RESUMEN
Yeast two-hybrid (YTH) technology is a powerful tool for studying protein interactions and has been widely used in various fields of molecular biology, including the study of antiviral innate immunity. This chapter presents detailed information and experimental procedures for identifying virus-host protein interactions involved in immune regulation using yeast two-hybrid technology.
Asunto(s)
Interacciones Huésped-Patógeno , Inmunidad Innata , Técnicas del Sistema de Dos Híbridos , Humanos , Interacciones Huésped-Patógeno/inmunología , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Saccharomyces cerevisiae/inmunología , Saccharomyces cerevisiae/genética , Unión Proteica , Mapeo de Interacción de Proteínas/métodosRESUMEN
This chapter describes the computational pipeline for the processing and visualization of Protec-Seq data, a method for purification and genome-wide mapping of double-stranded DNA protected by a specific protein at both ends. In the published case, the protein of choice was Saccharomyces cerevisiae Spo11, a conserved topoisomerase-like enzyme that makes meiotic double-strand breaks (DSBs) to initiate homologous recombination, ensuring proper segregation of homologous chromosomes and fertility. The isolated DNA molecules were thus termed double DSB (dDSB) fragments and were found to represent 34 to several hundred base-pair long segments that are generated by Spo11 and are enriched at DSB hotspots, which are sites of topological stress. In order to allow quantitative comparisons between dDSB profiles across experiments, we implemented calibrated chromatin immunoprecipitation sequencing (ChIP-Seq) using the meiosis-competent yeast species Saccharomyces kudriavzevii as calibration strain. Here, we provide a detailed description of the computational methods for processing, analyzing, and visualizing Protec-Seq data, comprising the download of the raw data, the calibrated genome-wide alignments, and the scripted creation of either arc plots or Hi-C-style heatmaps for the illustration of chromosomal regions of interest. The workflow is based on Linux shell scripts (including wrappers for publicly available, open-source software) as well as R scripts and is highly customizable through its modular structure.
Asunto(s)
Roturas del ADN de Doble Cadena , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Programas Informáticos , Meiosis/genética , Genoma Fúngico , Mapeo Cromosómico/métodos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Biología Computacional/métodos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismoRESUMEN
Epigenetic inheritance of heterochromatin requires transfer of parental H3-H4 tetramers to both daughter duplexes during replication. Three recent papers exploit yeast genetics coupled to inheritance assays and AlphaFold2-multimer predictions coupled to biochemistry to reveal that a replisome component (Mrc1/CLASPIN) is an H3-H4 tetramer chaperone important for parental histone transfer to daughters.
Asunto(s)
Replicación del ADN , Aprendizaje Profundo , Histonas , Saccharomyces cerevisiae , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Heterocromatina/metabolismo , Epigénesis GenéticaRESUMEN
Environmental conditions significantly impact the metabolism of Saccharomyces cerevisiae, a Crabtree-positive yeast that maintains a fermentative metabolism in high-sugar environments even in the presence of oxygen. Although the introduction of oxygen has been reported to induce alterations in yeast metabolism, knowledge of the mechanisms behind these metabolic adaptations in relation to redox cofactor metabolism and their implications in the context of wine fermentation remains limited. This study aimed to compare the intracellular redox cofactor levels, the cofactor ratios, and primary metabolite production in S. cerevisiae under aerobic and anaerobic conditions in synthetic grape juice. The molecular mechanisms underlying these metabolic differences were explored using a transcriptomic approach. Aerobic conditions resulted in an enhanced fermentation rate and biomass yield. Total NADP(H) levels were threefold higher during aerobiosis, while a decline in the total levels of NAD(H) was observed. However, there were stark differences in the ratio of NAD+/NADH between the treatments. Despite few changes in the differential expression of genes involved in redox cofactor metabolism, anaerobiosis resulted in an increased expression of genes involved in lipid biosynthesis pathways, while the presence of oxygen increased the expression of genes associated with thiamine, methionine, and sulfur metabolism. The production of fermentation by-products was linked with differences in the redox metabolism in each treatment. This study provides valuable insights that may help steer the production of metabolites of industrial interest during alcoholic fermentation (including winemaking) by using oxygen as a lever of redox metabolism.
