Los autores desean agradecer a los Dres. Philina S. Lee y Alan D. Grossman para la formación inicial en LAS microscopía de fluorescencia. Los autores también agradecen a los Dres. Melanie Berkmen y Kobayashi Hajime ayuda y consejos para la imagen. Este trabajo fue apoyado por la puesta en marcha de fondos de la Facultad de Literatura, Ciencias y Artes y del Departamento de Biología Molecular, Celular y del Desarrollo en la Universidad de Michigan.

Cultivos en crecimiento de las células para la microscopía 1. Uno o dos días antes de la imagen, preparar el B. subtilis cepa que contiene la proteína de fusión de traducción que desea visualizar. Rondas de prueba de los pasos 1A y 1B será necesario determinar qué condiciones de crecimiento ofrece las mejores imágenes de la cepa. En la mayoría de los casos, las células deben ser fotografiado durante la fase de crecimiento exponencial. <ol class="ualpha"><li> Para algunos B. cepas subtilis (en particular cepas, que crecen muy poco debido a la integración de la fusión traduccional entre el gen de interés y las buenas prácticas agrarias en su locus endógeno), el crecimiento inicial de dos días antes de la imagen es necesaria para imágenes de alta calidad. En el primer día, la racha de B. subtilis cepa a ser reflejado en agar LB con antibiótico de selección (s) e incubar durante la noche a 30 ° C. En el segundo día, inocular 100 l de solución salina 0,85% con una sola colonia y realizar tres diluciones seriadas de 10 veces en placas de agar LB (con antibióticos selectivos), revestimiento de 100 l de cada dilución, seguido de incubación durante la noche a 30 ° C. En el tercer día, seleccione la placa de la dilución con el crecimiento de la luz confluente de células. Crecimiento de la luz confluentes en un plato proporcionará el medio de cultivo con un inóculo de salud, crecimiento exponencial de arranque para producir potencialmente excelentes resultados de imagen (ver paso 4). Este paso es especialmente útil para las imágenes de una calidad más alta de membrana con la mancha FM4-64. </li><li> Otros B. subtilis cepas sólo se puede aplicar a un medio selectivo agar LB por las colonias solo con un palillo estéril el día anterior a la imagen y se incuban durante la noche a 30 ° C. Al día siguiente, las celdas de esta placa será suficiente para su uso como inóculo para la imagen (ver paso 4). NOTA: Para B. subtilis, cultivos de una noche en medio líquido no son apropiados debido a una combinación de la formación de esporas y las respuestas de quórum, lo que provocará un período prolongado de crecimiento de retraso de fase 1. </li></ol> 2. En la mañana de la imagen, introducir 10 ml de medio de S7 50 3.1 en un matraz Erlenmeyer de 125 mL estéril. 3. Resuspender las células en un máximo de 2 ml de medio de S7 50 de la placa de agar LB con colonias aisladas o las células confluentes luz para obtener un inóculo. 4. Añade suficiente S7 50 medio de las células del inóculo sobre el erlenmeyer que contiene 10 ml S7 50 por medio de una cultura con una densidad inicial óptica medida a 600 nm (OD 600) de ~ 0,08 a 0,1. En este paso es importante tener por lo menos un aire de 10 veces al cociente medio. Por lo tanto, se utiliza 10 a 12,5 ml de medio inoculado en un matraz Erlenmeyer de 125 ml. 5. Crecer cada cultivo a 30 ° C hasta que el cultivo llegue a un OD 600 ~ 0.5-0.8. Agitando los baños de agua son preferibles las revoluciones por minuto, 150 a 200. Culturas desafió con agentes inductores de dañar el ADN o la discrepancia se debe a una fase de crecimiento exponencial a principios OD 600 de tal manera que las células alcanzará la meta de 600 OD con el nuevo período de crecimiento que se requiere para el tratamiento (ver Tabla 