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At least 19 recordsLinked to original sources

PubMed2025

Investigating Fission Yeast Mutagenesis Using Canavanine Sensitivity Assays.

Fission yeast are genetically tractable and amenable to mutagenesis studies. Canavanine is a toxic antimetabolite that can be used to test mutation rate. Recent studies have shown that the molecular genetics of canavanine sensitivity are more complex than previously anticipated. However, genomics advances indicate that canavanine use to determine mutation remains an option. In this chapter, we provide methods to grow fission yeast and detect forward mutation in populations of canavanine-sensitive Schizosaccharomyces pombe. Wild-type S. pombe are functionally canavanine-sensitive and die in the presence of canavanine. These protocols use liquid cultures that are tested for density and viability through colony formation. The same cultures are plated onto canavanine-containing media. Cells are grown to find cells that can grow on the canavanine media. These resistant cells are compared to the number plated, and a mutation rate is calculated. While the protocol is straightforward, analysis and application of the data are evolving. These methods provide the ability to compare S. pombe mutant strains for the frequency and rate of mutation.

Schizosaccharomyces↗

PubMed2025

Measuring Cell Dimensions in Fission Yeast Using Machine Learning.

In fission yeast (Schizosaccharomyces pombe), cell length is a crucial indicator of cell cycle progression. Microscopy screens that examine the effect of agents or genotypes suspected of altering genomic or metabolic stability and thus cell size are crucial for studying disruptions to cell cycle dynamics. This method is based on using an automated cell segmentation algorithm to measure S. pombe cells imaged by brightfield (BF) microscopy methods. PhotoPhenosizer (PP) is a machine learning-based tool designed for automated cell measuring and dimensional analysis of morphology frequency distributions. Integration of this method into large-scale pipelines for tracking cell dimension change streamlines morphological measurements, which facilitates the examination of cellular responses to genomic and metabolic stresses. In this protocol, we use PP to observe the effect of genomic instability on cell size dynamics over a 12-day chronological lifespan assay. Our results show that relative to wild-type cells, a replication stress mutant shows larger cells during chronological aging in excess glucose media. Our results are consistent with activation of checkpoints that regulate cell morphology in response to DNA damage. This method's application highlights the relevance of its incorporation in experimental routines that require large-scale image processing and its adoption by users with routine needs in S. pombe molecular research projects.

Schizosaccharomyces↗

PubMed1979

Analysis of the significance of a periodic, cell size-controlled doubling in rates of macromolecular synthesis for the control of balanced exponential growth of fission yeast cells.

Mutant strains of the fission yeast Schizosaccharomyces pombe are available which divide at smaller mean sizes than wild type. Earlier work by the present authors has shown that all these strains double their rates of polyadenylated messenger RNA synthesis as a step once in each cell cycle. The smaller the cell, the later in the cycle is the doubling in rate of synthesis. Strains of all sizes, however, double their synthetic rate when at the same threshold size. We show here that the differences in cell cycle stage of doubling in rate of polyadenylated messenger RNA synthesis are enough to explain the reduced mean steady state polyadenylated messenger RNA contents of the smaller strains. The cell size-related control over doubling in rate of synthesis is also shown to maintain the mean polyadenylated messenger RNA content as a constant proportion of cell mass, irrespective of cell size. This control thus allows cells to maintain balanced exponential growth, even when absolute growth rate per cell is altered by mutation. It is also shown that the concentration of polyadenylated messenger RNA itself could act as a monitor of the threshold size triggering the doubling in rate of synthesis in each cell cycle.

Ascomycota↗

PubMed2025

Genome-Wide Profiling of Histone Modifications in Fission Yeast Using CUT&Tag.

Eukaryotic DNA is organized in the nucleus in the form of chromatin. Nucleosomes, the fundamental unit of chromatin, are subject to many posttranslational modifications (PTMs) as well as compositional variations through incorporation of histone variants. These alterations play important roles in regulation of genome structure and activity. Genome-wide profiling of these regulatory features is essential for understanding of genome function. Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-Seq) is a widely used method to assay genome-wide localization in fission yeast but suffers from the requirement for a large amount of input chromatin, antibodies, and a cumbersome experimental pipeline. New methods such as Cleavage Under Targets and Tagmentation (CUT&Tag), which combine the specificity of targeted cleavage and adapter insertion with the sensitivity of next-generation sequencing, enable identification and characterization of various epigenetic marks affording low input requirement as well as more streamlined protocols. However, these approaches have not been adapted for use in fission yeast, Schizosaccharomyces pombe. Here, we describe an adapted CUT&Tag protocol for epigenomic profiling in fission yeast using the heterochromatin-associated histone H3K9 methylation PTM for benchmarking.

