Ciliate Tetrahymena Thermophila Research Articles

DNA-[Adenine] Methylation in Lower Eukaryotes

DNA methylation in lower eukaryotes, in contrast to vertebrates, can involve modification of adenine to N6-methyladenine (m6A). While DNA-[cytosine] methylation in higher eukaryotes has been implicated in many important cellular processes, the function(s) of DNA-[adenine] methylation in lower eukaryotes remains unknown. I have chosen to study the ciliate Tetrahymena thermophila as a model system, since this organism is known to contain m6A, but not m5C, in its macronuclear DNA. A BLAST analysis revealed an open reading frame (ORF) that appears to encode for the Tetrahymena DNA-[adenine] methyltransferase (MTase), based on the presence of motifs characteristic of the enzymes in prokaryotes. Possible biological roles for DNA-[adenine] methylation in Tetrahymena are discussed. Experiments to test these hypotheses have begun with the cloning of the gene. Orthologous ORFs are also present in three species of the malarial parasite Plasmodium. They are compared to one another and to the putative Tetrahymena DNA-[adenine] MTase. The gene from the human parasite P. falciparum has been cloned.

Core Formation and the Acquisition of Fusion Competence are Linked During Secretory Granule Maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.

Elimination of Foreign DNA during Somatic Differentiation in Tetrahymena thermophila Shows Position Effect and Is Dosage Dependent

In the ciliate Tetrahymena thermophila, approximately 15% of the germ line micronuclear DNA sequences are eliminated during formation of the somatic macronucleus. The vast majority of the internal eliminated sequences (IESs) are repeated in the micronuclear genome, and several of them resemble transposable elements. Thus, it has been suggested that DNA elimination evolved as a means for removing invading DNAs. In the present study, bacterial neo genes introduced into the germ line micronuclei were eliminated from the somatic genome. The efficiency of elimination from two different loci increased dramatically with the copy number of the neo genes in the micronuclei. The timing of neo elimination is similar to that of endogenous IESs, and they both produce bidirectional transcripts of the eliminated element, suggesting that the deletion of neo occurred by the same mechanism as elimination of endogenous IESs. These results indicate that repetition of an element in the micronucleus enhances the efficiency of its elimination from the newly formed somatic genome of Tetrahymena thermophila. The implications of these data in relation to the function and mechanism of IES elimination are discussed.

Organization and pairing of meiotic chromosomes in the ciliateTetrahymena thermophila

During meiotic prophase in the ciliate Tetrahymena thermophila micronuclei dramatically elongate and form thread-like crescents. The arrangement of the chromosomes within the crescent as well as the timing of chromosome pairing and recombination with respect to the elongation process have been subjects of ongoing debate. Here, we addressed these issues by means of fluorescence in situ hybridization, labeling of individual chromosomes by BrdU (BrdU-painting) and by immunostaining of the recombination protein, Rad51. BrdU-painting indicated that chromosomes are arranged as parallel bundles within the crescent, and telomere-directed fluorescent in situ hybridization (FISH) revealed that most if not all telomeres are assembled near one end of the developing crescent. Prior to full crescent formation, Rad51 localizes to chromatin as numerous foci. Locus-specific FISH demonstrated that close pairing of homologues only occurs in the full crescent. Meiotic DNA double-strand break formation and the initiation of recombination thus seem to precede close pairing. A synaptonemal complex was not detected. We conclude that the chromosomes adopt a polarized arrangement within the crescent, probably resembling the classical bouquet arrangement. Furthermore, we propose that the elongated shape of meiotic micronuclei promotes the parallel arrangement of chromosomes and supports the juxtaposition of homologous regions in the absence of a synaptonemal complex. Several pieces of evidence indicate the presence of one to four chiasmata per bivalent, which would call for crossover interference to explain regular bivalent formation in spite of this low mean number. Tetrahymena might, therefore, pose a case of interference in the absence of a synaptonemal complex.

From endonucleases to transcription factors: evolution of the AP2 DNA binding domain in plants.

