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.     

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.     

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.     

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.     

Macronuclear DNA molecules of Tetrahymena thermophila.

The physical organization of the DNA in the macronuclei of Tetrahymena thermophila was investigated by using alternating-orthogonal-field gel electrophoresis. The genome consisted of a spectrum of molecules with lengths ranging from less than 100 to in excess of 1,500 kilobase pairs. There were about 270 different macronuclear DNA molecules, with an average size of about 800 kilobase pairs. Specific genes were mapped and were generally found on macronuclear DNA molecules of the same size in different strains of T. thermophila. This indicates that the molecular mechanisms giving rise to the macronuclear DNA molecules were precise. The fragmentation process that gave rise to macronuclear DNA molecules occurred between 11 and 19 h after the initiation of conjugation.     

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.     

An assessment of proteolytic enzymes in Tetrahymena thermophila.

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.     

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.     

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.     

嗜热四膜虫减数分裂研究进展

减数分裂是真核生物有性生殖过程的关键步骤,染色体的行为变化贯穿整个减数分裂的过程。近些年来,借助先进的分子生物学技术和细胞学实验手段,通过对突变细胞株的筛选和评价,单细胞真核模式生物原生动物嗜热四膜虫(Tetrahymena thermophila)减数分裂方面的研究取得了长足的进展。本文主要介绍嗜热四膜虫减数分裂的过程,以及在此过程中伴随染色体行为变化的相关基因的功能,从而为进一步探讨嗜热四膜虫减数分裂的分子机制提供有效信息。     

The Tetrahymena thermophila phagosome proteome

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.     

Oral assembly in left-handed Tetrahymena thermophila

We have investigated oral development in a non-genetically 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 degrees rotational permutation of the normal, resembling the inverted OAs of mirror-image doublets and LH cells of Glaucoma scintillans described by Suhama. We present a model 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.     

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.     

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.     

Transformation in Tetrahymena thermophila. Development of an inducible phenotype.

Tetrahymena thermophila transforms from a pyriform-shaped trophic form to an elongate rapidly swimming, dispersal form under the appropriate conditions of starvation [Nelsen, E. M., and DeBault, L. E. (1978). J. Protozool.25, 113–119]. The development and control of the dispersal phenotype are examined. After an initial starvation period, the cell replaces its oral structures. During oral replacement, a caudal cilium emerges at the posterior end of the cell. As oral replacement is completed, the cell becomes spindle shaped and the newly-formed oral membranelles are positioned beneath the surface of the cell with somatic ciliary rows exterior to them. The spindle-shaped cell then elongates to become the dispersal form. While the cell is developing the new oral structures, it is also drastically increasing its numbers of somatic basal bodies and cilia. The events in the transformation pathway may be arrested or reversed by feeding the cell, except that once oral replacement has begun, it is completed along with an associated streamlining of the cell. Refed cells revert to the normal vegetative phenotype, except that some shape changes persist for several hours, suggesting that they are compatible with, but independent of, growth. Blockage of protein synthesis with cycloheximide prevents all changes associated with transformation, including the shape changes and elongation of the caudal cilium. The relation between transformation and conjugation has also been examined. Less transformation takes place when mating is possible, but transformed cells may also mate.     

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.