RESUMEN
Epstein-Barr virus (EBV) infection has long been associated with the development of multiple sclerosis (MS). MS patients have elevated titers of EBV-specific antibodies in serum and show signs of CNS damage only after EBV infection. Regarding CD8+ T-cells, an elevated but ineffective response to EBV was suggested in MS patients, who present with a broader MHC-I-restricted EBV-specific T-cell receptor beta chain (TRB) repertoire compared to controls. It is not known whether this altered EBV response could be subject to dynamic changes, e.g., by approved MS therapies, and whether it is specific for MS. 1317 peripheral blood TRB repertoire samples of healthy donors (n=409), patients with MS (n=710) before and after treatment, patients with neuromyelitis optica spectrum disorder (n=87), myelin-oligodendrocyte-glycoprotein antibody-associated disease (n=64) and Susac's syndrome (n=47) were analyzed. Apart from MS, none of the evaluated diseases presented with a broader anti-EBV TRB repertoire. In MS patients undergoing autologous hematopoietic stem-cell transplantation, EBV reactivation coincided with elevated MHC-I-restricted EBV-specific TRB sequence matches. Therapy with ocrelizumab, teriflunomide or dimethyl fumarate reduced EBV-specific, but not CMV-specific MHC-I-restricted TRB sequence matches. Together, this data suggests that the aberrant MHC-I-restricted T-cell response directed against EBV is specific to MS with regard to NMO, MOGAD and Susac's Syndrome and that it is specifically modified by MS treatments interfering with EBV host cells or activated lymphocytes.
RESUMEN
Multidrug resistance is highly conserved in mammalian, fungal, and bacterial cells, is characterized by resistance to several unrelated xenobiotics, and poses significant challenges to managing infections and many cancers. Eukaryotes use a highly conserved set of drug efflux transporters that confer pleiotropic drug resistance (PDR). To interrogate the regulation of this critical process, here we developed a small molecule-responsive biosensor that couples transcriptional induction of PDR genes to growth rate in the yeast Saccharomyces cerevisiae Using diverse PDR inducers and the homozygous diploid deletion collection, we applied this biosensor system to genome-wide screens for potential PDR regulators. In addition to recapitulating the activity of previously known factors, these screens identified a series of genes involved in a variety of cellular processes with significant but previously uncharacterized roles in the modulation of yeast PDR. Genes identified as down-regulators of the PDR included those encoding the MAD family of proteins involved in the mitotic spindle assembly checkpoint (SAC) complex. Of note, we demonstrated that genetic disruptions of the mitotic spindle assembly checkpoint elevate expression of PDR-mediating efflux pumps in response to exposure to a variety of compounds that themselves have no known influence on the cell cycle. These results not only establish our biosensor system as a viable tool for investigating PDR in a high-throughput fashion, but also uncover critical control mechanisms governing the PDR response and a previously uncharacterized link between PDR and cell cycle regulation in yeast.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Técnicas Biosensibles , Puntos de Control del Ciclo Celular/genética , Resistencia a Múltiples Medicamentos/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Genoma Fúngico , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.
Asunto(s)
Productos Biológicos/metabolismo , Hongos/genética , Hongos/metabolismo , Expresión Génica , Ingeniería Genética , Vías Biosintéticas , Fermentación , Ingeniería Genética/métodos , Ensayos Analíticos de Alto Rendimiento , Regiones Promotoras Genéticas , Flujo de TrabajoRESUMEN
Progress achieved between 2014-2017 in the extraction and sample preparation of nucleic acid by isotachophoresis is reviewed in this paper. The isolation and purification of nucleic acids is very often compromised by a complex matrix such as blood and other bodily fluids, samples from the scene of crime, fossil samples, etc. While most of the common nucleic acids isolation techniques are based on extraction with inherent limitations with regard to quantitative results, isotachophoretic focusing is a quantitative process with a theoretically unlimited concentration factor. Since isotachophoresis belongs to less traditional approaches of nucleic acids purification, we present not only the latest developments in the application of isotachophoresis for the nucleic acids concentration but also a brief description of the principles of this method.
