Ingestion without inactivation of bacteriophages by Tetrahymena

Tetrahymena has been shown to ingest and inactivate bacteriophages, such as T4, in co-incubation experiments. In this study, Tetrahymena thermophila failed to inactivate phages PhiX174 and MS2 in co-incubations, although PhiX174 were ingested by T. thermophila, as demonstrated by: (1) recovery at defecation in a pulse-chase experiment, (2) recovery from Tetrahymena by detergent lysis, and (3) transmission electron microscopy. We conclude, therefore, that the phages must be digestion-resistant. Internalized PhiX174 were further shown to be partially protected from lethal damage by ultraviolet (UV) C and UVB irradiation. Finally, ingested PhiX174 were shown to be rapidly transported through buffer in a horizontal swimming, race tube-like assay. The transport and protection of phages may confer evolutionary advantages that explain the acquisition of digestion-resistance by some phages.     

Use of Tetrahymena thermophila to study the role of protozoa in inactivation of viruses in water

The ability of a ciliate to inactivate bacteriophage was studied because these viruses are known to influence the size and diversity of bacterial populations, which affect nutrient cycling in natural waters and effluent quality in sewage treatment, and because ciliates are ubiquitous in aquatic environments, including sewage treatment plants. Tetrahymena thermophila was used as a representative ciliate; T4 was used as a model bacteriophage. The T4 titer was monitored on Escherichia coli B in a double-agar overlay assay. T4 and the ciliate were incubated together under different conditions and for various times, after which the mixture was centrifuged through a step gradient, producing a top layer free of ciliates. The T4 titer in this layer decreased as coincubation time increased, but no decrease was seen if phage were incubated with formalin-fixed Tetrahymena. The T4 titer associated with the pellet of living ciliates was very low, suggesting that removal of the phage by Tetrahymena inactivated T4. When Tetrahymena cells were incubated with SYBR gold-labeled phage, fluorescence was localized in structures that had the shape and position of food vacuoles. Incubation of the phage and ciliate with cytochalasin B or at 4 degrees C impaired T4 inactivation. These results suggest the active removal of T4 bacteriophage from fluid by macropinocytosis, followed by digestion in food vacuoles. Such ciliate virophagy may be a mechanism occurring in natural waters and sewage treatment, and the methods described here could be used to study the factors influencing inactivation and possibly water quality.     

四膜虫种间骨架蛋白组分的比较研究

以上海四膜虫S1和嗜热四膜虫BF株和BT株为材料,结合显微观察,采用生化抽提、SDS-PAGE电泳、扫描及数据统计,分析与测定了三个不同株四膜虫对数生长期皮层骨架蛋白组分与含量,结果显示嗜热四膜虫的BF与BT株差异较小,两者与上海四膜虫S1株差异则较大,S1株细胞中有92KD、72KD、66KD、32KD、27KD,而BF和BT株细胞中没有,估计这些蛋白的不同与种间亲缘关系及株系、培养条件等有着密不可分的联系.       

Conserved cis- and trans-acting determinants for replication initiation and regulation of replication fork movement in tetrahymenid species.

The rDNA minichromosomes of Tetrahymena thermophila and Tetrahymena pyriformis share a high degree of sequence similarity and structural organization. The T.thermophila 5' non-transcribed spacer (5' NTS) is sufficient for replication and contains three repeated sequence elements that are conserved in T.pyriformis , including type I elements, the only known determinant for replication control. To assess the role of conserved sequences in replication control, structural and functional studies were performed on T.pyriformis rDNA. Similar to T.thermophila , replication initiates exclusively in the 5' NTS, localizing to a 900 bp segment. Elongating replication forks arrest transiently at one site which bears strong similarity to a tripartite sequence element present at fork arrest sites in T.thermophila rDNA. An in vitro type I element binding activity indistinguishable from the T.thermophila protein, ssA-TIBF, was detected in T.pyriformis extracts. The respective TIBF proteins bind with comparable affinity to type I elements from both species, suggesting that in vivo recognition could cross species boundaries. Despite these similarities, the T.pyriformis 5' NTS failed to support replication in transformed T.thermophila cells, suggesting a more complex genetic organization than previously realized.     

Small RNAs of Tetrahymena thermophila

Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination. In the nuclear RNA fraction we have detected at least 6 distinct snRNAs. Some of the RNA species showed microheterogeneity. SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned. Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently (18) as nuclear, particularly nucleolar RNAs. Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed.Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.     