Schizosaccharomyces↗

PubMed2026

Evolutionary diversification of invertase paralogs couples carbon metabolism and sexual reproduction in fission yeasts.

Dynamic patterns of gene gain and loss play a major role in the diversification of eukaryotes, reflecting adaptation to a broad range of ecological contexts. Reconstructing the evolutionary history of genes provides a powerful framework for understanding how functional innovation shapes life-history traits. Here we report a comprehensive analysis of gene gain and loss across the fission yeast clade, whose evolutionary trajectory remains elusive. Reductive evolution of metabolic genes is a major contributor to species diversification, as observed in other fungal taxa. Notably, we uncovered an evolutionary scenario in which an ancestral gene duplication was followed by lineage-specific loss of one or the other paralog, except in S. pombe, which retained both. We demonstrate that these paralogs encode catalytically-active invertases, named Inv1 and Inv2, with distinct enzymatic properties, localization, regulation, and physiological roles. Inv1 is a secreted enzyme subject to glucose catabolite repression and is the sole invertase required for sucrose assimilation, resembling canonical yeast invertases. In contrast, Inv2 is intracellular, constitutively expressed, and required for inducing sexual differentiation in response to nutrient availability. Overall, these findings reveal an unexpected role for carbon metabolism in modulating the haploid-diploid cycle of fission yeasts, suggesting that diversification of core metabolic functions may contribute to adaptation to environments with distinct sugar compositions.

Evolution↗

PubMed1979

The nucleotide sequence of tRNA tyrosine from the fission yeast Schizosaccharomyces pombe.

The sequence of tRNA tyrosine from the fission yeast Schizosaccharomyces pombe is pCUCCUGAUm1 GGUG psi AGDDGGDDAUCACACor (psi) CCGGUG psi Ai6 AACCGGUUGm7 GUm5C GCUAGT psi CGm1 AUUCUGGUCAGGAGACCAOH. This sequence differs in 30 nucleotides from the tRNA-Tyr seqence of the budding yeast Saccharomyces cerevisiae. It has a unique anticodon stem of only four GC base pairs. The normal fifth pair position of nucleotide 28-44 is occupied by a C-U and in 20% of the tRNA-Tyr molecules it is psi-U. This unusual feature and its implications are considered in the discussion.

Anticodon↗

PubMed2025

Macromolecular and cytological changes in fission yeast G0 nuclei.

When starved of nitrogen, cells of the fission yeast Schizosaccharomyces pombe enter a quiescent 'G0' state with smaller nuclei and transcriptional repression. The genomics of S. pombe G0 cells has been well studied, but much of its nuclear cell biology remains unknown. Here, we use confocal microscopy, immunoblots and electron cryotomography to investigate the cytological, biochemical and ultrastructural differences between S. pombe proliferating, G1-arrested and G0 cell nuclei, with an emphasis on the histone acetylation, RNA polymerase II fates and macromolecular complex packing. Compared to proliferating cells, G0 cells have lower levels of histone acetylation, nuclear RNA polymerase II and active transcription. The G0 nucleus has similar macromolecular crowding yet fewer chromatin-associated multi-megadalton globular complexes. Induced histone hyperacetylation during nitrogen starvation results in cells that have larger nuclei and therefore chromatin that is less compact. However, these histone-hyperacetylated cells remain transcriptionally repressed with similar nuclear crowding. Canonical nucleosomes - those that resemble the crystal structure - are rare in proliferating, G1-arrested and G0 cells. Our study therefore shows that extreme changes in nucleus physiology are possible without extreme reorganization at the macromolecular level.

Schizosaccharomyces↗

PubMed1979

The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast.

