Este trabalho foi financiado pelo Programa Ciência das Fronteiras Humanas concessão RGP0024.

1. Preparação de ADN de modelo e transcrição in vitro <ol><li> O gene de interesse é clonado sob T7 e / ou, por exemplo SP6 promotor transcricional. </li><li> Para transcrição in vitro do DNA molde é linearizado com uma enzima de restrição apropriada de corte a jusante do codão de paragem ORF e / ou mRNA extremidade 3 &#39;. Uma das necessidades para verificar a linearização completa do DNA de plasmídeo, executando o produto da digestão de restrição em electroforese em gel de agarose. </li><li> O plasmídeo linearizado é usado para reacção de transcrição in vitro. Concentração diferente de ADN molde pode ser testado para identificar a concentração de DNA óptima necessária para transcrição in vitro. Geralmente, com Kit Ambion da transcrição de alto rendimento MEGAscript (Ambion / Life Technologies, Grand Island, NY), 1 ug de DNA linearizado produz 40-60 ug de mRNA. Transcrição in vitro é efectuado de acordo com as instruções do fabricante (Ambion / Life Technologies, Grand Island, NY). </li><li> Após transcrição in vitro do ARNm é purificado por precipitação com cloreto de lítio de acordo com as instruções do fabricante (Kit Ambion da transcrição MEGAscript de alto rendimento). </li><li> integridade do mRNA é ainda verificado por electroforese em géis de acrilamida ou de agarose. </li></ol> 2. Tradução de células in vitro gratuita Para a tradução in vitro utilizando R abbit R eticulocyte Lysate, RRL (Promega, Madison, WI) siga os passos abaixo: <ol><li> Preparar 100 ul na instrução tradução in vitro fabricante seguinte reacção de. Resumidamente, em água livre de nuclease adicionar 2 ul mistura de ácido amino menos metionina (1 mM), 10 unidades de RNase inibidor (Invitrogen / Life Technologies, Grand Island, NY), 20 uCi de radioactivos [35 S]-Metionina Biomedicals (MP, Solon, OH), 70 ul de reticulócitos de coelho Lysate (Promega, Madison, WI). </li><li> Incubar ªreacção e, a 30 ° C durante 5 min de pré reacção a quente de tradução. Adicionar 2 ug de mRNA para a reacção de tradução e incubar a 30 ° C durante 5 min. </li><li> Parar a reacção através da colocação da reacção de tradução em gelo. </li></ol> 3. Isolamento de polipéptidos a partir de nascentes in vitro reacção de tradução <ol><li> Para isolar polipéptido nascente ligado a ribossomas (polissoma), a reacção de tradução é colocado em camadas sobre 4,5 ml de glicerol 30% em 10 mM de tampão Tris-HCl pH 7,6, contendo 100 mM de KCl, 10 mM de MgCI 2, e centrifugado a 100.000 xg em um TLA-110 do rotor (Beckman Coulter, Inc, Brea, CA) durante 1 hora a 4 ° C. </li><li> O sobrenadante é ainda mais cuidadosamente removido. </li><li> O sedimento contendo polipéptidos polissomal nascentes é ressuspenso em um pequeno volume (10-15 uL) de 1 mM de tampão Tris-HCl pH 7,6, contendo 0,5 mg / ml ribonuclease A, (Invitrogen / Life Technologies, Grand Island, NY) e incubadas durante 30 min a 37 ° C. A fimpara aumentar a hidrólise do peptidil-tRNA éster ligação, NaOH é adicionado a uma concentração final de 10 mM, ea incubação é continuada durante mais 30 min. </li></ol> In vitro reacção de tradução montado sem mRNA e executada sob as mesmas condições (seguido por isolamento de péptidos nascentes como foi descrito) serviria como um controlo principal. O tratamento da reacção de tradução (s) com puromicina antes de se submeter o extracto de centrifugação em gradiente de densidade pode servir como um controle adicional. 4. Resolvendo o Polipeptídeo Nascente em SDS-PAGE Tris-tricina <ol><li> Os polipéptidos isolados nascentes são resolvidos e analisados ​​em Tris-tricina SDS-PAGE. Note que a quantidade do material carregado no gel será dependente da eficiência de rotulagem de proteína, a eficiência de tradução da proteína e os parâmetros de muitos outros. A quantidade de material carregado deve ser ajustado empiricamente para permitir que os besvisualização T e separação de individuais nascentes cadeias polipeptídicas. </li><li> Depois de géis de electroforese são fixados, secou-se utilizando vácuo gel Dyer e submetido a autorradiografia. A distribuição de polipéptidos nascentes são observados utilizando phosphoimaging. </li></ol> 5. Os resultados representativos Bovina Gamma-B cristalina foi traduzido em lisado de reticulócitos de coelho, bem como em E. coli S30 Extracto Systems (Promega, Madison, WI), seguido por isolamento de nascentes cadeias polipeptídicas. A Figura 1 ilustra os passos envolvidos no isolamento de ribossomas ligados cadeias nascentes. No presente estudo o objectivo foi testar, se a alteração no ambiente de repertório tradução isto é, de tRNAs (conhecido como sendo substancialmente diferente em mamíferos (RRL) e procarióticos (E. coli) sistemas devido a diferenças na polarização codão entre os organismos) pode alterar movimento ribossomo ao longo do mRNA e efetuar a distribuição dos locais de pausa de tradução.Movimento