway contains a glucose-repressible protein and that the protein is located before the point where intracellular acidification triggers activation of the pathway.
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  • 36
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    Archives of microbiology 151 (1989), S. 198-202 
    ISSN: 1432-072X
    Keywords: Sexual agglutination ; Mating ; Saccharomyces cerevisiae ; Yeast
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    Topics: Biology
    Notes: Abstract Genetic regulation of the inducibility of sexual agglutination ability in the yeast Saccharomyces cerevisiae was studied. Detailed analysis of the degree of sexual agglutination was carried out; it showed that a greater number of genes are involved in the regulation of inducible sexual agglutination in strain H1-0 than previously assumed. Although dominancy of inducible phenotype over constitutive was confirmed, the effectiveness of one gene changing the constitutive phenotype to the inducible seemed to be somewhat low. Quantity per cell of agglutination substances responsible for sexual agglutination increased as the agglutination ability became greater.
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  • 37
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    Archives of microbiology 158 (1992), S. 115-126 
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Yeast cells ; Yeast protoplasts ; Cell wall ; Congo red ; (1 » 3)-β-d-glucan microfibrils ; Cytokinesis ; Reversion of walled protoplasts to cells
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    Topics: Biology
    Notes: Abstract Congo red was applied to growing yeast cells and regenerating protoplasts in order to study its effects on wall biogenesis and cell morphogenesis. In the presence of the dye, the whole yeast cells grew and divided to form chains of connected cells showing aberrant wall structures on both sides of the septum. The wall-less protoplasts in solid medium with the dye exhibited an abnormal increase in volume, regeneration of aberrant cell walls and inability to carry out cytokinesis or protoplast reversion to cells. In liquid medium, the protoplasts synthesized glucan nets composed mainly of thin fibrils orientated at random, whereas normally, in the absence of dye, the nets consist of rather thick fibrils, 10 to 20 nm in width, assembled into broad ribbons. These fibrils are known to consist of triple 6/1 helical strands of (1 » 3)-β-d-glucan aggregated laterally in crystalline packing. The thin fibrils (c. 4 to 8 nm wide) can contain only a few triple helical strands (c. 1.6 nm wide) and are supposed to be prevented from further aggregation and crystallization by complexing with Congo red on their surfaces. Some loose triple 6/1 helical strands (native elementary fibrils) are also discernible. They represent the first native (1 » 3)-β-d-glucan elementary fibrils depicted by electron microscopy. The effects of Congo red on growth and the wall structure in normal cells and regenerating protoplasts in solid medium can be explained by the presence of a complex which the dye forms with (helical) chain parts of the glucan network and which results in a loss of rigidity by a blocked lateral interaction between the helices.
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  • 38
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    Archives of microbiology 162 (1994), S. 211-214 
    ISSN: 1432-072X
    Keywords: Killer toxin ; Saccharomyces cerevisiae ; Toxin binding ; Cell wall receptor
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    Topics: Biology
    Notes: Abstract A recently described new method for determination of killer toxin activity was used for kinetic measurenments of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (K T1=2.6x109 L.U./ml, V max1=0.19 s-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (K T2=3.2x107 L.U./ml, V max2=0.03 s-1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occured within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.
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  • 39
    ISSN: 1432-072X
    Keywords: Saccharomyces cerevisiae ; Pyruvate decarboxylase ; Pyruvate kinase ; Signalling ; Glycolysis mutants
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    Notes: Abstract Pyruvate decarboxylase, PDCase, activity in wild-type yeast cells growing on ethanol is quite low but increases up to tenfold upon addition of glucose, less with galactose and only slightly with glycerol. PDCase levels in glycolysis mutant strains growing on ethanol or acetate were higher than in the wild-type strain. These levels correlated with the sum of the concentrations of three-carbon glycolytic metabolites. The highest accumulation was observed in a fructose bisphosphate aldolase deletion mutant concomintant with the highest PDCase activity wild-type level. On the other hand, the PDCase levels in the different mutants again correlated with the sum of the concentrations of the three-carbon glycolytic metabolites. This was interpreted to mean that full induction of PDCase activity requires the accumulation of hexose-and triosephosphates.
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  • 40
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    Current genetics 10 (1985), S. 171-177 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Nuclear mutations ; Mitochondrial DNA stability ; Uncoupling of phenotypes
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    Topics: Biology
    Notes: Summary We have studied a pleiotropic mutationpetD inS. cerevisiae which both confers the inability to grow on glycerol (Gly−) and greatly increases the frequency of cytoplasmic petites (Het). The first phenotype, Gly−, is recessive, whereas the second, Het, is dominant. Genetic and biochemical analysis showed that the majority of the petites inpetD strains are not of therho° type (completely lacking mit-DNA),but of therho − type (containing partially deleted mit-DNA). This finding and the fact that the phenotype Het is dominant argue in favour of the involvement of thepetD product in the excision process of the mit-DNA. Another nuclear mutation,mod, was shown to exhibit a dominant epistasy with respect to the Het phenotype of the mutationpetD. Two types of Gly+ revertants frompetD mutants were isolated:rpa revertants, which restore completely the wild-type phenotype, andrpb revertants, which restore only the growth on glycerol, but still allow the production of high frequencies of cytoplasmic petites. Thus the mutationsmod andrpb permit the genetic uncoupling of two phenotypes induced by the mutationpetD.
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  • 41
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    Current genetics 10 (1985), S. 187-195 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Inorganic mercury ; Catabolite regulation ; Sugar uptake
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    Topics: Biology
    Notes: Summary Saccharomyces cerevisiae strains sensitive to inorganic mercury (Ono and Sakamoto 1985) did not grow well on the medium rich in glucose and poor in peptone. This growth inhibition, like growth inhibition caused by inorganic mercury, was relieved by exogenous tyrosine. Sugars such as fructose and mannose were as inhibitory as glucose, but glycerol was not at all. Galactose was inhibitory but not so much as glucose. Agal2l mutation (defective in galactose uptake) partly relieved growth inhibition caused by excess galactose. Moreover, it was found that some of revertants which gained ability to grow well in the presence of excess glucose were defective in the glucose uptake. From these observations, we conclude that growth inhibition of the inorganic mercury sensitive strains by excess sugar is a consequence of the catabolite regulation. In other words, the inorganic mercury sensitive strains are hyper-sensitive to the catabolite regulation due to the presence of theHGS2-1 allele.
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  • 42
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    Current genetics 11 (1986), S. 193-200 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Ty elements ; Transposable elements ; Retroviruses ; tRNA genes
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    Topics: Biology
    Notes: Summary We have isolated and characterized a Ty element from a yeast cosmid library which exhibits several unsual features: it is flanked by non-homologous delta elements and directly associated with a singular delta element. A tRNA(Glu3) gene and tRNA(Cys) gene are found in conjunction with this element, located in opposite orientation on either end of it. The sequence information now available for several Ty elements has been used in a detailed comparative analysis to determine conserved features among the Ty elements, preferably between class I elements and a class II element. Highly conserved sequence motifs appear to be located at the borders of particular segments that correspond to the putative protein domains of the Tys. Furthermore, we include a comparison of the best-conserved amino acid homologies for these putative proteins of Ty elements, transposable elements from other organisms and several retroviral proviruses to confirm their close structural resemblance.
