A cycloheximide-resistant mutant of Tetrahymena pyriformis

A mutant of Tetrahymena pyriformis, syngen 1, resistant to cycloheximide was obtained after mutagenesis (with N-methyl-N′-nitro-N-nitrosoguanidine) followed by a cross (to obtain macro-nuclear expression of the mutant phenotype). A genetic analysis has shown that cycloheximide resistance in the mutant strain is due to a dominant nuclear allele, designated chx-1. Heterozygotes (chx-1/chx⁺) are initially resistant but segregate stable, sensitive cell lines during vegetative growth, demonstrating that allelic exclusion occurs with this determinant, as with many others in syngen 1. This feature, coupled with the selective advantage conferred by the chx-1 allele in the presence of cycloheximide, makes this mutation a useful genetic tool. A strain homozygous for the chx-1 allele exhibits an exponential growth rate identical to that of the wild type in proteose peptone-yeast extract medium in the absence of cycloheximide. In 10 μg/ml of the drug, the resistant cells grow at a somewhat lower rate, after an initial lag and adaptation to the presence of the drug. This concentration causes complete inhibition of growth and eventual lysis of wild-type cells. The cellular basis for cycloheximide resistance and adaptation in the mutant is presently under investigation.     

DNA replication in macronuclei of Tetrahymena pyriformis GL

DNA replication in macronuclei of Tetrahymena pyriformis GL has been studied to discriminate between hypotheses developed for the interpretation of intraclonal differentiation in ciliated protozoa (the diploid subnuclear, and the ‘master’-‘slave’ hypotheses). Tetrahymena cells were grown in a heavy ¹⁵N-³H medium and then transferred to a light ¹⁴N-¹⁴C medium. DNA was isolated after various periods following this transfer and studied in equilibrium CsCl density gradient centrifugation. Time-related changes in the DNA buoyant density pattern were investigated. The data obtained are interpreted to mean that all DNA in macronuclei of asynchronously growing Tetrahymena at exponential phase replicates semiconservatively once in a cell cycle. These data are in good agreement with the findings of Andersen & Zeuthen obtained on synchronous Tetrahymena cultures in the presence of BUdR.These results are not consistent with the ‘master’-‘slave’ hypothesis. The diploid subnuclear hypothesis is not in accord with other experimental evidence. An alternative hypothesis has been proposed concerning the nature of the macronuclear units and the process of determination. The two main points of this hypothesis are: (a) macronuclear units are diploid genome fragments (‘nucleosomes’); (b) determination is a process of haploidization by ‘allelic splitting’ at a definite macronuclear fission. Consistency with experimental data is discussed and some predictions of the hypothesis are given.     

Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis

A number of lysosomal enzymes are secreted from Tetrahymena pyriformis during growth and during starvation. The secretion is energy-dependent and kinetically different among hydrolases. On the basis of the secretion kinetics under starvation conditions, Tetrahymena hydrolases can be separated into three classes. The first group containing acid phosphatase, beta-glucosidase and alpha-galactosidase, are secreted slowly. Within this group about 4% of the initial cellular activity is released per hour. The second group of enzymes, including alpha-glucosidase, alpha-mannosidase and beta-galactosidase, exhibit moderate secretion (11-15% of the initial cellular activity per hour). The third group, N-acetyl-beta-hexosaminidase, has the highest rate of secretion (22% of the initial cellular activity per hour). N-Acetyl-beta-hexosaminidase shows a continuous increase in overall activity during starvation, which is completely blocked by adding cycloheximide; its secretion is also suppressed. Such involvement of enzyme biosynthesis was not seen in the first and second groups. Furthermore, treatment with weak bases caused inhibited secretion of differing degree among acid phosphatase (group I), alpha-glucosidase (group II) and N-acetyl-beta-hexosaminidase (group III).     

A physicochemical analysis of conjugation in Tetrahymena pyriformis

The process of conjugation in Tetrahymena pyriformis is a useful model system for investigating mechanisms of cellular recognition, adhesion and fusion. As a first step in the biochemical analysis of this process, we have examined the effects of (a) nutrients; (b) metal ions; (c) several pharmacological agents (actinomycin D, cycloheximide, colchicine, theophylline, dithiothreitol and caffeine); and (d) temperature. We find that:

1. 1. While the complete nutrient medium inhibits conjugation, no single compound or group of compounds of the defined medium [1]produces any inhibition.

2. 2. At least trace amounts of Ca²⁺ are required.

3. 3. All of the pharmacological agents tested, except actinomycin D, inhibit both the preparations for conjugation and pair formation itself, indicating a requirement for both protein synthesis and low intracellular levels of cAMP, as well as the involvement of microtubules.

