Name: rapid identification of chemical genetic interactions in saccharomyces cerevisiae
Uploaded: 2015-04-05T14:00:00.000+00:00
Duration: 12 min 13 s
Description: Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

Pesquisa no laboratório CJN é apoiado por subsídios operacionais de NSERC, o Instituto Sociedade Canadense do Câncer Research (CCSRI), eo Canadian Breast Cancer Foundation (BC-Yukon Branch).

NOTA: O fluxo de trabalho geral deste procedimento é descrito na Figura 2. 1. Determinação da dose de inibição de crescimento <ol><li> Preparação da cultura de levedura durante a noite NOTA: Os meios de crescimento de levedura-extract-peptone dextrose (YEPD) utilizados neste protocolo é uma receita padrão 13. <ol><li> Expelido BY4741 (MATa his3 Δ1 leu2Δ0 met15Δ0 ura3Δ0) as células em uma placa de agar de YEPD e incubar durante 48 horas a 30 ° C ou até formar colónias visíveis. </li><li> Prepare a culturas de uma noite inoculando 5 ml de meio líquido YEPD num vaso de cultura estéril com uma única colónia. Incubar durante a noite a 30 ° C com rotação contínua ou agitação. A cultura será tipicamente atingir a saturação da manhã (2 x 10 ~ 8 células / ml). </li></ol></li><li> Preparação ágar sólido contendo doses químicas diferentes <ol><li> Prepare diversas unidades populacionais deo produto químico a ser testado em 100x a concentração final desejada em um solvente apropriado. NOTA: As concentrações eficazes irão depender da natureza da substância, e deve ser determinada empiricamente. Por isso, é aconselhável testar inicialmente uma ampla gama de concentração final, ou seja, a partir de baixo ï¿½ a alta mM. </li><li> Para cada concentração de produto químico a ser testado, alíquota de 3 ml de agar de YEPD fundido a um tubo de cultura em vários estéril. Lugar no 55 ° C banho de água para manter-se de solidificação. </li><li> Para cada concentração de produto químico a ser testado adicionar 30 ul do composto a 100x meios fundidos, vortex 2-3 segundos para misturar, e pipeta de 1 ml em cada par de cavidades em duplicado de uma placa de 12 poços. Certifique-se de incluir um veículo único controle. Permitir placas para definir durante a noite à temperatura ambiente. Descarte qualquer mídia extra. </li></ol></li><li> Células se espalham chapeamento <ol><li> Inocular 10 ml de meio líquido YEPD com 2 ul de uma cultura saturada de levedura, cultivada durante a noite, como no Passo 1.1.2. </li><li> Placa de 25 ul de cultura de levedura diluída por poço, espalhe uniformemente com esferas de vidro estéreis ou vara, e permitir que o prato para secar sob uma chama. Incubar a placa a 30 ° C durante 48 h. </li><li> Avaliar o crescimento de leveduras (tanto o número total e tamanho de colónias) nas placas. Escolher a uma concentração sub-letal de composto que não iniba o crescimento em mais de 10% -15% para realizar a tela. </li></ol></li></ol> 2. Systematic Tela Chemical Genetic <ol><li> Manutenção de supressão-mutante-array (DMA) e Preparação de Fonte Placa <ol><li> Para uma descrição detalhada sobre a construção de matrizes de mutantes de deleção de stocks de glicerol consulte Baryshnikova et al. 14. Uma vez que os mutantes de deleção foram dispostas a uma densidade de 384 colónias por placa, o DMA podem ser armazenados durante vários meses a 4 ° C. Replicar em agar de YEPD contendo 200 ug / ml de G418, como necessário para prevenir crescimento de colónias em cadaoutro. NOTA: múltipla repetições em série deve ser evitada para evitar a perda de estirpes de crescimento lento e minimizar o aparecimento de mutantes supressores. Isto é particularmente relevante se a manutenção das coleções como haplóides. </li><li> Execute todas as etapas replica-chapeamento usando um microbiana arraying sistema robótico. Alternativamente, manipular colônias utilizando ferramentas manuais vestiu de aprisionamento. NOTA: A coleção de levedura eliminação está disponível como haplóides e diplóides de várias fontes comerciais e geralmente é enviado como stocks de glicerol em placas de 96 poços. </li></ol></li><li> Condensação da matriz mutante de deleção <ol><li> Preparar 250 ml de caldo YEPD meios de agar contendo 200 ug / ml de G418. A concentração eficaz de G418 pode variar e cada lote deve ser testada empiricamente. </li><li> Preparar cinco placas de agar de YEPD vertendo 50 ml de agar de YEPD contendo 200 ug / ml de G418 por placa. Faça isso um dia antes da condensação da matriz e permitir que as placas para esfriar à temperatura ambienteratura durante a noite em uma superfície lisa. NOTA: Quatro placas serão necessários para a recolha de densidade 1536-deformação, o quinto placa é derramado como um extra. </li><li> Remover os pratos de Petri contendo 16 a recolha mutante de deleção, a uma densidade de 384 colónias por placa e deixar vir à temperatura ambiente (~ 1 hora). </li><li> Limpe qualquer condensação que se formou sobre a tampa de cada placa usando uma tarefa delicada wipe. Não fazer isso pode resultar em gotas de água que está sendo depositado sobre a matriz e contaminação cruzada de mutantes dispostas. </li><li> Usando uma matriz fixando robô microbiana, condensar