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Fission yeast

Tiphanie Cavé, Marie-Chantal Grégoire, Marc-André Brazeau, Guylain Boissonneault
In mammals, spermiogenesis is characterized by transient formation of DNA double-strand breaks (DSBs) in the whole population of haploid spermatids. DSB repair in such haploid context may represent a mutational transition. Using a combination of pulsed-field gel electrophoresis and specific labelling of DSBs at 3'OH DNA ends, we showed that post-meiotic, enzyme-induced DSBs are also observed in the synchronizable pat1-114 mutant of Shizosaccharomyces pombe as well as in a wild-type strain, while DNA repair is observed at later stages...
February 20, 2018: International Journal of Biochemistry & Cell Biology
Anthony Perrot, Christopher Lee Millington, Blanca Gómez-Escoda, Diane Schausi-Tiffoche, Pei-Yun Jenny Wu
In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection...
February 2018: PLoS Genetics
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
S Richard, J M G C F Almeida, O H Cissé, A Luraschi, O Nielsen, M Pagni, P M Hauser
Fungi of the genus Pneumocystis are obligate parasites that colonize mammals' lungs and are host species specific. Pneumocystis jirovecii and Pneumocystis carinii infect, respectively, humans and rats. They can turn into opportunistic pathogens in immunosuppressed hosts, causing severe pneumonia. Their cell cycle is poorly known, mainly because of the absence of an established method of culture in vitro It is thought to include both asexual and sexual phases. Comparative genomic analysis suggested that their mode of sexual reproduction is primary homothallism involving a single mating type ( MAT ) locus encompassing plus and minus genes ( matMc , matMi , and matPi ; Almeida et al...
February 20, 2018: MBio
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
Laura Merlini, Bita Khalili, Omaya Dudin, Laetitia Michon, Vincent Vincenzetti, Sophie G Martin
In the fission yeast Schizosaccharomyces pombe , pheromone signaling engages a signaling pathway composed of a G protein-coupled receptor, Ras, and a mitogen-activated protein kinase (MAPK) cascade that triggers sexual differentiation and gamete fusion. Cell-cell fusion requires local cell wall digestion, which relies on an initially dynamic actin fusion focus that becomes stabilized upon local enrichment of the signaling cascade on the structure. We constructed a live-reporter of active Ras1 (Ras1-guanosine triphosphate [GTP]) that shows Ras activity at polarity sites peaking on the fusion structure before fusion...
February 16, 2018: Journal of Cell Biology
Kyoung Hwan Lee, Guidenn Sulbarán, Shixin Yang, Ji Young Mun, Lorenzo Alamo, Antonio Pinto, Osamu Sato, Mitsuo Ikebe, Xiong Liu, Edward D Korn, Floyd Sarsoza, Sanford I Bernstein, Raúl Padrón, Roger Craig
Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca 2+ binding and regulatory light-chain phosphorylation...
February 14, 2018: Proceedings of the National Academy of Sciences of the United States of America
Alba Timón-Gómez, David Sanfeliu-Redondo, Amparo Pascual-Ahuir, Markus Proft
Repair and removal of damaged mitochondria is a key process for eukaryotic cell homeostasis. Here we investigate in the yeast model how different protein complexes of the mitochondrial electron transport chain are subject to specific degradation upon high respiration load and organelle damage. We find that the turnover of subunits of the electron transport complex I equivalent and complex III is preferentially stimulated upon high respiration rates. Particular mitochondrial proteases, but not mitophagy, are involved in this activated degradation...
2018: Frontiers in Microbiology
Hemanth Noothalapati, Ryo Ikarashi, Keita Iwasaki, Tatsuro Nishida, Tomohiro Kaino, Keisuke Yoshikiyo, Keiji Terao, Daisuke Nakata, Naoko Ikuta, Masahiro Ando, Hiro-O Hamaguchi, Makoto Kawamukai, Tatsuyuki Yamamoto
α-lipoic acid (ALA) is an essential cofactor for many enzyme complexes in aerobic metabolism, especially in mitochondria of eukaryotic cells where respiration takes place. It also has excellent anti-oxidative properties. The acid has two stereo-isomers, R- and S- lipoic acid (R-LA and S-LA), but only the R-LA has biological significance and is exclusively produced in our body. A mutant strain of fission yeast, Δdps1, cannot synthesize coenzyme Q10, which is essential during yeast respiration, leading to oxidative stress...
