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Centrin is a member of the EF-hand super family of calcium-binding proteins, which can behave as a part of damage detector initiated the initiation of nucleotide excision repair (NER). Its self-assembly plays a causative role in fiber contraction associated with the cell division cycle and ciliogenesis. To explore the possible role of DNA in the process of centrin self-assembly, the aggregation properties of N-terminal domain of Euplotes octocarinatus centrin (N-EoCen) in the presence of DNA with or without metal ions are investigated. It is verified that metal ions, such as Ca2+ and Tb3+, can bind to N-EoCen with 2:1 stoichiometry by isothermal titration calorimetry (ITC). Importantly, this study reports that double strand DNA (dsDNA) is capable of binding N-EoCen, changing conformation of protein and modulating centrin aggregation, as demonstrated by extensive biophysical assays. Interestingly, the open conformation of protein induced by metal ions may be favour of the interaction of protein with dsDNA. Nevertheless, the randomly coiled single strand DNA (ssDNA) is completely inefficient to the aggregation regulation. Furthermore, results reveal that hydrophobic site could play important role in the process. This finding may link to the potent roles of centrin in the NER process.

Analysis of transcriptome revealed that a membrane occupation and recognition nexus (MORN) repeat protein-encoding gene of Euplotes octocarinatus (Eo-morn-9-31) was a candidate for programmed +1 ribosomal frameshifting (+1 PRF). In this study, a dual-luciferase assay was performed to detect its expression. The result showed that the MORN repeat protein (Eo-MORN-9-31) could be produced by the +1 PRF event during the process of translation in yeast and the frameshifting efficiency was about 4–5%. We further confirmed its reality by western blot and mass spectrometry. This study provided experimental evidence indicating that the expression of the Eo-MORN-9-31 of E. octocarinatus required the +1 PRF.

The C domain of eRF1 interacts with the C domain of eRF3, and the binding of both factors is essential for fast kinetics of the termination of protein translation. Analysis by computational simulation demonstrated that several peptides involved in Eo-eRF1/Eo-eRF3 interaction directly. Among these peptides, the two motifs GVEDT and GFGG were highly conserved, while the fragment aa338-346 of Eo-eRF1a/b was variable. In additional, I290 and D293 of Eo-eRF1 were also highly conserved. By the site-directed mutagenesis and pull-down analysis, the amino acid D293 in Eo-eRF1bC domain was conformed playing an important role in eRF1-eRF3 interaction. Eo-eRF1a and Eo-eRF1b may select different manners to interact with Eo-eRF3. These studies contribute to the better understanding the mode of eRF1-eRF3 interaction.

Defences induced against predators fall into three basic categories - behavioural, morphological and life history. Many species induce changes in more than one category. A theoretical advantage of a behavioural change is its potential for rapid induction compared to morphology or life history. We tested this theory by comparing modifications in behaviour and morphology along a time line in the hypotrich ciliate Euplotes octocarinatus exposed to chemical cues from Stenostomum virginianum, a predatory flatworm. Behavioural defences were induced much more rapidly than morphological, although as morphological defences became expressed changes in behaviour were slightly relaxed. This suggests a temporal compensatory relationship between the two traits. Behavioural defences are quickly induced to rapidly reduce predation risk, however they are relaxed as morphology changes are realised to avoid paying the cost of expressing both types of defence. © 2009 The Authors. Journal compilation © 2010 Oikos.

We describe one of the first examples of reciprocal phenotypic plasticity in a predator‐ prey system: the interaction between an inducible defence and an inducible offence. When confronted with the predatory ciliate Lembadion bullinum, the hypotrichous ciliate Euplotes octocarinatus develops protective lateral wings, which inhibit ingestion by the predator. We show that L. bullinum reacts to this inducible defence by expressing an inducible offence ‐ a plastic increase in cell size and gape size. This counteraction reduced the effect of the defence, but did not completely neutralize it. Therefore, the defence remained beneficial for E. octocarinatus. From L. bullinums point of view, the increase in feeding rate because of the offence was not larger than the increase in mean cell volume and apparently, did not increase the predator’s fitness. Therefore, the inducible offence of L. bullinum does not seem to be an effective counter-adaptation to the inducible defence of E. octocarinatus.

