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PubMed1994

Histidylation by yeast HisRS of tRNA or tRNA-like structure relies on residues -1 and 73 but is dependent on the RNA context.

Residue G-1 and discriminator base C73 are the major histidine identity elements in prokaryotes. Here we evaluate the importance of these two nucleotides in yeast histidine aminoacylation identity. Deletion of G-1 in yeast tRNA(His) transcript leads to a drastic loss of histidylation specificity (about 500-fold). Mutation of discriminator base A73, common to all yeast tRNA(His) species, into G73 has a more moderate but still significant effect with a 22-fold decrease in histidylation specificity. Changes at position 36 in the anticodon loop has negligible effect on histidylation. The role of residues -1 and 73 for specific aminoacylation by yeast HisRS was further investigated by studying the histidylation capacities of seven minihelices derived from the Turnip Yellow Mosaic Virus tRNA-like structure. Changes in the nature of nucleotides -1 and 73 modulate this activity but do not suppress it. The optimal mini-substrate for HisRS presents a G.A mismatch at the position equivalent to residues G-1.A73 in yeast tRNA(His), confirms the importance of this structural feature in yeast histidine identity. The fact that the minisubstrates contain a pseudoknot in which position -1 is mimicked by an internal nucleotide from the pseudoknot highlights further the necessity of a stacking interaction of this position over the amino acid accepting branch of the tRNA during the aminoacylation process. Individual transplantation of G-1 or A73 into yeast tRNA(Asp) transcript improves the histidylation efficiency of the engineered tRNA(Asp). However, a tRNA(Asp) transcript presenting simultaneously both residues G-1 and A73 becomes a less good substrate for HisRS, suggesting the importance of the structural context and/or the presence of antideterminants for an optimal expression of these two identity elements.

Anticodon↗

PubMed1990

Soluble factor requirements for the Tetrahymena peptide elongation system and the ribosomal ATPase as a counterpart of yeast elongation factor 3 (EF-3).

Peptide elongation factor 3 (EF-3), which is widely present in yeasts and fungi (Eumycota), does not occur in another lower eukaryote, the unicellular protozoan Tetrahymena pyriformis, as was shown by the following findings: (a) there is no activity to satisfy the EF-3 requirement of yeast ribosomes in the post-ribosomal supernatant fraction from Tetrahymena, and (b) the Tetrahymena ribosomes displayed their full capacity for polyphenylalanine synthesis with purified EF-1 alpha and EF-2 alone from either Tetrahymena or yeast, and their activity on the Tetrahymena ribosomes was not further enhanced by the addition of yeast EF-3, in contrast to the case of the yeast ribosomes. However, as a substitute for the ribosome-activated nucleotidase activity of EF-3, Tetrahymena ribosomes were shown to harbor strong, firmly bound ATPase and GTPase activities, which probably involve the same active site. The ribosome-bound ATPase activity was inhibited by a polyclonal antibody raised against yeast EF-3 with the same inactivation profile as that of polyphenylalanine synthesis on Tetrahymena ribosomes, indicating that the ribosomal ATPase plays an essential role in the elongation process on Tetrahymena ribosomes as previously revealed in the yeast system. It was also shown that the ribosomal nucleotidase plays a pivotal role in the elongation cycle in other eukaryotes.

Adenosine Triphosphatases↗

PubMed2006

Development of a novel strategy for engineering high-affinity proteins by yeast display.

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Cell Differentiation↗

PubMed1989

A yeast mitochondrial presequence functions as a signal for targeting to plant mitochondria in vivo.

To date, the presequence of the mitochondrial beta-subunit of ATPase from tobacco is the only signal sequence that has been shown to target a foreign protein into plant mitochondria in vivo. Here we report that the presequence of a yeast mitochondrial protein directs bacterial beta-glucuronidase (GUS) specifically into the mitochondrial compartment of transgenic tobacco plants. Fusions between the presequence of the mitochondrial tryptophanyl-tRNA-synthetase gene from yeast and the GUS gene have been introduced into tobacco plants and yeast cells. In both systems, proteins containing the complete yeast mitochondrial presequence are efficiently imported in the mitochondria. Measurements of GUS activity in different subcellular fractions indicate that there is no substantial misrouting of the chimeric proteins in plant cells. In vitro synthesized GUS fusion proteins have a higher molecular weight than those found inside yeast and tobacco mitochondria, suggesting a processing of the precursors during import. Interestingly, fusion proteins translocated across the mitochondrial membranes of tobacco have the same size as those that are imported into yeast mitochondria. We conclude that the processing enzyme in plant mitochondria may recognize a proximate or even the same cleavage site within the mitochondrial tryptophanyl-tRNA-synthetase presequence as the matrix protease from yeast.

