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Ciliates have a somatic and a germline nucleus; after sexual conjugation a new somatic nucleus forms from the new zygotic germline nucleus. Formation of the somatic nucleus involves precise elimination of a large portion of DNA sequences from the germline. Here we compare the architecture of the germline and somatic versions of the actin I gene in two geographically isolated strains of Stylonychia lemnae. We show that the structure of the germline gene is surprisingly mercurial, with the distinction between germline-limited and somatic sequences variable over the course of evolution. This is, to our knowledge, the first example of evolutionary swapping of retained versus deleted sequences during ciliate development, with sequences deleted during development that are specifically retained in another strain.

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.

A number of recent studies indicate that programmed + 1 ribosomal frameshifting is frequently required for the expression of genes in species of the genus Euplotes. In E. crassus, three genes encoding the telomerase reverse transcriptase (TERT) subunit have been previously found to possess one or two + 1 frameshift sites. To examine the origin of frameshift sites within the Euplotes group, we have isolated segments of the TERT gene from five Euplotes species. Coupled with phylogenetic analysis, the results indicate that one frameshift site in the TERT gene arose late in the evolution of the group. In addition, a novel frameshift site was identified in the TERT gene of E. minuta, a species where frameshifting has not been previously reported. Coupled with other studies, the results indicate that frameshift sites have arisen during the diversification of the euplotids. The results also are discussed in regard to the mutations necessary to generate frameshift sites, and the specialization of TERT protein function that has apparently occurred in E. crassus.

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.

During sexual reproduction, Euplotes crassus precisely fragments its micronuclear chromosomes and synthesizes new telomeres onto the resulting DNA ends to generate functional macronuclear minichromosomes. In the micronuclear chromosomes, the macronuclear-destined sequences are typically separated from each other by spacer DNA segments, which are eliminated following chromosome fragmentation. Recently, in vivo chromosome fragmentation intermediates that had not yet undergone telomere addition have been characterized. The ends of both the macronuclear-destined and eliminated spacers were found to consist of six-base, 3′ overhangs. As this terminal structure on the macronuclear-destined sequences serves as the substrate for de novo telomere addition, we sought to determine if the spacer DNAs might also undergo telomere addition prior to their elimination. Using a polymerase chain reaction approach, we found that at least some spacer DNAs undergo de novo telomere addition. In contrast to macronuclear-destined sequences, heterogeneity could be observed in the position of telomeric repeat addition. The observation of spacer DNAs with telomeric repeats makes it unlikely that differential telomere addition is responsible for differentiating between retained and eliminated DNA. The heterogeneity in telomere addition sites for spacer DNA also resembles the situation found for telomeric repeat addition to macronuclear-destined sequences in other ciliate species.

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.

Background Programmed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20–30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development. Methodology/Principal Findings To understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways. Conclusions/Significance This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

Telomere replication of eukaryotic chromosomes is achieved by a specialized enzyme, the telomerase. Although the biochemistry of end-replication is well understood, little is known about the organization of the end-replication machinery, its regulation throughout the cell cycle or the biological function of the telomerase-associated proteins. Here we investigate the function of the telomerase-associated protein p43 within the macronucleus of the ciliated protozoa Euplotes. It has been shown that p43 binds in vitro to the RNA subunit of telomerase and shares homology with the La autoantigen family. It therefore has been suggested that it is involved in the assembly and/or nuclear retention of telomerase. We show that the p43-telomerase complex is bound to a subnuclear structure in vivo and is resistant to electroelution. Upon inhibition of p43 or telomerase expression by RNAi, which in this study was used for the first time in spirotrichs, this complex is no longer retained in the nucleus. Further analysis revealed that the p43-telomerase complex is bound to the nuclear matrix in vivo and that after inhibition of p43 expression, telomerase is released from this structure, strongly suggesting that p43 is involved in anchoring of telomerase in the nucleus. This is the first in vivo demonstration of the biological function of this telomerase-associated component involved in telomere replication and allows us to propose a model for the organization of the end-replication machinery in the eukaryotic cell.