Two isotopic methods for measuring DNA replication in the fission yeast Schizosaccharomyces pombe are described. The first is a method for measuring the total quantity of [3H]uracil incorporated into DNA after pulse labelling. The second is a means of detecting DNA replication in single cells by autoradiography. Both of these techniques have been used to investigate the timing and duration of S-phase in a series of mutant strains whose cell mass at division varies over a 3-fold range. The results support the hypothesis that in S. pombe there are 2 different controls over the timing of S-phase: an attainment of a critical cell mass and a dependency upon the completion of the previous mitosis coupled with a short minimum time in G1. Strains whose cell mass at birth is above this critical level initiate DNA replication almost immediately after septation, that is, very soon after the previous mitosis. Strains whose cell mass at birth is below the critical level do not initiate replication until the critical cell mass is attained. The duration of S-phase has been estimated from the proportion of cells whose nuclei are labelled after a pulse of given duration. S-phase is short in S. pombe, lasting only about 0.1 of a cell cycle in wild type. Cell mass at S-phase does not have any consistent effect on this length. We have also investigated the degree of synchrony of S-phase initiation in daughter cells, and have found that, in a cell cycle 240 min long, their S-phases are initiated within 1--2 min of each other. This result indicates that between sisters variability in the duration of the G1 phase is small compared with variability in the total cell cycle time, and argues against the hypothesis that the rate of cell cycle traverse is determined by a random transition in G1.

Ascomycota↗

PubMed2026

Recurrent reversible mutations at gaf1 driving metastable TORC1 inhibitor resistance in fission yeast.

Metastable phenotypic inheritance is often attributed to epigenetic mechanisms, but reversible genetic alterations can produce similar instability. Here, we investigated the basis of unstable resistance to TORC1 inhibitor (rapamycin plus caffeine) in Schizosaccharomyces pombe. Six independent, metastable resistant mutants were isolated. Genetic mapping positioned the causal lesion to a single Mendelian locus, which sequencing identified as gaf1, encoding a GATA transcription factor and a key negative regulator of growth downstream of TORC1. In each mutant, distinct loss-of-function mutations (insertions, deletions, or point mutations) were found in gaf1 in the resistant state, and these mutations precisely reverted to the wild-type sequence upon loss of resistance. Restoring the wild-type gaf1 allele abolished resistance, indicating that reversible genetic disruption of gaf1 is both necessary and sufficient for the metastable phenotype. Furthermore, strong resistance in several strains from a genome-wide deletion library was due to secondary, inactivating mutations in gaf1, underscoring its role as a recurrent adaptive target under rapamycin plus caffeine treatment. Mechanistically, gaf1 inactivation established a distinct basal transcriptome and pronounced derepression of translation and metabolic programs upon drug treatment. While rapamycin plus caffeine triggered extensive chromatin remodeling and H3K9 methylation contributed partially to resistance, these epigenetic changes were most consistent with a downstream modifying layer. Our study shows that metastable drug resistance in fission yeast is predominantly associated with recurrent, reversible genetic inactivation of the central transcriptional regulator gaf1, demonstrating how rapidly reversible genetic switches can drive adaptive evolution.IMPORTANCEDistinguishing between genetic and epigenetic inheritance is fundamental to understanding how cells adapt to environmental stress. In the fission yeast Schizosaccharomyces pombe, rapid and reversible drug resistance is often assumed to be driven by epigenetic switches that change gene activity without altering DNA. However, our study reveals that this instability can be caused by physical mutations in a single gene, gaf1, which acts as a genetic toggle. These mutations appear under drug pressure and precisely revert to the original sequence when the drug is removed. We also demonstrate that these spontaneous mutations can contaminate standard laboratory yeast collections, leading to potential misinterpretation of experimental data. These findings broaden our understanding of unstable inheritance and show that DNA sequences can be far more dynamic than previously recognized during rapid evolution and the development of drug resistance.

TORC1 signaling↗

PubMed2025

Fast Fission Yeast Genome Editing by CRISPR/Cas9 Using Gap Repair and Fluoride Selection.

We present a protocol to perform CRISPR/Cas9-mediated genome editing in the fission yeast Schizosaccharomyces pombe that does not require cloning and uses the fluoride exporter channel Fex1 as the selection marker. Transformation is typically carried out on the same day of PCR primer arrival and successfully edited strains are selected 5 days after transformation. We expect the adoption of this protocol to further accelerate the throughput of genome editing in S. pombe.

Schizosaccharomyces↗

PubMed1978

Relationship between extracellular enzymes and cell growth during the cell cycle of the fission yeast Schizosaccharomyces pombe: acid phosphatase.

