Nous remercions Amy Chen pour son aide dans la préparation pour le tournage de la vidéo et pour démontrer la technique. Ce travail mené par ENIGMA: écosystèmes intégrés et des réseaux avec des gènes et des assemblages moléculaires (http://enigma.lbl.gov), un programme scientifique Secteur d&#39;intérêt au Lawrence Berkeley National Laboratory, a été pris en charge par le Bureau de la science, Bureau du biologique et recherche sur l&#39;environnement, du Département américain de l&#39;Énergie sous contrat DE-AC02-05CH11231.

<ol>
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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. 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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>

Les auteurs n&#39;ont pas de conflit d&#39;intérêt à divulguer.

La méthode DAP-puce décrit ici a été utilisé avec succès pour déterminer les cibles génétiques pour plusieurs RI dans Desulfovibrio vulgaris Hildenborough 7 dont un est représenté ici comme un résultat représentatif. Pour RR DVU3023, le choix d&#39;une cible de gène candidat était simple. DVU3025 est située immédiatement en aval du gène de la RR, et les gènes de la RR et cible sont conservées dans plusieurs espèces Desulfovibrio, et en outre DVU3025 a un promoteur de sig...

La capacité des bactéries à survivre et à prospérer dépend de façon critique sur la façon dont ils sont capables de percevoir et de répondre aux perturbations de leur environnement, et ce, à son tour dépend de leurs systèmes de transduction du signal. Le nombre de systèmes de signalisation une bactérie de encode a été appelé son &quot;QI microbienne&quot; et peut être une indication de la variabilité de son environnement et de sa capacité à détecter des signaux multiples et d&#39;affiner sa réponse 1. Deux systèmes de transduction de signaux composants (TCS) sont des systèmes de signalisation les plus répandues utilisées par les bactéries, et ils sont constitués d&#39;une histidine kinase (HK) qui détecte le signal externe et le transmet par l&#39;intermédiaire de la phosphorylation d&#39;un régulateur de réponse effectrice (RR) 2. RR peuvent avoir une variété de domaines de la production et donc différents modes effectrices, mais la réponse la plus fréquente est la régulation transcriptionnelle via un domaine 1 liaison à l&#39;ADN. Les signaux détectés et les fonctions correspondantes du canal déférentt majorité des TCS restent inconnus. Bien que des procédés in vivo tels que ChIP-chip sont couramment utilisés pour la détermination des sites de liaison de facteurs de transcription génomiques 3, ils ne peuvent être utilisés pour les deux RR bactériennes du système composant si les conditions ou les signaux d&#39;activation sont connus. Souvent, les signaux environnementaux qui activent un TCS sont plus difficiles à déterminer que leurs gènes cibles. La puce à ADN in vitro dosage à base décrit ici peut être utilisé pour déterminer rapidement et efficacement les gènes cibles et de prévoir des fonctions de TCS. Cet essai tire parti du fait que les RR peuvent être phosphorylés et donc activés in vitro à l&#39;aide de petites molécules telles que des donneurs de phosphate d&#39;acétyle 4. Dans ce procédé, appelé DAP-puce pour l&#39;ADN-affinité-puce purifié (Figure 1), le gène de la RR d&#39;intérêt est clone avec un His-tag dans E. coli, et une protéine marquée par la suite purifié est autorisé à bie à la tonte de l&#39;ADN génomique. L&#39;ADN lié à une protéine est ensuite enrichi par affinité purification, l&#39;ADN enrichi et entrée sont amplifiés, marqué par fluorescence, mis en commun et hybride à une gamme de carrelage qui est faite sur mesure pour l&#39;organisme d&#39;intérêt (figure 1). expériences de puces à ADN sont soumis à des artefacts et donc des mesures supplémentaires sont utilisés pour optimiser le dosage. Une telle démarche est de tenter de déterminer