ribossoma Altered deve conduzir a alterações na distribuição de tamanhos de polipéptidos nascentes que se acumulam durante a tradução, bem como as suas intensidades relativas. Intensidades relativas das bandas função da duração da pausa. Os polipéptidos nascentes foram isolados e resolvidos em 16,5% T, 6% C Tris-tricina SDS-PAGE (Figura 2). Gama-B cristalina é uma proteína de 20 kDa. A observação de polipéptidos nascentes de não idênticos comprimentos e intensidades em RRL e E. coli sistemas indica que o movimento de ribossomas ao longo do mRNA em estes dois sistemas segue uma cinética de tradução diferentes. Por exemplo, observamos uma banda de destaque de 17,7 kDa em E. coli lisado e não no sistema RRL, sugerindo que há um local de pausa adicional translacional na região 3&#39;-fim de ARNm (codificação região C-terminal da gama B cristalina), quando ela é traduzida em E. coli do sistema. Notamos que Gamma-B cristalina portos conjunto Arg códons (AGG 153 e AGA 154) que são frequentes em sistemas de mamíferos, mas são conhecidos por serem extremamente raro e lentamente traduzida em E. coli. um novo peptídeo nascente acumulando no E. sistema coli (~ 17,7 kDa Figura 2.2) é provavelmente causado pelo ribossomo pausa nesses códons raros em tandem. Note-se que nenhuma banda pode ser observado na ausência de mRNA (Figura 2.3) e que o tratamento puromicina remove a maioria das cadeias nascentes de polissomas (Figura 2.3). O tratamento prolongado com puromicina (&gt; min 15) remove todas as cadeias de nascente (dados não mostrados). Isto confirma que os produtos observados são polipeptídeos ribossomas associados cadeias nascentes, ao invés de mRNPs cosedimenting com polissomos. Como mencionado acima, a polarização codão é organismo, bem como o tecido específico. O exemplo representativo descrito aqui, indica claramente que a mudança do ambiente do repertório de tradução ou seja, de tRNAs / códonpolarização pode levar a circulação alterada do ribossoma ao longo do mRNA. Evidentemente, esta técnica simples não só pode permitir a identificação dos sítios de pausa de translação ao longo de ARNm, mas também permite a comparação rápida dos sítios de tradução de pausa entre sistemas diferentes. <img alt="A Figura 1" src="/files/ftp_upload/4026/4026fig1.jpg" /> Figura 1. Esquemático explicando as etapas envolvidas no isolamento ribossomas ligados polipeptídeos nascentes. Bovina Gamma-B cristalina seqüência ORF (528 pares de bases) foi clonado a jusante do promotor T7 em pBSKS +. Para transcrição in vitro do DNA molde foi linearizado por tratamento com EcoRI. Modelo linearizado foi utilizado para transcrição in vitro. T7 promoter em molde de ADN é reconhecido por T7 RNA polimerase que transcreve a jusante Gamma B-crystallin gene. mRNA é purificado utilizando lítio método de precipitação de cloreto. O mRNA purificado é utilizado para tradução in vitro. Seguintede incubação, a reacção de tradução é em camadas no topo de solução de glicerol a 30% e centrifugado durante 1 hr a 4 ° C a 100.000 xg para isolar bound ribossomas polipéptidos nascentes. <img alt="A Figura 2" src="/files/ftp_upload/4026/4026fig2.jpg" /> Figura 2. Isolamento de Bovinos polipeptídeos Gamma-B cristalina nascentes traduzidas em reticulócitos de coelho Lysate (RRL) e E. Lisado coli. 2 mRNA ug foi misturado em 100 ul de reacção contendo RRL ou E. coli S30 Extracto System (Promega, Madison, WI) de acordo com as instruções do fabricante, com 20 uCi [35 S]-metionina e incubadas a 30 ° C durante 5 min. Após a incubação a reacção de tradução foi derramado por cima de 4,5 ml de glicerol 30% em 10 mM de tampão Tris-HCl pH 7,6, contendo 100 mM de KCl, 10 mM de MgCI 2, e centrifugou-se durante 1 hora a 4 ° C e 100.000 X g em um TLA-110 do rotor (Beckman Coulter, Inc, Brea, CA). O sedimento foi isolado polyribosome resuspended em 20 uL de 1 mM de Tris-HCl pH 7,6, contendo 0,5 mg / ml ribonuclease A (Invitrogen / Life Technologies, Grand Island, NY), seguido por tratamento com NaOH (como descrito na secção de protocolo). Polipéptidos nascentes foram resolvidos em 16,5% T, 6% C Tris-Tricina gel SDS PAGE. O gel foi seco e exposto para autorradiografia. Após a exposição do gel foi digitalizada utilizando Scanner GE Healthcare Imaging da Typhoon. A Figura 2.1 mostra o gel com polipéptidos nascentes isoladas e resolvido depois reacções de tradução feito em dois sistemas diferentes (RRL e E. coli). Figura 2.2 O gel foi analisado por imagem Quant TL ., v2005 software e usado aqui como um exemplo para mostrar que o peso molecular ea intensidade dos polipéptidos nascentes que se acumulam em dois sistemas podem ser facilmente analisadas e comparadas Figura 2.3 mostra dois conjuntos de controlos: polipéptidos nascentes isolados e resolvidos em SDS-PAGE após a tradução As reacções foram montadassem ARNm e / ou após as reacções de tradução foram tratados com puromicina (5 min, conc final. 1 mM) antes da reacção foi submetida a centrifugação.

