Further Electrophoretic Characterization of Strains of Tetrahymena pyriformis*

Mobility patterns of 5 isozymes in strains of Tetrahymena pyriformis were demonstrated using polyacrylamide disc gel electrophoresis. Six stock strains were compared in these patterns to 4 strains representative of each of the previously described 4 major “phenotypic sets.” Stock strains segregated into predicted “phenosets,” and essentially confirmed validity and reproducibility of such a discrimination method. The proposal that new strain designations be assigned on a basis of “phenoset” conformity is reaffirmed.     

Isozymic Characterization of Three Mating Groups of the Tetrahymena pyriformis complex*

SYNOPSIS. Strains of 3 unnamed mating groups of the Tetrahymena pyriformis complex have been subjected to starch gel electrophoresis followed by staining the gels for the enzymes isocitrate dehydrogenase (NADP), tyrosine aminotransferase, and tetrazolium oxidase (superoxide dismutase). With respect to the electrophoretic mobilities of these enzyme systems, the mating groups referred to here as 5, 13 and 14 are very similar to Tetrahymena americanis (syngen 2), the most common North American species of the complex. Cultures in our collection labeled Tetrahymena cosmopolitans (formerly syngen 4) are either amicronucleate, with unique isozyme patterns, or micronucleate cells which mate with and have isozyme patterns similar to Tetrahymena canadensis (syngen 7). Immature progeny have been derived from crosses between the latter strains and T. canadensis recently collected in Colorado. The amicronucleate strains are now placed in the Tetrahymena sp. category, and we conclude that strains identifiable as T. cosmopolitanis are no longer available. The reliability of isozymes as characters in ciliate taxonomy was evaluated by comparing the present results for 3 enzymes in 15 groups of strains (syngens and phenosets) that had been compared in an earlier study. These enzyme systems gave correlation coefficients (r) of 0.75 or higher in the separate studies, and can be considered useful diagnostic traits. Other enzymes that were present at threshold levels of detectability or varied highly in concentration from species to species are too unreliable to be of diagnostic value. Some of the strains in the complex are so evolutionarily divergent at the molecular level that we have difficulty finding growth and electrophoretic conditions under which orthologous enzyme activities can be detected simultaneously for all the strains being compared.     

Variation in Glyoxylate Bypass Inducibility among Strains of Tetrahymena pyriformis

SYNOPSIS. Seven strains of Tetrahymena pyriformis were assayed for log phase activity of the glyoxylate bypass enzymes isocitrate lyase and malate synthase. In strains 6I, 6II, 6III, and W, isocitrate lyase was induced; in HS, neither enzyme was induced by acetate. During growth in glucose- or acetate-containing media, strains 6III and GL had 2 periods of increased glyoxylate bypass and isocitrate dehydrogenase enzyme activities. Enzyme activities reached a maximum at the end of log phase, declined until the middle of stationary phase, and then increased again to a maximum near the end of stationary phase.     

Isolation and Identification of Specific Cortical Proteins in Tetrahymena pyriformis strain GL*†

SYNOPSIS. Pellicles of the ciliate Tetrahymena pyriformis strain GL (phenoset A) were isolated by a new procedure. Oral apparatuses were also purified by a modification of a previous method. Both preparations were characterized by electron microscopy. Proteins of the isolates were separated by analytical SDS polyacrylamide gel electrophoresis. The isolated pellicles, which included oral apparatuses, contained only 6 major proteins (gel bands), designated A through F. Bands A, B, and C, were found in the pellicle fraction, but not in the oral apparatus fraction. Therefore, these proteins are believed to be present in the somatic cortex of Tetrahymena. Bands D and E were greatly enriched in the oral apparatus fraction; these proteins are therefore believed to be present primarily in the oral apparatus. Band F, identified as tubulin, was present in both preparations. Molecular weight determinations and some selective solubilization experiments are also presented.     