All members of the AP2/ERF family of plant transcription regulators contain at least one copy of a DNA binding domain called the AP2 domain. The AP2 domain has been considered plant specific. Here, we show that homologs are present in the cyanobacterium Trichodesmium erythraeum, the ciliate Tetrahymena thermophila, and the viruses Enterobacteria phage Rb49 and Bacteriophage Felix 01. We demonstrate that the T. erythraeum AP2 domain selectively binds stretches of poly(dG)/poly(dC) showing functional conservation with plant AP2/ERF proteins. The newly discovered nonplant proteins bearing an AP2 domain are predicted to be HNH endonucleases. Sequence conservation extends outside the AP2 domain to include part of the endonuclease domain for the T. erythraeum protein and some plant AP2/ERF proteins. Our phylogenetic analysis of the broader family of AP2 domains supports the possibility of lateral gene transfer. We hypothesize that a horizontal transfer of an HNH-AP2 endonuclease from bacteria or viruses into plants may have led to the origin of the AP2/ERF family of transcription factors via transposition and homing processes.

Transcriptional control of RAD51 expression in the ciliate Tetrahymena thermophila.

The expression of Rad51p, a DNA repair protein that mediates homologous recombination, is induced by DNA damage and during both meiosis and exconjugant development in the ciliate Tetrahymena thermophila. To completely investigate the transcriptional regulation of Tetrahymena RAD51 expression, reporter genes consisting of the RAD51 5' non-translated sequence (5' NTS) positioned upstream of either the firefly luciferase or green fluorescent protein coding sequences have been targeted for recombination at the macronuclear btu1-1 (K350M) locus of T. thermophila strain CU522. Expression from RAD51-luciferase reporter constructs has been directly quantified from transformant whole cell lysates. Luciferase is induced to maximum levels in transformants harboring the full-length RAD51-luciferase reporter gene following exposure to DNA damaging UV irradiation. A series of truncations, deletions, insertions, substitutions and inversions of the RAD51 5' NTS have led to the identification of three distinct transcriptional promoter elements. The first of these sequence elements is required for basal levels of transcription. The second modulates expression in the absence of DNA damage, whereas the third ensures increased RAD51 transcription in response to DNA damage and during meiosis. Tetrahymena RAD51 is tightly regulated through these transcriptional elements to produce the appropriate expression during conjugation, and in response to DNA damage.

Post-translational Tubulin Modifications in the Ciliate Tetrahymena thermophila: Role in Assembly and Maintenance of Complex Microtubular Organelles

Extended abstract of a paper presented at Microscopy and Microanalysis 2004 in Savannah, Georgia, USA, August 1–5, 2004.

Biochemical Characterization and Quantitative Gene Expression Analysis of the Multi-Stress Inducible Metallothionein from Tetrahymena thermophila

A cadmium-binding protein with biochemical features of a metallothionein (MT) has been isolated and purified to homogeneity from the ciliate Tetrahymena thermophila. N-terminal sequencing revealed the posttranslational cleavage of the first two amino acids and, in general, a high degree of identity with known MTs from other ciliates. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) analysis of the apothionein revealed a molecular mass (16,763 Da) higher to those of mammals and of other protozoa. Finally, quantitative real-time PCR has been used to investigate the susceptibility of this ciliate MT to gene activation in response to heavy metals and to other stressors. Our data indicate that while zinc is not effective at all and cadmium is the best inducer, other stress factors, such as mercury, copper, heat and hydrogen peroxide, also activated gene transcription. As in vertebrate cells, interleukin-6 (IL-6) that stimulates ciliate growth, was able to enhance MT gene synthesis. This complex of data seems to indicate a general role of this protein in stress response.

Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila.

Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision.

A non-long terminal repeat retrotransposon family is restricted to the germ line micronucleus of the ciliated protozoan Tetrahymena thermophila.

The ciliated protozoan Tetrahymena thermophila undergoes extensive programmed DNA rearrangements during the development of a somatic macronucleus from the germ line micronucleus in its sexual cycle. To investigate the relationship between programmed DNA rearrangements and transposable elements, we identified several members of a family of non-long terminal repeat (LTR) retrotransposons (retroposons) in T. thermophila, the first characterized in the ciliated protozoa. This multiple-copy retrotransposon family is restricted to the micronucleus of T. thermophila. The REP (Tetrahymena non-LTR retroposon) elements encode an ORF2 typical of non-LTR elements that contains apurinic/apyrimidinic endonuclease (APE) and reverse transcriptase (RT) domains. Phylogenetic analysis of the RT and APE domains indicates that the element forms a deep-branching clade within the non-LTR retrotransposon family. Northern analysis with a probe to the conserved RT domain indicates that transcripts from the element are small and heterogeneous in length during early macronuclear development. The presence of a repeated transposable element in the genome is consistent with the model that programmed DNA deletion in T. thermophila evolved as a method of eliminating deleterious transposons from the somatic macronucleus.

