Natural populations and inbred strains of Tetrahymena.

Tetrahymena typically is found in freshwater lakes, ponds, and streams in association with submerged or emergent vegetation. The genus consists of numerous breeding species with micronuclei and many asexual species without micronuclei. In summer months when most populations are at their peak, 30-50% of water samples may yield one or more species of Tetrahymena. This chapter describes both bulk and trapping procedures for collecting Tetrahymena and also evaluates barcode methods for species identification. The history and inbreeding of the laboratory model Tetrahymena thermophila is also discussed. There are numerous unresolved questions about Tetrahymena evolution and biogeography that may be solved by additional collecting.

Ciliate contributions to bioaggregation: laboratory assays with axenic cultures of Tetrahymena thermophila

Protists, mainly ciliates, play several essential roles in biological wastewater treatment, such as the transfer of matter and energy, bacterial predation, and the removal of organic material. Moreover, during the treatment process, the formation of bioaggregates-flocs and biofilms-is essential to obtaining high-quality effluents. In the present study, Tetrahymena thermophila was used as a model organism to demonstrate the contribution of ciliates to bioflocculation. Axenic cultures of this species were exposed to chemical and mechanical stimuli that promote bioaggregation. In either case, the secretion of a capsulate mucous material by the ciliates or by particle aggregation was detected. Numerous, small, loosely compacted flocs were observed under shaking conditions and in the presence of latex beads. The composition of the exopolymeric material secreted by ciliates was analyzed by a series of fluorochromes and colorimetric methods, which showed that carbohydrates and nucleic acids were the main components involved in matrix formation and particle adhesion (AU)

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Isolation of a Tetrahymena thermophila strain which induced metaphase I meiotic arrest: new pathway of abortive conjugation.

A hypodiploid strain of Tetrahymena thermophila has been obtained that shows arrest at the stage of condensed nuclei, corresponding to metaphase I of normal conjugants and induced arrest at meiotic metaphase I (i.e. at the stage of condensed, bivalent chromosomes) in its wt partner mate. The metaphase I arrested conjugants retained their old macronuclei and most of them underwent cell fusion, instead of separation of exconjugants. The doublets were viable and cortically integrated. When the arrest inducing strain was crossed to the haploid tester strain, the haploid micronuclei were arrested in the meiotic metaphase I as the diploid ones had been; the monovalent, chromosomes were condensed, the arms of sister chromatids were not separated, and they were not segregated. Separation of the arms of sister chromatids and disjunction of bivalent chromosomes were not prerequisite for the formation of microtubular spindles in those cells that were arrested in meiotic metaphase I. After re-feeding, the doublet cells resumed cell divisions, segregating two macronuclei and micronuclei at random. One macronucleus was derived from the arrest inducing strain and the other from the tester strain. Heterokaryon strains with macronuclei derived from the parental arrest inducing strain and with the micronucleus derived from the parental wt tester strain were obtained. Surprisingly, these heterokaryons did not induce meiotic arrest. Thus, the arrest in the melotic metaphase I was induced by the micronucleus and not by the macronucleus of the arrest inducing strain.

Screening for and characterization of phospholipase A1 hypersecretory mutants of Tetrahymena thermophila.

We have described a procedure for the isolation of mutants of Tetrahymena thermophila with hypersecretion of phospholipase A1 (PLA1). Using random chemical mutagenesis, uniparental cytogamy, genetic crossing and a new, fast and effective screening procedure, four PLA1-hypersecretory mutants were isolated. The screening procedure is based on the formation of a halo appearing around cylindrical holes in a lecithin-containing agar plate filled with cell-free supernatants. About 3,940 clones were tested with this procedure in primary screening for hypersecretory features, of which 60 putative hypersecretory mutants were isolated, subcloned and tested in a secondary screening. Of these, four selected mutants showed 1.8-2.2 more PLA1 activity in the cell-free supernatants compared to the wild-type strain CU 438.1. Hypersecretion was only observable for PLA1; no increased activity for two other lysosomal enzymes could be detected. These hypersecretory mutants of T. thermophila can be very useful for increasing the yield of PLA1 in fermentation processes. This is particularly relevant because, in contrast to other phospholipases, PLA1 is not available on the commercial market for fine chemicals and little is known about the role of PLA1 in cell signaling and metabolism.

Purification of Tetrahymena telomerase and cloning of genes encoding the two protein components of the enzyme.

Telomerase is a ribonucleoprotein DNA polymerase that catalyzes the de novo synthesis of telomeric simple sequence repeats. We describe the purification of telomerase and the cloning of cDNAs encoding two protein subunits from the ciliate Tetrahymena. Two proteins of 80 and 95 kDa copurified and coimmunoprecipitated with telomerase activity and the previously identified Tetrahymena telomerase RNA. The p95 subunit specifically cross-linked to a radiolabeled telomeric DNA primer, while the p80 subunit specifically bound to radiolabeled telomerase RNA. At the primary sequence level, the two telomerase proteins share only limited homologies with other polymerases and polymerase accessory factors.

A genetic screen for vegetative gene expression in the micronucleus of Tetrahymena thermophila.

The presence of a micronucleus with at least a small portion of the micronuclear genome appears to be indispensable for vegetative viability in the ciliate Tetrahymena thermophila. A genetic screen was devised to detect evidence of expression of essential genes in the vegetative micronucleus by identification of thermosensitive-lethal mutations expressed in the absence of nuclear reorganization. Although control experiments demonstrated the efficacy of the method for induction and recovery of thermosensitive lethal mutations in micronuclear genes, no expressed mutations were recovered in the absence of nuclear reorganization. This finding complements the existing lack of convincing biochemical evidence for gene expression in the vegetative micronucleus and suggests that the essential function may involve genomic DNA sequences for which thermosensitive mutant alleles are not recoverable, or perhaps a non-genomic component of the organelle.