Name: in vitro trna methylation assay with the entamoeba histolytica dna and trna methyltransferase dnmt2 (ehmeth) enzyme
Uploaded: 2010-10-19T17:00:00
Description: Watch this Scientific Journal Video about In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme at JoVE.com

Dit onderzoek werd ondersteund door subsidies van de Israel Science Foundation en het Rappaport Family Instituut voor Onderzoek in de Medische Wetenschappen, en de Deutsche Forschungsgemeinschaft (DFG).

Voorwaarde voor het begin van het experiment: <ul><li> RNase-vrij water wordt gebruikt in de behandeling van RNA in het laboratorium om het risico van RNA wordt afgebroken door RNases te verminderen. Voor dit doel, het water wordt meestal behandeld met 0,1% v / v diethylpyrocarbonate (DEPC) gedurende minstens een uur bij 37 ° C en vervolgens geautoclaveerd (minstens 15 minuten) naar sporen van DEPC inactiveren. </li><li> Pipetman pipetten worden gereinigd met een RNase decontaminatie-oplossing (RNase verdelger, biolog...

<ol>
	<li>Banerjee, S., Fisher, O., Lohia, A., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entamoeba+histolytica+DNA+methyltransferase+(Ehmeth)+is+a+nuclear+matrix+protein+that+binds+EhMRS2,+a+DNA+that+includes+a+scaffold/matrix+attachment+region+(S/MAR).">Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR).</a> Mol Biochem Parasitol. 139, 91-97 (2005).</li><li>Fisher, O., Siman-Tov, R., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cytosine+methylated+regions+and+5-cytosine+DNA+methyltransferase+(Ehmeth)+in+the+protozoan+parasite+Entamoeba+histolytica.">Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica.</a> Nucleic Acids Res. 32, 287-297 (2004).</li><li>Harony, H., Bernes, S., Siman-Tov, R., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+and+targeting+of+LINE+retrotransposons+in+Entamoeba+histolytica+and+Entamoeba+invadens.">DNA methylation and targeting of LINE retrotransposons in Entamoeba histolytica and Entamoeba invadens.</a> Mol Biochem Parasitol. 147, 55-63 (2006).</li><li>Kuhlmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+retrotransposons+in+Dictyostelium+by+DNA+methylation+and+RNAi.">Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi.</a> Nucleic Acids Res. 33, 6405-6417 (2005).</li><li>Katoh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmentally+regulated+DNA+methylation+in+Dictyostelium+discoideum.">Developmentally regulated DNA methylation in Dictyostelium discoideum.</a> Eukaryot Cell. 5, 18-25 (2006).</li><li>Phalke, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrotransposon+silencing+and+telomere+integrity+in+somatic+cells+of+Drosophila+depends+on+the+cytosine-5+methyltransferase+DNMT2.">Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2.</a> Nat Genet. 41, 696-702 (2009).</li><li>Tovy, A., Siman Tov, R., Gaentzsch, R., Helm, M., Ankri, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+nuclear+function+of+the+Entamoeba+histolytica+glycolytic+enzyme+enolase:+the+metabolic+regulation+of+cytosine-5+methyltransferase+2+(Dnmt2)+activity.">A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2) activity.</a> PLoS Pathog. 6, e1000775-e1000775 (2010).</li><li>Jeltsch, A., Nellen, W., Lyko, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+substrates+are+better+than+one:+dual+specificities+for+Dnmt2+methyltransferases.">Two substrates are better than one: dual specificities for Dnmt2 methyltransferases.</a> Trends Biochem Sci. 31, 306-308 (2006).</li><li>Fechter, P., Rudinger, J., Giege, R., Theobald-Dietrich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribozyme+processed+tRNA+transcripts+with+unfriendly+internal+promoter+for+T7+RNA+polymerase:+production+and+activity.">Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity.</a> FEBS Lett. 436, 99-103 (1998).</li><li>Hermann, A., Schmitt, S., Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+Dnmt2+has+residual+DNA-(Cytosine-C5)-methyltransferase+activity.">The human Dnmt2 has residual DNA-(Cytosine-C5)-methyltransferase activity.</a> J Biol Chem. 278, 31717-31721 (2003).</li><li>Narsa Reddy, M., Tang, L. Y., Lee, T. L., &amp;amp, T. L., James Shen, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+candidate+gene+for+Drosophila+genome+methylation.">A candidate gene for Drosophila genome methylation.</a> Oncogene. 22, 6301-6303 (2003).</li></ol>

In deze presentatie, we geïllustreerd hoe de actieve componenten voor te bereiden op de maat van het tRNA methyltransferase activiteit van E. histolytica Ehmeth enzym. Deze procedure kan dienen als een startpunt en gemakkelijk aangepast aan de maat van andere tRNA-methyl. Het is belangrijk dat het volgen van de procedures die de kwaliteit en de integriteit van het tRNA substraat en van de tRNA methyltransferase eiwit te behouden om een ​​optimale meting van de enzymatische activiteit te verzekeren.

