Haploid Yeast Strains Research Articles

Purification of Polysaccharide Produced by the Haploid Yeast Strain of Tremella sanguinea and Its Antioxidant and Prebiotic Activities.

Tremella sanguinea is a traditional Chinese medicinal and edible mushroom. Polysaccharides from Tremella mushrooms have received increasing amounts of research attention due to their diverse pharmacological activities. In this study, via the incubation of basidiospores collected from fresh artificially cultivated basidiocarps of T. sanguinea, a haploid yeast strain of T. sanguinea was obtained, and it was found to be a typical loose-slime-forming yeast capable of producing a large amount of exopolysaccharides (EPS). Using DEAE-52 cellulose column chromatography and Sephadex G-100 gel permeation chromatography, the major polysaccharide, named TSPS-1, was separated and purified from the EPS produced by the haploid yeast strain of T. sanguinea. TSPS-1 was a homogeneous polysaccharide with a molecular weight of 2.5 × 103 kDa and consisted of rhamnose, glucose, xylose, mannose and glucuronic acid at a molar ratio of 1: 0.7: 62.2: 24.6: 11.5. The bioactivity of the TSPS-1 polysaccharide was evaluated. The results show that TSPS-1 exhibited noticeable antioxidant activity by scavenging hydroxyl radicals (EC50 = 1.92 mg/mL) and superoxide radicals (EC50 = 1.33 mg/mL), and prebiotic activity by promoting the growth of different probiotic strains in the genus Lactobacillus and Bifidobacterium. These results suggest that the cultivation of the haploid yeast strain can be a promising alternative for the efficient production of valuable T. sanguinea polysaccharides with antioxidant and prebiotic potential.

Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.

A targeted double-strand break introduced into the genome of Saccharomyces cerevisiae is repaired by the relatively error-prone nonhomologous end joining (NHEJ) pathway when homologous recombination is not an option. A zinc finger nuclease cleavage site was inserted out-of-frame into the LYS2 locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5' overhangs. Repair events that destroyed the cleavage site were identified either as Lys+ colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+ events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol ζ and Pol η. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required translesion synthesis polymerases as well as the exonuclease activity of the replicative Pol δ DNA polymerase. Survivors were equally split between NHEJ events and 1.2 or 11.7 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3' tails. Finally, NHEJ was more efficient in nongrowing than in growing cells and was most efficient in G0 cells. These studies provide novel insights into the flexibility and complexity of error-prone DSB repair in yeast.

The bio3 mutation in sake yeast leads to changes in organic acid profiles and ester levels but not ethanol production

Biotin is an essential coenzyme that is bound to carboxylases and participates in fatty acid synthesis. The fact that sake yeast exhibit biotin prototrophy while almost all other Saccharomyces cerevisiae strains exhibit biotin auxotrophy, implies that biotin prototrophy is an important factor in sake brewing. In this study, we inserted a stop codon into the biotin biosynthetic BIO3 gene (cording for 7,8-diamino-pelargonic acid aminotransferase) of a haploid sake yeast strain using the marker-removable plasmid pAUR135 and investigated the fermentation profile of the resulting bio3 mutant. Ethanol production was not altered when the bio3 mutant was cultured in Yeast Malt (YM) medium containing 10% glucose at 15°C and 30°C. Interestingly, ethanol production was also not changed during the sake brewing process. On the other hand, the levels of organic acids in the bio3 mutant were altered after culturing in YM medium and during sake brewing. In addition, ethyl hexanoate and isoamyl acetate levels decreased in the bio3 mutant during sake brewing. Metabolome analysis revealed that the decreased levels of fatty acids in the bio3 mutant were attributed to the decreased levels of ethyl hexanoate. Further, the transcription level of genes related to the synthesis of ethyl hexanoate and isoamyl acetate were significantly reduced. The findings indicated that although the decrease in biotin biosynthesis did not affect ethanol production, it did affect the synthesis of components such as organic acids and aromatic compounds. Biotin biosynthesis ability is thus a key factor in sake brewing.