Asunto(s)
Isotacoforesis/métodos , Isotacoforesis/tendencias , Ácidos Nucleicos/aislamiento & purificación , Automatización , Líquidos Corporales/metabolismo , Tampones (Química) , ADN/análisis , Electrólitos , Humanos , MicroARNs/análisis , Microfluídica , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
Many cellular functions are mediated by protein-protein interaction networks, which are environment dependent. However, systematic measurement of interactions in diverse environments is required to better understand the relative importance of different mechanisms underlying network dynamics. To investigate environment-dependent protein complex dynamics, we used a DNA-barcode-based multiplexed protein interaction assay in Saccharomyces cerevisiae to measure in vivo abundance of 1,379 binary protein complexes under 14 environments. Many binary complexes (55%) were environment dependent, especially those involving transmembrane transporters. We observed many concerted changes around highly connected proteins, and overall network dynamics suggested that "concerted" protein-centered changes are prevalent. Under a diauxic shift in carbon source from glucose to ethanol, a mass-action-based model using relative mRNA levels explained an estimated 47% of the observed variance in binary complex abundance and predicted the direction of concerted binary complex changes with 88% accuracy. Thus, we provide a resource of yeast protein interaction measurements across diverse environments and illustrate the value of this resource in revealing mechanisms of network dynamics.
Asunto(s)
Mapas de Interacción de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Simulación por Computador , Código de Barras del ADN Taxonómico , Perfilación de la Expresión Génica , Modelos Biológicos , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Mapeo de Interacción de Proteínas/métodos , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Biología de SistemasRESUMEN
Single molecule sequencing (SMS) platforms enable base sequences to be read directly from individual strands of DNA in real-time. Though capable of long read lengths, SMS platforms currently suffer from low throughput compared to competing short-read sequencing technologies. Here, we present a novel strategy for sequencing library preparation, dubbed ConcatSeq, which increases the throughput of SMS platforms by generating long concatenated templates from pools of short DNA molecules. We demonstrate adaptation of this technique to two target enrichment workflows, commonly used for oncology applications, and feasibility using PacBio single molecule real-time (SMRT) technology. Our approach is capable of increasing the sequencing throughput of the PacBio RSII platform by more than five-fold, while maintaining the ability to correctly call allele frequencies of known single nucleotide variants. ConcatSeq provides a versatile new sample preparation tool for long-read sequencing technologies.
Asunto(s)
ADN Concatenado/análisis , ADN Concatenado/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Humanos , Masculino , Anotación de Secuencia MolecularRESUMEN
Several large-scale efforts have systematically catalogued protein-protein interactions (PPIs) of a cell in a single environment. However, little is known about how the protein interactome changes across environmental perturbations. Current technologies, which assay one PPI at a time, are too low throughput to make it practical to study protein interactome dynamics. Here, we develop a highly parallel protein-protein interaction sequencing (PPiSeq) platform that uses a novel double barcoding system in conjunction with the dihydrofolate reductase protein-fragment complementation assay in Saccharomyces cerevisiae. PPiSeq detects PPIs at a rate that is on par with current assays and, in contrast with current methods, quantitatively scores PPIs with enough accuracy and sensitivity to detect changes across environments. Both PPI scoring and the bulk of strain construction can be performed with cell pools, making the assay scalable and easily reproduced across environments. PPiSeq is therefore a powerful new tool for large-scale investigations of dynamic PPIs.
