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S. pombe

Simon David Brown, Olga Dorota Jarosinska, Alexander Lorenz
Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events...
March 17, 2018: Current Genetics
Vincent Normant, Thierry Mourer, Simon Labbe
In the fission yeast Schizosaccharomyces pombe , acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters promotes cellular heme import. Using a strain that cannot synthesize heme de novo ( hem1Δ ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a hem1Δ shu1Δ str3Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells...
March 16, 2018: Journal of Biological Chemistry
James L Kingsley, Jeffrey P Bibeau, S Iman Mousavi, Cem Unsal, Zhilu Chen, Xinming Huang, Luis Vidali, Erkan Tüzel
Fluorescence recovery after photobleaching (FRAP) is an important tool used by cell biologists to study the diffusion and binding kinetics of vesicles, proteins, and other molecules in the cytoplasm, nucleus, or cell membrane. Although many FRAP models have been developed over the past decades, the influence of the complex boundaries of 3D cellular geometries on the recovery curves, in conjunction with regions of interest and optical effects (imaging, photobleaching, photoswitching, and scanning), has not been well studied...
March 13, 2018: Biophysical Journal
Tomoyuki Hatano, Salvatore Alioto, Emanuele Roscioli, Saravanan Palani, Scott T Clarke, Anton Kamnev, Juan Ramon Hernandez-Fernaud, Lavanya Sivashanmugam, Bernardo Chapa-Y-Lazo, Alexandra M E Jones, Robert C Robinson, Karuna Sampath, Masanori Mishima, Andrew D McAinsh, Bruce L Goode, Mohan K Balasubramanian
Actins are major eukaryotic cytoskeletal proteins, which perform many important cell functions, including cell division, cell polarity, wound healing, and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively presently for biochemical studies of actin cytoskeleton from other organisms / cell types. Here we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin β4 and purified using an affinity tag introduced in the fusion...
March 13, 2018: Journal of Cell Science
Jessica Fletcher, Liam Griffiths, Thomas Caspari
The S. pombe checkpoint kinase, Cds1, protects the integrity of stalled DNA replication forks after its phosphorylation at threonine-11 by Rad3 (ATR). Modified Cds1 associates through its N-terminal forkhead-associated domain (FHA)-domain with Mrc1 (Claspin) at stalled forks. We report here that nutrient starvation results in post-translational changes to Cds1 and the loss of Mrc1. A drop in glucose after a down-shift from 3% to 0.1-0.3%, or when cells enter the stationary phase, triggers a sharp decline in Mrc1 and the accumulation of insoluble Cds1...
February 23, 2018: Cells
Alexis Zukowski, Aaron M Johnson
Mono-ubiquitinated histone H2B (H2B-Ub) is important for chromatin regulation of transcription, chromatin assembly, and also influences heterochromatin. In this review, we discuss the effects of H2B-Ub from nucleosome to higher-order chromatin structure. We then assess what is currently known of the role of H2B-Ub in heterochromatic silencing in budding and fission yeasts (S. cerevisiae and S. pombe), which have distinct silencing mechanisms. In budding yeast, the SIR complex initiates heterochromatin assembly with the aid of a H2B-Ub deubiquitinase, Ubp10...
February 20, 2018: Current Genetics
Jinyu Liu, Yan Li, Jie Chen, Yirong Wang, Mengting Zou, Ruyue Su, Ying Huang
Mitochondrial gene expression is essential for adenosine triphosphate synthesis via oxidative phosphorylation, which is the universal energy currency of cells. Here, we report the identification and characterization of a homologue of Saccharomyces cerevisiae Mtf2 (also called Nam1) in Schizosaccharomyces pombe. The Δmtf2 mutant with the intron-containing mitochondrial DNA (mtDNA) exhibited impaired growth on a rich medium containing the non-fermentable carbon source glycerol, suggesting that mtf2 is involved in mitochondrial function...