1) Por ejemplo, 2-aminopurine requiere una hora de tratamiento, y por lo tanto un cultivo tratado con 2-aminopurine debe estar en OD 600 ~ 0.3-0.4, de tal manera que una hora de tratamiento / los resultados de crecimiento en un 600 OD ~ 0.5-0.8, el cual las células están listas para la imagen. Preparación de la muestra 1. Pipetear 300 l de células cultivadas en un tubo de microcentrífuga de 1,5 ml. 2. Si las imágenes de las membranas celulares se desea, añadir la dilución 1:1000 de FM4-64 membrana mancha (Invitrogen) para cada muestra de una solución madre de 1 mg / ml. Una valoración de FM4-64 puede ser necesaria para lograr la señal de fluorescencia deseada. Una gama de titulación típica es de 1:100, 1:1000 y 1:10.000. 3. Permitir que las muestras permanezcan a temperatura ambiente hasta por diez minutos, mientras que la diapositiva se está preparando con agarosa al 1% en 1X medio Spizizens como se describe a continuación (ver paso 1 de &quot;preparación de diapositivas&quot;). 4. Si la concentración de las células es necesario, preferimos las células de filtrado mediante un filtro de 0,2 micras, con un aparato de vacío. Las células se pueden lavar con suavidad de la superficie del filtro con PBS, otro tampón apropiado o medio antes de su visualización. Preparación de diapositivas 1. Preparar agarosa al 1% con 1X Spizizens. Añadir 5 ml de caldo de Spizizens 10 veces a 45 ml ddH2O junto con 0,5 g de agarosa y microondas para derretir. Agarosa fundida en caliente es difícil de pipeta, y se solidificó de agarosa no entrará en la punta de la pipeta. La agarosa deben equilibrarse en un baño de agua a una temperatura de ~ 55-65 ° C, para entrar en la punta de la pipeta sin solidificar dentro de la<html> punta. 2. Sacar 20 l de Spizizens con el 1% de agarosa derretida en la punta de la pipeta. Pipeta una gota (aproximadamente 2.1 l) de la solución de agarosa en cada diapositiva y (nosotros usamos 15 y diapositivas, ver Tabla 2) de forma superficial las almohadillas de agarosa. La agarosa se solidifica inmediatamente. Diapositivas también se debe realizar inmediatamente antes de la aplicación de las células. Si las pastillas de agarosa se dejan a temperatura ambiente único, que puede deshidratar en cuestión de minutos y se pueden utilizar. Pastillas de agarosa debe estar centrado y llene cada uno bien, pero tienen una altura mínima para evitar interrumpir el cubreobjetos, y si las gotas son demasiado altos (tipo burbuja) o si la muestra se extienda más allá del límite, así, simplemente pase el limpia bien con un Kimwipe y aplicar una nueva plataforma de agarosa. 3. Aplicar la totalidad de los 300 l de la muestra (ver paso 6) a la diapositiva, distribuidos en la parte superior de cada pad de agarosa. Esta cantidad de células debería ser suficiente para cubrir cada uno y en la diapositiva. 4. Permitir la diapositiva cargado con células reposar a temperatura ambiente (23 ° C) durante aproximadamente diez minutos. Este paso permitirá que las células se depositan en la plataforma de agarosa ser inmóvil y listo para su imagen. Para necesidades especiales, esta temperatura puede ser necesario cambiar, por ejemplo, cuando se realizan experimentos que requieren un cambio de temperatura 4. 5. Aspirar el medio de distancia en exceso de cada pozo sin necesidad de retirar las pastillas de sí mismos. 