Os autores gostariam de agradecer as Dras. Philina S. Lee e Alan D. Grossman para a formação inicialmente LAS em microscopia de fluorescência. Os autores também agradecer as Dras. Melanie Berkmen e Hajime Kobayashi para a ajuda e dicas para a imagem latente. Este trabalho foi financiado por fundos start-up da Faculdade de Literatura, Ciência e Artes e do Departamento de Biologia Molecular, Celular e do Desenvolvimento na Universidade de Michigan.

Culturas em crescimento de células para microscopia 1. Um ou dois dias antes da imagem, prepare o B. subtilis cepa contendo a proteína de fusão traducional você deseja visualizar. Rounds julgamento de passos 1A e 1B será necessário determinar qual condição de crescimento fornece as melhores imagens da sua estirpe. Na maioria dos casos, as células devem ser trabalhada durante a fase de crescimento exponencial. <ol class="ualpha"><li> Para alguns B. subtilis cepas (em particular, cepas que crescem pouco devido à integração da fusão de translação entre o gene de interesse e GFP em seu lócus endógeno), o crescimento inicial de dois dias antes da imagem é necessário para imagens de alta qualidade. No primeiro dia raia, o B. subtilis estirpe a ser trabalhada em ágar LB com antibiótico seletivo (s) e incubar durante a 30 ° C. No segundo dia, inocular 100 ml de solução salina a 0,85%, com uma única colônia e realizar três de 10 diluições em placas de agar LB (com antibióticos seletiva), chapeamento de 100 L de cada diluição, seguido de incubação durante a noite a 30 ° C. No terceiro dia, selecione a placa de diluição com crescimento confluente luz de células. Crescimento confluente luz sobre uma placa irá fornecer o meio de crescimento com uma alimentação saudável, em crescimento exponencial de inóculo inicial para, potencialmente, produzir resultados excelentes imagens (veja o passo 4). Esta etapa é particularmente útil para a maior qualidade de imagem de membrana com a mancha FM4-64. </li><li> B. Outros cepas subtilis pode simplesmente ser aplicada a meio seletivo agar LB para colônias único com uma vara estéril do dia anterior à imagem e incubar durante a 30 ° C. No dia seguinte, as células nesta placa será suficiente para usar como um inóculo para geração de imagens (veja o passo 4). NOTA: Para B. subtilis, culturas durante a noite em meio líquido não são apropriados devido a uma combinação de formação de esporos e respostas quorum, o que irá causar um período prolongado de crescimento lag fase 1. </li></ol> 2. Na manhã de imagem, mL colocar 10 de média 50 S7 03/01 em um Erlenmeyer 125 mL estéril. 3. Ressuspender as células em até 2 mL de meio S7 50 da placa de ágar LB com colónias individuais ou as células confluentes luz para obter um inóculo. 4. Adicione o suficiente S7 50 inóculo médio célula para o Erlenmeyer contendo 10 mL 50 S7 meio de uma cultura com uma densidade óptica medida inicial em 600 nm (OD 600) de ~ ,08-,1. Nesta etapa é importante ter pelo menos o ar a 10 vezes a taxa média. Assim, usamos 10-12,5 mL de meio inoculado em 125 mL Erlenmeyer. 5. Crescer cada cultura a 30 ° C até a cultura atingir uma OD 600 ~ 0,5-0,8. Banhos de água balançando são preferidos com rotações por minuto 150-200. Culturas desafiados com agentes indutores danificar o DNA ou o desfasamento deve estar em uma fase inicial de crescimento exponencial OD 600 de tal forma que as células vão atingir a meta de OD 600 com o período adicional de crescimento que é necessário para o tratamento (ver Tabela 1) Por exemplo, 2-aminopurina requer uma hora de tratamento, e, portanto, uma cultura tratado com 2-aminopurina deve ser a ~ 0,3-0,4 OD 600, de modo que a uma hora de tratamento / crescimento resulta em uma OD 600 ~ 0,5-0,8, pelo qual as células estão prontos para a imagem. Preparação da Amostra 1. Pipetar 300 mL de células cultivadas em um tubo de microcentrífuga de 1,5 mL. 2. Se imagens das membranas celulares é desejada, adicione a diluição de 1:1000 FM4-64 membrana mancha (Invitrogen) para cada amostra a partir de uma solução estoque de 1 mg / mL. A titulação de FM4-64 pode ser necessária para atingir o desejado sinal de fluorescência. Uma gama de titulação típica é 1:100, 1:1000 e 1:10.000. 3. Deixe que as amostras à temperatura ambiente por até 10 minutos ou enquanto o slide está sendo preparada com agarose 1% em 1X médio Spizizens conforme descrito abaixo (veja o passo 1 de &quot;preparação da lâmina&quot;). 4. Se a concentração de células é necessário, nós preferimos células filtragem utilizando um filtro de 0,2 m com um aparelho de vácuo. Células podem então ser lavada suavemente a partir da superfície do filtro com PBS, outro tampão apropriado ou médio antes de visualização. Preparação de slides 1. Prepare agarose 1% com 1X Spizizens. Adicionar 5 mL de estoque Spizizens 10X a 45 ml ddH2O juntamente com 0,5 g de agarose, e microondas para derreter. Hot agarose fundida é difícil de pipeta, e solidificou agarose não vai entrar na ponta da pipeta. A agarose deve ser equilibrada em banho-maria a uma temperatura de ~ 55-65 ° C para que entre a ponta da pipeta sem se solidificar dentro do<html> ponta. 