4. 4. While actinomycin D inhibits the preparations for conjugation, its addition after cells have begun to pair does not block further pairing. This result suggests that a stable RNA which is required for conjugation is produced during the preparations for conjugation.

5. 5. Paired cells may be disrupted for the first i h after pairing by proteose peptone, cycloheximide, theophylline, and dithiothreitol. The cells undergo a transition h after pairing which renders them resistant to these agents.

6. 6. The period of initiation (the time of starvation required to make cells competent to conjugate, the period of costimulation (the lag time preceding cell pairing after competent cells are mixed), and the rate of cell pairing are all temperature sensitive. Large changes in these parameters occur over narrow temperature ranges, possibly as a result of temperature-induced changes in membrane lipid composition or structure.

     

Thymidine phosphorylation in synchronous cultures of Tetrahymena pyriformis GL

Recently it has been established that thymidine can be phosphorylated in two ways in Tetrahymena pyriformis

1. 1. by action of thymidine kinase

2. 2. by action of nucleoside phosphotransferase.

The present report confirms that thymidine kinase is a peak enzyme during S phase. It is suggested that a different thymidine concentration in the thymidine kinase assay might explain why previous workers have been unable to find thymidine kinase in Tetrahymena.     

Oral filament proteins and their regulation in Tetrahymena pyriformis

Two proteins from the Triton X-100-insoluble fraction of Tetrahymena pyriformis have been isolated and shown by immunological methods to be major components of a pervasive system of filaments localized within the oral apparatus. These proteins, OF-1 and OF-2, have apparent molecular weights (MWapp) in polyacrylamide gels of 87,000 and 80,000 D, respectively. Peptide maps obtained and the absence of immunological cross-reactivity suggest that these proteins are not closely related to each other. Indirect immunofluorescence studies on dividing cells have shown that the oral filament system forms late in the cell cycle. The filaments appeared first after the basal bodies in the oral primordium had organized into groups and the fission furrow had begun to form. Dedifferentiation of the oral filament system in the anterior (old) oral apparatus was also observed at this point in the cell cycle. Following this, the oral filament systems in both old and new oral apparatuses completed development synchronously. Proteins showing antigenic similarity to OF-1 were found in a number of other cell types. Tests with heterologous antisera failed to demonstrate a relationship between vertebrate cytoskeletal proteins and the oral filament proteins of Tetrahymena.     

梨形四膜虫衰老过程中DNA含量的变化

应用一种新型的细胞核内DNA含量测定方法──图像分析法,测定真核细胞梨形四膜虫衰老过程中DNA含量的变化.根据Beer-Lambert定律,以细胞核在不同生长期内的积分光密度的水平表示核内DNA含量的变化.该方法具有测量速度快,重复性好,操作简单,结果可靠等优点.实验结果表明:四膜虫在进入对数生长期时,DNA含量逐渐达到高峰,随着细胞逐渐老化,细胞分裂次数及核内DNA含量逐步减少.     

Evidence for existence of messenger ribonucleoprotein complexes in Tetrahymena pyriformis

When polysomes from Tetrahymena pyriformis pulse-labeled with ³²P-orthophosphate are dissociated and analysed on sucrose gradients, a large amount of labeled material is found in the 4–23 S region of the gradients.From labeling experiments a half-life of decay of 10.5 min is estimated for the 4–23S material. When cells are pulse-labeled with amino acids, no protein incorporation is found in the 4–23 S material but most of the material is retained on Millipore filters. The sedimentation values of the 4–23 S material are decreased after SDS-treatment. When polysomes from pulse-labeled cells are dissociated and analysed on CsCl-gradients, some rapidly labeled RNP-particles are observed with buoyant densities ranging from 1.51-1.47 g/cm³.     

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N₃CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membranes of Paracoccus denitrificans and Tetrahymena pyriformis. The N₃CCP uncouples respiration in P. denitrificans and T. pyriformis cells with $\text{U}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 1.05 μM and 0.24 μM, respectively. Binding studies show the presence of 0.65 ± 0.05 high affinity sites per cytochrome a with a K_d of 0.5 ± 0.1 μM in P. denitrificans membranes and 1.4 ± 0.2 sites per cytochrome a₂ with a K_d of 0.4 ± 0.1 μM in T. pyriformis membranes. Irradiation of [³H]-N₃CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10–15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647–656).     

