The hypotrichous ciliate Euplotes octocarinatus has only one type of tRNACys with GCA anticodon encoded on a single macronuclear DNA molecule

Deviations from the universal genetic code have evolved independently several times in ciliated protozoa, Thus, in some species UAA and UAG are no longer used as termination codons, but are read as glutamine, whereas in the genus Euplotes, UGA is translated as cysteine, We have investigated the nature of the tRNA(Cys) isoacceptor responsible for decoding UGA in Euplotes cells. Southern hybridization analyses indicated that a single DNA molecule of 630 bp encoding tRNA(Cys) exists in the macronucleus of Euplotes octocarinatus. Cloning and sequencing of th is fragment revealed that it contains only one copy of a tRNA(Cys) gene, which codes for a normal tRNA(Cys) with GCA anticodon. This is the first report of the characterization of a tRNA gene in any hypotrichous ciliate, It contains putative signals for initiation and termination of transcription by RNA polymerase ill and can be transcribed efficiently in vitro in HeLa cell nuclear extract. Intensive studies on the DNA and tRNA level involving PCR analyses have not disclosed the existence of any tRNA(Cys) isoacceptor with UCA or ICA anticodons. Translation of the UGA codon by tRNA(GCA)(Cys) GCA necessitates a G:A mispairing in the first anticodon position. We discuss a number of aspects which might contribute to the finding that a near-cognate tRNA isoacceptor efficiently translates the UGA stop codon.