Concanavalin A-Sepharose Chromatography of Hexosaminidase Isozymes Secreted By Tetrahymena*

Tetrahymena pyriformis strain HSM secretes 4 isozymes of hexosaminidase. Purified isozymes B₁ and B₂ are eluted from the void volume of a concanavalin A-Sepharose column, suggesting that they are not glycosylated. Purified isozymes A₁ and A₂ bind to the column and are eluted at ～0.1 M α-methylmannoside, suggesting that these isozymes are glycoproteins. In agreement with earlier deductions based on a differential kinetic assay for the A and B isozymes, the elution pattern of hexosaminidase activity from material secreted by cells grown to early and late stationary phase was consistent with these secretions containing primarily the B and the A isozymes, respectively.     

Sublethal Cytotoxic Effects of Mercuric Chloride on the Ciliate Tetrahymena pyriformis*

SYNOPSIS. The behavior and ultrastructure of Tetrahymena pyriformis was assessed after exposure to dosages of 8 and 16% of the lethal concentration of HgCl² (TLm 96 hr). The lower dosage caused no abnormal changes in cell motility, activity of the water explusion vesicles, or cell shape; the higher dosage caused deleterious changes in these parameters. The higher sublethal HgCl² concentration (0.50 mg/liter) elicited damage of several cell structures. This damage persisted and accumulated with time up to 24 hr. At the lower HgCl² dosage (0.25 mg liter) there were extensive changes after 1-hr exposure involving primarily mitochondria; however, all major changes were repaired after 24 hr of constant exposure to the HgCl², indicating adaptation to the toxicant. Based solely on cytotoxic evidence an attempt is made to apply the findings defining what constitutes a “safe'’concentration of HgCl₂ in the cell's environment.     

Morphometric Studies of Mitochondria in Tetrahymena pyriformis Exposed to Chloramphenicol or Ethidium Bromide*

Cultures of Tetrahymena pyriformis strain ST were exposed to 300 μg chloramphenicol/ml or 15 μg ethidium bromide/ml for 48 hr. Qualitative assessments of electron micrographs reveal that the abundance of mitochondrial cristae decreases greatly. By equating the spatial characteristics of the organism with those of a prolate spheroid, the distribution and abundance of mitochondria were quantified. Such characterizations reveal that the size of individual mitochondria decreases by 35–60% and that the number of mitochondria/cell increases ～8 fold. The observations are discussed in terms of coordinated mitochondrial and nuclear genetic activities.     

Temperature Effects on Maturity Periods in Tetrahymena pyriformis Syngen 1*

Cells of Tetrahymena pyriformis syngen 1 grown at 30 C after conjugation achieve sexual maturity more quickly than do cells grown at 19 C, whether time is measured in numbers of cell divisions or in terms of absolute time. This result is achieved regardless of the temperature at which conjugation and nuclear reorganization occur. These observations differ from those of other workers investigating Paramecium, and suggest that the long term “chronometer” is more tightly coupled to cell division in Paramecium multimicronucleatum and Paramecium caudatum than in Tetrahymena pyriformis.     

Cytotoxicity of Synthetic Fuel Products on Tetrahymena pyriformis. I. Phenol*

SYNOPSIS. Phenol is the major organic constituent of coal-conversion scrub water and is therefore a potential environmental contaminant. After Tetrahymena pyriformis strain GL-C, syngen 1 was exposed to phenol, its behavior, cytology, respiration, and growth rates were examined. Concentrations ≥ 75 mg/liter alter cell motility, shape, and contractile vacuole activity. O₂ uptake was abruptly reduced within 3 min of exposure to phenol in concentrations as low as 10 mg/liter. Concomitantly there was an increase in the electron density of the mitochondrial matrix. Recovery to normal rates of O₂ consumption was paralleled by a return to normal matrix density. Alterations of mucocysts, pellicle, and glycogen were also observed. The length of lag phase growth curves increased generally in proportion to concentration of toxicant. Phenol, however, did not affect the rate of cell multiplication during the exponential growth phase. The potential use of this system to examine the effects of other possible organic pollutants is discussed.     

