Name: a cell free assay to study chromatin decondensation at the end of mitosis
Uploaded: 2015-12-19T13:00:00
Description: Watch this Scientific Journal Video about A Cell Free Assay to Study Chromatin Decondensation at the End of Mitosis at JoVE.com

AKSは1＆2が発達セル31（3）、Magalskaから転載されている図にこの作品は、ドイツの研究財団とERC（WAにAN377 / 3-2と309528はCHROMDECON）とベーリンガーインゲルハイムフォンの博士フェローシップによってサポートされていましたら 、有糸分裂、305から318まで、2014年の終わりにクロマチン脱凝縮で、RuvBのようなATPアーゼ機能、エルゼビアからの親切な許可を得て。

HeLa細胞由来の有糸分裂クロマチンクラスターの単離 1.準備<ol><li> 細胞培養ソリューション <ol><li> DMEM、10％ウシ胎仔血清、100単位/ mlペニシリン、100μg/ mlのストレプトマイシンおよび2mMグルタミンを添加することにより、完全ダルベッコ改変イーグル培地（DMEM）を準備します。脱イオン水中に2.7 mMのKClを含むリン酸緩衝生理食塩水（PBS）、137mMの塩...

<ol>
	<li>Lohka, M. J., Masui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+in+vitro+of+sperm+pronuclei+and+mitotic+chromosomes+induced+by+amphibian+ooplasmic+components.">Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.</a> Science. 220, 719-721 (1983).</li><li>de la Barre, A. E., Robert-Nicoud, M., Dimitrov, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+mitotic+chromosomes+in+Xenopus+egg+extract.">Assembly of mitotic chromosomes in Xenopus egg extract.</a> Methods Mol Biol. 119, 219-229 (1999).</li><li>Maresca, T. 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. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+study+of+nuclear+assembly+and+nuclear+import+using+Xenopus+egg+extracts.">In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts.</a> Methods Mol Biol. 322, 289-300 (2006).</li><li>Gillespie, P. J., Gambus, A., Blow, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+use+of+Xenopus+egg+extracts+to+study+DNA+replication+and+chromatin+associated+proteins.">Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins.</a> Methods. 57, 203-213 (2012).</li><li>Gant, T. M., Wilson, K. 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. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+nucleoplasmin+from+Xenopus+laevis+oocytes+and+analysis+of+its+developmental+expression.">Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression.</a> Genes Dev. 1, 97-107 (1987).</li><li>Whitaker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+at+fertilization+and+in+early+development.">Calcium at fertilization and in early development.</a> Physiological reviews. 86, 25-88 (2006).</li><li>Gasser, S. M., Laemmli, U. 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. 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=The+condensin+complex+is+required+for+proper+spindle+assembly+and+chromosome+segregation+in+Xenopus+egg+extracts.">The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts.</a> J Cell Biol. 161, 1041-1051 (2003).</li><li>Forbes, D. J., Kirschner, M. W., Newport, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+formation+of+nucleus-like+structures+around+bacteriophage+DNA+microinjected+into+Xenopus+eggs.">Spontaneous formation of nucleus-like structures around bacteriophage DNA microinjected into Xenopus eggs.</a> Cell. 34, 13-23 (1983).</li><li>Hartl, P., Olson, E., Dang, T., Forbes, D. J. <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+with+lambda+DNA+in+fractionated+Xenopus+egg+extracts:+an+unexpected+role+for+glycogen+in+formation+of+a+higher+order+chromatin+intermediate.">Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: an unexpected role for glycogen in formation of a higher order chromatin intermediate.</a> J Cell Biol. 124, 235-248 (1994).</li><li>Sandaltzopoulos, R., Blank, T., Becker, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+repression+by+nucleosomes+but+not+H1+in+reconstituted+preblastoderm+Drosophila+chromatin.">Transcriptional repression by nucleosomes but not H1 in reconstituted preblastoderm Drosophila chromatin.</a> EMBO J. 13, 373-379 (1994).</li><li>Ulbert, S., Platani, M., Boue, S., Mattaj, I. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+membrane+protein-DNA+interactions+required+early+in+nuclear+envelope+assembly.">Direct membrane protein-DNA interactions required early in nuclear envelope assembly.</a> J Cell Biol. 173, 469-476 (2006).</li><li>Zhang, C., Clarke, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin-independent+nuclear+envelope+assembly+induced+by+Ran+GTPase+in+Xenopus+egg+extracts.">Chromatin-independent nuclear envelope assembly induced by Ran GTPase in Xenopus egg extracts.</a> Science. 288, 1429-1432 (2000).</li><li>Paulson, J. 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>

アフリカツメガエル卵抽出物を忠実にインビトロで細胞プロセスを再現するための非常に有用なツールであり、このシステムは正常細胞周期および細胞分裂事象2、3,5,6,17の特徴付けに使用したアフリカツメガエル 。原因卵形成中の卵に隔離核成分の大型店に、卵抽出物は、細胞成分の優れた供給源です。哺乳動物組織細胞株または遺伝子操作上のRNAiのよ...

