Questo lavoro è stato sostenuto dalla Fondazione tedesca per la ricerca e l&#39;ERC (AN377 / 3-2 e 309.528 CHROMDECON a WA) e un PhD Fellowship della Boehringer Ingelheim Fonds a AKS Figura 1 e 2 sono ristampati da Developmental Cell 31 (3), Magalska , RuvB-come la funzione ATPasi nella cromatina decondensazione alla fine della mitosi, 305-318, 2014 et al., gentile concessione di Elsevier.

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J., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+spindle+assembly+and+chromosome+condensation+in+Xenopus+egg+extracts.">Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.</a> Methods Mol Biol. 322, 459-474 (2006).</li><li>Galy, V., 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=A+role+for+gp210+in+mitotic+nuclear-envelope+breakdown.">A role for gp210 in mitotic nuclear-envelope breakdown.</a> J Cell Sci. 121, 317-328 (2008).</li><li>Chan, R. C., Forbes, D. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+assembly.">Nuclear assembly.</a> Annu Rev Cell Dev Biol. 13, 669-695 (1997).</li><li>Magalska, A., 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=RuvB-like+ATPases+function+in+chromatin+decondensation+at+the+end+of+mitosis.">RuvB-like ATPases function in chromatin decondensation at the end of mitosis.</a> Developmental Cell. 31, 305-318 (2014).</li><li>Belmont, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+chromosome+structure+and+condensation.">Mitotic chromosome structure and condensation.</a> Curr Opin Cell Biol. 18, 632-638 (2006).</li><li>Lavoie, B. D., Tuffo, K. M., Oh, S., Koshland, D., Holm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitotic+chromosome+condensation+requires+Brn1p,+the+yeast+homologue+of+Barren.">Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren.</a> Mol Biol Cell. 11, 1293-1304 (2000).</li><li>Philpott, A., Leno, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleoplasmin+remodels+sperm+chromatin+in+Xenopus+egg+extracts.">Nucleoplasmin remodels sperm chromatin in Xenopus egg extracts.</a> Cell. 69, 759-767 (1992).</li><li>Philpott, A., Leno, G. H., Laskey, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+decondensation+in+Xenopus+egg+cytoplasm+is+mediated+by+nucleoplasmin.">Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin.</a> Cell. 65, 569-578 (1991).</li><li>Burglin, T. R., Mattaj, I. W., Newmeyer, D. D., Zeller, R., De Robertis, E. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+the+isolation+of+individual+and+clustered+mitotic+chromosomes.">Improved methods for the isolation of individual and clustered mitotic chromosomes.</a> Exp Cell Res. 173, 85-98 (1987).</li><li>Eisenhardt, N., Schooley, A., Antonin, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+in+vitro+assays+to+analyze+the+function+of+transmembrane+nucleoporins+and+targeting+of+inner+nuclear+membrane+proteins.">Xenopus in vitro assays to analyze the function of transmembrane nucleoporins and targeting of inner nuclear membrane proteins.</a> Methods Cell Biol. 122, 193-218 (2014).</li><li>Wignall, S. M., Deehan, R., Maresca, T. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+chromosome+clusters+from+metaphase-arrested+HeLa+cells.">Isolation of chromosome clusters from metaphase-arrested HeLa cells.</a> Chromosoma. 85, 571-581 (1982).</li><li>Ramadan, K., 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=Cdc48/p97+promotes+reformation+of+the+nucleus+by+extracting+the+kinase+Aurora+B+from+chromatin.">Cdc48/p97 promotes reformation of the nucleus by extracting the kinase Aurora B from chromatin.</a> Nature. 450, 1258-1262 (2007).</li></ol>

The authors have nothing to disclose.

Xenopus laevis estratti a base di uova sono uno strumento molto utile per riprodurre fedelmente i processi cellulari in vitro, e questo sistema è stato utilizzato con successo nella caratterizzazione degli eventi del ciclo cellulare e la divisione cellulare 2, 3,5,6,17. A causa di grandi negozi di componenti nucleari sequestrati nell&#39;uovo durante oogenesi, estratti a base di uova sono una fonte eccellente di componenti cellulari. Rispetto ad altri approcci come RNAi su linee ...

