Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi.
Discovery of a novel restriction endonuclease by genome comparison and application of a wheat-germ-based cell-free translation assay: PabI (5'-GTA/C) from the hyperthermophilic archaeon Pyrococcus abyssi.
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DOI：
10.1093/nar/gni113
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发表时间：
2005-07-21
影响因子：
14.9
通讯作者：
Kobayashi, Ichizo
中科院分区：
文献类型：
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作者：
Ishikawa, Ken;Watanabe, Miki;Kuroita, Toshihiro;Uchiyama, Ikuo;Bujnicki, Janusz M;Kawakami, Bunsei;Tanokura, Masaru;Kobayashi, Ichizo
为了寻找限制性内切酶，我们使用了一种新的基于植物的无细胞翻译程序，绕过了这些酶的毒性。为了确定候选基因，对超嗜热古菌深海热球菌和堀越热球菌的相关基因组进行了比较。与限制修饰系统的自私移动的基因假说一致，假定限制基因周围的明显基因组重排作为选择标准。几个候选限制性基因被鉴定，然后以这样一种方式扩增，即它们从自己的翻译信号中被去除。在将它们克隆到质粒中的过程中，这些基因与植物翻译信号连接起来。通过T7 RNA聚合酶体外转录后，将mRNA从模板DNA中分离出来，并在基于小麦胚芽的无细胞蛋白质合成系统中翻译。可以直接测定所得溶液的限制活性。我们鉴定了两种脱氧核糖核酸酶。该酶被命名为PabI，纯化后发现其识别5′-GTAC并留下一个3′-TA突出端（5′-GTA/C），这是一种新的限制性内切酶产生的末端。PabI在高达90°C的温度下具有活性，在pH约为6和NaCl浓度为100至200 mM时具有最佳活性。我们预测它具有新颖的3D结构。
To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction–modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5′-GTAC and leave a 3′-TA overhang (5′-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90°C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.