Small RNAs in genome rearrangement in Tetrahymena

Small RNAs produced by an RNAi-related mechanism are involved in DNA elimination during development of the somatic macronucleus from the germline micronucleus in Tetrahymena. The properties of these small RNAs can explain how the primary sequence of the parental macronucleus epigenetically controls genome rearrangement in the new macronucleus and provide the first demonstration of an RNAi-mediated process that directly alters DNA sequence organization. Methylation of histone H3 on lysine 9 and accumulation of chromodomain proteins, hallmarks of heterochromatin, also occur specifically on sequences undergoing elimination and are dependent on the small RNAs. These findings contribute to a new paradigm of chromatin biology: regulation of heterochromatin formation by RNAi-related mechanisms in eukaryotes.     

Experimental identification and analysis of macronuclear non-coding RNAs from the ciliate Tetrahymena thermophila

The ciliate Tetrahymena thermophila is an important eukaryotic model organism that has been used in pioneering studies of general phenomena, such as ribozymes, telomeres, chromatin structure and genome reorganization. Recent work has shown that Tetrahymena has many classes of small RNA molecules expressed during vegetative growth or sexual reorganization. In order to get an overview of medium-sized (40-500 nt) RNAs expressed from the Tetrahymena genome, we created a size-fractionated cDNA library from macronuclear RNA and analyzed 80 RNAs, most of which were previously unknown. The most abundant class was small nucleolar RNAs (snoRNAs), many of which are formed by an unusual maturation pathway. The modifications guided by the snoRNAs were analyzed bioinformatically and experimentally and many Tetrahymena-specific modifications were found, including several in an essential, but not conserved domain of ribosomal RNA. Of particular interest, we detected two methylations in the 5'-end of U6 small nuclear RNA (snRNA) that has an unusual structure in Tetrahymena. Further, we found a candidate for the first U8 outside metazoans, and an unusual U14 candidate. In addition, a number of candidates for new non-coding RNAs were characterized by expression analysis at different growth conditions.     

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax.

The processing of phagosomes containing Legionella pneumophila and Escherichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22 degrees C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium. T. vorax ingested L. pneumophila normally, but by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.     

The macronuclear polyubiquitin gene of the ciliate Tetrahymena pyriformis.

The presence of ubiquitin in ciliates was first demonstrated in Tetrahymena pyriformis. One clone--pTU2--presents two incomplete open reading frames and the putative polyubiquitin genes have been shown to be highly similar to those of other organisms. To further analyze the organization of this multigene family, several fragments of macronuclear DNA were cloned. We report here the isolation and characterization of one genomic clone (pTU20) that encodes a polyubiquitin gene (TU20) with five tandem repeats and presenting only one extra triplet CAA (Gln) upstream from the TGA. The promoter region of TU20 also presents a consensus heat shock element. The specific detection of RNA species with a synthetic oligonucleotide probe reveals that it corresponds to the 1.8 kb mRNA species whose expression is increased by temperature stress.     

Methylation of replicating and nonreplicating DNA in the ciliate Tetrahymena thermophila.

Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila was examined. In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, but methylation was also detectable on the parental strand. Methylation of nonreplicating DNA from starved cells was demonstrated.     

Gene network landscape of the ciliate Tetrahymena thermophila

Background

Genome-wide expression data of gene microarrays can be used to infer gene networks. At a cellular level, a gene network provides a picture of the modules in which genes are densely connected, and of the hub genes, which are highly connected with other genes. A gene network is useful to identify the genes involved in the same pathway, in a protein complex or that are co-regulated. In this study, we used different methods to find gene networks in the ciliate Tetrahymena thermophila, and describe some important properties of this network, such as modules and hubs.

Methodology/Principal Findings

Using 67 single channel microarrays, we constructed the Tetrahymena gene network (TGN) using three methods: the Pearson correlation coefficient (PCC), the Spearman correlation coefficient (SCC) and the context likelihood of relatedness (CLR) algorithm. The accuracy and coverage of the three networks were evaluated using four conserved protein complexes in yeast. The CLR network with a Z-score threshold 3.49 was determined to be the most robust. The TGN was partitioned, and 55 modules were found. In addition, analysis of the arbitrarily determined 1200 hubs showed that these hubs could be sorted into six groups according to their expression profiles. We also investigated human disease orthologs in Tetrahymena that are missing in yeast and provide evidence indicating that some of these are involved in the same process in Tetrahymena as in human.

Conclusions/Significance

This study constructed a Tetrahymena gene network, provided new insights to the properties of this biological network, and presents an important resource to study Tetrahymena genes at the pathway level.     

Surface display of a parasite antigen in the ciliate Tetrahymena thermophila.

The ciliated protozoan, Tetrahymena thermophila, offers an attractive medium for the expression of heterologous proteins and could prove particularly useful for the display of foreign proteins on the cell surface. Although progress has been made in transformation of Tetrahymena with heterologous DNA, methods that permit reliable expression of foreign genes have been lacking. Using a mutant strain of T. thermophila carrying a negatively selectable allele of a beta-tubulin gene, we have been able to direct foreign genes to this locus by homologous recombination. Transformed cell lines producing foreign proteins were readily identified and, in at least one case, targeting of proteins to the plasma membrane was accomplished.     