Biotechnology: use of available carbon sources on the planet to generate alternatives energy

Biotechnology has been an essential tool in the search for solutions and in the optimization of bioprocesses associated with issues of human, plant, animal, energy and also the balance of ecosystems on planet Earth. The objective of this research was to present an unconventional substrate (cellulose), in abundance on the planet, to be used as a substitute source of carbon and energy for biotechnology processes, with the possibility of increasing industrial production of biomass and energy. As basis for the research, an extensive literature review and quantitative and qualitative analyzes were carried out. Genetic Engineering techniques were used to enable the yeast Saccharomyces cerevisiae for partial cellulose degradation, through the use of genetic transformation methods to insert a plasmid carrying the cellobiohydrolase cDNA. It was found that the recombinant and biologically active cellobiohydrolase protein was expressed and excreted in haploid and diploid laboratory yeast strains. The analyzes allowed the visualization of cellulolysis halos around colonies of recombinant strains grown in solid YPD medium with 1% microgranular cellulose. The recombinant clones derived from the haploid lineage yielded in average of 1.70 mg ART/mL, while recombinant clones derived from the diploid lineage produced in average of 2.05 mg ART/mL.

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and β-alanine metabolism.

SummaryMevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3‐Hydroxy‐3‐methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431‐fold and 9‐fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and β‐alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3‐4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl‐CoA supply and β‐alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3‐4‐CPS generated from expressing β‐caryophyllene synthase in SQ3‐4 produced 11.86 ± 0.09 mg l−1 β‐caryophyllene, while strain SQ3‐5 resulted from down‐regulation of ERG1 in SQ3‐4 produced 408.88 ± 0.09 mg l−1 squalene in shake flask cultivations. Strain SQ3‐5 produced 4.94 g l−1 squalene in fed‐batch fermentation in cane molasses medium, indicating the promising potential for cost‐effective production of squalene.

Deletion of Ch. IV Telomeres via Genetic Engineering of a Circular Chromosome in the Budding Yeast S. cerevisiae

Chromosome structure varies from a single circular chromosome in prokaryotes to multiple linear chromosomes in eukaryotes. Linear chromosomes differ in that they possess specialized ends, called telomeres, that play important roles in protecting the ends from degradation. Furthermore, telomeres cannot be fully copied during DNA replication without the enzyme complex telomerase. Since telomerase is not expressed in all cells, telomeres shorten with aging. On the other hand, overexpression of telomerase is observed in >85% of human cancers. Given that telomeres create this end‐replication problem, our lab is interested in ultimately understanding why linear chromosomes evolved in the first place. To address this broad question, we are taking a novel genetic approach to systematically convert each of the linear chromosomes into circularized versions using the single‐celled eukaryote Saccharomyces cerevisiae. Our approach involves building two DNA cassettes with selectable marker elements that are integrated into the left and right arms of each chromosome in a haploid yeast strain. The cassettes are designed so that recombination between the two regions will reconstitute a functional marker gene, allowing genetic selection of circularized chromosomes. To date, we have isolated a circularized version of Ch. IV, the longest of the 16 yeast chromosomes. We have verified recombination between the cassette via PCR across the newly formed junction. The circularized Ch. IV yeast form colonies slightly smaller in size and exhibit a ~10% decrease in doubling time relative to wild‐type. Together, these data demonstrate that our approach can generate viable yeast with individual chromosomes circularized. We are currently analyzing cellular morphology using light microscopy, testing the ability of the circularized yeast to undergo mating and sporulation, and analyzing impacts on gene expression. A longer‐term goal of the lab is to use this approach to attempt to circularize the other 15 chromosomes and explore how linear chromosomes evolved.