2. Desenhe 20 l de Spizizens com 1% de agarose derretida na ponta da pipeta. Pipetar uma gota (cerca de 1-2 mL) da solução de agarose em cada slide bem (nós usamos 15 slides bem, ver Tabela 2) para formar blocos de agarose rasas. A agarose solidifica imediatamente. Slides também deve ser feita imediatamente antes da aplicação das células. Se as almofadas de agarose são deixados à temperatura ambiente por si só, podem desidratar em minutos e se tornar inutilizável. Almofadas de agarose deve ser centrado e preencha cada bem, mas tem altura mínima para evitar interromper as lamelas, se gotículas são muito altos (tipo bolha) ou se a amostra se espalha além do limite bem, passe simplesmente o limpa bem com um Kimwipe e aplicar um nova agarose pad. 3. Aplicar a amostra 300 mL inteira (consulte a etapa 6) para o slide, distribuídos na parte superior de cada bloco de agarose. Esta quantidade de células deve ser suficiente para cobrir cada bem no slide. 4. Permitir que o slide carregado com células a sentar-se à temperatura ambiente (23 ° C) por aproximadamente 10 minutos. Essa etapa permitirá que para as células a se estabelecer na almofada agarose tornando-se imóvel e pronto para geração de imagens. Para necessidades específicas esta temperatura pode ter de ser alterado, por exemplo, quando a realização de experimentos que requerem uma mudança de temperatura 4. 5. Aspirar o meio afastado o excesso de cada poço, sem retirar as almofadas si. 6. Aplicar a lamínula para o slide, pressionando com firmeza e de maneira uniforme, mas com cuidado, em toda a área do slide. Certifique-se que a lamínula é contra o slide e que a lamínula não é aumentado acima do slide. Visualização de células 1. Muitos diferentes microscópios de fluorescência vai funcionar bem. É fundamental ter uma lente de imersão em óleo de 100X. O Laboratório de Simmons usa: <ul><li> Olympus BX61 equipado com microscópio de 1,45 TIRFM 100X óleo NA lente objetiva de imersão </li><li> Hamamatsu ORCAR 2 CCD refrigerado câmera </li><li> Lumen arco metálico 200 (prévia) fonte de luz. </li><li> Para a detecção de GFP (FITC), excitação e emissão de filtro 460-500 510-560 </li><li> Para a detecção de FM4-64 (TRITC), filtro de excitação e emissão 510-560 572-648. </li><li> As imagens foram capturadas SlideBook 4.2 (Figura 1) </li></ul> 2. Trazer as células para o foco usando luz branca. 3. Capturar a imagem usando filtros adequados para cada fluoróforo. Resultados representante Imagens representativas são mostradas (Figura 1). GFP focos devem ser bem definidos, enquanto FM4-64 coloração deve ser brilhante e claro 4-7. Imagens GFP ou membrana pode ser pseudo-colorida com qualquer cor, para permitir a imagem da mais alta qualidade a serem apresentados sem perder quaisquer dados. <img src="/files/ftp_upload/1736/1736fig1.jpg" alt="Figura 1" /> Figura 1: Imagens GFP Representante do B. subtilis proteínas de fusão de translação. proteínas GFP são mostrados em verde, enquanto a coloração FM4-64 membrana é mostrado em vermelho. A barra de escala branca indica 3 mm (A) mutS-GFP [genótipo relevantes: mutS - gfp (SPC), amyE:: Pspac mutL (cat)]. Na presença de 600 mcg / mL 2 aminopurina e 1 mM IPTG. FM4-64 do sinal não é apresentada, a fim de mostrar mais claramente os focos mutS-GFP (B) MutL-GFP [genótipo relevantes: mutL:: mutL-GFP (spc)].. Na presença de 2-aminopurina (C) DNAX-GFP [genótipo relevantes: DNAX:: DNAX-GFP (spc)]. (D) RecA-GFP [genótipo relevantes: recA:: recA-GFP (spc)] na presença de 20 ng / mL mitomicina C. (E) TAGC-GFP genótipo [relevantes: TAGC:: TAGC-GFP (spc) ] na presença de 1 mg / mL mitomicina C. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tratamento / concentração ou quantidade </td><td> Função </td><td> Tempo de incubação </td></tr><tr><td> Mitomicina C (MMC) [20 e 150 ng / mL] </td><td> DNA agente alquilante </td><td> Uma hora </td></tr><tr><td> 2 aminopurina-(2-AP) [600 mcg / ml] </td><td> indução de incompatibilidade </td><td> Uma hora </td></tr><tr><td> Hidroxiureia (HU) </td><td> esgota dNTPs </td><td> 3 horas </td></tr><tr><td> luz ultravioleta (UV) 20 J/m2 </td><td> Timina-timina dímeros e 6-4 fotoprodutos </td><td> varia dependendo da fonte, geralmente 20 segundo </td></tr><tr><td> grays (radiação ionizante) 5 a 100 Gy </td><td> Dupla vertente quebra, rupturas dos filamentos simples e sites dano base </td><td> varia dependendo da fonte </td></tr></tbody></table>&quot;&gt; Tabela 1. Resumo de danificar o DNA e tratamentos de indução de incompatibilidade <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Multiteste slide, 15 bem </td><td> Biomedicals MP </td><td> 6041505E </td><td></td></tr><tr><td> Tampa de vidro de microscópio </td><td> Pescador </td><td> 12-544-B </td><td></td></tr><tr><td> Agarose </td><td> Pescador </td><td> BP160-500 </td><td></td></tr><tr><td> Mitomicina C </td><td> Sigma </td><td> M0503-2mg </td><td> mutagênico, usar luvas </td></tr><tr><td> 2-aminopurina </td><td> Sigma </td><td> A3509-250mg </td><td> luvas </td></tr><tr><td> Hidroxiuréia </td><td> Sigma </td><td> H8627-25G </td><td> luvas </td></tr><tr><td> FM4-64 </td><td> Invitrogen </td><td> T13320 </td><td> sensível à luz </td></tr></tbody></table> Tabela 2. Lista de reagentes e equipamentos específicos:

<ol>
	<li>Hardwood, C. R., Cutting, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Biological Methods for Bacillus. , (1990).</li><li>Berkmen, M. B., Grossman, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+organization+of+the+Bacillus+subtilis+replication+cycle.">Spatial and temporal organization of the Bacillus subtilis replication cycle.</a> Mol. Microbiol. 62, 57-71 (2006).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clp+and+Lon+proteases+occupy+distinct+subcellular+positions+in+Bacillus+subtilis.">Clp and Lon proteases occupy distinct subcellular positions in Bacillus subtilis.</a> J. Bacteriol. 190, 6758-6768 (2008).</li><li>Simmons, L. A., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+is+required+for+the+RecA+localization+response+to+DNA+damage+in+Bacillus+subtilis.">Replication is required for the RecA localization response to DNA damage in Bacillus subtilis.</a> Proc. Natl. Acad. Sci. U S A. 104, 1360-1365 (2007).</li><li>Simmons, L. A., Davies, B. W., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=b+Clamp+Directs+Localization+of+Mismatch+Repair+in+Bacillus+subtilis.">b Clamp Directs Localization of Mismatch Repair in Bacillus subtilis.</a> Mol. Cell. 29, 291-301 (2008).</li><li>Simmons, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+responses+to+double-strand+breaks+between+Escherichia+coli+and+Bacillus+subtilis+reveals+different+requirements+for+SOS+induction.">Comparison of responses to double-strand breaks between Escherichia coli and Bacillus subtilis reveals different requirements for SOS induction.</a> J. Bacteriol. 191, 1152-1161 (2009).</li><li>Smith, B. T., Grossman, A. D., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+mismatch+repair+in+bacterial+cells.">Visualization of mismatch repair in bacterial cells.</a> Mol. Cell. 8, 1197-1206 (2001).</li></ol>

Tentativa e erro são necessários para encontrar as condições de exposição de imagens da mais alta qualidade para cada cepa, e supomos que 1 milissegundo é apropriado para imagens com luz branca, enquanto exposições de 100 a 2000 ms são apropriados para GFP (FITC) e FM4-64 (TRITC ) imagens. Tempo de exposição vai variar dependendo do equipamento de imagem utilizada. Recomendamos o uso de uma cepa por 15 bem-lâmina de microscópio para a imagem mais simples, como a qualidade pad e difusão de células da fron...

