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1.
Genetics ; 225(3)2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37758508

RESUMEN

Standardized nomenclature for genes, gene products, and isoforms is crucial to prevent ambiguity and enable clear communication of scientific data, facilitating efficient biocuration and data sharing. Standardized genotype nomenclature, which describes alleles present in a specific strain that differ from those in the wild-type reference strain, is equally essential to maximize research impact and ensure that results linking genotypes to phenotypes are Findable, Accessible, Interoperable, and Reusable (FAIR). In this publication, we extend the fission yeast clade gene nomenclature guidelines to support the curation efforts at PomBase (www.pombase.org), the Schizosaccharomyces pombe Model Organism Database. This update introduces nomenclature guidelines for noncoding RNA genes, following those set forth by the Human Genome Organisation Gene Nomenclature Committee. Additionally, we provide a significant update to the allele and genotype nomenclature guidelines originally published in 1987, to standardize the diverse range of genetic modifications enabled by the fission yeast genetic toolbox. These updated guidelines reflect a community consensus between numerous fission yeast researchers. Adoption of these rules will improve consistency in gene and genotype nomenclature, and facilitate machine-readability and automated entity recognition of fission yeast genes and alleles in publications or datasets. In conclusion, our updated guidelines provide a valuable resource for the fission yeast research community, promoting consistency, clarity, and FAIRness in genetic data sharing and interpretation.


Asunto(s)
Schizosaccharomyces , Humanos , Schizosaccharomyces/genética , Alelos , Comprensión , Bases de Datos Genéticas , Fenotipo
3.
Methods Mol Biol ; 2579: 169-179, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36045206

RESUMEN

The fission yeast, Schizosaccharomyces pombe, is a genetically tractable model organism for cell cycle and molecular genetics research. We describe methods to synchronize S. pombe cultures, and the benefits and limitations of each. Drug-induced synchrony is a convenient method to arrest the cell cycle. An example of the drug hydroxyurea is shown, which arrests cells in S-phase. Environmental modulation through media composition or growth conditions may also be used to synchronize cultures, most commonly with nitrogen depletion to arrest in G1-phase. Finally, examples of temperature-sensitive conditional alleles are shown which arrest the cell cycle at key transition points. Each of these methods must be assessed relative to the desired effect and the process being studied, providing the best synchrony with the fewest off-target effects.


Asunto(s)
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , División Celular , Micología/métodos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
4.
Methods Mol Biol ; 2579: 235-246, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36045211

RESUMEN

We have previously described methods to synchronize cultures of fission yeast, Schizosaccharomyces pombe. In this chapter, we provide methods to detect cell cycle stage in cells and populations of S. pombe. These protocols used fixed samples. First, we describe sample preparation for flow cytometry of bulk DNA content. This technique allows users to monitor progression of DNA replication and detect any perturbation during the synthesis (S) phase of the cell cycle. Second, we describe methods to stain nuclei and septa of fixed yeast cells, and monitor proportions of cell cycle stages within cultures. Together, these methods provide the ability to compare cell cycle progression or delay between cultures, making use of the powerful molecular genetics tool that is S. pombe.


Asunto(s)
Schizosaccharomyces , Ciclo Celular , División Celular , Replicación del ADN , Fase S , Schizosaccharomyces/metabolismo
5.
Sci Rep ; 11(1): 23315, 2021 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-34857794

RESUMEN

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.


Asunto(s)
Antivirales/farmacología , Coronavirus/efectos de los fármacos , Coronavirus/metabolismo , Desarrollo de Medicamentos/métodos , Reposicionamiento de Medicamentos/métodos , Benzamidas/farmacología , Línea Celular , Simulación por Computador , Coronavirus/química , Bases de Datos Farmacéuticas , Descubrimiento de Drogas/métodos , Interacciones Huésped-Patógeno , Humanos , Imidazoles/farmacología , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , SARS-CoV-2/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Triazinas/farmacología , Tratamiento Farmacológico de COVID-19
6.
Cannabis Cannabinoid Res ; 5(1): 105-116, 2020 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-32322682