January 12, 2018: Microbiology
Caia D S Duncan, María Rodríguez-López, Phil Ruis, Jürg Bähler, Juan Mata
Eukaryotes respond to amino acid starvation by enhancing the translation of mRNAs encoding b-ZIP family transcription factors (GCN4 in Saccharomyces cerevisiae and ATF4 in mammals), which launch transcriptional programs to counter this stress. This pathway involves phosphorylation of the eIF2 translation factor by Gcn2-protein kinases and is regulated by upstream ORFs (uORFs) in the GCN4/ATF4 5' leaders. Here, we present evidence that the transcription factors that mediate this response are not evolutionarily conserved...
February 5, 2018: Proceedings of the National Academy of Sciences of the United States of America
Ángel Benito, Fernando Calderón, Santiago Benito
This chapter describes a methodology to isolate yeast strains from Schizosaccharomyces pombe species. The method is based on a selective-differential medium that notably facilitates the isolation of S. pombe. The main difficulty in isolating microorganisms from this genus is their extremely low incidence in nature when they are compared to other microorganisms. The proposed methodology allows isolating and selecting strains from this species for industrial purposes. Methodologies allows detecting the presence of those yeasts when they are considered spoilage microorganisms...
2018: Methods in Molecular Biology
Ángel Benito, Fernando Calderón, Santiago Benito
The traditional way of producing wine is through the use of Saccharomyces cerevisiae in order to convert glucose and fructose into alcohol. In the case of red wines, after this alcoholic fermentation lactic bacteria Oenococus oeni is used to stabilize wine from a microbiological point of view by converting malic acid into lactic acid that it is not a microbiological substract. The yeast species Schizosaccharomyces pombe was traditionally considered spoilage yeast. Nevertheless, during the last decade it started to be used due to its unique malic acid deacidification ability to reduce the harsh acidity of wines from northern Europe, by converting malic acid to ethanol and CO2 without producing lactic acid as lactic bacteria does...
2018: Methods in Molecular Biology
Sudhir Kumar Rai, Angela Atwood-Moore, Henry L Levin
The introduction of ectopic DNA, such as plasmids, into yeast cells has for decades been a critical protocol for the study of this eukaryotic model system. We describe here an efficient transformation procedure for use in the fission yeast Schizosaccharomyces pombe. This method relies on chemical agents (lithium acetate, and polyethylene glycol) and temperature stresses, which ultimately facilitate transfer of the genetic material through the cell wall and plasma membrane without significant impact on the transferred DNA or the recipient cell...
2018: Methods in Molecular Biology
Jinpu Yang, Fei Li
Chromatin-associated proteins play critical roles in many cellular processes, including gene expression, epigenetic regulation, DNA repair, recombination, and replication. Especially, epigenetic landscape, shaped by a variety of chromatin-binding proteins, is dynamic and regulated in a context-dependent manner. In situ chromatin-binding assay is a powerful but simple tool to investigate how proteins, such as epigenetic components, associate with chromatin. This approach relies on the fact that chromatin bound proteins are more resistant to detergent extraction...
2018: Methods in Molecular Biology
Zhen Zhu, Olivier Frey, Andreas Hierlemann
This chapter describes a microfluidic device that enables immobilization and culturing of single rod-shaped S. pombe cells in a stand-up mode. The wide-band electrical impedance spectroscopy (EIS) has been integrated in the microfluidic device to continuously measure cell growth of single S. pombe cells. Cell growth curves showing cellular and intracellular features at high spatiotemporal resolution can be obtained from EIS signals. The features include longitudinal cell elongation in the G2 phase, mitosis, and cell division during an entire cell cycle of S...
2018: Methods in Molecular Biology
Robert Roth, Hiten D Madhani, Jennifer F Garcia
The fission yeast, Schizosaccharomyces pombe, is an important model organism for investigations of gene regulation. Essential to such studies is the ability to quantify the levels of a specific RNA. We describe a protocol for the isolation and quantification of RNA in S. pombe using reverse-transcription followed by quantitative PCR. In this procedure, the cells are lysed using zirconia beads, then total RNA is selectively isolated away from proteins and DNA using the Trizol reagent. Contaminating DNA is then removed from the RNA by using TURBO DNase, which is easily inactivated and requires no subsequent clean-up step...