アフリカツメガエルの卵抽出物は、無細胞アッセイのシンプルで複雑 ​​な細胞事象を研究するための強力かつ広く適用するツールです。 Lohka＆増井1によるそれらの最初の記述以来、彼らは広く、このようなクロマチン凝縮2、スピンドルアセンブリ3、核膜崩壊4だけでなく、核-細胞質間輸送5またはDNA複製6などの有糸分裂のプロセスを研究するために使用されています。例えば、核エンベロープ改革と核膜孔複合体の再構築などinterphasic核の改革のために必要な有糸分裂の終わりで行われているイベントは、はるかに少ない初期の有糸分裂のイベントに比べて理解されているが、同様にアフリカツメガエルの卵抽出物7を使用して研究することができます。我々は最近、有糸分裂8の最後にクロマチン脱凝縮を研究するためにアフリカツメガエルの卵抽出物に基づくアッセイを確立した、アンダー調べプロセスは、私が待っています詳細な特徴づけをtsで。 後生動物では、クロマチンは高度に遺伝物質の忠実な分離を行うために、有糸分裂のエントリに集光されます。クロマチンは、間期中の遺伝子発現およびDNA複製のためにアクセス可能であることを保証するために、それは、有糸分裂の終了時に脱圧縮される必要があります。脊椎動物では、クロマチンは 、Sにおける有糸分裂圧縮は通常はるかに低い酵母、 例えば、2つだけ倍とは対照的に、相間9に比べて有糸分裂の間に複数の圧縮された50倍までですセレビシエ 10。脊椎動物クロマチン脱凝縮は、主に卵の受精後の精子DNAの再編との関連で検討されています。分子機構は、どのヌ、豊富な卵母細胞のタンパク質で、卵に保存されたヒストンH2AとH2Bに精子固有のプロタミンを交換します。このプロセスはまた、 アフリカツメガエル卵抽出物11、12を用いて解明されました。しかし、表現ヌの卵母細胞13と、有糸分裂クロマチンに限定されているこれらの精子固有のプロタミンが含まれていません。したがって、有糸分裂の最後に脱凝縮クロマチンは、8つの独立したヌクレオです。 インビトロ脱凝縮反応のために我々は同期のHeLa細胞から単離された活性化したアフリカツメガエル の卵とクロマチンクラスターから生成されたエキスを使用しています。カルシウムイオノフォアと卵の治療は、受精時の精子のエントリによって生成された卵母細胞へのカルシウム放出を模倣。カルシウム波は最初相間14に進行し、減数分裂の第二中期で逮捕され、細胞周期の再開と卵をトリガします。そのため、フォームアクティブに卵準備卵抽出物は、有糸分裂終了/相間状態を表し、クロマチン脱凝縮、核膜などの有糸分裂終了のための特定のイベントを誘導する能力があると複雑な改革を細孔。有糸分裂CHの単離のためにクラスタromatin我々は、染色体のクラスターが有糸分裂に同期したHeLa細胞からの溶解により放出され、勾配遠心分離によってバッファを含むポリアミンで単離されるガッサー＆レムリ15、によって公開されたプロトコルを少し変更したバージョンを使用していました。

<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.

脱凝縮反応の時間依存性 図1は、脱凝縮アッセイの典型的な時間経過を示します。反応の開始時に目に見える染色体のクラスターがdecondenses、シングル、ラウンドと滑らかな核に合流。卵抽出物は、スクロースで置き換えられたときに染色体クラスタバッファ脱凝縮活性は卵抽出物中に存在することを示唆している、縮合したままです。 ?...

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.

無細胞アッセイは、有糸分裂の終了時クロマチン脱凝縮を勉強します

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

Research

JoVE Journal

Biology

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

トカゲの運動の速度と方向の視覚感度のテスト

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

A Chromatin Assay for Human Brain Tissue

人間の脳組織のためのクロマチンアッセイ

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

トルクメーターでのショウジョウバエのオペラント学習

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

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

細胞間の高分子輸送アッセイプランタで微粒子銃砲撃を活用

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

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

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

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

クロマチンの相互作用のマッピングと転写調節を理解するためのペアエンドタグシーケンシング（CHIA-PET）によるクロマチンの相互作用の解析

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

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

事前のmRNA基質の3 &#39;末端切断：無細胞系を用いたRNAプロセシング反応の解析<em&gt; in vitroで</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

In vitro Methylation Assay to Study Protein Arginine Methylation

タンパク質アルギニンメチル化を研究するためのin vitroでメチル化アッセイで

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

中心体におけるタンパク質レベルの変化を測定するための定量的免疫蛍光アッセイ

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

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

シロイヌナズナタンパク質-DNA相互作用の同定のための免疫沈降アッセイクロマチン<em&gt;インビボ</em

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