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Código de Barras del ADN Taxonómico/métodos , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
The low costs of array-synthesized oligonucleotide libraries are empowering rapid advances in quantitative and synthetic biology. However, high synthesis error rates, uneven representation, and lack of access to individual oligonucleotides limit the true potential of these libraries. We have developed a cost-effective method called Recombinase Directed Indexing (REDI), which involves integration of a complex library into yeast, site-specific recombination to index library DNA, and next-generation sequencing to identify desired clones. We used REDI to generate a library of ~3,300 DNA probes that exhibited > 96% purity and remarkable uniformity (> 95% of probes within twofold of the median abundance). Additionally, we created a collection of ~9,000 individually accessible CRISPR interference yeast strains for > 99% of genes required for either fermentative or respiratory growth, demonstrating the utility of REDI for rapid and cost-effective creation of strain collections from oligonucleotide pools. Our approach is adaptable to any complex DNA library, and fundamentally changes how these libraries can be parsed, maintained, propagated, and characterized.
Asunto(s)
Análisis de Secuencia de ADN/métodos , Levaduras/genética , Sistemas CRISPR-Cas , Biología Computacional/métodos , ADN de Hongos/genética , Biblioteca de GenesRESUMEN
The Yeast Knockout Collection is a complete set of gene deletion strains for the budding yeast, Saccharomyces cerevisiae In each strain, one of approximately 6000 open-reading frames is replaced with a dominant selectable marker flanked by two DNA barcodes. These barcodes, which are unique to each gene, allow the growth of thousands of strains to be individually measured from a single pooled culture. The collection, and other resources that followed, has ushered in a new era in chemical biology, enabling unbiased and systematic identification of chemical-genetic interactions (CGIs) with remarkable ease. CGIs link bioactive compounds to biological processes, and hence can reveal the mechanism of action of growth-inhibitory compounds in vivo, including those of antifungal, antibiotic, and anticancer drugs. The chemogenomic profiling method described here measures the sensitivity induced in yeast heterozygous and homozygous deletion strains in the presence of a chemical inhibitor of growth (termed haploinsufficiency profiling and homozygous profiling, respectively, or HIPHOP). The protocol is both scalable and amenable to automation. After competitive growth of yeast knockout collection cultures, with and without chemical inhibitors, CGIs can be identified and quantified using either array- or sequencing-based approaches as described here.
Asunto(s)
Antifúngicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Eliminación de Gen , Genes Fúngicos , Inhibidores de Crecimiento/farmacología , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Biblioteca de Genes , Pruebas Genéticas , Saccharomyces cerevisiae/genéticaRESUMEN
Chemical-genetic interactions (CGIs) describe a phenomenon where the effects of a chemical compound (i.e., a small molecule) on cell growth are dependent on a particular gene. CGIs can reveal important functional information about genes and can also be powerful indicators of a compound's mechanism of action. Mapping CGIs can lead to the discovery of new chemical probes, which, in contrast to genetic perturbations, operate at the level of the gene product (or pathway) and can be fast-acting, tunable, and reversible. The simple culture conditions required for yeast and its rapid growth, as well as the availability of a complete set of barcoded gene deletion strains, facilitate systematic mapping of CGIs in this organism. This process involves two basic steps: first, screening chemical libraries to identify bioactive compounds affecting growth and, second, measuring the effects of these compounds on genome-wide collections of mutant strains. Here, we introduce protocols for both steps that have great potential for the discovery and development of new small-molecule tools and medicines.
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Antifúngicos/farmacología , Evaluación Preclínica de Medicamentos/métodos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrollo , Bibliotecas de Moléculas Pequeñas/farmacología , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Genome-scale CRISPR interference (CRISPRi) has been used in human cell lines; however, the features of effective guide RNAs (gRNAs) in different organisms have not been well characterized. Here, we define rules that determine gRNA effectiveness for transcriptional repression in Saccharomyces cerevisiae. RESULTS: We create an inducible single plasmid CRISPRi system for gene repression in yeast, and use it to analyze fitness effects of gRNAs under 18 small molecule treatments. Our approach correctly identifies previously described chemical-genetic interactions, as well as a new mechanism of suppressing fluconazole toxicity by repression of the ERG25 gene. Assessment of multiple target loci across treatments using gRNA libraries allows us to determine generalizable features associated with gRNA efficacy. Guides that target regions with low nucleosome occupancy and high chromatin accessibility are clearly more effective. We also find that the best region to target gRNAs is between the transcription start site (TSS) and 200 bp upstream of the TSS. Finally, unlike nuclease-proficient Cas9 in human cells, the specificity of truncated gRNAs (18 nt of complementarity to the target) is not clearly superior to full-length gRNAs (20 nt of complementarity), as truncated gRNAs are generally less potent against both mismatched and perfectly matched targets. CONCLUSIONS: Our results establish a powerful functional and chemical genomics screening method and provide guidelines for designing effective gRNAs, which consider chromatin state and position relative to the target gene TSS. These findings will enable effective library design and genome-wide programmable gene repression in many genetic backgrounds.