Asunto(s)
Fermentación , Oxidación-Reducción , Oxígeno , Saccharomyces cerevisiae , Vino , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Oxígeno/metabolismo , Vino/microbiología , Vino/análisis , Anaerobiosis , Vitis/microbiología , Vitis/metabolismo , NAD/metabolismo , Etanol/metabolismo , NADP/metabolismo , Aerobiosis , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Coenzimas/metabolismoRESUMEN
Considerable effort is required to build mathematical models of large protein regulatory networks. Utilizing computational algorithms that guide model development can significantly streamline the process and enhance the reliability of the resulting models. In this article, we present a perturbation approach for developing data-centric Boolean models of cell cycle regulation. To evaluate networks, we assign a score based on their steady states and the dynamical trajectories corresponding to the initial conditions. Then, perturbation analysis is used to find new networks with lower scores, in which dynamical trajectories traverse through the correct cell cycle path with high frequency. We apply this method to refine Boolean models of cell cycle regulation in budding yeast and mammalian cells.
Asunto(s)
Algoritmos , Ciclo Celular , Modelos Biológicos , Humanos , Animales , Redes Reguladoras de Genes , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomycetales/metabolismoRESUMEN
BACKGROUND: Metabolic engineering enables the sustainable and cost-efficient production of complex chemicals. Efficient production of terpenes in Saccharomyces cerevisiae can be achieved by recruiting an intermediate of the mevalonate pathway. The present study aimed to evaluate the engineering strategies of S. cerevisiae for the production of taxadiene, a precursor of taxol, an antineoplastic drug. RESULT: SCIGS22a, a previously engineered strain with modifications in the mevalonate pathway (MVA), was used as a background strain. This strain was engineered to enable a high flux towards farnesyl diphosphate (FPP) and the availability of NADPH. The strain MVA was generated from SCIGS22a by overexpressing all mevalonate pathway genes. Combining the background strains with 16 different episomal plasmids, which included the combination of 4 genes: tHMGR (3-hydroxy-3-methylglutaryl-CoA reductase), ERG20 (farnesyl pyrophosphate synthase), GGPPS (geranyl diphosphate synthase) and TS (taxadiene synthase) resulted in the highest taxadiene production in S. cerevisiae of 528 mg/L. CONCLUSION: Our study highlights the critical role of pathway balance in metabolic engineering, mainly when dealing with toxic molecules like taxadiene. We achieved significant improvements in taxadiene production by employing a combinatorial approach and focusing on balancing the downstream and upstream pathways. These findings emphasize the importance of minor gene expression modification levels to achieve a well-balanced pathway, ultimately leading to enhanced taxadiene accumulation.
Asunto(s)
Ingeniería Metabólica , Ácido Mevalónico , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ingeniería Metabólica/métodos , Ácido Mevalónico/metabolismo , Alquenos/metabolismo , Fosfatos de Poliisoprenilo/metabolismo , Diterpenos/metabolismo , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , NADP/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , SesquiterpenosRESUMEN
Microbial secretions, such as metabolic enzymes, are often considered to be cooperative public goods as they are costly to produce but can be exploited by others. They create incentives for the evolution of non-producers, which can drive producer and population productivity declines. In response, producers can adjust production levels. Past studies suggest that while producers lower production to reduce costs and exploitation opportunities when under strong selection pressure from non-producers, they overproduce secretions when these pressures are weak. We challenge the universality of this trend with the production of a metabolic enzyme, invertase, by Saccharomyces cerevisiae, which catalyses sucrose hydrolysis into two hexose molecules. Contrary to past studies, overproducers evolve during evolutionary experiments even when under strong selection pressure from non-producers. Phenotypic and competition assays with a collection of synthetic strains - engineered to have modified metabolic attributes - identify two mechanisms for suppressing the benefits of invertase to those who exploit it. Invertase overproduction increases extracellular hexose concentrations that suppresses the metabolic efficiency of competitors, due to the rate-efficiency trade-off, and also enhances overproducers' hexose capture rate by inducing transporter expression. Thus, overproducers are maintained in the environment originally thought to not support public goods production.
