Establishment of a Cre/loxP recombination system for N-terminal epitope tagging of genes in <Emphasis Type="Italic">Tetrahymena</Emphasis>

Background  

Epitope tagging is a powerful strategy to study the function of proteins. Although tools for C-terminal protein tagging in the ciliated protozoan Tetrahymena thermophila have been developed, N-terminal protein tagging in this organism is still technically demanding.     

A phylogenetic analysis based on the gene encoding phosphoglycerate kinase

Summary We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.Offprint requests to: R.E. Pearlman     

Lysosomal enzymes produced by immobilized Tetrahymena thermophila

Summary The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 × 10⁷ cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions -glucosidase, -glucosidase, -hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes. Offprint requests to: A. Tiedtke     

Restricted distribution and limited gene flow in the model ciliate Tetrahymena thermophila

The biogeography of microbial eukaryotes has long been debated, but few phylogeographic data have been available to assess whether protists tend to have ubiquitous or endemic distributions. We addressed this issue in the ciliate Tetrahymena thermophila, a highly successful model system in cell and molecular biology. We found that this species has a distribution that is restricted to the Eastern United States, with high diversity in the northeast and low diversity across the rest of its distribution. We find high levels of population subdivision, low rates of migration and significant isolation by distance, supporting the moderate endemicity model of protist biogeography. This restricted gene flow may be a result of small population size, which would reduce the probability of migration events, or the inability to establish after migration. This work lays the foundation for T. thermophila to become a valuable model system for studying population biology.     

Ontogenetic Shifts in the Ability of the Cladoceran,Moina macrocopa Straus and Ceriodaphnia cornuta Sars to Utilize Ciliated Protists as Food Source

The ontogenetic diet shifts and age specific ability of the two cladoceran species Moina macrocopa and Ceriodaphnia cornuta to derive energy from ciliated protists have been investigated in laboratory. The postembryonic developmental rates and life table demography (longevity, age and size at first reproduction, fecundity and intrinsic rate of natural increase) of the cladocerans have been elucidated on algae (Chlorella vulgaris) and the ciliated protists (Tetrahymena pyriformis, Colpoda (c.f.) steini) as food. For either of the cladoceran, the somatic growth rate and average body size at first reproduction were higher with algal diet. During initial stages of development (0–5 days), either cladoceran realized higher rate of somatic growth on algal diet, subsequently ciliated protists supported significantly higher growth rate than the alga. Algal and ciliate diets did not differ in maximum body size (C. cornuta: 539–554 μm; M. macrocopa: 1274.8–1309 μm) reached by either of the cladocerans. The maximum body sizes were larger than size at first reproduction with either of the ciliated protists, however, with algal diet the maximum body sizes did not differ from the size at first reproduction in each case. In case of C. cornuta the generation time (20.5 ± 0.3 days on ciliate; 15.6 ± 0.17 days on algal diet), reproductive rates (net reproductive rate: 20.05 ± 3.2 on ciliate; 15.5 ± 1.2 on algal diet), and average life expectancy at hatching (27 ± 0.8 days on ciliate; 22.7 ± 0.71 days on alga) were higher, whereas the size at first reproduction (482 μm on ciliate; 521 μm on alga) was smaller with the ciliate than with an algal diet. The algal and the ciliate diets did not differ in survival (life expectancy at hatching: 9.2 ± 0.7 days) and fecundity (NRR: 23.6 ± 2.4) for M. macrocopa. The two ciliates used in the experiment did not differ in their performance as food source for either cladoceran species. Our results suggest that both the cladoceran species are able to utilize smaller ciliate (e.g., T. pyriformis, C. (c.f.) steini) as food; however with differential ability to derive energy from the ciliate diet and this ability is size and age structured in both cases. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Eliminated sequences with different copy numbers clustered in the micronuclear genome of Tetrahymena thermophila

