In vitro tRNA methylation assay with the Entamoeba histolytica DNA and tRNA methyltransferase Dnmt2 (Ehmeth) enzyme.
In vitro tRNA methylation assay with the Entamoeba histolytica DNA and tRNA methyltransferase Dnmt2 (Ehmeth) enzyme.
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DOI：
10.3791/2390
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发表时间：
2010-10
期刊：
影响因子：
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通讯作者：
A. Tovy;B. Hofmann;M. Helm;S. Ankri
中科院分区：
文献类型：
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作者：
A. Tovy;B. Hofmann;M. Helm;S. Ankri
原生动物寄生虫是人类最具破坏性的感染源之一，可导致包括阿米巴病在内的各种疾病，阿米巴病是寄生虫病导致死亡的三大最常见原因之一。阿米巴病的病原体是阿米巴寄生虫溶组织内阿米巴，它存在于两个阶段：在食物或水中发现的感染性包囊和生活在肠道中的侵袭性滋养体。阿米巴病的临床表现从无症状到结肠炎、痢疾或肝脓肿。溶组织棘球绦虫是一种基因组中含有5-甲基胞嘧啶(5mC)的罕见单细胞寄生虫。它含有一个DNA甲基转移酶，Ehmeth，属于DNMT2家族。DNMT2在组织裂殖酵母、盘基网柄菌和果蝇中对重复元件的调控作用已得到证实。我们最近的工作表明，Ehmeth甲基化tRNA(Asp)，这一发现表明，该酶具有双重DNA/tRNA(Asp)甲基转移酶活性。这一观察结果与已报道的盘状葡萄球菌和黑腹葡萄球菌的双重活性是一致的。DNMT2酶的DNA/tRNA特异性的功能意义尚不清楚。为了解决这个问题，建立了一种测定DNMT2蛋白tRNA甲基转移酶活性的方法。在这段视频中，我们描述了一种直接的方法来为DNMT2制备足够的tRNA底物，并描述了一种测量其tRNA甲基转移酶活性的方法。
Protozoan parasites are among the most devastating infectious agents of humans responsible for a variety of diseases including amebiasis, which is one of the three most common causes of death from parasitic disease. The agent of amebiasis is the amoeba parasite Entamoeba histolytica that exists under two stages: the infective cyst found in food or water and the invasive trophozoite living in the intestine. The clinical manifestations of amebiasis range from being asymptomatic to colitis, dysentery or liver abscesses. E. histolytica is one of the rare unicellular parasite with 5-methylcytosine (5mC) in its genome. It contains a single DNA methyltransferase, Ehmeth, that belongs to the Dnmt2 family. A role for Dnmt2 in the control of repetitive elements has been established in E. histolytica, Dictyostelium discoideum and Drosophila. Our recent work has shown that Ehmeth methylates tRNA(Asp), and this finding indicates that this enzyme has a dual DNA/tRNA(Asp) methyltransferase activity. This observation is in agreement with the dual activity that has been reported for D. discoideum and D. melanogaster. The functional significance of the DNA/tRNA specificity of Dnmt2 enzymes is still unknown. To address this question, a method to determine the tRNA methyltransferase activity of Dnmt2 proteins was established. In this video, we describe a straightforward approach to prepare an adequate tRNA substrate for Dnmt2 and a method to measure its tRNA methyltransferase activity.