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  • 43
    ISSN: 1432-0983
    Keywords: Rho°-petites ; Lycorine ; Mitochondrial DNA replication ; Saccharomyces cerevisiae
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    Notes: Summary This paper describes the isolation and characterization of mutants affected in the maintenance of the mitochondrial (mt) genome. The rationale of the screening procedure is based on the observation that the alkaloid lycorine inhibits growth of rho −-mutants, whereas rho°-mutants, devoid of mt DNA, are resistant to this drug (Del Giudice et al. 1984). Fourteen temperature sensitive mutants have been isolated that display the following phenotype: -Growth on fermentable medium at 23°C and 35°C (exclusion of general temperature-sensitive mutants). -no growth at 23°C and growth at 35°C on fermentable medium containing lycorine (selection for mutants producing rho°-petites). -growth at 23°C and no growth an 35°C on non-fermentable medium (selection for temperature-dependent loss of respiratory competence). These mutants were termed tmm (for temperature sensitive maintenance of mt genome). Mutant tmm1-1 was analyzed genetically and biochemically. It carries a recessive nuclear mutation which gives rise to 90–95% cytoplasmic petites at the non-permissive temperature. The population of petites consists of more than 95% rho°-petites as shown by their resistance to lycorine, by staining with 4′,6-diamidino-2-phenylindole (DAPI), and by Southern hybridization with mt DNA probes. Wild-type control cultures produced approximately 1% petites with less than 10% rho°-mutants.
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  • 44
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Inducible repair ; Plasmid transformation
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    Notes: Summary Many reports show that resistance of Saccharomyces cerevisiae to a large UV dose can be enhanced by pre-induction with a smaller one given some hours before. This work tests if such increased cell survival is associated with increased DNA repair on UV damaged plasmid transformed into yeast. There was no change in transformation efficiency of UV-damaged plasmid DNA under conditions where RAD cell survival increased 5-fold, and where rad1-1 and rad6-1 survival increased 2-fold. It is concluded that DNA repair activity involving the RAD6 and RAD3 pathways is either not inducible or is unable to work on plasmid DNA. It is suggested that the enhancement of cellular survival detected may be based on changes in cell-cycle behaviour which permit cells generally proficient in repair a greater chance to recover.
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  • 45
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Nucleo-mitochondrial interactions ; Mitochondrial status ; Dominant lycorine resistance
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    Notes: Summary Mutants resistant to 200 µg/ml of the alkaloid lycorine (LYC R) in non-fermentable substrate were isolated after nitrosoguanidine mutagenesis. Tetrad analysis and growth of heterozygous (LYC R/lyc s) diploids from two different mutants revealed that a single nuclear and dominant mutation is responsible for the resistant phenotype. In the wild type total protein synthesis is only slightly inhibited, whereas DNA and RNA synthesis is lowered to about 30% of the control. In the lycorine resistant mutants all macromolecular syntheses are unaffected by the drug.
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  • 46
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; 2 μ plasmids ; Plasmid free segregants
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    Notes: Summary The maximum specific growth rates (μmax) of 2 μ-plasmid-free ([cir°]) segregants of three haploid and one diploid strain of Saccharomyces cerevisiae have been determined and compared with the μmax of their 2 μ-plasmid-containing ([cir +]) progenitors. Two classes of [cir°] strains have been examined: those induced by transformation with a 2 μ-based recombinant plasmid according to the method of Dobson et al. (1980) and those isolated as spontaneous [cir°] segregants from glucose-limited continuous cultures. The μmax of the spontaneous [cir°] segregants was not found to differ significantly from that of their [cir +] parents. In all cases, however, the induced [cir°] strains had a μmax which was significantly less than that of their [cir +] counterparts. This effect was particularly marked in the case of the diploid strain where a 34% reduction in μmax was observed. The implications of these results are discussed in terms of the effect of the transformation process on host yeast cells.
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  • 47
    ISSN: 1432-0983
    Keywords: β-glucosidase ; Kluyveromyces fragilis ; DNA sequence ; Saccharomyces cerevisiae
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    Notes: Summary The complete nucleotide sequence of the β-glucosidase gene of Kluyveromyces fragilis has been determined. This sequence contains an open reading frame of 2535 base pairs encoding a protein of 845 amino acids. Analysis of the transcription products revealed only one transcript of about 3 kb identical in both Kluyveromyces fragilis and in the expression host Saccharomyces cerevisiae. The protein molecular weight of 93,811 Kd deduced from the sequence is consistent with the 90,000 Kd determined by SDS polyacrylamide gel electrophoresis with the purified protein. Mapping of the starts of transcription shows that two starting points are used in the natural host Kluyveromyces fragilis. A comparison of the amino acid sequence with that of other β-glucosidases revealed three regions of homology. One of these regions contains an amino acid sequence very similar to a peptide isolated from the active site of β-glucosidase A3 from Aspergillus wentii and could be implicated in the catalytic mechanism of these glucolytic enzymes.
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  • 48
    ISSN: 1432-0983
    Keywords: Glucanolytic brewer's yeast ; Endo-β-1,4-glucanase ; Chromosomal integration ; Transformation ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Summary Barley β-glucans present in wort reduce beer filtrability and cause hazes and precipitates in the finished beer. The endo-β-1,4-glucanase enzyme, EGI, found in the filamentous fungus Trichoderma reesei, is capable of efficiently hydrolyzing these β-glucans. The cDNA copy of the eg11 gene, which codes for the EGI enzyme, was coupled to yeast regulatory sequences and transferred to a brewer's yeast using the yeast copper chelatin gene CUP1 as a selection marker in the transformation. The eg11 gene was transferred to the yeast both on a multicopy plasmid and on an integrating plasmid. In both cases, highly glycosylated, active EGI enzyme was secreted into the medium. Barley β-glucans present in wort were efficiently hydrolyzed by the recombinant brewer's yeast.
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  • 49
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    Current genetics 12 (1987), S. 511-517 
    ISSN: 1432-0983
    Keywords: Mating-type switching ; Cytoduction ; Saccharomyces cerevisiae
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    Notes: Summary Use of a selective system for cytoduction in Saccharomyces cerevisiae allowed us to monitor hybrid formation and to clone the haploid nuclei of cells which have participated in illegitimate matings: a × a, α × α. Our approach has made it possible to select nuclei with mating-type switches and mutations within the MAT locus. It was shown that matings in α × α crosses often proceed through nonheritable genetic changes located within chromosome III. We suggest that these non-heritable genetic changes are due to premutational lesions, expressed phenotypically as transient α-matingtype. After a mating event these lesions are either repaired or converted to true mutations within the MAT locus.
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  • 50
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Minor tRNAs ; Codon usage ; Transposable elements ; Delta ; Tau
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    Notes: Summary During characterization of the whole tRNA(Glu) family from the yeast, Saccharomyces cerevisiae, we isolated one cosmid clone bearing a tRNA(Glu) gene copy that is deviant from the major tRNA(Glu3) gene members in only five positions. This divergent tRNA(Glu) is a minor species and is represented by a single gene copy. One of the nucleotide exchanges concerns the anticodon which is modified from T-T-C in the tRNA(Glu3) gene to C-T-C which implies that this tRNA serves the codon triplet G-A-G. Two other minor yeast tRNA species have been reported which appear to be particularly designed for the translation of those codons that have a G in its third (Wobble) position. The low abundance of such minor tRNA species correlates positively to the low occurrence of most of the N-N-G codons in yeast. Furthermore, the GAGtRNA(Glu) locus represents another case of the general phenomenon in which the majority of the tRNA genes in yeast are associated with one or several transposable elements forming complex patterns. In this particular case, divergent segments of delta and tau are present in the 5′ flanking region of the tRNA gene and arranged in a novel configuration. The sequence data lend support to the view that tau is not an evolutionary young element as was earlier anticipated.