Application of the yeast comet assay in Testing some food additives for genotoxicity by comet assay in yeast

Food additives of various types are frequently used in the food sector. Their use is justified by the need to preserve, color, or sweeten a variety of foods. Despite the fact that some additives have been demonstrated to be cytotoxic, they are nevertheless utilized in practice. The effects of monosodium glutamate (MSG), sodium benzoate (SB), and saffron on several yeast haploid knockout strains were studied in this study (YKO). We used the Comet test method to find the optimum amounts at which this set of dietary additives could cause DNA damage. Three regularly used dietary additives were found to efficiently damage DNA. We also evaluated the sensitivity of higher eukaryotic cells to the genotoxic effects of these chemicals with yeast. The comet assay exhibited a better sensitivity of yeast cells, which was undeniably confirmed. The genotypes of haploid (knockout) yeast strains were chosen based on the (Clustal Omega Multiple Sequence Alignment EMBL-EBI) alignment of human and yeast gene sequence homology.

Saccharomyces cerevisiae quiescent cells: cadmium resistance and adaptive response

The budding yeast Saccharomyces cerevisiae is a widely used model organism to investigate the changes occurring in the eukaryotic cell and to predict its possible ‘reaction’ to different environmental factors. Recently it was also shown that these microorganisms possess another advantageous ability: to ‘enter’ into quiescent state (G0). Yeast G0 cells have similar physiological characteristics to those of higher eukaryotes making them a better model for toxicology studies. As cadmium could affect severely human health, the main aim of the present study was to use Saccharomyces cerevisiae quiescent cells to investigate the resistance potential and corresponding adaptive response of eukaryotic cells to elevated cadmium concentrations. Both diploid and haploid yeast strains in logarithmic, quiescent and non-quiescent state were exposed to different concentrations of Cd(NO3)2. The half-maximal inhibitory concentration (IC50) for all tested cell types was 100 µmol/L Cd(NO3)2. The deleterious effects of cadmium on intracellular macromolecule structures were analyzed through evaluating the levels of accumulated reactive oxygen species (ROS) and carbonylated proteins. The highest ROS concentration was measured in logarithmic cells: up to 50% versus about 8% in G0 cells. Significant damage in protein molecules was observed in haploid cells where the protein carbonylation reached levels of 25 µmol/mg. Studying the adaptive response to elevated Cd2+ concentrations revealed that quiescent and non-quiescent cells respond with increased expression of key elements from the antioxidant defense system: reduced glutathione, superoxide dismutase (SOD) and catalase. Furthermore, an additional SOD izoenzyme was detected when diploid and haploid cell populations were exposed to Cd2+.

Structural evidence for an essential Fe-S cluster in the catalytic core domain of DNA polymerase ϵ.

DNA polymerase ϵ (Pol ϵ), the major leading-strand DNA polymerase in eukaryotes, has a catalytic subunit (Pol2) and three non-catalytic subunits. The N-terminal half of Pol2 (Pol2CORE) exhibits both polymerase and exonuclease activity. It has been suggested that both the non-catalytic C-terminal domain of Pol2 (with the two cysteine motifs CysA and CysB) and Pol2CORE (with the CysX cysteine motif) are likely to coordinate an Fe–S cluster. Here, we present two new crystal structures of Pol2CORE with an Fe–S cluster bound to the CysX motif, supported by an anomalous signal at that position. Furthermore we show that purified four-subunit Pol ϵ, Pol ϵ CysAMUT (C2111S/C2133S), and Pol ϵ CysBMUT (C2167S/C2181S) all have an Fe–S cluster that is not present in Pol ϵ CysXMUT (C665S/C668S). Pol ϵ CysAMUT and Pol ϵ CysBMUT behave similarly to wild-type Pol ϵ in in vitro assays, but Pol ϵ CysXMUT has severely compromised DNA polymerase activity that is not the result of an excessive exonuclease activity. Tetrad analyses show that haploid yeast strains carrying CysXMUT are inviable. In conclusion, Pol ϵ has a single Fe–S cluster bound at the base of the P-domain, and this Fe–S cluster is essential for cell viability and polymerase activity.

