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PubMed1990

Affinity chromatography of mammalian and yeast nucleosomes. Two modes of binding of transcriptionally active mammalian nucleosomes to organomercurial-agarose columns, and contrasting behavior of the active nucleosomes of yeast.

The reasons for the selective binding of nucleosomes from transcriptionally active genes to the organomercurial-agarose columns have been investigated. At least two modes of binding are identified by a new two-stage elution procedure that discriminates between nucleosomes which are retained by the Hg-column because of their salt-labile associations with SH-reactive non-histone proteins, and nucleosomes in which a conformational change has made the thiol groups of histone H3 accessible to SH-reagents. The first class is released from the column in 0.5 M NaCl; the second class is eluted in 10 mM dithiothreitol which displaces the bound H3-thiols. In mammalian cells, both classes of Hg-bound nucleosomes are enriched in the DNA sequences being transcribed at the time, and their histones H3 and H4 are hyperacetylated. In yeast cells, in which histone H3 lacks cysteinyl residues, only a small fraction of nucleosomes binds to the mercury column, and it has no enrichment of DNA sequences derived from the actively transcribed GAL, HIS4, and ACT1 genes. Since few nucleosomes remain on the column after elution in 0.5 M NaCl, the bound nucleosomes of yeast are retained primarily because of salt-labile associations with thiol-reactive nonhistone proteins. Thus, the presence of histone H3-thiol groups appears to be essential for the mercury binding of the second class of nucleosomes which, in mammalian cells, is derived from the transcriptionally active genes. The results support models of reversible nucleosome unfolding during transcription in mammalian cells to reveal previously inaccessible H3-SH groups, and they also indicate that other thiol-containing proteins, including high mobility group 1 and 2, become closely but transiently associated with the chromatin subunits during their transcription.

Acetylation↗

PubMed1989

[Historic development of yeast genetics from the beginning to the first gene transformation in brewing yeast strains].

A more intensive use of the potential of brewing yeasts in the biotechnological process of brewing is based on the knowledge of the genetic background of these microorganisms. It is given a review on the stages of genetic manipulation of brewing yeasts including recombinant DNA technology which has proved to be the most successful method for a further improvement of strains.

DNA, Fungal↗

PubMed1988

[Mutagenesis on cloned yeast genes. The mutation of the yeast gene comprising the plasmid and chromosome].

The cells of Saccharomyces cerevisiae were transformed by plasmid pYG-007 treated in vitro with o-methylhydroxylamine. The plasmid consists of a portion of the bacterial plasmid with genes of resistance to ampicillin, chloramphenicol and tetracycline, 2 mkm yeast DNA and yeast genes ADE2 and LEU2. The collection of mutants containing a mutant allele of ADE2 gene within the plasmid was obtained. Interallelic complementation and that induced by suppression were studied in these ade 2 mutants. It was shown that all these induced ade 2 mutations were base-pair substitutions. Using the mechanism of conversion we managed to transfer the plasmid ade 2 mutations into the chromosome. Three pairs of strains carrying similar mutation in plasmid and chromosome were created. Analysis of frequency of reversions induced by UV-light and hydroxylaminopurine in the mutant ade2 locus comprised in the plasmid and chromosome showed that the former induced reversions in plasmid alleles less effectively than the latter.

Alleles↗

PubMed1987

[Genetic study of plasmid integration into yeast chromosomes. IV. Integration of the plasmid pYF91 into different yeast chromosomes].

Integration of the episomic chimeric plasmid pYF91 into yeast chromosomes has been studied. Plasmid insertion into the chromosomes was observed to occur with the frequency of 4 X 10(-8). 379 integrants were selected from the highly unstable (cir0) transformants. The fact of plasmid integration into particular chromosomes was confirmed for 318 integrants. Genetic analysis showed that the plasmid can integrate into the region of LEU2 gene or into another arm of chromosome III (227 integrants), and also into other chromosomes: I, II, IV, V, VI, VII, VIII, IX, XII, XV (91 integrants). It is suggested that integration is the result of recombination between yeast chromosomes and homologous plasmid regions carrying LEU2 gene or Ty element and "delta" sequence.