RESUMEN

Introduction: Understanding the inheritance of cannabinoid compounds in Cannabis sativa will facilitate effective crop breeding and careful regulation of controlled substances. The production of two key cannabinoids, Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), is partially controlled by two additive loci. Here, we present the first study to search for evidence of alternate genetic models describing the inheritance and expression of cannabinoids. Materials and Methods: Using an information-theoretic approach, we estimated composite genetic effects (CGEs) of four cultivars with pure CBD or pure THC chemotypes, their F1 and F2 hybrid progeny, to identify genetic models that explain cannabinoid inheritance patterns. We also estimated the effective number of genetic factors that control differences in cannabinoid concentration (THC, CBD, and cannabichromene [CBC]). Results: Unlike previous research, we note nonadditive components of cannabinoid inheritance. Concentration of THC is a polygenic trait (three to four genetic factors). Both additive and dominance CGEs best explained THC expression patterns. In contrast, cytoplasmic genomes and additive genes may influence CBD concentration. Maternal additive effects and additive genetic effects apparently influence CBC expression. Conclusions: Cannabinoid inheritance is more complex than previously appreciated; among other genetic effects, cytogenetic and maternal contributions may be undervalued influences on cannabinoid ratios and concentrations. Further research on the environmental sensitivity of cannabinoid production is advised.

7.
G3 (Bethesda) ; 8(6): 1993-2006, 2018 05 31.
Artículo en Inglés | MEDLINE | ID: mdl-29661843

RESUMEN

The Hif1 protein in the yeast Saccharomyces cerevisie is an evolutionarily conserved H3/H4-specific chaperone and a subunit of the nuclear Hat1 complex that catalyzes the acetylation of newly synthesized histone H4. Hif1, as well as its human homolog NASP, has been implicated in an array of chromatin-related processes including histone H3/H4 transport, chromatin assembly and DNA repair. In this study, we elucidate the functional aspects of Hif1 Initially we establish the wide distribution of Hif1 homologs with an evolutionarily conserved pattern of four tetratricopeptide repeats (TPR) motifs throughout the major fungal lineages and beyond. Subsequently, through targeted mutational analysis, we demonstrate that the acidic region that interrupts the TPR2 is essential for Hif1 physical interactions with the Hat1/Hat2-complex, Asf1, and with histones H3/H4. Furthermore, we provide evidence for the involvement of Hif1 in regulation of histone metabolism by showing that cells lacking HIF1 are both sensitive to histone H3 over expression, as well as synthetic lethal with a deletion of histone mRNA regulator LSM1 We also show that a basic patch present at the extreme C-terminus of Hif1 is essential for its proper nuclear localization. Finally, we describe a physical interaction with a transcriptional regulatory protein Spt2, possibly linking Hif1 and the Hat1 complex to transcription-associated chromatin reassembly. Taken together, our results provide novel mechanistic insights into Hif1 functions and establish it as an important protein in chromatin-associated processes.


Asunto(s)
Chaperonas de Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Secuencia Conservada , Técnicas de Inactivación de Genes , Chaperonas de Histonas/química , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Mutación/genética , Filogenia , Unión Proteica , Dominios Proteicos , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/química
8.
Methods Mol Biol ; 1672: 509-526, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29043645

RESUMEN

Chromatin fibers, first described by Jackson and Pombo (J Cell Biol 140(6):1285-1295, 1998) are prepared from cells lysed on glass coverslips, and require minimal equipment to produce. Since the DNA is not previously treated with denaturing agents, proteins are left intact and may be used to model other DNA-based processes. Such an analysis can be daunting, without a rigorous method for analysis. We describe a pipeline for chromatin fiber use to model DNA replication complexes. Full protocols for chromatin fiber preparation and staining are presented. Further, we have developed an analysis algorithm for One Dimensional Data-Boolean Logic Operations Binning System (ODD-BLOBS). This freely available software defines replication and protein tracts, measures their lengths, and then correlates replicated areas with protein distributions. Our methods and analysis are tested in Schizosaccharomyces pombe (fission yeast) but may be applied to model replication structures across multiple organisms.


Asunto(s)
Cromatina/genética , Replicación del ADN , ADN de Hongos/química , ADN de Hongos/genética , Conformación de Ácido Nucleico , Schizosaccharomyces/genética , Técnica del Anticuerpo Fluorescente , Microscopía Fluorescente
9.
Biomolecules ; 5(3): 2123-39, 2015 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-26393661

RESUMEN

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.


Asunto(s)
Replicación del ADN , ADN de Hongos , ADN de Cadena Simple , Schizosaccharomyces/genética , Daño del ADN , Humanos
10.
Mol Biol Cell ; 26(19): 3439-50, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26246602

RESUMEN

DNA replication stress causes genome mutations, rearrangements, and chromosome missegregation, which are implicated in cancer. We analyze a fission yeast mutant that is unable to complete S phase due to a defective subunit of the MCM helicase. Despite underreplicated and damaged DNA, these cells evade the G2 damage checkpoint to form ultrafine bridges, fragmented centromeres, and uneven chromosome segregations that resembles micronuclei. These micronuclei retain DNA damage markers and frequently rejoin with the parent nucleus. Surviving cells show an increased rate of mutation and chromosome rearrangement. This first report of micronucleus-like segregation in a yeast replication mutant establishes underreplication as an important factor contributing to checkpoint escape, abnormal chromosome segregation, and chromosome instability.