High-Throughput Yeast One-Hybrid Screens Using a Cell Surface gLUC Reporter.

Gene-centered yeast one-hybrid (Y1H) screens using arrayed genome-wide transcription factor (TF) clone collections provide a simple and effective strategy to identify TF-promoter interactions using a DNA fragment as bait. In an effort to improve the assay we recently developed a Y1H system that uses a cell surface Gaussia luciferase reporter (gLUC59). Compared to other available methods, this luciferase-based strategy requires a shorter processing time, enhances the throughput and improves result analysis of gene-centered Y1H screens. Here, we described the procedure to perform high-throughput screens using this novel strategy, which involves a protocol for mating two haploid yeast strains carrying an arrayed TF clone collection and a promoter::gLUC59 reporter, respectively, and a protocol for analyzing gLUC59 activity in the resulting diploid cells. © 2019 by John Wiley & Sons, Inc.

Impact of several PD‐associated genes on the toxicity of a‐synuclein in a yeast model

Parkinson's disease (PD) is characterized by a‐synuclein misfolding and the death of midbrain neurons. PD can be described as familial, or sporadic, both of which are influenced by a multitude of environmental and genetic factors. Familial PD is directly caused by a mutation in one of at least ten genes, including SNCA, DJ‐1, VPS35, and ATP13A2. SNCA, which encodes a‐synuclein, has six identified missense mutations (A30P, E46K, H50Q, G51D, A53E, and A53T) that each cause autosomal dominant PD. Sporadic PD is linked with several risk genes and loci, including VPS13, the Sac I domain of SYNJ1, and the Swa2 domain of DNAJC6. Using our previously established budding yeast model system for α‐synuclein, we first show that wild‐type (WT), E46K, A53T, H50Q, and A53E a‐synuclein are toxic to yeast and show varying degrees of membrane binding and aggregation, while A30P and G51D a‐synuclein are relatively non‐toxic and shows cytoplasmic diffuse localization. What is still not well understood is whether the other PD‐causing and risk genes mentioned above can influence toxicity and localization properties of WT a‐synuclein and these six familial PD mutants. To test the hypothesis that they do influence a‐synuclein, WT and familial mutant forms of a‐synuclein were studied in haploid yeast strains that were singly deleted for these six PD‐linked genes (all of which are linked to loss‐of‐function in PD). Results show that some gene deletions increase (Δhsp31) or decrease (Δatp13, Δvps35) a‐synuclein toxicity and alter its localization in a highly familial mutant specific way, while others more broadly increase a‐synuclein toxicity or aggregation (Δvps13, Δsac1), while still others no effect (Δswa2). Our findings suggest that WT and each familial mutant of a‐synuclein create cellular toxicity and alter localization in distinct ways and that each is likely regulated by different subsets of genes, opening doors for mutant‐specific mechanistic insight into the varying modes of a‐synuclein toxicity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A Perfect Match Genomic Landscape Provides a Unified Framework for the Precise Detection of Variation in Natural and Synthetic Haploid Genomes.

We present a conceptually simple, sensitive, precise, and essentially nonstatistical solution for the analysis of genome variation in haploid organisms. The generation of a Perfect Match Genomic Landscape (PMGL), which computes intergenome identity with single nucleotide resolution, reveals signatures of variation wherever a query genome differs from a reference genome. Such signatures encode the precise location of different types of variants, including single nucleotide variants, deletions, insertions, and amplifications, effectively introducing the concept of a general signature of variation. The precise nature of variants is then resolved through the generation of targeted alignments between specific sets of sequence reads and known regions of the reference genome. Thus, the perfect match logic decouples the identification of the location of variants from the characterization of their nature, providing a unified framework for the detection of genome variation. We assessed the performance of the PMGL strategy via simulation experiments. We determined the variation profiles of natural genomes and of a synthetic chromosome, both in the context of haploid yeast strains. Our approach uncovered variants that have previously escaped detection. Moreover, our strategy is ideally suited for further refining high-quality reference genomes. The source codes for the automated PMGL pipeline have been deposited in a public repository.