Asunto(s)
Sistemas CRISPR-Cas/genética , Genoma Fúngico , ARN Guía de Kinetoplastida/genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Cromatina/genética , Humanos , Oxigenasas de Función Mixta/genética , Nucleosomas/genética , Proteínas de Saccharomyces cerevisiae/genética , Sitio de Iniciación de la TranscripciónRESUMEN
Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke.
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Mastocitos/inmunología , Meninges/inmunología , Accidente Cerebrovascular/inmunología , Animales , Encéfalo/inmunología , Encéfalo/patología , Modelos Animales de Enfermedad , Citometría de Flujo , Técnicas de Sustitución del Gen , Imagen por Resonancia Magnética , Masculino , Mastocitos/patología , Meninges/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Accidente Cerebrovascular/patologíaRESUMEN
Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.
Asunto(s)
Células/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Resistencia a Medicamentos/genética , Redes Reguladoras de Genes , Estudio de Asociación del Genoma Completo/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Línea Celular Tumoral , Haploinsuficiencia , Humanos , Farmacogenética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genéticaRESUMEN
BACKGROUND: Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. RESULTS: We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu's positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson's disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. CONCLUSIONS: A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations.
Asunto(s)
Cobre/metabolismo , Homeostasis/genética , Levaduras/genética , Levaduras/metabolismo , Respiración de la Célula , Análisis por Conglomerados , Cobre/deficiencia , Medios de Cultivo , Disulfiram/farmacología , Perfilación de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Redes Reguladoras de Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Homeostasis/efectos de los fármacos , Humanos , Hidrazinas/farmacología , Concentración de Iones de Hidrógeno , Fenotipo , Vacuolas/genética , Vacuolas/metabolismo , Levaduras/efectos de los fármacosRESUMEN
The many virtues that made the yeast Saccharomyces cerevisiae a dominant model organism for genetics and molecular biology, are now establishing its role in chemical genetics. Its experimental tractability (i.e., rapid doubling time, simple culture conditions) and the availability of powerful tools for drug-target identification, make yeast an ideal organism for high-throughput phenotypic screening. It may be especially applicable for the discovery of chemical probes targeting highly conserved cellular processes, such as metabolism and bioenergetics, because these probes would likely inhibit the same processes in higher eukaryotes (including man). Importantly, changes in normal cellular metabolism are associated with a variety of diseased states (including neurological disorders and cancer), and exploiting these changes for therapeutic purposes has accordingly gained considerable attention. Here, we review progress and challenges associated with forward chemical genetic screening in yeast. We also discuss evidence supporting these screens as a useful strategy for discovery of new chemical probes and new druggable targets related to cellular metabolism.