6. Aplicar el cubreobjetos a la diapositiva, presionando con firmeza y de manera uniforme, pero con cuidado, a través del área de la diapositiva. Asegúrese de que la hoja de la cubierta está en contra de la diapositiva y que la hoja de la cubierta no se eleva por encima de la diapositiva. La visualización de las células 1. Muchos microscopios de fluorescencia diferentes va a funcionar bien. Es fundamental contar con una lente de aceite de inmersión 100X. El Laboratorio de Simmons utiliza: <ul><li> Olympus BX61 microscopio equipado con lentes de 1,45 NA TIRFM petróleo objetivo de inmersión 100X </li><li> Hamamatsu ORCAR 2 CCD refrigerado por cámara </li><li> Lumen 200 de arco de metal (antes) la fuente de luz. </li><li> Para la detección de buenas prácticas agrarias (FITC), la excitación y la emisión de filtro 460-500 510-560 </li><li> Para la detección de FM4-64 (TRITC), la excitación y la emisión de filtro 510-560 572-648. </li><li> Las imágenes fueron capturadas utilizando SlideBook 4,2 (Figura 1) </li></ul> 2. Traiga las células en el foco con luz blanca. 3. Capturar la imagen con los filtros adecuados para cada fluoróforo. Resultados representante Imágenes representativas se muestran (Figura 1). GFP focos deben estar bien definidos, mientras que FM4-64 tinción debe ser brillante y claro 4-7. Imágenes de las buenas prácticas agrarias o de la membrana puede ser pseudo-color con cualquier color, para permitir la mejor calidad de imagen que se presenta sin perder ningún dato. <img src="/files/ftp_upload/1736/1736fig1.jpg" alt="Figura 1" /> Figura 1: Imágenes representativas de las buenas prácticas agrarias B. subtilis proteínas de fusión de traslación. proteínas GFP se muestran en verde, mientras que FM4-64 tinción de membrana se muestra en rojo. La barra de escala blanco indica 3 m (A) MutS-GFP [genotipo relevantes: mutS - GFP (CPE), amyE:: Pspac mutL (cat)]. En presencia de 600 mg / ml de 2-aminopurina y 1 mM IPTG. FM4-64 de la señal no se presenta con el fin de mostrar más claramente los focos MutS-GFP (B) MutL-GFP [genotipo relevantes: mutL:: mutL-GFP (spc)].. En presencia de 2-aminopurine (C) DNAX-GFP [genotipo relevantes: dnaX:: dnaX-GFP (spc)]. (D) Reca-GFP [pertinentes genotipo: Reca:: Reca-GFP (spc)] en presencia de 20 ng / ml de mitomicina C (E) TAGC-GFP [genotipo relevantes: TAGC:: TAGC-GFP (SPC) ] en presencia de 1 mg / ml de mitomicina C. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Tratamiento / concentración o la cantidad </td><td> Función </td><td> Tiempo de incubación </td></tr><tr><td> La mitomicina C (MMC) [20 a 150 ng / ml] </td><td> ADN con fármacos alquilantes </td><td> 1 hora </td></tr><tr><td> 2-aminopurine (2-AP) [600 mg / ml] </td><td> desajuste inducir </td><td> 1 hora </td></tr><tr><td> La hidroxiurea (HU) </td><td> agota dNTPs </td><td> 3 horas </td></tr><tr><td> la luz ultravioleta (UV) 20 J/m2 </td><td> Timina-timina dímeros y fotoproductos 6-4 </td><td> varía dependiendo de la fuente, unos 20 segundos </td></tr><tr><td> grises (radiación ionizante) 5 a 100 Gy </td><td> Doble cadena se rompe, se rompe una sola cadena y sitios de base de los daños </td><td> varía dependiendo de la fuente </td></tr></tbody></table>&quot;&gt; La Tabla 1. Resumen de dañar el ADN y los tratamientos de inducción de desajuste <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Multitest de diapositivas, de 15, así </td><td> MP Biomedicals </td><td> 6041505E </td><td></td></tr><tr><td> Microscopio de vidrio cubierta </td><td> Pescador </td><td> 12 a 544-B </td><td></td></tr><tr><td> Agarosa </td><td> Pescador </td><td> BP160-500 </td><td></td></tr><tr><td> Mitomicina C </td><td> Sigma </td><td> M0503-2MG </td><td> mutágeno, use guantes </td></tr><tr><td> 2-aminopurine </td><td> Sigma </td><td> A3509-250MG </td><td> guantes </td></tr><tr><td> Hidroxiurea </td><td> Sigma </td><td> H8627-25G </td><td> guantes </td></tr><tr><td> FM4-64 </td><td> Invitrogen </td><td> T13320 </td><td> sensible a la luz </td></tr></tbody></table> Tabla 2. Lista de los reactivos y equipos 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>