Xenopus laevis estratto uovo è uno strumento potente e ampiamente applicato per studiare eventi cellulari complicati nella semplicità di un test cell-free. Fin dalla loro prima descrizione da Lohka &amp; Masui 1 che sono stati ampiamente utilizzati per studiare i processi mitotici quali la condensazione della cromatina 2, del fuso 3, rottura membrana nucleare 4, ma anche il trasporto nucleocytoplasmic 5 o 6 replicazione del DNA. Gli eventi che si svolgono al termine della mitosi, necessari per la riforma del nucleo interfasico quali riforma membrana nucleare e poro nucleare complesso rimontaggio sono molto meno inteso rispetto ai primi eventi mitotico ma possono essere studiati in modo simile utilizzando Xenopus estratto uovo 7. Abbiamo recentemente stabilito un saggio basato su estratto uovo Xenopus per studiare cromatina decondensazione alla fine della mitosi 8, un processo sotto-indagato che attende its caratterizzazione dettagliata. In metazoi, cromatina è altamente condensato all&#39;entrata mitotica per eseguire fedelmente la segregazione del materiale genetico. Per garantire che la cromatina è accessibile per l&#39;espressione genica e la replicazione del DNA durante interfase, deve essere de-compattato alla fine della mitosi. Nei vertebrati, cromatina è fino a cinquanta volte più compatta durante la mitosi rispetto interfase 9, in contrasto con lieviti dove la compattazione mitotico è generalmente molto inferiore, ad esempio, solo due volte nella S. cerevisiae 10. Vertebrato cromatina decondensazione è stata per lo più studiata nel contesto della riorganizzazione del DNA spermatico dopo la fecondazione delle uova. Un meccanismo molecolare, in cui nucleoplasmin, una proteina ovocita abbondante, scambia protamine specifici sperma di istoni H2A e H2B memorizzati nel uovo. Questo processo è stato anche chiarito utilizzando Xenopus estratto uovo 11, 12. Tuttavia, l&#39;espressionedi nucleoplasmin è limitata a ovociti 13 e cromatina mitotico non contiene queste protamine specifici sperma. Pertanto cromatina decondensazione alla fine della mitosi è nucleoplasmin indipendente 8. Per la reazione decondensazione in vitro impieghiamo estratto generato da attivi X. laevis uova e cluster cromatina isolate da cellule HeLa sincronizzate. Il trattamento di uova con un ionoforo del calcio imita il rilascio del calcio nel citoplasma dell&#39;ovocita generato da ingresso dello spermatozoo durante la fecondazione. L&#39;onda di calcio innesca la ripresa del ciclo cellulare e l&#39;uovo, arrestato nella seconda metafase della meiosi, progredisce alla prima interfase 14. Pertanto, gli estratti di uova preparati forma attivata uova rappresentano lo stato uscita / interfase mitosi e sono competenti per indurre eventi specifici per l&#39;uscita mitosi come cromatina decondensazione, membrana nucleare e poro riforma complessa. Per l&#39;isolamento di ch mitoticaromatin cluster abbiamo usato una versione leggermente modificata del protocollo pubblicato da Gasser &amp; Laemmli 15, dove i cluster cromosomiche sono rilasciate dai lisi da cellule HeLa sincronizzate in mitosi e isolati in poliammine contenenti buffer da centrifugazioni gradiente.

