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At least 217 records · Page 12Linked to original sources
PubMed2007

Revised procedures for yeast metabolites extraction: application to a glucose pulse to carbon-limited yeast cultures, which reveals a transient activation of the purine salvage pathway.

In this study we have revised our original procedure of yeast metabolites extraction. We showed that: (a) less than 5% of intracellular metabolites leaks out during the step of rapid arrest of cellular metabolism by quenching yeast cells into a 60% methanol solution kept at -40 degrees C; and (b) with a few exception, the stability of metabolites were not altered during the 3 min boiling procedure in a buffered ethanol solution. However, there was a loss of external added metabolites of 5-30%, depending on the type of metabolites. This was mainly attributable to their retention on cellular debris after ethanol treatment, which prevented centrifugation of the cellular extracts before evaporation of ethanol. We further simplified our previous high-performance ionic chromatography (HPIC) techniques for easier, more reliable and robust quantitative measurements of organic acids, sugar phosphates and sugar nucleotides, and extended these techniques to purine and pyrimidine bases, using a variable wavelength detector set at 220 and 260 nm in tandem with a pulsed electrochemical or suppressed conductivity detector. These protocols were successfully applied to a glucose pulse to carbon-limited yeast cultures on purines metabolism. This study showed that glucose induced a fast activation of the purine salvage pathway, as indicated by a transient drop of ATP and ADP with a concomitant rise of IMP and inosine. This metabolic perturbation was accompanied by a rapid increase in the activity of the ISN1-encoded specific IMP-5'-nucleotidase. The mechanism of this activation remains to be determined.

Adenine Nucleotides↗
PubMed1995

Physiological and molecular characterization of flor yeasts: polymorphism of flor yeast populations.

Yeast strains which form velum on the surface of Sherry wine during the aging process have been isolated and characterized. According to their metabolic and molecular features most of the yeasts that were isolated belong to different races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis and rouxii). Due to the conditions under which these yeasts were isolated, all strains have in common the capacity to develop a film as an adaptive mechanism which allows them to grow and survive in 15.5% vol. ethanol. All strains were prototrophs for amino acids and most vitamins but they gave different responses to the killer factor. However, whereas their physiological features were similar, they showed a great heterogeneity with regards to the nuclear and mitochondrial genome (mtDNA): DNA content per cell was quite variable (1.3 to 2n), electrophoretic karyotypes of nuclear genomes indicated a main pattern with some variations, and polymorphism shown by the mtDNA was very high. Under extreme conditions such as Sherry wine with 15.5% vol. ethanol, no fermentable sugar and an exclusively oxidative metabolism, cells hardly grow and the maintenance of a live population depends on survival and respiration, which in turn depend on the mtDNA. At the same time these environmental conditions are mutagenic for the mtDNA, causing an increase in variation. Thus, the polymorphism observed might reflect the enormous variability induced by the ethanol followed by the selection of those mtDNA sequences which make the mitochondria metabolically active under these conditions.

DNA, Fungal↗
PubMed2000

Coexpression of genetically engineered fused enzyme between yeast NADPH-P450 reductase and human cytochrome P450 3A4 and human cytochrome b5 in yeast.

Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH-P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH-P450 reductase. To enhance the efficiency of electron transfer from NADPH-P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH-P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6beta-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6beta-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.

Base Sequence↗
PubMed1997

Isolation of human and fission yeast homologues of the budding yeast origin recognition complex subunit ORC5: human homologue (ORC5L) maps to 7q22.

Orc5p is a subunit of the origin recognition complex in the budding yeast Saccharomyces cerevisiae, which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing. We have cloned cDNAs from both human and fission yeast Schizosaccharomyces pombe that encode proteins homologous to the budding yeast and Drosophila Orc5p. Human Orc5p showed 35.1, 22.3, and 19.4% identity to the Drosophila, S. pombe, and S. cerevisiae Orc5p, respectively. We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescence in situ hybridization. We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1. We have identified the S. pombe ORC5 gene and located it in a cosmid mapped on chromosome II.

Amino Acid Sequence↗
PubMed1992

The tomato nia gene promoter functions in fission yeast but not in budding yeast.

A fragment comprising 1 kb of the 5' region and the 81 first nucleotides of the coding region of the tomato nitrate reductase nia gene was placed in translational fusion with the lacZ reporter gene. This construct was introduced in budding and in fission yeast using a derivative of the Saccharomyces cerevisiae/Schizosaccharomyces pombe autonomously replicating vector pUZL. Beta-galactosidase activity was detected in S. pombe but not in S. cerevisiae. Primer extension experiments show that in fission yeast transcripts are initiated at the same starting point as in tomato, indicating for the first time that a plant promoter can be correctly recognized in fission yeast.

