Showing papers in "Yeast in 2000"

Tailoring wine yeast for the new millennium: novel approaches to the ancient art of winemaking

Abstract: Yeasts are predominant in the ancient and complex process of winemaking. In spontaneous fermentations, there is a progressive growth pattern of indigenous yeasts, with the final stages invariably being dominated by the alcohol-tolerant strains of Saccharomyces cerevisiae. This species is universally known as the ‘wine yeast’ and is widely preferred for initiating wine fermentations. The primary role of wine yeast is to catalyze the rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important, metabolites without the development of off-flavours. However, due to the demanding nature of modern winemaking practices and sophisticated wine markets, there is an ever-growing quest for specialized wine yeast strains possessing a wide range of optimized, improved or novel oenological properties. This review highlights the wealth of untapped indigenous yeasts with oenological potential, the complexity of wine yeasts’ genetic features and the genetic techniques often used in strain development. The current status of genetically improved wine yeasts and potential targets for further strain development are outlined. In light of the limited knowledge of industrial wine yeasts’ complex genomes and the daunting challenges to comply with strict statutory regulations and consumer demands regarding the future use of genetically modified strains, this review cautions against unrealistic expectations over the short term. However, the staggering potential advantages of improved wine yeasts to both the winemaker and consumer in the third millennium are pointed out. Copyright # 2000 John Wiley & Sons, Ltd.

Rapid and reliable protein extraction from yeast.

Abstract: The methods currently used for protein extraction from yeast are either laborious or insufficiently reliable. Here I report a method for protein extraction for electrophoretic analysis that is both easy and reliable. In this method, yeast cells are subjected to mild alkali treatment and then boiled in a standard electrophoresis loading buffer. The method was tested for different strains of Saccharomyces cerevisiae and for yeast Hansenula polymorpha DL-1. It yields virtually complete extraction independently of the strain, growth conditions and protein molecular weight and allows working with small amounts of yeast cells grown on agar plates. Copyright © 2000 John Wiley & Sons, Ltd.

Mitochondria-targeted green fluorescent proteins : convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae

Abstract: We describe the construction and characterization of a novel set of plasmids for expression of mitochondria-targeted green fluorescent protein (GFP) in Saccharomyces cerevisiae. The vectors include constructs with strong regulatable and constitutive promoters, four different auxotrophic markers for yeast transformation, and a green (S65T) and a blue-shifted (P4-3) variant of GFP. Mitochondria are brightly fluorescent in living yeast cells grown on different carbon sources and at different temperatures, with virtually no background staining. Specific staining of mitochondria is also shown for a respiratory-deficient mutant with abnormal mitochondrial morphology. The plasmids facilitate convenient analysis of mutants defective in mitochondrial morphology or inheritance and, at the same time, are suitable vectors for easy construction of different kinds of GFP fusion proteins to study various aspects of organelle biogenesis in living yeast cells.

CIp10, an efficient and convenient integrating vector for Candida albicans

Abstract: Candida albicans is the major fungal pathogenin humans (Odds, 1994). Despite its medicalimportance, the molecular dissection of thisfungus has progressed relatively slowly. This isbecause (a) it is diploid, (b) a sexual cycle has notbeen elaborated, and (c) the alternative usage ofthe CTG codon renders most reporter genes non-functional (Santos et al., 1993; Magee, 1998). TheC. albicans molecular toolbox has expandedsignificantly within the last 10 years, thanks tothe development of auxotrophic markers, genomicand cDNA libraries, a robust gene disruptiontechnology, specialized reporter genes and regula-table promoters (Smith et al., 1992; Fonzi andIrwin, 1993; Swoboda et al., 1993; Pla et al., 1996;Srikantha et al., 1996; Cormack et al., 1997;Leuker et al., 1997; Care et al., 1999). Further-more, the isolation of C. albicans genes has beenfacilitated by rapid advancements in C. albicansgenome mapping and sequencing (Tait et al.,1997; Chibana et al., 1998; Magee, 1998; http://alces.med.umn.edu:80/bin/genelist?seqs). How-ever, a limited number of reliable and robustplasmids are available to support genetic screensand functional analyses in C. albicans. Here wedescribe a convenient and efficient integratingvector for C. albicans.Most plasmids transform C. albicans veryinefficiently, and genes carried on these vectorstend to be expressed inefficiently (see e.g. Baileyet al., 1996). Also, episomal plasmids tend torearrange or are unstable in C. albicans (Pla et al.,1996). Therefore, we constructed the vector CIp10(Candida integrating plasmid 10). The plasmid isbased on pBluescript KS

The Kyoto encyclopedia of genes and genomes--KEGG.

