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Concept: Paul Nurse

170

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.

Concepts: Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Model organisms, Schizosaccharomyces, Paul Nurse

2

Glucose is the fundamental energy source for life; thus cells need to respond appropriately to changes in available glucose concentration. We investigated the relationship between media glucose concentration and respiration-dependency of proliferation, using Schizosaccharomyces pombe. In media containing ≥0.2% glucose, neither antimycin A, an inhibitor of Complex III, nor gene deletions of essential electron transfer chain components, impaired cell division, while these factors completely inhibited cell division in media containing ≤0.1% glucose. These results indicate the existence of a threshold in glucose concentration that governs respiration-dependency of S. pombe proliferation.

Concepts: Protein, Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

Mitogen Activated Protein Kinases (MAPKs) play vital roles in multiple cellular processes and represent prominently pursued targets for development of therapeutic regimes. The MAPK Spc1 (p38 homolog) is known to be very important for both mitotic promotion and delay in Schizosaccharomyces pombe. However, the mechanism responsible for mitotic inhibition has remained elusive. Cdc25 (Cdc2 activator) and Wee1 (Cdc2 inhibtor) are important determinants of mitotic timing in all eukaryotes. Our results show that Spc1 can sense the perturbations in the balance of Cdc25 and Wee1 activities in S. pombe and that its function as a mitotic inhibitor is very important for controlling the same. An Spc1-Srk1-Rad24 dependent pathway for mitotic inhibition has been reported earlier.Here we report the presence of an alternative mechanism wherein Spc1 targets the 14-3-3 protein, Rad24, independently of Srk1, leading to relocalisation of Cdc25 and mitotic inhibition. Our observations suggest that this pathway can serve as a backup mechanism for Cdc2 inactivation in absence of Wee1.

Concepts: Signal transduction, Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

The fission yeast Schizosaccharomyces pombe undergoes a switch from yeast to filamentous invasive growth in response to certain environmental stimuli. Among them is ammonium limitation. Amt1, one of the three ammonium transporters in this yeast, is required for the ammonium limitation-induced morphological transition; however, the underlying molecular mechanism remains to be understood. Cells lacking Amt1 became capable of invasive growth upon increasing concentrations of ammonium in the medium, suggesting that the ammonium taken up into the cell or a metabolic intermediate in ammonium assimilation might serve as a signal for the ammonium limitation-induced morphological transition. To investigate the possible role of ammonium-metabolizing enzymes in the signaling process, deletion mutants were constructed for the gdh1, gdh2, gln1, and glt1 genes, which were demonstrated by enzyme assays to encode NADP-specific glutamate dehydrogenase, NAD-specific glutamate dehydrogenase, glutamine synthetase, and glutamate synthase, respectively. Growth tests on various nitrogen sources revealed that a gln1Δ mutant was a glutamine auxotroph and that a gdh1Δ mutant had a defect in growth on ammonium, particularly at high concentrations. The latter observation indicates that the NADP-specific glutamate dehydrogenase of S. pombe plays a major role in ammonium assimilation under high ammonium concentrations. Invasive growth assays showed that gdh1Δ and glt1Δ mutants underwent invasive growth to a lesser extent than did wild-type strains. Increasing the ammonium concentration in the medium suppressed the invasive growth defect of the glt1Δ mutant, but not the gdh1Δ mutant. These results suggest that the nitrogen status of the cell is important in the induction of filamentous invasive growth in S. pombe.

Concepts: Enzyme, Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

In this introduction we discuss some basic genetic tools and techniques that are used with the fission yeast Schizosaccharomyces pombe Genes commonly used for selection or as reporters are discussed, with an emphasis on genes that permit counterselection, intragenic complementation, or colony-color assays. S. pombe is most stable as a haploid organism. We describe its mating-type system, how to perform genetic crosses and methods for selecting and propagating diploids. We discuss the relative merits of tetrad dissection and random spore preparation in strain construction and genetic analyses. Finally, we present several types of mutant screens, with an evaluation of their respective strengths and limitations in the light of emerging technologies such as next-generation sequencing.

Concepts: Gene, Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

Schizosaccharomyces pombe cells initiate a sexual differentiation program, which comprises meiosis and spore formation, on nitrogen starvation. This protocol describes a simple procedure to induce meiosis and sporulation semisynchronously in heterozygous diploid S. pombe cells. The procedure is appropriate for a variety of applications, including fluorescence-activated cell sorting (FACS) and northern and western blotting. Zygotic meiosis can also be induced by the same procedure, although less synchronously.

Concepts: Yeast, Model organism, Meiosis, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

This protocol describes chemical transformation of Schizosaccharomyces pombe with linear DNA in a 96-well format. This procedure has been successfully used for large-scale strain construction in fission yeast.

Concepts: Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

The uncapping of telomeres induces a DNA damage response. In Schizosaccharomyces pombe, deletion of pot1+ causes telomere uncapping and rapid telomere resection, resulting in chromosome fusion. Using the nmt-pot1-aid strain, we previously reported that Pot1 shut-off causes telomere loss and chromosome fusion in S. pombe. However, the factors responsible for the resection of uncapped telomeres remain unknown. In this study, we investigated these factors and found that concomitant deletion of rqh1+ and exo1+ alleviated the loss of telomeres following Pot1 shut-off, suggesting that Rqh1 and Exo1 are redundantly involved in the resection of uncapped telomeres. We also investigated the role of Rqh1 helicase activity and found it to be essential for the resection of uncapped telomeres. Moreover, we found that Dna2 and Exo1 function redundantly in the resection of uncapped telomeres. Taken together, these results suggest that Exo1 and Rqh1-Dna2 redundantly contribute to the resection of uncapped telomeres. Therefore, our results demonstrate that nmt-pot1-aid is an important model strain to study the role of helicases and nucleases in the resection of uncapped telomeres and to improve our understanding of DNA double-strand break repair.

Concepts: DNA, Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Concepts: Enzyme, Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse

0

Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast.

Concepts: Gene, Yeast, Model organism, Ascomycota, Schizosaccharomyces pombe, Yeasts, Schizosaccharomyces, Paul Nurse