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PubMed2005

Yeast DEL assay detects clastogens.

Chromosomal rearrangements, including DNA deletions are involved in carcinogenesis. The deletion (DEL) assay scoring for DNA deletions in the yeast Saccharomyces cerevisiae is able to detect a wide range of carcinogens. Among approximately 60 compounds of known carcinogenic activity, the DEL assay detected 86% correctly whereas the Ames Salmonella assay detected only 30% correctly [R.J. Brennan, R.H. Schiestl, Detecting carcinogens with the yeast DEL assay, Methods Mol. Biol. 262 (2004) 111-124]. Since the DEL assay is highly inducible by DNA double strand breaks, this study examined the utility of the DEL assay for detecting clastogens. Ten model compounds, with varied mechanisms of genotoxicity, were examined for their effect on the frequency of DNA deletions with the DEL assay. The compounds tested were: actinomycin D, camptothecin, methotrexate and 5-fluorodeoxyuridine, which are anticancer agents, noscapine and furosemide are therapeutics, acridine, methyl acrylate and resorcinol are industrial chemicals and diazinon is an insecticide. The in vitro micronucleus assay (IVMN) in CHO cells, a commonly used tool for detection of clastogens, was performed on the same compounds and the results of the two assays were compared. The results of our study show that there is 70% concordance in the presence of metabolic activation (rat liver S9) and 80% concordance in the absence of metabolic activation between the DEL assay and the standard in vitro micronucleus assay. The lack of cytotoxicity observed for four of the ten compounds examined indicates limited diffusion of lipophilic compounds across the yeast cell wall. Thus, the development of a more permeable yeast tester strain is expected to greatly improve concordance of the DEL assay with the IVMN assay. The yeast DEL assay is inexpensive, amenable to automation and requires less expertise to perform than the IVMN. Thus, it has a strong potential as a robust, fast and economical screen for detecting clastogens in vitro.

Animals↗

PubMed1999

Restriction enzyme analysis of PCR amplified rDNA as a taxonomic tool in yeast identification.

A method has been developed to simplify the identification of yeast strains. We used the restriction fragment patterns of PCR-amplified 18S rRNA-coding DNA with the neighbouring ITS1 region for differentiation and identification of 169 yeast strains representing 128 species associated mainly with food, wine, beer, and soft drinks. The amplicons were digested with four different four-base-cutting restriction enzymes. To construct a database of restriction fragment patterns, the gels have been scanned and analyzed using the Molecular Analyst Fingerprint 2.0 software. The use of four enzymes proved to be sufficient for strain identification.

Beer↗

PubMed2003

Development of a yeast bioassay to characterize G protein-coupled receptor kinases. Identification of an NH2-terminal region essential for receptor phosphorylation.

G protein-coupled receptor kinases (GRKs) specifically bind and phosphorylate the agonist-occupied form of G protein-coupled receptors. To further characterize the mechanism of GRK/receptor interaction, we developed a yeast-based bioassay using strains engineered to functionally express the somatostatin receptor subtype 2 and exhibit agonist-dependent growth. Here, we demonstrate that agonist-promoted growth was effectively inhibited by co-expression with either wild type GRK2 or GRK5, whereas catalytically inactive forms of these kinases were without effect. In an effort to identify residues involved in receptor interaction, we generated a pool of GRK5 mutants and then utilized the bioassay to identify mutants selectively deficient in inhibiting agonist-promoted growth. This resulted in the identification of a large number of mutants, several of which were expressed, purified, and characterized in more detail. Two of the mutants, GRK5-L3Q/K113R and GRK5-T10P, were defective in receptor phosphorylation and also exhibited a partial defect in phospholipid binding and phospholipid-stimulated autophosphorylation of the kinase. In contrast, these mutants had wild type activity in phosphorylating the non-receptor substrate tubulin. To further characterize the function of the NH2-terminal region of GRK5, we generated a deletion mutant lacking residues 2-14 and found that this mutant was also severely impaired in receptor phosphorylation and phospholipid-promoted autophosphorylation. In addition, an NH2-terminal 14-amino acid peptide from GRK5 selectively inhibited receptor phosphorylation by GRK5 but had minimal effect on GRK2 activity. Based on these findings, we propose a model whereby the extreme NH2 terminus of GRK5 mediates phospholipid binding and is required for optimal receptor phosphorylation.

