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1.
Mol Cell ; 33(1): 124-35, 2009 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-19150434

RESUMO

Systematic functional genomics approaches were used to map a network centered on the small ubiquitin-related modifier (SUMO) system. Over 250 physical interactions were identified using the SUMO protein as bait in affinity purification-mass spectrometry and yeast two-hybrid screens. More than 500 genes and 1400 synthetic genetic interactions were mapped by synthetic genetic array (SGA) analysis using eight different SUMO pathway query genes. The resultant global SUMO network highlights its role in 15 major biological processes and better defines functional relationships between the different components of the SUMO pathway. Using this information-rich resource, we have identified roles for the SUMO system in the function of the AAA ATPase Cdc48p, the regulation of lipid metabolism, localization of the ATP-dependent endonuclease Dna2p, and recovery from the DNA-damage checkpoint.


Assuntos
Redes Reguladoras de Genes , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Núcleo Celular , Cromatografia de Afinidade , Dano ao DNA , Reparo do DNA , Replicação do DNA , Genes Fúngicos , Metabolismo dos Lipídeos , Espectrometria de Massas , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo
2.
Science ; 353(6306)2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27708008

RESUMO

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing more than 23 million double mutants, identifying about 550,000 negative and about 350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.


Assuntos
Redes Reguladoras de Genes , Genes Fúngicos/fisiologia , Pleiotropia Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Epistasia Genética , Genes Essenciais
3.
PLoS One ; 9(1): e85177, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24465499

RESUMO

High-throughput genetic screens in model microbial organisms are a primary means of interrogating biological systems. In numerous cases, such screens have identified the genes that underlie a particular phenotype or a set of gene-gene, gene-environment or protein-protein interactions, which are then used to construct highly informative network maps for biological research. However, the potential test space of genes, proteins, or interactions is typically much larger than current screening systems can address. To push the limits of screening technology, we developed an ultra-high-density, 6144-colony arraying system and analysis toolbox. Using budding yeast as a benchmark, we find that these tools boost genetic screening throughput 4-fold and yield significant cost and time reductions at quality levels equal to or better than current methods. Thus, the new ultra-high-density screening tools enable researchers to significantly increase the size and scope of their genetic screens.


Assuntos
Regulação Fúngica da Expressão Gênica , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Algoritmos , Contagem de Colônia Microbiana , Ensaios de Triagem em Larga Escala , Mutação , Proteômica/estatística & dados numéricos , Saccharomyces cerevisiae/crescimento & desenvolvimento
4.
Science ; 337(6100): 1353-6, 2012 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-22984072

RESUMO

The dimorphic switch from a single-cell budding yeast to a filamentous form enables Saccharomyces cerevisiae to forage for nutrients and the opportunistic pathogen Candida albicans to invade human tissues and evade the immune system. We constructed a genome-wide set of targeted deletion alleles and introduced them into a filamentous S. cerevisiae strain, Σ1278b. We identified genes involved in morphologically distinct forms of filamentation: haploid invasive growth, biofilm formation, and diploid pseudohyphal growth. Unique genes appear to underlie each program, but we also found core genes with general roles in filamentous growth, including MFG1 (YDL233w), whose product binds two morphogenetic transcription factors, Flo8 and Mss11, and functions as a critical transcriptional regulator of filamentous growth in both S. cerevisiae and C. albicans.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/genética , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/genética , Alelos , Biofilmes/crescimento & desenvolvimento , Candida albicans/citologia , Análise Mutacional de DNA , Deleção de Genes , Hifas/genética , Hifas/crescimento & desenvolvimento , Proteínas Nucleares/genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica
5.
Nat Biotechnol ; 29(6): 505-11, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21572441

RESUMO

Dosage suppression is a genetic interaction in which overproduction of one gene rescues a mutant phenotype of another gene. Although dosage suppression is known to map functional connections among genes, the extent to which it might illuminate global cellular functions is unclear. Here we analyze a network of interactions linking dosage suppressors to 437 essential genes in yeast. For 424 genes, we curated interactions from the literature. Analyses revealed that many dosage suppression interactions occur between functionally related genes and that the majority do not overlap with other types of genetic or physical interactions. To confirm the generality of these network properties, we experimentally identified dosage suppressors for 29 genes from pooled populations of temperature-sensitive mutant cells transformed with a high-copy molecular-barcoded open reading frame library, MoBY-ORF 2.0. We classified 87% of the 1,640 total interactions into four general types of suppression mechanisms, which provided insight into their relative frequencies. This work suggests that integrating the results of dosage suppression studies with other interaction networks could generate insights into the functional wiring diagram of a cell.


Assuntos
Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Supressão Genética , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Árvores de Decisões , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Biblioteca Gênica , Genes Essenciais , Genes Fúngicos , Sequenciamento de Nucleotídeos em Larga Escala , Cinetocoros/metabolismo , Mutação , Fases de Leitura Aberta , Fenótipo , Mapeamento de Interação de Proteínas/métodos , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transcrição Gênica
6.
Nat Biotechnol ; 29(4): 361-7, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21441928

RESUMO

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (∼45%) of the 1,101 essential yeast genes, with ∼30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.


Assuntos
Genes Essenciais , Genoma Fúngico , Saccharomyces cerevisiae/genética , Temperatura , Alelos , Bases de Dados Genéticas , Genes Fúngicos , Genes Letais , Engenharia Genética/métodos , Loci Gênicos , Espectrometria de Massas/métodos , Análise em Microsséries/métodos , Microscopia Confocal , Mutação , Fenótipo , Plasmídeos , RNA Mensageiro , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de Célula Única , Tubulina (Proteína)/análise
7.
Science ; 327(5964): 425-31, 2010 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-20093466

RESUMO

A genome-scale genetic interaction map was constructed by examining 5.4 million gene-gene pairs for synthetic genetic interactions, generating quantitative genetic interaction profiles for approximately 75% of all genes in the budding yeast, Saccharomyces cerevisiae. A network based on genetic interaction profiles reveals a functional map of the cell in which genes of similar biological processes cluster together in coherent subsets, and highly correlated profiles delineate specific pathways to define gene function. The global network identifies functional cross-connections between all bioprocesses, mapping a cellular wiring diagram of pleiotropy. Genetic interaction degree correlated with a number of different gene attributes, which may be informative about genetic network hubs in other organisms. We also demonstrate that extensive and unbiased mapping of the genetic landscape provides a key for interpretation of chemical-genetic interactions and drug target identification.


Assuntos
Redes Reguladoras de Genes , Genoma Fúngico , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Biologia Computacional , Duplicação Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Aptidão Genética , Redes e Vias Metabólicas , Mutação , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética
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