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  • 51
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    Current genetics 12 (1987), S. 405-411 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Adenylate kinase ; Nucleotide sequence
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    Topics: Biology
    Notes: Summary The structural gene for yeast adenylate kinase (AKY) has been isolated and analyzed with respect to its nucleotide sequence. Southern and northern analyses imply that the gene is single copy and is transcribed into an mRNA of about 1,100 bases. The flanking regions of the gene contain the canonical elements typical for initiation and termination of transcription of yeast protein coding genes. The amino acid primary structure deduced from the open reading frame is identical with the protein sequence reported for yeast adenylate kinase (Tomasselli et al. 1986) with the exception of an extension of two amino acids (Met-Ser) at the N-terminus and aspartic acid instead of asparagine at the carboxyl end. Yeast adenylate kinase reveals a striking homology with both the mammalian cytosolic and, particularly, with the mitochondrial isozyme. It has an insertion of 31 amino acids in the middle segment of the protein, when compared to the cytosolic version of the mammalian enzyme. A strikingly conserved insert sequence of the same length and at exactly the same position is present in the mammalian mitochondria) isozyme. The question of the subcellular location of the yeast enzyme is discussed.
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  • 52
    ISSN: 1432-0983
    Keywords: Yeast ; Saccharomyces cerevisiae ; Nonsense suppression ; Omnipotent suppressors ; Gene mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Ten dominant omnipotent suppressors of Saccharomyces cerevisiae, which were previously shown to be different from SUP46, have been examined. Nine are mapped in a region between lys5 and cyh2 on the left arm of chromosome VII. These suppressors, like SUP46, manifest sensitivity to increased temperature and the antibiotics paromomycin and hygromycin B. In addition, they have an identical action spectrum. These results strongly suggest that they are allelic to each other and they are designated SUP138. The tenth is mapped to a position between his1 and arg6 on the right arm of chromosome V. This suppressor, named SUP139, does not manifest temperature sensitivity nor antibiotic sensitivity. SUP139 and SUP138, which are clearly distinguished by means of action spectrum, act on much fewer nonsense mutations than SUP46. It is now clear that dominant omnipotent suppressors arising at a single locus are homogeneous and that their efficiency is locus-dependent. The order of efficiency is SUP46〉SUP138〉SUP139.
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  • 53
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    Current genetics 13 (1988), S. 21-23 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Transformation ; Plasmid ; Colony ; Polyethylene glycol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A rapid and simple yeast transformation procedure has been developed using colonies on agar plates. Saccharomyces cerevisiae SHY3 cells were picked up from colonies on YPD plates grown freshly or stored at 4 °C and incubated with M13RK9-T DNA at 30 °C for 1–2 h in a solution of Li+, Ca2+, Mg2+, triacetin and polyethylene glycol. About 3,500 transformants were obtained per µg of double stranded M13RK9-T DNA. Unlike the existing spheroplast techniques, single stranded M13RK9-T DNA transformed intact cells below one-hundredth frequency of the duplex form.
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  • 54
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    Current genetics 14 (1988), S. 331-335 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Meiosis ; Deletion mutations ; Sequence dissimilarities
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A diploid yeast strain with extensive sequence dissimilarity in homologous regions near the LYS2 locus was sporulated, and spontaneous lys2 and lys5 mutant spores, selected on α-amino adipate, were analyzed. As many as 50% of the mutant spores contained a deletion in LYS2. These deletions occurred at a frequency of 5.0 × 10−7. While deletions of various sizes and endpoints were obtained, all the deletions recovered in this study included the border between homologous and non-homologous sequences located 4 kb upstream of LYS2. Large lys2 deletions that extended into an adjacent CYH2 duplication occurred at a frequency of 2.0 × 10−7, more than 1,000 times the frequency of the CYH2-LYS2 deletions found in a related haploid strain. This high frequency of CYH2-LYS2 deletions was observed only after sporulation of the diploid strain, and was dependent upon extensive sequence dissimilarity near the LYS2 locus.
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  • 55
    ISSN: 1432-0983
    Keywords: Schizosaccharomyces pombe ; Saccharomyces cerevisiae ; Argininosuccinate lyase ; Gene cloning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A gene bank of Sau3A partially restricted Schizosaccharomyces pombe DNA in YEp13 was used to transform an arg4 mutant of Saccharomyces cerevisiae. One colony was recovered which contained the YEp13 plasmid bearing a large insert complementing the argininosuccinate lyase (ASL) mutation. As shown by restriction mapping and subcloning experiments, the DNA sequence required for complementation is localized on a 2 kb BamHI-BamHI fragment. The plasmid complemented several S. cerevisiae arg4 mutants of independent origin and a S. pombe arg7 mutant lacking ASL. Low but significant ASL activities were detected in crude extracts of these transformants. No complementation of the E. coli argH mutant was observed. Southern blot hybridizations showed that the insert originates from the S. pombe genome. No cross-hybridization was found between this sequence and S. cerevisiae DNA. It can be concluded that the cloned DNA fragment bears the S. pombe ARG7 gene coding for ASL.
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  • 56
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Allosuppressor ; Translation ; Fidelity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Allosuppressor (sal) mutations enhance the efficiency of the yeast ochre suppressor SUQ5 and define five unlinked loci, SALT-SALS. A number of sal4 mutants were isolated and found to have pleiotropic, allele;specific phenotypes, including hypersensitivity in vivo to paromomycin and other antibiotics that stimulate translational errors in yeast. To examine further the nature of the SAL4 gene product, the wild type SAL4 gene was isolated by complementation of a conditional lethal allele sal4-2, and demonstrated to be a single copy gene encoding a single 1.6 kb transcript. Restriction mapping and DNA hybridisation analysis were used to demonstrate that the SAL4 gene is identical to the previously identified omnipotent suppressor gene SUP45 (SUPT). Our results implicate the SAL4 gene product as playing a major role in maintaining translational accuracy in yeast.
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  • 57
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Ribosomal protein genes ; CYH2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A diploid strain of the yeast Saccharomyces cerevisiae has been constructed that has one copy of the ribosomal protein gene CYH2 completely deleted and replaced with the TRP1 gene using the method of Rothstein (1983). There are only small differences in growth rate and no detectable difference in steady state level of CYH2 mRNA between the diploid that is heterozygous for the CYH2 deletion and the parent diploid with two normal copies of this gene. This suggests that the diploid must partially compensate for the loss of one CYH2 gene. Tetrad dissection shows that haploid spores lacking the CYH2 gene cannot germinate. The lethality of this deletion can be rescued by a CYH2 cDNA on a low copy vector. Haploids which lack the genomic copy of the CYH2 gene, but contain a plasmid copy of the CYH2 cDNA are able to grow normally. These CYH2 deleted yeast haploids should be useful to analyze mutationally altered CYH2 genes and genes homologous to CYH2 from other organisms without interference from a genomic copy.
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  • 58
    ISSN: 1432-0983
    Keywords: Yeast ; Gene regulation ; Saccharomyces cerevisiae ; PDCI promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A 870 by promoter fragment of the PDC1 gene that includes the carbon source dependent regulatory regions was investigated using 5′ and 3′ promoter deletions. The results indicate that glucose and ethanol regulation of PDC1 transcription are independently controlled by distinct cis-acting regions. The consensus sequence AAATCGATA may play a role in this regulation, while the sequence (ATCA)AACCT may be important in transcription initiation.