Animals↗

PubMed2003

Microbial biosynthesis and making of pigment melanin.

The efficient strain and the technology of biosynthesis and extraction process from a biomass received in process cultivation of new yeast strain Saccharomyces neoformans var. nigricans are developed. The influence of a carbon substrates (glucose, spirit, milk whey) and conditions of fermentation process on an output and chromatically received melanin are investigated. It is is established, that for the given strain the fermentation time makes 36-40 hours.

Animals↗

PubMed1983

Aeromonas hydrophila typing scheme based on patterns of agglutination with erythrocytes and yeast cells.

An agglutination typing scheme has been developed for strains of Aeromonas hydrophila. Primary agglutination typing is based on testing agar-grown A. hydrophila cells with human, horse, rat, and guinea pig erythrocytes and Saccharomyces cerevisiae cells. Further subdivision of primary groups is based firstly on whether yeast cell agglutination is inhibited by a D-mannose polymer, yeast mannan, and secondly on patterns of inhibition of hemagglutination by yeast mannan and the monomeric sugars L-fucose, D-galactose, and D-mannose. A total of 320 isolates were tested, and these were divisible into 39 distinct types on the basis of this scheme. Application of this typing scheme in the future to isolates of A. hydrophila known to be associated with human infection may enable correlations to be made between particular agglutination types and human pathogenicity.

Aeromonas↗

PubMed2002

Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains.

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.

Aquaporin 1↗

PubMed1995

Tandem integration of multiple ILV5 copies and elevated transcription in polyploid yeast.

An industrial yeast strain was modified by introducing DNA into brewing yeast such that the derived cells contain only yeast DNA. Thus selectable markers and bacterial sequences are not present in the final strain, making this procedure attractive for the development of generally acceptable brewing yeast. Linear DNA containing the cloned ILV5 gene was introduced into lager yeast along with an unlinked circular bifunctional plasmid containing a dominant resistance marker. Resistant colonies were screened for site-directed integration of the ILV5 DNA. Candidates were examined by several methods including Southern transfer and polymerase chain reaction. In this way, a strain WM56 was identified containing three tandem copies of ILV5. The amplified ILV5 region is stable during repeated subculturing in the absence of selective pressure. Correspondingly elevated levels of ILV5 transcript in strain WM56 compared to the control (i.e. non-tandem) parental strain led to increased amounts of encoded acetohydroxyacid reductoisomerase as evidenced by significantly lower diacetyl production. WM56 appears to be identical to the parental strain judged by CHEF, total restriction digestion patterns, and probing, but differs in the ILV5 region of the chromosome. The method is generally applicable to other yeast strains, and if desired, is amenable to iterated cycles of integration to increase the number of copies.

Genes, Fungal↗

PubMed2006

Analytical approach for monitoring endocrine-disrupting compounds in urban waste water treatment plants.

The presence of endocrine-disrupting compounds in influent and effluent water samples from four waste water treatment plants located in Italy was studied. The estrogen-like activity of the water samples was measured using a chemiluminescent recombinant yeast assay which is based on genetically engineered yeast cells that express the human estrogen receptor. This receptor, once activated, elicits the expression of the reporter gene lac-Z and, consequently, the production of beta-galactosidase, which is then measured by chemiluminescence. To control and minimize sample matrix effects, an external control based on a modified yeast strain stably expressing beta-galactosidase was developed and also used in the assay. Rapid and sensitive chemiluminescent enzyme immunoassays were also developed and validated for the quantification of 17beta-estradiol, estrone, and estriol in waste water samples. Results from both methods were compared with a reference high-performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC ESI-MS-MS) method developed for the quantification of natural estrogens. The recombinant yeast assay revealed a significant estrogenic activity in the influent samples, ranging from 80 to 400 pmol/L 17beta-estradiol equivalents (EEQ), which was reduced by 70-95% in the effluent samples. The yeast assay also showed a systematic 20-30% overestimation of estrogenic activity relative to the HPLC ESI-MS-MS method, suggesting the presence of other compounds in the samples with estrogenic activity. The chemiluminescent enzyme immunoassays showed the presence of estrogens in the influent samples (mean concentrations: 350-450 pmol/L for estrone, 5-100 pmol/L for 17beta-estradiol, 25-300 pmol/L for estriol), with significantly lower concentrations detected in the respective effluent samples. The waste water treatment was able to reduce natural estrogen concentrations by 40-95%, although a high variability was observed. The enzyme immunoassay data correlated well with data obtained by the HPLC ESI-MS-MS method. Although the recombinant yeast assay represents a useful tool for a first-level screening of estrogenic activity due to its simplicity and high analytical throughput, sample matrix effects observed in waste water of industrial origin were found to strongly affect the yeast cells response, even when properly corrected for using the external control, thereby limiting its use to urban waste water. Its integration with chemiluminescent enzyme immunoassays would improve its performance by reducing false negative results, thereby enabling its use in extensive studies monitoring for the presence of endocrine-disrupting compounds in urban treatment plant effluents.