Abstract: The aim of the present study was to explore the function and interaction of differentially expressed genes (DEGs) in pulmonary embolism (PE). The gene expression profile GSE13535, was downloaded from the Gene Expression Omnibus database. The DEGs 2 and 18 h post-PE initiation were identified using the affy package in R software. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the DEGs were analyzed using Database for Annotation Visualization and Integrated Discovery (DAVID) online analytical tools. In addition, protein-protein interaction (PPI) networks of the DEGs were constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins. The PPI network at 18 h was modularized using ClusterONE, and a functional enrichment analysis of the DEGs in the top three modules was performed with DAVID. Overall, 80 and 346 DEGs were identified 2 and 18 h after PE initiation, respectively. The KEGG pathways, including chemokine signaling and toll‐like receptor signaling, were shown to be significantly enriched. The five highest degree nodes in the PPI networks at 2 or 18 h were screened. The module analysis of the PPI network at 18 h revealed 11 hub nodes. A Gene Ontology terms analysis demonstrated that the DEGs in the top three modules were associated with the inflammatory, defense and immune responses. The results of the present study suggest that the DEGs identified, including chemokine‐related genes TFPI2 and TNF, may be potential target genes for the treatment of PE. The chemokine signaling pathway, inflammatory response and immune response were explored, and it may be suggested that these pathways have important roles in PE.

Distinct requirements for zebrafish angiogenesis revealed by a VEGF-A morphant.

Abstract: Angiogenesis is a fundamental vertebrate developmental process that requires signalling by the secreted protein vascular endothelial growth factor-A (VEGF-A). VEGF-A functions in the development of embryonic structures, during tissue remodelling and for the growth of tumour-induced vasculature. The study of the role of VEGF-A during normal development has been significantly complicated by the dominant, haplo-insufficient nature of VEGF-A-targeted mutations in mice. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish VEGF-A morphant loss of function model. Zebrafish VEGF-A morphant embryos develop with an enlarged pericardium and with major blood vessel deficiencies. Morphological assessment at 2 days of development indicates a nearly complete absence of both axial and intersegmental vasculature, with no or reduced numbers of circulating red blood cells. Molecular analysis using the endothelial markers fli-1 and flk-1 at 1 day of development demonstrates a fundamental distinction between VEGF-A requirements for axial and intersegmental vascular structure specification. VEGF-A is not required for the initial establishment of axial vasculature patterning, whereas all development of intersegmental vasculature is dependent on VEGF-A signalling. The zebrafish thus serves as a quality model for the study of conserved vertebrate angiogenesis processes during embryonic development.

Relationship of codon bias to mRNA concentration and protein length in Saccharomyces cerevisiae

Abstract: In 1982, Ikemura reported a strikingly unequal usage of different synonymous codons, in five Saccharomyces cerevisiae nuclear genes having high protein levels. To study this trend in detail, we examined data from three independent studies that used oligonucleotide arrays or SAGE to estimate mRNA concentrations for nearly all genes in the genome. Correlation coefficients were calculated for the relationship of mRNA concentration to four commonly used measures of synonymous codon usage bias: the codon adaptation index (CAI), the codon bias index (CBI), the frequency of optimal codons (F(op)), and the effective number of codons (N(c)). mRNA concentration was best approximated as an exponential function of each of these four measures. Of the four, the CAI was the most strongly correlated with mRNA concentration (r(s)=0.62+/-0.01, n=2525, p<10(-17)). When we controlled for CAI, mRNA concentration and protein length were negatively correlated (partial r(s)=-0.23+/-0.01, n=4765, p<10(-17)). This may result from selection to reduce the size of abundant proteins to minimize transcriptional and translational costs. When we controlled for mRNA concentration, protein length and CAI were positively correlated (partial r(s)=0.16+/-0.01, n=4765, p<10(-17)). This may reflect more effective selection in longer genes against missense errors during translation. The correlation coefficients between the mRNA levels of individual genes, as measured by different investigators and methods, were low, in the range r(s)=0.39-0.68.