Asunto(s)
Saccharomyces cerevisiae , beta-Fructofuranosidasa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Fructofuranosidasa/metabolismo , beta-Fructofuranosidasa/genética , Sacarosa/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Evolución Biológica , Hexosas/metabolismo , Regulación hacia Arriba , Regulación Fúngica de la Expresión GénicaRESUMEN
In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA. Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA ( >10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability.
Asunto(s)
ADN Mitocondrial , Inestabilidad Genómica , Retroelementos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Retroelementos/genética , Núcleo Celular/metabolismo , Núcleo Celular/genética , Reparación del ADN por Unión de Extremidades , Roturas del ADN de Doble Cadena , Meiosis/genética , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genéticaRESUMEN
Chromatin remodelling complexes (CRC) are ATP-dependent molecular machines important for the dynamic organization of nucleosomes along eukaryotic DNA. CRCs SWI/SNF, RSC and INO80 can move positioned nucleosomes in promoter DNA, leading to nucleosome-depleted regions which facilitate access of general transcription factors. This function is strongly supported by transcriptional activators being able to interact with subunits of various CRCs. In this work we show that SWI/SNF subunits Swi1, Swi2, Snf5 and Snf6 can bind to activation domains of Ino2 required for expression of phospholipid biosynthetic genes in yeast. We identify an activator binding domain (ABD) of ATPase Swi2 and show that this ABD is functionally dispensable, presumably because ABDs of other SWI/SNF subunits can compensate for the loss. In contrast, mutational characterization of the ABD of the Swi2-related ATPase Sth1 revealed that some conserved basic and hydrophobic amino acids within this domain are essential for the function of Sth1. While ABDs of Swi2 and Sth1 define separate functional protein domains, mapping of an ABD within ATPase Ino80 showed co-localization with its HSA domain also required for binding actin-related proteins. Comparative interaction studies finally demonstrated that several unrelated activators each exhibit a specific binding pattern with ABDs of Swi2, Sth1 and Ino80.
Asunto(s)
Adenosina Trifosfatasas , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN , Unión Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Factores de Transcripción , Activación Transcripcional , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación Fúngica de la Expresión Génica , Dominios Proteicos , Proteínas Nucleares , Proteínas de Ciclo Celular , Factores de Transcripción con Motivo Hélice-Asa-Hélice BásicoRESUMEN
BACKGROUND: Novel protein-coding genes were considered to be born by re-organization of pre-existing genes, such as gene duplication and gene fusion. However, recent progress of genome research revealed that more protein-coding genes than expected were born de novo, that is, gene origination by accumulating mutations in non-genic DNA sequences. Nonetheless, the in-depth process (scenario) for de novo origination is not well understood. RESULTS: We have conceived bioinformatic analysis for sketching a scenario for de novo origination of protein-coding genes. For each de novo protein-coding gene, we firstly identified an edge of a given phylogenetic tree where the gene was born based on parsimony. Then, from a multiple sequence alignment of the de novo gene and its orthologous regions, we constructed ancestral DNA sequences of the gene corresponding to both end nodes of the edge. We finally revealed statistical features observed in evolution between the two ancestral sequences. In the analysis of the Saccharomyces cerevisiae lineage, we have successfully sketched a putative scenario for de novo origination of protein-coding genes. (1) In the beginning was GC-rich genome regions. (2) Neutral mutations were accumulated in the regions. (3) ORFs were extended/combined, and then (4) translation signature (Kozak consensus sequence) was recruited. Interestingly, as the scenario progresses from (2) to (4), the specificity of mutations increases. CONCLUSION: To the best of our knowledge, this is the first report outlining a scenario of de novo origination of protein-coding genes. Our bioinformatic analysis can capture events that occur during a short evolutionary time by directly observing the evolution of the ancestral sequences from non-genic to genic. This property is suitable for the analysis of fast evolving de novo genes.
