Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 146
Filtrar
1.
Sci Adv ; 9(21): eadg5702, 2023 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-37235661

RESUMO

Genome-wide phenotypic screens in the budding yeast Saccharomyces cerevisiae, enabled by its knockout collection, have produced the largest, richest, and most systematic phenotypic description of any organism. However, integrative analyses of this rich data source have been virtually impossible because of the lack of a central data repository and consistent metadata annotations. Here, we describe the aggregation, harmonization, and analysis of ~14,500 yeast knockout screens, which we call Yeast Phenome. Using this unique dataset, we characterized two unknown genes (YHR045W and YGL117W) and showed that tryptophan starvation is a by-product of many chemical treatments. Furthermore, we uncovered an exponential relationship between phenotypic similarity and intergenic distance, which suggests that gene positions in both yeast and human genomes are optimized for function.


Assuntos
Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética
2.
Elife ; 112022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-35119359

RESUMO

The process wherein dividing cells exhaust proliferative capacity and enter into replicative senescence has become a prominent model for cellular aging in vitro. Despite decades of study, this cellular state is not fully understood in culture and even much less so during aging. Here, we revisit Leonard Hayflick's original observation of replicative senescence in WI-38 human lung fibroblasts equipped with a battery of modern techniques including RNA-seq, single-cell RNA-seq, proteomics, metabolomics, and ATAC-seq. We find evidence that the transition to a senescent state manifests early, increases gradually, and corresponds to a concomitant global increase in DNA accessibility in nucleolar and lamin associated domains. Furthermore, we demonstrate that senescent WI-38 cells acquire a striking resemblance to myofibroblasts in a process similar to the epithelial to mesenchymal transition (EMT) that is regulated by t YAP1/TEAD1 and TGF-ß2. Lastly, we show that verteporfin inhibition of YAP1/TEAD1 activity in aged WI-38 cells robustly attenuates this gene expression program.


Assuntos
Senescência Celular , Transição Epitelial-Mesenquimal , Idoso , Envelhecimento/fisiologia , Linhagem Celular , Senescência Celular/genética , Fibroblastos/metabolismo , Humanos
3.
Mol Biol Cell ; 32(22): ar39, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34668730

RESUMO

The electron transport chain (ETC) is a well-studied and highly conserved metabolic pathway that produces ATP through generation of a proton gradient across the inner mitochondrial membrane coupled to oxidative phosphorylation. ETC mutations are associated with a wide array of human disease conditions and to aging-related phenotypes in a number of different organisms. In this study, we sought to better understand the role of the ETC in aging using a yeast model. A panel of ETC mutant strains that fail to survive starvation was used to isolate suppressor mutants that survive. These suppressors tend to fall into major nutrient sensing and signaling pathways, suggesting that the ETC is involved in proper starvation signaling to these pathways in yeast. These suppressors also partially restore ETC-associated gene expression and pH homeostasis defects, though it remains unclear whether these phenotypes directly cause the suppression or are simply effects. This work further highlights the complex cellular network connections between metabolic pathways and signaling events in the cell and their potential roles in aging and age-related diseases.


Assuntos
Transporte de Elétrons/genética , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Citosol/química , Citosol/metabolismo , Transporte de Elétrons/fisiologia , Regulação Fúngica da Expressão Gênica , Genoma Mitocondrial , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Transcrição/genética
4.
Mol Syst Biol ; 17(6): e10207, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34096681

RESUMO

The ability to switch a gene from off to on and monitor dynamic changes provides a powerful approach for probing gene function and elucidating causal regulatory relationships. Here, we developed and characterized YETI (Yeast Estradiol strains with Titratable Induction), a collection in which > 5,600 yeast genes are engineered for transcriptional inducibility with single-gene precision at their native loci and without plasmids. Each strain contains SGA screening markers and a unique barcode, enabling high-throughput genetics. We characterized YETI using growth phenotyping and BAR-seq screens, and we used a YETI allele to identify the regulon of Rof1, showing that it acts to repress transcription. We observed that strains with inducible essential genes that have low native expression can often grow without inducer. Analysis of data from eukaryotic and prokaryotic systems shows that native expression is a variable that can bias promoter-perturbing screens, including CRISPRi. We engineered a second expression system, Z3 EB42, that gives lower expression than Z3 EV, a feature enabling conditional activation and repression of lowly expressed essential genes that grow without inducer in the YETI library.