Summary As the ciliated protozoan Tetrahymena thermophila develops a new macronucleus (MAC) from products of its micronucleus (MIC), several repetitive sequences are eliminated from the MAC genome. Four MIC DNA clones containing repetitive sequences that are eliminated from the MAC were obtained. One clone contains a representative from each of three families of eliminated sequences. One, present in 200–300 copies in the MIC, is almost completely eliminated from the MAC. A second, present in approximately 50 copies in the MIC, is scattered throughout the genome, although up to half of the family members examined could be localized to chromosome 2. Approximately one tenth of the members of this less repetitive family persist in the MAC while the rest are eliminated. The third type of eliminated sequence has three to four members, all of which are eliminated from the MAC. Three of the members are located on three of the five MIC chromosomes, and one could not be mapped. This sequence is clustered with the other two families of sequences in at least three of the four sites. All three types of eliminated sequences are found in similar arrangements in the MIC of several different inbred strains of T. thermophila.     

Some but not All Tetrahymena Species Destroy Monolayer Cultures of Cells from a Wide Range of Tissues and Species

The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not.     

A micronucleus-limited sequence family in Tetrahymena thermophila: Organization and sequence conservation

During mocronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronu-cleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited. © 1992 Wiley-Liss, Inc.     

Circular rDNA Replicons Persist in Tetrahymena thermophila Transformants Synthesizing GGGGTC Telomeric Repeats

Site-directed mutagenesis of the telomerase RNA from Tetrahymena thermophila was used previously to demonstrate the templating function of a sequence within this RNA; this sequence specifies the sequence of telomeric DNA in vivo. The possible functional importance of a phylogenetically conserved nucleotide outside the telomerase RNA template region was investigated by a similar experimental approach. The telomerase RNA gene was altered by site-directed mutagenesis, cloned in a circular selectable transformation vector consisting of an rRNA gene carrying a selectable drug resistance marker, and introduced into macronuclei of vegetatively dividing Tetrahymena thermophila by microinjection. Changing an invariant A to U at position 16 of the telomerase RNA (A16U) had no effect detectable by phenotype on telomerase function in vivo. However these experiments showed that a telomerase template alteration that dictates the synthesis of the mutant telomeric DNA sequence GGGGTC leads to a profound change in the population of rDNA replicons. The addition of GGGGTC mutant repeats leads to selective pressure for the loss of high copy linear rDNA, and the rRNA genes are maintained in the form of the circular rDNA replicons introduced during transformation.     

Sequence homology near the center of the extrachromosomal rDNA palindrome in Tetrahymena

The nucleotide sequence in the central region of the extrachromosomal ribosomal DNA palindrome of Tetrahymena pigmentosa has been determined. The sequence data show that 26 nucleotides at the very center are not palindromic. A segment of 34 base-pairs just outside the non-palindromic region is highly conserved between Tetrahymena pigmentosa and Tetrahymena thermophila, while the rest of the central regions show little sequence homology.     

A new method using enhanced green fluorescent protein (EGFP) to determine grazing rate on live bacterial cells by protists

A new method was developed for estimating the grazing rate of live bacteria by protists. Bacterial cells (Escherichia coli bearing plasmid pEGFP) expressing enhanced green fluorescent protein (EGFP) were used as a live bacterial tracer. Ciliates (Tetrahymena thermophila) were fed with EGFP-tagged bacterial cells, and the individual cells taken up by the ciliates were detected by epifluorescence microscopy. The EGFP fluorescence was stable during the storage of samples fixed with glutaraldehyde. Comparison of clearance rates based on the uptake of EGFP-tagged live cells and fluorescently-labeled heat-killed cells suggested that the use of heat-killed cells underestimates the clearance rates. We suggest that EGFP-tagged bacteria are a useful tracer for determining protist bacterivory in culture and aquatic environments. Received: January 22, 2001 / Accepted: October 27, 2001     

