Versatile vectors to study recoding: conservation of rules between yeast and mammalian cells

Abstract

In many viruses and transposons, expression of some genes requires alternative reading of the genetic code, also called recoding. Such events depend on specific mRNA sequences and can lead to read through of an in-frame stop codon or to +1 or -1 frameshifting. Here, we addressed the issue of conservation of recoding rules between the yeast Saccharomyces cerevisiae and mammalian cells by establishing a versatile vector that can be used to study recoding in both species. We first assessed this vector by analysing the site of +1 frameshift of the Ty1 transposon. Two sequences from higher organisms were then tested in both yeast and mammalian cells: the gag-pol junction of human immunodeficiency virus type 1 (HIV-1) (a site of -1 frameshift), and the stop codon region of the replicase cistron from the tobacco mosaic virus (a site of UAG read through). We show that both sequences direct a high level of recoding in yeast. Furthermore, different mutations of the target sequences have similar effects on recoding in yeast and in mouse cells. Most notably, a strong decrease of frameshifting was observed in the absence of the HIV-1 stem-loop stimulatory signal. Taken together, these data suggest that mechanisms of some recoding events are conserved between lower and higher eukaryotes, thus allowing the use of S. cerevisiae as a model system to study recoding on target sequences from higher organisms.

Premature stop codons involved in muscular dystrophies show a broad spectrum of readthrough efficiencies in response to gentamicin treatment

“…14,15 In this study, we analyzed the gentamicin susceptibility of individual primary stop mutations concerning Duchenne muscular dystrophy (DMD) and congenital muscular dystrophy (CMD) patients, using a very sensitive dual reporter gene system. 16 This experimental model closely corresponds to the physiological situation, in which a given mutation is embedded in a unique nucleotide context, and differs between patients. Several reasons indicated an investigation of gentamicin: its effect on readthrough is the best documented to date, and it is known to present a much lower toxicity than other suppressive molecules like G418.…”

“…A dual gene reporter system 16 was used to quantify the effect of gentamicin on stop mutation readthrough in culture cells (Figure 1). Sequences to be analyzed, spanning 27 nucleotides centered on the different stop mutations (Table 1), were inserted in-frame between the lacZ and luc coding sequences; every translating ribosome would thus result in b-galactosidase synthesis, but only those reading through the stop codon would yield active luciferase.…”

Sen1p Performs Two Genetically Separable Functions in Transcription and Processing of U5 Small Nuclear RNA inSaccharomyces cerevisiae

“…Protein and immunological methods: b-Galactosidase activity was measured as described previously (Stahl et al 1995). Immunoprecipitation (IP) and Western blotting were described previously (Ursic et al 2004).…”

Control of mRNA Export and Translation Termination by Inositol Hexakisphosphate Requires Specific Interaction with Gle1

“…We speculated that IP 6 specifically targets Gle1 during translation termination. To test this hypothesis, we utilized read-through termination assays as described previously (33,44). A reporter containing a ␤-galactosidase/luciferase fusion separated by a linker sequence with an in-frame stop codon was transformed into the respective wild type and mutant strains.…”