DNA, Fungal↗

PubMed1985

Inhibition of the reductive activation of a valyl-tRNA synthetase from yeast by unsaturated fatty acids and associated observations on newly found lipophilic substances from yeast.

The reductive activation of a valyl-tRNA synthetase from yeast is strongly inhibited by 1-30 microM unsaturated fatty acids, and the inhibition is antagonized by 10-100 microM saturated fatty acids. Diethylstilbestrol also inhibits the activation. The possibility that unesterified palmitoleic and oleic acids are bona fide regulatory effectors is supported by a dramatic inverse relation between their cellular content and the growth rate of commercial bakers' yeast. An increase in the ratio of unsaturated to saturated acids with slowing growth in a laboratory strain, S288C, also supports the regulatory hypothesis. The free fatty acids are extracted into slightly acidified 50% alcohol together with traces of numerous novel lipophilic substances. One of these is suggested to function as a cofactor in conjunction with a heat-stable polypeptide that activates valyl-tRNA synthetase.

Amino Acyl-tRNA Synthetases↗

PubMed1983

[Genetic study of saccharomycete yeast sensitivity to the lethal and mutagenic action of nitrous acid. III. Relation between mitochondrial genome variability and the unstable sensitivity of yeast cells to inactivation by nitrous acid].

The sensitivity of the yeast Saccharomyces cerevisiae to nitrous acid (NA) is significantly influenced by various spontaneous mutations of the mitochondrial (mt) genome as well as by the nuclear mutation mmg 1 leading to a decrease in the spontaneous mutability of the mt genome. The mmg 1 locus and the mt genome most probably interact and this nucleo-cytoplasmic interaction plays a role in determining the NA sensitivity of yeast cells. A significant subclonal variation of the NA sensitivity has already been reported for the strains under study. Here we show this variability to decrease significantly when the cells are devoid of the mt DNA or carry the mmg 1 mutation. These data suggest a direct relation between the unstable NA sensitivity and the variability of the mt genome.

Genes↗

PubMed1984

Chromatographic differentiation of the mitochondrial and cytosolic fumarases of rat liver and Baker's yeast and differential induction of two fumarases of Baker's yeast.

The mitochondrial and cytosolic fumarases of rat liver could be separated from each other by Bio-Gel HTP column chromatography, showing that there are some conformational differences between the native proteins of the mitochondrial and cytosolic fumarases which have been indistinguishable by their physicochemical, catalytic, and immunochemical properties. The fumarase associated with the mitochondrial fraction of Baker's yeast could also be separated from that located in the cytosol by the Bio-Gel HTP column chromatography. By applying this chromatography to the differential determination of the fumarase activities in the Baker's yeast cultured under various conditions, it was shown that the mitochondrial and cytosolic fumarases in the cell are regulated by independent control systems.

Animals↗

PubMed1996

Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF).

In Saccharomyces cerevisiae, vacuoles are inherited by the formation of tubular and vesicular structures from the mother vacuole, the directed projection of these structures into the bud and the homotypic fusion of these vesicles. We have previously exploited a cell-free inheritance assay to show that the fusion step of vacuole inheritance requires cytosol, ATP and the GTPase Ypt7p. Here we demonstrate, using affinity-purified antibodies and purified recombinant proteins, a requirement for Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF) in homotypic vacuole fusion in vitro. Thus, Sec17p and Sec18p, which are typically involved in heterotypic transport steps, can also be involved in homotypic organelle fusion. We further show that vacuole-to-vacuole fusion is stimulated by certain fatty acyl-coenzyme A compounds in a Sec18p-dependent fashion. Finally, our data suggest the presence of a cytosolic factor which activates vacuole membrane-bound Sec18p.

Acyl Coenzyme A↗

PubMed1975

[Interaction of yeasts with tannins. II. Study of various yeasts hydrolysing tannic acid in tannin culture media].

Growth and hydrolytic action on tannins of 6 strains of yeasts (isolated from tanning liquors and xylophagous insects) are studied in culture media containing various concentrations of tannic acid. The influence of medium acidity is also considered. According to the strains, growth is more or less restrained and hydrolytic activity is variable. Except for gallotannins, hydrolysable tannins are not hydrolysed.

Candida↗

PubMed1993

Alternate interactions of the D-galactose-specific yeast lectin Kb-CWL I with sensitive yeast strains.