une cible pour le RR à l&#39;étude en utilisant des tests électrophorétique de décalage de mobilité (EMSA) (voir flux de travail dans la figure 2). Puis, après liaison à l&#39;ADN génomique et les étapes de la DAP, l&#39;ADN lié à une protéine et d&#39;entrée sont examinés par qPCR pour déterminer si la cible positif est enrichi dans la fraction liée aux protéines par rapport à la fraction d&#39;entrée, confirmant ainsi les conditions optimales de liaison pour l&#39; RR (figure 2). Après tableau hybridation, les données sont analysées pour trouver des pics de signal d&#39;intensité plus élevée indiquant loci génomiques où la protéine hannonce lié. Les fonctions peuvent être prévus pour la RR en fonction des objectifs de gènes obtenus. Les loci génomiques cibles sont utilisés pour prédire motifs de sites de liaison, qui sont ensuite validés expérimentalement à l&#39;aide EMSA (Figure 2). Les prédictions fonctionnelles et cibles génétiques pour la RR peuvent ensuite être étendues aux espèces qui codent RR orthologues en scannant les génomes pour des motifs de liaison similaires (figure 2) étroitement liés. La méthode DAP-puce peut fournir une mine d&#39;informations pour un TCS où il n&#39;y avait auparavant aucune. Le procédé peut également être utilisé pour n&#39;importe quel régulateur transcriptionnel si la protéine peut être purifiée et des conditions de liaison d&#39;ADN peut être déterminée, et pour n&#39;importe quel organisme d&#39;intérêt avec une séquence génomique disponible. <img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig1highres.jpg" width="500" /> Figure 1. L&#39;-puce ADN purifié par affinité (DAP-puce) stratégie 7. Le gène de la RR de l&#39;organisme d&#39;intérêt est cloné avec un His-tag carboxy-terminale dans un E. coli souche d&#39;expression. Protéine purifiée marquée par His est activée par phosphorylation avec du phosphate d&#39;acétyle, et on le mélange avec de l&#39;ADN génomique cisaillé. Une aliquote de la réaction de liaison est enregistré sous forme d&#39;ADN d&#39;entrée, tandis que le reste est soumis à une purification par affinité en utilisant une résine Ni-NTA. L&#39;entrée et l&#39;ADN de RR lié sont génome amplifié, et marqué avec Cy3 et Cy5, respectivement. L&#39;ADN marqué est mis en commun et hybridée à une matrice de carrelage, qui est ensuite analysé afin de déterminer les gènes cibles. Figure modifiée et réimprimée en utilisant la licence creative commons de 7. <img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51715/51715fig2highres.jpg" width="500" /> Figure 2. Sommaire des flux de travail. Pour toute protei marqué purifién, commencer par déterminer une cible en utilisant l&#39;EMSA. Autoriser protéine à lier l&#39;ADN génomique et l&#39;ADN purifier par affinité (DAP) et l&#39;ensemble du génome amplifier (WGA), le ADN enrichi et entrée. Si une cible du gène est connue, utiliser qPCR pour s&#39;assurer que la cible connue est enrichi dans la fraction liée aux protéines. Si aucune cible pourrait être déterminée, passez directement à l&#39;étiquetage de l&#39;ADN et l&#39;hybridation tableau. Si l&#39;enrichissement par qPCR pas pu être observée, puis répétez la liaison protéine-ADNg et étapes DAP-WGA utilisant différentes quantités de protéines. Utiliser l&#39;analyse de réseau pour trouver les pics et les faire correspondre à cibler des gènes. Utiliser les régions en amont de gènes cibles pour prédire motifs de site de liaison. Valider les motifs expérimentalement à l&#39;aide EMSA. Utilisez le motif de scanner les génomes d&#39;espèces apparentées codant orthologues de la RR à l&#39;étude, et de prévoir les gènes ciblés dans ces espèces. Sur la base des cibles de gènes obtenus, la fonction physiologique de la RR et ses orthologues peut être prédite. Figure modifié et reproduit à l&#39;aide du c créativeommons licence 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