Asunto(s)
Ensayos de Selección de Medicamentos Antitumorales , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Animales , Antimetabolitos Antineoplásicos/farmacología , Glucólisis/efectos de los fármacos , Glucólisis/genética , Humanos , Leucovorina/farmacología , Metotrexato/farmacología , Terapia Molecular Dirigida , Mutagénesis , Neoplasias/tratamiento farmacológico , Fenotipo , Saccharomyces cerevisiae/metabolismoRESUMEN
OBJECTIVE: To advance our understanding of disease biology, the characterization of the molecular target for clinically proven or new drugs is very important. Because of its simplicity and the availability of strains with individual deletions in all of its genes, chemogenomic profiling in yeast has been used to identify drug targets. As measurement of drug-induced changes in cellular metabolites can yield considerable information about the effects of a drug, we investigated whether combining chemogenomic and metabolomic profiling in yeast could improve the characterization of drug targets. BASIC METHODS: We used chemogenomic and metabolomic profiling in yeast to characterize the target for five drugs acting on two biologically important pathways. A novel computational method that uses a curated metabolic network was also developed, and it was used to identify the genes that are likely to be responsible for the metabolomic differences found. RESULTS AND CONCLUSION: The combination of metabolomic and chemogenomic profiling, along with data analyses carried out using a novel computational method, could robustly identify the enzymes targeted by five drugs. Moreover, this novel computational method has the potential to identify genes that are causative of metabolomic differences or drug targets.
Asunto(s)
Redes y Vías Metabólicas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biología Computacional , Sistemas de Liberación de Medicamentos , Perfilación de la Expresión Génica , Metabolómica , Saccharomyces cerevisiae/efectos de los fármacosRESUMEN
Meiosis is a highly regulated developmental process that occurs in all eukaryotes that engage in sexual reproduction. Previous epidemiological work shows that male and female infertility is rising and environmental factors, including pollutants such as organic solvents, are thought to play a role in this phenomenon. To better understand how organic compounds interfere with meiotic development, the model organism Saccharomyces cerevisiae was exposed to 446 bioactive molecules while undergoing meiotic development, and sporulation efficiency was quantified employing two different high-throughput assays. 12 chemicals were identified that strongly inhibited spore formation but did not interfere with vegetative growth. Many of these chemicals are known to bind to monoamine-receptors in higher eukaryotes and are cationic amphiphilic drugs. A detailed analysis of one of these drugs, tripelennamine, revealed that it induces sporulation-specific cytotoxicity and a strong inhibition of meiotic M phase. The drug, however, only mildly interfered with pre-meiotic DNA synthesis and the early meiotic transcriptional program. Chemical-genomic screening identified genes involved in autophagy as hypersensitive to tripelennamine. In addition, we found that growing and sporulating yeast cells heterozygous for the aminophospholipid translocase, NEO1, are haploinsufficient in the presence of the drug.
Asunto(s)
Cationes/química , Saccharomyces cerevisiae/metabolismo , Esporas Fúngicas/fisiología , Tensoactivos/farmacología , Antifúngicos/farmacología , Autofagia , ADN/biosíntesis , Diseño de Fármacos , Eliminación de Gen , Regulación Fúngica de la Expresión Génica , Genómica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Meiosis , Metabolómica/métodos , Modelos Genéticos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Esporas Fúngicas/metabolismo , Factores de Tiempo , Transcripción Genética , Tripelenamina/farmacologíaRESUMEN
Changes in protein-protein interactions that occur in response to environmental cues are difficult to uncover and have been poorly characterized to date. Here we describe a yeast-based assay that allows many binary protein interactions to be assessed in parallel and under various conditions. This method combines molecular bar-coding and tag array technology with the murine dihydrofolate reductase-based protein-fragment complementation assay. A total of 238 protein-fragment complementation assay strains, each representing a unique binary protein complex, were tagged with molecular barcodes, pooled, and then interrogated against a panel of 80 diverse small molecules. Our method successfully identified specific disruption of the Hom3:Fpr1 interaction by the immunosuppressant FK506, illustrating the assay's capacity to identify chemical inhibitors of protein-protein interactions. Among the additional findings was specific cellular depletion of the Dst1:Rbp9 complex by the anthracycline drug doxorubicin, but not by the related drug idarubicin. The assay also revealed chemical-induced accumulation of several binary multidrug transporter complexes that largely paralleled increases in transcript levels. Further assessment of two such interactions (Tpo1:Pdr5 and Snq2:Pdr5) in the presence of 1,246 unique chemical compounds revealed a positive correlation between drug lipophilicity and the drug response in yeast.
