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

Bu çalışma, İsrail Bilim Vakfı ve Rappaport Aile Enstitüsü Tıp Bilimleri Araştırma ve Deutsche Forschungsgemeinschaft (DFG) hibe tarafından desteklenen oldu.

Deney başlamadan önce Önkoşul: <ul><li> RNaz içermeyen su RNA RNases tarafından bozulmuş olma riskini azaltmak için laboratuvarda RNA alınmasında kullanılır. Bu amaçla, su genellikle 37 en az 1 saat için 0.1% v / v diethylpyrocarbonate (DEPC) ile tedavi edilir ° C ve daha sonra DEPC izleri inaktive etmek için otoklava (en az 15 dk). </li><li> Pipetman pipetler kullanmadan önce bir RNaz dekontaminasyon solüsyonu (RNaz Exterminator, Biyolojik Sanayi Beit Haemek) ile temizlenir. </li><li> Eppendorf tüpl...

<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>

Bu sunumda, E. tRNA metiltransferaz aktivite ölçmek için aktif bileşenleri nasıl hazırlanacağını gösterildiği histolytica Ehmeth enzim. Bu prosedür, bir başlangıç ​​noktası olarak hizmet edebilir ve diğer tRNA methyltransferases ölçmek için kolayca adapte edilebilir. TRNA substrat ve tRNA metiltransferaz protein kalitesini ve bütünlüğünü korumak enzimatik aktivite optimal bir ölçüsü sigorta işlemlerini takip etmek önemlidir.

<!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

Protozoa parazitler amebiasis, paraziter hastalık ölüm üç en yaygın nedenlerinden biri de dahil olmak üzere çeşitli hastalıkların sorumlu insanların en yıkıcı enfeksiyöz ajanlar arasında yer almaktadır. Amebiyazis ajan iki aşamada altında var olan amip parazit Entamoeba histolytica: enfektif kisti, bağırsak gıda ya da su ve invaziv trophozoite yaşam bulundu . Asemptomatik kolit, dizanteri veya karaciğer abseleri amebiasis aralığının klinik bulgular E. histolytica nadir tek hücreli parazitin biri kendi genom 5-methylcytosine (5MC) ile 1, 2 Dnmt2 ailesine ait metiltransferaz tek bir DNA Ehmeth, içerir. 2 tekrarlayan elemanlarla kontrol Dnmt2 bir rol E. kurulmuştur histolytica, 3 Diktiyostelyum diskodeyum 4,5 ve Drosophila 6 Bizim son iş bu Ehmeth metiller tRNA Asp gösterdi ve bu bulgu, bu enzim bir çift DNA / tRNA Asp metiltransferaz aktivitesi olduğunu göstermektedir. 7 Bu gözlem çift aktivite ile anlaşma D. bildirilmiştir diskodeyum ve D. melanogaster. Dnmt2 enzimler DNA / tRNA özgüllüğü 8 fonksiyonel önemi hala bilinmiyor. Bu soruya cevap için, Dnmt2 proteinler tRNA metiltransferaz etkinliğini belirlemek için bir yöntem kurulmuştur. Bu video, Dnmt2 için yeterli bir tRNA substrat hazırlamak için basit bir yaklaşımdır ve tRNA metiltransferaz aktivitesini ölçmek için bir yöntem açıklanmaktadır.

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

Bu protokol hazırlanması için sentetik bir tRNA substrat açıklar<em&gt; Entamoeba histolytica</em&gt; DNA / tRNA metiltransferaz 2 (Dnmt2) homolog Ehmeth ve metil faaliyet ölçüsüdür. Bu deneysel bir yaklaşım, diğer Dnmt2 proteinlerin aktivitesini araştırmak için kullanılabilir.

Entamoeba histolytica DNA ve tRNA Metiltransferaz Dnmt2 (Ehmeth) Enzim ile in vitro tRNA Metilasyon Testi

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

Negatif Seçilebilir Marker Geliştirme 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

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

Nicel<em&gt; In vitro</emStatik Koşullarında Account İlköğretim İnsan mikrovasküler endotel hücreleri için Nötrofil yapışma Tedbir&gt; Testi

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

Vitro Testi HIV-spesifik CD4 T hücre efektör işlevi üzerinde immünoregülatör yolları etkisini değerlendirmek için

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

Bir kullanma Pnömokok Kolonizasyon üzerinde Probiyotiklerin Etkileri incelenmesi<em&gt; In Vitro</em&gt; Yapışma Deneyi

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

Bir Kısıtlama Enzim Temelli Klonlama Yöntemi değerlendirin<em&gt; In vitro</em&gt; HIV-1 Tipi C Gag-MJ4 Kimerik Virüslerin Çoğaltma Kapasitesi

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

Yapımcı Epitel Hasar Analizi<em&gt; Entamoeba histolytica</em&gt; Enfeksiyon

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

Bir Geliştirilmesi<em&gt; In vitro</emEtkileşimini incelemek için&gt; bir model sistem<em&gt; Equus</em&gt;<em&gt; Caballus</emYüksek afiniteli reseptör FcεRI&gt; ile IgE

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

Enzim-linked Lektin Testi tarafından Grip Nöraminidaz İnhibisyon antikor titreleri Ölçme

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

Bir kullanma Lenfosit Ekstravazasyonunun analizi<em&gt; İn Vitro</em&gt; İnsan Kan-beyin bariyeri Model

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