Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system

Abstract

Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.