Damos las gracias a Amy Chen por su ayuda en la preparación para la filmación del video y para la demostración de la técnica. Este trabajo realizado por ENIGMA: Ecosistemas y redes integradas con Genes y Asambleas Molecular (http://enigma.lbl.gov), un programa científico del área de focalización en el Laboratorio Nacional Lawrence Berkeley, con el apoyo de la Oficina de Ciencia, Oficina de Biológica y Investigación del Medio Ambiente, del Departamento de Energía de los EE.UU. bajo el Contrato No. DE-AC02-05CH11231.

<ol>
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V., Struhl, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation+for+determining+the+association+of+proteins+with+specific+genomic+sequences+in+vivo.">Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo.</a> Curr Protoc Cell Biol. 17 (17), (2004).</li><li>McCleary, W. R., Stock, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetyl+phosphate+and+the+activation+of+two-component+response+regulators.">Acetyl phosphate and the activation of two-component response regulators.</a> J Biol Chem. 269, 31567-31572 (1994).</li><li>Novichkov, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RegPrecise+web+services+interface:+programmatic+access+to+the+transcriptional+regulatory+interactions+in+bacteria+reconstructed+by+comparative+genomics.">RegPrecise web services interface: programmatic access to the transcriptional regulatory interactions in bacteria reconstructed by comparative genomics.</a> Nucleic Acids Res. 40, 604-608 (2012).</li><li>Studholme, D. J., Buck, M., Nixon, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+sigma(N)-dependent+promoters+in+bacterial+genomes.">Identification of potential sigma(N)-dependent promoters in bacterial genomes.</a> Microbiology. 146 (12), 3021-3023 (2000).</li><li>Rajeev, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+mapping+of+two+component+response+regulators+to+gene+targets+in+a+model+sulfate+reducing+bacterium.">Systematic mapping of two component response regulators to gene targets in a model sulfate reducing bacterium.</a> Genome Biol. 12, 99 (2011).</li><li>Crooks, G. E., Hon, G., Chandonia, J. M., Brenner, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WebLogo:+a+sequence+logo+generator.">WebLogo: a sequence logo generator.</a> Genome Res. 14, 1188-1190 (2004).</li><li>Barbieri, C. M., Mack, T. R., Robinson, V. L., Miller, M. T., Stock, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+response+regulator+autophosphorylation+through+interdomain+contacts.">Regulation of response regulator autophosphorylation through interdomain contacts.</a> J Biol Chem. 285, 32325-32335 (2010).</li><li>Bourret, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receiver+domain+structure+and+function+in+response+regulator+proteins.">Receiver domain structure and function in response regulator proteins.</a> Curr Opin Microbiol. 13, 142-149 (2010).</li><li>Gao, R., Stock, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+strategies+for+phosphorylation-mediated+regulation+of+response+regulator+activity.">Molecular strategies for phosphorylation-mediated regulation of response regulator activity.</a> Curr Opin Microbiol. 13, 160-167 (2010).</li><li>Hung, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+of+the+response+regulator+ComE+from+Streptococcus+mutans+is+affected+by+phosphorylation.">Oligomerization of the response regulator ComE from Streptococcus mutans is affected by phosphorylation.</a> J Bacteriol. 194, 1127-1135 (2012).</li><li>Ladds, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+regulator+Spo0A+from+Bacillus+subtilis+is+efficiently+phosphorylated+in+Escherichia+coli.">The response regulator Spo0A from Bacillus subtilis is efficiently phosphorylated in Escherichia coli.</a> FEMS Microbiol Lett. 223, 153-157 (2003).</li><li>Wen, Y., Feng, J., Scott, D. R., Marcus, E. A., Sachs, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+the+HP0165-HP0166+two-component+system+in+expression+of+some+acidic-pH-upregulated+genes+of+Helicobacter+pylori.">Involvement of the HP0165-HP0166 two-component system in expression of some acidic-pH-upregulated genes of Helicobacter pylori.</a> J Bacteriol. 188, 1750-1761 (2006).</li><li>Liu, X., Noll, D. M., Lieb, J. D., Clarke, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIP-chip:+rapid+and+accurate+determination+of+DNA-binding+specificity.">DIP-chip: rapid and accurate determination of DNA-binding specificity.</a> Genome Res. 15, 421-427 (2005).</li><li>Gossett, A. J., Lieb, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+Immunoprecipitation+(DIP)+for+the+Determination+of+DNA-Binding+Specificity.">DNA Immunoprecipitation (DIP) for the Determination of DNA-Binding Specificity.</a> CSH Protoc. 2008, (2008).</li><li>Mascher, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Streptococcus+pneumoniae+cia+regulon:+CiaR+target+sites+and+transcription+profile+analysis.">The Streptococcus pneumoniae cia regulon: CiaR target sites and transcription profile analysis.</a> J Bacteriol. 185, 60-70 (2003).</li><li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nat Rev Genet. 10, 669-680 (2009).</li><li>Lefrancois, P., Zheng, W., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-Seq+using+high-throughput+DNA+sequencing+for+genome-wide+identification+of+transcription+factor+binding+sites.">ChIP-Seq using high-throughput DNA sequencing for genome-wide identification of transcription factor binding sites.</a> Methods Enzymol. 470, 77-104 (2010).</li><li>Ho, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-chip+versus+ChIP-seq:+lessons+for+experimental+design+and+data+analysis.">ChIP-chip versus ChIP-seq: lessons for experimental design and data analysis.</a> BMC Genomics. 12, 134 (2011).</li><li>Liu, X., Lee, C. K., Granek, J. A., Clarke, N. D., Lieb, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-genome+comparison+of+Leu3+binding+in+vitro+and+in+vivo+reveals+the+importance+of+nucleosome+occupancy+in+target+site+selection.">Whole-genome comparison of Leu3 binding in vitro and in vivo reveals the importance of nucleosome occupancy in target site selection.</a> Genome Res. 16, 1517-1528 (2006).</li></ol>