Study of the role of the covalently linked cell wall protein (Ccw14p) and yeast glycoprotein (Ygp1p) within biofilm formation in a flor yeast strain.

Flor yeasts are Saccharomyces cerevisiae strains noted by their ability to create a type of biofilm in the air-liquid interface of some wines, known as 'flor' or 'velum', for which certain proteins play an essential role. Following a proteomic study of a flor yeast strain, we deleted the CCW14 (covalently linked cell wall protein) and YGP1 (yeast glycoprotein) genes-codifying for two cell surface glycoproteins-in a haploid flor yeast strain and we reported that both influence the weight of the biofilm as well as cell adherence (CCW14).

A novel one-step approach for the construction of yeast surface display Fab antibody libraries

BackgroundYeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating.ResultsWithin this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties.ConclusionWe demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.

A Streamlined Approach for the Construction of Large Yeast Surface Display Fab Antibody Libraries.

Yeast surface display is a versatile platform technology for antibody discovery. Nevertheless, the construction of antibody Fab libraries typically is a tedious multistep process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Here, we present a focused one-step Golden Gate cloning approach for the generation of yeast surface display Fab libraries that allows for simultaneous introduction of heavy-chain and light-chain variable regions into one single display vector. Thereby, the overall time as well as the materials needed for library generation can be reduced significantly.

Disruption of the cell wall integrity gene ECM33 results in improved fermentation by wine yeast

Severe oenological conditions, such as limited assimilable nitrogen and high sugar contents restrict yeast’s ability to successfully complete fermentation. In the absence of a comprehensive commercially available deletion collection in a wine yeast background, a screening approach was applied to a transposon library in a wine yeast derivative to identify clones with superior fermentation performance. Five candidate genes, when disrupted by Ty insertion, were identified as enabling yeast to efficiently complete a model oenological fermentation with limited nitrogen availability. Analogous single gene disruptions were subsequently constructed in the haploid wine yeast strain C911D, and the performance of these during fermentation was analysed. Deletion of ECM33 resulted in the shortest fermentation (up to 31% reduction) in both synthetic medium and grape juice. Interestingly, no significant differences were found in nitrogen utilization, cell viability or biomass yield between ∆ecm33 and the wild type. ∆ecm33 did, however, display growth hypersensitivity to the dyes Calcofluor White and Congo Red, suggesting a link to cell wall integrity. Transcriptional profiling of ∆ecm33 during fermentation demonstrated the up-regulation of SLT2 and HOG1, encoding mitogen activated protein kinases involved in the cell wall integrity (CWI) and high osmolarity glycerol (HOG) pathways, respectively. CHS3 a major chitin synthase gene was also found to be upregulated, and the transcript abundance of key genes of central nitrogen metabolism, GLN1, GLT1, GDH1 and GDH2 in mutant ∆ecm33 were also altered. The findings highlight the complexity of the robust fermentation phenotype and provide clues for further improvement of industrial strains.

Construction of low-ethanol\u2013wine yeasts through partial deletion of the Saccharomyces cerevisiae PDC2 gene

We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduction in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Compared to the control, the diploid BY4743pdc2Δ519 mutant strain reduced up to 7.4% the total ethanol content in lab scale-vinifications. The residual sugar and volatile acidity was not significantly affected by this ethanol reduction. Remarkably, we got a much higher ethanol reduction of 10 and 15% when the pdc2Δ519 mutation was tested in a native and a commercial wine yeast strain against their respective controls. Our results demonstrate that the insertion of the pdc2Δ519 mutation in wine yeast strains can reduce the ethanol concentration up to 1.89% (v/v) without affecting the fermentation performance. In contrast to non-GMO based strategies, our approach permits the insertion of the pdc2Δ519 mutation in any locally selected wine strain, making possible to produce quality wines with regional characteristics and lower alcohol content. Thus, we consider our work a valuable contribution to the problem of high ethanol concentration in wine.

