Expression vectors for quantitating in vivo translational ambiguity: their potential use to analyse frameshifting at the HIV gag-pol junction.
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.
Cassan
M
M
Groupe Fidélité de la Traduction et Différenciation Cellulaire, URA CNRS 1354, Université Paris, Orsay, France.
Berteaux
V
V
Angrand
P O
PO
Rousset
J P
JP
D000595:Amino Acid Sequence; D000617:Aminoglycosides; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D016368:Frameshift Mutation; D015685:Fusion Proteins, gag-pol / Q000096:biosynthesis / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D010303:Paromomycin / Q000494:pharmacology; D014176:Protein Biosynthesis; D015203:Reproducibility of Results
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D004279:DNA, Viral; D015685:Fusion Proteins, gag-pol; D010303:Paromomycin; C100305:kasugamycin