Base Sequence↗
PubMed1984

Expression of an artificial yeast TRP-gene cluster in yeast and Escherichia coli.

All five tryptophan biosynthetic genes of Saccharomyces cerevisiae were unified on plasmid pME554, which is based on 2 micrometer DNA and pBR322 sequences allowing for autonomous replication in yeast and E. coli. Homologous and heterologous expression of this artificial yeast TRP-gene cluster was studied. Plasmid pME554 allowed for nearly normal growth of a yeast strain bearing auxotrophic mutations in all five TRP-genes. The plasmid-borne genes TRP2 to TRP5 were expressed and regulated normally in the frame of the general control. Gene TRP1, carried on an EcoRI/Bg/II fragment lacking the ARS1 function, was expressed poorly and did not respond to the general control like the chromosomally-borne TRP1 gene. Plasmid pME554 allowed for poor growth of E. coli strain W3110 tna- delta trpEA2 on minimal medium. Marked stimulation was observed, however, when anthranilic acid or indole were added. Accordingly, poor expression of the first Trp-enzyme anthranilate synthase and the last enzyme tryptophan synthase was found, whereas the other three genes were moderately well expressed in E. coli.

Base Sequence↗
PubMed1988

The 5'-upstream region of the yeast 25S rRNA gene contains a promoter element allowing expression in yeast and E. coli.

The 25S rRNA gene of Saccharomyces cerevisiae is preceded by a bona fide TATA sequence which allows the initiation of transcription--presumably by polymerase II--from the same strand as the 25S rRNA gene. When the promoter fragment is cloned in front of a lacZ gene equipped with an initiation codon but lacking a promoter, this element permits formation of beta-galactosidase both in yeast and E. coli. Using RNA from yeast transformed with the fusion plasmid, we mapped by primer elongation a single initiation site 63 bp downstream from the presumed TATA sequence, i.e. about 53 bp 5' of the 25S rRNA gene. A similar signal at about the same position was observed when RNA from untransformed wild-type yeast was used as a template for primer elongation. These results suggest that transcription from this polymerase II promoter-like element occurs in vivo. A regulatory function could not be assigned to this transcript. Its initiation is not significantly influenced by heme or carbon source, although two boxes of high homology with upstream activation sequences (UAS) mediating heme dependent expression of the iso-1-cytochrome c gene (CYC1) precede the promoter at the appropriate distance.

Amino Acid Sequence↗
PubMed1976

Assimilation spectrum of the yeast Candida utilis 49 used for producing fodder yeast from synthetic ethanol.

Oxidizing and assimilating ability of the yeast Candida utilis 49 was tested with 21 different low-boiling organic compounds which come as components of raw synthetic ethanol. The highest yields of yeast dry weight were obtained with ethanol (72.0%), propanol (48.2%), ethyl acetate (43.4%) and acetic acid (34.2%). To a minor extent, the yeast was capable of utilizing also 2-propanol, butanol and 2-butanol; it oxidized most of the compounds tested.

1-Propanol↗
PubMed1984

Possible energization of K+ accumulation into metabolizing yeast by the protonmotive force. Binding correction to be applied in the calculation of the yeast membrane potential from tetraphenylphosphonium distribution.

Membrane potentials of yeast cells, Saccharomyces cerevisiae, calculated from the equilibrium distribution of tetraphenylphosphonium (TPP) between cell-water and medium should be corrected for a contribution due to binding of TPP to intracellular constituents. The magnitude of this correction depends upon the way in which it is determined. In cells permeabilized by boiling, cell-binding is much higher than in cells permeabilized by repeated freezing and thawing. The binding corrections are 75 +/- 1 mV and 49 +/- 7 mV, respectively. The binding correction obtained from TPP distribution between deenergized cells and medium is much lower and amounts to 19 +/- 9 mV. The latter value is probably more reliable. It is supposed that permeabilization of the cells by boiling or repeated freezing and thawing unmasks potential TPP binding groups in the cell. The K+ accumulation into anaerobically metabolizing yeast cells can be accounted for almost quantitatively by a cotransport of protons and K+ ions if the lower binding correction is applied. This means that K+ accumulation into the yeast cell may be driven by the sum of the protonmotive force and the membrane potential.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed1981

Degradation of nucleic acids with ozone. II. Degradation of yeast RNA, yeast phenylalanine tRNA and tobacco mosaic virus RNA.