Los autores no tienen ningún conflicto de intereses a revelar.

El método DAP-chip descrito aquí se utilizó con éxito para determinar los objetivos de genes durante varios RR en Desulfovibrio vulgaris Hildenborough 7 de los cuales uno se muestra aquí como un resultado representativo. Para DVU3023 RR, la elección de un objetivo de genes candidatos fue sencillo. DVU3025 está situada inmediatamente aguas abajo del gen RR, y los genes RR y de destino se conservan en varias especies Desulfovibrio, y adicionalmente DVU3025 tiene un promotor sigma54 depe...

La capacidad de las bacterias para sobrevivir y prosperar depende críticamente de lo bien que son capaces de percibir y responder a las perturbaciones en sus ambientes, y esto a su vez depende de los sistemas de transducción de señales. El número de sistemas de señalización A codifica bacteria que se ha llamado su &quot;IQ microbiana&quot; y puede ser una indicación de la variabilidad de su medio ambiente y su capacidad de detectar señales múltiples y afinar su respuesta 1. Sistemas de dos componentes de transducción de señales (TCS) son los sistemas de señalización más prevalentes utilizados por las bacterias, y que consisten en una histidina quinasa (HK) que detecta la señal externa y transmite a través de la fosforilación de un regulador de la respuesta efectora (RR) 2. RR puede tener una variedad de dominios de salida y por lo tanto diferentes modos efectoras, pero la respuesta más común es la regulación transcripcional a través de un ADN de dominio de unión 1. Las señales detectadas y las funciones correspondientes de los conductos deferentest mayoría de TCSs siguen siendo desconocidos. Aunque los métodos in vivo, tales como chip-chip se utilizan rutinariamente para la determinación de sitios de unión de factores de transcripción genómica 3, que sólo se pueden utilizar para bacterianas RR sistema de dos componentes si se conocen las condiciones de activación o señales. A menudo, las señales ambientales que activan una TCS son más difíciles de determinar que sus genes diana. El microarray de ensayo in vitro basado descrito aquí se puede utilizar para determinar eficaz y rápidamente los objetivos de genes y predecir funciones de TCSs. Este ensayo se aprovecha del hecho de que los RR se pueden fosforilar y por lo tanto activan in vitro utilizando pequeñas moléculas donantes como fosfato de acetilo 4. En este método, llamado DAP-chip para ADN-afinidad-chip purificado (Figura 1), el gen RR de interés se clona con una etiqueta de His en E. coli, y una proteína marcada posteriormente purificado se permite a Bind a esquilada de ADN genómico. El ADN unido a la proteína se enriquecen entonces por purificación por afinidad, el ADN enriquecido y de entrada se amplifican, marcado con fluorescencia, agrupados juntos y se hibridó a una matriz de mosaico que está hecho a medida para el organismo de interés (Figura 1). Microarray experimentos están sujetos a los artefactos, por lo que se emplean pasos adicionales para optimizar el ensayo. Una de esas medidas es tratar de determinar un objetivo para el RR en estudio el uso de ensayos de cambio de movilidad electroforética (EMSA) (véase el flujo de trabajo en la Figura 2). Entonces, después de la unión al ADN genómico y los pasos de DAP, el ADN unido a proteína y de entrada se examinó por qPCR para ver si el objetivo positivo se enriquece en la fracción