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The genetic code of nuclear genes in some ciliates was found to differ from that of other organisms in the assignment of UGA, UAG, and UAA codons, which are normally assigned as stop codons. In some ciliate species, the universal stop codons UAA and UAG instead encode glutamine. In some other ciliates, the universal stop codon UGA appears to be translated as cysteine or tryptophan. Eukaryotic release factor 1 (eRF1) is a key protein in stop codon recognition, thus, the protein is believed to play an important role in the stop codon reassignment in ciliates. We have cloned, sequenced, and analyzed the cDNA of eRF1 from four ciliate species of three different classes: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Phylogenetic analysis of these eRF1s supports the hypothesis that the genetic code in ciliates has deviated independently several times from the universal genetic code, and that different ciliate eRF1s may have undergone different processes to change the codon specificity. Using computational methods, we have also suggested areas on the surface of eRF1s that are important for stop codon recognition in ciliate eRF1s.

We have cloned, sequenced, and characterized cDNA of actins from five ciliate species of three different classes of the phylum Ciliophora: Karyorelictea (Loxodes striatus), Heterotrichea (Blepharisma japonicum, Blepharisma musculus), and Litostomatea (Didinium nasutum, Dileptus margaritifer). Loxodes striatus uses UGA as the stop codon and has numerous in-frame UAA and UAG, which are translated into glutamine. The other four species use UAA as the stop codon and have no in-frame UAG nor UGA. The putative amino acid sequences of the newly determined actin genes were found to be highly divergent as expected from previous findings of other ciliate actins. These sequences were also highly divergent from other ciliate actins, indicating that actin genes are highly diverse even within the phylum Ciliophora. Phylogenetic analysis showed high evolutionary rate of ciliate actins. Our results suggest that the evolutionary rate was accelerated because of the differences in molecular interactions.

Most ciliates use a deviant genetic code. Eukaryotic release factor (eRF1) appears to play an important role in the process of reassignment of stop codons. Although the precise site on eRF1 for recognition of stop codons remains obscure, studies have suggested that the tip region NIKS and its adjacent YxCxxxF motifs in domain 1 are important for stop codon recognition. Litostomatea is a class of ciliate that appears to use the standard genetic code. We used Dileptus (Litostomatea) eRF1 in this study to identify key residues located in or near the YxCxxxF motif. We predicted sites involving stop codon recognition by computational calculation of RNA–protein interaction propensity. We introduced mutations at the predicted sites of Dileptus eRF1 and examined the activity of the mutated Dileptus eRF1 using in vivo assay systems. The results show that the single mutation R128I ( Dileptus eRF1 numbering) in the YxCxxxF motif converted the omnipotent recognition of Dileptus eRF1 to Euplotes -type dualpotent eRF1. Our results indicate that R128 is one of the key residues preserving the ability to recognize all three stop codons, especially UGA, in Dileptus . We discuss a possible advantage that ciliates from the Litostomatea class may gain from using the standard genetic code.

Stop codon reassignments have occurred very frequently in ciliates. In some ciliate species, the universal stop codons UAA and UAG are translated into glutamine, while in some other species, the universal stop codon UGA appears to be translated into cysteine or tryptophan. The class Litostomatea has been hypothesized to be the only group of ciliates using the universal genetic code. However, the hypothesis was based on a statistical analysis of quite small sequence dataset which was insufficient to elucidate the codon usage of the class among such highly deviated phylum. In this study, together with the updated database sequence analysis for the class, we approached the problem of stop codon usage by examining the capacity of the translation termination factor eRF1 for recognizing stop codons. Using in vivo assay systems in budding yeast, we estimated the activity of eRF1 from two litostome species Didinium nasutum and Dileptus margaritifer. The results clearly showed that Didinium and Dileptus eRF1s efficiently recognize all three stop codons. This is the first experimental evidence that strongly supports the hypothesis that litostome ciliates use universal genetic code.