Engineering of ribozyme-based riboswitches for mammalian cells

Artificial RNA riboswitches--apart from protein-based gene regulation systems, which have been known about for a long time--have become increasingly important in biotechnology and synthetic biology. Aptamer-controlled hammerhead ribozymes (so-called aptazymes) have been shown to be a versatile platform for the engineering of novel gene regulators. Since aptazymes are cis-acting elements that are located in the untranslated regions of a gene of interest, their application does not need any further protein co-factor. This presents the opportunity to simplify complex gene networks while simultaneously expanding the repertoire of available parts. Nevertheless, the generation of novel aptazymes requires a functional aptamer-ribozyme connection, which can be difficult to engineer. This article describes a novel approach for using fluorescence activated cell sorting (FACS) in order to identify functional aptazymes in bacteria and their subsequent transfer into mammalian cells.     

The ciliated protozoa Tetrahymena thermophila as a bionsensor to detect mycotoxins

The cytotoxic effect of four mycotoxins (patulin, diacetoxyscirpenol, roquefortine and T-2 toxin) has been tested on the ciliate Tetrahymena thermophila. This ciliate has been shown to be a very sensitive biosensor to patulin, diacetoxyscirpenol and T-2 toxin. With respect to the roquefortine, this is the first reported bioassay using eukaryotic cells, and results show a lower sensitivity than tests using bacteria. Results are compared with those obtained using other biosensors.     

Conjugation involving dividing cells of Tetrahymena thermophila

Feulgen-stained preparations of mixtures of starved Tetrahymena thermophila cells of complementary mating types have revealed an atypical form of conjugation involving cells which have completed the nuclear events of cell division, but have not undergone cytokinesis. Both micronuclei in the dividing cells are induced to undergo meiosis, but in 21 of 23 cases, the anterior micronucleus was activated 1st, suggesting that the meiotic inducer is synthesized near the mating junction and diffuses posteriad. Despite the induction of two micronuclei, "triad" conjugants appear to regulate nuclear events so as to produce a normal outcome.     

The dynein heavy chain gene family in Tetrahymena thermophila

The dynein ATPases are a family of motor enzymes that drive microtubule sliding in cilia and flagella and contribute to microtubule-based transport inside cells. The multi-dynein hypothesis makes two predictions: 1) Axonemes contain multiple dynein heavy chain (DHC) isoforms, each encoded by a different gene; 2) Each isoform performs a specific role in ciliary beating. We used PCR-based techniques to clone thirteen different DHC sequences from Tetrahymena genomic DNA. All thirteen genes appeared to be expressed in growing cells. Comparisons of the deduced amino acid sequences of the thirteen DHCs with other known DHCs suggested that we have cloned three outer arm DHCs, two cytoplasmic DHCs, and eight inner arm DHCs.     

Multiple introns in a conjugation-specific gene from Tetrahymena thermophila

Multiple introns have been found in a gene from a ciliated protozoan. This Tetrahymena thermophila gene (cnjB) is large (7.5 kb mRNA) and active only during conjugation, the organism's sexual cycle. Six introns ranging in size from 62 bp to 676 bp were found when we sequenced a 3.1 kb segment of the cnjB gene together with its corresponding cDNA. We estimate, by extrapolation of our current data, a total of approximately 30 introns in this gene with a total gene size (introns plus exons) of 15 kb or more. The number of introns is surprising given the scarcity of introns in ciliate genes examined to date. Our findings constitute the first example of multiple introns in a ciliate gene. Having the sequence of several introns has allowed us to construct consensus sequences for T. thermophila mRNA introns. The 5' and 3' intron junctions resemble those of general nuclear mRNA (GT/AG rule is followed) but differences are seen. In particular, stretches of 10 or more adenines and thymines are found adjacent to the conserved GT and AGs at the junctions. Unusual aspects of the coding region of this gene are discussed.     

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.     