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  • 59
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Sporulation ; Inessential genes ; Genome organization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The SPR6 gene of Saccharomyces cerevisiae encodes a moderately abundant RNA that is present at high levels only during sporulation. The gene contains a long open reading frame that could encode a hydrophilic protein approximately 21 kDa in size. This protein is probably produced by the yeast, because the lacZ gene of Escherichia coli is expressed during sporulation when fused to SPR6 in the expected reading frame. SPR6 is inessential for sporulation; mutants that lack SPR6 activity sporulate normally and produce viable ascospores. Nonetheless, the SPR6 gene encodes a function that is relevant to sporulating cells; the wild-type allele can enhance sporulation in strains that are defective for several SPR functions. SPR6 is located on chromosome V, 14.4 centimorgans centromere-distal to MET6.
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  • 60
    ISSN: 1432-0983
    Keywords: Peroxisomes ; Protein import ; Saccharomyces cerevisiae ; Hansenula polymorpha
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The DAS gene of Hansenula polymorpha was expressed in Saccharomyces cerevisiae under the control of different promoters. The heterologously synthesized dihydroxyacetone synthase (DHAS), a peroxisomal enzyme in H. polymorpha, shows enzymatic activity in baker's yeast. The enzyme was imported into the peroxisomes of S. cerevisiae not only under the appropriate physiological conditions for peroxisome proliferation (oleic acid media), but also in glucose-grown cells where it induced the enlargement of the few peroxisomes present. This growth process was not accompanied by an increase in the number of microbodies, which suggests a separate control mechanism for peroxisomal proliferation.
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  • 61
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; SKI3 ; SKI5 ; M1 dsRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have identified a mutant strain of the yeast Saccharomyces cerevisiae which overproduces killer toxin. This strain contains a single mutation which fails to complement defects in both the SKI3 and SKI5 genes, while a cloned copy of this gene complements both the ski3 and ski5 defects. The level of secreted toxin from a cDNA based plasmid is not increased in a ski3 strain, showing that the overproduction phenotype is dependent upon an increased level of M1 dsRNA.
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  • 62
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Psoralen photoaddition ; Interstrand cross-link ; Repair deficiency ; Genotoxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Two different UVA irradiation systems were initially biologically calibrated with two haploid yeast strains proficient and deficient, respectively, in nucleotide excision repair. The number of DNA lesions introduced into the cell's genome by the photoactivated bifunctional furocoumarin 8-MOP was then calculated by means of the applied UVA exposure doses. At LD37 the repair-proficient wild type had about 14 ICL and 34 furan-side monoadducts in its DNA, while doubly blocked repair mutant rad3-12 pso1-1 had 2 ICL and 3 monoadducts. Locus-specific reversion of lys1-1 followed two-hit kinetics in the repair-proficient wild type and one-hit kinetics in an excision-deficient rad2-20 mutant, as would be expected if ICL was the main type of mutagenic lesion in the wild type and monoadducts the main mutagenic lesion type in the excision-deficient strain. Quantitative comparison of 8-MOP + UVA-induced ICL with those induced by bifunctional mustard revealed the former to have a much higher genotoxicity.
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  • 63
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Nucleo-mitochondrial interactions ; Mitochondrial status ; Lycorine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In a previous paper we have shown that the alkaloid lycorine inhibits growth of rho +, mit - and rho -, strains of Saccharomyces cerevisiae, whereas strains devoid of mitochondrial DNA (rho o) are resistant to more than 200 μg/ml of the alkaloid. In this report we show that hypersuppressive petites are almost as resistant as rho o mutants, whereas isogenic rho - petites, which have retained tained longer segments of the genome, are sensitive to the drug.
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  • 64
    ISSN: 1432-0983
    Keywords: Mutagen hyper-resistance ; Nitrogen mustard ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A screening of haploid yeast strains for enhanced resistance to nitrogen mustard (HN2) yielded a recessive mutant allele, hnm1, that conferred hyper-resistance (HYR) to HN2. Diploids, homo- or heterozygous for the HNM1 locus, exhibit normal wild-type like resistance while homozygosity for hnm1 leads to the phenotype HYR to HN2. The hnm1 mutation could be found in yeast strains proficient or deficient in different DNA repair systems. In these mostly HN2-sensitive haploid repair-deficient mutants, hnm1 acted as a partial suppressor of HN2 sensitivity. All isolated recessive mutations conferring hyper-resistance belonged to a single complementations group. The HYR to HN2 phenotype was maximally expressed in growing cells and was associated with reduced mutability by HN2. HNM1 most probably controls uptake of HN2 which would be impaired in the hnm1 mutants.
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  • 65
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; G418 resistance ; Gene cartridges ; Heterologous Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36–48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.
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  • 66
    ISSN: 1432-0983
    Keywords: Gene cloning ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have carried out experiments aimed at explaining the observed variations in transformation frequencies when Saccharomyces cerevisiae or Saccharomyces carlbergensis are transformed with chimeric plasmids that contain one of 4 possible EcoRI fragments of the yeast 2-μm circle. These plasmids fall into 2 classes when used to transform 2 different yeast his3 auxotrophs, one (strain LL20) harbours indigenous 2-μm circle, and the other (strain YF233) is devoid of this plasmid. Hybrid plasmids containing either the 2.4 mega-dalton (mD) R-form EcoRI fragment (pYF88) or the l.4 mD L-form EcoRI fragment (pYF177) of 2-μm circle transform either of the two hosts at a high frequency (50,000 colonies per Mg in LL20 and 10,000 colonies per μg in YF233). Hybrid plasmids containing the 1.5 mD R-form EcoRI fragment (pYF87) or the 2.5 mD L-form EcoRI fragment (pYF178) of the 2-μm circle transform LL20 at a reduced frequency (6,000–16,000 colonies per μg) and YF233 at extremely low frequencies (1–5 colonies per μg). All plasmids retrieved from strain YF233 that had been transformed with pYF88 or pYF177 were identical to the original transforming plasmid. Of the plasmids retrieved from strain LL20 that had been transformed with pYF87 and pYF178, approximately half had acquired an extra copy of the 2-μm circle. Of the plasmids retrieved from strain LL20 that had been transformed with pYF88 and pYF177, an average of only approximately 13% had acquired an extra copy of 2-μm circle. Taken together, these observations indicate that the transformation of yeast by a plasmid lacking the ability to replicate (pYF87 and pYF1780) occurs by the recombinational acquisition of 1 copy of the host 2-μm circle, which serves to supply the incoming plasmid with missing essential sequences. A comparison of 2-μm circle DNA fragments carried by pYF88 and pYF177 indicates that the region of 2-μm circle required for high frequency transformation is a 1.2 mD segment that is common to the 2.4 mD R-form and 1.4 ml) L-form EcoRI fragments. This region extends from the EcoRI cut site adjacent to the PstI site, through to the end of the inverted repeat. However, the inverted repeat sequence alone is not sufficient to bestow high frequency transformation of yeast.
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  • 67
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    Current genetics 20 (1991), S. 181-184 
    ISSN: 1432-0983
    Keywords: Alpha amylase ; Secretion ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Extracellular glucoamylase activity was increased by a gene, which is present in super-secretor, but absent in low-secretor, strains of the yeast Saccharomyces cerevisiae. Genetic data indicated that this super-secretor gene is linked to the STA3 structural gene for glucoamylase. This gene appears to act specifically since it increased the secretion of glucoamylase but not of other secreted enzymes like acid phosphatase and invertase.