Chromatography, High Pressure Liquid↗

PubMed2002

Development of a simple screening system for endocrine disruptors.

BACKGROUND: An endocrine disruptor is a synthetic chemical, which causes adverse effects in an organism, or its progeny, after causing perturbations in the endocrine system. It is important to know which synthetic chemicals have endocrine-disrupting action. However, an increasing number of synthetic chemicals are being produced by modern synthetic chemistry, and the examination of endocrine disruptor potential has not yet caught up with the advances in synthetic chemistry. In this study, we have developed such a screening system for detecting synthetic chemicals with estrogen-like effects. MATERIAL/METHODS: The system was based on the yeast one-hybrid system. Both HIS3 and lacZ reporter genes connected to three tandem copies of the estrogen response element were prepared. Gal4-estrogen receptor is a fusion protein made from the activation domain (AD) of the yeast GAL4 transactivator gene and then incorporated into a plasmid, which was transfected into the YM4271 yeast cell strain. The estrogen effect was judged by this developed screening system. RESULTS: A dual reporter assay-system was established by transfection of the both HIS3 and lacZ reporter genes into the yeast cells. This screening system enabled the detection of as little as 10-12 mol of beta-estradiol. CONCLUSIONS: These results show that this newly developed dual assay is useful for the screening of endocrine-disruptors that have estrogen-like action.

Dose-Response Relationship, Drug↗

PubMed2006

Summary of recombinant human serum albumin development.

The methylotrophic yeast Pichia pastoris is well known as a host strain for the production of a variety of heterologous proteins. We have used P. pastoris for the production of recombinant human serum albumin (rHSA), for which we have developed efficient and specialized downstream processes. Results from structural analysis suggest that purified rHSA possesses an identical conformation to plasma derived human albumin (pdHA) and no difference from pdHA has been observed in neo-antigenicity. Host-cell-derived impurities (i.e. Pichia yeast component, DNA and mannan) have been evaluated in the purification process as well as in the drug substance and relevant specifications established. The efficacy and safety of rHSA have been tested in clinical studies and no difference from pdHA has been found in comparative study. Such studies have confirmed rHSA to have high efficacy with little or no adverse reaction.

Clinical Trials as Topic↗

PubMed1992

Isolation and characterization of RAT1: an essential gene of Saccharomyces cerevisiae required for the efficient nucleocytoplasmic trafficking of mRNA.