The Conversion of Phenylalanine to Tyrosine in Tetrahymena pyriformis*

SYNOPSIS. Phenylalanine hydroxylase could not be assayed in extracts of Tetrahymena pyriformis strain W in a system by which the enzyme could be assayed in rat liver extracts. Isotopically labelled phenylalanine, however, was converted to tyrosine by growing or washed cells. Growth conditions which allowed limited synthesis of unconjugated tetrahydropteridine severely reduced the ability of the cells to synthesize tyrosine from phenylalanine. The presence of glucose and acetate in the growth medium resulted in elevated free tyrosine pools and an increased capacity of washed cell suspensions to convert phenylalanine to tyrosine. It would appear that the putative phenylalanine hydroxylation system is not subject to the repressive effects of glucose and acetate which apply to the enzymes of tyrosine catabolism. The significance of this distinction is discussed.     

The Induction of Endocytosis in Starved Tetrahymena pyriformis

SYNOPSIS. The formation of digestive vacuoles by starved Tetrahymena pyriformis could be induced by mixtures of latex particles and a variety of potentially digestible solutes. Latex particles themselves had little effect in inducing vacuole formation. Protein, polypeptide, and RNA were highly effective inducers, while glutamate, amino acid mixtures, polysacharides, and glucose were moderately effective. Sodium-β-glycerophosphate had a slight effect and sodium acetate was ineffective. The possible stimulus to endocytosis is discussed. The endocytic response to inducers does not appear to be an all-or-none phenomenon and varies with the concentration of inducer. The stimulatory effect for protein-related inducers seems to be produced by a large number of stimulatory molecules acting upon a single cell and the magnitude of the response appears to be related to molecular size.     

Monoamine Oxidase and Catechol-O-Methyl Transferase Activity in Tetrahymena*

SYNOPSIS Tetrahymena pyriformis strain HSM was found to have monoamine oxidase (MAO) and a catechol-O-methyl transferase-like (COMT) activity. As in mammalian tissues, the MAO activity is predominantly localized in the mitochondrial pellet and COMT in the cytosol. The COMT-like activity was present in amounts comparable to several mouse tissues and was inhibited by tropolone. MAO activity was much lower than in any of the mouse tissues tested, and its activity varied greatly from preparation to preparation. The substrate preference of Tetrahymena MAO was tryptamine > serotonin > dopamine, and activity increased with increasing pH from pH 6.5 to pH 7.8, as does that of mouse liver MAO. The K_m of Tetrahymena MAO for tryptamine was 4 μM, an order of magnitude lower than that of mouse liver MAO. Sensitivity to inhibition by MAO inhibitors was variable. In some preparations, no inhibition was observed. In others clear inhibition was obtained, harmine and clorgyline being among the most potent inhibitors.     

Partial Purification and Characterization of a Bacteriolytic Enzyme Secreted by Tetrahymena*

Synopsis. Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3–4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded “C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacterio-lytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.     

Mirex and Aroclor® 1254: Effect on and Accumulation by Tetrahymena pyriformis Strain W*

Effects of 2 toxicants, Mirex and Aroclor 1254, on Tetrahymena pyriformis strain W in axenic cultures were investigated. Mirex is a chlorinated hydrocarbon effective against the fire ant, and Aroclor 1254 is a compound structurally related to DDT and used extensively in various industrial processes. Both toxicants reduced growth rates and population densities of T. pyriformis grown at 26 C generally in proportion to concentrations of the chemicals, their effects becoming statistically significant (P < 0.05) at 0.9 μg/liter for Mirex and 1.0 and 10.0 μg/liter for Aroclor 1254. Ciliates exposed to the toxicants for 7 days concentrated Mirex 193 × and Aroclor 60 × as compared to the initial concentrations of these compounds. It is suggested that the chief effect of the 2 toxicants on populations of T. pyriformis and of similarly responding ciliates in nature would be to reduce the availability of these protozoa as food organisms and nutrient regenerators. The ability of the ciliates to concentrate the tested compounds would permit the toxicants to enter into and to be translocated through aquatic food chains. In this manner the compounds could exert toxic effects at higher trophic levels.     

