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

מחקר זה נתמך על ידי מענקי הקרן הלאומית למדע ומכון רפפורט למחקר במדעי הרפואה, ואת Forschungsgemeinschaft דויטשה (DFG).

דרישות מוקדמות לפני ביצוע הניסוי: <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> RNase ללא מים משמש בטיפול של רנ&quot;א במעבדה כדי להפחית את הסיכון של רנ&quot;א להיות מושפל על ידי RNases. לענין זה, המים מטופלים בדרך כלל במשך שעה לפחות 1 עם 0.1% diethylpyrocarbonate v / v (DEPC) בשעה 37 ° C ו autoclaved ?...

<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>

במצגת זו, הדגמנו איך להכין את הרכיבים הפעילים במשך למדוד את הפעילות של ה tRNA methyltransferase histolytica אנזים Ehmeth. הליך זה יכול לשמש כנקודת פתיחה ו להתאים בקלות מידה של methyltransferases tRNA אחרים. חשוב לבצע את ההליכים לשמר את איכות על שלמות המצע tRNA של החלבון tRNA methyltransferase כדי להבט...

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

טפילים protozoan הם בין גורמים מדבקים הרסנית ביותר של בני אדם אחראי מגוון רחב של מחלות כולל amebiasis, שהוא אחד משלושת הגורמים השכיחים ביותר למוות ממחלות טפיליות. הסוכן של amebiasis הוא טפיל האמבה Entamoeba histolytica שקיים תחת שני שלבים: ציסטה זיהומיות למצוא מזון או מים חיים trophozoite פולשנית במעי. הביטויים הקליניים של מגוון amebiasis מלהיות אסימפטומטיים עד מורסות, קוליטיס דיזנטריה או כבד. E. histolytica הוא אחד הטפיל unicellular נדיר עם 5-methylcytosine (5mC) בגנום שלו. 1, 2 הוא מכיל דנ&quot;א יחיד methyltransferase, Ehmeth, אשר שייך למשפחת Dnmt2. 2 Dnmt2 תפקיד בשליטה על אלמנטים חוזרים יש הוקמו E. histolytica, 3 discoideum Dictyostelium 4,5 ו תסיסנית. 6 העבודה האחרונה שלנו הוכיח כי Ehmeth methylates tRNA ASP, ממצא זה מראה כי אנזים זה יש DNA כפול / tRNA פעילות methyltransferase-ASP. 7 תצפית זו עולה בקנה אחד עם פעילות כפולה כך דווחה עבור ד discoideum וד melanogaster. 8 משמעות תפקודית של סגוליות DNA / tRNA של Dnmt2 אנזימים עדיין לא ידוע. כדי לענות על שאלה זו, שיטה כדי לקבוע את פעילות tRNA methyltransferase של Dnmt2 חלבונים הוקמה. בסרטון הזה, אנו מתארים גישה פשוטה להכין מצע tRNA מתאים Dnmt2 ו שיטה למדידת פעילות tRNA methyltransferase שלה.

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

פרוטוקול זה מתאר את הכנת המצע tRNA סינתטי עבור<em&gt; Entamoeba histolytica</em&gt; הדנ&quot;א / tRNA methyltransferase 2 (Dnmt2) Ehmeth homolog ואת מידת הפעילות methyltransferase שלה. גישה זו הניסוי יכול לשמש חוקרת את פעילותו של Dnmt2 חלבונים אחרים.

ב Assay tRNA מתילציה במבחנה עם histolytica Entamoeba דנ&quot;א Dnmt2 tRNA methyltransferase (Ehmeth) אנזימים

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

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