Assuntos
Genes Essenciais , Saccharomyces cerevisiae , Biblioteca Gênica , Plasmídeos , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/genética
5.
Genetics ; 216(2): 261-262, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33023927

RESUMO

The Thomas Hunt Morgan Medal recognizes lifetime contributions to the field of genetics. The 2020 recipient is David Botstein of Calico Labs and Princeton University, recognizing his multiple contributions to genetics, including the collaborative development of methods for defining genetic pathways, mapping genomes, and analyzing gene expression.


Assuntos
Bases de Dados Genéticas/história , Ligação Genética , Genética/história , Distinções e Prêmios , Comunicação , História do Século XX , História do Século XXI
6.
G3 (Bethesda) ; 10(9): 2911-2925, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32631951

RESUMO

In recent years, improved sequencing technology and computational tools have made de novo genome assembly more accessible. Many approaches, however, generate either an unphased or only partially resolved representation of a diploid genome, in which polymorphisms are detected but not assigned to one or the other of the homologous chromosomes. Yet chromosomal phase information is invaluable for the understanding of phenotypic trait inheritance in the cases of compound heterozygosity, allele-specific expression or cis-acting variants. Here we use a combination of tools and sequencing technologies to generate a de novo diploid assembly of the human primary cell line WI-38. First, data from PacBio single molecule sequencing and Bionano Genomics optical mapping were combined to generate an unphased assembly. Next, 10x Genomics linked reads were combined with the hybrid assembly to generate a partially phased assembly. Lastly, we developed and optimized methods to use short-read (Illumina) sequencing of flow cytometry-sorted metaphase chromosomes to provide phase information. The final genome assembly was almost fully (94%) phased with the addition of approximately 2.5-fold coverage of Illumina data from the sequenced metaphase chromosomes. The diploid nature of the final de novo genome assembly improved the resolution of structural variants between the WI-38 genome and the human reference genome. The phased WI-38 sequence data are available for browsing and download at wi38.research.calicolabs.com. Our work shows that assembling a completely phased diploid genome de novo from the DNA of a single individual is now readily achievable.


Assuntos
Diploide , Genoma Humano , DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA
7.
PLoS One ; 15(5): e0233779, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470059

RESUMO

Trehalose metabolism in yeast has been linked to a variety of phenotypes, including heat resistance, desiccation tolerance, carbon-source utilization, and sporulation. The relationships among the several phenotypes of mutants unable to synthesize trehalose are not understood, even though the pathway is highly conserved. One of these phenotypes is that tps1Δ strains cannot reportedly grow on media containing glucose or fructose, even when another carbon source they can use (e.g. galactose) is present. Here we corroborate the recent observation that a small fraction of yeast tps1Δ cells do grow on glucose, unlike the majority of the population. This is not due to a genetic alteration, but instead resembles the persister phenotype documented in many microorganisms and cancer cells undergoing lethal stress. We extend these observations to show that this phenomenon is glucose-specific, as it does not occur on another highly fermented carbon source, fructose. We further demonstrate that this phenomenon appears to be related to mitochondrial complex III function, but unrelated to inorganic phosphate levels in the cell, as had previously been suggested. Finally, we found that this phenomenon is specific to S288C-derived strains, and is the consequence of a variant in the MKT1 gene.