Asunto(s)
Evolución Molecular , Sistemas de Lectura Abierta , Filogenia , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Biología Computacional/métodos , Mutación , Genoma FúngicoRESUMEN
Expressing plant metabolic pathways in microbial platforms is an efficient, cost-effective solution for producing many desired plant compounds. As eukaryotic organisms, yeasts are often the preferred platform. However, expression of plant enzymes in a yeast frequently leads to failure because the enzymes are poorly adapted to the foreign yeast cellular environment. Here, we first summarize the current engineering approaches for optimizing performance of plant enzymes in yeast. A critical limitation of these approaches is that they are labour-intensive and must be customized for each individual enzyme, which significantly hinders the establishment of plant pathways in cellular factories. In response to this challenge, we propose the development of a cost-effective computational pipeline to redesign plant enzymes for better adaptation to the yeast cellular milieu. This proposition is underpinned by compelling evidence that plant and yeast enzymes exhibit distinct sequence features that are generalizable across enzyme families. Consequently, we introduce a data-driven machine learning framework designed to extract 'yeastizing' rules from natural protein sequence variations, which can be broadly applied to all enzymes. Additionally, we discuss the potential to integrate the machine learning model into a full design-build-test cycle.
Asunto(s)
Ingeniería Metabólica , Ingeniería Metabólica/métodos , Plantas , Enzimas/genética , Enzimas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Aprendizaje Automático , Redes y Vías Metabólicas/genéticaRESUMEN
The fermentative model yeast Saccharomyces cerevisiae has been extensively used to study the genetic basis of stress response and homeostasis. In this study, we performed quantitative trait loci (QTL) analysis of the high-temperature fermentation trait of the progeny from the mating of the S. cerevisiae natural isolate BCC39850 (haploid#17) and the laboratory strain CEN.PK2-1C. A single QTL on chromosome X was identified, encompassing six candidate genes (GEA1, PTK2, NTA1, NPA3, IRT1, and IML1). The functions of these candidates were tested by reverse genetic experiments. Deletion mutants of PTK2, NTA1, and IML1 showed growth defects at 42 °C. The PTK2 knock-out mutant also showed significantly reduced ethanol production and plasma membrane H+ ATPase activity and increased sensitivity to acetic acid, ethanol, amphotericin B (AMB), and ß-1,3-glucanase treatment. The CRISPR-Cas9 system was used to construct knock-in mutants by replacement of PTK2, NTA1, IML1, and NPA3 genes with BCC39850 alleles. The PTK2 and NTA1 knock-in mutants showed increased growth and ethanol production titers at 42 °C. These findings suggest an important role for the PTK2 serine/threonine protein kinase in regulating plasma membrane H+ ATPase activity and the NTA1 N-terminal amidase in protein degradation via the ubiquitin-proteasome system machinery, which affects tolerance to heat stress in S. cerevisiae.
Asunto(s)
Etanol , Fermentación , Calor , Sitios de Carácter Cuantitativo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Etanol/metabolismoRESUMEN
Aminoacyl-transfer RiboNucleic Acid synthetases (ARSs) are essential enzymes that catalyze the attachment of each amino acid to their cognate tRNAs. Mitochondrial ARSs (mtARSs), which ensure protein synthesis within the mitochondria, are encoded by nuclear genes and imported into the organelle after translation in the cytosol. The extensive use of next generation sequencing (NGS) has resulted in an increasing number of variants in mtARS genes being identified and associated with mitochondrial diseases. The similarities between yeast and human mitochondrial translation machineries make yeast a good model to quickly and efficiently evaluate the effect of variants in mtARS genes. Genetic screening of patients with a clinical suspicion of mitochondrial disorders through a customized gene panel of known disease-genes, including all genes encoding mtARSs, led to the identification of missense variants in WARS2, NARS2 and RARS2. Most of them were classified as Variant of Uncertain Significance. We exploited yeast models to assess the functional consequences of the variants found in these genes encoding mitochondrial tryptophanyl-tRNA, asparaginyl-tRNA, and arginyl-tRNA synthetases, respectively. Mitochondrial phenotypes such as oxidative growth, oxygen consumption rate, Cox2 steady-state level and mitochondrial protein synthesis were analyzed in yeast strains deleted in MSW1, SLM5, and MSR1 (the yeast orthologues of WARS2, NARS2 and RARS2, respectively), and expressing the wild type or the mutant alleles. Pathogenicity was confirmed for most variants, leading to their reclassification as Likely Pathogenic. Moreover, the beneficial effects observed after asparagine and arginine supplementation in the growth medium suggest them as a potential therapeutic approach.
