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Showing papers on "Yeast published in 2001"


Journal ArticleDOI
TL;DR: A comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents is presented, and specific features of the gene expression responses that are dependent on the Mec1 pathway are identified.
Abstract: Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. The human ATR kinase and its homolog in yeast, MEC1, play central r...

572 citations


Journal ArticleDOI
TL;DR: The unexpected roles of glycogen and trehalose found in the control of glycolytic flux, stress responses and energy stores for the budding process, demonstrate that their presence confers survival and reproductive advantages to the cell.
Abstract: Glycogen and trehalose are the two glucose stores of yeast cells. The large variations in the cell content of these two compounds in response to different environmental changes indicate that their metabolism is controlled by complex regulatory systems. In this review we present information on the regulation of the activity of the enzymes implicated in the pathways of synthesis and degradation of glycogen and trehalose as well as on the transcriptional control of the genes encoding them. cAMP and the protein kinases Snf1 and Pho85 appear as major actors in this regulation. From a metabolic point of view, glucose-6-phosphate seems the major effector in the net synthesis of glycogen and trehalose. We discuss also the implication of the recently elucidated TOR-dependent nutrient signalling pathway in the control of the yeast glucose stores and its integration in growth and cell division. The unexpected roles of glycogen and trehalose found in the control of glycolytic flux, stress responses and energy stores for the budding process, demonstrate that their presence confers survival and reproductive advantages to the cell. The findings discussed provide for the first time a teleonomic value for the presence of two different glucose stores in the yeast cell.

560 citations


Journal ArticleDOI
02 Feb 2001-Science
TL;DR: It is shown that bakers' yeast Saccharomyces cerevisiae can initiate biofilm formation and that attachment to plastic and mat formation require Flo11p, a member of a large family of fungal cell surface glycoproteins involved in adherence.
Abstract: Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Here we show that bakers' yeast Saccharomyces cerevisiae can initiate biofilm formation. When grown in low-glucose medium, the yeast cells adhered avidly to a number of plastic surfaces. On semi-solid (0.3% agar) medium they formed "mats": complex multicellular structures composed of yeast-form cells. Both attachment to plastic and mat formation require Flo11p, a member of a large family of fungal cell surface glycoproteins involved in adherence. The ability to study biofilm formation in a tractable genetic system may facilitate the identification of new targets for antifungal therapy.

554 citations


Journal ArticleDOI
TL;DR: It is shown by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria, pointing to a role for oxygen in the yeast ageing process.
Abstract: Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT-mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro-apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild-type yeast mother cells.

418 citations


Journal ArticleDOI
TL;DR: ‘management’ of the energy pool (especially ATP) seems to constitute an ethanol stress response and to involve different mechanisms.

385 citations


Journal ArticleDOI
TL;DR: Partial models may be proposed for the interaction of the regulatory pathways, both in the case of S. cerevisiae and in that of C. albicans, for Morphogenesis in different yeast genera share common elements, but there are also important differences.
Abstract: Pseudohyphal growth in both haploid and diploid strains of Saccharomyces cerevisiae reflects concerted changes in different cellular processes: budding pattern, cell elongation and cell adhesion. These changes are triggered by environmental signals and are controlled by several pathways which act in parallel. Nitrogen deprivation, and possibly other stresses, activate a MAP kinase cascade which has the transcription factor Ste12 as its final target. A cAMP-dependent pathway, in which the protein kinase Tpk2 plays a specific role, is also required for the morphogenetic switch. Both pathways contribute to modulate the expression of the MUC1/FLO11 gene which encodes a cell-surface flocculin required for pseudohyphal and invasive growth. The MAP kinase cascade could also control the activity of the cyclin/Cdc28 complexes which affect both the budding pattern of yeast and cell elongation. A further protein which stimulates filamentous growth in S. cerevisiae is Phd1; although its mode of action is unknown, it may be regulated by a cAMP-dependent protein kinase, as occurs with the homologous protein Efg1 from Candida albicans, which is required for the formation of true hyphae. Morphogenesis in different yeast genera share common elements, but there are also important differences. Although a complete picture cannot yet be drawn, partial models may be proposed for the interaction of the regulatory pathways, both in the case of S. cerevisiae and in that of C. albicans.