U17/snR30 is a ubiquitous snoRNA with two conserved sequence motifs essential for 18S rRNA production.

Saccharomyces cerevisiae snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) required for the processing of 18S rRNA. Here, we show that the previously characterized human, reptilian, amphibian, and fish U17 snoRNAs represent the vertebrate homologues of yeast snR30. We also demonstrate that U17/snR30 is present in the fission yeast Schizosaccharomyces pombe and the unicellular ciliated protozoan Tetrahymena thermophila. Evolutionary comparison revealed that the 3'-terminal hairpins of U17/snR30 snoRNAs contain two highly conserved sequence motifs, the m1 (AUAUUCCUA) and m2 (AAACCAU) elements. Mutation analysis of yeast snR30 demonstrated that the m1 and m2 elements are essential for early cleavages of the 35S pre-rRNA and, consequently, for the production of mature 18S rRNA. The m1 and m2 motifs occupy the opposite strands of an internal loop structure, and they are located invariantly 7 nucleotides upstream from the ACA box of U17/snR30 snoRNAs. U17/snR30 is the first identified box H/ACA snoRNA that possesses an evolutionarily conserved role in the nucleolytic processing of eukaryotic pre-rRNA.

Histone H3 lysine 9 methylation is required for DNA elimination in developing macronuclei in Tetrahymena.

Genome-wide DNA elimination accompanies development of the somatic macronucleus from the germ-line micronucleus during the sexual process of conjugation in the ciliated protozoan Tetrahymena thermophila. Small RNAs, referred to as "scan RNAs" (scnRNAs), that accumulate only during conjugation are highly enriched in the eliminated sequences, and mutations that prevent DNA elimination also affect the accumulation of scnRNAs, suggesting that an RNA interference (RNAi)-like mechanism is involved in DNA elimination. Histone H3 that is methylated at lysine 9 (K9) is a hallmark of heterochromatin and, in Tetrahymena, is found only in developing macronuclei (anlagen) in association with chromatin containing the sequences undergoing elimination. In this article, we demonstrate that a mutation in the TWI1 gene that eliminates the accumulation of scnRNAs also abolishes H3 methylation at K9. We created mutant strains of Tetrahymena in which the only major H3 contained a K9Q mutation. These mutants accumulated scnRNAs normally during conjugation but showed dramatically reduced efficiency of DNA elimination. These results provide strong genetic evidence linking an RNAi-like pathway, H3 K9 methylation, and DNA elimination in Tetrahymena.

EVOLUTION OF HSP90 EXPRESSION IN TETRAHYMENA THERMOPHILA (PROTOZOA, CILIATA) POPULATIONS EXPOSED TO THERMALLY VARIABLE ENVIRONMENTS

Evolutionary consequences of thermally varying environments were studied in the ciliated protozoan Tetrahymena thermophila. Replicated lines were propagated for 60 days, a maximum of 500 generations, in stable, slowly fluctuating (red spectrum), and rapidly fluctuating (blue spectrum) temperatures. The red and blue fluctuations had a dominant period length of 15 days and two hours, respectively. The mean temperature of all time series was 25°C and the fluctuating temperatures had the same minimum (10°C), maximum (40°C), and variance. During the experiment, population sizes and biomasses were monitored at three-day intervals. After the experiment, carrying capacity and maximum growth rate were measured at low (15°C), intermediate (25°C), and high (35°C) temperatures for each experimental line. Physiological changes in the lines were assessed by measuring the expression of stress-induced heat shock protein Hsp90 at 25°C, 35°C, and 39°C. Population sizes and biomasses showed no differences between stable, blue, or red temperature treatments during the experiment. Also, after the experiment, mean carrying capacities and maximum growth rates were comparable in the stable, blue, and red temperature treatments. The expression of Hsp90 was higher in lines from the blue environment than in lines from the stable environment. Lines from the red environment had an intermediate level of Hsp90 expression. This supports the hypothesis that inducible thermotolerance and expression of canalizing genes can evolve in response to rapidly varying environments. Furthermore, we found correlative evidence of benefits and disadvantages of high Hsp90 expression. Lines with high expression of Hsp90 had an increased growth rate at the highest temperature when food resources were not limiting growth. At low and intermediate temperatures the same lines had the lowest carrying capacities.

