在体外的tRNA与阿米巴 DNA和tRNA甲基Dnmt2（Ehmeth）酶甲基化含量
Summary
这个协议描述为合成tRNA的衬底准备<em>阿米巴</em>的tRNA / DNA的甲基转移酶2（Dnmt2）的同源性Ehmeth和甲基转移酶活性的措施。这种实验方法可用于调查其他Dnmt2蛋白的活性。
Abstract
原虫是最具毁灭性的传染性病原体对人类负责各种疾病，包括阿米巴病，寄生虫病的三种最常见的死亡原因之一。阿米巴剂是阿米巴寄生虫阿米巴存在下两个阶段：发现感染性囊肿的食物或水，并在肠道内侵入滋养体生活。阿米巴范围的临床表现从无症状大肠杆菌肠炎，痢疾或肝脓肿。 阿米巴是一个罕见的单细胞寄生虫，它的基因组与5 -甲基胞嘧啶（5mC）1，2，它包含一个单一的DNA甲基化，Ehmeth，属于Dnmt2家庭。2一个Dnmt2在重复元素的控制作用建立了在大肠杆菌中3， 阿米巴粘菌 4,5和果蝇 。6我们最近的工作表明，Ehmeth甲基化tRNA 的 ASP，这一发现表明，这种酶具有双重DNA / tRNA 的 ASP甲基转移酶活性。7这个观察的双重活动的协议已报告为D。 discoideum和 D。 果蝇 。8 Dnmt2酶的DNA / tRNA的特异性的功能意义，仍是未知数。为了解决这个问题，成立了一个方法来确定的tRNA Dnmt2蛋白甲基转移酶活性。在这段视频中，我们描述了一个简单的方法，准备Dnmt2足够的tRNA基板和方法来衡量它的tRNA甲基转移酶活性。
Protocol
然后才开始实验的前提条件： 无RNase的水是用于在实验室中的RNA处理，以减少风险的RNA被RNase的降解。为此，水通常是治疗至少1小时，0.1％V / V酸二乙酯（DEPC）在37℃，然后高压灭菌（至少15分钟），灭活的DEPC的痕迹。 Pipetman移液器核糖核酸酶净化解决方案（核糖核酸灭虫，生物产业拜特哈Haemek）在使用前清洗。 Eppendorf管和提示应认证DNase和RNase的自由，以保持样品的完整性?…
Discussion
在此演示中，我们说明如何准备tRNA 的 E.甲基转移酶活性的措施中的活性成分阿米巴 Ehmeth酶。这个程序可以作为一个起点和衡量其他的tRNA甲基很容易适应。重要的是要遵循的程序，维护tRNA的衬底和tRNA的甲基蛋白质的质量和完整性，以确保酶活性的最佳措施。
Declarações
The authors have nothing to disclose.
Acknowledgements
这项研究是由以色列科学基金会和拉帕波特医学科学院研究家庭研究所和德意志研究联合会（DFG）的资助的支持。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Taq Polymerase (5 U/μl) | Rapidozym, Berlin | GEN-003-1000 | ||
| 10X Gentherm PCR Buffer | Rapidozym, Berlin | With GEN-003-1000 | ||
| MgCl2 (50 mM) | Rapidozym, Berlin | With GEN-003-1000 | ||
| dNTP mix (10 mM) | Fermentas, St. Leon-Rot | R0191 | ||
| Oligo nucleotides | IBA, Göttingen | Customized | ||
| NTP mix (25mM) | Fermentas, St. Leon-Rot | R0481 | ||
| GelRed Nucleic Acid Stain 3X in water | Biotium | 41001 | ||
| Dithiotreitol | Fermentas, St. Leon-Rot | R0861 | ||
| Spermidine | Sigma, Germany | S0266 | ||
| Pall Nanosep 0.45 μm | Sigma, Germany | Z722014 | ||
| Dichlorodimethylsilane | Sigma, Germany | 40140 | ||
| Ethanol (Ph. Eur.) | Roth, Karlsruhe | 5054.3 | ||
| Tris HCl | Roth, Karlsruhe | 9090.3 | ||
| Tris | Roth, Karlsruhe | AE15.1 | ||
| BSA | Fermentas, St. Leon-Rot | B14 | ||
| Triton X-100 | Sigma, Germany | T8787 | ||
| TEMED | Roth, Karlsruhe | 2367.1 | ||
| Ammonium Peroxodisulfate | Roth, Karlsruhe | 9592.2 | ||
| Rotiphorese Sequencing gel concentrate | Roth, Karlsruhe | 3043.1 | ||
| Rotiphorese Sequencing gel diluent | Roth, Karlsruhe | 3047.1 | ||
| Rotiphorese Sequencing gel buffer concentrate | Roth, Karlsruhe | 3050.1 | ||
| Rotiphorese 10X TBE-buffer | Roth, Karlsruhe | 3061.2 | ||
| DEPC | Sigma | D5758 | ||
| RNAse ExTERMINATOR | Biological Industry Beit Haemek | 01-895-1B | ||
| Ampicillin | Sigma | A0166 | ||
| IPTG | Sigma | I6758 | ||
| PMSF | Sigma | P7626 | ||
| Lysozyme | Sigma | L7651 | ||
| Leupeptine | Sigma | L2884 | ||
| Benzonase Nuclease | Novagen | 70746-3 | ||
| BugBuster protein extraction reagent | Novagen | 70921-3 | ||
| Glutathione-agarose resin | Sigma | G4510 | ||
| L-glutathione reduced | Sigma | G4251 | ||
| AdoMet | New England Biolabs | B9003 | ||
| AdoMet 3H | PerkinElmer | NET155250UC | ||
| Scintillation Cocktail | CytoScint | 882453 |
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Tags
In Vitro TRNA Methylation AssayEntamoeba HistolyticaDNA Methyltransferase Dnmt2Ehmeth EnzymeProtozoan ParasitesAmebiasisInfectious AgentsClinical Manifestations5-methylcytosine (5mC)Single DNA MethyltransferaseControl Of Repetitive ElementsDual DNA/tRNAAsp Methyltransferase ActivityFunctional SignificanceTRNA Methyltransferase ActivityDnmt2 Proteins
Citar este artigo
Tovy, A., Hofmann, B., Helm, M., Ankri, S. In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme. J. Vis. Exp. (44), e2390, doi:10.3791/2390 (2010).