A contractile ring and cortical changes found in the dividing Tetrahymena pyriformis

A contractile ring consisting mainly of microfilaments was found in the fission zone of dividing Tetrahymena pyriformis. Diameters of the microfilaments were widely distributed from 2.5 to 15 nm. Ring-associated structures such as lateral stripes, linkers and beads with siender tails were recognized. Lateral stripes arranged at regular intervals of about 84 nm on some parts of microfilament bundles were found in both tangential and transverse sections, suggesting that they correspond to bands fastening the contractile ring microfilaments. Linkers that connect individual lateral stripes to the epiplasmic layer were present. Beads or beads with slender tails were found to be arranged on some microfilaments.The results of the present paper also indicate that drastic morphological changes occur in the cortex of the fission zone, especially in the epiplasmic layer, accompanying contraction of the division furrow. The epiplasmic layer which was proved to be a compact filamentous network in this study has been known to exist at the periphery of cytoplasm in immediate contact with one of the cell surface membranes, the inner alveolar membrane; however, in the fission zone of the dividing ceil, it was frequently separated from the membrane and subsided into the cytoplasm. The subsided epiplasmic layer was then loosened and dispersed. The subsidence of the epipiasmic layer appears to be caused by the force generated by the contraction of the contractile ring and transmitted with the linkers to the epiplasmic layer. The changes observed in the epiplasmic layer are presumably indispensable for the rigid cortical layer contraction involved in cytokinesis of Tetrahymena.     

Timing of tRNA and 5S rRNA gene replication in Tetrahymena pyriformis

The timing for replication of the genes coding for tRNA and 5S rRNA has been studied in Tetrahymena pyriformis. The cells were synchronized by two different procedures, known to synchronize not only cell divisions but also the macronuclear DNA replication, namely (1) the heat-shock procedure described by Zeuthen [12] and (2) the starvation/refeeding procedure described by Cameron & Jeter [13]. The DNA replication was followed by addition of 5-bromodeoxyuridine (BUdR) prior to a synchronous DNA replication. DNA was isolated at various times during replication, analysed by CsCl gradient centrifugation and hybridization with tRNA and 5S rRNA. The results show that the replication of the genes for tRNA and 5S rRNA follows the replication of the bulk macronuclear DNA.     

Conventional and confocal microscopic studies of insulin receptor induction in Tetrahymena pyriformis

Tetrahymena pyriformis reportedly possesses binding structures for the vertebrate hormone insulin that are amplified in cells having prior exposure to the hormone. Conventional and confocal microscopic studies were conducted to verify the validity of the reports and to localize the binding sites. Logarithmic cultures were exposed to insulin concentrations of 0, 3, 6, and 12 μg/ ml for 1 h (receptor induced, RI). After an additional culture period the cells were fixed, exposed to porcine insulin (antigen), immunocytochemically processed, and examined for staining intensity by video image analysis. Observations indicate that T. pyriformis does bind insulin whether or not the cells have prior exposure to insulin. Staining intensity increased at the two highest RI concentrations over 0 μg/ml (P < 0.01) but the staining intensity at 0 μg/ml was not different from that at 3 μg/ml. The results confirm that T. pyriformis does bind insulin and that prior exposure to insulin increases the binding capacity for insulin in what may be a concentration-dependent manner. Confocal microscopy of RI cells that had been labeled with either fluorescein isothiocyanate-insulin or the immunocytochemical technique outlined above revealed labeling of the cytoplasm that appeared to be vesicular. Both techniques produced very similar labeling patterns when optical sections through the cells were viewed. Conventional fluorescence revealed ciliary labeling that could be decreased by incubation with excess unlabeled insulin. Further studies with the exo⁻ mutant of T. thermophila, SB 255, showed that mucocyst discharge and capsule formation are not involved in insulin binding.     

Fluorometric estimation of surface potential change associated with chemotactic stimulation in Tetrahymena pyriformis

For the quantitative estimation of surface potential change in intact cells a method was devised with the use of fluorescent probes, 8-anilino-1-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPN). Estimated values in liposomes were compared with changes in the zeta potential determined from electrophoresis. Both values agreed within the experimental variation, showing the usefulness of the method. The method was also applied to Tetrahymena pyriformis, which exhibits chemotaxis to various chemical stimuli. The surface potential change was observed when the cell was stimulated not only by inorganic salts but also by electrically neutral, hydrophobic compounds. The surface potential started to change in accordance with the depolarization of the membrane potential, except for the case of K⁺. Changes in the surface potential of T. pyriformis in response to Ca²⁺ and K⁺ were compared with those in the membrane potential. The quantitative contribution of the surface potential to cell depolarization associated with chemoreception is discussed.     