Les méthodes in vivo tels que ChIP-chip sont des techniques bien établies utilisées pour déterminer les cibles mondiales de gènes pour des facteurs de transcription. Cependant, ils sont d&#39;une utilité limitée dans l&#39;exploration de deux systèmes de réglementation composants bactériens avec des conditions d&#39;activation non caractérisés. De tels systèmes régulent la transcription seulement quand elle est activée en présence de signaux uniques. Etant donné que ces signaux sont souvent inconnus, le procédé in vitro de puces à ADN située à décrit dans cet article vidéo peut être utilisée pour déterminer des cibles de gènes et des sites de liaison pour les régulateurs de réponse. Cette méthode puce ADN purifié par affinité peut être utilisée pour n&#39;importe quel régulateur purifiée dans un organisme avec un génome séquencé. Le protocole consiste à permettre la protéine marquée purifiée à se lier à l&#39;ADN génomique cisaillée et ensuite la purification par affinité de l&#39;ADN lié à une protéine, suivie par le marquage fluorescent de l&#39;ADN et l&#39;hybridation à un réseau de carrelage personnalisé. Précédentes étapes qui peuvent être utilisés pour optimiser le dosage de spécificationc régulateurs sont également décrits. Les pics générés par l&#39;analyse des données du tableau sont utilisés pour prédire motifs de sites de liaison, qui sont ensuite validés expérimentalement. Les prédictions de motifs peuvent être utilisés en outre pour déterminer les gènes cibles de régulateurs de réponse orthologues d&#39;espèces étroitement apparentées. Nous démontrons l&#39;applicabilité de ce procédé en déterminant les gènes cibles et de motifs de site de liaison et la prédiction ainsi la fonction d&#39;un régulateur de réponse DVU3023 de sigma54-dépendante de l&#39;environnement dans la bactérie Desulfovibrio vulgaris Hildenborough.

La méthode ci-dessus a été appliquée pour déterminer les gènes cibles mondiales des RR dans le sulfate de modèle réduisant bactérie Desulfovibrio vulgaris Hildenborough 7. Ce micro-organisme a un grand nombre de TCS représenté par plus de 70 RI, ce qui indique la grande variété de signaux possibles qui détecte et répond à. Analyses in vivo sur les fonctions de ces systèmes de signalisation sont difficiles à réaliser étant donné que leurs signaux, et donc les conditions d...

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

Cet article vidéo décrit une méthode de puces à ADN sur la base in vitro afin de déterminer les gènes cibles et des sites de liaison pour deux régulateurs de réponse du système de composants.

ADN purifié par affinité Chip (DAP-puce) Méthode pour déterminer les cibles génétiques pour bactérienne Deux systèmes de réglementation composants

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 vues.  Lawrence Berkeley National Laboratory.  Cet article vidéo décrit une méthode de puces à ADN sur la base in vitro afin de déterminer les gènes cibles et des sites de liaison pour deux régulateurs de réponse du système de composants. 

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

Bacterial Gene Expression Analysis Using Microarrays

Analyse bactérienne expression génique par puces à ADN

Recherche

Biologie

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.

Salut-C: une méthode d&#39;étude de l&#39;architecture tridimensionnelle des génomes.

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.

Immunoprécipitation de la chromatine (ChIP) pour doser modification des histones dynamique dans l&#39;expression génique dans les cellules humaines Activé

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.

Analyse du génome entier en utilisant ChIP d&#39;identifier les gènes cibles spécifiques Isoform

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.

Utilisation de la séquence d&#39;arrêt SecM comme un outil pour isoler les polypeptides Ribosome Bound

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.

L&#39;utilisation de<em&gt; Ex Vivo</em&gt; Imagerie entier orgue et histologie tissulaire quantitative du Bio-distribution des molécules marquées par fluorescence

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.

Utilisation d&#39;une méthode immunohistochimique intégrale pour étudier l&#39;inervation du tractus biliaire dans<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.

Le test de fixation du Lactate déshydrogénase — Une méthode Simple et fiable pour déterminer l’activité de séquestration autophagique en vrac dans des cellules de mammifères

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.

Imagerie en direct à haut débit des microcolonies pour mesurer l’hétérogénéité de la croissance et de l’expression des gènes

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.

Profilage de la modification H3K4me3 dans les usines à l’aide du clivage sous cibles et de la marquage

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