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Não há conflitos de interesse declarados.

Para obter resultados reprodutíveis, a qualidade ea concentração dos componentes utilizados para transcrição in vitro e reacções de tradução são críticos. No presente estudo, utilizamos kits disponíveis comercialmente e extratos que fornecem dados altamente reprodutíveis, se manuseados com cuidado. No entanto, a tradução-competentes extractos podem ser preparados a partir da célula da sua escolha, se necessário. Qualidade de ARNm pode afectar a tradução, por isso, é de extrema impor...

<table border="1"><tbody><tr><td> Nome de reagente / Kit </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> MEGAscript T7 Kit de Transcrição de alto rendimento </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> Inibidor da ribonuclease </td><td> Invitrogen </td><td> 15518012 </td></tr><tr><td> Trans [35 S]-Label </td><td> Biomedicals MP </td><td> 0151006 </td></tr><tr><td> Ribonuclease-A </td><td> Invitrogen </td><td> 12091 </td></tr><tr><td> Sistema de Lisado de reticulócitos de coelho, Nuclease Tratada </td><td> Promega </td><td> L4960 </td></tr><tr><td> E. coli S30 Sistema de Extrato de Modelos Lineares </td><td> Promega </td><td> L1030 </td></tr><tr><td> Centrifugação </td><td> Beckman Coulter </td><td> Optima ultracentrífuga TLX </td></tr><tr><td> Armazenamento de fósforo autorradiografiay </td><td> GE Healthcare </td><td> Typhoon 9410 imager modo variável </td></tr><tr><td> Software para análise polipeptídeo nascente </td><td> GE Healthcare </td><td> Imagem Quant TL, v2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

