A novel robust heat-inducible promoter for heterologous gene expression in Tetrahymena thermophila

An increasing amount of data has revealed the importance of inducible promoters in ciliate research and in ciliate-related industries. However, knowledge about these promoters and related genes is relatively sparse. Here we report a novel inducible promoter from a Tetrahymena cytoplasmic Hsp70 gene member, HSP70-2. The reported promoter was able to induce the endogenous gene up to ～9000-fold after a short heat shock treatment and this remarkable feature has been retained when a relatively short region of the promoter was introduced into a reporter construct followed by transformation. During the recovery period following a short heat shock, both the mRNA and protein levels of the reporter gene were maintained high up to two hours. A constant heat shock treatment to the transformed cells led to a stabilization of the reporter mRNA up to at least six hours and the reporter protein continued to accumulate up to around three hours. The promoter strength appears to be similar to that of the cadmium-induced metallothionein gene (MTT1) promoter. Therefore, the HSP70-2 promoter represents an attractive alternative for the over-expression of proteins in Tetrahymena, and the promoter-reporter gene construct used in this study is an ideal tool to help in understanding the regulation mechanisms of heat shock genes in ciliates.     

Glutathione in Tetrahymena thermophila

In Tetrahymena, glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

A novel chromodomain protein, pdd3p, associates with internal eliminated sequences during macronuclear development in Tetrahymena thermophila

Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.     

Oral Assembly in Left-Handed Tetrahymena thermophila

We have investigated oral development in a non-genically derived left-handed (LH) form of Tetrahymena thermophila , in which the large-scale asymmetry of arrangement of cortical structures is reversed whereas the local asymmetry of ciliary architecture remains normal. Approximately 1/2 of the oral apparatuses (OAs) of LH cells develop in the form of superficial mirror-images of OAs of RH cells. In most of these OAs, membranelles are assembled from the cells'anterior to posterior. Nonetheless, the posterior ends of these membranelles undergo the basal body displacements that lead to a sculptured appearance, so that the membranelles of LH OAs become organized as rotational permutations of membranelles of normal RH OAs. Many of these membranelles re-orient to a normal orientation near the end of oral development. Membranelles and undulating membranes (UMs) may develop independently of each other, and formation of postciliary microtubules of UMs is separate from that of ribbed wall microtubules. In some cases, the entire OA develops and remains as a 180° rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama [36, 37]. We present a model (Fig. 37) for these complex developmental outcomes. These developmental patterns resemble those described previously and less completely for secondary OAs of cells with mirror-image global patterns, including janus cells. The present study demonstrates that such alterations in oral development are not a direct outcome of genotypic changes.     

A pseudo-phytochelatin synthase in the ciliated protozoan Tetrahymena thermophila

Phytochelatins (PCs) and metallothioneins (MTs) are the two major heavy metal chelating peptides in eukaryotes. We report here on the identification of a biosynthetically inactive pseudo-phytochelatin synthase enzyme (TtψPCS) in the ciliate Tetrahymena thermophila, the first of this kind (pseudo-PCS) to be described in eukaryotes. TtψPCS which resembles a true PCS at the N-terminal region, while it is most divergent in its Cys-poor C-terminal region, was found to be up-regulated under cadmium stress conditions. However, only glutathione (GSH) hydrolysis products, but not PCs, could be detected in extracts from Cd-treated cells. The latter feature is reminiscent of pseudo-PCS enzymes recently identified in cyanobacteria, which are also biosynthetically inactive, but capable to hydrolyze GSH.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.     

New class of cargo protein in Tetrahymena thermophila dense core secretory granules

Regulated exocytosis of dense core secretory granules releases biologically active proteins in a stimulus-dependent fashion. The packaging of the cargo within newly forming granules involves a transition: soluble polypeptides condense to form water-insoluble aggregates that constitute the granule cores. Following exocytosis, the cores generally disassemble to diffuse in the cell environment. The ciliates Tetrahymena thermophila and Paramecium tetraurelia have been advanced as genetically manipulatable systems for studying exocytosis via dense core granules. However, all of the known granule proteins in these organisms condense to form the architectural units of lattices that are insoluble both before and after exocytosis. Using an approach designed to detect new granule proteins, we have now identified Igr1p (induced during granule regeneration). By structural criteria, it is unrelated to the previously characterized lattice-forming proteins. It is distinct in that it is capable of dissociating from the insoluble lattice following secretion and therefore represents the first diffusible protein identified in ciliate granules.     

A novel family of mobile genetic elements is limited to the germline genome in Tetrahymena thermophila

In the ciliated protozoan Tetrahymena thermophila, extensive DNA elimination is associated with differentiation of the somatic macronucleus from the germline micronucleus. This study describes the isolation and complete characterization of Tlr elements, a family of approximately 30 micronuclear DNA sequences that are efficiently eliminated from the developing macronucleus. The data indicate that Tlr elements are comprised of an ~22 kb internal region flanked by complex and variable termini. The Tlr internal region is highly conserved among family members and contains 15 open reading frames, some of which resemble genes encoded by transposons and viruses. The Tlr termini appear to be long inverted repeats consisting of (i) a variable region containing multiple direct repeats which differ in number and sequence from element to element and (ii) a conserved terminal 47 bp sequence. Taken together, these results suggest that Tlr elements comprise a novel family of mobile genetic elements that are confined to the Tetrahymena germline genome. Possible mechanisms of developmentally programmed Tlr elimination are discussed.     