<table>
	<tbody>
		<tr>
			<td>spermine tetrahydrochloride</td>
			<td>Fluka analytical</td>
			<td>85610-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>spermidine trihydrochloride</td>
			<td>Sigma&#160;</td>
			<td>S2501-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>high-purity digitonin</td>
			<td>Millipore</td>
			<td>300410-1GM</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Applichem</td>
			<td>A0999,0100</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>thymidine</td>
			<td>Calbiochem</td>
			<td>6060</td>
			<td></td>
		</tr>
		<tr>
			<td>nocodazole</td>
			<td>Calbiochem</td>
			<td>487928</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>37 % formaldehyde solution</td>
			<td>Roth</td>
			<td>7398-1</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>trypan blue solution (0.4%)</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>1,4-dithiothreitol (DTT)</td>
			<td>Roth</td>
			<td>6908.2</td>
			<td></td>
		</tr>
		<tr>
			<td>AEBSF hydrochloride</td>
			<td>Applichem</td>
			<td>A1421,0001</td>
			<td></td>
		</tr>
		<tr>
			<td>pepstatin</td>
			<td>Roth</td>
			<td>2936.1/2/3</td>
			<td></td>
		</tr>
		<tr>
			<td>leupeptin</td>
			<td>Roth</td>
			<td>CN334</td>
			<td></td>
		</tr>
		<tr>
			<td>aprotinin</td>
			<td>Roth</td>
			<td>A162.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll (colloidal silica particles solution)</td>
			<td>GE Healthcare</td>
			<td>17-0891-01</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamine</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>75 cm&#178; tissue culture flasks</td>
			<td>Greiner Bio-one</td>
			<td>658175</td>
			<td></td>
		</tr>
		<tr>
			<td>heat-inactivated fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td>10500-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Homogenizer (40 mL tissue grinder)</td>
			<td>Wheaton</td>
			<td>357546</td>
			<td></td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Assistent</td>
			<td>441/1</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Oak Ridge Centrifuge Tubes, polycarbonate (50 ml)</td>
			<td>Nalgene</td>
			<td>3118-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m cell strainer, nylon</td>
			<td>BD Falcon</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr>
			<td>cytochalasin B</td>
			<td>Applichem</td>
			<td>A7657,0010</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Roth</td>
			<td>8682.3</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>L-cystein</td>
			<td>Merck</td>
			<td>1,028,381,000</td>
			<td></td>
		</tr>
		<tr>
			<td>hCG available as Ovogest</td>
			<td>MSD</td>
			<td>1431593</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG available as Intergonan</td>
			<td>MSD</td>
			<td>1431015</td>
			<td></td>
		</tr>
		<tr>
			<td>A23187 (mixed calcium-magnesium-salt)</td>
			<td>Enzo</td>
			<td>ALX-450-002-M010</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>syringe needles (1.20 x 40 mm, 18 G x 1 1/2&#34;)</td>
			<td>Braun</td>
			<td>4665120</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Serva</td>
			<td>10920.03</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP, 2 Na x 3 H20</td>
			<td>Roth</td>
			<td>K056.1/2/3/4</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphat disodium salt</td>
			<td>Calbiochem</td>
			<td>2380</td>
			<td></td>
		</tr>
		<tr>
			<td>creatine phosphokinase</td>
			<td>Sigma</td>
			<td>C3755-35KU</td>
			<td></td>
		</tr>
		<tr>
			<td>DMAP</td>
			<td>Sigma</td>
			<td>D2629-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI&#160;</td>
			<td>Roche</td>
			<td>10236276001</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma</td>
			<td>P-6148</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>centrifugation tubes for TLA 100 (7 x 10 mm, 5/16 x 13/16 in.)</td>
			<td>Beckman Coulter</td>
			<td>343775</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Cell-Saver&#34; (tips with wide opening, 1000 &#181;L)</td>
			<td>Biozym</td>
			<td>729065</td>
			<td></td>
		</tr>
		<tr>
			<td>50 % glutaraldehyde solution, grade I</td>
			<td>Sigma alderich</td>
			<td>G7651-10 mL</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>0.1 % (w/v) poly-L-lysine solution</td>
			<td>Sigma</td>
			<td>P8920-100 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>flat-bottom tubes (6 mL, 16.0/55 mm)</td>
			<td>Greiner Bio-one</td>
			<td>145211</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield mounting medium</td>
			<td>Vector laboratories</td>
			<td>H1000</td>
			<td></td>
		</tr>
		<tr>
			<td>tubes for TLA120 (11 x 34 mm, 7/16 x 1 3/8 in.)</td>
			<td>Beckman Coulter</td>
			<td>343778</td>
			<td></td>
		</tr>
		<tr>
			<td>&#34;Cell-Saver&#34; (tips with wide opening, 200 &#181;L)</td>
			<td>Biozym</td>
			<td>729055</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm coverslips</td>
			<td>Thermo Scientific</td>
			<td>0784 #1</td>
			<td></td>
		</tr>
	</tbody>
</table>

a cell free assay to study chromatin decondensation at the end of mitosis

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod shaped chromosomes which are moved individually by the mitotic spindle apparatus. Mitotic chromatin condensation yields chromosomes compacted fifty-fold denser as in interphase. During exit from mitosis, chromosomes have to re-establish their functional interphase state, which is enclosed by a nuclear envelope and is competent for replication and transcription. The decondensation process is morphologically well described, but in molecular terms poorly understood: We lack knowledge about the underlying molecular events and to a large extent the factors involved as well as their regulation. We describe here a cell-free system that faithfully recapitulates chromatin decondensation in vitro, based on mitotic chromatin clusters purified from synchronized HeLa cells and X. laevis egg extract. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its co-ordination with processes simultaneously occurring during mitotic exit such as nuclear envelope and pore complex re-assembly.

Ora la dipendenza della reazione decondensazione La figura 1 mostra un tipico andamento temporale del dosaggio decondensazione. Il cluster di cromosomi visibili all&#39;inizio della reazione decondenses e si fonde in un unico, rotondo e liscio nucleo. Quando l&#39;estratto uovo è sostituito dal saccarosio tampone cluster cromosoma rimane condensata, che suggeriscono che l&#39;attività decondensazione è presente nell&#39;estratto uovo. Deconden...

Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

Un Free Cell Assay per studiare cromatina decondensazione alla fine del mitosi

During the vertebrate cell cycle chromatin undergoes extensive structural and functional changes. Upon mitotic entry, it massively condenses into rod ...

a-cell-free-assay-to-study-chromatin-decondensation-at-end

Research

JoVE Journal

Biology

10.2K Views.  Max Planck Society. The molecular mechanisms of the decondensation of highly compacted mitotic chromatin are ill-defined. We present a cell-free assay based on mitotic chromatin clusters isolated from HeLa cells and Xenopus laevis egg extract that faithfully reconstitutes the decondensation process in vitro.