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Taq Polymerase (5 U/&mu;l)</td>
<td>&nbsp;</td>
<td>Rapidozym, Berlin</td>
<td>GEN-003-1000</td>
<td>&nbsp;</td>
<tr>
<td>10X Gentherm PCR Buffer</td>
<td>&nbsp;</td>
<td>Rapidozym, Berlin</td>
<td>With GEN-003-1000</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2 (50 mM)</td>
<td>&nbsp;</td>
<td>Rapidozym, Berlin</td>
<td>With GEN-003-1000</td>
<td>&nbsp;</td>
<tr>
<td>dNTP mix (10 mM)</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>R0191</td>
<td>&nbsp;</td>
<tr>
<td>Oligo nucleotides</td>
<td>&nbsp;</td>
<td>IBA, G&ouml;ttingen</td>
<td>Customized</td>
<td>&nbsp;</td>
<tr>
<td>NTP mix (25mM)</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>R0481</td>
<td>&nbsp;</td>
<tr>
<td>GelRed Nucleic Acid Stain 3X in water</td>
<td>&nbsp;</td>
<td>Biotium</td>
<td>41001</td>
<td>&nbsp;</td>
<tr>
<td>Dithiotreitol</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>R0861</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>S0266</td>
<td>&nbsp;</td>
<tr>
<td>Pall Nanosep 0.45 &mu;m</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>Z722014</td>
<td>&nbsp;</td>
<tr>
<td>Dichlorodimethylsilane</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>40140</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol (Ph. Eur.)</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>5054.3</td>
<td>&nbsp;</td>
<tr>
<td>Tris HCl</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>9090.3</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>AE15.1</td>
<td>&nbsp;</td>
<tr>
<td>BSA</td>
<td>&nbsp;</td>
<td>Fermentas, St. Leon-Rot</td>
<td>B14</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>&nbsp;</td>
<td>Sigma, Germany</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>TEMED</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>2367.1</td>
<td>&nbsp;</td>
<tr>
<td>Ammonium Peroxodisulfate</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>9592.2</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese Sequencing gel concentrate</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3043.1</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese Sequencing gel diluent</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3047.1</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese Sequencing gel buffer concentrate</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3050.1</td>
<td>&nbsp;</td>
<tr>
<td>Rotiphorese 10X TBE-buffer</td>
<td>&nbsp;</td>
<td>Roth, Karlsruhe</td>
<td>3061.2</td>
<td>&nbsp;</td>
<tr>
<td>DEPC</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5758</td>
<td>&nbsp;</td>
<tr>
<td>RNAse ExTERMINATOR</td>
<td>&nbsp;</td>
<td>Biological Industry Beit Haemek</td>
<td>01-895-1B</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0166</td>
<td>&nbsp;</td>
<tr>
<td>IPTG</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I6758</td>
<td>&nbsp;</td>
<tr>
<td>PMSF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P7626</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L7651</td>
<td>&nbsp;</td>
<tr>
<td>Leupeptine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L2884</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase Nuclease</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70746-3</td>
<td>&nbsp;</td>
<tr>
<td>BugBuster protein extraction reagent</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70921-3</td>
<td>&nbsp;</td>
<tr>
<td>Glutathione-agarose resin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G4510</td>
<td>&nbsp;</td>
<tr>
<td>L-glutathione reduced</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G4251</td>
<td>&nbsp;</td>
<tr>
<td>AdoMet</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>B9003</td>
<td>&nbsp;</td>
<tr>
<td>AdoMet 3H</td>
<td>&nbsp;</td>
<td>PerkinElmer</td>
<td>NET155250UC</td>
<td>&nbsp;</td>
<tr>
<td>Scintillation Cocktail</td>
<td>&nbsp;</td>
<td>CytoScint</td>
<td>882453</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vitro trna methylation assay with the entamoeba histolytica dna and trna methyltransferase dnmt2 (ehmeth) enzyme

Protozoaire parasieten behoren tot de meest verwoestende ziekteverwekkers van de mens verantwoordelijk is voor een verscheidenheid van ziekten, waaronder amebiasis, dat is een van de drie meest voorkomende oorzaken van overlijden als gevolg van parasitaire ziekten. De agent van amebiasis is de amoebe parasiet Entamoeba histolytica die er bestaat in twee fasen: de besmettelijke cyste gevonden in voedsel of water en de invasieve trophozoite leven in de darm. De klinische manifestaties van amebiasis variëren van asymptomatisch tot colitis, dysenterie of lever abcessen. E. histolytica is een van de zeldzame eencellige parasiet met 5-methylcytosine (5mC) in zijn genoom. 1, 2 Het bevat een enkel DNA-methyltransferase, Ehmeth, dat behoort tot de Dnmt2 familie. 2 Een rol voor Dnmt2 in de controle van repetitieve elementen heeft is opgericht in E. histolytica, 3 Dictyostelium discoideum 4,5 en Drosophila. zes Onze recente werk heeft aangetoond dat Ehmeth methyleert tRNA Asp, en deze bevinding geeft aan dat dit enzym een dubbele DNA / tRNA Asp methyltransferase-activiteit heeft. 7 Deze waarneming is in overeenstemming met de dubbele activiteit dat is gemeld voor D. discoideum en D. melanogaster. 8 De functionele betekenis van de DNA / tRNA specificiteit van Dnmt2 enzymen is nog onbekend. Om deze vraag was een methode om de tRNA methyltransferase activiteit van eiwitten te bepalen Dnmt2 vastgesteld. In deze video, beschrijven we een rechttoe rechtaan aanpak om een ​​adequaat tRNA substraat voor te bereiden op Dnmt2 en een methode om de tRNA methyltransferase activiteit te meten.

Watch this Scientific Journal Video about In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme at JoVE.com

In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 Ehmeth Enzyme

Dit protocol beschrijft de bereiding van een synthetisch tRNA substraat voor de<em&gt; Entamoeba histolytica</em&gt; DNA / tRNA methyltransferase 2 (Dnmt2) homoloog Ehmeth en de maatstaf van de methyltransferase activiteit. Deze experimentele benadering kan worden gebruikt voor het onderzoeken van de activiteit van andere Dnmt2 eiwitten.

In vitro tRNA methylatie test met de Entamoeba histolytica DNA en tRNA methyltransferase Dnmt2 (Ehmeth) Enzym

Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of ...

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