o DMA de uma densidade de 384 colônias por placa (16 placas de Petri) a 1.536 colônias por placa (4 placas de Petri). Realizar isso para que a 1ª colônia de placa 1 pins para a linha 1 coluna 1 da matriz condensado, o 1º colônia de placa 2 para a linha 1 coluna 2, o 1º colônia de placa 3 a linha 2 e coluna 1, e a colônia 1º de placa 4 a linha 2coluna 2. </li><li> Incubar a matriz consolidada a 30 ° C durante a noite. </li><li> Examine a matriz para assegurar a transferência uniforme de colônias a partir de placas de origem. Haverá várias posições em branco, propositadamente incorporados na matriz, que servem como guia para assegurar o DMA foi condensada correctamente (Figura 4A). NOTA: Estas serão as placas de base para a réplica de placas em meios contendo o composto de teste. A chapa única fonte pode ser usado para várias pinnings. Além disso, muitos robôs terão um recurso de compensação, o que irá garantir um número semelhante de levedura estão sendo transferidos de cada vez. </li></ol></li><li> Placa Replica variedade mutante de deleção <ol><li> Prepare 700 ml de controle (veículo só) e 700 ml de mídia experimental (contendo química). Isto é suficiente para realizar o ensaio em triplicado (12 placas por condição, além de dois extras). </li><li> Despeje 50 ml de agar YEPD contendo substância química de ensaio ou de controlo por placa. Permitir que as placas to esfriar à temperatura ambiente durante a noite em uma superfície lisa. </li><li> Usando o robô para placa réplica, o DMA para inocular três conjuntos de placas que contêm produtos químicos com a concentração determinada experimentalmente, bem como três conjuntos de placas contendo o controlo do veículo. Incubar as placas à temperatura ambiente, durante 24-48 h. </li></ol></li></ol> 3. As placas de imagem e análise de dados <ol><li> Retirar as placas da incubadora e permitir-lhes atingir a temperatura ambiente (~ 1 hora) antes da imagem. Isso vai evitar a formação de condensação enquanto a imagem das placas. </li><li> Placas de imagem em 24 e 48 horas por remover a tampa e colocar o rosto para baixo em um scanner de mesa. Capturar imagens com uma resolução de pelo menos 300 dpi. Como alternativa, use uma câmera digital para capturar imagens. Se fazê-lo, placas lugar enfrentam-se em um fundo escuro e remover as tampas para a imagem. NOTA: O formato de arquivo e de posicionamento das placas dependerá do programa de quantificação utilizados. </li&gt; <li> Execute quantificação e comparação dos tamanhos colônia matriz usando um dos vários programas de código aberto, incluindo Balony 15, SGAtools 16, e ScreenMill 17. </li></ol> 4. Validação de Tela NOTA: Nesta fase, várias exclusões de leveduras mutantes vai marcar como hipersensibilidade à substância química de interesse. Essas interações químicas-genético deve ser validado junto de duas maneiras. Primeiro a identidade de cepas sensíveis devem ser confirmados por PCR. Em segundo lugar, a sensibilidade química deve ser marcado de forma independente. Descritos abaixo é um breve protocolo para realização de ensaios mancha para validar hipersensibilidade tensão, uma técnica comum em biologia levedura. <ol><li> Streak fora desejado (hipersensibilidade) mutantes da coleção eliminação fermento em agar YEPD contendo 200 ug / ml de G418. Incubar a 30 ° C até formar colónias. Verifique se o genótipo da estirpe por PCR com primers de diagnóstico de acordo com ao protocolo do fabricante. </li><li> Inocular 5 ml de líquido YEPD para cada estirpe e incubar durante a noite a 30 ° C com rotação ou agitação. </li><li> Meça OD 600 e diluir cada cepa de DO600 = 1 em dH estéril 2 O. Estes são agora as culturas de levedura normalizados. </li><li> Dispensar 100 ul de normalizada levedura do tipo selvagem (BY4741) a cultura bem A1 de uma placa de 96 poços. Dispensar 100 l de até sete estirpes adicionais para poços B1-H1. </li><li> Use uma pipeta multicanal para dispensar 90 ul de dH 2 O estéril para poços A2-H2 através A6-H6. Finalmente, preparar uma série de diluições 1:10 de culturas de levedura normalizados. Comece serialmente pipetando 10 ul de coluna 1 para a coluna 2 com um pipetador multicanal, e continuar ao longo da placa de 96 poços até seis diluições são feitas (isto é, 10 0 a 10 -5). </li><li> Transferir 2-5 ul de culturas de levedura diluídos em uma grade usando um multicanalpipettor em meios sólidos YEPD contendo controle químico e do veículo. Incubar as placas à temperatura apropriada, durante 24-48 h. </li><li> Inspeccionar placas e comparar a sensibilidade de escolha para mutantes química (em relação ao controle BY4741). Placas de imagem às 24 e 48 horas como no passo 3.2. NOTA: Embora a identificação de várias cepas de hipersensibilidade com ontologias ou funções de genes relacionados empresta confiança para a validade da tela, a transformação de uma estirpe eliminação hipersensibilidade com um plasmídeo contendo uma cópia do tipo selvagem do gene é necessário para estabelecer formalmente que um gene eliminação de interesse (e não escondidos segundo mutações no local) faz com que a sensibilidade de drogas. </li></ol>

