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

Denna studie har finansierats med bidrag från Israel Science Foundation och Rappaport Familj Institutet för forskning inom medicinska vetenskaper, och Deutsche Forschungsgemeinschaft (DFG).

Förutsättning innan inleda experiment: <ul><li> RNas-fria vatten används i hanteringen av RNA i laboratoriet för att minska risken av RNA som bryts ned av RNaser. För detta ändamål är vatten behandlas vanligtvis med 0,1% v / v diethylpyrocarbonate (DEPC) i minst 1 timme vid 37 ° C och därefter autoklaveras (minst 15 min) för att inaktivera spår av DEPC. </li><li> Pipetman pipetter rengörs med en RNas sanering lösning (RNase UTROTARE, biologiska industri Beit Haemek) innan användning. </li><li> Eppendorf-...

<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>

I denna presentation illustrerade vi hur man förbereder aktiva komponenter för mätning av tRNA metyltransferas aktivitet E. histolytica Ehmeth enzym. Detta förfarande kan fungera som en utgångspunkt och lätt anpassas till åtgärden av andra tRNA metyltransferaser. Det är viktigt att följa de förfaranden som bevarar kvaliteten och integriteten i tRNA-substrat och tRNA metyltransferas protein för att försäkra en optimal mått på den enzymatiska aktiviteten.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<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

Protozo parasiter är bland de mest förödande infektioner av människor med ansvar för en rad olika sjukdomar, bland annat amebiasis, som är en av de tre vanligaste dödsorsakerna från parasitsjukdom. Agenten av amebiasis är amöba parasit Entamoeba histolytica som finns i två steg: det smittsamma cysta som finns i mat eller vatten och invasiva trophozoite lever i tarmen. Den kliniska manifestationer av amebiasis variera från att vara asymtomatiska till kolit, dysenteri eller lever abscesser. E. histolytica är en av de få encelliga parasiten med 5-methylcytosine (5mC) i sin arvsmassa. 1, 2 Det innehåller en enda DNA-metyltransferas, Ehmeth, som tillhör Dnmt2 familjen. 2 En roll för Dnmt2 i kontrollen av repetitiva element har fastställts i E. histolytica, 3 Dictyostelium discoideum 4,5 och Drosophila. 6 Vårt senaste arbete har visat att Ehmeth methylates tRNA Asp, och detta fynd tyder på att detta enzym har en dubbel DNA / tRNA Asp metyltransferas aktivitet. 7 Detta konstaterande är i överensstämmelse med den dubbla verksamheten som har rapporterats för D. discoideum och D. melanogaster. 8 Den funktionella betydelsen av DNA / tRNA-specificiteten hos Dnmt2 enzymer är fortfarande okänd. För att lösa denna fråga, var en metod för att bestämma tRNA metyltransferas aktivitet Dnmt2 proteiner fastställts. I denna video beskriver vi en okomplicerad metod för att förbereda en lämplig tRNA substrat för Dnmt2 och en metod för att mäta sin tRNA metyltransferas aktivitet.

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

Detta protokoll beskriver förberedelserna av ett syntetiskt tRNA substrat för den<em&gt; Entamoeba histolytica</em&gt; DNA / tRNA metyltransferas 2 (Dnmt2) homolog Ehmeth och mått på dess metyltransferas aktivitet. Denna experimentella metod kan användas för att undersöka aktiviteten hos andra Dnmt2 proteiner.

In vitro-tRNA Metylering Analysera med Entamoeba histolytica DNA och tRNA metyltransferas Dnmt2 (Ehmeth) Enzyme

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

Research

JoVE Journal

Immunology and Infection

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

Development of a Negative Selectable Marker for Entamoeba histolytica

Development of a Negative Selectable Marker for Entamoeba histolytica

Utveckling av en negativ markör för Entamoeba histolytica</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Inrättande av en<em&gt; In vitro</em&gt; System för att studera intracellulära Beteende<em&gt; Candida glabrata</em&gt; I Human THP-1 makrofager

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Kvantitativ<em&gt; In vitro</em&gt; Analys för att mäta neutrofilvidhäftning till Aktiverade primära humana mikrovaskulära endotelceller under statiska förhållanden

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In vitro-analys för att utvärdera effekten av immunPathWays på HIV-specifika CD4 T-cell effektorfunktion

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Undersöka effekterna av probiotika på Pneumococcal Colonization Använda en<em&gt; In Vitro</em&gt; Adherensanalysen

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

Ett restriktionsenzym Based Kloning metod för att bedöma<em&gt; In vitro</em&gt; Replikering Kapacitet på HIV-1 subtyp C Gag-MJ4 Chimära virus

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analys av epitelskada Producerad av<em&gt; Entamoeba histolytica</em&gt; Infektion

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcÃƒÅ½Ã‚ÂµRI

Utveckling av ett<em&gt; In vitro</em&gt; Modellsystem för att studera interaktionen mellan<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE med sin högaffinitetsreceptor FcsRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Mätning influensaneuraminidas Inhibition Antikroppstitrar genom enzymkopplad lektin analys

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analys av Lymphocyte Extravasation Med användning av ett<em&gt; In Vitro</em&gt; Modell av det mänskliga blod-hjärnbarriären

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...