unida a proteínas en relación con la fracción de entrada, confirmando de este modo las condiciones de unión óptimas para el RR (Figura 2). Después de la gama de hibridación, los datos se analizan para encontrar picos de señal de mayor intensidad que indican loci del genoma en donde la proteína had obligado. Las funciones pueden ser predichos para el RR en base a los objetivos de genes obtenidos. Los loci genómicos de destino se utilizan para predecir motivos sitio de unión, que luego son validados experimentalmente utilizando EMSAs (Figura 2). Las predicciones funcionales y objetivos de genes para el RR pueden entonces extenderse a especies que codifican los RR ortólogos mediante el escaneo de los genomas de los motivos de unión similares (Figura 2), estrechamente relacionado. El método DAP-chip puede proporcionar una gran cantidad de información de un TCS donde antes no había ninguna. El método también puede utilizarse para cualquier regulador transcripcional si la proteína se puede purificar y condiciones de unión de ADN se puede determinar, y para cualquier organismo de interés con una secuencia del genoma disponible. <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig1highres.jpg" width="500" /> Figura 1. El chip purificado-ADN-afinidad (DAP-chip) estrategia 7. El gen RR a partir del organismo de interés se clona con un carboxi-terminal de His-tag en una E. cepa de expresión coli. Se purificó la proteína marcada con His se activa por fosforilación con fosfato de acetilo, y se mezcla con el ADN genómico cortado. Una alícuota de la reacción de unión se guarda como ADN de entrada, mientras que el resto se sometió a purificación de afinidad utilizando resina de Ni-NTA. La entrada y el ADN RR-unidos se todo el genoma amplificado, y marcado con Cy3 y Cy5, respectivamente. El ADN marcado se agrupen y se hibridó a una matriz de suelo de baldosas, que luego se analizó para determinar los objetivos de genes. Figura modificado y reimpreso con el licencia creative commons de 7. <img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig2highres.jpg" width="500" /> Figura 2. Resumen de flujo de trabajo. Para cualquier protei etiquetados purificadon, comenzar por la determinación de un destino utilizando la EMSA. Permitir proteínas se unen al ADN genómico y ADN-purificar por afinidad (DAP) y la totalidad de su genoma amplificar (WGA) y la entrada de ADN enriquecido. Si se conoce un gen objetivo, utilizar la qPCR para asegurar que el objetivo conocido se enriquece en la fracción unida a proteínas. Si se pudo determinar ningún objetivo, pase directamente al marcaje de ADN y la gama de hibridación. Si no se pudo observar el enriquecimiento por qPCR, a continuación, repetir la proteína de unión-ADNg y pasos DAP-WGA utilizando diferentes cantidades de proteína. Utilice el análisis conjunto para encontrar picos y asignarlos a los genes objetivo. Utilice las regiones aguas arriba de los genes diana de predecir motivos sitio de unión. Validar los motivos experimentalmente utilizando EMSAs. Utilice el motivo para escanear los genomas de las especies relacionadas que codifican ortólogos de la RR en estudio, y predecir determinados genes en estas especies también. Sobre la base de los objetivos de genes obtenidos, la función fisiológica de la RR y sus ortólogos se puede predecir. Figura modificado y reimpreso usando el C creativoommons licencia de 7.

