Analysis of gene expression of Tetrahymena thermophila treated with Panax japonicas / 中国中药杂志

Panax japonicus is a traditional Chinese medicine,and its principle components have shown certain pharmacological activities for cell damage,aging and cell apoptosis. In order to clarify the pharmacological mechanism and involved metabolic pathways of P. japonicas,the gene expression of Tetrahymena thermophila under P. japonicus treatment was analyzed through high-throughput transcriptome sequencing in this study. Based on the transcriptome analysis,3 544 differentially expressed genes were identified in control group,of which 1 945 genes showed up-regulated expression and 1 599 genes showed down-regulated expression. Under P. japonicas treatment in the experiment group,3 312 differentially expressed genes were screened,of which 1 `493 genes showed up-regulated expression and 1 819 genes showed down-regulated expression. GO enrichment analysis indicated that in control group,the genes in the cells in a series of fundamental biological process were down-regulated,such as DNA replication and protein synthesis; while the signal transduction process and fatty acids oxidizing process were enriched. Whereas in the experiment group,down-regulated genes were mainly enriched in oxidation-reduction,cofactor metabolic process and vitamin metabolic process; up-regulated genes were enriched in signal transduction process and protein modification process. In the analysis using KEGG database,cell cycle pathway was enhanced and autophagy pathway was inhibited under the condition of P. japonicas treatment. Real-time quantitative polymerase chain reaction( RT-qPCR) was used to detect the expression differences between 6 up-regulated and 4 down-regulated genes in related metabolic pathways. The RT-q PCR results and RNA-Seq data were highly correlated and consistent with each other. This study could provide important direction and basis for further study on the mechanism of cell growth regulation with the treatment of P. japonica.

The key role of CYC2 during meiosis in Tetrahymena thermophila

Meiotic recombination is carried out through a specialized pathway for the formation and repair of DNA double-strand breaks (DSBs) made by the Spo11 protein. The present study shed light on the functional role of cyclin, CYC2, in Tetrahymena thermophila which has transcriptionally high expression level during meiosis process. Knocking out the CYC2 gene results in arrest of meiotic conjugation process at 2.5-3.5 h after conjugation initiation, before the meiosis division starts, and in company with the absence of DSBs. To investigate the underlying mechanism of this phenomenon, a complete transcriptome profile was performed between wild-type strain and CYC2 knock-out strain. Functional analysis of RNA-Seq results identifies related differentially expressed genes (DEGs) including SPO11 and these DEGs are enriched in DNA repair/mismatch repair (MMR) terms in homologous recombination (HR), which indicates that CYC2 could play a crucial role in meiosis by regulating SPO11 and participating in HR.

Functional Modification of a Specific RNA with Targeted Trans-Splicing

The self-splicing group I intron from Tetrahymena thermophila has been demonstrated to perform splicing reaction with its substrate RNA in the trans configuration. In this study, we explored the potential use of the trans-splicing group I ribozymes to replace a specific RNA with a new RNA that exerts any new function we want to introduce. We have chosen thymidine phosphorylase (TP) RNA as a target RNA that is known as a valid cancer prognostic factor. Cancer-specific expression of TP RNA was first evaluated with RT-PCR analysis of RNA from patients with gastric cancer. We determined next which regions of the TP RNA are accessible to ribozymes by employing an RNA mapping strategy, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. A specific ribozyme recognizing the most accessible sequence in the TP RNA with firefly luciferase transcript as a 3' exon was then developed. The specific trans-splicing ribozyme transferred an intended 3' exon tag sequence onto the targeted TP transcripts, resulting in a more than two fold induction of the reporter activity in the presence of TP RNA in mammalian cells, compared to the absence of the target RNA. These results suggest that the Tetrahymena ribozyme can be a potent anti-cancer agent to modify TP RNAs in tumors with a new RNA harboring anti-cancer activity.

Phototoxicity evaluation--Tetrahymena thermophila as an alternative model.

Interest in utilizing an alternative to animal method for toxicological evaluation has received considerable attention due to cost effectiveness and the ethical issues involving animal experimentation. Alternative methods for phototoxicity evaluation are significant because of growing concern over increasing health effects due to stratospheric ozone depletion resulting in an increasing penetration of ultraviolet light-B radiation (UVB, 290-320 nm) which contributes to activation of chemical and biological molecules to potential phototoxic agents. The classic rabbit eye-irritancy test referred to as Draize test has been the subject of severe criticism by animal welfare groups. Dermal toxicity test using guinea pigs and mouse tail phototoxicity test is time consuming and requires a large number of laboratory animals. In photohaemolysis assay some of the phototoxic agents (such as riboflavin) react with the membrane proteins of the erythrocyte. However, in vitro test system using protozoa offers a promising alternative means of phototoxicity evaluation. Our previous studies have demonstrated that synergistic action of photochemically reactive agents and sunlight produces lethal effects to Paramecium but the protozoan has not received serious consideration for use as an alternative model for phototoxicity evaluation. In the present communication we have described the potential application of Tetrahymena as an alternative model to study the radiation-induced changes both in the presence or absence of photoreactive chemical agents. This model is likely to provide scope for studying the biological effects of environmental UVB radiation, DNA damage and defence against oxidative stress.

Recent progress in understanding the temporal behavior of unicellular organisms

This survey summarizes the findings concerning endogenous oscillations of three unicellular organisms: the dinophyte Gonyaulax polyedra, the ciliate Tetrahymena thermophila and the euglenophyte Euglena gracilis. All of them behave rhythmically and show the common features of zeitgeber action, differential sensitivity and temperature compensation; however, they exhibit some species-specific peculiarities that make each of them suitable for addressing particular chronobiological questions. Although ultradian rhythms have been described for Tetrahymena thermophila and Euglena gracilis, they appear under different conditions: in the first case, a modulation of the period in relation to the concentration of nutrients is observed, whereas Euglena oscillates in an ultradian and circadian fashion simultaneously. Transitions between periodic and aperiodic states can be induced in Euglena gracilis and Gonyaulax polyedra: Euglena gracilis can enter an aperiodic state after repeated exposure to short light pulses (up to I 0 sec) given at intervals of 40 min or less, whereas in Gonyaulax polyedra the circadian oscillator is arrested at temperatures below 12 degrees Celsius. In the arrhythmic state, the oscillator might be driven into singularity within the phase space of a limit cycle attractor; re-initiation from the holding point occurs by transition to a relatively precisely defined new phase. Photoperiodism as another important chronobiological phenomenon can be studied in Gonyaulax polyedra: cells enter the dormant stage of an asexual cyst under short days and a temperature below 16 degrees Celsius. This response can be mimicked by 5-methoxylated indoleamines such as melatonin and 5-methoxytryptamine, which are synthesized by this organism. Melatonin concentration exhibits an endogenous circadian rhythm characterized by a rapid increase shortly after the onset of darkness. Encystment, as induced by indoleamines, is associated with stimulations of bioluminescence. The coupling of the two processes involves, as a common element, the release of protons from an acidic vacuole.