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Whole-cell-based biosensors represent an innovative and efficient class of tools useful for environmental monitoring. The majority of these biosensors that have been developed up to now are mainly based on bacteria. However, bacteria sense toxicity in a different way from eukaryotes, thus limiting the extrapolation of results to higher eukaryotes and humans. So far, the most used eukaryotic sensing element is represented by yeast, which is generally viewed as the benchmark eukaryotic microorganism. We propose, as a valid alternative to the yeast-based sensors, the use of genetically engineered cell lines of the ubiquitous freshwater ciliated protozoa Tetrahymena thermophila. Ciliates, and in particular Tetrahymena species, offer a number of suitable characteristics to be used as environmental sensors: (a) they occupy the first trophic levels and consequently are early warning indicators of cellular suffering; (b) they are available for most of the newly developed molecular genetic techniques; (c) they can be easily cultured and maintained in small volumes; and (d) their cultures can be frozen and maintained in liquid nitrogen. Moreover, the analysis of the recently sequenced macronuclear genome of Tetrahymena thermophila has revealed that this ciliate shares a degree of sequence conservation with human genes higher than that showed by other single-celled eukaryotic model organisms, yeast included. These considerations make Tetrahymena an appealing bio-system for toxicity assessment since it can provide information of direct relevance to human health. In this study, Tetrahymena thermophila cells were transfected with the circular vector pD5H8 containing the coding sequence of the reporter gene “green fluorescent protein” (GFP) under the control of the homologous hsp70 stress promoter, to generate a fluorescent-inducible bioreporter strain able to detect general toxicity. Several classes of chemicals (inorganic and organic), physical stressors and also real environmental samples from contaminated sites were assayed using the bioreporter strain. The sensitivity of this assay was significantly higher than that measured by lethality tests using the same organism.

Fenestrin is a cytoskeletal protein, which participates in conjugation and cell cycle regulation of Tetrahymena by formation of specific structures during these processes. In cell cycle it appears at the anterior poles of future proter and opisth cells. By 2-D electrophoresis, we found that in starved T. thermophila cells fenestrin appears in four isoforms that have the pH in the range of 5.2–5.5. The differences of pH of about 0.1–0.3 between isoforms could suggest that they differ by the phosphorylation state. By mass spectrometry, we identified some of the peptides derived form the enzyme digestion of fenestrin. These peptides were used for searching through T. thermophila genome, available at the site http://www.tigr.org. A nucleotide sequence coding one of the peptides was initially found. Then, by extension, the sequence of all fenestrin gene appeared, containing the other peptides identified by spectrometry. By RT-PCR we characterized one intron in this gene. The analysis of the aminoacid sequence of the exons revealed that fenestrin is composed from at least two domains: N-terminal leucine-rich domain and C-terminal glutamic acid-rich domain. The BLAST search on general databases showed the existence of human and mouse proteins homologous to fenestrin in sequence, molecular mass and domain organization. Thus, fenestrin appears to be a protein correlated to cell polarity with an evolutionarily conserved structure. Its function should be cleared by mutational analysis and gene knock out.

In euplotid ciliates, the cortex is reinforced by alveolar plates—proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed α-, β-, and γ-plateins) have been identified. Using anti-platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with microsequences obtained directly from purified plateins identified this gene as encoding one of the closely related β- or γ-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (pI = 4.88). Microsequences from authentic α-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences o...

Pheromones of Euplotes raikovi form a homologous family of proteins with 37- to 40-amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er-23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl-terminal tail following the last cysteine of the sequence. The elongation of the Er-23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx2–4-Cys-Xxx5–7 -Cys.

For long, our knowledge of the biology of ciliate pheromones has long relied solely upon the study of the two structurally unrelated "gamones" identified in culture filtrates of a Blepharisma species. However, the characterization of a number of polypeptide pheromones secreted by Euplotes raikovi and E. octocarinatus has now established that structural relationships of homology usually link these molecules, which is consistent with the genetic basis of the mating type systems evolved by these species. In this context, our growing appreciation of the conserved and variable elements of the pheromone architecture should foster progress in the understanding of pheromone-receptor interactions and thus, provide important clues into pheromone mechanisms of action.