2018: Methods in Molecular Biology
Valerie Migeot, Damien Hermand
The distribution of modified histones within the fission yeast Schizosaccharomyces pombe genome is ultimately dependent upon the transcriptional activity and in turn influences the ability of the polymerases to bind and progress through the chromatin template. The Chromatin Immunoprecipitation-Polymerase Chain Reaction (ChIP-PCR) method currently provides the highest resolution, accuracy, and reproducibility to characterize histones modifications within a defined region of the genome. The following protocol details the method applied to S...
2018: Methods in Molecular Biology
Angad Garg, Ana M Sanchez, Stewart Shuman, Beate Schwer
The expression of the phosphate transporter Pho84 in fission yeast Schizosaccharomyces pombe is repressed in phosphate-rich medium and induced during phosphate starvation. Two other phosphate-responsive genes in S. pombe (pho1 and tgp1) had been shown to be repressed in cis by transcription of a long noncoding (lnc) RNA from the upstream flanking gene, but whether pho84 expression is regulated in this manner is unclear. Here we show that repression of pho84 is enforced by transcription of the SPBC8E4.02c locus upstream of pho84 to produce a lncRNA that we name prt2 (pho-repressive transcript 2) We identify two essential elements of the prt2 promoter: HomolD box and TATA box, mutations of which inactivate the prt2 promoter and derepress the downstream pho84 promoter under phosphate-replete conditions...
February 2, 2018: Journal of Biological Chemistry
Paweł Satora, Dorota Semik-Szczurak, Tomasz Tarko, Andrzej Bułdys
Currently in apple winemaking, pure cultures of Saccharomyces cerevisiae and S. bayanus strains are mainly used. The aim of this study was to determine the influence of Saccharomyces cerevisiae (Johannisberg Riesling - LOCK 105), S. bayanus (DSMZ 3774), S. paradoxus (CBS 7302), and Schizosaccharomyces pombe (DSMZ 70576) applied in pure and mixed cultures on the chemical composition and sensory profile of apple wines. Pasteurized Gloster apple musts with addition of sucrose (up to 22°Blg) were inoculated with specific volume (0...
January 25, 2018: Journal of Food Science
Matthew T Swulius, Lam T Nguyen, Mark S Ladinsky, Davi R Ortega, Samya Aich, Mithilesh Mishra, Grant J Jensen
Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that "saddles" the inward-bending membrane at the leading edge of the division septum...
January 18, 2018: Proceedings of the National Academy of Sciences of the United States of America
Yan Sha, Vladimir Vartanian, Nichole Owen, Stephanie J Mengden Koon, Marcus J Calkins, Courtney S Thompson, Zahra Mirafzali, Sara Mir, Lisa E Goldsmith, Huaping He, Chun Luo, Scott M Brown, Paul W Doetsch, Andy Kaempf, Jeong Y Lim, Amanda K McCullough, R Stephen Lloyd
The molecular basis for ultraviolet (UV) light-induced nonmelanoma and melanoma skin cancers centers on cumulative genomic instability caused by inefficient DNA repair of dipyrimidine photoproducts. Inefficient DNA repair and subsequent translesion replication past these DNA lesions generate distinct molecular signatures of tandem CC to TT and C to T transitions at dipyrimidine sites. Since previous efforts to develop experimental strategies to enhance the repair capacity of basal keratinocytes have been limited, we have engineered the N-terminally truncated form (Δ228) UV endonuclease (UVDE) from Schizosaccharomyces pombe to include a TAT cell-penetrating peptide sequence with or without a nuclear localization signal (NLS): UVDE-TAT and UVDE-NLS-TAT...
January 15, 2018: Scientific Reports
Hana Raschmanova, Astrid Weninger, Anton Glieder, Karin Kovar, Thomas Vogl
Within five years, the CRISPR-Cas system has emerged as the dominating tool for genome engineering, while also changing the speed and efficiency of metabolic engineering in conventional (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and non-conventional (Yarrowia lipolytica, Pichia pastoris syn. Komagataella phaffii, Kluyveromyces lactis, Candida albicans and C. glabrata) yeasts. Especially in S. cerevisiae, an extensive toolbox of advanced CRISPR-related applications has been established, including crisprTFs and gene drives...
January 10, 2018: Biotechnology Advances
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