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A previous survey of Euplotes genes available in GenBank indicated that >5% required a +1 translational frameshift for expression, much higher than observed in other organisms. Because this sample of sequences was non-random and biased towards highly expressed genes, we have randomly selected and sequenced 25 Euplotes crassus macronuclear DNA molecules to more accurately assess the frequency of frameshifting. Three new +1 frameshift genes were identified, and all have sequence features similar to the previously postulated frameshift sites. The results indicate that the frequency of Euplotes+1 frameshift genes is indeed high, and likely >10%. We have also examined the evolution of one of two frameshift sites present in the telomerase reverse transcriptase (TERT) genes of E. crassus. TERT gene segments were isolated from five additional Euplotes species. FS2 was found only in E. crassus and the closely related E. vannus, and phylogenetic analyses indicate that the site arose late in the evolution of the Euplotes group. Moreover, a novel frameshift site (FS3) was identified solely in the TERT gene of E. minuta. These results indicate that frameshift sites have arisen within genes during the recent history of Euplotes, and that it may be an ongoing process. Overall, the data suggest to us that the appearance of a frameshift site in a Euplotes gene is selectively neutral, or near-neutral. This would require an efficient mechanism of frameshifting in Euplotes. A model will be presented linking frequent frameshifting to a second unusual genetic feature of Euplotes, stop codon reassignment.

The use of small double-stranded RNA to inhibit expression of genes has proven to be a powerful tool to analyze the biological function of genes in various eukaryotic organisms. We have established the RNAi technology in three genera of spirotrichous ciliates, Euplotes, Oxytricha and Stylonychia . Here we describe this method to silence gene expression in these three genera. Expression of the following genes was inhibited: the catalytic subunit of telomerase (TERT) and the telomerase-associated protein p43 in Euplotes and the a-subunit of the telomere-binding protein (αTeBP) of Stylonychia and Oxytricha . In addition, we report how the biological function of a developmentally regulated gene can be elucidated by this technique in Stylonychia.

Ciliates show two forms of reproduction: asexual (vegetative) and sexual reproduction (conjugation). Conjugation in Euplotes octocarinatus is induced by pheromones secreted by cells of one mating type binding to corresponding receptors on cells of a different mating type. In this study, we used heterologously-expressed pheromone 2 (Phr2) in affinity chromatography to isolate pheromone 2 binding proteins in Euplotes octocarinatus syngen 1. Using this approach, we isolated a 42-kDa protein (Phr2B) that bound Phr2. A tryptic fragment of Phr2B was used to construct degenerate oligonucleotides for PCR amplification of the macronuclear gene. The gene is predicted to encode a 58 amino acid protein. The N-terminus of the deduced protein shows significant similarity to membrane bound protein kinases of such diverse organisms as Xenopus laevis , Mus musculus and Homo sapiens , while the C-terminus contains a region which is similar to an extracellular region within chemosensory receptors of Caenorhabditis elegans . Western blotting analysis demonstrated that Phr2B specifically recognizes Phr2 and preliminary results indicate that antibodies raised against a C-terminal peptide of Phr2B inhibit cell mating. Further studies should be undertaken to investigate the precise role that Phr2B plays in preconjugative intercellular communication in E. octocarinatus.

Summary Two new stocks of the hypotrichous ciliate Euplotes octocarinatus isolated in Pennsylvania and Florida/USA were identified as members of a new mating type XI (mt5mt5) secreting a so far unknown pheromone 5. As deduced from their pheromone encoding genes, the pheromones secreted by the two stocks differ from each other by 14 amino acids and a few silent mutations. Their 5′-non-coding sequences are almost identical with the 5′-non-coding sequences of the pheromone genes 1 to 4. This holds also true for the prepro-sequences which contain a 16 amino acids long ER-import signal. The 3′-non-coding sequences are also highly conserved. Like the other pheromone encoding genes the two mt5 alleles contain in-frame TGA stop codons, probably encoding cysteines. Both genes are interrupted by three introns in positions which are homologous to those in the other pheromone genes. Intron 1 of the stock from Florida is 143 bp longer than intron 1 of the stock from Pennsylvania. Indirect evidence suggests that the two stocks do not belong to E. octocarinatus syngen 1. In this respect the two stocks resemble two other stocks isolated in North America which we have previously investigated. Whether the two stocks belong to the same syngens as the other two North American stocks is unclear.

Summary Two new stocks of the hypotrichous ciliate Euplotes octocarinatus isolated in Florida and Maryland/USA were identified as members of mating type VI (mt 2 mt 3 ), secreting pheromones 2 and 3 and mating type VII (mt 1 mt 1 ), secreting pheromone 1. These are two out of the ten already known mating types of Euplotes octocarinatus. Results of breeding experiments suggest that the new stocks belong to new syngens of this species complex. The analysis of the macronuclear molecules containing the new pheromone 1, 2 and 3-encoding genes show that their structure is very similar to those isolated before. Like in these genes the coding regions are interrupted by three introns with a high degree of homology. A comparison of the deduced amino acid sequences of the new pheromones with the known versions of the corresponding pheromones reveals homologies of 76 to 93% for the secreted proteins whereas the leader sequences are 98 to 100% identical. In both versions of pheromones 1, 2 and 3 there are ten cysteine residues which are located at strictly conserved positions. In spite of their differences both versions of pheromones 1, 2 and 3 show the same specific biological activity. This might be an indication that the unchanged residues participate in receptor binding.