A taxa de alongamento de translação é não-uniforme. mRNA estrutura secundária, uso de códon mRNA e proteínas associadas podem alterar movimento ribossomo na mensagem para revisão ver 1. No entanto, é agora amplamente aceito que o uso de codons sinônimos é a principal causa da não-uniformes taxas de alongamento de translação 1. Codons sinônimos não são utilizados com freqüência idêntica. Um viés existe no uso de codons sinônimos com alguns códons utilizados com mais freqüência do que os outros 2. Códon viés é organismo, assim como tecidos específicos 2,3. Além disso, a frequência de utilização de codões é directamente proporcional à concentração de tRNAs cognato 4. Assim, um codão frequentemente utilizadas terão maior multiplicidade de tRNAs correspondentes, o que implica ainda que um codão frequente será traduzida mais rápido do que um um pouco frequentes. Assim, as regiões de mRNA enriquecido em códons raros locais (pausa potenciais), regra geral diminuam o movimento ribossomo na message e causa acúmulo de peptídeos nascentes dos respectivos tamanhos 5-8. Estes sites de pausa pode ter impacto funcional sobre a expressão de proteínas, a estabilidade do mRNA e enovelamento de proteínas para revisão ver 9. Com efeito, foi demonstrado que a atenuação dos sítios de pausa tais pode alterar movimento ribossoma em ARNm e, subsequentemente, pode afectar a eficiência da co-translacional enrolamento de proteínas (in vivo) 1,7,10,11. Para compreender o processo de enrolamento de proteínas in vivo, na célula, que é finalmente acoplado ao processo de síntese de proteína que é essencial para obter uma percepção abrangentes para o impacto do codão de conteúdo de uso / tRNA sobre o movimento dos ribossomas ao longo do mRNA durante o alongamento translacional . Descrevemos aqui uma técnica simples que pode ser usado para localizar locais de tradução importantes de pausa para uma dada mRNA traduzido em vários sistemas isentos de células de 6-8. Este procedimento é baseado no isolamento de polipéptidos nascente accumulating em ribossomas durante a tradução in vitro de um mRNA alvo. A razão é que a baixa frequência codões, o aumento no tempo de residência dos resultados ribossomas em quantidades aumentadas de péptidos nascentes dos tamanhos correspondentes. Em mRNA transcrito in vitro é utilizado para reacções de translação in vitro na presença de marcados radioactivamente aminoácidos para permitir a detecção das cadeias nascentes. A fim de isolar bound ribossomas nascentes complexos polipeptídicas a reacção de tradução é em camadas no topo de solução de glicerol a 30%, seguido por centrifugação. Polipéptidos nascente na polissomal pelete são ainda tratadas com ribonuclease A e resolvidos por SDS-PAGE. Esta técnica pode ser potencialmente usada para qualquer proteína e permite a análise de movimento ao longo do mRNA ao ribossoma e à detecção dos sítios de pausa principais. Além disso, este protocolo pode ser adaptado para estudar factores e condições que podem alterar o movimento ribossoma e assim potencialmente também podem alterar the função / conformação da proteína.

Watch this Scientific Journal Video about Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA at JoVE.com

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

Uma técnica para identificar locais de pausa de translação sobre mRNA é descrito. Este procedimento é baseado no isolamento de polipéptidos nascentes que se acumulam nos ribossomas durante in vitro tradução de um ARNm alvo, seguido de análise de tamanho das cadeias nascentes utilizando uma electroforese em gel de desnaturação.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

isolation-ribosome-bound-nascent-polypeptides-vitro-to-identify

Research

JoVE Journal

Biology

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

16.2K visualizações.  Cleveland State University. Uma técnica para identificar locais de pausa de translação sobre mRNA é descrito. Este procedimento é baseado no isolamento de polipéptidos nascentes que se acumulam nos ribossomas durante in vitro tradução de um ARNm alvo, seguido de análise de tamanho das cadeias nascentes utilizando uma electroforese em gel de desnaturação.

Vídeo: Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Clip - um método para identificar em toda a Transcriptoma Sítios de Ligação de Proteínas RNA Binding

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biologia

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

Isolamento e In vitro A ativação do Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

Usando SCOPE para identificar potenciais Motivos regulamentação em Genes Coregulated

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Transcrição in vitro e nivelamento de Gaussia mRNA luciferase Seguido por HeLa transfecção celular

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

In Vitro Síntese de ARNm de modificação para indução da expressão da proteína em células humanas

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Medição de resistência de Estresse Oxidativo<em&gt; Caenorhabditis elegans</em&gt; Em placas de 96 poços de microtitulação

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Exame de proteínas ligadas ao DNA em células de mamíferos Nascente usar a técnica de-BrdU-chip-Slot Ocidental

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

Um protocolo para o uso de Gene definir análise de enriquecimento para identificar o modelo Animal adequado para investigação de translação

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

In Vitro Ensaio de SUMOylation para estudar a atividade de Ligase E3 sumô

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...