Adenosine Triphosphatases↗

PubMed2006

Influence of grape treatment on the wine yeast populations isolated from spontaneous fermentations.

AIM: To study the influence of different methods of grape treatment in wineries on the diversity of the yeast species in spontaneous fermentations. METHODS AND RESULTS: Grapes were crushed and pressed in three different ways followed by spontaneous fermentation. The same grape material picked and crushed aseptically directly in the vineyard served as control. Yeasts isolated at different stages of the fermentation were characterized by 5.8S-ITS-RFLP. Yeasts of the Saccharomyces sensu stricto complex were additionally analysed by microsatellite polymerase chain reaction fingerprinting. The diversity of yeast species isolated from winery fermentations was much greater than from the vineyard fermentation in respect to yeasts of the genus Saccharomyces as well as non-Saccharomyces. CONCLUSIONS: Oenonogical methods alter significantly the yeast diversity in spontaneous fermentations of grape juice. SIGNIFICANCE AND IMPACT OF THE STUDY: Managing spontaneous fermentations successfully depends not only on choosing the suitable grapes but also on the crushing and pressing techniques leading to different yeast populations.

Fermentation↗

PubMed2005

Contaminant yeast detection in industrial ethanol fermentation must by rDNA-PCR.

AIMS: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5.8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. METHODS AND RESULTS: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. CONCLUSIONS: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.

DNA, Ribosomal↗

PubMed1997

The molecular genetics of hexose transport in yeasts.

Transport across the plasma membrane is the first, obligatory step of hexose utilization. In yeast cells the uptake of hexoses is mediated by a large family of related transporter proteins. In baker's yeast Saccharomyces cerevisiae the genes of 20 different hexose transporter-related proteins have been identified. Six of these transmembrane proteins mediate the metabolically relevant uptake of glucose, fructose and mannose for growth, two others catalyze the transport of only small amounts of these sugars, one protein is a galactose transporter but also able to transport glucose, two transporters act as glucose sensors, two others are involved in the pleiotropic drug resistance process, and the functions of the remaining hexose transporter-related proteins are not yet known. The catabolic hexose transporters exhibit different affinities for their substrates, and expression of their corresponding genes is controlled by the glucose sensors according to the availability of carbon sources. In contrast, milk yeast Kluyveromyces lactis contains only a few different hexose transporters. Genes of other monosaccharide transporter-related proteins have been found in fission yeast Schizosaccharomyces pombe and in the xylose-fermenting yeast Pichia stipitis. However, the molecular genetics of hexose transport in many other yeasts remains to be established. The further characterization of this multigene family of hexose transporters should help to elucidate the role of transport in yeast sugar metabolism.

Biological Transport↗

PubMed1987

Screening of caries in children through salivary lactobacilli and yeasts.

The caries predictive value of salivary counts of lactobacilli and yeasts was evaluated in 298 children (from 6 to 11 yr of age) concurrently to a 3-yr xylitol field study in Hungary. On a group level the salivary yeasts and the combined information of lactobacilli and yeasts predicted the 3-yr caries increment acceptably. On an individual basis salivary yeasts had more power than salivary lactobacilli. At best the sensitivity and specificity of yeasts were 74% and 75%, and the combined information of lactobacilli and yeasts, 69% and 83%, respectively. Although the results were not that good in all subgroups of the study, they indicate the value of salivary yeasts in caries prediction either as a sole test or together with salivary lactobacilli.

Child↗

PubMed2006

Real-time quantitative PCR (QPCR) and reverse transcription-QPCR for detection and enumeration of total yeasts in wine.

Real-time PCR, or quantitative PCR (QPCR), has been developed to rapidly detect and quantify the total number of yeasts in wine without culturing. Universal yeast primers were designed from the variable D1/D2 domains of the 26S rRNA gene. These primers showed good specificity with all the wine yeasts tested, and they did not amplify the most representative wine species of acetic acid bacteria and lactic acid bacteria. Numerous standard curves were constructed with different strains and species grown in yeast extract-peptone-dextrose medium or incubated in wine. The small standard errors with these replicas proved that the assay is reproducible and highly robust. This technique was validated with artificially contaminated and natural wine samples. We also performed a reverse transcription-QPCR (RT-QPCR) assay from rRNA for total viable yeast quantification. This technique had a low detection limit and was more accurate than QPCR because the dead cells were not quantified. As far as we know, this is the first time that RT-QPCR has been performed to quantify viable yeasts from rRNA. RT-QPCR is a rapid and accurate technique for enumerating yeasts during industrial wine fermentation and controlling the risk of wine spoilage.

DNA Primers↗

PubMed1975

Evaluation of antibody coating of yeasts in urine as an indicator of the site of urinary tract infection.

