Grentzmann G, Ingram J A, Kelly P J, Gesteland R F, Atkins J F
Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112, USA.
RNA. 1998 Apr;4(4):479-86.
A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.
一种用于测量移码或通读等重编码机制翻译偶联效率的新型报告系统已被开发出来。一个重编码测试序列被克隆到海肾荧光素酶和萤火虫荧光素酶报告基因之间，随后在同一试管中测量两种荧光素酶的活性。两种活性的标准化比率与核糖体“读取”重编码信号从而从一个开放阅读框转换到下一个开放阅读框的效率成正比。测量两种活性的内部对照提供了一种方便且可靠的效率测定方法。这是首个适用于体外翻译的酶促双报告基因测定法。翻译信号可在体内和体外从单个构建体进行测试，这使得能够对两个系统进行密切比较。该测定法适用于高通量筛选程序。双荧光素酶报告系统已应用于HIV-1 gag-pol、MMTV gag-pro、MuLV gag-pol和人类抗酶的体内和体外重编码。