Alcohol acetyltransferases and the significance of ester synthesis in yeast

Abstract: This paper reviews our current knowledge of yeast alcohol acyltransferases. Much of this information has been gathered over the past 10 years through the application of powerful yeast molecular biology techniques. Evidence from gene disruption and expression analysis of members of the alcohol acyltransferase (ATF) gene family indicates that different ester synthases are involved in the synthesis of esters during alcoholic fermentation. The natural physiological rationale behind these enzyme activities remains unclear. However, it is believed that these enzymes may be involved in very different functions, including cellular fatty acid homeostasis and detoxification mechanisms. Insights into the regulation of yeast ester synthesis by oxygen and unsaturated fatty acids have contributed to our understanding of the general mechanisms of gene regulation. In particular, control mechanisms that underpin the oxygen-mediated regulation of ATF1 gene transcription appear to be closely linked to those involved in the regulation of fatty acid metabolism. Data pertaining to the regulation of ATF1 gene transcription have been integrated into a working model for future research. Copyright © 2000 John Wiley & Sons, Ltd.

A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions

Abstract: For some time, gene disruptions in Candida albicans have been made with the hisG-URA3-hisG (‘Ura-blaster’) cassette, which can be re-used in successive transformations of a single strain after homologous excision of URA3. However, the hisG repeats are too large for efficient PCR amplification of the entire cassette, so it cannot be used for PCR product-directed gene disruptions. We describe here a gene disruption cassette, URA3-dpl200, with 200 bp flanking repeats that permit efficient PCR amplification. After transformation and integration to produce both arg5::URA3-dpl200 and rim101::URA3-dpl200 alleles, we find that arg5::dpl200 and rim101::dpl200 segregants, respectively, can be obtained. We have used the cassette to create rim101::dpl200/rim101::URA3-dpl200 mutants exclusively through PCR product-directed disruption. Copyright © 2000 John Wiley & Sons, Ltd.

Biotransformation of monoterpene alcohols by Saccharomyces cerevisiae, Torulaspora delbrueckii and Kluyveromyces lactis.

Abstract: Monoterpenoids are important flavour compounds produced by many plant species, including grapes (Vitis vinifera) and hops (Humulus lupulus). Biotransformation reactions involving monoterpenoids have been characterized in filamentous fungi, but few examples have been observed in yeasts. As monoterpenoids are in contact with yeasts during beer and wine production, biotransformation reactions may occur during the fermentation of these beverages. This paper describes the biotransformation of monoterpene alcohols, of significance in the alcoholic beverage industries, by three yeast species. All three species analysed had the ability to convert monoterpenoids. Saccharomyces cerevisiae and Kluyveromyces lactis reduced geraniol into citronellol, whilst all three yeasts produced linalool from both geraniol and nerol. Monocyclic alpha-terpineol was formed from both linalool and nerol, by all three yeasts. alpha-Terpineol was then converted into the diol cis-terpin hydrate. K. lactis and Torulaspora delbrueckii also had the ability to form geraniol from nerol. Finally, the stereospecificity of terpenoid formation was analysed. Both (+) and (-) enantiomers of linalool and alpha-terpineol were formed in roughly equal quantities, from either geraniol or nerol.

Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins.

Abstract: A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein-protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.

A web site for the computational analysis of yeast regulatory sequences.

Abstract: A series of computer programs were developed for the analysis of regulatory sequences, with a special focus on yeast. These tools are publicly available on the web (http://copan.cifn.unam. mx/Computational_Biology/yeast-tools or http://www.ucmb.ulb.ac. be/bioinformatics/rsa-tools/). Basically, three classical problems can be addressed: (a) search for known regulatory patterns in the upstream regions of known genes; (b) discovery of unknown regulatory patterns within a set of upstream regions known to be co-regulated; (c) search for unknown genes potentially regulated by a known transcription factor. Each of these tasks can be performed on basis of a simple (string) or more refined (matrix) description of the regulatory patterns. A feature-map program automatically generates visual representations of the positions at which patterns were found. The site also provides a series of general utilities, such as generation of random sequence, automatic drawing of XY graphs, interconversions between sequence formats, etc. Several tools are linked together to allow their sequential utilization (piping), but each one can also be used independently by filling the web form with external data. This widens the scope of the site to the analysis of non-regulatory and/or non-yeast sequences.