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  • 68
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Transcriptional activator ; Oxidative stress ; Glutathione
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The PAR1/SNQ3 gene of S. cerevisiae, which increases resistance to iron chelators in multi-copy transformants, is identical to the YAP1 gene, a yeast activator protein isolated as a functional homologue of the human c-jun oncogene by binding specifically to the AP-1 consensus box. The observed H2O2-sensitivity of par1 mutants has been attributed to an increased sensitivity to reduced oxygen intermediates. Accordingly, par1 mutants did not survive an elevated oxygen pressure and were very sensitive to menadione and methylviologene, two chemicals enhancing the deleterious effects of oxygen. The specific activities of enzymes involved in oxygen detoxification, such as superoxide dismutase, glucose 6-phosphate dehydrogenase and glutathione reductase, were decreased in par1 mutants and increased after PAR1 over-expression. As in the case of oxygen detoxification enzymes, the cellular levels of glutathione were similarly affected. These observations indicate that PAR1/YAP1/SNQ3 is involved in the gene regulation of certain oxygen detoxification enzymes. The finding that H2O2 promotes DNA-binding of human c-jun is consistent with a similar function for PAR1/YAP1/SNQ3 and c-jun in cellular metabolism.
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  • 69
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mitochondrial trp-tRNA synthetase ; Nuclear mutation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The conditional respiratory-deficient Saccharomyces cerevisiae mutant pet-ts2281 was complemented by an yeast genomic DNA library. The gene thus isolated was sequenced and proved to be identical to the known MSW1 sequence encoding mitochondrial tryptophanyl-tRNA synthetase (Myers and Tzagoloff 1985). Compared to the wild-type, the ts2281 mutant allele of MSW1 contained a single T→C transition leading to a Leu→Ser replacement at position 294 of the protein sequence. In addition to this mutational alteration, our sequence data for the wild-type gene differ from the originally published MSW1 sequence at five other DNA positions which affect two locally restricted regions of the polypeptide chain. As expected, at the non-permissive temperature ts2281 cells are specifically defective in mitochondrial trp-tRNA formation and, thus, in overall mitochondrial protein synthesis. In addition, the patterns of cytochrome b mRNA maturation intermediates were distinctly different in ts2281 and wild-type yeast cells. The mutational effect of the observed amino-acid substitution in ts2281 is discussed in terms of weakened hydrogen bonding in the C-terminal half of the MSW1-encoded protein.
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  • 70
    ISSN: 1432-0983
    Keywords: Psoralen sensitivity ; Saccharomyces cerevisiae ; DNA repair ; Oxidative stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The complementation and genetical analysis of yeast mutants sensitive to photoactivated 3-carbethoxy-psoralen define three novel recessive mutant alleles pso-5-1, pso6-1, and pso7-1. Their cross-sensitivity to UV254nm, radiomimetic mutagens, and to chemicals enhancing oxidative stress suggest that these mutants are either impaired in metabolic steps protecting from oxidative stress or in mechanisms of the repair of oxygen-dependent DNA lesions. None of the three novel mutant alleles block the induction of reverse mutation by photoactivated mono- and bi-functional psoralens, nitrogen mustards, or UV254nm.
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  • 71
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; recA gene expression ; UV radiation ; Mitotic gene conversion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated. The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region. Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants. We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation. The results indicate that a RecA-like activity could participate in steps preceeding mitotic conversion events in yeast.
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  • 72
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Kluyveromyces lactis ; Transcriptional regulation ; Catabolite repression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Promoter regions of the KlQCR7, KlQCR8 and KlCYC1 genes, coding for subunits of the bc 1-complex and cytochrome c respectively, in the shortterm Crabtree-negative yeast Kluyveromyces lactis differ markedly in sequence from their Saccharomyces cerevisiae counterparts. They have, however, conserved very similar configurations of binding-site motifs for various transcription factors known to be involved in global and carbon-source regulation in S. cerevisiae. To investigate the carbon source-dependent expression of these genes in K. lactis, we have carried out medium-shift experiments and determined transcript levels during the shifts. In sharp contrast to the situation in S. cerevisiae, the level of expression in K. lactis is not affected when glucose is added to a non-fermentable carbon-source medium. However, the genes are not constitutively expressed, but become significantly induced when the cells are shifted from glucose to a nonfermentable carbon source. Finally, induction of transcriptional activation does not occur in media containing both glucose and non-femmentable carbon sources.
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  • 73
    ISSN: 1432-0983
    Keywords: Glycolysis ; Transcriptional activation ; Saccharomyces cerevisiae ; Chromatin structure ; Glucose induction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 74
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; pso4-1 mutant Sporulation ; DNA repair ; Meiotic recombination Induced mutagenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have evaluated the effect of the Saccharomyces cerevisiae pso4-1 mutation in sporulation and DNA repair during meiosis. We have found that pso4-1 cells were arrested in an early step of meiosis, before premeiotic DNA synthesis, and hence did not produce spores. These results suggest that the PSO4 gene may act at the start point of the cell cycle, as do some SPO and CDC genes. The pso4-1 mutant cells are specifically sensitive to 8-MOP- and 3-CPs-photoinduced lesions, and are found to be severely affected in meiotic recombination as well as impaired in the mutagenic response, as previously described for mitosis. This means that the PSO4 gene is important for the repair 8-MOP-photoinduced lesions, mainly double-strand breaks, and the processing of these lesions into recombinogenic intermediates.
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  • 75
    ISSN: 1432-0983
    Keywords: Heat-shock response ; Multidrug resistance ; AP-1 homolog ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have examined whether the stress-induced transcriptional activation ofYDR1/PDR5/STS1 is mediated by yAP-1 and yAP-2. Of the stresses examined, heat shock-induced, rapid and transient PDR5 expression became very low in ayap1 yap2 double-gene disruptant, indicating that the yAP proteins mediate the response. Similar results were obtained withSNQ2, a close homologue ofPDR5. A set of 5′-truncation derivatives of thePDR5 gene identified the region from −484 to −434 as being sufficient for the response. A sequence similar to the yAP-1 recognition element recently identified in the stress-responsive yeast genes was found in this region and in the 5′-flanking sequences ofSNQ2.
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  • 76
    ISSN: 1432-0983
    Keywords: Key words Citrinin ; Pet mutants ; Mitochondrial biogenesis ; Vacuolar ATPase ; YKL118W disruption ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In countries with a hot climate the mycotoxin citrinin represents a serious problem in fungal food-poisoning. In humans the renal system is affected the most and the mitochondrial respiratory chain was identified as a possible sensitive target for this toxin. In addition, citrinin has an antifungal activity that also inhibits the growth of the yeast Saccharomyces cerevisiae. So far the precise mode of action and the subcellular targets for citrinin have not been identified. Therefore, we decided to use the model organism yeast for a genetic approach to identify genes that play a role in the sensitivity against this mycotoxin. A large collection of conditional respiratory deficient yeast mutants was screened for sensitivity against citrinin. One special pet-ts mutant was identified that exhibited a higher sensitivity against citrinin. The genetic system of yeast allowed the isolation of the respective wild-type gene. This yeast gene encodes the Vph2p subunit that is essential for the correct assembly of the vacuolar ATPase. Isolation of the mutated gene and gene-disruption experiments of VPH2 and the partially overlapping small YKL118W gene verified this finding. The wild-type VPH2 gene restores all defects of the mutants. In contrast to this, YKL118W gave no complementation and the null mutant showed no phenotype. Thereby the yeast vacuolar ATPase was found to be important for the toxic effect of citrinin in yeast cells. The consequences of this finding for the molecular mechanism of citrinin action and its relation to the mitochondrial respiratory chain are discussed.