Asunto(s)
Replicación del ADN/fisiología , Componente 4 del Complejo de Mantenimiento de Minicromosoma/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/citología , Schizosaccharomyces/genética , Alelos , Centrómero/metabolismo , Segregación Cromosómica , Reordenamiento Génico , Micronúcleos con Defecto Cromosómico , Pruebas de Micronúcleos , Fase S/fisiología , Schizosaccharomyces/metabolismo , Estrés Fisiológico/genética
11.
Methods Mol Biol ; 1300: 13-41, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25916703

RESUMEN

Temporal and spatial visualization of replication proteins and associated structures within the narrow confines of a yeast nucleus is technically challenging. Choosing the appropriate method depends upon the parameters of the experiment, the nature of the molecules to be observed, and the hypothesis to be tested. In this chapter, we review three broad types of visualization: whole-cell fluorescence or immunofluorescence, which is useful for questions of timing and chromatin association; nuclear spreads, which provide greater resolution within the chromatin for co-localization and region-specific effects; and chromatin fibers, which allow observation of labeled proteins and newly synthesized DNA on a linear chromosome. We also suggest a mounting procedure for live fission yeast with fluorescent proteins. We discuss applications of these protocols and some considerations for choosing methods and fluorophores.


Asunto(s)
Replicación del ADN , Microscopía/métodos , Schizosaccharomyces/metabolismo , Bromodesoxiuridina/metabolismo , Cromosomas Fúngicos/metabolismo , ADN de Hongos , Técnica del Anticuerpo Fluorescente , Imagenología Tridimensional
12.
Methods Mol Biol ; 1300: 79-97, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25916706

RESUMEN

Flow cytometry is an essential tool to monitor DNA content and determine cell cycle distribution. Its utility in fission yeast reflects the ease of sample preparation, the stochiometric binding of the most popular DNA dyes (propidium iodide and Sytox Green), and ability to monitor cell size. However, the study of DNA replication with multicolour flow analysis has lagged behind its use in mammalian cells. We present basic and advanced protocols for analysis of DNA replication in fission yeast by flow cytometry including whole cell, nuclear "ghosts," two-color imaging with BrdU, and estimates of DNA synthesis using EdU.


Asunto(s)
ADN de Hongos/metabolismo , Citometría de Flujo/métodos , Schizosaccharomyces/metabolismo , Bromodesoxiuridina/metabolismo , Núcleo Celular/metabolismo , Fase S , Coloración y Etiquetado
13.
PLoS One ; 8(12): e81434, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24353754

RESUMEN

Schizosaccharomyces pombe shares many genes and proteins with humans and is a good model for chromosome behavior and DNA dynamics, which can be analyzed by visualizing the behavior of fluorescently tagged proteins in vivo. Performing a genome-wide screen for changes in such proteins requires developing methods that automate analysis of a large amount of images, the first step of which requires robust segmentation of the cell. We developed a segmentation system, PombeX, that can segment cells from transmitted illumination images with focus gradient and varying contrast. Corrections for focus gradient are applied to the image to aid in accurate detection of cell membrane and cytoplasm pixels, which is used to generate initial contours for cells. Gradient vector flow snake evolution is used to obtain the final cell contours. Finally, a machine learning-based validation of cell contours removes most incorrect or spurious contours. Quantitative evaluations show overall good segmentation performance on a large set of images, regardless of differences in image quality, lighting condition, focus condition and phenotypic profile. Comparisons with recent related methods for yeast cells show that PombeX outperforms current methods, both in terms of segmentation accuracy and computational speed.