The dual origin of the yeast mitochondrial proteome.

Abstract: We propose a scheme for the origin of mitochondria based on phylogenetic reconstructions with more than 400 yeast nuclear genes that encode mitochondrial proteins. Half of the yeast mitochondrial proteins have no discernable bacterial homologues, while one-tenth are unequivocally of α-proteobacterial origin. These data suggest that the majority of genes encoding yeast mitochondrial proteins are descendants of two different genomic lineages that have evolved in different modes. First, the ancestral free-living α-proteobacterium evolved into an endosymbiont of an anaerobic host. Most of the ancestral bacterial genes were lost, but a small fraction of genes supporting bioenergetic and translational processes were retained and eventually transferred to what became the host nuclear genome. In a second, parallel mode, a larger number of novel mitochondrial genes were recruited from the nuclear genome to complement the remaining genes from the bacterial ancestor. These eukaryotic genes, which are primarily involved in transport and regulatory functions, transformed the endosymbiont into an ATP-exporting organelle.

Destabilized green fluorescent protein for monitoring dynamic changes in yeast gene expression with flow cytometry

Abstract: Green fluorescent protein (GFP) has many advantages as a reporter molecule, but its stability makes it unsuitable for monitoring dynamic changes in gene expression, among other applications Destabilized GFPs have been developed for bacterial and mammalian systems to counter this problem Here, we extend such advances to the yeast model We fused the PEST-rich 178 carboxyl-terminal residues of the G(1) cyclin Cln2 to the C terminus of yEGFP3 (a yeast- and FACS-optimized GFP variant), creating yEGFP3-Cln2(PEST) We tested the hybrid protein after integrating modules harbouring the yEGFP3 or yEGFP3-CLN2(PEST) ORFs into the Saccharomyces cerevisiae genome yEGFP3- Cln2(PEST) had a markedly shorter half-life (t(1/2)) than yEGFP3; inhibition of protein synthesis with cycloheximide lead to a rapid decline in GFP content and fluorescence (t(1/2) approximately 30 min) in cells expressing yEGFP3-Cln2(PEST), whereas these parameters were quite stable in yEGFP3-expressing cells (t(1/2) approximately 7 h) We placed yEGFP3-CLN2(PEST) under the control of the CUP1 promoter, which is induced only transiently by copper This transience was readily discernible with yEGFP3-Cln2(PEST), whereas yEGFP3 reported only on CUP1 switch-on, albeit more slowly than yEGFP3-Cln2(PEST) Cell cycle-regulated transcriptional activation/inactivation of the CLN2 promoter was also discernible with yEGFP3- Cln2(PEST), using cultures that were previously synchronized with nocodazole In comparison to CLN2, expression from the ACT1 promoter was stable after release from nocodazole We also applied a novel flow-cytometric technique for cell cycle analysis with asynchronous cultures The marked periodicities of CLN2 and CLB2 (mitotic cyclin) transcription were readily evident from cellular yEGFP3-Cln2(PEST) levels with this non-perturbing approach The results represent the first reported successful destabilization of a yeast-GFP This new construct expands the range of GFP applications open to yeast workers

Functional coupling of mammalian receptors to the yeast mating pathway using novel yeast/mammalian G protein alpha-subunit chimeras.

Abstract: The expression of mammalian G protein coupled receptors (GPCRs) in S. cerevisiae provides a powerful assay system for functional analysis, ligand identification and pharmaceutical screening. However, relatively few receptors have been coupled to the pheromone response pathway via the yeast G(alpha), Gpa1p, or chimeric yeast/mammalian G(alpha) subunits containing long C-terminal regions of mammalian G(alpha) proteins. We tested an extended range of seven such chimeras for G(alpha) sub-types of three major classes (G(alphai/o), G(alphas) and G(alphaq)), against eight human GPCRs (SST(2), SST(5), 5-HT(1A), 5-HT(1Dalpha), ML(1B), P2Y(1) and P2Y(2)). Although the G(alphai/o) chimeras increased the range of receptors that coupled efficiently, the G(alphas) and G(alphaq) chimeras were inactive when expressed using the GPA1 promoter. We describe 10 novel Gpa1p chimeras, designated 'transplants', in which the C-terminal five amino acids of Gpa1p were exchanged with mammalian residues. Coupling efficiency and ligand sensitivity improved significantly using the transplants. For the P2Y purinergic receptors, coupling could only be detected with the transplants; this is the first report of G(q) specificity coupling in yeast. Thus, the transplants offer major advantages over previously described approaches, in terms of both the range of receptors coupled and the efficiency of coupling.