The first information system emerged on the earth as primordial version of the genetic code and genetic texts. The natural appearance of arithmetic power in such a linguistic milieu is theoretically possible and practical for producing information systems of extremely high efficiency. In this case, the arithmetic symbols should be incorporated into an alphabet, i.e. the genetic code. A number is the fundamental arithmetic symbol produced by the system of numeration. If the system of numeration were detected inside the genetic code, it would be natural to expect that its purpose is arithmetic calculation e.g., for the sake of control, safety, and precise alteration of the genetic texts. The nucleons of amino acids and the bases of nucleic acids seem most suitable for embodiments of digits. These assumptions were used for the analyzing the genetic code. The compressed, life-size, and split representation of the Escherichia coli and Euplotes octocarinatus code versions were considered simultaneously. An exact equilibration of the nucleon sums of the amino acid standard blocks and/or side chains was found repeatedly within specified sets of the genetic code. Moreover, the digital notations of the balanced sums acquired, in decimal representation, the unique form 111, 222...., 999. This form is a consequence of the criterion of divisibility by 037. The criterion could simplify some computing mechanism of a cell if any and facilitate its computational procedure. The cooperative symmetry of the genetic code demonstrates that possibly a zero was invented and used by this mechanism. Such organization of the genetic code could be explained by activities of some hypothetical molecular organelles working as natural biocomputers of digital genetic texts. It is well known that if mutation replaces an amino acid, the change of hydrophobicity is generally weak, while that of size is strong. The antisymmetrical correlation between the amino acid size and the degeneracy number is known as well. It is shown that these and some other familiar properties may be a physicochemical effect of arithmetic inside the genetic code. The "frozen accident" model, giving unlimited freedom to the mapping function, could optimally support the appearance of both arithmetic symbols and physicochemical protection inside the genetic code.

eRF1a, one of the class-I release factors from ciliate Euplotes octocarinatus, has been crystallized by the vapor-diffusion method using polyethylene glycol 4000 as the precipitant at pH 7.5. The crystal belongs to space group P2(1) and the unit-cell parameters are a=90.4, b=107.9, c=114.8A, beta=94.2 degrees. There appear to be four eRF1a molecules in the asymmetric unit.

1. The level of antipredator defence should be proportional to the actual attack probability to minimize the cost of defence and maximize the net benefit. 2. The hypothesis that the induced antipredator morphology of Euplotes octocarinatus is a graded response to the actual risk of predation by Stylonychia mytilus was tested by manipulating the density of both prey and predator populations. 3. The magnitude of the response was graded according to both predator and prey density. A dense prey population may be protective since a prey is more exposed to a predator's attack as a solitary individual. 4. The results suggest that Euplotes is able to 'estimate' the real risk of predation and respond appropriately, without mobilizing more resources than needed. 5. Separation of the prey and predator with a nylon net revealed that the response was not induced by a water-transmitted factor but that direct cell-to-cell contacts were important. This finding departs from those of other studies.

In laboratory populations and in a model of prey population growth, the freshwater ciliate prey Euplotes octocarinatus (Carter) showed long-term advantages from predator-induced defense, whereas th...

Centrins are four-EF-hand Ca(2+)-binding proteins, which belong to the CaM super family. The centrin from ciliate Euplotes Octocarinatus has been expressed in Escherichia coli, purified and crystallized using the hanging-drop method. Rod-like crystals were grown and diffracted to 2.0 angstroms. The crystals belong to space group P2(1)2(1)2(1) and the unit-cell parameters are a=34.442 angstroms, b=48.954 angstroms, c=72.583 angstroms.