325 citations


Journal ArticleDOI
TL;DR: The results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.
Abstract: To improve production of fuel ethanol from renewable raw materials, laccase from the white rot fungus Trametes versicolor was expressed under control of the PGK1 promoter in Saccharomyces cerevisiae to increase its resistance to phenolic inhibitors in lignocellulose hydrolysates. It was found that the laccase activity could be enhanced twofold by simultaneous overexpression of the homologous t-SNARE Sso2p. The factors affecting the level of active laccase obtained, besides the cultivation temperature, included pH and aeration. Laccase-expressing and Sso2p-overexpressing S. cerevisiae was cultivated in the presence of coniferyl aldehyde to examine resistance to lignocellulose-derived phenolic fermentation inhibitors. The laccase-producing transformant had the ability to convert coniferyl aldehyde at a faster rate than a control transformant not expressing laccase, which enabled faster growth and ethanol formation. The laccase-producing transformant was also able to ferment a dilute acid spruce hydrolysate at a faster rate than the control transformant. A decrease in the content of low-molecular-mass aromatic compounds, accompanied by an increase in the content of high-molecular-mass compounds, was observed during fermentation with the laccase-expressing strain, illustrating that laccase was active even at the very low levels of oxygen supplied. Our results demonstrate the importance of phenolic compounds as fermentation inhibitors and the advantage of using laccase-expressing yeast strains for producing ethanol from lignocellulose.

311 citations


Journal ArticleDOI
TL;DR: Cl cloning and characterization of the IRT2 cDNA are reported, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level, and therefore provide the first tissue localization of a plant metal transporter.
Abstract: Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters. One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast. Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level. IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation. However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast. IRT2 expression is detected only in roots of A. thaliana plants, and is upregulated by iron deficiency. By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter. Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions.

299 citations


Journal ArticleDOI
TL;DR: Budding and fission yeasts provide ideal model systems for eukaryotic cell biology and classical yeast genetics remains an essential element in discovering and characterizing the genes that make up a eukARYotic cell.
Abstract: Understanding the biology of complex systems is facilitated by comparing them with simpler organisms. Budding and fission yeasts provide ideal model systems for eukaryotic cell biology. Although they differ from one another in terms of a range of features, these yeasts share powerful genetic and genomic tools. Classical yeast genetics remains an essential element in discovering and characterizing the genes that make up a eukaryotic cell.

271 citations


Journal ArticleDOI
TL;DR: The quinoid nucleus of the benzoquinone, ubiquinone (coenzyme Q; Q), is derived from the shikimate pathway in bacteria and eukaryotic microorganisms and is considered a vitamin since mammals synthesize it from the essential amino acid tyrosine.
Abstract: The quinoid nucleus of the benzoquinone, ubiquinone (coenzyme Q; Q), is derived from the shikimate pathway in bacteria and eukaryotic microorganisms. Ubiquinone is not considered a vitamin since mammals synthesize it from the essential amino acid tyrosine. Escherichia coli and other Gram-negative bacteria derive the 4-hydroxybenzoate required for the biosynthesis of Q directly from chorismate. The yeast, Saccharomyces cerevisiae, can either form 4-hydroxybenzoate from chorismate or tyrosine. However, unlike mammals, S. cerevisiae synthesizes tyrosine in vivo by the shikimate pathway. While the reactions of the pathway leading from 4-hydroxybenzoate to Q are the same in both organisms the order in which they occur differs. The 4-hydroxybenzoate undergoes a prenylation, a decarboxylation and three hydroxylations alternating with three methylation reactions, resulting in the formation of Q. The methyl groups for the methylation reactions are derived from S-adenosylmethionine. While the prenyl side chain is formed by the 2-C-methyl-D-erythritol 4-phosphate (non-mevalonate) pathway in E. coli, it is formed by the mevalonate pathway in the yeast.