The degradation of a mixture of four 5'-ribonucleotides (AMP, GMP, CMP and UMP), yeast RNA, yeast phenylalanine tRNA, and tobacco mosaic virus RNA (TMV-RNA) with ozone (concentration in inlet gas, 0.1-0.5 mg/l) was examined in a phosphate buffer (pH 6.9). In the case of the mixture, GMP alone was degraded in the initial stage. In the ozonization of yeast RNA, the guanine moiety was less vulnerable to attack by ozone than in the case of free GMP, but it again degraded most rapidly among the four nucleotides. In the treatment of tRNA with ozone, the guanine moiety degraded first. When the numbers of degraded nucleotides reached 4.8 (remaining amino acid acceptor activity was 3.6%), the polyacrylamide gel electrophoresis of the ozonized tRNA gave a single band with the same mobility as that of the intact tRNA. It is evident that ozonolysis of tRNA proceeded without cleavage of the polynucleotide chain. In the case of TMV-RNA, the loss of the infectivity by ozone proceeded rapidly within 30 min and was followed by preferential degradation of the guanine moiety. The outstanding lability of the guanine moiety observed in each case is discussed in connection with the inactivation of tRNA and TMV-RNA.

Chemical Phenomena↗
PubMed1989

Domains of the SFL1 protein of yeasts are homologous to Myc oncoproteins or yeast heat-shock transcription factor.

We identified a yeast suppressor gene for flocculation (SFL1), which complemented a newly isolated sfl1 mutant. This mutation causes asexual cell aggregation. SFL1 encodes a 767-amino acid protein which has two domains significantly homologous to Myc oncoproteins and the yeast heat shock transcription factor (HSTF). The Myc homologous region in SFL1 overlaps with the conserved region in a series of interesting proteins: MyoD1, Drosophila achaete-scute, twist, daughterless gene products and immunoglobulin enhancer-binding proteins. In addition, the N-terminal region of the SFL1 gene product shows extensive homology to the DNA-binding domain of HSTF. Mutational analysis of SFL1 demonstrates that it is required for normal cell-surface assembly in vegetative growth. We propose that the SFL1 gene product may be a transcription factor which is involved in regulation of the gene(s) related to yeast flocculation.

Amino Acid Sequence↗
PubMed2004

Comparative analysis of cytokinesis in budding yeast, fission yeast and animal cells.

Cytokinesis is a temporally and spatially regulated process through which the cellular constituents of the mother cell are partitioned into two daughter cells, permitting an increase in cell number. When cytokinesis occurs in a polarized cell it can create daughters with distinct fates. In eukaryotes, cytokinesis is carried out by the coordinated action of a cortical actomyosin contractile ring and targeted membrane deposition. Recent use of model organisms with facile genetics and improved light-microscopy methods has led to the identification and functional characterization of many proteins involved in cytokinesis. To date, this analysis indicates that some of the basic components involved in cytokinesis are conserved from yeast to humans, although their organization into functional machinery that drives cytokinesis and the associated regulatory mechanisms bear species-specific features. Here, we briefly review the current status of knowledge of cytokinesis in the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe and animal cells, in an attempt to highlight both the common and the unique features. Although these organisms diverged from a common ancestor about a billion years ago, there are eukaryotes that are far more divergent. To evaluate the overall evolutionary conservation of cytokinesis, it will be necessary to include representatives of these divergent branches. Nevertheless, the three species discussed here provide substantial mechanistic diversity.

Animals↗
PubMed1992

The 4 beta-methyl group of substrate does not affect the activity of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast: difference between the substrate recognition by yeast and plant sterol 14 alpha-demethylases.

Interaction of obtusifoliol and 24,28-dihydroobtusifoliol with yeast lanosterol 14 alpha-demethylase (P-450(14)DM) was studied to elucidate the role of the 4 beta-methyl group of substrate. P-450(14)DM of Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of obtusifoliol. Apparent Vmax of obtusifoliol demethylation (15.4 nmol/min/nmol P-450) was similar to that of 24-methylene-24,25-dihydrolanosterol demethylation and was a little higher than those of lanosterol and 24,25-dihydrolanosterol demethylations. Apparent Km for obtusifoliol (12.0 microM) was higher than those for lanosterol and 24-methylene-24,25-dihydrolanosterol but was lower than that for 24,25-dihydrolanosterol. 24,28-Dihydroobtusifoliol was a very poor substrate for yeast P-450(14)DM. These facts suggest that the 4 beta-methyl group of sterol slightly affects the activity of yeast P-450(14)DM, while hydrogenation of a double bond in the sterol side-chain considerably impairs the activity. This finding is a contrast to the fact that the plant P-450(14)DM could not catalyze demethylation of sterols having 4 beta-methyl group, but favorably interacts with sterols having saturated side chain.

Cholestadienols↗
PubMed1985

Yeast sterols: yeast mutants as tools for the study of sterol metabolism.