RNA splicing in the T-even bacteriophage

Group 1 introns, first demonstrated in the nuclear large rRNA of Tetrahymena thermophila and subsequently in many yeast, fungal mitochondrial, and chloroplast precursor RNAs, are capable of intron excision and exon ligation in vitro, although this process occurs much more rapidly in vivo. The discovery and characterization of a similar intron in the T4 phage thymidylate synthase gene (td) led to the finding of additional group 1 introns in other T4 genes and in genes of the related T2 and T6 phages. Because protein factors are not required in the splicing of group 1 introns in vitro, it has been postulated that the precursor RNA can assume a critical conformation enabling it to undergo site-specific autocatalytic cleavage and ligation (self-splicing). By means of site-directed mutation, it has been shown unequivocally that several sequence elements in the Tetrahymena rRNA intron are involved in the formation of base-paired stem structures that are essential for the self-splicing process. These sequence elements have been demonstrated in other eukaryotic group 1 introns, as well as in the td intron. In this brief review we shall describe the biochemical and structural properties of the td intron in relation to other newly found phage introns. The interesting implications arising from these revelations will also be discussed.     

Hydrolysis of 2-arachidonoylglycerol in Tetrahymena thermophila. Identification and partial characterization of a Monoacylglycerol Lipase-like enzyme

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1μM and V(max) of 5.8nmol/min×mg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at ～45 and ～40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.     

High efficiency transformation of Tetrahymena using a codon-optimized neomycin resistance gene

Tetrahymena thermophila is a useful model for the study of eukaryotic biology. A neomycin resistance gene (neo) has been developed that was optimized for the codon usage of T. thermophila. Using this codon-optimized neo gene (neoTet), a new drug resistance marker cassette, neo4, has been constructed. The neo4 cassette resulted in about ten times more drug resistant transformants than a cassette containing the non-codon-optimized original neo gene. The new cassette enables transgenic Tetrahymena strains to be created with high efficiency. This study also emphasizes the importance of codon optimization in transgene expression in Tetrahymena.     

Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena.

The codon usage of Tetrahymena thermophila and other ciliates deviates from the 'universal genetic code' in that UAA and probably UAG are not translational termination signals but code for glutamine. Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA. We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins. The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s). The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.     

诱导嗜热四膜虫(Tetrahymena thermophila)细胞凋亡样变化的研究

本文以单细胞真核生物嗜热四膜虫(Te-trahymena thermophila)作为实验材料以抗肿瘤药物高三尖杉脂碱(Homoharringtonine,HHT)、糖皮质激素类药物地塞米松(9α-Fluo-ro-16α-methylprednisolone,Dex)和抗生素类药物放线菌素D(Actinomycin D)诱导嗜热四膜虫凋亡并研究其细胞凋亡过程的生物化学特性。结果表明抗肿瘤药物及抗生素类药物均不能明显地诱导嗜热四膜虫细胞凋亡。但糖皮质激素类药物在含一定量的Ca~(2 )、Mg~(2 )离子时能诱导嗜热四膜虫发生凋亡。作者认为诱导嗜热四膜虫凋亡过程可能与糖皮质激素类药物诱导鼠胸腺细胞凋亡的机制是类似的,嗜热四膜虫与胸腺细胞的凋亡过程可能同样被Ca~(2 )、Mg~(2 )离子依赖性的核酸内切酶的活化机制所控制着。     

Developmentally regulated telomere addition in Tetrahymena thermophila.

To investigate the developmentally programmed telomere addition that accompanies chromosome fragmentation during macronuclear differentiation in Tetrahymena thermophila, five representative telomeric regions from the macronucleus were cloned and characterized in detail. The sequences adjacent to the telomeric (C4A2:T2G4) repeats on these five macronuclear ends had no significant sequence homology or shared secondary structure. Two developmentally independent examples of one macronuclear telomere had a 5 base pair difference in the position of the junction between the telomeric repeats and the adjacent sequences. A telomere-adjacent sequence, in the form of a synthetic oligonucleotide, was unable to prime the addition of telomeric repeats in vitro. The implications of these results for the mechanisms underlying developmentally programmed chromosome fragmentation and telomere addition in Tetrahymena are discussed.     

Single-cell isolation and cloning of Tetrahymena thermophila cells with a fluorescence-activated cell sorter

We developed a method for cloning cells of the ciliate Tetrahymena thermophila in chemically defined medium (CDM) using a fluorescence-activated cell sorter (FACS). Although T. thermophila is a model unicellular eukaryote, two major technical difficulties remain in its cloning. First, T. thermophila fails to proliferate from low density in CDM, particularly if the inoculum contains single cells. Second, general cloning methods are time consuming and have low throughput. Here, we modified the CDM by addition of bovine serum albumin that helped growth from an inoculum with a density of 10 cell/ml (1 cell/100 μl). In addition, we applied a FACS for isolation of single cells. We showed that it is possible to separate cell populations based on the presence or absence of phagocytosed fluorescent beads and to isolate single cells in a modified CDM by FACS. Our techniques allow the direct isolation of single cells and facilitate the establishment of clonal strains.     