Morphants: A New Systematic Vertebrate Functional Genomics Approach

Abstract: The vertebrate genome contains a predicted 50 000 100 000 genes, many of unknown function. The recent development of morpholino-based gene knock-down technology in zebrafish has opened the door to the genome-wide assignment of function based on sequence in a model vertebrate. This review describes technical aspects of morpholino use for functional genomics applications, including the potential for multigene targeting and known methodological limitations. The result of successful gene inactivation by this agent is proposed to yield embryos with a ‘morphant’ phenotypic designation. The establishment of a morphant database opens the door to true functional genomics using the vertebrate, Danio rerio.

Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

Abstract: Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD(+). The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and biomass under anaerobic and aerobic growth conditions, respectively. This was done by deletion of GPD1 and GPD2, encoding two isoenzymes of glycerol 3-phosphate dehydrogenase, and expression of a cytoplasmic transhydrogenase from Azotobacter vinelandii, encoded by cth. In anaerobic batch fermentations of strain TN5 (gpd2-Delta1), formation of glycerol was significantly impaired, which resulted in reduction of the maximum specific growth rate from 0.41/h in the wild-type to 0.08/h. Deletion of GPD2 also resulted in a reduced biomass yield, but did not affect formation of the remaining products. The modest effect of the GPD1 deletion under anaerobic conditions on the maximum specific growth rate and product yields clearly showed that Gdh2p is the important factor in glycerol formation during anaerobic growth. Strain TN6 (gpd1-Delta1 gpd2-Delta1) was unable to grow under anaerobic conditions due to the inability of the strain to reoxidize NADH to NAD(+) by synthesis of glycerol. Also, strain TN23 (gpd1-Delta1 gpd2-Delta1 YEp24-PGKp-cth-PGKt) was unable to grow anaerobically, leading to the conclusion that the NAD(+) pool became limiting in biomass synthesis before the nucleotide levels favoured a transhydrogenase reaction that could convert NADH and NADP(+) to NADPH and NAD(+). Deletion of either GPD1 or GPD2 in the wild-type resulted in a dramatic reduction of the glycerol yields in the aerobic batch cultivations of strains TN4 (gpd1-Delta1) and TN5 (gpd2-Delta1) without serious effects on the maximum specific growth rates or the biomass yields. Deletion of both GPD1 and GPD2 in strain TN6 (gpd1-Delta1 gpd2-Delta1) resulted in a dramatic reduction in the maximum specific growth rate and in biomass formation. Expression of the cytoplasmic transhydrogenase in the double mutant, resulting in TN23, gave a further decrease in micromax from 0.17/h in strain TN6 to 0.09/h in strain TN23, since the transhydrogenase reaction was in the direction from NADPH and NADP(+) to NADH and NADP(+). Thus, it was not possible to introduce an alternative pathway for reoxidation of NADH in the cytoplasm by expression of the transhydrogenase from A. vinelandii in a S. cerevisiae strain with a double deletion in GPD1 and GPD2.

Yeast two-hybrid systems and protein interaction mapping projects for yeast and worm.

Abstract: The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as 'functional genomics'. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein-protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein-protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.

SSU1 mediates sulphite efflux in Saccharomyces cerevisiae.

Abstract: Ssu1p, a plasma membrane protein involved in sulphite metabolism in Saccharomyces cerevisiae, was found to be required for efficient sulphite efflux. An SSU1 null mutant accumulated significantly more sulphite than wild-type, whereas cells expressing multicopy SSU1 accumulated significantly less. Cells expressing FZF1-4, a dominant allele of a transcriptional activator of SSU1 that confers sulphite resistance, also accumulated less sulphite. beta-galactosidase activity in the FZF1-4 strain carrying an SSU1::lacZ fusion was found to be 8.5-fold higher than in a strain carrying wild-type FZF1, confirming that the heightened resistance was correlated with hyperactivation of SSU1. Multicopy SSU1 was also found to increase the sulphite resistance of a number of unrelated sulphite-sensitive strains by a factor of 3- to 8-fold. Rates of efflux of free sulphite from cells expressing multicopy SSU1 or FZF1-4 were significantly greater than that from wild-type or from a SSU1 null mutant. Rates of efflux of bound sulphite from wild-type, a SSU1 null mutant, a FZF1-4 mutant, or cells expressing multicopy SSU1 were not significantly different, suggesting that Ssu1p specifically mediates efflux of the free form of sulphite.