270 citations


Book
18 Oct 2001
TL;DR: This chapter discusses the brewing process, yeast management, and the biochemistry of fermentation as well as some of the techniques used in fermentation management.
Abstract: Preface. Acknowledgements. 1. Beer and brewing. 2. The brewing process. 3. The biochemistry of fermentation. 4. Brewing yeast. 5. Fermentation systems. 6. Fermentation management. 7. Yeast management. 8. Microbiology.References. Index.

Journal ArticleDOI
TL;DR: Intracellular metabolite analyses and in vitro enzyme activities suggest that the control of flux in a strain that overexpresses XKS1 has shifted to the nonoxidative steps of the pentose phosphate pathway (i.e., downstream of xylose 5-phosphate), and enzymatic steps in the lower part of glycolysis and ethanol formation pathways do not have a high flux control in this recombinant strain.

Journal ArticleDOI
TL;DR: Simultaneous saccharification and fermentation studies were carried out to produce ethanol from lignocellulosic wastes (sugar cane leaves and Antigonum leptopus leaves) using Trichoderma reesei cellulase and yeast cells and K. fragilis was found to perform better in the SSF process and result in high yields of ethanol.

Journal ArticleDOI
TL;DR: The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years and confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomycles dominated towards the end of the alcoholic fermentation.
Abstract: Yeast ecology, biogeography and biodiversity are important and interesting topics of research The population dynamics of yeasts in several cellars of two Spanish wine-producing regions was analysed for three consecutive years (1996 to 1998) No yeast starter cultures had been used in these wineries which therefore provided an ideal winemaking environment to investigate the dynamics of grape-related indigenous yeast populations Non-Saccharomyces yeast species were identified by RFLPs of their rDNA, while Saccharomyces species and strains were identified by RFLPs of their mtDNA This study confirmed the findings of other reports that non-Saccharomyces species were limited to the early stages of fermentation whilst Saccharomyces dominated towards the end of the alcoholic fermentation However, significant differences were found with previous studies, such as the survival of non-Saccharomyces species in stages with high alcohol content and a large variability of Saccharomyces strains (a total of 112, all of them identified as Saccharomyces cerevisiae) with no clear predominance of any strain throughout all the fermentation, probably related to the absence of killer phenotype and lack of previous inoculation with commercial strains

Journal ArticleDOI
TL;DR: Two cDNAs (LeIRT1 and LeIRT2) are isolated from a library constructed from roots of iron-deficient tomato plants, using the Arabidopsis iron transporter cDNA, IRT1, as a probe and, surprisingly, both genes are arranged in tandem with a tail-to-tail orientation.
Abstract: Although iron deficiency poses severe nutritional problems to crop plants, to date iron transporters have only been characterized from the model plant Arabidopsis thaliana. To extend our molecular knowledge of Fe transport in crop plants, we have isolated two cDNAs (LeIRT1 and LeIRT2) from a library constructed from roots of iron-deficient tomato (Lycopersicon esculentum) plants, using the Arabidopsis iron transporter cDNA, IRT1, as a probe. Their deduced polypeptides display 64% and 62% identical amino acid residues to the IRT1 protein, respectively. Transcript level analyses revealed that both genes were predominantly expressed in roots. Transcription of LeIRT2 was unaffected by the iron status of the plant, while expression of LeIRT1 was strongly enhanced by iron limitation. The growth defect of an iron uptake-deficient yeast (Saccharomyces cerevisiae) mutant was complemented by LeIRT1 and LeIRT2 when ligated to a yeast expression plasmid. Transport assays revealed that iron uptake was restored in the transformed yeast cells. This uptake was temperature-dependent and saturable, and Fe2+ rather than Fe3+ was the preferred substrate. A number of divalent metal ions inhibited Fe2+ uptake when supplied at 100-fold or 10-fold excess. Manganese, zinc and copper uptake-deficient yeast mutants were also rescued by the two tomato cDNAs, suggesting that their gene products have a broad substrate range. The gene structure was determined by polymerase chain reaction experiments and, surprisingly, both genes are arranged in tandem with a tail-to-tail orientation.

