A positive feedback vector for identification of nucleotide sequences that enhance translation.
A positive feedback vector for identification of nucleotide sequences that enhance translation.
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用于鉴定增强翻译的核苷酸序列的正反馈载体。
DOI：
10.1073/pnas.0409892102
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Mauro,VincentP
中科院分区：
文献类型：
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作者：
Zhou,Wei;Edelman,GeraldM;Mauro,VincentP
在早期的研究中，我们发现了作为内部核糖体进入位点(IRES)和增强翻译功能的短序列(6-到22-nt)。这些IRES元件的大小表明它们可能普遍存在于信使群体中，个别元件可能影响不同组mRNAs的翻译。为了开始评估哺乳动物细胞中不同IRES元件的数量，我们开发了一种强大的方法，它使用正反馈机制来放大单个IRES元件的活性。该方法使用编码双顺反子的载体作为第一顺反子，酵母Gal4/病毒蛋白16(VP16)转录因子作为第二顺反子。这种mRNA的转录是由一个最小的启动子驱动的，该启动子包含四个Gal4上游激活序列的拷贝。在这种方法中，IRES在顺反子间区的存在促进了Gal4/VP16的翻译，后者与上游激活序列结合，并触发了一个正反馈环，从而促进了双顺反子mRNA和Gal4/VP16的产生。通过测量荧光素酶活性或使用FACS来监测第一顺反子(荧光素酶或EGFP)翻译的相应增加。后者使IRES阳性细胞得以分离。我们使用Gtx同源结构域基因的IRES模块和丙型肝炎病毒的IRES对反馈机制进行了测试，并展示了该载体系统在筛选、鉴定和分析IRES元件方面的有效性。
In earlier studies, we identified short (6- to 22-nt) sequences that functioned as internal ribosome entry sites (IRESes) and enhanced translation. The size of these IRES elements suggested that they might be prevalent within the messenger population and that individual elements might affect the translation of different groups of mRNAs. To begin to assess the number of different IRES elements in mammalian cells, we have developed a powerful method that uses a positive feedback mechanism to amplify the activities of individual IRES elements. This method uses a vector that encodes a dicistronic mRNA with a reporter gene (Renillaluciferase or the EGFP) as the first cistron and the yeast Gal4/viral protein 16 (VP16) transcription factor as the second cistron. Transcription of this mRNA is driven by a minimal promoter containing four copies of the Gal4 upstream activation sequence. In this method, the presence of an IRES in the intercistronic region facilitates the translation of Gal4/VP16, which binds to the upstream activation sequences and triggers a positive feedback loop that escalates the production of dicistronic mRNA and Gal4/VP16. A corresponding increase in the translation of the first cistron (luciferase or EGFP) is monitored either by measuring luciferase activity or by using FACS. The latter enables IRES-positive cells to be isolated. We present tests of the feedback mechanism by using an IRES module fromGtxhomeodomain mRNA and an IRES from hepatitis C virus and demonstrate the utility of this vector system for the screening, identification, and analysis of IRES elements.