these analyses reveal that no single À1 RF signal is completely conserved among the budding yeasts (Supplementary Table S4) . However, closer analysis shows that strong candidate À1 RF signals can be identified in the orthologs of all of these genes, although not in every species. For example, as noted above, the S. cerevisiae EST2 mRNA contains five potential À1 RF signals. Similarly, the S. paradoxus ortholog contains five potential À1 RF signals, although none are identical to the S. cerevisiae elements. S. mikatae EST2 appears to harbor two potential À1 RF signals, S. bayanus has three, and S. castelli contains two, and S. kluyveri has three. None were identified in the S. kudriavzevii EST2 ortholog. Turning to SPR6, the S. cerevisiae mRNA contains a second potential À1 RF signal beginning at nucleotide 348 in addition to that identified beginning at nucleotide 279 (see http://cbmgintra.umd.edu/prfdb/index .cgi/detail?id=1755&accession=SGDID:S0000917& slipstart=348). Both the S. paradoxus and S. kudriavzevii SPR6 orthologs contain three potential À1 RF signals, but none were identified in the S. mikatae S. bayanus or S. castelli orthologs. Interestingly, the S. kluyveri SPR6 ortholog contains a slippery site followed by a strong stem-loop structure; while this may or may not constitute a À1 RF signal, it does suggest the presence of a rapidly evolving cis-acting element (see Discussion below). S. cerevisiae BUB3 contains the operational À1 RF signal at nucleotide 858, plus potential À1 RF signals beginning at nucleotides 27 and 732. The orthologous mRNAs in S. paradoxus, S. bayanus, S. castelli and S. kudriavzevii each appear to have one potential À1 RF signal, but none were identified in S. mikatae or S. kluyveri. Lastly, the S. cerevisiae TBF1 mRNA has only the single confirmed À1 RF signal. The S. castelli, S. kudriavzevii and S. kluyveri orthologs contain two each, and the S. mikatae has one. No potential À1 RF signals were identified in either S. paradoxus or S. bayanus. As a control, six S. cerevisiae genes lacking predicted À1 RF signals were selected (PGK1, HHT1, TEF2, MIC14, CMD1 and GRX1), orthologs from the six other yeast species identified, and these were in turn queried for the presence of putative À1 RF signals. These analyses revealed that none of the orthologs of these six genes contain predicted À1 RF signals (Supplementary Table S5 , and hyperlinked data therein). The potential evolutionary significance these observations are discussed below.