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The authors declare they have no competing financial interests.

Esboçado aqui é uma abordagem para a geração de perfis de interação química-genética. O método é simples: comparando dimensões das colónias de cada estirpe em colecções de deleção de genes completos, na presença e ausência de uma substância química, todos os genes necessários para tolerar uma agressão química são identificados. Análise anotação enriquecimento da lista resultante de estirpes sensíveis pode então ser usada para fornecer uma visão sobre o modo de acção de produtos químicos....

As ferramentas e os recursos disponíveis no organismo modelo Saccharomyces cerevisiae genéticas permitiram grande escala genômica funcional estudos que fornecem coletivamente uma nova visão sobre como os genes funcionam como redes de cumprir os requisitos de sistemas biológicos. A pedra angular dessas ferramentas foi a criação colaborativa de um conjunto completo de deleções de genes não essenciais de todos os quadros de leitura aberta em levedura 1,2. Uma observação interessante foi que apenas ~ 20% dos genes de levedura são necessários para a viabilidade, quando cultivada como haplóides, sob condições laboratoriais padrão. Isto realça a capacidade de uma célula para o tampão contra perturbações genómicas, através da utilização de vias alternativas biológicas. Mutantes genéticos que sejam viáveis ​​individualmente, mas em combinação letal, sinalizar caminhos biológicos paralelas ligadas ou convergentes e formam redes de interação genéticas que descrevem a função biológica. Com o desenvolvimento do te condicionalalelos mperatura-sensíveis e hipomórfico de genes essenciais da tecnologia não tem sido limitada para o estudo dos genes não essenciais 3,4. Este conceito foi aplicado em escala genômica produzindo um mapa imparcial interação genética que ilustra como os genes envolvidos em processos celulares similares agrupam 5. Perturbações químicos de redes genéticas deleções de genes imitam (Figura 1) 6. Consultando os compostos inibidores do crescimento contra uma matriz de alta densidade de estirpes de deleção de hipersensibilidade identifica um perfil de interacção química-genético, isto é, uma lista de genes que são necessários para tolerar o stress químico. Como interacções genéticas, telas em larga escala de bibliotecas químicas têm mostrado que os compostos com um modo semelhante de acção de cluster em conjunto 7. Portanto, ao estabelecer o perfil de interação química, genética de um composto o modo de ação pode ser deduzida por comparação com larinteração genética genética e química sintética ge-escala conjuntos de dados de 8,9. Telas químico-genética em larga escala, onde dezenas de compostos são interrogados, foram realizados ensaios de competição por código de barras. Nesta abordagem, o conjunto reunido de estirpes de deleção é cultivado em massa por várias gerações em um pequeno volume de um meio contendo uma substância química. Uma vez que cada mutante de deleção abriga um código de barras genético único, a viabilidade / crescimento de mutantes individuais dentro da piscina de estirpes de deleção é rastreado por microarray ou de alto rendimento seqüenciamento 10. Deduzir aptidão monitorando tamanho da colônia de mutantes fisicamente vestiu cultivadas em agar sólido contendo um composto bioativo também é um método eficaz para identificar as interações químicas genético 11,12. Esta abordagem fornece uma alternativa de baixo custo para triagem baseado na competição e é bem adequado para ensaiar pequenas bibliotecas de produtos químicos.Esboçado aqui é uma metodologia simples para a produção de uma lista de interações químicas-genética em S. cerevisiae que não confia em molecular manipulações de biologia ou de infra-estrutura. Ela exige apenas uma coleção de levedura eliminação, um aparelho de pinagem robótico ou manual e software de análise de imagens disponíveis gratuitamente.