Unusual distribution of mitochondrial large subunit rRNA in the cytosol during conjugation in Tetrahymena thermophila

The distribution of mitochondria during conjugation of the ciliated protozoan Tetrahymena thermophila was surveyed using a mitochondrial stain and fluorescence in situ hybridization (FISH). When the mitochondria-specific stain, Mito-Tracker, was used, the majority of mitochondria were detected in the cortex; their distribution was not changed during conjugation. On the other hand, FISH using mitochondrial large subunit (LSU) rRNA as a probe showed an unusual distribution of signals during conjugation. Unexpectedly, the signals were detected throughout the cytoplasm of conjugating cells. These signals were not observed in pre-mating cells and in exconjugants. The cytosolic localization of mitochondrial rRNA was supported by northern blot analysis using post-mitochondrial RNA fraction at the later stages of conjugation. These observations suggest selective mitochondrial breakdown or transport of LSU rRNA into cytosol. The biological significance of the conjugation-specific appearance of the cytosolic mitochondrial rRNA is discussed.

Profilin functions in cytokinesis, nuclear positioning, and stomatogenesis in Tetrahymena thermophila.

Expression of the actin-binding protein profilin was disrupted in the ciliate Tetrahymena thermophila by an antisense ribosome method. In cells with the antisense disruption no profilin protein was detected. Cultures of cells with the antisense disruption could be maintained, indicating that profilin was not essential for cytokinesis or vegetative growth. Disruption of the expression of profilin resulted in many cells that were large and abnormally shaped. Formation of multiple micronuclei, which divide mitotically, was observed in cells with a single macronucleus, indicating a defect in early cytokinesis. Some cells with the antisense disruption contained multiple macronuclei, which in Tetrahymena may indicate a function late in cytokinesis. The lack of profilin also affected cytokinesis in the cells that could divide. Normal-sized and normal-shaped cells with the antisense disruption took significantly longer to divide than control cell types. The profilin disruption revealed two new processes in which profilin functions. In cells lacking profilin, micronuclei were not positioned at their normal site on the surface of the macronucleus and phagocytosis was defective. The defect in phagocytosis appeared to be due to disruption of the formation of oral apparatuses (stomatogenesis) and a possible failure in the internalization of phagocytic vacuoles.

The Fenestrin Antigen in Submembrane Skeleton of the Ciliate Tetrahymena thermophila is Proposed as a Marker of Cell Polarity during Cell Division and in Oral Replacement

Tetrahymena thermophila cells have two types of polarized morphogenesis: divisional morphogenesis and oral reorganization (OR). The aim of this research is the analysis of cortical patterns of immunostaining during cell division and in OR using previously characterized antibodies against fenestrin and epiplasm B proteins. During cell division, the anarchic field of basal body proliferation of the new developing oral apparatus (AF) showed concomitant strong binding of the fenestrin antigen and withdrawal of a signal of the epiplasm B antigen. At a specific stage, the fenestrin antigen also appeared as a character of the anterior cortex pole, with a co-localized decrease in the detected epiplasm B antigen. The fenestrin antigen also showed a polarity of duplicating basal bodies in ciliary rows. Indirect immunofluorescence and immunogold labeling experiments were performed in the absence and presence of an inhibitor of activity of serine/threonine kinases, 6-dimethylaminopurine (6-DMAP) as an inducer of the oral replacement process. In the presence of 6-DMAP, one class of cells started OR, and some others were trapped and affected in cell division. Both types of cells showed an instability of oral structures and formed enlarged primordial oral fields. These anarchic fields (AFs) bind the fenestrin antigen, with disappearance of epiplasmic antigen staining. Only one protein (about 64 kDa) is detected in western blots by the anti-fenestrin antibody and it accumulated in 6-DMAP-treated cells that are involved in uncompleted morphogenetic activity. At a defined stage of oral development, both during cell division and in OR, the fenestrin antigen served as a marker of polarity of the cell of the anterior pole character.