We have combined techniques of genetics and histochemistry to identify genes required for the nucleocytoplasmic export of mRNA in the budding yeast Saccharomyces cerevisiae. We adapted in situ hybridization using a digoxigenin-labeled oligo(dT)50 probe to localize poly(A)+ RNA in fixed yeast cells and used yeast strains carrying the rna1-1 mutation to develop an assay. The rna1-1 mutation is the only previously described mutation that causes defects in mRNA export. As visualized with this RNA localization assay, rna1-1 strains accumulated poly(A)+ RNA at the nuclear periphery at the nonpermissive temperature. This was in contrast to the RNA localization pattern of wild-type cells or rna1-1 cells grown at permissive temperature. Wild-type cells showed bright uniform cytoplasmic staining with little detectable RNA in the nuclei. We used this RNA localization assay to screen a bank of temperature-sensitive yeast strains for mutants with inducible defects in mRNA trafficking. Strains identified in this manner are designated RAT mutants for ribonucleic acid trafficking. The rat1-1 allele conferred temperature-sensitive accumulation of poly(A)+ RNA in one to several intranuclear spots that appear to lie at the nuclear periphery. RNA processing was unaffected in rat1-1 strains, except for an inducible defect in trimming the 5' end of the 5.8S rRNA. The wild-type RAT1 gene was cloned by complementation; it encodes an essential 116-kD protein with regions of homology to the protein encoded by SEP1 (also known as DST2, XRN1, KEM1, and RAR5). Sep1p is a nucleic acid binding protein, a 5'----3' exonuclease, and catalyzes DNA strand transfer reactions in vitro. We discuss the possible significance of the Rat1p/Sep1p homology for RNA trafficking. We also discuss the potential of this RNA localization assay to identify genes involved in nuclear structure and RNA metabolism.

Amino Acid Sequence↗

PubMed2004

A proteomic approach to understanding the development of multidrug-resistant Candida albicans strains.

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by the overexpression of genes that encode multidrug efflux pumps ( CDR1, CDR2, or MDR1). We have undertaken a proteomic approach to gain further insight into the regulatory network controlling efflux pump expression and drug resistance in C. albicans. Three pairs of matched fluconazole-susceptible and resistant clinical C. albicans isolates, in which drug resistance correlated with stable activation of MDR1 or CDR1/2, were analyzed for differences in their protein expression profiles. In two independent, MDR1-overexpressing, strains, additional up-regulated proteins were identified, which are encoded by the YPR127 gene and several members of the IFD ( YPL088) gene family. All are putative aldo-keto reductases of unknown function. These proteins were not up-regulated in a fluconazole-resistant strain that overexpressed CDR1 and CDR2 but not MDR1, indicating that expression of the various efflux pumps of C. albicans is controlled by different regulatory networks. To investigate the possible role of YPR127 in the resistance phenotype of the clinical isolates, we constitutively overexpressed the gene in a C. albicans laboratory strain. In addition, the gene was deleted in a C. albicans laboratory strain and in one of the drug-resistant clinical isolates in which it was overexpressed. Neither forced overexpression nor deletion of YPR127 affected the susceptibility of the strains to drugs and other toxic substances, suggesting that the regulatory networks which control the expression of efflux pumps in C. albicans also control genes involved in cellular functions not related to drug resistance.

Antifungal Agents↗

PubMed1998

A simple and reliable method for hybridization of homothallic wine strains of saccharomyces cerevisiae

A procedure was developed for the hybridization and improvement of homothallic industrial wine yeasts. Killer cycloheximide-sensitive strains were crossed with killer-sensitive cycloheximide-resistant strains to get killer cycloheximide-resistant hybrids, thereby enabling hybrid selection and identification. This procedure also allows backcrossing of spore colonies from the hybrids with parental strains.

Journal Article↗

PubMed2006

Microarray-based method for monitoring yeast overexpression strains reveals small-molecule targets in TOR pathway.

Identification of the cellular targets of small-molecule hits in phenotypic screens is a central challenge in the development of small molecules as biological tools and potential therapeutics. To facilitate the process of small-molecule target identification, we developed a global, microarray-based method for monitoring the growth of pools of yeast strains, each overexpressing a different protein, in the presence of small molecules. Specifically, the growth of Saccharomyces cerevisiae strains harboring approximately 3,900 different overexpression plasmids was monitored in the presence of rapamycin, which inhibits the target of rapamycin (TOR) proteins. TOR was successfully identified as a candidate rapamycin target, and many additional gene products were implicated in the TOR signaling pathway. We also characterized the mechanism of LY-83583, a small-molecule suppressor of rapamycin-induced growth inhibition. These data enabled functional links to be drawn between groups of genes implicated in the TOR pathway, identified several candidate targets for LY-83583, and suggested a role for mitochondrial respiration in mediating rapamycin sensitivity.

Aminoquinolines↗

PubMed1999

Selective detection of inactivating mutations of the tumor suppressor gene p53 in bladder tumors.