By using the intact cells of the fission yeast Schizosaccharomyces pombe, the activity of acid phosphatase (EC 3.1.3.2) was compared through the cell cycle with the growth in cell length as a measure of cell growth. The cells of a growing asynchronous culture increased exponentially in number and in total enzyme activity, but remained constant in average length and in specific activity, In a synchronous culture prepared by selection or by induction, the specific activity was periodic in parallel with the increase in average cell length. When hydroxyurea was added to an asynchronous or a synchronous culture by selection, both specific and total activity followed the same continuous pattern as the growth in cell length after the stoppage of cell division. When oversized cells produced by a hydroxyurea pulse treatment to the culture previously syndronized by selection were transferred to a poor medium, they divided synchronously but could hardly grow in the total cell length. In this experimental situation, the total enzyme activity also scarcely increased through three division cycles. These results suggested that the increase in acid phosphatase in dependent on cell elongation.

Acid Phosphatase↗

PubMed1978

Oxygen uptake during the cell cycle of the fission yeast Schizosaccharomyces pombe.

Oxygen uptake was measured in synchronous cultures of the fission yeast Schizosaccharomyces pombe. The rate of oxygen uptake was found to increase in a step-wise manner at the beginning of the cycle and again in the middle of the cycle. The increases in rate were such that overall, oxygen uptake doubled in rate once per cell cycle. Addition of inhibitors of DNA synthesis or nuclear division to a synchronous culture did not affect the uptake of oxygen. In an induced synchronous culture, in which DNA synthesis, cell division, and nuclear division, but not 'growth' were synchronized, oxygen uptake increased continuously in rate and did not show the step-wise rises which were shown in the selection-synchronized culture. These results were compared with previous measurements of oxygen uptake in yeast and an explanation is suggested for the many different patterns which have been reported.

Ascomycota↗

PubMed1978

Carbon dioxide evolution during the cell cycle of the fission yeast Schizosaccharomyces pombe.

The rate of CO2 evolution was measured in synchronous cultures of the fission yeast Schizosaccharomyces pombe growing in a minimal medium. The rate of CO2 evolution was found to double sharply at about the time of nuclear division (0.75 of the way through the cell cycle). For the remainder of the cell cycle the rate remained constant. Addition of inhibitors of DNA synthesis or nuclear division did not affect the pattern of CO2 evolution in synchronous cultures. Similarly, in an induced synchronous culture, in which DNA synthesis, nuclear division and cell division--but not growth, were synchronized, CO2 evolution showed a continuous pattern and not the step-wise increase associated with the normal synchronous cultures. When S. pombe was grown in a complete medium, the evolution of CO2 in a synchronous cultures was shown to increase in a continuous manner but at a rate faster than the growth of the culture.

Ascomycota↗

PubMed2025

ChIPmentation for Epigenomic Analysis in Fission Yeast.

Histone modifications and transcription factor-DNA interactions regulate vital processes such as transcription, recombination, repair, and accurate chromosome segregation. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been instrumental in studying genome-wide distribution of DNA-bound or chromatin-associated factors and histone posttranslational modifications (PTMs). Here, we describe a ChIPmentation protocol adapted for fission yeast, Schizosaccharomyces pombe. This method merges Tn5 mediated tagmentation with existing ChIP protocols, resulting in lower sample input requirements with significant reduction in hands-on time and sample preparation costs.

Schizosaccharomyces↗

PubMed1978

Mutants altered in the control co-ordinating cell division with cell growth in the fission yeast Schizosaccharomyces pombe.

The control co-ordinating cell division with cell growth has been investigated in the fission yeast Schizosaccharomyces pombe. Twenty-five mutants altered in this control have been isolated which have the same growth rate as wild type but divide at a smaller cell size. The mutants define two genes wee 1 and wee 2, both of which are involved in a control initiating mitosis when the cell attains a critical size.

Cell Count↗

PubMed2026

Glutathione acts as an exometabolite that promotes growth recovery in fission yeast with defects in amino acid metabolism and cell polarity.