Asunto(s)
Aminoacil-ARNt Sintetasas , Mitocondrias , Enfermedades Mitocondriales , Saccharomyces cerevisiae , Humanos , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Enfermedades Mitocondriales/genética , Saccharomyces cerevisiae/genética , Mitocondrias/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación MissenseRESUMEN
Double-strand breaks (DSBs) in DNA are challenging to repair. Cells employ at least three DSB-repair mechanisms, with a preference for non-homologous end joining (NHEJ) over homologous recombination (HR) and microhomology-mediated end joining (MMEJ). While most eukaryotic DNA is transcribed into RNA, providing complementary genetic information, much remains unknown about the direct impact of RNA on DSB-repair outcomes and its role in DSB-repair via end joining. Here, we show that both sense and antisense-transcript RNAs impact DSB repair in a sequence-specific manner in wild-type human and yeast cells. Depending on its sequence complementarity with the broken DNA ends, a transcript RNA can promote repair of a DSB or a double-strand gap in its DNA gene via NHEJ or MMEJ, independently from DNA synthesis. The results demonstrate a role of transcript RNA in directing the way DSBs are repaired in DNA, suggesting that RNA may directly modulate genome stability and evolution.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ARN/metabolismo , ARN/genética , Inestabilidad Genómica , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ARN sin Sentido/genética , ARN sin Sentido/metabolismoRESUMEN
Transposable elements (TEs) are ubiquitous in the eukaryote genomes, but their evolutionary and functional significance remains largely obscure and contentious. Here, we explore the evolution and functional impact of TEs in two model unicellular eukaryotes, the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae, which diverged around 330 to 420 million years ago. We analyze the distribution of LTR retrotransposons (LTR-RTs, the only TE order identified in both species) and their solo-LTR derivatives in 35 strains of S. pombe and 128 strains of S. cerevisiae. We find that natural LTR-RT and solo-LTR insertions exhibit high presence-absence polymorphism among individuals in both species. Population genetics analyses show that solo-LTR insertions experienced functional constraints similar to synonymous sites of host genes in both species, indicating a majority of solo-LTR insertions might have evolved in a neutral manner. When knocking out nine representative solo-LTR insertions separately in the S. pombe strain 972h- and 12 representative solo-LTR insertions separately in the S. cerevisiae strain S288C, we find that one solo-LTR insertion in S. pombe has a significant effect on the fitness and transcriptome of its host. Together, our findings indicate that a fraction of natural TE insertions likely shape their host transcriptomes and thereby contribute to their host fitness, with implications for understanding the functional significance of TEs in eukaryotes.
Asunto(s)
Aptitud Genética , Retroelementos , Saccharomyces cerevisiae , Schizosaccharomyces , Schizosaccharomyces/genética , Saccharomyces cerevisiae/genética , Elementos Transponibles de ADN , Evolución Molecular , Secuencias Repetidas Terminales , Mutagénesis Insercional , Genoma FúngicoRESUMEN
In eukaryotes, the origin recognition complex (ORC) faciliates the assembly of pre-replicative complex (pre-RC) at origin DNA for replication licensing. Here we show that the N-terminal intrinsically disordered region (IDR) of the yeast Orc2 subunit is crucial for this process. Removing a segment (residues 176-200) from Orc2-IDR or mutating a key isoleucine (194) significantly inhibits replication initiation across the genome. These Orc2-IDR mutants are capable of assembling the ORC-Cdc6-Cdt1-Mcm2-7 intermediate, which exhibits impaired ATP hydrolysis and fails to be convered into the subsequent Mcm2-7-ORC complex and pre-RC. These defects can be partially rescued by the Orc2-IDR peptide. Moreover, the phosphorylation of this Orc2-IDR region by S cyclin-dependent kinase blocks its binding to Mcm2-7 complex, causing a defective pre-RC assembly. Our findings provide important insights into the multifaceted roles of ORC in supporting origin licensing during the G1 phase and its regulation to restrict origin firing within the S phase.