<table border="0" cellpadding="0" cellspacing="0" width="719">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Name of Material/Equipment</td>
			<td style="width:248px;">Company</td>
			<td style="width:113px;">Catalog number</td>
			<td style="width:104px;">Comments</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">HisTrapFF column (Ni-Sepharose column)</td>
			<td>GE Lfe Sciences, Pittsburgh, PA, USA</td>
			<td>17-5255-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Akta explorer (FPLC instrument)</td>
			<td colspan="2">GE Lfe Sciences, Pittsburgh, PA, USA</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">HiPrep 26/10 Desalting column</td>
			<td>GE Lfe Sciences, Pittsburgh, PA, USA</td>
			<td>17-5087-01</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Qiaquick Gel extraction kit</td>
			<td>Qiagen Inc, Valencia, CA, USA</td>
			<td align="right">28704</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Biotin-labeled oligonucleotides</td>
			<td>Integrated DNA Technologies</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">6% polyacrylamide-0.5X TBE precast mini DNA retardation gel</td>
			<td>Life Technologies, Grand Island, NY, USA</td>
			<td>EC63652BOX</td>
			<td>Alternately, you can pour your own gel.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Nylon membrane</td>
			<td>EMD Millipore, Billerica, MA, USA</td>
			<td>INYC00010</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Trans-Blot SD Semi-dry electrophoretic transfer cell</td>
			<td>Biorad, Hercules, CA, USA</td>
			<td>170-3940</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Extra thick blot paper, 8 x 13.5 cm</td>
			<td>Biorad, Hercules, CA, USA</td>
			<td>170-3967</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">UV crosslinker Model XL-1000</td>
			<td>Fisher Scientific</td>
			<td style="width:113px;">11-992-89</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Nucleic Acid chemiluminescent detection kit (Pierce)</td>
			<td>Thermo fisher Scientific, Rockford, IL, USA</td>
			<td align="right">89880</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Ni-NTA agarose resin</td>
			<td>Qiagen Inc, Valencia, CA, USA</td>
			<td align="right">30210</td>
			<td></td>
		</tr>
		<tr height="119">
			<td height="119" style="height:119px;width:255px;">GenomePlex Whole genome amplification kit (Fragmentation buffer, library preparation buffer, library stabilization solution, library preparation enzyme, 10X amplification master mix, WGA polymerase )</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>WGA2-50RXN</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Nanodrop ND-1000</td>
			<td colspan="2">Thermo Scientific, Wilmington, DE, USA</td>
			<td>For quantitation of DNA</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Perfecta Sybr Green SuperMix, with ROX</td>
			<td>Quanta biosciences</td>
			<td>95055-500</td>
			<td>Any Sybr Green PCR mix may be used</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">PlateMax Ultra clear heat sealing film for qPCR</td>
			<td>Axygen</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">96 well clear low profile PCR microplate</td>
			<td>Life Technologies, Grand Island, NY, USA</td>
			<td colspan="2">PCR-96-LP-AB-C</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Applied Biosystems StepOne Plus Real time PCR system</td>
			<td>Life Technologies, Grand Island, NY, USA</td>
			<td align="right">4376600</td>
			<td>Any real time PCR system may be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Qiaquick PCR purification kit</td>
			<td>Qiagen Inc, Valencia, CA, USA</td>
			<td align="right">28104</td>
			<td>Any PCR clean up kit may be used</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Cy3/Cy5-labeled nonamers</td>
			<td>Trilink biotechnologies, San Diego, CA, USA</td>
			<td colspan="2">N46-0001, N46-0002</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Klenow polymerase 50,000U/ml, 3&#39;-5&#39; exo-</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>M0212M</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">Hybridization system</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td>This company no longer makes arrays or related items, so alternate sources such as Agilent or Affymetrix will need to be used,</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Custom printed microarrays and mixers</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:255px;">Hybridization kit (2X Hybridization buffer, Hybridization component A, Alignment oligo)</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Wash buffer kit (10X Wash buffer I, II, III, 1 M DTT)</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">GenePix 4200A microarray scanner</td>
			<td colspan="2">Molecular Devices, Sunnyvale CA, USA</td>
			<td>This model has been replaced by superior ones</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:255px;">GenePix Pro microarray software</td>
			<td colspan="2">Molecular Devices, Sunnyvale CA, USA</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:255px;">Nimblescan v.2.4, ChIP-chip analysis software</td>
			<td>Roche-Nimblegen, Madison, WI, USA</td>
			<td>N/A</td>
			<td></td>
		</tr>
	</tbody>
</table>