Asunto(s)
Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Schizosaccharomyces/citología , Programas Informáticos , Inteligencia Artificial , Compartimento Celular , Fluorescencia
14.
Genetics ; 193(1): 143-57, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23150603

RESUMEN

Nucleoside analogs are frequently used to label newly synthesized DNA. These analogs are toxic in many cells, with the exception of the budding yeast. We show that Schizosaccharomyces pombe behaves similarly to metazoans in response to analogs 5-bromo-2'-deoxyuridine (BrdU) and 5-ethynyl-2'-deoxyuridine (EdU). Incorporation causes DNA damage that activates the damage checkpoint kinase Chk1 and sensitizes cells to UV light and other DNA-damaging drugs. Replication checkpoint mutant cds1Δ shows increased DNA damage response after exposure. Finally, we demonstrate that the response to BrdU is influenced by the ribonucleotide reductase inhibitor, Spd1, suggesting that BrdU causes dNTP pool imbalance in fission yeast, as in metazoans. Consistent with this, we show that excess thymidine induces G1 arrest in wild-type fission yeast expressing thymidine kinase. Thus, fission yeast responds to nucleoside analogs similarly to mammalian cells, which has implications for their use in replication and damage research, as well as for dNTP metabolism.


Asunto(s)
Daño del ADN/efectos de los fármacos , Nucleósidos/toxicidad , Schizosaccharomyces/efectos de los fármacos , Schizosaccharomyces/genética , Bromodesoxiuridina/toxicidad , Replicación del ADN , Mutagénesis/efectos de los fármacos , Mutación/efectos de los fármacos , Tasa de Mutación , Fase S/efectos de los fármacos , Schizosaccharomyces/metabolismo , Transducción de Señal/efectos de los fármacos
15.
Mol Cell Biol ; 32(24): 4986-97, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-23045396

RESUMEN

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork "collapse point" in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.


Asunto(s)
ADN de Hongos/biosíntesis , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa de Punto de Control 2 , Daño del ADN , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Fúngicos , Hidroxiurea/farmacología , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Modelos Biológicos , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Schizosaccharomyces/efectos de los fármacos , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
16.
Methods Enzymol ; 470: 759-95, 2010.
Artículo en Inglés | MEDLINE | ID: mdl-20946835

RESUMEN

In this chapter we present basic protocols for the use of Schizosaccharomyces pombe, commonly known as fission yeast, in molecular biology and genetics research. Fission yeast is an increasingly popular model organism for the study of biological pathways because of its genetic tractability and as a model for metazoan biology. It provides an alternative and complimentary approach to Saccharomyces cerevisiae for addressing questions of cell biology, physiology, genetics, and genomics/proteomics. We include details and considerations for growing fission yeast, information on crosses and genetics, gene targeting and transformation, cell synchrony and analysis, and molecular biology protocols.


Asunto(s)
Biología Molecular/métodos , Schizosaccharomyces/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Schizosaccharomyces/citología
17.
Methods Mol Biol ; 521: 449-61, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19563122

RESUMEN

Flow cytometry is an essential tool to monitor DNA content and determine cell cycle distribution. Its utility reflects the relative ease of sample preparation and the stochiometric nature of the most popular DNA-binding dyes (propidium iodide and Sytox Green). Mammalian precedents using flow cytometry for replication and cell biology studies are attractive examples for S. pombe researchers. However, the study of DNA replication with multicolor analysis has lagged behind that in mammalian cells. We present basic and advanced protocols for analysis of DNA replication in fission yeast by flow cytometry including whole cell, nuclear "ghosts," and two-color imaging with BrdU.


Asunto(s)
ADN de Hongos/análisis , Citometría de Flujo/métodos , Schizosaccharomyces/química , Bromodesoxiuridina/metabolismo , Ciclo Celular , Replicación del ADN , ADN de Hongos/biosíntesis , ADN Mitocondrial/análisis , ADN Mitocondrial/biosíntesis , Micología/métodos , Fase S , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo
18.
Methods Mol Biol ; 521: 463-82, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19563123

RESUMEN

Temporal and spatial visualization of replication proteins and associated structures within the narrow confines of a yeast nucleus is technically challenging. Choosing the appropriate method depends upon the parameters of the experiment, the nature of the molecules to be observed, and the hypothesis to be tested. In this chapter, we review three broad types of visualization: whole cell fluorescence or immunofluorescence, which is useful for questions of timing and chromatin association; nuclear spreads, which provide greater resolution within the chromatin for colocalization and region-specific effects; and chromatin fibers, which allow observation of labeled proteins and newly synthesized DNA on a linear chromosome. We discuss applications of these protocols and some considerations for choosing methods and fluorophores.


Asunto(s)
Replicación del ADN , ADN de Hongos/biosíntesis , Microscopía Fluorescente/métodos , Schizosaccharomyces/metabolismo , Bromodesoxiuridina/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Cromosomas Fúngicos/metabolismo , Colorantes Fluorescentes , Micología/métodos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Schizosaccharomyces/citología , Proteínas de Schizosaccharomyces pombe/metabolismo
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