Ensayo y error están obligados a encontrar las condiciones de exposición de las imágenes de mayor calidad para cada cepa, nos encontramos con que una milésima de segundo es apropiado para las imágenes de luz blanca, mientras que las exposiciones de 100 a 2000 ms son apropiados para las buenas prácticas agrarias (FITC) y FM4 64 (TRITC ) imágenes. Tiempo de exposición puede variar en función de los equipos de imágenes utilizados. Se recomienda el uso de una cepa por portaobjetos de microscopio de 15 y para el m?...

Recetas específicas solución 1: S7 10x 50 sales 0,5 M MOPS 100 mM de sulfato de amonio 50 mM fosfato de potasio monobásico Filtro de esterilización, y el abrigo S7 50 medios de comunicación en papel de aluminio antes de su almacenamiento Metales 100x 0,2 M de MgCl2 70 mM de CaCl2 5 mM MnCl2 0,1 mM ZnCl2 100 mg / ml Tiamina HCl 2 mM HCl 0,5 mM FeCl 3 * dH 2 O al volumen final * FeCl 3 se añade al final, para evitar la precipitación. Después de la esterilización de filtros, envuelva 100x solución de metal en lámina. S7 50 medios de comunicación 1x S7 50 sales 1x metal 1% de glucosa 0,1% de glutamato 40 mg / mL de triptófano 40 mg / mL de fenilalanina H 2 O destilada a volumen final Spizizens 10x (g / L) 151,4 mM de sulfato de amonio (20 g / L) 803,8 mM fosfato de potasio monobásico (140 g / L) 440,9 mM fosfato de potasio dibásico (60 g / L) 34,0 mM citrato de sodio (10 g / L) 16,6 mM MgSO 4 (2 g / L) dH 2 O al volumen final Filtro de esterilización

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

Tanto en procariotas y eucariotas responden al daño del ADN a través de una compleja serie de cambios fisiológicos. Alteraciones en la expresión génica, la redistribución de las proteínas existentes, y el montaje de nuevos complejos de proteínas puede ser estimulada por una variedad de lesiones en el ADN y no coincidentes de pares de bases del ADN. Microscopía de fluorescencia se ha utilizado como una poderosa herramienta experimental para la visualización y cuantificación de estas y otras respuestas a las lesiones del ADN y para supervisar el estado de replicación del ADN dentro de la compleja estructura subcelular de una célula viva. Fusiones traduccionales entre las proteínas fluorescentes reportero y los componentes de la replicación del ADN y la maquinaria de reparación se han utilizado para determinar las señales que se dirigen a proteínas de reparación del ADN de sus lesiones afines<em&gt; In vivo</em&gt; Y para entender cómo estas proteínas se organizan dentro de las células bacterianas. Además, las fusiones de transcripción y traducción vinculados a los promotores de ADN inducible daños han revelado que las células dentro de una población ha activado las respuestas de estrés genotóxico. En esta revisión, nos proporcionan un protocolo detallado para el uso de microscopía de fluorescencia a la imagen de la asamblea de la reparación del ADN y los complejos de replicación del ADN en las células bacterianas individuales. En particular, este trabajo se centra en las proteínas de reparación del ADN de imagen, la recombinación homóloga, la replicación del ADN y una proteína de SOS-inducible en<em&gt; Bacillus subtilis</em&gt;. Todos los procedimientos aquí descritos son fácilmente susceptibles de complejos de proteínas de imágenes en una variedad de especies 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

Un protocolo detallado se describe para la formación de imágenes en tiempo real de los complejos de reparación del ADN en<em&gt; Bacillus subtilis</em&gt; Células.

Imágenes de reparación del ADN y la respuesta celular al daño del ADN en 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 vistas.  University of Michigan-Ann Arbor. Un protocolo detallado se describe para la formación de imágenes en tiempo real de los complejos de reparación del ADN en Bacillus subtilis Células.

Video: 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.

De dos y tres dimensiones imágenes de células vivas de las proteínas de respuesta al daño del ADN

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

Investigación

Biología

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.

Imágenes en tiempo real de Soltero Engineered ARN transcritos en células vivas utilizando Ratiometric bimoleculares Balizas

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 Enriquecimiento por una tecnología basada en la transposasa de Análisis NGS de la secuencia entera de<em&gt; BRCA1</em&gt;,<em&gt; BRCA2</em&gt;, y 9 genes involucrados en la reparación del ADN daños

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.

Visualización de miniSOG Tagged proteínas de reparación de ADN en combinación con Electron espectroscópico Imaging (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.

Medición de daños y reparación del ADN en esplenocitos de ratón Después crónica<em&gt; En Vivo</em&gt; La exposición a dosis muy bajas de beta y gamma Radiación

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.

En Vivo alcalina Comet Assay y Comet Assay alcalina modificada con enzimas para la medición de roturas en el ADN Strand y daño oxidativo del ADN en el hígado de rata

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.

Investigar los efectos perjudiciales de baja presión Plasma esterilización sobre la supervivencia de vivir el Bacillus subtilis esporas usando el 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.

Sola célula microfluídicos análisis 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.

Visualización Y Cuantificación De La Endonucleasa Basada En El Daño Específico Del ADN Específico Del Sitio

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 Análisis de la función E3 Ubiquitina Ligasa

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