dna-affinity-purified chip (dap-chip) method to determine gene targets for bacterial two component regulatory systems

Los métodos in vivo como chip-chip son técnicas bien establecidas que se utilizan para determinar dianas génicas globales para los factores de transcripción. Sin embargo, son de uso limitado en la exploración de dos sistemas de regulación componente bacteriano con condiciones de activación no caracterizados. Tales sistemas regulan la transcripción sólo cuando se activa en la presencia de señales únicas. Puesto que estas señales son a menudo desconocida, el método in vitro de microarrays basado en describe en este artículo de vídeo se puede utilizar para determinar objetivos de genes y sitios de unión para los reguladores de respuesta. Este método chip purificado-ADN-afinidad puede ser utilizado para cualquier regulador purificado en cualquier organismo con un genoma secuenciado. El protocolo consiste en permitir que la proteína marcada purificada para unirse al ADN genómico cortado y después de purificación por afinidad del ADN a las proteínas, seguido de marcaje fluorescente del ADN y la hibridación con un suelo de baldosas gama personalizado. Precediendo pasos que se pueden usar para optimizar el ensayo para la especificaciónTambién se describen los reguladores c. Los picos generados por el análisis de datos de la matriz se utilizan para predecir motivos sitio de unión, que luego se validan experimentalmente. Los motivo de las predicciones se pueden usar además para determinar objetivos de genes de respuesta de los reguladores ortólogos en especies estrechamente relacionadas. Se demuestra la aplicabilidad de este método mediante la determinación de los objetivos de genes y vinculante motivos sitio y predecir así la función de un regulador de respuesta DVU3023 sigma54 dependiente en la bacteria Desulfovibrio vulgaris ambiental Hildenborough.

El método anterior se aplicó para determinar los genes objetivos globales de los RR en el modelo de reducción de sulfato bacteria Desulfovibrio vulgaris Hildenborough 7. Este organismo tiene un gran número de TCSs representado por más de 70 RR, lo que indica la amplia variedad de posibles señales que detecta y responde a. Los análisis in vivo en las funciones de estos sistemas de señalización son difíciles de realizar, ya que sus señales y por lo tanto sus condiciones de activaci?...

Watch this Scientific Journal Video about DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems at JoVE.com

DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

Este artículo de vídeo describe un método de microarrays basado en vitro para determinar los objetivos de genes y sitios de unión para dos reguladores de respuesta del sistema de componentes.

Purificado de ADN afinidad Chip (DAP-chip) Método para determinar los objetivos de genes para bacteriana Dos Sistemas Regulatorios componentes

In vivo methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of ...

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Research

JoVE Journal

Biology

DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

16.6K vistas.  Lawrence Berkeley National Laboratory.  Este artículo de vídeo describe un método de microarrays basado en vitro para determinar los objetivos de genes y sitios de unión para dos reguladores de respuesta del sistema de componentes. 

Video: DNA-affinity-purified Chip DAP-chip Method to Determine Gene Targets for Bacterial Two component Regulatory Systems

Bacterial Gene Expression Analysis Using Microarrays

Gen bacteriano y Análisis de Expresión El uso de microarrays

Investigación

Biología

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: Un método para estudiar la arquitectura tridimensional de los genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

Inmunoprecipitación de cromatina (CHIP) para el ensayo dinámico de modificación de las histonas en la expresión génica activado en las células humanas

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me33. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform4. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner.
To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.