The cytochrome P450ALK multigene family of an n-alkane-assimilating yeast, Yarrowia lipolytica: cloning and characterization of genes coding for new CYP52 family members

Abstract: Genes encoding cytochromes P450 of the CYP52 family in the n-alkane-assimilating yeast Yarrowia lipolytica have been cloned and analyzed. Degenerate PCR primers which were designed for the conserved amino acid sequences of cytochromes P450ALK of Candida species were used for amplification and isolation of genes encoding P450ALK from a genomic DNA library of Y. lipolytica CX161–1B. Seven new genes (YlALK2–YlALK8) were isolated. Five of the seven YlALK genes were induced by n-alkane under the culture conditions used in this study, whereas their expression was strictly repressed by glycerol but not by glucose, similar to the case of YlALK1, reported previously. Disruption of YlALK2, YlALK3, YlALK4 or YlALK6 did not change the growth of Y. lipolytica on medium containing n-alkanes of various chain lengths. A mutant with disruptions in both YlALK1 and YlALK2 did not grow well on n-hexadecane, whereas one with disruptions in both YlALK1 and YlALK3, which has the same phenotype as the YlALK1 singly disrupted mutant, grew well on n-hexadecane. These results suggest that the presence of multiple P450ALK species is a rather common phenomenon among the n-alkane-assimilating yeasts and that in the n-alkane assimilation of Y. lipolytica, YlALK1 functions to assimilate n-decane and longer molecules, whereas YlALK2 is involved in the assimilation of molecules longer than n-dodecane; other YlALKs are not significantly involved in the assimilation of C10–C16 n-alkanes. Copyright © 2000 John Wiley & Sons, Ltd.

A counterselection for the tryptophan pathway in yeast: 5-fluoroanthranilic acid resistance

Abstract: The ability to counterselect, as well as to select for, a genetic marker has numerous applications in microbial genetics. Described here is the use of 5-fluoroanthranilic acid for the counterselection of TRP1, a commonly used genetic marker in the yeast Saccharomyces cerevisiae. Counterselection using 5-fluoroanthranilic acid involves antimetabolism by the enzymes of the tryptophan biosynthetic pathway, such that trp1, trp3, trp4 or trp5 strains, which lack enzymes required for the conversion of anthranilic acid to tryptophan, are resistant to 5-fluoroanthranilic acid. Commonly used genetic procedures, such as selection for loss of a chromosomally integrated plasmid, and a replica-plating method to rapidly assess genetic linkage in self-replicating shuttle vectors, can now be carried out using the TRP1 marker gene. In addition, novel tryptophan auxotrophs can be selected using 5-fluoroanthranilic acid.

A history of research on yeasts 2: Louis Pasteur and his contemporaries, 1850–1880

Abstract: Introduction . . . . . . . . . . . . . . . . . . . . . . . 755 Pasteur's passage from chemistry to microbiology . . . . . . . . . . . . . . . . . . . . . . . . . 756 Pasteur on alcoholic fermentation . . . . . . . . 758 Problem of distinguishing between activities of whole organisms and of enzymes . . . . . . . 760 Quantitative differences between aerobic and anaerobic sugar utilization . . . . . . . . . . . . . . . 762 Yeasts of wine and beer . . . . . . . . . . . . . . . 763 References . . . . . . . . . . . . . . . . . . . . . . . . 769

Comparative Genomics Uncovers Large Tandem Chromosomal Duplications in Mycobacterium bovis BCG Pasteur

Abstract: On direct comparison of minimal sets of ordered clones from bacterial artificial chromosome (BAC) libraries representing the complete genomes of Mycobacterium tuberculosis H37Rv and the vaccine strain, Mycobacterium bovis BCG Pasteur, two major rearrangements were identified in the genome of M. bovis BCG Pasteur. These were shown to correspond to two tandem duplications, DU1 and DU2, of 29 668 bp and 36 161 bp, respectively. While DU1 resulted from a single duplication event, DU2 apparently arose from duplication of a 100 kb genomic segment that subsequently incurred an internal deletion of 64 kb. Several lines of evidence suggest that DU2 may continue to expand, since two copies were detected in a subpopulation of BCG Pasteur cells. BCG strains harbouring DU1 and DU2 are diploid for at least 58 genes and contain two copies of oriC, the chromosomal origin of replication. These findings indicate that these genomic regions of the BCG genome are still dynamic. Although the role of DU1 and DU2 in the attenuation and/or altered immunogenicity of BCG is yet unknown, knowledge of their existence will facilitate quality control of BCG vaccine lots and may help in monitoring the efficacy of the world's most widely used vaccine.