UNLABELLED: Microorganisms in nature form communities through diverse interactions, such as mutualism and competition, to adapt to their ecological environments. These interactions seem to be mediated by extracellular metabolites (exometabolites), yet the chemical and biological diversity underlying these processes remains largely unexplored. In this study, we examined the chemical basis of exometabolite-mediated interactions in the fission yeast Schizosaccharomyces pombe by a genome-wide screen employing 3,420 viable gene deletion mutants. We identified 37 strains that exhibited growth defects in monoculture on a minimal medium but exhibited growth recovery in the vicinity of wild-type colonies (co-culture), suggesting that exometabolites derived from wild-type cells compensated for the gene deletion. Both lipophilic and water-soluble fractions obtained by solvent partitioning of the wild-type culture supernatant promoted growth recovery. Among the 11 mutants rescued by the water-soluble fraction, 6 were cysteine auxotrophs, prompting analyses of thiol-containing metabolites by liquid chromatography-mass spectrometry (LC-MS), revealing the presence of glutathione (GSH) in the culture supernatant. GSH restored growth in most strains as a nutrient source. In contrast, GSH rescued cell morphology defects in the hob3∆ mutant, lacking the Bin/amphiphysin/Rvs (BAR) adaptor protein Hob3, through a mechanism independent of nutrition. This research advances understanding of exometabolite-mediated interactions in S. pombe by identifying GSH as an exometabolite that influences cellular processes and potentially shapes microbial communities. IMPORTANCE: Microorganisms secrete a wide range of metabolites that control microbial community behavior. These extracellular metabolites (exometabolites) include not only well-studied signaling molecules but also diverse primary and secondary metabolites, suggesting complex interactions among microbes. However, the molecular basis of these interactions remains poorly understood, partly due to challenges in detecting them experimentally. In this study, we surveyed exometabolites involved in cell-cell interactions in the model eukaryotic microorganism Schizosaccharomyces pombe. S. pombe releases a wide variety of metabolites outside the cells, including previously reported nitrogen signaling factors (NSFs) and glutathione (GSH) identified in this work. By analyzing gene deletion mutants whose growth is supported by extracellular GSH, we provide new insights into how secreted primary exometabolites compensate for specific genetic defects and influence cell physiology in microbial populations.

exometabolite↗

PubMed2026

Integrative modeling of the genome structure and dynamics in fission yeast.

Genome organization in the nucleus is highly structured and dynamic. Recent advances in genomic technology have enabled the measurement of genome-wide architecture and locus-specific motion, yielding contact maps and live-cell trajectories. However, these outcomes are derived from different modalities and are not directly comparable, with their quantitative integration being a key challenge. Here we establish a genome-wide live-cell imaging platform in fission yeast Schizosaccharomyces pombe, tracking 131 chromosomal loci, along with the spindle pole body (SPB) and nucleolus, to construct a quantitative map of locus dynamics. By integrating these dynamics with contact data through polymer modeling of Hi-C data, we build a physics-based "digital twin" of the S. pombe genome consistent with the spatiotemporal dynamics of interphase chromatin. We validate it against genome-wide mobility patterns and known architectural features, including centromere and telomere clustering. The model also identifies distinct dynamical regimes: centromere- and telomere-proximal loci relax within [Formula: see text]150 s, whereas the remaining loci relax within [Formula: see text]70 s. We measure semiperiodic dynamics of SPB motion, including a characteristic peak near 225 s and [Formula: see text] fluctuations. We use the model with SPB-directed forcing to show how these low-frequency components propagate through the genome to drive genome-wide chromatin displacements. Together, this predictive physics-based modeling framework integrates genome structure and dynamics to reveal how nuclear mechanical driving forces shape chromosome motion, linking mechanically driven chromatin responses to genome maintenance and regulation.

Schizosaccharomyces↗

PubMed1979

The reaction of cytochrome oxidase with oxygen in the fission yeast Schizosaccharomyces pombe 972h-. Studies at subzero temperatures and measurement of apparent oxygen affinity.

1. Cytochrome alpha 3 in whole-cell suspensions of the fission yeast Schizosaccharomyces pombe reacted in the reduced form with CO to give a photodissociable CO complex with absorption maxima at 429, 543 and 591 nm in CO-liganded reduced-minus-reduced difference spectra. 2. Other CO-bound haemoproteins, cytochromes P-420 and P-450, were not photodissociated under the conditions employed. 3. Measurements of the rates of reassociation of CO with cytochrome alpha 3 after flash photolysis over the temperature range from -101 to -109 degrees C gave a value for Eact. of 28.6 kJ/mol. 4. Between -94 and -106 degrees C, O2 reacted with cytochrome oxidase in intact cells to give an oxygenated intermediate (compound A). 5. At -70 degrees C compound A was converted into a second spectrally distinct intermediate (compound B). 6. Electron transport, indicated by the oxidation of cytochromes alpha + alpha 3 and cytochrome c, did not occur until the temperature was raised to -50 degrees C. 7. At room temperature cytochfome oxidase was oxidized to 50% of its steady-state concentration by 0.35 microM-O2.

Ascomycota↗