Septins stabilize mitochondria in Tetrahymena thermophila

We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8:81-93, 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.     

Rate of phenotypic assortment in Tetrahymena thermophila

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf/ the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published R_f values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of R_f. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with ～45 assorting units, as has been asserted. © 1992 Wiley-Liss, Inc.     

A Simple, Efficient Technique for Freezing Tetrahymena thermophila

ABSTRACT. We have developed a simple, efficient procedure for the long term freezing of Tetrahymena thermophila in liquid nitrogen. This technique yields excellent recovery of viable cells with all strains tested and does not require the use of a controlled rate low temperature freezer. To optimize the freezing technique, we have examined the effects of varying a number of parameters, including the physiological state of the cells prior to freezing, the time of exposure to cryoprotectant, and the rate of freezing and thawing. the frequency of viable cell recovery following freezing using this technique has been tested for a variety of different cell lines.     

A cyclin-dependent protein kinase homologue associated with the basal body domains in the ciliate Tetrahymena thermophila

The tight coupling between cell cycle progression and morphogenetic development in the unicellular ciliates presents a unique model system for examination of the roles of Cdks in developmental processes. We here describe the isolation and characterization of the first cyclin-dependent kinase (Cdk) homologue, TtCdk1, from Tetrahymena thermophila. TtCdk1 corresponds to the larger of the two polypeptides recognized by anti-PSTAIRE antibody in a whole cell lysate, which differ from each other in their affinity for yeast p13(suc1) protein. In contrast to the constant protein expression levels of typical eukaryotic Cdks, the TtCdk1 protein level fluctuates periodically over the vegetative cell cycle, reaching a maximum at the end of the cell cycle, correlating with its histone H1 kinase activity. Its association with the membrane-skeletal domains that surround mature, but not nascent, basal bodies in the cell cortex suggests that TtCdk1 plays a role in the regulation of cortical morphogenesis in T. thermophila. A partial TtCDK1 knockout cell line constructed through somatic biolistic transformation resulted in a reduction of the regularity of the rows of basal bodies plus an additional effect on chromatin condensation in both macro- and micronuclei. Unlike the situations in higher eukaryotic cells, no apparent effect on basal body duplication was found upon disruption of the TtCDK1 gene.     

Conjugation in Tetrahymena thermophila: A temporal analysis of cytological stages

The time course and synchrony of the stages of conjugation in Tetrahymena thermophila as defined by cytologically observable changes in the morphology and position of the nuclei were established. The time required for 50% of the pairs to enter or pass a particular stage, as well as the duration of each stage were determined. The relative synchrony of the pairs as they went through conjugation was followed by correlating the maximum percentage of the population found in a stage with the duration of that stage. The degree of synchrony between the pairs was found to be high under the conditions of this study, with very little decrease in synchrony seen during the initial 9 h of conjugation. Although some variability in the degree of synchrony was seen between different matings, there was little change detected in the duration of each cytological stage. Prolonged starvation of the cells prior to their mating resulted in a gradual loss of synchrony.     

Membrane protein differences correlated with the development of mating competence in Tetrahymena thermophila

Initiation is the contact-independent phase of sexual conjugation which occurs when mature cells of Tetrahymena thermophila are shifted from growth medium to a low-salt starvation buffer. Immaturity, like high-salt starvation, restricts the ability of cells to conjugate; immature cells do not conjugate in either low- or high-salt buffers. Comparisons between sexually mature cells starved in initiation-restrictive and initiation-permissive buffers, and between immature and mature cells starved in an initiation-permissive buffer permitted the analysis of membrane protein expression correlated with mating competence. No polypeptides identified by lactoperoxidase-catalyzed iodination were found to be specific to mating-competent cells; however, several polypeptides not present in initiated cells were found to be common to the cell surfaces of immature and non-initiated cells which suggests that (1) initiation involves the removal of specific proteins from the cell surface, and (2) immaturity may be due to an inability to initiate.     

Kinetics of the DFPase activity in Tetrahymena thermophila

Crude homogenates of the ciliate protozoon, Tetrahymena thermophila, can hydrolyze the potent acetylcholinesterase inhibitors O,O-diisopropylphosphorofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoride (soman). Characterization of the enzymatic activity of the homogenate has been performed. The DFPase operates over a pH range of 4 to 10 and an ionic range of 0-500 mM NaCl. Rate of reaction increases three- to four-fold from 25 degrees C to 40 degrees C and is still present at 55 degrees C. These results indicate that the enzymatic activity operates over a broad range of environmental conditions, making it an attractive material for use in the detoxification and detection of organofluorophosphates. DFPases may be important in the metabolism of naturally occurring organophosphates.