Cytomembrane differentiation in a ciliate,Tetrahymena pyriformis

Summary Two special kinds of smooth surfaced differentiations of the endoplasmic reticulum (ER) of the ciliate Tetrahymena pyriformis are described. (A) A novel type of cytomembrane structure is represented by localized bifacial regions in which one side of the cisterna is studded with ribosomes, flexible in outline and of a cytomembraneous ultrastructure and the other side has a smooth, straight profile and a plasma membrane-like triple-layered appearance. Such smooth patches of predominantly rough ER-cisternae have a tendency to pair with a separation of ca. 250 Å. The micrographs suggest a participation of such patches in the formation of vesicles and/or dictyosomes. (B) Tubular structures, including those with microtubular as well as with macrotubular (300–650 Å) diameters, can be in continuity with ER profiles. Possible origins and functions of these tubular forms are discussed.The work was supported in part by the Deutsche Forschungsgemeinschaft.The authors are indebted to Miss Sigrid Krien for skilful technical assistance as well as to Drs. Ch. Bracker, D. J. Morré (both Purdue University, Lafayette, U.S.A), and H. Falk (this institute) for helpful discussions.     

Selenocysteyl-tRNA occurs in the diatom Thalassiosira and in the ciliate Tetrahymena

Selenocysteyl-tRNAs that decode UGA were identified previously in animal and bacterial cells and the genes for these tRNAs have been shown to be widespread in animals and eubacteria. In the present study, we identify a selenocysteyl-tRNA that codes for UGA in Thalassiosira pseudonana, which is a diatom, and in Tetrahymena borealis, which is a ciliate. The fact that these very diverse unicellular organisms also contain a selenocysteyl-tRNA suggests that selenocysteine-containing proteins and the use of UGA as a codon for selenocysteine are widespread, if not ubiquitous, in nature.     

Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T. thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two of these occur between U2 and U6, whereas the third occurs between U1 and U2. The proposed interactions locate the intron 5' splice-site close to the intron branch-site nucleotide as well as to the most highly conserved domain of U6. We envisage that these interactions may facilitate the first step of pre-mRNA splicing.     

Small nuclear RNAs from Drosophila KC-H cells; characterization and comparison with mammalian RNAs

Small molecular weight nuclear RNAs were extracted from cultured Drosophila KC-H cells and characterized by their electrophoretic mobilities in 5–15% gradient acrylamide gels or in 10% acrylamide-7 M urea gels. Comparison between the electrophoretic profiles of these SnRNAs with those from human and mouse cells revealed striking similarities and allowed for assignation of band nomenclatures as established for mammalian cells. Comparison of mobilities in the two gel systems also permitted correspondence between the different nomenclatures established by various groups for this class of RNAs, as well as an approximate estimate of their molecular sizes.     

A novel class of nucleolar RNAs from Tetrahymena.

We describe a family of at least four nucleolar RNAs (snoRNAs) from the ciliate, Tetrahymena. The snoRNAs are 120-140 nucleotides long, moderately AU-rich and contain no modified nucleotides. Their 5' ends are blocked by a cap of unknown nature. The snoRNAs can be folded into similar secondary structures consisting of two hairpins separated by a single-stranded AU-rich spacer. The sequences and secondary structures show no extensive sequence or secondary structure resemblance to any other small RNAs in the public databases.     

Polyterpenoids as cholesterol and tetrahymanol surrogates in the ciliate Tetrahymena pyriformis

The tetracyclic sterol precursors, cyclolaudenol, cycloartenol and lanosterol, inhibit efficiently the tetrahymanol biosynthesis in the ciliate Tetrahymena pyriformis, as reported earlier for cholesterol and other sterols. The prokaryotic bacteriohopanetetrols have little effect, and diplopterol, another hopanoid, as well as the carotenoid, canthaxanthin, have no effect. In the presence of triparanol, a hypocholesterolemic drug inhibiting the squalene cyclase of T. pyriformis and modifying the fatty acid metabolism, the cells do not grow further, but growth can be restored by the addition to the culture medium of suitable polyterpenoids. Thus, growth in presence of triparanol (13 microM) is almost normal after addition of a sterol such as sitosterol and cyclolaudenol, and longer lag times and lower absorbances than those of untreated cultures are observed in presence of cyclartenol, lanosterol, euphenol (a lanosterol isomer), bacteriohopanetetrols and three carotenoids. No growth at all is observed in the presence of tetrahymanol and diplopterol, although these triterpenoids are the normal reinforcers of the ciliate, probably because of a poor bioavailability. Thus, structurally different polyterpenoids are (at least partially) functionally equivalent and capable of replacing tetrahymanol or sterols and might act as membrane reinforcers in T. pyriformis cells.     

Arsenate toxicity and stress responses in the freshwater ciliate Tetrahymena pyriformis

The arsenic metabolism in different biological organisms has been studied extensively. However, little is known about protozoa. Herein, we investigated the cell stress responses of the freshwater ciliate Tetrahymena pyriformis to arsenate toxicity. An acute toxicity assay revealed an 18-h EC(50) arsenate concentration of ca. 40 μM, which caused significant changes in the cell shape, growth and organism mobility. Whereas, under exposure to 30 μM arsenate, T. pyriformis could grow reasonably well, indicating a certain resistance of this organism. Arsenic speciation analysis revealed that 94-98% of the total arsenate in cells of T. pyriformis could be transformed to monomethylarsonic acid, dimethylarsinic acid and a small proportion of arsenite after 18 h of arsenate exposure, thus indicating the major detoxification pathway by arsenic oxidation/reduction and biomethylation. Finally, comparative proteomic analysis unveiled significant changes in the expression of multiple proteins involved in anti-oxidation, sugar and energy metabolism, proteolysis, and signal transduction. Our results revealed multiple pathways of arsenate detoxification in T. pyriformis, and indicated that protozoa may play important roles in the biogeochemical cycles of arsenic.     

Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila.

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.