Here we are presenting a chromatin immunoprecipitation (ChIP) procedure for genome-wide location analysis of protein isoforms that differ in a histone-binding domain. We are applying it to ChIP-Seq analysis to identify the targets of the KDM5A/JARID1A/RBP2 histone demethylase.

Análisis de todo el genoma con chip para identificar objetivos específicos para cada isoforma Gene

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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.

Usar secuencias de detención SecM como una herramienta para aislar Ribosome polipéptidos enlazados

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

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro and in vivo. Fluorescent probes are particularly useful in determining the bio-distribution of administered large molecules, where the addition of a small-molecule fluorescent label is unlikely to affect the kinetics or bio-distribution of the compound. A variety of methods exist to examine bio-distribution that vary significantly in the amount of effort required and whether the resulting measurements are fully quantitative, but using multiple methods in conjunction can provide a rapid and effective system for analyzing bio-distributions.
Ex vivo whole-organ imaging is a method that can be used to quickly compare the relative concentrations of fluorescent molecules within tissues and between multiple types of tissues or treatment groups. Using an imaging platform designed for live-animal or whole-organ imaging, fluorescence within intact tissues can be determined without further processing, saving time and labor while providing an accurate picture of the overall bio-distribution. This process is ideal in experiments attempting to determine the tissue specificity of a compound or for the comparison of multiple different compounds. Quantitative tissue histology on the other hand requires extensive further processing of tissues in order to create a quantitative measure of the labeled compounds. To accurately assess bio-distribution, all tissues of interest must be sliced, scanned, and analyzed relative to standard curves in order to make comparisons between tissues or groups. Quantitative tissue histology is the gold standard for determining absolute compound concentrations within tissues.
Here, we describe how both methods can be used together effectively to assess the ability of different administration methods and compound modifications to target and deliver fluorescently labeled molecules to the central nervous system1.

The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules

Ex vivo whole-organ imaging is a rapid method for determining the relative concentrations of fluorescently labeled compounds within and between tissues or treatment groups. Conversely, quantitative fluorescence histology, while more labor intensive, allows for the quantification of the absolute tissue levels of labeled molecules.

El uso de<em&gt; Ex Vivo</em&gt; Imaging entero de órganos y de tejidos Histología cuantitativa para determinar la bio-distribución de las moléculas marcadas con fluorescencia

Fluorescent labeling is a well-established process for examining the fate of labeled molecules under a variety of experimental conditions both in vitro ...

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the biliary tract in Suncus murinus (S. murinus&#160;). First, the specimen was dissected from S. murinus and fixed in 4% paraformaldehyde (PFA). Second, an enzymatic treatment and potential endogenous peroxidase inactivation were performed. The specimen was then exposed to the primary antibody, anti-neurofilament protein antibody, for 3-6 days. It was then incubated with the secondary antibody conjugated with horseradish peroxidase. The color reaction was revealed by reacting the specimen with a 3,3&#39;-diaminobenzidine (DAB) substrate. This method can be applied to analyze the innervation of all visceral organs. Furthermore, this protocol can also be adapted to test other neuronal antibodies, but optimization of the antibodies should be done first. This method was originally introduced by Kuratani and Tanaka1,2,3.

Using a Whole-mount Immunohistochemical Method to Study the Innervation of the Biliary Tract in Suncus murinus

A whole-mount immunohistochemical approach, to visualize neurofilament protein expression in the extrahepatic biliary tract in Suncus murinus. is presented here. This protocol can be used to analyze the innervation of all visceral organs in S. murinus or other species.

Uso de un método inmunohistoquímico de montaje completo para estudiar la inervación del tracto biliar en<em&gt; Suncus murinus</em

This work describes the whole-mount immunohistochemistry staining method in detail, using neurofilament protein antibody to label the innervation of the ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

El ensayo de secuestro del lactato deshidrogenasa, Es un método Simple y confiable para determinar la actividad de autofagia secuestro a granel en células de mamíferos

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

Imágenes en vivo de alto rendimiento de microcolonias para medir la heterogeneidad en crecimiento y expresión génica

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Elaboración de perfiles de modificación de H3K4me3 en plantas utilizando escisión bajo objetivos y etiquetado

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...