Asunto(s)
Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas/genética , Bibliotecas de Moléculas Pequeñas/metabolismo , Tetrahidrofolato Deshidrogenasa/metabolismo , Animales , Biología Computacional , Proteínas de Unión al ADN , Ratones , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Formil Péptido/metabolismo , Proteínas de Saccharomyces cerevisiae , Tacrolimus , LevadurasRESUMEN
Candida albicans is the most common human fungal pathogen, causing infections that can be lethal in immunocompromised patients. Although Saccharomyces cerevisiae has been used as a model for C. albicans, it lacks C. albicans' diverse morphogenic forms and is primarily non-pathogenic. Comprehensive genetic analyses that have been instrumental for determining gene function in S. cerevisiae are hampered in C. albicans, due in part to limited resources to systematically assay phenotypes of loss-of-function alleles. Here, we constructed and screened a library of 3633 tagged heterozygous transposon disruption mutants, using them in a competitive growth assay to examine nutrient- and drug-dependent haploinsufficiency. We identified 269 genes that were haploinsufficient in four growth conditions, the majority of which were condition-specific. These screens identified two new genes necessary for filamentous growth as well as ten genes that function in essential processes. We also screened 57 chemically diverse compounds that more potently inhibited growth of C. albicans versus S. cerevisiae. For four of these compounds, we examined the genetic basis of this differential inhibition. Notably, Sec7p was identified as the target of brefeldin A in C. albicans screens, while S. cerevisiae screens with this compound failed to identify this target. We also uncovered a new C. albicans-specific target, Tfp1p, for the synthetic compound 0136-0228. These results highlight the value of haploinsufficiency screens directly in this pathogen for gene annotation and drug target identification.
Asunto(s)
Candida albicans/genética , Elementos Transponibles de ADN , Descubrimiento de Drogas/métodos , Anotación de Secuencia Molecular/métodos , Terapia Molecular Dirigida/métodos , Organismos Modificados Genéticamente , Candida albicans/fisiología , Elementos Transponibles de ADN/genética , Alimentos , Perfilación de la Expresión Génica , Biblioteca Genómica , Haploinsuficiencia/genética , Análisis por Micromatrices , Modelos Biológicos , Mutagénesis Insercional/métodos , Organismos Modificados Genéticamente/genética , Lugares Marcados de SecuenciaRESUMEN
Spo13 is a key meiosis-specific regulator required for centromere cohesion and coorientation, and for progression through two nuclear divisions. We previously reported that it causes a G2/M arrest and may delay the transition from late anaphase to G1, when overexpressed in mitosis. Yet its mechanism of action has remained elusive. Here we show that Spo13, which is phosphorylated and stabilized at G2/M in a Cdk/Clb-dependent manner, acts at two stages during mitotic cell division. Spo13 provokes a G2/M arrest that is reversible and largely independent of the Mad2 spindle checkpoint. Since mRNAs whose induction requires Cdc14 activation are reduced, we propose that its anaphase delay results from inhibition of Cdc14 function. Indeed, the Spo13-induced anaphase delay correlates with Cdc14 phosphatase retention in the nucleolus and with cyclin B accumulation, which both impede anaphase exit. At the onset of arrest, Spo13 is primarily associated with the nucleolus, where Cdc14 accumulates. Significantly, overexpression of separase (Esp1), which promotes G2/M and anaphase progression, suppresses Spo13 effects in mitosis, arguing that Spo13 acts upstream or parallel to Esp1. Given that Spo13 overexpression reduces Pds1 and cyclin B degradation, our findings are consistent with a role for Spo13 in regulating APC, which controls both G2/M and anaphase. Similar effects of Spo13 during meiotic MI may prevent cell cycle exit and initiation of DNA replication prior to MII, thereby ensuring two successive chromosome segregation events without an intervening S phase.