Journal ArticleDOI
TL;DR: The results suggest that Gdn-HCl cures prions by inhibiting Hsp104 activity in vivo, since replication of yeast prions is dependent on HSp104.
Abstract: The presence of millimolar concentrations of guanidine hydrochloride (Gdn-HCl) in growth media causes efficient loss of the normally stable [PSI + ] element from yeast cells. Although it has become common practice to include 5 mm Gdn-HCl in growth media to cure [PSI + ] and other prions of yeast, the biochemical mechanism by which it cures is unknown. We find that 5 mm Gdn-HCl significantly reduces Hsp104-mediated basal and acquired thermotolerance. Gdn-HCl also reduced the ability of Hsp104 to restore activity of thermally denatured luciferase in vivo. The abundance of Hsp104 was not reduced in cells grown in the presence of Gdn-HCl, ruling out negative effects on expression or stability of Hsp104. We therefore conclude that Gdn-HCl inhibits Hsp104 activity in vivo. Since replication of yeast prions is dependent on Hsp104, our results suggest that Gdn-HCl cures prions by inhibiting Hsp104 activity.

Journal ArticleDOI
TL;DR: In this paper, the authors studied the release of polysaccharides by three strains of Saccharomyces cerevisiae during alcoholic fermentation in synthetic medium and red wine and found that Mannose was the main component (90%) of these poly-scharides, with the exception of yeast strain BM45 which contained approximatively 50% glucose and 50% mannose.
Abstract: Release of polysaccharides by three strains of Saccharomyces cerevisiae during alcoholic fermentation in synthetic medium and red wine was studied. Polysaccharides after isolation from media by ethanol precipitation were quantified by a colourimetric method as well as by HPLC assay in wines. Yeast strains differed in their capacity to release polysaccharides into the medium and a maximum concentration of 100mg/L was observed. For all strains, release varied according to metabolic phase, with greater release occurring when the yeast mortality rate was high. Mannose was the main component (90%) of these polysaccharides, with the exception of yeast strain BM45 which contained approximatively 50% glucose and 50% mannose. This composition may be related to cell wall composition. The results suggest that polysaccharides can combine with anthocyanins and tannins in wine. This combination seems to increase colour stability and decrease astringency.

Journal ArticleDOI
TL;DR: Yeast plasma membrane nutrient sensors discovered indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought.
Abstract: It has been known for a long time that yeast are capable of making rapid metabolic adjustments in response to changing extracellular nutrient conditions. Until recently it was thought that yeast, in contrast to mammalian cells, primarily monitored nutrient availability through the activity of intracellular sensors. Recent advances in our understanding of nutrient sensing indicate that yeast cells possess several nutrient-sensing systems localized in the plasma membrane that transduce information regarding the presence of extracellular amino acids, ammonium. and glucose. Strikingly, the transmembrane components of several of these sensors, Ssylp, Mep2p, Snf3p. and Rgt2p, are unique members of nutrient-transport protein families. Perhaps with the exception of Mep2p, the ability of these transporter homologues to transduce nutrient-(ligand)-induced signals across the plasma membrane appears to be independent of nutrient uptake; and thus these sensor components may function analogously to traditional ligand-dependent receptors. Additionally, the G protein-coupled receptor Gpr1p has been shown to exhibit properties consistent with it being a sensor. These recent advances indicate that yeast cells obtain information regarding their growth environments using sensing systems that are more similar to those present in mammalian cells than previously thought. The fact that yeast plasma membrane nutrient sensors have only recently been discovered reveals how little is understood regarding the molecular signals that enable eukaryotic cells to adapt to changing environments.

Journal ArticleDOI
TL;DR: The results suggest that Aft2p and Aft1p have overlapping roles in the control of iron-regulated pathway(s) connected to oxidative stress resistance in yeast.