Caspase-like activity in programmed nuclear death during conjugation of Tetrahymena thermophila.

Apoptosis, or programmed cell death, is common in a variety of eucaryotes, from unicellular protozoa to vertebrates. The ciliated protozoan Tetrahymena thermophila has a unique apoptosis-like nuclear death during conjugation, called programmed nuclear death. This death program involves nuclear condensation (pyknosis) and oligonucleosomal DNA fragmentation in the parental macronucleus. Subsequently, the condensed nucleus is entirely resorbed in the autophagosome. Here we demonstrate that caspase-8- and -9-like activity was detected, but no caspase-3-like activity, by in vitro assay during the nuclear resorption process, suggesting that caspase-like activity is associated with both programmed cell death and apoptosis-like nuclear death in Tetrahymena. The use of indicator dye to detect the loss of mitochondrial membrane potential suggested the uptake of mitochondria and the degenerating macronucleus by the autophagosome. An involvement of mitochondria in the programmed nuclear death is discussed.

Proprotein Processing within Secretory Dense Core Granules ofTetrahymena thermophila

In the ciliate Tetrahymena thermophila, the polypeptides stored in secretory dense core granules (DCGs) are generated by proteolytic processing of precursors, and the mature products assemble as a crystal. Previous observations suggested that this maturation involves precise cleavage at distinct motifs by a small number of enzymes. To test these inferences, we analyzed the determinants for site-specific processing of pro-Grl1p (Granule lattice protein 1) by complete gene replacement with modified alleles. Contrary to the predictions of previous models, none of the component amino acids in a putative processing motif was necessary for targeted cleavage. Indeed, cleavage at a range of alternative positions near the native site was consistent with normal DCG assembly. Furthermore, substitution of other classes of processing site motifs did not perturb DCG structure or function. These results suggest that processing can be catalyzed by multiple proteases, for which substrate accessibility may be the prime determinant of site specificity. Consistent with this, inhibition of either subtilisin or cathepsin family proteases resulted in delayed processing of pro-Grl1p.

Ecological effects of various toxic agents on the aquatic microcosm in comparison with acute ionizing radiation

The purpose of this study was an evaluation of the effect levels of various toxic agents compared with acute doses of ionizing radiation for the experimental model ecosystem, i.e., microcosm mimicking aquatic microbial communities. For this purpose, the authors used the microcosm consisting of populations of the flagellate alga Euglena gracilis as a producer, the ciliate protozoan Tetrahymena thermophila as a consumer and the bacterium Escherichia coli as a decomposer. Effects of aluminum and copper on the microcosm were investigated in this study, while effects of γ-rays, ultraviolet radiation, acidification, manganese, nickel and gadolinium were reported in previous studies. The microcosm could detect not only the direct effects of these agents but also the community-level effects due to the interspecies interactions or the interactions between organisms and toxic agents. The authors evaluated doses or concentrations of each toxic agent which had the following effects on the microcosm: (1) no effects; (2) recognizable effects, i.e., decrease or increase in the cell densities of at least one species; (3) severe effects, i.e., extinction of one or two species; and (4) destructive effects, i.e., extinction of all species. The resulting effects data will contribute to an ecological risk assessment of the toxic agents compared with acute doses of ionizing radiation.

Bioconversion of Egg Cholesterol to Pro‐vitamin D Sterols with Tetrahymena Thermophila

ABSTRACT: The potential use of the non‐pathogenic ciliate Tetrahymena thermophila for converting cholesterol into pro‐vitamin D sterols in egg suspensions was investigated. Conditions screened for efficient conversion included inoculum size and age, egg and egg yolk dilution, initial pH, and aeration. By optimizing these parameters, 55%± 6 cholesterol reduction was obtained in 24 hours incubation. Tetrahymena converted cholesterol into three unsaturated sterols, D7,22 bis‐dehydrocholesterol, an ergosterol analog (27%), D7‐dehydrocholesterol, also known as pro‐vitamin D3 (4%), and Δ22‐ dehydrocholesterol (8%). Despite significant proteolysis, culturing with Tetrahymena did not result in net consumption of total amino acids content, which was in fact increased by 16%± 2. These findings emphasize the potential use of Tetrahymena in the food industry.