Receitas específicas solução 1: 10x S7 50 sais 0,5 M MOPS 100 Sulfato de Amônio mM 50 mM fosfato de potássio monobásico Filtro de esterilizar, e enrole S7 50 media em folha antes do armazenamento Metais 100x 0,2 M MgCl 2 70 mM CaCl 2 5 mM MnCl 2 0,1 mM ZnCl 2 100 mcg / mL Tiamina HCl 2 mM HCl 0,5 mM FeCl 3 * dH 2 O para volume final * FeCl 3 deve ser adicionado por último, para evitar precipitação. Após a esterilização do filtro, embrulhe solução de metal em folha de 100x. S7 50 media 1x S7 50 sais 1x de Metal A glicose 1% O glutamato 0,1% 40 mcg / mL Triptofano 40 mcg / mL Fenilalanina H 2 O destilada para volume final Spizizens 10x (g / L) 151,4 mM Sulfato de Amônio (20g / L) 803,8 mM fosfato de potássio monobásico (140g / L) 440,9 mM de potássio dibásico (60g / L) 34,0 mM citrato de sódio (10g / L) 16,6 mM MgSO 4 (2g / L) dH 2 O para volume final Filtro de esterilizar

imaging mismatch repair and cellular responses to dna damage in bacillus subtilis

Ambos os procariotas e eucariotas responder a danos no DNA através de um complexo conjunto de alterações fisiológicas. Alterações na expressão gênica, a redistribuição de proteínas existentes, ea montagem de complexos de proteínas novo pode ser estimulada por uma variedade de lesões de DNA e de pares de bases incompatíveis DNA. Microscopia de fluorescência tem sido utilizada como uma poderosa ferramenta experimental para visualizar e quantificar as respostas para essas e outras lesões de DNA e para monitorar o status de replicação de DNA dentro da complexa arquitetura subcelular de uma célula viva. Fusões de translação entre proteínas fluorescentes e componentes repórter da replicação do DNA e máquinas de reparação têm sido usados ​​para determinar as sugestões que visam as proteínas do reparo do DNA de suas lesões cognate<em&gt; In vivo</em&gt; E para entender como essas proteínas são organizados dentro das células bacterianas. Além disso, fusões de transcrição e translação ligadas a promotores de DNA induzida danos revelaram que as células dentro de uma população de ter activado as respostas ao estresse genotóxico. Nesta revisão, nós fornecemos um protocolo detalhado para o uso de microscopia de fluorescência para a imagem da montagem de reparo de DNA e complexos de DNA de replicação em single células bacterianas. Em particular, este trabalho se concentra em proteínas incompatibilidade imagem reparo, recombinação homóloga, a replicação de DNA e uma proteína induzível SOS-in<em&gt; Bacillus subtilis</em&gt;. Todos os procedimentos aqui descritos são facilmente passíveis de complexos de proteínas de imagens em uma variedade de espécies bacterianas.

Watch this Scientific Journal Video about Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis at JoVE.com

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Um protocolo detalhado é descrito para a formação de imagem em tempo real de complexos de reparo de DNA em<em&gt; Bacillus subtilis</em&gt; Células.

Mismatch Repair imagem e respostas celulares a danos no DNA em Bacillus subtilis</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

imaging-mismatch-repair-cellular-responses-to-dna-damage-bacillus

Research

JoVE Journal

Biology

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

10.9K visualizações.  University of Michigan-Ann Arbor. Um protocolo detalhado é descrito para a formação de imagem em tempo real de complexos de reparo de DNA em Bacillus subtilis Células.

Vídeo: Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Duas e três dimensões imagens de células vivas de Proteínas de DNA resposta a danos

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Biologia

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

gDNA Enriquecimento por uma tecnologia baseada em transposase para NGS Análise de toda a sequência de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt; e 9 genes envolvidos na reparação de danos no DNA

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

Visualização de miniSOG Proteínas Tagged reparo de DNA em combinação com Electron espectroscópica Imagem (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of &#947;- and &#946;-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called &#947;H2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled &#947;H2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors.

Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation

A protocol to evaluate changes in DNA damage levels and DNA repair capacity that may be induced by chronic in vivo low dose irradiation in mouse spleen lymphocytes, by measuring phosphorylated histone H2AX, a marker of DNA double-strand breaks, using flow cytometry is presented.

Medindo danos DNA e reparar no mouse Esplenócitos Depois crônica<em&gt; In Vivo</em&gt; A exposição a doses muito baixas de beta e gama-radiação

Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high ...

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

In Vivo Alkaline Ensaio Cometa e Enzyme-modificado Alkaline Comet Ensaio para medição Breaks DNA Strand e dano oxidativo ao DNA em Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Investigando os prejudiciais efeitos de baixa pressão Plasma esterilização na sobrevivência de esporos de Bacillus subtilis usando Live celular microscopia

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

Célula única Microfluidic análise de Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

Visualizando e quantificando danos de DNA específicos do local baseados em endonuclease

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análise da Função Ligase E3 Ubiquitin

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...