Journal ArticleDOI
TL;DR: It is reported that yeast Hst2p and a mammalian Hst 2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear.
Abstract: In budding yeast, the silent information regulator Sir2p is a nuclear NAD-dependent deacetylase that is essential for both telomeric and rDNA silencing. All eukaryotic species examined to date have multiple homologues of Sir two (HSTs), which share a highly conserved globular core domain. Here we report that yeast Hst2p and a mammalian Hst2p homologue, hSirT2p, are cytoplasmic in yeast and human cells, in contrast to yHst1p and ySir2p which are exclusively nuclear. Although yHst2p cannot restore silencing in a sir2 deletion, overexpression of yHst2p influences nuclear silencing events in a SIR2 strain, derepressing subtelomeric silencing while increasing repression in the rDNA. In contrast, a form of ySir2p carrying a point mutation in the conserved core domain disrupts both telomeric position effect (TPE) and rDNA repression at low expression levels. This argues that non-nuclear yHst2p can compete for a substrate or ligand specifically required for telomeric, and not rDNA repression.

Journal ArticleDOI
TL;DR: In this paper, a flexible engagement member with an engagement projection of the panel is formed in a portion of the wall by cutting a part of a wall, and the member may be provided with lock release portions engageable with a lock release instrument inserting into the inside of the housing.
Abstract: A connector having an improved mechanism for its lock onto a panel. A housing of the connector has a wall on which a sliding rail capable of slidingly fitting onto guide rails of the panel is provided. A flexible engagement member having an engagement projection engageable with an engagement projection of the panel is formed in a portion of the wall by cutting a part of the wall. When the sliding rail is slid on the guide rail to fit thereon, the flexible engagement member is deformed and has its position changed toward the inside of the housing while its engagement projection abuts on the engagement projection of the panel until the member recovers its original position upon the engagement between the projections. The arrangement of the flexible engagement member is advantageous in that it does not require any deformation distance to be provided on the outside of the housing, thereby enabling the entire connector to be compact. The member may be provided with lock release portions engageable with a lock release instrument inserting into the inside of the housing. With this arrangement, the connector can be easily released from its state of being locked.

Journal ArticleDOI
TL;DR: Among various treatment sequences using multiple enzymes, treatment with protease followed by nuclease resulted in the highest 5'-guanosine monophosphate (5'-GMP) content.

Journal ArticleDOI
TL;DR: The purified C-terminal domain of yeast Sco1 binds one Cu(I)/monomer, and data obtained from size-exclusion chromatography experiments with mitochondrial lysates suggest that full-length Sco1 may be oligomeric in vivo.

Journal ArticleDOI
TL;DR: It can be concluded that the eca40 mutation has a drastic effect on the production of isobutanol, and the results suggest that another route could lead to the formation of these two higher alcohols.
Abstract: Organoleptic compounds produced by yeast during the fermentation of wort have a great impact on beer smell and taste. Among them, fusel alcohols are the major abundant volatile compounds. The availability of Saccharomyces cerevisiae mutants in which the genes coding for the two branched-chain amino acid aminotransferases have been deleted offers the possibility of further defining the role of these enzymes in the formation of higher alcohols. Comparing the production profiles of different strains, it is clear that they are not all influenced in the same way by branched-chain amino acid aminotransferase mutations. First of all, as propanol is synthesised from α-ketobutyrate, the first metabolic intermediate in the anabolic pathway of isoleucine, neither the eca39 nor eca40 mutations have any effect on the production of this higher alcohol. On the other hand, it can be concluded that the eca40 mutation has a drastic effect on the production of isobutanol. To a certain extent, the same conclusion can be made for the production of active amyl alcohol and isoamyl alcohol, although the results suggest that another route could lead to the formation of these two higher alcohols.

Journal ArticleDOI
30 Apr 2001-Oncogene
TL;DR: In yeast members of the AP-1 family of proteins are required for the transcriptional response to oxidative stress and many of the features appear to be evolutionarily conserved and consequently the study of model organisms, such as yeast, will have widespread utility.
Abstract: Cells have evolved complex and efficient strategies for dealing with variable and often-harsh environments. A key aspect of these stress responses is the transcriptional activation of genes encoding defense and repair proteins. In yeast members of the AP-1 family of proteins are required for the transcriptional response to oxidative stress. This sub-family of AP-1 (called yAP-1) proteins are sensors of the redox-state of the cell and are activated directly by oxidative stress conditions. yAP-1 proteins are bZIP-containing factors that share homology to the mammalian AP-1 factor complex and bind to very similar DNA sequence sites. The generation of reactive oxygen species and the resulting potential for oxidative stress is common to all aerobically growing organisms. Furthermore, many of the features of this response appear to be evolutionarily conserved and consequently the study of model organisms, such as yeast, will have widespread utility. The important structural features of these factors, signaling pathways controlling their activity and the nature of the target genes they control will be discussed.