Yeast mutants defective in ergosterol synthesis are valuable tools for investigating sterol metabolism. Both sterol mutants and sterol auxotrophs have been utilized in determining what sterol structural features are required for yeast cell viability. Both types of mutants can also be studied to ascertain how changes in sterol structure affect membrane properties. Other aspects of sterol metabolism, such as the specificity of sterol esterification, have been elucidated by the sterol auxotrophs. In broader applications, interrelationships between sterol metabolism and other cellular functions (e.g., heme metabolism) may also be examined with these mutants. By analyzing the lipid composition of the sterol mutants, on the other hand, much of the ergosterol biosynthetic pathway has been delineated. The unusual sterols of the mutants can also be obtained to develop assays for the enzymes involved in ergosterol synthesis. Thus, by utilizing mutants, the simple eukaryotic system of yeast may be extended to explore the entire field of sterol metabolism and its relationship to cellular physiology.

Chromatography, High Pressure Liquid↗
PubMed2002

DNA chips for yeast biotechnology. The case of wine yeasts.

The yeast Saccharomyces cerevisiae is one of the most popular model organisms. It was the first eukaryote whose genome was sequenced. Since then many functional analysis projects have tried to find the function of many genes and to understand its metabolism in a holistic way. Apart from basic science this microorganism is of great interest in several biotechnology processes, such as winemaking. Only global studies of the cell as a whole can help us to understand many of the technical problems facing winemaking. DNA chip technology is one of the most promising tools for the analysis of cell physiology. Yeast has been the model organism for the development of this technique. Many of the studies can be applied to improve our knowledge of wine strains. Nevertheless wine strains are quite different in some aspects from the laboratory reference strains so a particular study of wine strains and especially during the winemaking process is needed. During the past two years some groups have started this study and the first results have been published. We review here the current state of the knowledge of wine yeast and the capacity of DNA chip technology for its improvement.

Biological Evolution↗
PubMed2001

Construction of an efficient amylolytic industrial yeast strain containing DNA exclusively derived from yeast.

An amylolytic industrial yeast strain of Saccharomyces cerevisiae containing the Schwanniomyces occidentalis SWA2 amylase gene was generated. The new strain contains DNA derived exclusively from yeast and expresses a high starch hydrolyzing activity. Yeast transformation was carried out by an integrative process targeted to a dispensable upstream region of the ILV2 locus, which determines sulfometuron resistance. The SWA2 enzyme was constitutively expressed under the ADH1 promoter. The growth, substrate utilization and fermentative capacity of this organism are described.

Biotechnology↗
PubMed2001

Utilization of brewer's yeast cells for the production of food-grade yeast extract. Part 1: Effects of different enzymatic treatments on solid and protein recovery and flavor characteristics.

Yeast extract was produced from brewer's yeast of a beer factory by combined enzymatic treatments using endoprotease, exoprotease, 5'-phosphodiesterase, and adenosine monophosphate (AMP)-deaminase. Effects of enzyme combination, enzyme dosages and treatment sequence on the recovery of solid and protein, flavor and compositional characteristics were investigated. Exoprotease dosage strongly affected the recovery of protein and degree of hydrolysis (DH) and sensory characteristics. When the yeast cells were treated using optimal combination of endoprotease and exoprotease (0.6% Protamex and 0.6% Flavourzyme), high solid recovery (48.3-53.1%) and the best flavor profile were obtained. Among various treatment sequences using multiple enzymes, treatment with protease followed by nuclease resulted in the highest 5'-guanosine monophosphate (5'-GMP) content. The optimal concentrations of both 5'-phosphodiesterase and AMP-deaminase were found to be 0.03%. After treatments using optimal combination of enzyme, enzyme dosages and treatment sequence for four enzymes, a high solid yield of 55.1% and 5'-nucleotides content of 3.67% were obtained.

AMP Deaminase↗
PubMed1991

Generating yeast transcriptional activators containing no yeast protein sequences.

We previously reported that roughly 1% of the short peptides encoded by Escherichia coli genomic DNA fragments act as transcriptional activating regions in yeast when fused to GAL4(1-147), a DNA-binding portion of the yeast transcriptional activator GAL4 (ref. 1). Struhl questioned the conclusion that we had identified new transcriptional activating sequences that function in the absence of yeast transcriptional activating sequences. His criticism was based on two considerations: first, GAL4(1-147) contains an acidic segment (and subsequent experiments have shown that this region contains a weak activating region in vitro); second, attempts to isolate new activating regions failed when the DNA-binding domain of a bacterial repressor, LexA(1-87), was used as the DNA-binding unit. We report here a repeat of our original experiment using the complete LexA molecule LexA(1-202) as the DNA-binding region, instead of GAL4(1-147) or LexA(1-87). We find that, as in the original experiment, about 1% of the short peptides encoded by E. coli genomic fragments act as transcriptional activating regions when fused to intact LexA. All of the new activating regions whose sequences we determined bore an excess of acidic amino acids (see Table 1).

Amino Acid Sequence↗