Isolation of a Δ7-cholesterol desaturase from Tetrahymena thermophila

Cell-free preparations of Tetrahymena thermophila catalyze the direct desaturation of cholesterol to Δ⁷-dehydrocholesterol (provitamin D₃). The activity was isolated in the microsomal fraction from Tetrahymena homogenates. Δ⁷-Desaturase activity was stimulated fivefold by the addition of 6 mM ATP. Other cofactors assayed, including NAD, NADP, NADH or NADPH, had no significant effect. The activity was found in microsomes prepared from stationary-phase cultures of the ciliate, grown either with or without added cholesterol, thus indicating that it is constitutively expressed in T. thermophila cells. Received: 17 May 1999 / Accepted: 24 September 1999     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Biochemical evidence for a P2Y-like receptor in Tetrahymena thermophila

Extracellular nucleotides are ubiquitous signaling molecules. ATP signals through two receptor types: the ionotropic P2X receptors, and the metabotropic P2Y receptors. ATP acts as a chemorepellent in Tetrahymena thermophila, where it causes a distinct avoidance response. The intracellular mechanisms by which ATP causes avoidance in this organism, however, are unknown. In this study, we use in vivo pharmacological assays along with enzyme immuno-assays to obtain information about the ATP chemorepellent pathway and its associated second messenger systems. Our data show strong similarities between the presumed ATP receptor of T. thermophila and members of the P2Y family of receptors. The ATP response of T. thermophila appears to be coupled to phospholipase C, a defining characteristic of the P2Y receptor family. In addition, the ATP chemoresponse appears to be linked to a G_i/o protein, nitric oxide synthase, and adenylyl cyclase, all of which are characteristic of some P2Y receptors. This is an important first step in describing the pathways involved in ATP chemoresponse of this organism.Abbreviations cAMP adenosine 3'5'-monophosphate - ATP--S adenosine-5'-O-(3-thiotriphosphate) - EIA enzyme immunoassay - GDP--S guanosine 5'-O-(2-thiodiphosphate) - cGMP guanosine 3'5'-monophosphate - IMP 2-imino-4-methylpiperidine - IP₃ inositol 1,4,5-trisphosphate - NO nitric oxide - iNOS inducible nitric oxide synthase - PACAP pituitary adenylate cyclase activating polypeptide - PKA cAMP-dependent protein kinase - PKC protein kinase C - PKG cGMP-dependent protein kinase - Rp-cAMPs Rp-adenosine-3',5' cyclic monophosphorothioate     

Water-soluble vitamins in cells and spent culture supernatants of Poteriochromonas stipitata,Euglena gracilis,and Tetrahymena thermophila

Vitamins B₆ and B₁₂, biotin, folates, riboflavin, nicotinate, pantothenate, biopterin, and vitamin C (l-ascorbate) were assayed in Poteriochromonas stipitata, Euglena gracilis, and Tetrahymena thermophila cells grown in defined media and in spent culture supernatants. P. stipitata and E. gracilis synthesized, stored and excreted folates (mainly as 5-methyltetrahydrofolate), B₆, riboflavin, pantothenate, nicotinate, biopterin, and ascorbate. E. gracilis synthesized and stored biotin. T. thermophila did not synthesize the above vitamins except for B₁₂, biopterin, and ascorbate; it excreted biopterin and stored B₁₂ and ascorbate. Thiamin was left of consideration because all 3 organisms are thiamin auxotrophs. Possible ecological implications of these findings are considered.     

Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena

A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2000 μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.     

Autoradiographic evidence for self-fertilization in Tetrahymena thermophila

Conjugation in Tetrahymena thermophila consists of a sequence of nuclear events, including meiosis and reciprocal cross-fertilization, which result in biparental genetic endowment of the sexual progeny. Genetic evidence was recently provided that the normal exchange of gametic nuclei between conjugating cells can be efficiently blocked by hyperosmotic shock. In this paper we confirm this finding autoradiographically. We also report that the inhibitor of microtubule assembly, vinblastine, also blocks this step, as well as the subsequent fusion of gametic nuclei. The ability of conjugating cells to survive and continue more or less normally after blocks of self-fertilization and pro-nuclear fusion demonstrates a surprisingly high degree of developmental regulation during conjugation. Self-fertilization has proven useful for the isolation of recessive mutants in T. thermophila.