Journal ArticleDOI
TL;DR: In this paper, the authors reported the identification of two key fatty acyltransferases of the glycerolipid biosynthesis pathway in Saccharomyces cerevisiae, which can effectively utilize a broad range of fatty acids as acyl donors.

Journal ArticleDOI
TL;DR: The findings demonstrate the utility of the yeast recombinant assay for analyzing complex natural samples and stress the necessity of a panel of different yeast systems to adequately describe endocrine‐disruptor activities.
Abstract: Endocrine disruptors exert physiological effects at very low concentrations. Surface waters present often a mixture of high concentrations of low-potency disruptors and low amounts of very powerful ones, making their chemical analysis complicated and expensive. We developed a recombinant yeast assay (RYA) for estrogenic compounds using 96-well microtiter plates. This assay is based on three yeast strains, transformed with self-propagating plasmids. One strain contains an expression plasmid for the human estrogen hormone receptor and an appropriate reporter; it detects estrogenic and antiestrogenic activities. The two other yeast strains, one expressing the human progesterone receptor and a second based on the yeast activator Gal4p, served to analyze the nature of antiestrogenic activities. We applied this technique to water samples from two tributaries of the Llobregat river (NE Spain) as well as from four sewage treatment plants discharging on them. Our results indicate that the efficiency of sewage treatment plants for eliminating estrogenic compounds varied notably, being in at least one case completely inefficient. We also observed a prevalence of an inhibitory activity all through the two rivers; this inhibition was hormone specific. These results were consistent to previously obtained chemical analyses of the same samples, although chemical and in vivo analyses showed rather different levels of sensitivity for some compounds. Our findings demonstrate the utility of the yeast recombinant assay for analyzing complex natural samples; at the same time, they stress the necessity of a panel of different yeast systems to adequately describe endocrine-disruptor activities.

Journal ArticleDOI
TL;DR: Growth experiments suggest that this substance excreted in C. albicans does not inhibit yeast cell growth but inhibits filamentous growth, which has implications for developmental signaling by the fungus and might have medicinal value in the development of antifungal therapies.
Abstract: The yeast Candida albicans has a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a budding yeast form and a multicellular invasive filamentous form. This ability has been postulated to contribute to the virulence of this organism. Studies on the morphological transition from a filamentous to a budding yeast form in C. albicans have shown that this organism excretes an autoregulatory substance into the culture medium. This substance was extracted and purified by normal-phase and reversed-phase HPLC. The autoregulatory substance was structurally identified as 3,7,11-trimethyl-2,6,10-dodecatrienoate (farnesoic acid) by NMR and mass spectrometry. Growth experiments suggest that this substance does not inhibit yeast cell growth but inhibits filamentous growth. These findings have implications for developmental signaling by the fungus and might have medicinal value in the development of antifungal therapies.

Journal ArticleDOI
TL;DR: Yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles, according to kinetics of induction of two genes activated by the A FT2-1up allele, which are consistent with Aft2p acting as a direct transcriptional factor.
Abstract: Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1Δaft2Δ deletion was generated. Growth assays established that the single aft2Δ strain exhibited no iron-dependent phenotype. However, the double-mutant aft1Δaft2Δ strain was more sensitive to low-iron growth conditions than the single-mutant aft1Δ strain. A mutant allele of AFT2 (AFT2-1up), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory-deficient phenotype of the aft1Δ strain. The AFT2-1up allele also increased the uptake of 59Fe in an aft1Δ strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1up-dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1up allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles.

Journal ArticleDOI
TL;DR: Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and an aminoacyled tRNAs that function efficiently in suppression of amber codons, and two such aaRS–suppressor tRNA pairs are described.
Abstract: Two critical requirements for developing methods for the site-specific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRS–suppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The “21st synthetase–tRNA pairs” include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyl-tRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using error-prone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2- to 6.8-fold.