<table><tbody><tr><td>Yeast Extract</td><td>BioBasic</td><td>G0961</td><td>For YEPD liquid/solid media add to 1% final concentration (w/v)</td></tr><tr><td>Tyrptone Powder</td><td>BD Biosciences</td><td>211820</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v)</td></tr><tr><td>Dextrose</td><td>Anachemia</td><td>31096-380</td><td>For YEPD liquid/solid media add to 2% final concentration (w/v) &amp;#8212; do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving.</td></tr><tr><td>Agar A</td><td>Bio Basic</td><td>FB0010</td><td>For YEPD solid media add to 2% final concentration (w/v)</td></tr><tr><td>G418</td><td>A.G. Scientific Inc.</td><td>G-1033</td><td>Prepare 1,000x stock at 200&amp;nbsp;mg/ml in dH&lt;sub&gt;2&lt;/sub&gt;O and filter sterilize.&amp;nbsp;</td></tr><tr><td>12-well plate</td><td>Greiner Bio One</td><td>655180</td><td></td></tr><tr><td>5 ml culture tubes</td><td>Evergreen Scientific</td><td>222-2376-080</td><td></td></tr><tr><td>10 cm Petri Dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>ROTOR HDA</td><td>Singer Instruments&amp;nbsp;</td><td>ROT-001</td><td>high-throughput microbial array pinning robot</td></tr><tr><td>PLUSPLATE&amp;copy; Petri Dish</td><td>Singer Instruments&amp;nbsp;</td><td>PLU-001</td><td>Box of 200 dishes</td></tr><tr><td>384 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-384</td><td>Box of 1,000 pads</td></tr><tr><td>1536 Short-Pin RePad</td><td>Singer Instruments&amp;nbsp;</td><td>RP-MP-1536</td><td>Box of 1,000 pads</td></tr><tr><td colspan="4">Alternative Pinning Tools:</td></tr><tr><td>Fully Automated Robtic Systems</td><td>S&amp;amp;P robotics</td><td>http://www.sprobotics.com</td><td>Several automated colony handling robitic and imagining systems available.</td></tr><tr><td>Manual Pinning Tools</td><td>V&amp;amp;P Scientific</td><td>http://www.vp-scientific.com</td><td>Handheld replication tools and accessories.&amp;nbsp;</td></tr></tbody></table>

rapid identification of chemical genetic interactions in saccharomyces cerevisiae

Determinando o modo de acção de produtos químicos bioactivos é de interesse para uma larga gama de académico, farmacêuticos, industriais e cientistas. Saccharomyces cerevisiae, ou levedura de brotamento, é um modelo eucariota para que um conjunto completo de ~ 6000 mutantes de deleção do gene e gene essencial hipomórfico mutantes estão disponíveis comercialmente. Estas colecções de mutantes pode ser usado para detectar interacções químicas sistematicamente-gene, isto é, genes necessários para tolerar um produto químico. Esta informação, por sua vez, comunica com o modo provável de acção do composto. Aqui nós descrevemos um protocolo para a rápida identificação de interações químicas-genética em brotamento da levedura. Demonstramos o método usando o agente quimioterapêutico 5-fluorouracilo (5-FU), que tem um mecanismo bem definido de acção. Os nossos resultados mostram que o TRAMP nuclear RNA e de exossoma enzimas de reparação de ADN são necessários para a proliferação na presença de 5-FU, o que é consistente com m anterioricroarray abordagens genéticas baseadas em códigos de barras de produtos químicos e o conhecimento de que 5-FU afeta adversamente o metabolismo de RNA e DNA. Os protocolos de validação necessárias dessas telas de alto rendimento são também descritos.