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  • 77
    ISSN: 1432-0983
    Keywords: Key words Adenine biosynthesis ; ade8-18 ; ade2 mutations ; Red/white colony color assay ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the yeast Saccharomyces cerevisiae the ade2, and/or the ade1, mutation in the adenine biosynthetic pathway leads to the accumulation of a cell-limited red pigment, while epistatic mutations in the same pathway, i.e. ade8, preclude this phenomenon, resulting in normal white colonies. The shift in color from red to white (or vice versa) with a combination of appropriate wild-type and mutant alleles of the adenine-pathway genes has been widely utilized as a non-selective phenotype to visualise and quantify the occurrence of various genetic events such as recombination, conversion and aneuploidy. It has provided an invaluable tool for the study of gene dosage and plasmid stability. In competition experiments between disrupted ade2, ade8-18 transformants carrying either a functional or non-functional episomal ADE8 gene, we verified that white ade8 ade2 cells show a remarkable selective advantage over red ade2 cells, with important implications on the use of this assay for the monitoring of genetic events. The accumulation of the red pigment in ade2 cells is likely to be the cause for impaired growth in these cells.
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  • 78
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Multiple mutants of DNA repair ; Sensitivity to nitrogen mustard and to radiation ; Thermoconditional DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Three haploid yeast mutants (snm) sensitive or thermoconditionally sensitive to the DNA cross-linking agent nitrogen mustard (HN2) were crossed with four rad strains representing mutations in the three pathways of DNA dark repair. The resulting haploid double and triple mutant strains were tested for their sensitivity to UV, HN2 and HN1. From the observed epistatic or synergistic interactions of the combinations of mutant alleles we could derive the relation of the SNM1 and SNM2 genes to the postulated repair pathways. Alleles snm1-1 and snml-2 ts were found epistatic to genes of the rad3 group, whereas snm2-1 ts was epistatic to rad6. The snm1 and snm2 mutant alleles interacted synergistically. From these data it is concluded that the SNM1 gene product plays a cross-link specific role in excision repair while the SNM2 gene product may be involved in a system of error-prone repair.
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  • 79
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    Current genetics 6 (1982), S. 163-165 
    ISSN: 1432-0983
    Keywords: Hybridization ; Polyethylene glycol ; Nuclear transfer ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Viable hybrids of Saccharomyces cerevisiae were obtained by transfer of isolated diploid nuclei into haploid protoplasts using a polyethylene glycol (PEG) fusion procedure.
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  • 80
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; DEL1 ; rad ; ste7
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In DEL1 strains of the yeast, Saccharomyces cerevisiae, the iso-1-cytochrome c (CYC1) region is flanked on either side by Tyl elements in direct orientation which promote cyc1 deletions of the bracketed DNA in the haploid cell. In this study, we asked which genes might control this event by testing the possibility that the DEL1 mutation mechanism requires an enzyme (or enzymes) that is also utilized in the repair of damaged DNA. To this end, we independently coupled eight repair mutations, rad3–2, rad4–4, rad6–1, rad6–3, rad9–1, rev3–1, rad50–1, and rad51-1, toDEL1 and asked whether DEL1 was still functional. We found that none of these rad mutations significantly affects the mutation frequency of 10−6-10−5 established in DEL1 strains for the CYC1 locus. Furthermore, we determined that ste7, a temperature-sensitive sterile allele known to alter gene regulation in Ty-mediated mutations, is not required for DEL1 function. Finally, DEL1 is not temperature-sensitive at 23° or 37 °C.
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  • 81
    ISSN: 1432-0983
    Keywords: Oversecretion mutants ; Protease defect ; Wall glucan defect ; Saccharomyces cerevisiae
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    Notes: Summary Two chromosomal mutations in yeast that result in oversecretion of the K1 killer toxin protein were examined. A recessive mutation in gene ski5 appears to lead to toxin oversecretion through a defect in a cell surface, PMSF-inhibited protease. A wild type killer strain degraded toxin following synthesis, and degradation could be partially prevented by addition of PMSF to the growth medium. The ski5 mutation caused an approximate ten fold oversecretion of toxin, similar to that seen in a PMSF-treated wild type culture, and no increased oversecretion in the presence of PMSF. The ski5 mutation caused oversecretion of other low molecular weight secreted proteins and appeared to oversecrete the α-factor pheromone, as judged by activity tests. The ski5 mutation was complemented by mutations in ski genes 1–4, and the mutant was not supersensitive to mating pheromones or K2 killer toxin. We also examined killer strains with a mutation in the nuclear gene krel which results in a defective (1→6)-β-D-glucan cell wall receptor for killer toxin. Such strains oversecrete toxin into the growth medium, but also, unexpectedly, oversecrete most other secreted proteins. The defect in (1→6)-β-D-glucan in these mutants appears to perturb the partitioning of secreted proteins between the cell wall and the medium.
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  • 82
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    Current genetics 6 (1982), S. 159-162 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Transformation ; Gene subcloning
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We have developped a procedure to delimit the boundaries of a cloned gene carried on a DNA fragment as large as 4 to 5 kilobases. The method consists in the following. Two series of limit digest products generated with a tetranucleotide recognition sequence endonuclease and originating from either of the two ends of this DNA segment are tested for their complementing capacity by yeast transformation. The gene is then delimited by the overlap of the two shortest complementing fragments.
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  • 83
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    Current genetics 7 (1983), S. 235-237 
    ISSN: 1432-0983
    Keywords: DNA replication ; Shuttle vectors ; Saccharomyces cerevisiae
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    Notes: Summary Mitotic segregation of three 2 μm-pBR322 chimaeric plasmids (YEp6, YEp21, and YEp24) was studied in yeast. Each displayed a characteristic rate of loss: YEp6 was lost at approximately twice the rate of YEp21 and YEp24. The loss rates were not significantly increased when two chimaeric plasmids were coresident, nor was the endogenous 2 μm plasmid itself displaced. Therefore these plasmids appear to be compatible in yeast.
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  • 84
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    Current genetics 8 (1984), S. 81-84 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Mitochondrial genes ; Vegetative segregation ; Uniparental inheritance
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Summary Zygotes of Saccharomyces cerevisiae that are heteroplasmic for mitochondrial alleles produce diploid progeny that are homoplasmic for one allele or the other, judged by the criterion that upon further subcloning they produce daughter cells of only one phenotype or the other. Here we show that when such cells are subjected to strong selection for the missing allele, it cannot be detected, so that it is probably not present in even a single copy.
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  • 85
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Yeast transformation ; Centromere-containing plasmids ; Mitotic stability of minichromosomes
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    Topics: Biology
    Notes: Summary A mutant with unstable maintenance of hybrid plasmids containing either one of the centromeric loci CEN3, CEN6, CEN11 and arsl or the replicator of the 2 μ plasmid has been obtained. The frequency of loss of hybrid plasmids in the mutant was up to 3 · 10−1 per one generation versus 10−2 in the original strain. The unstable maintenance of minichromosomes in the mutant is controlled by a recessive nuclear gene, named SMC for stability of minichromosomes. Loss of some minichromosomes is connected with impairment of their segregation in cell division. In diploids homozygous for smc mitotic chromosomal segregation is not affected but sporulation is impaired. The question of adequacy of usage of minichromosomes for selection of mutants with impaired function of centromeric loci is discussed.