Antibody coating of yeasts (Candida sp. and Torulopsis sp.) found in urine specimens was investigated to ascertain whether the presence of such coating might identify the site of urinary tract infection. Washed yeast cells obtained by centrifugation of fresh urine specimens were reacted with fluorescein-conjugated goat antihuman immunoglobulins (Ig) G, A, and M and examined by fluorescent microscopy. IgG was found on the surface of all species of yeast encountered in all urine specimens evaluted, whereas there was variability of IgA AND IgM coating. Antibody coating with IgG, IgA, AND IgM was also demonstrated on yeasts from other body sites (sputum, gastrostomy, oral, etc.). Control experiments confirmed the specificity of the reactions. Thus, it appears that yeasts from any body site are coated with antibodies. These results are in contrast to recent work with bacteria which showed that the presence of antibody-coated (IgG) bacteria indicates upper urinary tract infection (pyelonephritis) while bacteria are not coated with antibodies in lower urinary tract infection. Since all yeasts from all body sites tested were found to be coated with antibody regardless of the clinical situation, the presence of surface antibody has no diagnostic value in identifying the site of urinary tract infection with yeasts.

Antibodies, Fungal↗

PubMed1987

Comparison of Sabouraud dextrose and Pagano-Levin agar media for detection and isolation of yeasts from oral samples.

The sensitivities of Sabouraud dextrose agar and modified Pagano-Levin agar for the primary isolation of yeasts and the recovery of multiple yeast species from single clinical samples were compared by using oral-rinse samples. Although there was a highly significant positive correlation between the numbers of yeasts recovered from both media, modified Pagano-Levin agar was far superior in detecting multiple yeast species in a single sample. Of 150 oral samples containing yeasts, 23 (15.3%) contained more than one yeast species. The most frequent combination of different yeasts was Candida albicans and Torulopsis glabrata.

Candida↗

PubMed1992

Value of extended agitation and subculture of BACTEC NR 660 aerobic resin blood culture bottles for clinical yeast isolates.

From 10,351 blood cultures, we prospectively studied 1,000 BACTEC NR 660 aerobic resin blood culture bottles (26+ and Peds Plus) for patients suspected of having yeast septicemia to determine whether extended agitation and subculturing would increase the recovery of yeasts. Aerobic bottles were agitated continuously for 144 h. On day 7, 1,000 culture-negative aerobic bottles which had fungal blood culture requests were agitated for an additional 14 days. During this time they were subcultured twice and read twice by BACTEC NR 660. ON days 1 to 7, 81 bottles were cultured positive for yeasts from 36 patients, which included 44 isolates of Candida albicans, averaging 1.4 days to detection, and 12 isolates of Cryptococcus neoformans, averaging 3.8 days to detection. The average detection time for all yeasts was 2.2 days. On days 7 to 21, no yeasts were detected by BACTEC or recovered from the subcultures. We conclude that when continuously agitated for at least 5 full days (120 h), the BACTEC NR 660 aerobic resin bottles reliably isolate yeasts, and it is unnecessary to subculture or hold these bottles beyond 5 days. It also eliminates the need for an additional blood culture system for yeast detection, thus saving (i) confusion in the collection process, (ii) patients' blood and money, and (iii) laboratory technologists' time.

Blood↗

PubMed2003

Vulvovaginal carriage of yeasts other than Candida albicans.

AIMS: We investigated the spectrum of yeasts isolated, and compared the epidemiological and laboratory characteristics of women carrying vulvovaginal Candida albicans with those carrying yeasts other than C. albicans. METHOD: Between April and June 2001, 5802 consecutively received genital swabs from women were plated onto Candida ID chromogenic media (BioMerieux). Blue colonies were reported as C. albicans; all other colonies (white and pink) were identified to species level using the Vitek YBC card (BioMerieux). In vitro susceptibility to amphotericin (AMB), fluconazole (FLU), itraconazole (ITZ), and voriconazole (VOR) was determined for approximately 40% of non-C. albicans yeasts using a standardised microdilution method. RESULTS: Yeast was isolated from 1221 women (21%). Of these, C. albicans only was isolated from 1087 (89%) and yeasts other than C. albicans from 129 (11%) women. C. glabrata comprised 89 (69%) of the latter. Women in whom other yeasts were recovered were older than those with C. albicans (mean 43, versus 33 years, p <0.001). All isolates tested (n=53) were susceptible to AMB and VOR. Seven (24%) C. glabrata strains were susceptible to FLU with 21 (72%) testing susceptible-dose dependent. CONCLUSION: Yeasts other than C. albicans are common vaginal isolates even in a primary care population. The species isolated are less susceptible to FLU than most C. albicans.