Summary Physical contact between the freshwater ciliate Euplotes octocarinatus and its turbellarian predator, Stenostomum sphagnetorum , elicits a behavioural response of the potential prey which is different from the normal “avoiding reaction” of Euplotes . Because of its defensive character it is called “escape response”. The response was analysed by the use of a video camera and recorder system. It was found that the escape response takes a regular course, beginning with sudden backward moving of Euplotes , followed by a rapid turn around and a subsequent forward movement. Velocities during the escape response are 1 to 5 times higher than calculated for control cells. The whole reaction lasts about 2 s. Within this time a Euplotes cell moves more than 1,000 µm away from its predator. While 90% of well-fed Euplotes cells demonstrated the defensive response, starved cells cultivated separately from their predators revealed fewer escape responses, however, several of the starved cells regained the capacity for the escape response after exposure to Stenostomum or Stenostomum -conditioned medium for 20 h. It is discussed whether or not the inducible behavioural response is triggered by the same predator-released substances that are known to induce defensive morphological changes in Euplotes .

Maintenance of accuracy during translation is important for optimal fitness of the cell. The protein synthetic machinery of the cell, including the ribosome, ensures fidelity during decoding. Structural components of the ribosome play an important role in maintaining fidelity. Decoding is also affected by trans- acting factors such as tRNAs, elongation factors and peptide release factors. The concentration of substrates probing the A site also influences the rate of decoding. Slow decoding at the A site not only increases misreading but also promotes the occurrence of alternate recoding events like frameshifting. To understand how different factors affect translational accuracy, I have studied the roles of ribosomal protein- protein interactions near the decoding center and that of variant code release factors in decoding. The binding of a cognate tRNA at the A site induces a closed conformation of the ribosomal small subunit. The closure of the small subunit was suggested to disrupt the interface between two accuracy modulating proteins rpS4 and rpS5. Mutations in either protein confer an error- prone or ram phenotype. Mutations in these proteins have been proposed to promote disruption of the interface, facilitating domain closure even in the absence of the cognate tRNA leading to error- prone phenotype. I used a yeast two hybrid system to look at the effects of the ram mutations on the interactions between rpS4 and rpS5. My results confirm the predicted interactions between rpS4 and rpS5. But the fact that some of the error- prone mutations do not affect the protein- protein interaction at the interface contradicts the proposed model. I have also studied the efficiency of stop codon recognition by variant- code ciliate chimeric release factors. Stop codon recognition at a shifty stop frameshift site influences frameshifting efficiency. Using a yeast genetic system, I showed that the chimeric yeast release factors with domain 1 replaced by that of Euplotes and Tetrahymena, impaired recognition of all three stop codons. My results support our hypothesis that poor recognition of UAA and UAG stop codons at the shifty stop frameshift site AAA-UAA/ UAG- A promotes frequent frameshifting in Euplotes spp.

Organisms that use the standard genetic code recognize UAA, UAG, and UGA as stop codons, whereas variant code species frequently alter this pattern of stop codon recognition. We previously demonstrated that a hybrid eRF1 carrying the Euplotes octocarinatus domain 1 fused to Saccharomyces cerevisiae domains 2 and 3 (Eo/Sc eRF1) recognized UAA and UAG, but not UGA, as stop codons. In the current study, we identified mutations in Eo/Sc eRF1 that restore UGA recognition and define distinct roles for the TASNIKS and YxCxxxF motifs in eRF1 function. Mutations in or near the YxCxxxF motif support the cavity model for stop codon recognition by eRF1. Mutations in the TASNIKS motif eliminated the eRF3 requirement for peptide release at UAA and UAG codons, but not UGA codons. These results suggest that the TASNIKS motif and eRF3 function together to trigger eRF1 conformational changes that couple stop codon recognition and peptide release during eukaryotic translation termination.

In 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes), pH 7.4, 25 degrees C, the conformational change of the truncated form of ciliate Euplotes Octocarinatus centrin (P23) induced by metal ions were investigated using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a probe. The results show that upon metal ions binding, P23 undergo a conformational change and the contributions to the conformational change from the two EF-hands are different, and Tb3+ has more larger influence than Ca2+ with the same concentration metal ions, which provide possible the evidence that the different EF-hands play distinct biological functions. Meanwhile, the conditional binding constants of TNS and Ca2-loaded or Tb2-loaded P23 were obtained, K (Ca2-P23+TNS)=(7.49+/-0.88)x10(5) mol-1 L, K (Tb2-P23+TNS)=(8.24+/-0.49)x10(5) mol-1 L.