In vitro growth assays with a purified D-galactose-specific lectin Kb-CWL I extracted from Kluyveromyces bulgaricus showed marked antifungal effects on 9 of 12 test strains belonging to the genera Kluyveromyces, Saccharomyces, Pichia, Candida, Rhodotorula and Schizosaccharomyces. The inhibition of growth was proportional to the lectin concentration (0.04 and 0.08 mg ml-1) in the culture medium. Under the test conditions, K. bulgaricus, K. lactis and S. bayanus were consistently agglutinated without growth inhibition. D-Galactose, the inhibitory sugar of the cell aggregation activity, did not abolish the antifungal effects of the lectin Kb-CWL I on other yeast strains tested. In C. albicans and C. tropicalis, the lectin influenced the dimorphism of these strains and stimulated germ tube formation.

Antifungal Agents↗

PubMed2006

Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells.

During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chili pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H(+)-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.

Acids↗

PubMed1996

A complete mechanism for steady-state oxidation of yeast cytochrome c by yeast cytochrome c peroxidase.

Steady-state oxidation of yeast cytochrome c (yCc) was monitored as a function of ionic strength (mu) for mutants of a cloned cytochrome c peroxidase [CcP(MI)]. The data are best interpreted in the context of a two binding site model, where the affinity of the two sites for yCc differs by approximately 1000-fold and rapid intracomplex electron transfer (ET) occurs only at the high-affinity site identified in the crystal structure. At low mu, catalysis is apparently limited by the rate of yCc dissociation from the reactive high-affinity site (koff). Binding of yCc at the low-affinity site increases koff and therefore increases the rate of catalysis. Mutations at the high-affinity site also increase the rate of catalysis by the 1:1 CcP(MI):yCc complex by increasing koff. Mutations at residues that interact strongly with yCc at the high-affinity site (Asp 34, Glu 290, and Ala 193) cause the greatest increase in koff (25-38-fold at mu = 20 mM). Mutations at residues that interact less strongly with yCc (Glu 32 and Glu 291) cause smaller increases in koff (10- and 3-fold, respectively, at mu = 20 mM). The results provide additional evidence that the high-affinity site formed in solution is similar to the one identified in the crystal structure and that yCc dissociation from this site limits enzyme turnover at low ionic strength. Numerical integration simulations show that the model accurately predicts enzyme turnover rates at the high-affinity site, using published rate constants for the elementary reaction steps.

Binding Sites↗

PubMed1995

Chromosome bisection in the yeast Saccharomyces cerevisiae facilitated by yeast artificial chromosomes bearing a site-specific recombination system.

A chromosome bisection method was constructed using yeast artificial chromosomes (YAC) and a GAL1-promoted site-specific recombination system. This method was applied to bisect chromosome IV into the left and right parts of the centromere region. The bisection occurred at frequencies of about 10% when the recombination site DNAs were integrated onto YAC and chromosome IV in the same direction, but were less than 10(-3) when they were in opposite directions. Reconstruction of the original chromosome IV from the bisected chromosomes was also induced by galactose at high frequencies. Loss of the left part chromosome was found at the frequencies of 0.9 x 10(-3) after hybrid cells between the chromosome-bisected strain and a normal haploid were subcultivated in a complete medium. The bisection and reconstruction of chromosome IV and deletion of the left part chromosome were demonstrated by electrophoretical karyotyping.

Chromosome Deletion↗

PubMed1999

[Distinction of yeast flocculent phenotypes and studies of the physiological and biochemical characteristics of yeast flocculation].

More than 400 yeast strains were examined for their flocculation, and five of them displayed strong flocculation. The 5 strains were divided into two groups which are Flo 1 and NewFlo phenotypes on the basis of their response to sugar inhibition. Related physiological and biochemical characteristics of the two phenotypes' strains were studied. The results showed that the flocculation of Flo 1 phenotype was only inhibited by mannose; it was sensitive to high temperature (70 degrees C), pronase E, trypsin, whereas tolerant to pronase K, chymotrypsin, Ca2+, pH; the flocculation of NewFlo was inhibited by many sugars, such as glucose, maltose, sucrose, mannose; it was sensitive to high temperature (70 degrees C), pronase, Ca2+, pH. The Calcium concentration and pH value of the optimum flocculaton of the Flo1 and NewFlo phenotypes strains were respectively 10 mmol/L-1 mol/L; 3.0-4.5.