Assuntos
Glucose/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Fermentação , Frutose/metabolismo , Glucosiltransferases/genética , Mutação com Perda de Função , Trealose/biossíntese
8.
mSystems ; 4(1)2019.
Artigo em Inglês | MEDLINE | ID: mdl-30834327

RESUMO

Isozymes are enzymes that differ in sequence but catalyze the same chemical reactions. Despite their apparent redundancy, isozymes are often retained over evolutionary time, suggesting that they contribute to fitness. We developed an unsupervised computational method for identifying environmental conditions under which isozymes are likely to make fitness contributions. This method analyzes published gene expression data to find specific experimental perturbations that induce differential isozyme expression. In yeast, we found that isozymes are strongly enriched in the pathways of central carbon metabolism and that many isozyme pairs show anticorrelated expression during the respirofermentative shift. Building on these observations, we assigned function to two minor central carbon isozymes, aconitase 2 (ACO2) and pyruvate kinase 2 (PYK2). ACO2 is expressed during fermentation and proves advantageous when glucose is limiting. PYK2 is expressed during respiration and proves advantageous for growth on three-carbon substrates. PYK2's deletion can be rescued by expressing the major pyruvate kinase only if that enzyme carries mutations mirroring PYK2's allosteric regulation. Thus, central carbon isozymes help to optimize allosteric metabolic regulation under a broad range of potential nutrient conditions while requiring only a small number of transcriptional states. IMPORTANCE Gene duplication is one of the main evolutionary paths to new protein function. Typically, duplicated genes either accumulate mutations and degrade into pseudogenes or are retained and diverge in function. Some duplicated genes, however, show long-term persistence without apparently acquiring new function. An important class of isozymes consists of those that catalyze the same reaction in the same compartment, where knockout of one isozyme causes no known functional defect. Here we present an approach to assigning specific functional roles to seemingly redundant isozymes. First, gene expression data are analyzed computationally to identify conditions under which isozyme expression diverges. Then, knockouts are compared under those conditions. This approach revealed that the expression of many yeast isozymes diverges in response to carbon availability and that carbon source manipulations can induce fitness phenotypes for seemingly redundant isozymes. A driver of these fitness phenotypes is differential allosteric enzyme regulation, indicating isozyme divergence to achieve more-optimal control of metabolism.

9.
Methods Mol Biol ; 1819: 317-333, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30421411

RESUMO

This chapter describes sequencing-based methods for profiling dynamic changes in DNA accessibility and gene expression in Saccharomyces cerevisiae. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) is a powerful technique for identifying nucleosome-free regions of the genome. Combining ATAC-Seq with RNA Sequencing (RNA-Seq) is a rapid approach for studying the relationship between genome structure and changes in global patterns of gene expression from a single experiment. A laboratory protocol is presented for these methods as well as examples of typical results and visualizations.


Assuntos
DNA Fúngico/genética , Perfilação da Expressão Gênica/métodos , Genoma Fúngico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Fúngico/genética , Saccharomyces cerevisiae/genética , DNA Fúngico/metabolismo , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/metabolismo
10.
ACS Chem Biol ; 13(10): 3011-3020, 2018 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-30240188

RESUMO

Sugar alcohols (polyols) exist widely in nature. While some specific sugar alcohol phosphatases are known, there is no known phosphatase for some important sugar alcohols (e.g., sorbitol-6-phosphate). Using liquid chromatography-mass spectrometry-based metabolomics, we screened yeast strains with putative phosphatases of unknown function deleted. We show that the yeast gene YNL010W, which has close homologues in all fungi species and some plants, encodes a sugar alcohol phosphatase. We term this enzyme, which hydrolyzes sorbitol-6-phosphate, ribitol-5-phosphate, and (d)-glycerol-3-phosphate, polyol phosphatase 1 or PYP1. Polyol phosphates are structural analogs of the enediol intermediate of phosphoglucose isomerase (Pgi). We find that sorbitol-6-phosphate and ribitol-5-phosphate inhibit Pgi and that Pyp1 activity is important for yeast to maintain Pgi activity in the presence of environmental sugar alcohols. Pyp1 expression is strongly positively correlated with yeast growth rate, presumably because faster growth requires greater glycolytic and accordingly Pgi flux. Thus, yeast express the previously uncharacterized enzyme Pyp1 to prevent inhibition of glycolysis by sugar alcohol phosphates. Pyp1 may be useful for engineering sugar alcohol production.


Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fosfatos Açúcares/metabolismo , Deleção de Genes , Glucose-6-Fosfato Isomerase/antagonistas & inibidores , Hidrólise , Monoéster Fosfórico Hidrolases/genética , Proteínas de Saccharomyces cerevisiae/genética , Fosfatos Açúcares/química
12.
Mol Biol Cell ; 29(8): 897-910, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29444955

RESUMO

Metabolic dysregulation leading to sugar-phosphate accumulation is toxic in organisms ranging from bacteria to humans. By comparing two models of sugar-phosphate toxicity in Saccharomyces cerevisiae, we demonstrate that toxicity occurs, at least in part, through multiple, isomer-specific mechanisms, rather than a single general mechanism.


Assuntos
Frutosefosfatos/toxicidade , Galactosefosfatos/toxicidade , Genes Supressores , Saccharomyces cerevisiae/metabolismo , Meios de Cultura/química , Frutosefosfatos/metabolismo , Galactosefosfatos/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Mutação , Saccharomyces cerevisiae/genética
13.
Science ; 354(6311)2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27789812

RESUMO

Cellular metabolic fluxes are determined by enzyme activities and metabolite abundances. Biochemical approaches reveal the impact of specific substrates or regulators on enzyme kinetics but do not capture the extent to which metabolite and enzyme concentrations vary across physiological states and, therefore, how cellular reactions are regulated. We measured enzyme and metabolite concentrations and metabolic fluxes across 25 steady-state yeast cultures. We then assessed the extent to which flux can be explained by a Michaelis-Menten relationship between enzyme, substrate, product, and potential regulator concentrations. This revealed three previously unrecognized instances of cross-pathway regulation, which we biochemically verified. One of these involved inhibition of pyruvate kinase by citrate, which accumulated and thereby curtailed glycolytic outflow in nitrogen-limited yeast. Overall, substrate concentrations were the strongest driver of the net rates of cellular metabolic reactions, with metabolite concentrations collectively having more than double the physiological impact of enzymes.


Assuntos
Redes e Vias Metabólicas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Regulação Alostérica , Citratos/metabolismo , Glicólise , Cinética , Nitrogênio/deficiência , Piruvato Quinase/antagonistas & inibidores , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/química
14.
J Vis Exp ; (110): e54239 |, 2016 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-27077531

RESUMO

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment was required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by φ29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 10(10) cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30, HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.


Assuntos
DNA Circular/isolamento & purificação , DNA Fúngico/isolamento & purificação , Herança Extracromossômica/genética , Saccharomyces cerevisiae/genética , DNA Circular/genética , DNA Fúngico/genética , Células Eucarióticas , Genoma , Genoma Fúngico
15.
Genetics ; 201(2): 599-612, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26281848

RESUMO

Studies of natural populations of many organisms have shown that traits are often complex, caused by contributions of mutations in multiple genes. In contrast, genetic studies in the laboratory primarily focus on studying the phenotypes caused by mutations in a single gene. However, the single mutation approach may be limited with respect to the breadth and degree of new phenotypes that can be found. We have taken the approach of isolating complex, or polygenic mutants in the lab to study the regulation of transcriptional activation distance in yeast. While most aspects of eukaryotic transcription are conserved from yeast to human, transcriptional activation distance is not. In Saccharomyces cerevisiae, the upstream activating sequence (UAS) is generally found within 450 base pairs of the transcription start site (TSS) and when the UAS is moved too far away, activation no longer occurs. In contrast, metazoan enhancers can activate from as far as several hundred kilobases from the TSS. Previously, we identified single mutations that allow transcription activation to occur at a greater-than-normal distance from the GAL1 UAS. As the single mutant phenotypes were weak, we have now isolated polygenic mutants that possess strong long-distance phenotypes. By identification of the causative mutations we have accounted for most of the heritability of the phenotype in each strain and have provided evidence that the Mediator coactivator complex plays both positive and negative roles in the regulation of transcription activation distance.