OBJECTIVE: Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors. In bladder tumors they are usually detected by immunohistochemistry. The assumption underlying protein analysis is that high level p53 expression is a consequence of mutations, but numerous exceptions have been reported. We describe the detection of p53 mutations in bladder cancer using a functional assay in yeast. MATERIALS AND METHODS: The prospective study consisted of 60 consecutive patients with bladder tumors (7 pT0, 2 CIS, 23 pTa, 24 pT1 and 4 pT2). High grade 3 was observed in primary carcinoma in situ, in 75% of pT1 tumors and in all pT2 tumors. The p53 mRNA extracted from endoscopic resection tissue was reverse transcribed and PCR-amplified. The transcriptional competence of the p53 cDNA was then tested in a yeast reporter strain. A simple functional assay was developed for p53 mutation in which human p53 is expressed in Saccharomyces cerevisiae which activates transcription of the ADE2 gene. Colonies containing wild type p53 are white and colonies containing mutant p53 are red. RESULTS: As this assay evaluates the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations. In pTo and pTa bladder tumors, no p53 mutations were detected. In contrast, the functional assay permitted us to detect p53 mutations in 66% of patients with stage T1 tumors (72% of case of high grade 3) and in all cases with primary carcinoma in situ and in 4 cases of stage T2 tumors. CONCLUSION: This preliminary study demonstrates that this functional assay method is a simple and efficient procedure to detect p53 mutations in bladder cancers and suggests that p53 mutations seem to be associated with invasive bladder tumors.

Chromosomes, Artificial, Yeast↗

PubMed2006

Population genomic analysis of outcrossing and recombination in yeast.

The budding yeast Saccharomyces cerevisiae has been used by humans for millennia to make wine, beer and bread. More recently, it became a key model organism for studies of eukaryotic biology and for genomic analysis. However, relatively little is known about the natural lifestyle and population genetics of yeast. One major question is whether genetically diverse yeast strains mate and recombine in the wild. We developed a method to infer the evolutionary history of a species from genome sequences of multiple individuals and applied it to whole-genome sequence data from three strains of Saccharomyces cerevisiae and the sister species Saccharomyces paradoxus. We observed a pattern of sequence variation among yeast strains in which ancestral recombination events lead to a mosaic of segments with shared genealogy. Based on sequence divergence and the inferred median size of shared segments (approximately 2,000 bp), we estimated that although any two strains have undergone approximately 16 million cell divisions since their last common ancestor, only 314 outcrossing events have occurred during this time (roughly one every 50,000 divisions). Local correlations in polymorphism rates indicate that linkage disequilibrium in yeast should extend over kilobases. Our results provide the initial foundation for population studies of association between genotype and phenotype in S. cerevisiae.

Crosses, Genetic↗

PubMed2006

Transgenic wine yeast technology comes of age: is it time for transgenic wine?

Saccharomyces cerevisiae is the main yeast responsible for alcoholic fermentation of grape juice during wine making. This makes wine strains of this species perfect targets for the improvement of wine technology and quality. Progress in winemaking has been achieved through the use of selected yeast strains, as well as genetic improvement of wine yeast strains through the sexual and pararexual cycles, random mutagenesis and genetic engineering. Development of genetically engineered wine yeasts, their potential application, and factors affecting their commercial viability will be discussed in this review.

Fermentation↗

PubMed1995

Genetically-modified brewing yeasts for the 21st century. Progress to date.

Academic studies and traditional breeding of yeasts depend upon their sporulation lifestyle. The strains used have been specially selected to sporulate readily and to mate producing new yeast types. Unfortunately brewing yeast strains do not behave in this way. They sporulate poorly, any spores which are formed are usually non-viable and any haploid strains produced are invariably non-maters. Only in recent years, with the development of recombinant-DNA techniques, has the specific breeding of new brewing yeast strains become widespread. Strains have been produced with the ability to ferment a wider range of carbohydrates, with altered flocculation properties and which produce beers with modified flavours. Many have been tested on the pilot scale and one, an amylolytic brewing yeast, has received approval for commercial use.

Carbohydrate Metabolism↗