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  • 86
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    Current genetics 10 (1986), S. 491-494 
    ISSN: 1432-0983
    Keywords: Protoplast fusion ; Saccharomyces cerevisiae ; Schwanniomyces castellii ; Starch fermentability
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Prototrophic hybrids have been obtained by the fusion of various auxotrophic haploid strains of Saccharomyces cerepisiae and Schwanniomyces castellii. The fusion hybrids showed starch fermentability which derived from one of the fusion parents, S. castellii. Surprisingly, these fusion hybrids were found to exhibit excellent sporulation and spore germination. The progenies of these fusion hybrids showed a few aberrant segregations, but mostly normal segregation for auxotrophic genetic markers. They also showed many tetrads with an apparently digenic segregation (2:2, 3:1 and 4:0) for starch fermentation. On the other hand, mating types of segregants of the fusion hybrids were determined by the prototrophic recovery method. Consequently, tetrad types for mating type were mostly 2a:1α:1 non-mater and several asci showed tetrad types of 2a:2 non-mater and 2a:2α. The 60 prototrophic fusion hybrids and its segregants did not secrete α-amylase on the starch agar plate. However, all of the data suggested that fusion hybrid could carry two dominant genes (STAB and STAC) to ferment starch, and that the two genes STAB and STA2 may be identical or allelic as may be the genes STAC and STA3.
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  • 87
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    Current genetics 1 (1979), S. 63-74 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Translation ; Coordinate regulation ; Electrophoresis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The products of protein synthesis from exponential phase cultures of Saccharomyces cerevisiae grown at 23 °C or at 36 °C appear to be essentially identical. However, yeast cells respond to a shift in culture temperature from 23 °C to 36 °C with the rapid de novo synthesis of a polypeptide species of molecular weight 100,000. Within 60–90 min after the shift this polypeptide represents approximately 2.5% of the total cellular protein, a 5–10 fold increase over the preshift level. The level of this polypeptide then decreases with continued growth of the cells at 36 °C. Analyses by SDS-polyacrylamide gel electrophoresis of polypeptides obtained from cells pulse labeled with [35S]methionine demonstrate that following a temperature shift from 23 °C to 36 °C the synthetic rate of the 100,000 molecular weight polypeptide (as well as a number of other polypeptide species) increases to a level at least 10 fold higher than that observed prior to the shift. A concomittant decrease is observed in the synthesis of a large number of polypeptide species which were actively synthesized before the shift. Maximum changes in synthetic rates are observed 20–30 min after the shift and preshift synthetic patterns are regained within 60–90 min. Synthetic changes of the same magnitude and time course can be produced by short (20–30 min) exposures to 36 °C implicating a heat shock response. Several of the transiently induced polypeptides, including the 100,000 molecular weight species, show an affinity for DNA as determined by DNA-cellulose chromatography.
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  • 88
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    Current genetics 10 (1986), S. 943-945 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; α-factor ; Protoplasts
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    Topics: Biology
    Notes: Summary When Mat a cells are treated with α-factor prior to being protoplasted and fused, the frequency of karyogamy is higher than in unarrested controls.
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  • 89
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    Current genetics 10 (1985), S. 179-185 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Resistance ; Mercury ; Tyrosine
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    Topics: Biology
    Notes: Summary From a cross of two strains ofSaccharomyces cerevisiae, both of which had the same (wild type or normal) level of resistance to inorganic mercury, segregants having three distinguishable resistance levels, normal, sensitive and semi-sensitive, were obtained. Genetic analyses of the parents and the progeny indicated that the levels of inorganic mercury sensitivity were determined by three distinct loci,HGS1, HGS2 andMSM1. The recessive allele of theHGS1 locus,hgsl-1, and the codominant allele of theHGS2 locus,HGS2-1, were necessary for the sensitive phenotypes, and alleles in theMSM1 locus,MSMI-1 andmsml-2, were responsible for the different sensitivity levels. In short, the strains of genotypeshgs1-1 HGS2-1 msml-2 andhgsl-1 HGS2-1 MSMI-1 were sensitive and semi-sensitive, respectively, while the strains of all other genotypes were normal. Although thehgs1-1 allele was identified as thearo7-1 mutation which confers deficiency of tyrosine and phenylalanine, mutations such asaro1B (deficiency of tyrosine, phenylalanine and tryptophan) andtyr1 (deficiency of tyrosine) had similar effects asaro7-1 on inorganic mercury sensitivity. From these results we conclude that theHGS2-1 allele causes inorganic mercury sensitivity when the cells are defective in the tyrosine biosynthesis. In fact, addition of tyrosine to the growth medium containing inorganic mercury resulted in increase of colony forming ability of the sensitive strains.
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  • 90
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; DNA replication origins ; GC clusters ; Genome rearrangements
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    Topics: Biology
    Notes: Summary We determined the restriction maps and primary structures of two as yet poorly characterized regions of the mitochondrial genomes of different wildtype strains of Saccharomyces cerevisiae. These regions respectively comprised the ori1 sequence and the newly identified ori8 sequence. Ori1 and ori8, together with their flanking sequences, exhibit a large polymorphism, resulting from specific variations due to insertions or deletions of optional GC clusters at different locations. The mechanisms underlying such sequence rearrangements are discussed.
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  • 91
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Cytochrome c ; Regulatory gene
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    Topics: Biology
    Notes: Summary The CYP1 gene has previously been identified as coding for a positive trans active factor that activates the expression of CYC1 and CYP3, which are the structural genes for isol- and iso2-cytochrome c. Two phenotypically distinct classes of CYP1 mutations can be obtained indicating that CYC1 and CYP3 are differentially regulated by the product of CYP1. The HAP1 gene codes for a product which has previously been proved to be necessary for the expression of the heme dependent CYC1-UAS1 cis regulatory sequence. In this article, we show by complementation and recombination that CYP1 and HAP1 are the same gene, moreover we identify hap1-1 as an iso2-cytochrome c underproducer mutation of the CYP1 gene.
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  • 92
    ISSN: 1432-0983
    Keywords: agα1 Mutant ; Agglutination ; Gene dose effect ; Mapping ; Saccharomyces cerevisiae
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    Notes: Summary A recessive agα1 mutation leads to specific defect in sexual agglutinability specifically in α cells of the yeast Saccharomyces cerevisiae. The cryptopleurine resistance gene cryR 1, closely linked to the mating type locus, was used to select α/α strains which emerged from α/α strains by mitotic nonreciprocal recombination, to genetically analyse agα1, since agα1 is expressed only in α mating type. The agα1 gene was found to be linked to the centromere tightly, to met3 at 4.4 cM, and to ilv3 at 12 cM on chromosome X. Sexual agglutinability of α cells was shown to be dependent on the dose of the AGα1 gene, using α/α isogenic strains carrying AGα1/AGα1, AGα1/agα1 or agα1/agα1. The sst2-1 mutation did not suppress the agα1 mutation. Based on these results, function of the AGα1 gene is discussed.
