Programmed À1 ribosomal frameshifting, but not specific À1 RF signals appears to be conserved and rapidly evolving in budding yeasts If regulation of gene expression through À1 RF is biologically significant, then À1 RF signals should be present in orthologous mRNAs from other budding yeast species. To address this, the BUB3, EST2, SPR6 and TBF1 orthologs were identified in S. paradoxus, S. mikatae, S. bayanus, S. castellii, S. kudriavzevii and S. kluyveri, and analyzed for potentially significant À1 RF signals as previously described (15) . At first glance, Positions of the slippery sites of five predicted À1 RF signals and their sequences are indicated. The full-length gene including native 5 0 and 3 0 UTR sequences were cloned into a low-copy yeast vector to create pEST2. Silent coding mutations that are predicted to inactivate À1 RF were introduced to produce pEST2ssÁ. (B) pEST2 or pEST2ssÁ were introduced into est2D or est2D upf1D cells and EST2 mRNA steady state abundances were determined by quantitative real-time PCR. (C) Quantitative real-time PCR was used to monitor steady-state abundance of the endogenous EST2 mRNA in isogenic cells expressing three different forms of ribosomal protein L3: Wild-Type RPL3 (WT); the R247A mutant which promotes decreased rates of À1 RF and the W255C/P257S mutant which promotes increased À1 RF efficiency. EST2 mRNA abundances were normalized to U3 snoRNA abundance for each sample, and the values shown are relative to wild-type cells.