Positional reorganization in compound janus cells of Tetrahymena thermophila

The janus mutations of Tetrahymena thermophila convert the large-scale organization of the dorsal surface of the cell into a mirror-image of the ventral surface, which is characterized by a second, abnormal, oral apparatus and by contractile vacuole pores to the left of the second oral area rather than the usual right. This conversion could be due either to a local change in the response to an unaltered positional system or to a more global reorganization of the system itself. janus homopolar doublets were used to distinguish between these two alternatives. Homopolar doublets can be made by fusing two similarly oriented cells in side-by-side parabiosis. Non-janus homopolar doublets typically possess two sets of normal oral structures with contractile vacuole pores to the right of each of them. In janus doublets, there are up to four sets of oral structures, with the abnormal oral structures located between the two sets of normal oral structures; contractile vacuole pores are situated to the right of the normal oral areas and to the left of the abnormal oral structures. Non-janus homopolar doublets are known to propagate their compound condition for a number of cell divisions, but also to regulate toward the singlet state through a progressive reduction in number of ciliary rows followed by loss of one of the two sets of major cell surface structures. janus homopolar doublets go through a corresponding regulation. As a consequence, the location of the abnormal oral structures relative to the normal ones is more variable in janus doublets than in janus singlets. Sometimes the abnormal oral structures shift to a position close to their normal counterparts and then the intervening CVP sets disappear. There is evidence for occasional fusion of an abnormal oral area with an adjacent normal oral apparatus, a condition that may be transitional to the singlet state. These observations are inconsistent with the idea of a fixed positional system and strongly suggest a global reorganization of the surface pattern in a manner consistent with predictions of an intercalation model that was first proposed to explain the regulation of non-janus doublets to singlets.     

Targeted gene knockout of inner arm 1 in Tetrahymena thermophila

Cilia and flagella contain at least eight different types of dynein arms. It is not entirely clear how the different types of arms are organized along the axoneme. In addition, the role each different type of dynein plays in ciliary or flagellar motility is not known. To initiate studies of dynein organization and function in cilia, we have introduced a mutation into one dynein heavy chain gene (DYH6) in Tetrahymena themophila by targeted gene knockout. We have generated mutant cells that lack wild-type copies of the DYH6 gene. We have shown that the DYH6 gene encodes one heavy chain (HC2) of Tetrahymena 18S dynein and that 18S dynein occupies the I1 position in the ciliary axoneme. We have also shown that Tetrahymena I1 is required for normal motility, normal feeding and normal doubling rate.     

Genome-wide analysis of the Tetrahymena thermophila glutathione S-transferase gene superfamily

The ciliate Tetrahymena thermophila has a rapid response to detoxify xenobiotics, which presents opportunity to study the diversification of Glutathione S-Transferase superfamily. In-silico identification of putative GST genes were resulted with 70 GST genes; 49 TtGSTmu, 7 TtGSTomega, 5 TtGSTtheta, 2 TtGSTzeta, 4 TtMAPEG and 3 TtEF1G. TtGST superfamily has short intron carrying or intronless genes. The most expressed mRNAs of TtGST are limited to 4 members at all life stages. TtGST genes are widely distributed to all five micronuclear chromosomes with the highest diversified members from different classes in chromosome 4. The clustering and the orientation of some TtGSTs in the T. thermophila genome give clues about the recent gene duplication. Analysis of GSH affinity-purified GST proteins with Western blot and activity assay showed GST activity carrying purified TtGST populations. In conclusion, the enhanced genome capacity of TtGST superfamily may have evolved through improved GST enzymatic activity.     

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.     

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.     

Phenotypic responses to temperature in the ciliate Tetrahymena thermophila

Understanding the effects of temperature on ecological and evolutionary processes is crucial for generating future climate adaptation scenarios. Using experimental evolution, we evolved the model ciliate Tetrahymena thermophila in an initially novel high temperature environment for more than 35 generations, closely monitoring population dynamics and morphological changes. We observed initially long lag phases in the high temperature environment that over about 26 generations reduced to no lag phase, a strong reduction in cell size and modifications in cell shape at high temperature. When exposing the adapted populations to their original temperature, most phenotypic traits returned to the observed levels in the ancestral populations, indicating phenotypic plasticity is an important component of this species thermal stress response. However, persistent changes in cell size were detected, indicating possible costs related to the adaptation process. Exploring the molecular basis of thermal adaptation will help clarify the mechanisms driving these phenotypic responses.     