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  • 93
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    Current genetics 10 (1986), S. 657-664 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Amino acid biosynthesis ; General control ; GCD-genes ; GCN-genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mutant strains, resistant against the amino acid analogues 5-methyltryptophan, 5-fluorotryptophan and canavanine were isolated, starting with a trp2 leaky auxotrophic strain. Of 10 such strains, only four turned out to be of the “general control derepressed” (gcd) mutant type. Three other isolates were shown to be defective in the general amino acid permease system, while the remaining three strains displayed low spore viability and were not further investigated. Complementation tests amongst the four new gcd-mutant strains, including strain RH558 gcd2-1 isolated earlier, yielded five complementation groups: GCD2, GCD3, GCD4, GCD5, and GCD6. All mutant strains showed a dual phenotype, which was not separable by wild type backcrosses: “constitutive derepression” and “slow growth”. Epistatis of all gcd mutations over gcn1-1, gcn2-1 and gcn3-1 was found with respect to both phenotypes, except for gcd5-1, which was lethal in these combinations. On the other hand gcn4-101 was found to be epistatic over all gcd mutations, but only with respect to the “constitutive derepression” phenotype, and not to “slow growth”; again the combination with gcd5-1 was lethal. Mutation gcd2-1 was mapped on chromosome VII, 50 cM from leu1 and 22 cM from ade6. A new model is discussed, in which GCD-genes are involved in the amino acid uptake into the vacuoles.
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  • 94
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    Current genetics 16 (1989), S. 315-321 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Antisuppressor ; ADE3 ; Translation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Mutations in a known yeast gene, ADE3, were shown to act as an antisuppressor, reducing the efficiency of the omnipotent suppressor, sup45-2. The ADE3 locus encodes the trifunctional enzyme C1-tetrahydrofolate synthase, which is required for the biosynthesis of purines, thymidylate, methionine, histidine, pantothenic acid and formylmethionyl-tRNAfmet. The role of this enzyme in translational fidelity had not previously been suspected.
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  • 95
    ISSN: 1432-0983
    Keywords: Formaldehyde ; DNA-protein cross-links ; Repair ; Saccharomyces cerevisiae ; Hyperresistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The formation and removal of formaldehyde-mediated DNA protein cross-linking was measured by CsCI density gradient analysis in yeast strains of differing resistance to formaldehyde. Wild-type cells and transformants made hyperresistant to formaldehyde by a multi-copy vector containing the yeast SFA gene were specifically labeled in their DNA and incubated in the presence of formaldehyde. Treatment with formaldehyde lead to the formation of equal amounts of DNA protein cross-links; subsequent liquid holding of cells for 24 h resulted in the removal of nearly all DNA protein crosslinks regardless of the original formaldehyde resistance status of the strains.
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  • 96
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    Current genetics 13 (1988), S. 283-289 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Inducible antisense gene ; Acetolactate synthase ; Bradytrophic phenocopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary We report for the first time on the regulation of gene expression in yeast by antisense RNA. Chimaeric genes were constructed containing the 5′ upstream and partial coding sequence of SMR1 — a sulfometuron methyl resistant allele of the ILV2 locus. Such fragments were placed 5′ to 3′ and 3′ to 5′ under control of the GAL10 promoter and CYCl terminator in a high copy YEp plasmid. Following galactose induction only transformants containing antisense RNA genes showed biological activity against SMR1 gene expression. Antisense RNA inhibited synthesis of the SMR1 gene product acetolactate synthase and thus repressed cellular growth which resulted in a bradytrophic auxotroph revertable by addition of isoleucine and valine. Antisense RNA inhibition was enhanced in galactose medium containing sulfometuron methyl and in gcn4 cells deficient for positive regulation of the ILV2 locus. This system can be used to study factors that interfere with antisense RNA function and to assign biological function to randomly cloned DNA fragments.
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  • 97
    ISSN: 1432-0983
    Keywords: Aspergillus terreus clonotheque ; Saccharomyces cerevisiae ; Homologous integration ; 2 μ circledirected chromosome destabilization
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    Topics: Biology
    Notes: Summary A genome clonotheque consisting of 25- to 40-kb Sau3A1 fragments of Aspergillus terreus DNA was constructed in the episomal cosmid vector pES33 containing the yeastARG4 gene. From the 475 transformants of cir° yeast strain ESH-0, 23 stable Arg + transformants were independently selected. Genetic and Southern analysis of these stable transformants showed that 39% arose as a result of recombination between cloned A. terreus DNA sequences and yeast chromosome XII. The recombination events most likely occurred in the regions of homology within the rDNA clusters of A. terreus and Saccharomyces cerevisiae.
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  • 98
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Site specific recombination ; 2 μ DNA plasmid
    Source: Springer Online Journal Archives 1860-2000
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    Notes: Summary The purpose of this work is to identify and quantitate in vivo 2 μ plasmid FLP-independent recombination in yeast, using a nonselective assay system for rapid detection of phenotypic expression of the recombination events. A tester plasmid was constructed such that in vivo recombination between 2 μ direct repeat sequences produces the resolution of the plasmid into two circular DNA molecules. This recombinational event is detected as a phenotypic shift from red to white colonies, due to the mitotic loss of the plasmid portion containing the yeast ADE8 gene in a recipient ade1 ade2 ade8 genetic background. In the absence of the 2 μ FLP recombinase and/or its target DNA sequence, recombination is not abolished but rather continues at a high frequency of about 17%. This suggests that the FLP-independent events are mediated by the chromosomally-encoded general homologous recombination system. We therefore conclude that the totality of 2 μ DNA recombination events occurring in FLP+ cells is the contribution of both FLP-mediated and FLP-independent events.
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  • 99
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    Current genetics 14 (1988), S. 413-418 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Chromosome length polymorphisms ; FIGE ; OFAGE
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Although Saccharomyces cerevisiae strains generally have similar chromosomal band patterns as revealed by pulsed field gel electrophoresis, individual bands often move slightly differently from one strain to the other. Surveying strains from our stock collection, we found that nearly all the bands of a certain pair of strains differed in their mobility. Some of these chromosome length polymorphisms segregated in a 2:2 ratio, indicating that they resulted from single structural alterations (i.e. additions or deletions). One of these was mapped on the right arm of chromosome 1. Others did not segrate in a simple 2:2 ratio. That is, there were progenies which had bands not present in either parent. We suggest that these new bands are the products of recombination between homologous chromosomes having two or more structural alterations.
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  • 100
    ISSN: 1432-0983
    Keywords: DNA transformation ; Saccharomyces cerevisiae ; Site-specific recombination ; 2μ DNA plasmid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The 2μ DNA plasmid of the yeast Saccharomyces cerevisiae does not confer any known selectable phenotype to the host cell carrying it. Selection of cells transformed with purified 2μ DNA therefore cannot be achieved, and the intracellular presence of 2μ can only be assessed by molecular analysis of the DNA complement. In addition, 2μ alone does not replicate in bacterial hosts, thus rendering its amplification by conventional methods impossible. We have isolated a shuttle plasmid, pBH-2L, generated by in vivo sites-pecific recombination between the endogenous 2μ DNA plasmid and pRL, a pBR322 derivative containing the yeast LEU2 gene and one 2μ repeat sequence associated with the origin of replication. This new shuttle plasmid has the property, when transformed into yeast, of undergoing site-specific recombinational resolution between its two direct repeat sequences. This releases 2μ plasmid and pRL as individual molecules. The latter can undergo progressive mitotic loss during growth in nonselective medium, ultimately leaving leucine auxotrophic transformants that contain only 2μ DNA plasmid. This system can be utilized to introduce 2μ DNA alone into cells lacking it, thereby providing a novel means to study the biology and the molecular genetics of the plasmid and its potential practical applications as a vector.
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