Proteome profiling—pitfalls and progress

Abstract: In this review we examine the current state of analytical methods in proteomics. The conventional methodology using two-dimensional electrophoresis gels and mass spectrometry is discussed, with particular reference to the advantages and shortcomings thereof. Two recently published methods which offer an alternative approach are presented and discussed, with emphasis on how they can provide information not available via two-dimensional gel electrophoresis. These two methods are the isotope-coded affinity tags approach of Gygi et al. and the two-dimensional liquid chromatography–tandem mass spectrometry approach as presented by Link et al. We conclude that both of these new techniques represent significant advances in analytical methodology for proteome analysis. Furthermore, we believe that in the future biological research will continue to be enhanced by the continuation of such developments in proteomic analytical technology.

Danio rerio, the Zebrafish

Abstract: The zebrafish has long been a favourite model for the study of vertebrate development. Here we provide an overview of the current state of knowledge and resources for the study of this fish, with comments on the future direction of zebrafish genomics from Professor Mark Fishman and Dr Stephen Wilson.

Sorbic acid resistance: the inoculum effect.

Abstract: Zygosaccharomyces is a genus associated with the more extreme spoilage yeasts. Zygosaccharomyces spoilage yeasts are osmotolerant, fructophiles (preferring fructose), highly-fermentative and extremely preservative-resistant. Zygosaccharomyces bailii can grow in the presence of commonly-used food preservatives, benzoic, acetic or sorbic acids, at concentrations far higher than are legally permitted or organolepically acceptable in foods. An inoculum effect has been described for many micro-organisms and antimicrobial agents. The minimum inhibitory concentration (MIC) increases with the size of the inoculum; large inocula at high cell density therefore require considerably higher concentrations of inhibitors to prevent growth than do dilute cell suspensions. A substantial inoculum effect was found using sorbic acid against the spoilage yeast Zygosaccharomyces bailii NCYC 1766. The inoculum effect was not caused by yeasts metabolizing or adsorbing sorbic acid, thereby lowering the effective concentration; was not due to absence of cell-cell signals in dilute cell suspensions; and was not an artefact, generated by insufficient time for small inocula to grow. The inoculum effect appeared to be caused by diversity in the populations of yeast cells, with higher probability of sorbic acid-resistant cells being present in large inocula. It was found that individual cells of Zygosaccharomyces bailii populations, grown as single cells in microtitre plate wells, were very diverse, varying enormously in resistance to sorbic acid. 26S ribosomal DNA sequencing did not detect differences between the small fraction of resistant 'super cells' and the average population. Re-inoculation of the 'super cells' after overnight growth on YEPD showed a normal distribution of resistance to sorbic acid, similar to that of the original population. The resistance phenotype was therefore not heritable and not caused by a genetically distinct subpopulation. It was concluded that resistance of the spoilage yeast Zygosaccharomyces bailii to sorbic acid was due to the presence of small numbers of phenotypically resistant cells in the population.

Expression of the AZR1 gene (ORF YGR224w), encoding a plasma membrane transporter of the major facilitator superfamily, is required for adaptation to acetic acid and resistance to azoles in Saccharomyces cerevisiae.