镉和铜对嗜热四膜虫金属硫蛋白基因的诱导表达

本文在荧光定量PCR优化的基础上,利用该技术考察了不同浓度的重金属镉和铜对嗜热四膜虫金属硫蛋白基因(MTT1)诱导表达的变化规律。结果表明MTT1基因的表达对镉离子的诱导更灵敏,且在一定阈值浓度(≤35.2μmol/L)范围内,镉离子浓度升高会增加MTT1基因表达量,超过该阈值后表达量迅速下降;镉与铜同时诱导时MTT1基因的表达情况与镉单独诱导的类似,但阈值浓度减小为22μmol/L,表明二者的联合毒性为协同作用。镉离子浓度低于22μmol/L时,与铜离子的共同作用会大大增加MTT1基因的表达量,从而增强了四膜虫的解毒能力。     

The in vivo Tetrahymena thermophila extracellular glucose drop assay for characterization of mammalian insulin activity

Insulin activity is generally determined by an in vivo rabbit blood glucose drop assay in research and industriel laboratories. The humane experimental techniques imply the use of alternative invertebrate organisms in place of animals, known as replacement rule of the 3Rs. In this study, we report an alternative in vivo extracellular glucose drop assay using unicellular invertebrate Tetrahymena thermophila to replace the use of rabbit and mouse. This assay has four major steps; growing cells, starving cells, treatment of cells and measurement of glucose drop. In this assay, 0.2 mg/ml of human, porcine and bovine insulins dropped extracellular glucose level to 16%, 14% and 12%, respectively in ten minutes. In addition, mammalian insulins respectively increased the cell area about 19%, 15%, and 16% at 6th hour with statistically significant effect on the cell growth, but not in the cell viability. The results showed that the in vivo Tetrahymena thermophila extracellular glucose drop assay could be used as an alternative assay to replace the mouse or the rabbit insulin blood glucose drop assay.     

Protein binding to expanded telomere repeats in Tetrahymena thermophila

The ends of eukaryotic chromosomes are protected by DNA-protein structures called telomeres. Telomeric DNA is highly conserved, usually consisting of long tracts of a repeating G-rich sequence. Tetrahymena thermophila telomeric DNA consists of alternating blocks of GGGG and TT sequences (i.e. a G4T2 repeat sequence). We examined the relative importance of the guanine and thymine elements of the repeat sequence in promoting in vitro binding by T. thermophila proteins. We identified single- and, for the first time, double-stranded telomere binding activities from a crude T. thermophila protein extract and tested the binding of these activities to altered telomere repeat sequences. All deletions or substitutions made to the guanine element virtually abolished binding, indicating that four G's are essential for recognition by the binding activity. However, G's alone are not sufficient for efficient binding, as elimination of the thymine element dramatically reduced binding. By contrast, substantial expansion of the thymine element was well tolerated, even though one such change, G4T4, is lethal in vivo. We tested up to a four-fold expansion of the thymine element and found that highly efficient binding was still achieved. These results suggest a minimal recognition sequence for T. thermophila proteins, with the T element providing an important spacer between essential G elements.     

Development in Electrofused Conjugants of Tetrahymena thermophila

Electric shock can create parabiotic fusions of living Tetrahymena cells. In this study, cells were mated and successful pairs were electrofused with either vegetatively growing cells or other mating pairs. In particular, we electrofused pairs from normal [diploid x diploid] matings with vegetatively dividing cells in G- or M-phase of the cell cycle. We also fused [diploid x diploid] conjugants with mating pairs involving an aneuploid partner [diploid x "star"], which typically undergo an abortive conjugal pathway termed genomic exclusion. Using such parabiotic fusions we identified and characterized two developmentally critical landmarks: 1) the "abort" signal, which is initiated in pairs with nuclear defects (this first becomes evident soon after the completion of Meiosis I or the beginning of Meiosis II); and 2) the "terminal commitment point", a developmental stage in normal [diploid x diploid] pairs after which conjugation no longer responds to a parabiotically transmitted abort signal (this correlates with the onset of the second postzygotic nuclear division). Finally we demonstrate that a conjugal-arrest-activity varies with the vegetative cell cycle, reaching its highest level of activity during M-phase and dropping just after cytokinesis.