Video: A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test di sensibilità visiva alla velocità e direzione del movimento in lucertole

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Ricerca

Biologia

A Chromatin Assay for Human Brain Tissue

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental factors that might result in epigenetic alterations of gene expression and other molecular pathology. Traditionally, however, expression studies in postmortem brain were confined to quantification of mRNA or protein. The limitations encountered in postmortem brain research   such as variabilities in autolysis time and tissue integrities   are also likely to impact any studies of higher order chromatin structures. However, the nucleosomal organization of genomic DNA   including DNA:core histone binding - appears to be largely preserved in representative samples provided by various brain banks. Therefore, it is possible to study the methylation pattern and other covalent modifications of the core histones at defined genomic loci in postmortem brain. Here, we present a simplified native chromatin immunoprecipitation (NChIP) protocol for frozen (never-fixed) human brain specimens. Starting with micrococcal nuclease digestion of brain homogenates, NChIP followed by qPCR can be completed within three days. The methodology presented here should be useful to elucidate epigenetic mechanisms of gene expression in normal and diseased human brain.

Until recently, expression studies on human brain were limited to quantification of RNA or protein. With the chromatin immunoprecipitation techniques described in this paper, it will be possible to map histone methylation and other epigenetic regulators of gene expression in postmortem brain.

Un saggio cromatina per tessuto cerebrale umano

Chronic neuropsychiatric illnesses such as schizophrenia, bipolar disease and autism are thought to result from a combination of genetic and environmental ...

Operant Learning of Drosophila at the Torque Meter

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly's intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly's control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.

Measuring the yaw torque of tethered Drosophila with the torque meter allows the neuroscientist exquisite control of the stimulus situation of the experimental animal. Together with the unique genetic tools available in the fruit fly, this paradigm is used for a wide variety of neurobiological research.

L&#39;apprendimento della Drosophila operante presso il misuratore di coppia

For experiments at the torque meter, flies are kept on standard fly medium at 25&deg;C and 60% humidity with a 12hr light/12hr dark regime. A standardized ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Una cellula-cellula Macromolecole saggio di trasporto In Planta Utilizzando Bombardamento Biolistic

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Saggio di immunoprecipitazione della cromatina per geni tessuto-specifici utilizzando nella fase iniziale di embrioni di topo

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Chromatin Interaction Analysis with Paired-End Tag Sequencing (ChIA-PET) for Mapping Chromatin Interactions and Understanding Transcription Regulation

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such architectures are not random and involve interactions between gene promoters and regulatory elements 13. The binding of transcription factors to specific regulatory sequences brings about a network of transcription regulation and coordination 1, 14. 
Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) was developed to identify these higher-order chromatin structures 5,6. Cells are fixed and interacting loci are captured by covalent DNA-protein cross-links. To minimize non-specific noise and reduce complexity, as well as to increase the specificity of the chromatin interaction analysis, chromatin immunoprecipitation (ChIP) is used against specific protein factors to enrich chromatin fragments of interest before proximity ligation. Ligation involving half-linkers subsequently forms covalent links between pairs of DNA fragments tethered together within individual chromatin complexes. The flanking MmeI restriction enzyme sites in the half-linkers allow extraction of paired end tag-linker-tag constructs (PETs) upon MmeI digestion. As the half-linkers are biotinylated, these PET constructs are purified using streptavidin-magnetic beads. The purified PETs are ligated with next-generation sequencing adaptors and a catalog of interacting fragments is generated via next-generation sequencers such as the Illumina Genome Analyzer. Mapping and bioinformatics analysis is then performed to identify ChIP-enriched binding sites and ChIP-enriched chromatin interactions 8. 
We have produced a video to demonstrate critical aspects of the ChIA-PET protocol, especially the preparation of ChIP as the quality of ChIP plays a major role in the outcome of a ChIA-PET library. As the protocols are very long, only the critical steps are shown in the video.

Chromatin Interaction Analysis with Paired-End Tag Sequencing ChIA-PET for Mapping Chromatin Interactions and Understanding Transcription Regulation

Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a method for de novo detection of chromatin interactions, for better understanding of transcriptional control.

Cromatina Analisi Interazioni con Tag Sequencing appaiati-End (Chia-PET) per la mappatura Interazioni cromatina e comprensione del regolamento Trascrizione

Genomes are organized into three-dimensional structures, adopting higher-order conformations inside the micron-sized nuclear spaces 7, 2, 12. Such ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Analisi delle reazioni RNA di trasformazione che utilizzano cellulari Sistemi gratis: 3 &#39;Fine Decolleté di pre-mRNA Substrati<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Quantitative Immunofluorescence Assay misurare la variazione dei livelli di proteine ​​ad centrosomi

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...