Transformation in Tetrahymena pyriformis: Description of an Inducible Phenotype*†

SYNOPSIS. Transformation of Tetrahymena pyriformis to a rapid-swimming (presumably dispersal) form can be induced by washing cells and suspending them in distilled H₂O, Dryl's solution or 10 mm Tris. Transformation is possible with high efficiency in mass cultures of axenically grown cells within ～ 5 h at 30 C. The radically different phenotype produced during transformation is characterized by a more elongate body form, increased numbers of somatic basal bodies and cilia, a long caudal cilium and oral membranelles positioned beneath the cell surface. DNA quantities characteristic of G1, S, and G2 cells are found in these transformed ciliates, suggesting that achievement of a particular stage in the DNA-division cycle is not a prerequisite for transformation. Preliminary observations on cells belonging to syngens 2–12 indicate that they also have a capacity to form a caudal cilium, but that the amicronucleate strain GL-C does not. Possible relevance of the transformed phenotype for taxonomy of Tetrahymena is discussed.     

The Methylated Purines of Tetrahymena pyriformis Transfer Ribonucleic Acid*

SYNOPSIS. By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N²-DMG. Also present were 1-MG, N²-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N⁶-methyladenines were detected.     

An Ultrastructural Investigation of 180°-Rotated Ciliary Meridians in Tetrahymena pyriformis*

SYNOPSIS. An ultrastructural investigation has been carried out on 180°-rotated ciliary meridians (inverted meridians) in Tetrahymena pyriformis temperature-sensitive mutant (mol^b/mol^b), syngen 1, strain B. The longitudinal, transverse and postciliary microtubular bands, the kinetodesmal fiber, and the parasomal sac, are shown to be disposed at a 180° angle to their normal positions or orientations. Other abnormalities are as folows: the first 2 basal bodies of the inverted meridian fail to organize into “couplets” and the inverted meridian intrudes into the anterior pole region; an extra longitudinal microtubular band is found in one of the cell lines.     

Growth Kinetics of Three Species of Tetrahymena On Solid Agar*

A nutrient-agar method without liquid overlay has been developed for cultivation of ciliates. Three species of Tetrahymena-T. pyriformis strain W, T. rostrata strain UNI, and T. vorax strain V₂S, representing the 3 main groups of Tetrahymena species, were used; however the method should apply to other ciliates. Growth on the surface of the agar was facilitated by an optimal surface-to-volume ratio yielding a high density of ciliates (5.8 × 10₅ cells/cm² for T. pyriformis at 25 C) and short generation times (3 h for T. pyriformis at 30 C). At the highest density achieved, the cells became irregularly hexagonal and formed a monolayer “tissue” on the agar. Ciliates grown on agar were like those in liquid culture, typical oral ciliature, food-vacuole formation, and typical cortical patterns being retained. Advantages of this method include high cell density, easy recovery, and optimal O₂ supply. the organisms can also be cultivated on the surface of sterile cellulose-nitrate filters, facilitating in situ fixation and staining as well as transfer into different media by transfer of filters with cells, without prior centrifugation and resuspension.     

Effects of Dimethyl Sulfoxide on Tetrahymena pyriformis GL. Fine Structural Changes and Their Reversibility*

SYNOPSIS. Fine-structural changes are induced in Tetrahymena by exposure to 7.5% dimethyl sulfoxide (DMSO) in the presence of growth medium. Some of these changes (nucleolar, mitochondrial, peroxisomal) resemble those seen during starvation, in agreement with the previously reported inhibitory effect of DMSO on food-vacuole formation; however, changes such as helical formations of polyribosomes indicate additional internal actions of the reagent. The effects vary to some extent within the same group of cells, suggesting that sensitivity to the reagent may differ with the stage in the cell cycle. The structural changes induced by a 1-hr exposure to DMSO are reversible, but recovery of the cells after removal of the reagent is slower than that seen after starvation. The observations suggest that the recovery is associated with renewed synthesis.     

Studies of Membrane Formation in Tetrahymena pyriformis. VIII. On the Origin of Membranes Surrounding Food Vacuoles*†

SYNOPSIS. A procedure was devised for the isolation of purified food vacuoles from Tetrahymena pyriformis fed particles of ferric oxide. Phospholipids extracted from vacuolar membranes were more similar in composition to the lipids of microsomes than to lipids of whole cells, cilia or post-microsomal supernatant. Fractionation of cells grown in the presence of [¹⁴C]palmitic acid or [³²P]inorganic phosphate also revealed similarities in the specific radioactivities of microsomes and vacuolar membranes. The data suggested that vacuolar membranes arise from a pool of cytoplasmic membranes.