We propose that the amino acid residues 57/58 and 60/61 of eukaryotic release factors (eRF1s) (counted from the N-terminal Met of human eRF1) are responsible for stop codon recognition in protein synthesis. The proposal is based on amino acid exchanges in these positions in the eRF1s of two ciliates that reassigned one or two stop codons to sense codons in evolution and on the crystal structure of human eRF1. The proposed mechanism of stop codon recognition assumes that the amino acid residues 57/58 interact with the second and the residues 60/61 with the third position of a stop codon. The fact that conventional eRF1s recognize all three stop codons but not the codon for tryptophan is attributed to the flexibility of the helix containing these residues. We suggest that the helix is able to assume a partly relaxed or tight conformation depending on the stop codon recognized. The restricted codon recognition observed in organisms with unconventional eRF1s is attributed mainly to the loss of flexibility of the helix due to exchanged amino acids.

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.

A polypeptide, termed gamone 1, was isolated and purified to homogeneity from culture filtrates of mating type VII of the freshwater ciliate Euplotes octocarinatus. The gamone induces intraclonal conjugation in cells of certain other mating types. The isolation and purification of the gamone was carried out by a combination of two chromatographic steps. The purified gamone was found to be still effective in a concentration of approx. 10−15M.

Ten mating types are known for the hypotrich ciliate Euplotes octocarinatus. The mating types are determined by four codominant alleles that control the production of four gamones. The gamones induce mature cells of certain other mating types to get ready for conjugation. That the cells respond only to gamones that they do not synthesize themselves is rationalized by assuming that mating competence is specified by gamone-specific receptors and that cells express receptors only for those gamones they do not synthesize themselves. Here we show that exconjugant clones are initially composed of cells that neither release gamones nor respond to gamones. If cells are heterozygous and therefore capable of producing two gamones, they start at a clonal age of about 15 fissions to synthesize first one gamone and five to ten fissions later a second gamone. Later still, their capacity to respond to gamones develops, first for one gamone and then five to ten fissions later for a second gamone. Cells responding in maturity to three gamones often acquire this capacity in three steps. These results support our assumption that in E. octocarinatus the gamones are perceived by gamone-specific receptors. We propose that the genes coding for these gamone receptors are derepressed individually in clonal development. The same is assumed for the derepression of the genes that code for the four gamones. We further propose that the cells have the capacity to synthesize receptors for all four gamones, but they down-regulate those receptors to which they synthesize a corresponding gamone.

Gamones 3 and 4 of the ciliate Euplotes octocarinatus were isolated and purified to chromatographic and electrophoretic homogeneity. They are secreted into the culture medium by cells of certain mating types and induce cells of other mating types to unite in pairs and exchange gametic nuclei. The purified gamones are biologically active at concentrations as low as 0.1-1 pM. Both are polypeptides with unusually low pI values of approximately 3.2, and both have a carbohydrate content of less than 2%. Gamone 3 has an Mr of 18,800 and is slightly smaller than gamone 4, which has an Mr of 23,500. Gamone 3 was isolated from the starvation medium of cells homozygous for the mating type allele mt3 and gamone 4 was isolated from the medium of cells homozygous for the mating type allele mt4. Each of the two homozygous cell lines was found to secrete one gamone only.

To determine whether the micronucleus makes essential contributions during asexual reproduction, observations were made on cells of Euplotes octocarinatus from which the micronucleus had been removed with a micropipette. Most cells underwent one postenucleation division, then became arrested in macronuclear G1, slowed down in food uptake, developed macronuclear deformations, and finally died. Such cells could be rescued if a micronucleus was reimplanted before macronuclear deformations had developed. When provided with a new micronucleus, cells initiated macronuclear DNA synthesis about 12–16 h later. The data suggest that the micronucleus is involved in the control of the cell's transition from macronuclear G1 to S, and a model is proposed which postulates that in Euplotes macronuclear DNA synthesis is initiated when a micronucleus-encoded “initiator protein” has accumulated to a critical amount.