Adult↗

PubMed2002

The effects of fungicides on the phylloplane yeast populations of creeping bentgrass.

The effects of fungicides on population size and the development of fungicide resistance in the phylloplane yeast flora of bentgrass was investigated. In the spring of 2001, azoxystrobin, chlorothalonil, flutolanil, and propiconazole were applied separately over a 6-week period to creeping bentgrass (Agrostis palustris Huds.). Total and fungicide-resistant yeast populations were assessed by dilution plating onto either potato dextrose agar or potato dextrose agar amended with the test fungicides. Total yeast populations in the fungicide-treated plots were significantly lower than the check plots on three out of four sample dates. In the fall, azoxystrobin or propiconazole were applied twice to the bentgrass over 3 weeks. Significantly larger total yeast populations were observed compared with resistant or highly resistant populations for each treatment on every sample date. Total yeast populations were significantly higher in the check plots compared with either the propiconazole- or azoxystrobin-treated plots on the first three of five sample dates. A collection of yeasts (N = 114) with no prior exposure to fungicides were more sensitive to chlorothalonil, propiconazole, flutolanil, and iprodione than a second group (N = 115) isolated from fungicide-treated turfgrass. These results suggest that fungicide resistance among phylloplane yeasts is widespread and could be an important factor in the development of biological control agents for turfgrass diseases.

Drug Resistance, Microbial↗

PubMed2003

Shifts in community composition provide a mechanism for maintenance of activity of soil yeasts in the presence of elevated copper levels.

Soil dilution plates were prepared from different soil samples using a solid synthetic selective medium containing (i). glucose as carbon source, (ii). thymine as nitrogen source, (iii). vitamins, (iv). minerals, and (v). chloramphenicol as antibacterial agent. Using the Diazonium Blue B colour reaction, it was found that both ascomycetous and basidiomycetous yeasts were able to grow on this medium. Subsequently, the medium was used to enumerate yeasts in soil microcosms prepared from four different soil samples, which were experimentally treated with the fungicide copper oxychloride, resulting in copper (Cu) concentrations of up to 1000 ppm. The selective medium supplemented with 32 ppm of Cu was used to enumerate Cu-resistant yeasts in the microcosms. The results showed that the addition of Cu at concentrations >or=approximately 1000 ppm did not have a significant effect on total number of yeasts in the soil. Furthermore, it was found that Cu-resistant yeasts were present in all the soil samples, regardless of the amount of Cu that the soil was challenged with. At the end of the incubation period, yeasts in the microcosms with zero and approximately 1000 ppm of additional Cu were enumerated, isolated, and identified with sequence analyses of the D1/D2 600-650 bp region of the large subunit of ribosomal DNA. Hymenomycetous species dominated in the control soil, while higher numbers of the urediniomycetous species were found in the soil that received Cu. These observations suggest that urediniomycetous yeasts may play an important role in re-establishing overall microbial activity in soils, following perturbations, such as the addition of Cu-based fungicides.

Copper↗

PubMed2006

Cell organisation, sulphur metabolism and ion transport-related genes are differentially expressed in Paracoccidioides brasiliensis mycelium and yeast cells.

BACKGROUND: Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. RESULTS: In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation - cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-beta-glucosidase) in mycelium cells; and ags (an alpha-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport - two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells - isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. CONCLUSION: Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.

Biological Transport↗

PubMed2004

Rapid isolation of yeast genomic DNA: Bust n' Grab.

BACKGROUND: Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates. RESULTS: Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng--3 microg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase cocktail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human beta-globin locus YAC. CONCLUSION: An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

Blotting, Southern↗

PubMed1977

[Studies on brewer's yeast (II). Phlogistic action (author's transl)].

Yeast has a phlogistic effect, and the inflammatory action of 10 brewer's yeast samples fractionated by autolysis and/or destruction of the cell wall was studied in rat hind paw. The yeast samples produced a dose dependent edema when given subcutaneously into the paw. The samples obtained by dissolution or destruction of the cell wall produced the most prominent edema and the phlogistic effect was more potent than commerical yeast powder. Peak of the response was noted 1 hour after injection and thereafter the grade of edema rapidly decreased. The pattern of swelling resembled that by dextran. The edema was partially (less than 40%) inhibited by anti-inflammatory drugs such as aspirin, aminopyrine, phenylbutazone, and indomethacin. Mannan, a main constituent of the yeast cell wall, produced a swelling similar to that observed with yeast samples and this edema was almost completely inhibited by cyprohepatdine, an anti-serotonin drug, but was not inhibited by diphenhydramine, an anti-histamine drug. The results suggest that there is a release of serotonin when brewer's yeast is given subcutaneously, thus producing a phlogistic effect.

Animals↗