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

Surface display of a parasite antigen in the ciliate Tetrahymena thermophila.

The ciliated protozoan, Tetrahymena thermophila, offers an attractive medium for the expression of heterologous proteins and could prove particularly useful for the display of foreign proteins on the cell surface. Although progress has been made in transformation of Tetrahymena with heterologous DNA, methods that permit reliable expression of foreign genes have been lacking. Using a mutant strain of T. thermophila carrying a negatively selectable allele of a beta-tubulin gene, we have been able to direct foreign genes to this locus by homologous recombination. Transformed cell lines producing foreign proteins were readily identified and, in at least one case, targeting of proteins to the plasma membrane was accomplished.     

D-3 phosphoinositides of the ciliate Tetrahymena: characterization and study of their regulatory role in lysosomal enzyme secretion

Phosphatidylinositol 3-phosphate, PtdIns3P, is a phosphoinositide which is implicated in regulating membrane trafficking in both mammalian and yeast cells. It also serves as a precursor for the synthesis of phosphatidylinositol 3,5-bisphosphate, PtdIns3,5P2, a phosphoinositide, the exact functions of which remain unknown. In this report, we show that these two phosphoinositides are constitutive lipid components of the ciliate Tetrahymena. Using HPLC analysis, PtdIns3P and PtdIns3,5P2 were found to comprise 16% and 30-40% of their relevant phosphoinositide pools, respectively. Treatment of Tetrahymena cells with wortmannin (0.1-10 microM) resulted in the depletion of PtdIns3P and PtdIns3,5P2 without any effect on D-4 phosphoinositides. Wortmannin was further used for the investigation of D-3 phosphoinositide involvement in the regulation of lysosomal vesicular trafficking. Incubation of Tetrahymena cells with wortmannin resulted in enhanced secretion of two different lysosomal enzymes without any change in their total activities. Experiments performed with a T. thermophila secretion mutant strain verified that the wortmannin-induced secretion is specific and it is not due to a diversion of lysosomal enzymes to other secretory pathways. Moreover, experiments performed with a phagocytosis-deficient T. thermophila strain showed that a substantial fraction of wortmannin-induced secretion was dependent on the presence of functional phagosomes/phagolysosomes.     

Spliceosomal small nuclear RNAs of Tetrahymena thermophila and some possible snRNA-snRNA base-pairing interactions

We have identified and characterized the full set of spliceosomal small nuclear RNAs (snRNAs; U1, U2, U4, U5 and U6) from the ciliated protozoan Tetrahymena thermophila. With the exception of U4 snRNA, the sizes of the T. thermophila snRNAs are closely similar to their metazoan homologues. The T. thermophila snRNAs all have unique 5' ends, which start with an adenine residue. In contrast, with the exception of U6, their 3' ends show some size heterogeneity. The primary sequences of the T. thermophila snRNAs contain the sequence motifs shown, or proposed, to be of functional importance in other organisms. Furthermore, secondary structures closely similar to phylogenetically proven models can be inferred from the T. thermophila data. Analysis of the snRNA sequences identifies three potential snRNA-snRNA base-pairing interactions, all of which are consistent with available phylogenetic data. Two of these occur between U2 and U6, whereas the third occurs between U1 and U2. The proposed interactions locate the intron 5' splice-site close to the intron branch-site nucleotide as well as to the most highly conserved domain of U6. We envisage that these interactions may facilitate the first step of pre-mRNA splicing.     

Preparation and Biological Effect of Nucleotide-Capped CdSe/ZnS Quantum Dots on Tetrahymena thermophila

In this paper, we described the preparation and characterization of different types of modified CdSe/ZnS quantum dots (QDs) and explored the biological effects of QDs with different surface modifications on the whole growth of unicellular protozoan Tetrahymena thermophila BF(5) using a thermal activity monitor air isothermal microcalorimeter. Our results demonstrated that adenosine 5'-monophosphate (AMP) showed stronger interaction with QDs than other types of nucleotide. AMP-QDs could stimulate the growth of T. thermophila while mercaptoacetic acid-capped CdSe/ZnS quantum dots inhibited it. In addition, the population density determination and fluorescence imaging of T. thermophila BF(5) also confirmed the results obtained from microcalorimetry. It is believed that this approach will provide a more convenient methodology for the kinetics and thermodynamics of microorganism when coexisting with QDs in real time, and all of which are very significant to understanding the effect of QDs to organism.     

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.