Asunto(s)
Replicación del ADN , Complejo de Reconocimiento del Origen , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Complejo de Reconocimiento del Origen/metabolismo , Complejo de Reconocimiento del Origen/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Fosforilación , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Origen de Réplica/genética , Unión Proteica , Mutación , Fase G1 , Proteínas Intrínsecamente Desordenadas/metabolismo , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/química , Secuencias de AminoácidosRESUMEN
Transcriptional silencing in Saccharomyces cerevisiae is a persistent and highly stable form of gene repression. It involves DNA silencers and repressor proteins that bind nucleosomes. The silenced state is influenced by numerous factors including the concentration of repressors, nature of activators, architecture of regulatory elements, modifying enzymes and the dynamics of chromatin.Silencers function to increase the residence time of repressor Sir proteins at silenced domains while clustering of silenced domains enables increased concentrations of repressors and helps facilitate long-range interactions. The presence of an accessible NDR at the regulatory regions of silenced genes, the cycling of chromatin configurations at regulatory sites, the mobility of Sir proteins, and the non-uniform distribution of the Sir proteins across the silenced domain, all result in silenced chromatin that only stably silences weak promoters and enhancers via changes in transcription burst duration and frequency.These data collectively suggest that silencing is probabilistic and the robustness of silencing is achieved through sub-optimization of many different nodes of action such that a stable expression state is generated and maintained even though individual constituents are in constant flux.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Saccharomyces cerevisiae , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae/genética , Transcripción Genética , Cromatina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Proteínas Represoras/genéticaRESUMEN
Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1. This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis.
Asunto(s)
Mutación , Trastornos del Neurodesarrollo , Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trastornos del Neurodesarrollo/genética , Mutación/genética , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Femenino , Sistemas CRISPR-Cas/genéticaRESUMEN
Redox homeostasis is the balance between oxidation and reduction reactions. Its maintenance depends on glutathione, including its reduced and oxidized form, GSH/GSSG, which is the main intracellular redox buffer, but also on the nicotinamide adenine dinucleotide phosphate, including its reduced and oxidized form, NADPH/NADP+. Under conditions that enable yeast cells to undergo fermentative metabolism, the main source of NADPH is the pentose phosphate pathway. The lack of enzymes responsible for the production of NADPH has a significant impact on yeast cells. However, cells may compensate in different ways for impairments in NADPH synthesis, and the choice of compensation strategy has several consequences for cell functioning. The present study of this issue was based on isogenic mutants: Δzwf1, Δgnd1, Δald6, and the wild strain, as well as a comprehensive panel of molecular analyses such as the level of gene expression, protein content, and enzyme activity. The obtained results indicate that yeast cells compensate for the lack of enzymes responsible for the production of cytosolic NADPH by changing the content of selected proteins and/or their enzymatic activity. In turn, the cellular strategy used to compensate for them may affect cellular efficiency, and thus, the ability to grow or sensitivity to environmental acidification.
Asunto(s)
Fermentación , Homeostasis , NADP , Oxidación-Reducción , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , NADP/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Glutatión/metabolismo , Vía de Pentosa FosfatoRESUMEN
The antibacterial, antibiofilm, and cytotoxicity activity of cell-free supernatants (CFSs) from probiotics, including Lactobacillus plantarum, Bifidobacterium bifidum, and Saccharomyces cerevisiae against multi-drug resistant Escherichia coli evaluated in current research. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the CFSs were determined by analyzing inhibition zone formation using agar disk diffusion for antibacterial activity, microtiter plate for biofilm analysis, and auto-aggregation were done. CFSs substances were analyzed by GC-MS. The MTT assay on HEK293 cells investigated CFS's influence on cell viability. CFSs were examined for biofilm-related virulence genes, including aggR and fimH using real-time polymerase chain reaction (real-time PCR). All CFSs had bacteriostatic and bactericidal effects. The B. bifidum exhibited the highest antibiofilm activity compared to the others. Bifidobacterium bifidum, L. plantarum, and S. cerevisiae produce 19, 16, and 11 mm inhibition zones against E. coli, respectively. GC-MS indicated that Hydroxyacetone, 3-Hydroxybutyric acid, and Oxime-methoxy-phenyl-dominated CFSs from L. plantarum, B. bifidum, and S. cerevisiae CFSs, respectively. The MTT test demonstrated a cell viability rate of over 90%. Statistically, adding all CFSs lowered the relative expression of both aggR and fimH virulence genes.