Cassan M, Berteaux V, Angrand P O, Rousset J P
Groupe Fidélité de la Traduction et Différenciation Cellulaire, URA CNRS 1354, Université Paris, Orsay, France.
Res Virol. 1990 Nov-Dec;141(6):597-610. doi: 10.1016/0923-2516(90)90033-f.
Translational errors are necessary so as to allow gene expression in various organisms. In retroviruses, synthesis of pol gene products necessitates either readthrough of a stop codon or frameshifting. Here we present an experimental system that permits quantification of translational errors in vivo. It consists of a family of expression vectors carrying different mutated versions of the luc gene as reporter. Mutations include both an in-frame stop codon and 1-base-pair deletions that require readthrough or frameshift, respectively, to give rise to an active product. This system is sensitive enough to detect background errors in mammalian cells. In addition, one of the vectors contains two unique cloning sites that make it possible to insert any sequence of interest. This latter vector was used to analyse the effect of a DNA fragment, proposed to be the target of high level slippage at the gag-pol junction of HIV. The effect of paromomycin and kasugamycin, two antibiotics known to influence translational ambiguity, was also tested in cultured cells. The results indicate that paromomycin diversely affects readthrough and frameshifting, while kasugamycin had no effect. This family of vectors can be used to analyse the influence of structural and external factors on translational ambiguity in both mammalian cells and bacteria.
翻译错误对于在各种生物体中实现基因表达是必要的。在逆转录病毒中，pol基因产物的合成需要通读终止密码子或发生移码。在此，我们展示了一种能够在体内对翻译错误进行定量的实验系统。它由一族携带不同突变版本的荧光素酶（luc）基因作为报告基因的表达载体组成。突变包括一个框内终止密码子和分别需要通读或移码才能产生活性产物的单碱基对缺失。该系统灵敏度足以检测哺乳动物细胞中的背景错误。此外，其中一个载体包含两个独特的克隆位点，使得插入任何感兴趣的序列成为可能。后一种载体被用于分析一个DNA片段的作用，该片段被认为是HIV的gag-pol连接处高水平滑动的靶点。还在培养细胞中测试了已知会影响翻译歧义性的两种抗生素——巴龙霉素和春日霉素的作用。结果表明，巴龙霉素对通读和移码有不同影响，而春日霉素没有作用。这一族载体可用于分析结构和外部因素对哺乳动物细胞和细菌中翻译歧义性的影响。