Abstract: In this work, we report results on the functional analysis of Saccharomyces cerevisiae ORF YGR224w, predicted to code for an integral membrane protein, with 14 potential transmembrane segments, belonging to the major facilitator superfamily (MFS) of transporters which are required for multiple-drug resistance (MDR). This MFS-MDR homologue is required for yeast adaptation to high stress imposed by low-chain organic acids, in particular by acetic acid, and for resistance to azoles, especially to ketoconazole and fluconazole; the encoding gene was thus named the AZR1 gene. These conclusions were based on the higher susceptibility to these compounds of an azr1Delta deletion mutant strain compared with the wild-type and on the increased resistance of both azr1Delta and wild-type strains upon increased expression of the AZR1 gene from a centromeric plasmid clone. AZR1 gene expression reduces the duration of acetic acid-induced latency, although the growth kinetics of adapted cells under acetic acid stress is apparently independent of AZR1 expression level. Fluorescence microscopy observation of the distribution of the Azr1-GFP fusion protein in yeast living cells indicated that Azr1 is a plasma membrane protein. Studies carried out to gain some understanding of how this plasma membrane putative transporter facilitates yeast adaptation to acetic acid did not implicate Azr1p in the alteration of acetic acid accumulation into the cell through the active efflux of acetate.

The level of cAMP-dependent protein kinase A activity strongly affects osmotolerance and osmo-instigated gene expression changes in Saccharomyces cerevisiae.

Abstract: The influence of cAMP-dependent protein kinase (PKA) on protein expression during exponential growth under osmotic stress was studied by two-dimensional polyacrylamide gel electrophoresis (2D–PAGE). The responses of isogenic strains (tpk2Δtpk3Δ) with either constitutively low (tpk1w1), regulated (TPK1) or constitutively high (TPK1bcy1Δ) PKA activity were compared. The activity of cAMP-dependent protein kinase (PKA) was shown to be a major determinant of osmotic shock tolerance. Proteins with increased expression during growth under sodium chloride stress could be grouped into three classes with respect to PKA activity, with the glycerol metabolic proteins GPD1, GPP2 and DAK1 standing out as independent of PKA. The other osmotically induced proteins displayed a variable dependence on PKA activity; fully PKA-dependent genes were TPS1 and GCY1, partly PKA-dependent genes were ENO1, TDH1, ALD3 and CTT1. The proteins repressed by osmotic stress also fell into distinct classes of PKA-dependency. Ymr116c was PKA-independent, while Pgi1p, Sam1p, Gdh1p and Vma1p were fully PKA-dependent. Hxk2p, Pdc1p, Ssb1p, Met6p, Atp2p and Hsp60p displayed a partially PKA-dependent repression. The promotors of all induced PKA-dependent genes have STRE sites in their promotors suggestive of a mechanism acting via Msn2/4p. The mechanisms governing the expression of the other classes are unknown. From the protein expression data we conclude that a low PKA activity causes a protein expression resembling that of osmotically stressed cells, and furthermore makes cells tolerant to this type of stress. Copyright © 2000 John Wiley & Sons, Ltd.

Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift

Abstract: During wine fermentation yeasts quickly reach a stationary phase, where cells are metabolically active by consuming sugars present in grape must. It is, consequently, of great interest at this stage to identify suitable gene promoters that may be used to induce the expression of genes with enological applications. With this aim, we have studied a group of genes showing an induction peak at the diauxic shift, and possessing stress response elements (STRE) at their promoters. We have determined their induction levels under individualized stress conditions, such as carbon source starvation or high salt concentrations. In all the cases studied, the activation and/or basal transcription are dependent on the transcriptional factors Msn2p and Msn4p. We have analysed the expression patterns and mRNA levels during wine fermentation, and have found that they are all activated at the stationary phase. Finally, we have identified SPI1, a new highly expressed yeast gene which is specifically induced at the stationary phase of both microvinification and laboratory growth conditions. Copyright © 2000 John Wiley & Sons, Ltd.

Ultradian oscillation of Saccharomyces cerevisiae during aerobic continuous culture: hydrogen sulphide mediates population synchrony

Abstract: Saccharomyces cerevisiae showed an ultradian respiratory oscillation during aerobic continuous culture. Analysis of the off-gas revealed that hydrogen sulphide production also oscillated. Production was first detected at the onset of low respiration and reached a maximum (1.5 µM) prior to minimum respiratory activity. Then H2S concentration fell rapidly to below 0.2 µM before the onset of high respiration. Injection of respiratory oscillation perturbation agents, such as glutathione (50 µM), NaNO2 (50 µM) or acetaldehyde (4.5 mM), transiently increased H2S production above 6 µM. The synchronization properties of H2S were analysed to reveal that changes of oscillation period and amplitude were dependent on H2S concentration in culture. It is concluded that H2S produced during oscillation produces population synchrony by respiratory chain inhibition. Copyright © 2000 John Wiley & Sons, Ltd.