Genomic saturation mutagenesis and polygenic analysis identify novel yeast genes affecting ethyl acetate production, a non-selectable polygenic trait.

Isolation of mutants in populations of microorganisms has been a valuable tool in experimental genetics for decades. The main disadvantage, however, is the inability of isolating mutants in non-selectable polygenic traits. Most traits of organisms, however, are non-selectable and polygenic, including industrially important properties of microorganisms. The advent of powerful technologies for polygenic analysis of complex traits has allowed simultaneous identification of multiple causative mutations among many thousands of irrelevant mutations. We now show that this also applies to haploid strains of which the genome has been loaded with induced mutations so as to affect as many non-selectable, polygenic traits as possible. We have introduced about 900 mutations into single haploid yeast strains using multiple rounds of EMS mutagenesis, while maintaining the mating capacity required for genetic mapping. We screened the strains for defects in flavor production, an important non-selectable, polygenic trait in yeast alcoholic beverage production. A haploid strain with multiple induced mutations showing reduced ethyl acetate production in semi-anaerobic fermentation, was selected and the underlying quantitative trait loci (QTLs) were mapped using pooled-segregant whole-genome sequence analysis after crossing with an unrelated haploid strain. Reciprocal hemizygosity analysis and allele exchange identified PMA1 and CEM1 as causative mutant alleles and TPS1 as a causative genetic background allele. The case of CEM1 revealed that relevant mutations without observable effect in the haploid strain with multiple induced mutations (in this case due to defective mitochondria) can be identified by polygenic analysis as long as the mutations have an effect in part of the segregants (in this case those that regained fully functional mitochondria). Our results show that genomic saturation mutagenesis combined with complex trait polygenic analysis could be used successfully to identify causative alleles underlying many non-selectable, polygenic traits in small collections of haploid strains with multiple induced mutations.

Yeast Replicator: A High-Throughput Multiplexed Microfluidics Platform for Automated Measurements of Single-Cell Aging.

The yeast Saccharomyces cerevisiae is a model organism for replicative aging studies; however, conventional lifespan measurement platforms have several limitations. Here, we present a microfluidics platform that facilitates simultaneous lifespan and gene expression measurements of aging yeast cells. Our multiplexed high-throughput platform offers the capability to perform independent lifespan experiments using different yeast strains or growth media. Using this platform in minimal media environments containing glucose, we measured the full lifespan of individual yeast cells in wild-type and canonical gene deletion backgrounds. Compared to glucose, in galactose we observed a 16.8% decrease in replicative lifespan accompanied by an ∼2-fold increase in single-cell oxidative stress levels reported by PSOD1-mCherry. Using PGAL1-YFP to measure the activity of the bistable galactose network, we saw that OFF and ON cells are similar in their lifespan. Our work shows that aging cells are committed to a single phenotypic state throughout their lifespan.

Adenine auxotrophy - be aware: some effects of adenine auxotrophy inSaccharomyces cerevisiaestrain W303-1A

Adenine auxotrophy is a commonly used genetic marker in haploid yeast strains. Strain W303-1A, which carries the ade2-1 mutation, is widely used in physiological and genetic research. Yeast extract-based rich medium contains a low level of adenine, so that adenine is often depleted before glucose. This could affect the cell physiology of adenine auxotrophs grown in rich medium. The aim of our study was to assess the effects of adenine auxotrophy on cell morphology and stress physiology. Our results show that adenine depletion halts cell division, but that culture optical density continues to increase due to cell swelling. Accumulation of trehalose and a coincident 10-fold increase in desiccation stress tolerance is observed in adenine auxotrophs after adenine depletion, when compared to prototrophs. Under adenine starvation, long-term survival of W303-1A is lower than during carbon starvation, but higher than during leucine starvation. We observed drastic adenine-dependent changes in cell stress physiology, suggesting that results may be biased when adenine auxotrophs are grown in rich media without adenine supplementation.