February 6, 2018: Spectrochimica Acta. Part A, Molecular and Biomolecular Spectroscopy
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
Yuichi Shichino, Yoko Otsubo, Yoshitaka Kimori, Masayuki Yamamoto, Akira Yamashita
Accurate and extensive regulation of meiotic gene expression is crucial to distinguish germ cells from somatic cells. In the fission yeast Schizosaccharomyces pombe, a YTH family RNA-binding protein, Mmi1, directs the nuclear exosome-mediated elimination of meiotic transcripts during vegetative proliferation. Mmi1 also induces the formation of facultative heterochromatin at a subset of its target genes. Here, we show that Mmi1 prevents the mistimed expression of meiotic proteins by tethering their mRNAs to the nuclear foci...
February 9, 2018: ELife
Wilber Escorcia, Susan L Forsburg
Random spore analysis (RSA) is a tool that allows for the screening of a large number of meiotic products. It requires only a limited effort, and is often the method of choice for constructing strains with unambiguous genotypes. It is also useful to identify the frequency of rare events. Strains are crossed on a nitrogen-limiting medium for three days. Mated cells are observed under the microscope to check for the presence of ripe asci. To release spores from their ascus, a sample of the cross is taken from the mating plate and resuspended in an enzyme solution overnight at 25-29 °C...
2018: Methods in Molecular Biology
Wilber Escorcia, Susan L Forsburg
Tetrad dissection is a powerful tool in yeast genetics that allows the analysis of products of a single meiosis. With just a few tetrads, it is possible to determine linkage, identify unique phenotypes associated with double mutants, or assess specific meiotic defects. Strains are crossed on nitrogen-limiting medium for 3 days. With the help of a micromanipulator, ripe asci are isolated to spots 5 mm apart on a YES plate. Incubation at 36 °C for about 3-5 h is necessary for the ascus walls to break down...
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
Akihisa Matsuyama, Atsuko Shirai, Minoru Yoshida
Immunoprecipitation is one of the most important and widely used techniques for the detection and purification of a protein of interest. Thanks to highly specific interaction between antigen and antibody, a target protein is purified and concentrated effectively. To obtain reasonable amounts of a target protein, it is crucially important to prepare total cell lysates in which the target protein is present in a soluble form. Here, we describe methods to prepare total cell lysates of fission yeast, which are then used directly for immunoprecipitation...
2018: Methods in Molecular Biology
Qianhua Dong, Fei Li
Proteins act as executors for almost all kinds of cellular processes. The majority of proteins achieve their proper functions through interacting with other proteins. Knowing the binding partners of a protein is instrumental for understanding its function. The antibody pull-down method is a powerful and common approach to detect protein-protein interactions. Here, an antibody pull-down protocol is described for detecting protein-protein interactions in fission yeast Schizosaccharomyces pombe.
2018: Methods in Molecular Biology
Haruhiko Asakawa, Yasushi Hiraoka, Tokuko Haraguchi
Cellular structures and biomolecular complexes are not simply assemblies of proteins, but are organized with defined numbers of protein molecules in precise locations. Thus, evaluating the spatial localization and numbers of protein molecules is of fundamental importance in understanding cellular structures and functions. The amounts of proteins of interest have conventionally been determined by biochemical methods. However, biochemical measurements based on the population average have limitations: it is sometimes difficult to determine the amounts of insoluble proteins or low expression proteins localized in small portions of the cell...
2018: Methods in Molecular Biology
Pranjal Biswas, Uddalak Majumdar, Sanjay Ghosh
The patterns of gene expression in the fission yeast Schizosaccharomyces pombe under various experimental conditions form the basis of any transcriptomic study. We describe a method involving reverse transcription of the mRNA, Polymerase Chain Reaction (PCR), and the subsequent separation of the products onto Urea-Polyacrylamide gel that can be used to study the gene expression patterns in the fission yeast. The method described is cost effective and reproducible with satisfactory resolution of expressed transcripts in the gel...
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
Ge Li, Richard Y Zhao
Fission yeast is a single-cell eukaryote that has been used extensively as a model organism to study cell biology and virology of higher eukaryotes including plants and humans. In particular, it is a very well-tested model to study evolutionary highly conserved cellular activities such as cell proliferation, cell cycle regulation, and cell death. Some of the advantages of using fission yeast as a surrogate system: easy to carry out functional and genome-wide analysis of small viral genome, easy to maintain in the laboratory with a relatively short doubling time...
2018: Methods in Molecular Biology
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