Assuntos
Proteínas de Ligação a DNA/genética , Complexo Mediador/genética , Transcrição Gênica , Ativação Transcricional/genética , Regulação Fúngica da Expressão Gênica , Mutação , Fenótipo , Sequências Reguladoras de Ácido Nucleico/genética , Saccharomyces cerevisiae/genética , Sítio de Iniciação de Transcrição
16.
Proc Natl Acad Sci U S A ; 112(24): E3114-22, 2015 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-26038577

RESUMO

Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥ 15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution.


Assuntos
DNA Circular/genética , DNA Fúngico/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA Circular/isolamento & purificação , DNA Fúngico/isolamento & purificação , Evolução Molecular , Herança Extracromossômica , Variação Genética , Genoma Fúngico , Modelos Genéticos , Mutação , Origem de Replicação
17.
Proc Natl Acad Sci U S A ; 112(19): 6116-21, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918382

RESUMO

Trehalose is a highly stable, nonreducing disaccharide of glucose. A large body of research exists implicating trehalose in a variety of cellular phenomena, notably response to stresses of various kinds. However, in very few cases has the role of trehalose been examined directly in vivo. Here, we describe the development and characterization of a system in Saccharomyces cerevisiae that allows us to manipulate intracellular trehalose concentrations independently of the biosynthetic enzymes and independently of any applied stress. We found that many physiological roles heretofore ascribed to intracellular trehalose, including heat resistance, are not due to the presence of trehalose per se. We also found that many of the metabolic and growth defects associated with mutations in the trehalose biosynthesis pathway are not abolished by providing abundant intracellular trehalose. Instead, we made the observation that intracellular accumulation of trehalose or maltose (another disaccharide of glucose) is growth-inhibitory in a carbon source-specific manner. We conclude that the physiological role of the trehalose pathway is fundamentally metabolic: i.e., more complex than simply the consequence of increased concentrations of the sugar and its attendant physical properties (with the exception of the companion paper where Tapia et al. [Tapia H, et al. (2015) Proc Natl Acad Sci USA, 10.1073/pnas.1506415112] demonstrate a direct role for trehalose in protecting cells against desiccation).


Assuntos
Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Simportadores/metabolismo , Trealose/metabolismo , Transporte Biológico , Carbono/metabolismo , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Temperatura Alta , Maltose/metabolismo , Metabolômica
18.
Cell ; 158(6): 1230-1232, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25215481

RESUMO

The 2014 Lasker∼Koshland Special Achievement Award will be presented to Mary-Claire King, a pioneer and visionary who revolutionized the use of genetics to identify disease genes, provide insights into human evolution, and champion human rights causes.


Assuntos
Distinções e Prêmios , Doença/genética , Genética Médica/história , Animais , Evolução Biológica , Feminino , História do Século XX , Direitos Humanos , Humanos , Estados Unidos
19.
Nucleic Acids Res ; 42(6): e48, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24445804

RESUMO

A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of ß-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input-output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.


Assuntos
Regulação Fúngica da Expressão Gênica , Engenharia Genética , Saccharomyces cerevisiae/genética , Regiões 5' não Traduzidas , Sítios de Ligação , Estradiol/farmacologia , Galactoquinase/genética , Ligantes , Regiões Promotoras Genéticas , Receptores de Estrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Biologia Sintética/métodos , Fatores de Transcrição/metabolismo
20.
J Vis Exp ; (81): e51153, 2013 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-24300440

RESUMO

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.


Assuntos
Proteínas de Fluorescência Verde/genética , Engenharia de Proteínas/métodos , Biologia Sintética/métodos , Fatores de Transcrição/genética , Sequência de Bases , Conexina 43/genética , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/química , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Plasmídeos/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/química , Leveduras/genética , Leveduras/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...