Dynamic aspects of cytoplasmic poly(A) RNA of Tetrahymena pyriformis

In the present work a study was made of the compartmentalization of the poly(A)+ RNA populations during the cultural development of cells of T. pyriformis that were pre-starved or derived from stationary cultures. It was found that the poly(A)+ RNA content increases when the cells change from stationary to lag phase. The increase in RNA poly(A)+ is manifested exclusively in the polysome compartment. The level of poly(A)+ RNA in the cytoplasmic non-polysomal compartment does not change. The increase in poly(A)+ RNA is concomitant with an expansion of the polysomes. Pre-starved cells initiate polysome formation soon after being transferred to a growing medium. During this time the poly(A)+ RNA content of the non-polysomal compartment decreases and that of polysomes increases in close proportion. Not only in the starved but also in stationary cells and in those that are beginning to grow, the proportion of poly(A)+ RNA in mRNP is higher than in the polysomes. These data are interpreted as indicating that cells of T. pyriformis, derived from stationary cultures are dependent on RNA synthesis for polysome formation; on the other hand, pre-starved cells use preformed non-polysomal poly(A)+ RNA for the same purpose, in the beginning of the cultural development.     

Inhibition of conjugation in Tetrahymena pyriformis by ConA : Localization of ConA-binding sites

Two patterns of ConA binding to starved mating types of Tetrahymena pyriformis were observed depending on its time of addition. When ConA was added upon mixing of the mating types, at zero time of conjugation, it was first bound to the oral region and subsequently was taken into intracellular vacuoles. When it was added to conjugants, it was specifically bound as a ring around the conjugation area. The ability of the cells to form vacuoles, assayed by addition of carmine particles, declined prior to pair formation. The relationship between the above phenomena and the ability of ConA to inhibit conjugation is discussed.     

Nucleoside phosphotransferase activity through the growth and cell cycle of Tetrahymena pyriformis GL-I

Tetrahymena pyriformis GL-I were synchronized by three different techniques and nucleoside phosphotransferase activity measured through the different cell cycles obtained. In cells that were starved and then refed, activity did not increase until 75 min after refeeding. This increase in activity occurred well before nuclear DNA synthesis and was not blocked by hydroxyurea. In cells synchronized by the induction technique of one heat shock per generation and the selection technique of differential density labelling, enzyme activity increased continuously over the cell cycle but did not double. However, during early logarithmic growth nucleoside phosphotransferase activity more than doubled over one cell cycle time while late in log growth phase less than a doubling was observed. Cycloheximide and mixed extract experiments suggest that the patterns of activity observed reflect the patterns of enzyme synthesis. These results are discussed with respect to the pattern of activity observed for thymidine kinase in other organisms.     

Isolation and lipid composition of nuclear membranes from macronuclei of Tetrahymena pyriformis

A procedure was developed for isolation of macronuclei and nuclear membranes from the ciliated protozoan Tetrahymena pyriformis E, and the lipid composition of the isolated nuclear membranes was determined.This method involves cell lysis with octanol, separation of the nuclear membrane with 0.2 M phosphate–1M NaCl and purification on a discontinuous sucrose gradient. By phase-contrast and electron microscopic examinaton, our preparations were pure and preserved the typical nuclear membrane morphology: inner and outer nuclear membranes, and nuclear pore complexes. As for lipid distribution, the three major phospholipids in the membranes were phosphatidylcholine (31.0%), phosphatidylethanolamine (26.1%) and 2-aminoethylphosphonolipids (23.3%) and the molar ratio of a sterol-like lipid, tetrahymanol to phospholipid phosphorus was 0.036. These results were compared to other membrane fractions of Tetrahymena.     

Effects of cytochalasin B on cell division and vacuole formation in Tetrahymena pyriformis GL

Cytochalasin-B (Cyt-B) was tested for its effect on cell division and vacuole formation in Tetrahymena. Little effect was found on cell division in the synchronous cell system in concentrations up to 37 μg/ml; however, slight delay was caused by 71 μg/ml. As measured by particle uptake, much lower concentrations, 7–8 μg/ml, caused significant inhibition of vacuole formation in exponentially multiplying and in starved cells, 16.6 or 37 μg/ml caused strong inhibition. This effect was immediate and completely reversible. The presence of Cyt-B caused starvation of Tetrahymena. The essential absence of inhibition of cell division by Cyt-B may reflect that the drug can enter the cell only by way of vacuoles.     

Inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis

An inhibitory effect of inorganic phosphate on the axonemal ATPase of cilia from Tetrahymena pyriformis was shown. P_i inhibited the terminal phosphate liberation from [γ-³²P]ATP by 30-S dynein and inhibited the conversion of [8-¹⁴C]ATP to ADP and AMP by digitonin-extracted cilia.