Culture Media↗

PubMed1985

Phosphorylation and inactivation of yeast fructose-1,6-bisphosphatase by cyclic AMP-dependent protein kinase from yeast.

Purified fructose-1,6-bisphosphatase from Saccharomyces cerevisiae was phosphorylated in vitro by purified yeast cAMP-dependent protein kinase. Maximal phosphorylation was accompanied by an inactivation of the enzyme by about 60%. In vitro phosphorylation caused changes in the kinetic properties of fructose-1,6-bisphosphatase: 1) the ratio R(Mg2+/Mn2+) of the enzyme activities measured at 10 mM Mg2+ and 2 mM Mn2+, respectively, decreased from 2.6 to 1.2; 2) the ratio R(pH 7/9) of the activities measured at pH 7.0 and pH 9.0, respectively, decreased from 0.62 to 0.38, indicating a shift of the pH optimum to the alkaline range. However, the affinity of the enzyme for its inhibitors fructose-2,6-bisphosphate (Fru-2,6-P2) and AMP, expressed as the concentration required for 50% inhibition, was not changed. The maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase was 0.6-0.75 mol/mol of the 40-kDa subunit. Serine was identified as the phosphate-labeled amino acid. The initial rate of in vitro phosphorylation of fructose-1,6-bisphosphatase, obtained with a maximally cAMP-activated protein kinase, increased when Fru-2,6-P2 and AMP, both potent inhibitors of the enzyme, were added. As Fru-2,6-P2 and AMP did not affect the phosphorylation of histone by cAMP-dependent protein kinase, the inhibitors must bind to fructose-1,6-bisphosphatase in such a way that the enzyme becomes a better substrate for phosphorylation. Nevertheless, Fru-2,6-P2 and AMP did not increase the maximum amount of phosphate incorporated into fructose-1,6-bisphosphatase beyond that observed in the presence of cAMP alone.

Adenosine Monophosphate↗

PubMed1984

[Yeast resistance to polyene antibiotics. II. An analysis of the sterol composition of Saccharomyces cerevisiae yeasts resistant to nystatin].

The recessive yeast mutations nys providing resistance to polyene antibiotics in Saccharomyces cerevisiae are described. The analysis of UV-absorbtion spectra of sterols from cells of different mutants allows to assume that NYS genes control synthesis of ergosterol. The lack of alterations in the sterol composition of the same strains carrying dominant nystatin resistance mutations points to the existence of another mechanisms of resistance to polyene antibiotics, in addition to the sterol mechanism.

Anti-Bacterial Agents↗

PubMed1997

Sequencing of a 9.9 kb segment on the right arm of yeast chromosome VII reveals four open reading frames, including PFK1, the gene coding for succinyl-CoA synthetase (beta-chain) and two ORFs sharing homology with ORFs of the yeast chromosome VIII.

A 9.9 kb DNA fragment from the right arm of chromosome VII of Saccharomyces cerevisiae has been sequenced and analysed. The sequence contains four open reading frames (ORFs) longer than 100 amino acids. One gene, PFK1, has already been cloned and sequenced and the other one is the probable yeast gene coding for the beta-subunit of the succinyl-CoA synthetase. The two remaining ORFs share homology with the deduced amino acid sequence (and their physical arrangement is similar to that) of the YHR161c and YHR162w ORFs from chromosome VIII.

Amino Acid Sequence↗

PubMed2005

A novel test for identifying genes involved in aldehyde detoxification in the yeast. Increased sensitivity of superoxide-deficient yeast to aldehydes and their metabolic precursors.

A novel test for the identification of genes involved in aldehyde metabolism is proposed, based on detection of altered sensitivity of the yeast to corresponding alcohols, metabolic precursors of the aldehydes. This attitude enabled to an unexpected detection increased sensitivity of mutants devoid of CuZn-superoxide dismutase (CuZnSOD) to allyl alcohol (precursor of acrolein) and nonenol. We interpret this finding as due to inactivation of some important element of aldehyde detoxification by increased flux of superoxide in DeltaCuZnSOD mutants.

Acrolein↗