Como uma validação desta abordagem foi realizado um rastreio genético química representativa interacção do agente quimioterapêutico 5-fluorouracilo (5-FU), seguindo o protocolo descrito acima. 5-FU é conhecido por perturbar a timidilato-sintase, assim como o metabolismo do ADN e ARN 18. As interações químicas genéticos de 5-FU são bem estudados e foram investigados por meio de técnicas de levedura de código de barras de microarranjos utilizando ambas as coleções de deleção heterozigotos e h...

Watch this Scientific Journal Video about Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae at JoVE.com

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Identificação rápida de químicos Genéticos Interações em<em&gt; Saccharomyces cerevisiae</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

rapid-identification-chemical-genetic-interactions-saccharomyces

Research

JoVE Journal

Biology

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

10.3K visualizações.  University of Victoria. Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Vídeo: Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident processes. Formaldehyde crosslinking followed by chromatin immunoprecipitation and high-throughput sequencing (X-ChIP-seq) has been used to gain many valuable insights into genome biology. However, X-ChIP-seq has notable limitations linked to crosslinking and sonication. Native ChIP avoids these drawbacks by omitting crosslinking, but often results in poor recovery of chromatin-bound proteins. In addition, all ChIP-based methods are subject to antibody quality considerations. Enzymatic methods for mapping protein-DNA interactions, which involve fusion of a protein of interest to a DNA-modifying enzyme, have also been used to map protein-DNA interactions. We recently combined one such method, chromatin endogenous cleavage (ChEC), with high-throughput sequencing as ChEC-seq. ChEC-seq relies on fusion of a chromatin-associated protein of interest to micrococcal nuclease (MNase) to generate targeted DNA cleavage in the presence of calcium in living cells. ChEC-seq is not based on immunoprecipitation and so circumvents potential concerns with crosslinking, sonication, chromatin solubilization, and antibody quality while providing high resolution mapping with minimal background signal. We envision that ChEC-seq will be a powerful counterpart to ChIP, providing an independent means by which to both validate ChIP-seq findings and discover new insights into genomic regulation.

Genome-wide Mapping of Protein-DNA Interactions with ChEC-seq in Saccharomyces cerevisiae

We describe chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq), a chromatin immunoprecipitation (ChIP)-orthogonal method for mapping protein binding sites genome-wide with micrococcal nuclease (MNase) fusion proteins.

Mapeamento genômico de interações proteína-DNA com ChEC-seq em<em&gt; Saccharomyces cerevisiae</em

Genome-wide mapping of protein-DNA interactions is critical for understanding gene regulation, chromatin remodeling, and other chromatin-resident ...

Genética

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

Uma tela de supressor de Deep-sequenciamento-assistida, espontânea na fissão levedura Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

Mapeamento genético de diferenças de termotolerância entre espécies de levedura Saccharomyces via análise de Hemizygosidade recíproca em todo o genoma

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificação de complexos de proteínas com proteômica quantitativa em S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biologia

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

Detecção de Intermediários de Recombinação Homóloga via Ligadura de Proximidade e PCR Quantitativa em Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, and release of the complex components. Glycerol gradient centrifugation has been used for the separation of protein or RNP (RiboNucleoProtein) complexes for functional and structural studies. Here, we describe the utilization of Grafix (Gradient Fixation), which was first developed to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy, to identify interactions between splicing factors that bind transiently to the spliceosome complex. This method is based on the centrifugation of samples into an increasing concentration of a fixation reagent to stabilize complexes. After centrifugation of yeast total extracts loaded on glycerol gradients, recovered fractions are analyzed by dot blot for the identification of the spliceosome sub-complexes and determination of the presence of individual splicing factors.

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Here, we describe the utilization of Grafix (Gradient Fixation), a glycerol gradient centrifugation in the presence of a crosslinker, to identify interactions between splicing factors that bind transiently to the spliceosome complex.

Utilização de Grafix para a detecção de Interagentes Transintes de Saccharomyces cerevisiae Subcomplexos Spliceosome

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Bioquímica

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Microarray Analysis for Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Competitiva Screens Genomic de Bibliotecas Yeast Barcoded

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Determinação baseado no crescimento e confirmação bioquímica de Requisitos Genéticos para a degradação de proteínas em<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Triagem Genética para Identificação de Supressores Multicópia em Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Identificação rápida de químicos Genéticos Interações em

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