A thermal denaturation study of macronuclear chromatin in Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida)

The macronuclear chromatin of the ciliate Blepharisma japonicum, in two starvation states, was studied by thermal denaturation analysis. The behaviour of B. japonicum chromatin, native and reconstituted with Tetrahymena pyriformis H1 histone, was analysed. The data obtained are consistent with the hypothesis that B. japonicum macronuclear chromatin contains a H1-like peptide associated with the linker DNA, although this peptide is reduced in amount and/or chromatin stabilising ability when compared to Tetrahymena macronuclear H1.     

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.     

Extrusion of DNA from the Macronucleus of Tetrahymena pyriformis GL

SYNOPSIS Administration of the thymidine analog 5-bromodeoxyuridine to exponentially growing cultures of Tetrahymena pyriformis GL in chemically defined medium results in inhibition of cell multiplication by at least one generation before DNA synthesis stops. Cell multiplication can be restored in these cultures, if they are transferred to fresh growth medium, but although most of the cells in the culture contain close to a G2-amount of DNA, a full DNA replication round is a prerequisite for renewed cell multiplication. Large extrusion bodies are found at the first division after transfer to fresh growth medium. Autoradiographic analysis has revealed that the DNA in the extrusion body is a representative of the DNA in the macronucleus indicating a random distribution of DNA between daughter nuclei and extrusion body.     

Electrophoretic Characterization of Classical Tetrahymena pyriformis Strains*

The electrophoretic mobility patterns of 8 enzymes have been examined in 43 classical strains of Tetrahymena pyriformis. The strains may be assorted into sets on the basis of a high degree of similarity of their mobility patterns. Strains of similar designation are frequently in different sets, whereas differently labeled strains may be in the same set. It is proposed that new strain designations be made on the basis of phenotypic similarity.     

Purification and characterization of membrane-bound CO-reactive hemoprotein from Tetrahymena pyriformis mitochondria

Abstract A CO-reactive hemoprotein was purified from the mitochondrial membrane fraction of Tetrahymena pyriformis . It showed absorption peaks at 615 and 455 nm in the reduced form and an α peak at 565 nm in the pyridine ferrohemochrome spectrum. Although the spectral properties were apparently similar to those of 'cytochrome a ₆₂₀' which was previously proposed as a mitochondrial terminal oxidase in T. pyriformis , it did not contain any molecules of heme a or copper atoms. Further, it showed neither cytochrome c oxidase nor cytochrome c peroxidase activity. These results suggest that 'cytochrome a ₆₂₀' may not be the terminal oxidase in the mitochondrial respiratory chain of T. pyriformis .     

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.     

Evidence for Chromosomal Macronuclear Substructures in Tetrahymena*†

SYNOPSIS.
Under the growth conditions employed, the G₁ macronucleus of Tetrahymena pyriformis HSM contains 7.4 × 10^-12 g DNA, the G₂ micronucleus 0.42 × 10^-12 g. DNA content from the Tetrahymena thermophila macronucleus did not significantly differ from that of HSM, but the micronucleus contained about twice as much DNA as the micronucleus of the HSM cells. The T. thermophila macronucleus contained on average enough DNA for ˜ 35 haploid micronuclear copies. A new spreading technic allowed separation of macronuclear substructures from cells of late G₂ to early G₁. Photometric determination of DNA content of 345 individual structures suggested the existence of 5 different-sized macronuclear structures with a DNA content corresponding to 2, 4, 8, and 16 × the basic values. Comparison of the DNA content of these structures with (a) mitotic micronuclear chromosomes and (b) meiotic micronuclear chromosomes of T. thermophila cells suggests that the 5 basic values of macronuclear structures derive from structures of micronuclear chromosomes. The micronuclear chromosomes of T. pyriformis may be oligotenic. It is suggested that these results further our understanding of macronuclear organization.     

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.     

RNA Synthesis in Division Synchronized Tetrahymena: Macronuclear and Cytoplasmic RNA*

SYNOPSIS. Synthesis of RNA in the macronucleus and appearance of RNA in the cytoplasm were studied in heat synchronized Tetrahymena pyriformis GL and compared to those found under conditions of logarithmic growth (28 C) and during heat shocks (34 C). In macronuclei of logarithmically growing cells precursors were processed to 2 rRNA species (25S and 17S). In addition, another RNA (15S), more homogeneous than the RNA (8-15S) in the cytoplasm, was observed in the macronucleus. Both 17S and 25S rRNA species were found in the cytoplasm, 17S rRNA appearing more rapidly than 25S rRNA. Synthesis of rRNA was suppressed at 34 C in cells subjected to heat synchronization; 8-15S RNA synthesis appeared to be inhibited to a lesser extent. During the time preceding the first synchronized division, the synthesis of rRNAs in the macronucleus slowly recovered. Early in the cycle, almost no newly synthesized rRNAs were extracted. By 30 min after the last heat shock (EH), most of the RNA synthesized was not identified as rRNA. By 60 min after EH, the pattern of RNA synthesis had not returned to that observed in logarithmically growing cells.     

Relationship of Cellular Energetics to RNA Metabolism in Tetrahymena pyriformis W.*

Cultures of Tetrahymena pyriformis in a non-nutrient buffer degrade RNA and excrete hypoxanthine, uracil and orthophosphate. Glucose addition leads to the retention of a portion of the purine, pyrimidine, and orthophosphate by the cells; however, the hexose has little influence on the RNA level. Acetate supplementation has no effect on RNA degradation or on the distribution of the catabolic products between the cells and the environment. Interruption of oxidative phosphorylation by 2,4-dinitrophenol results in an increase in RNA degradation. This action is annulled by the glycolytic substrate, glucose, but not by acetate. A combination of iodoacetic acid and glucose blocks glycolysis and increases cellular RNA loss which can be reversed by the addition of the citric acid cycle substrate, acetate. These findings suggest that the available cellular energy supply in starved cells is sufficient to regulate the rate of RNA degradation. Disruption of ATP generation by the appropriate inhibitors, however, allows the demonstration of the importance of energy-yielding reactions in the determination of the amount of nucleic acid loss. It appears that glycolysis and oxidative phosphorylation are equally efficient in sustaining the regulatory process. RNA synthesis during starvation conditions is a discontinuous process with a sharp rate change after 30 min of incubation. 2,4-Dinitrophenol inhibits [2-¹⁴C] uracil incorporation into the nucleic acid. Glucose does not annul the inhibition of synthesis in contrast to the influence of the hexose on RNA degradation. This observation demonstrates that the synthetic and degradative processes are not directly coupled. Glycogen synthesis and RNA degradation appear to compete for the available energy supply and respond in a similar fashion to the metabolic inhibitors and carbon sources.     

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.     

On Food Vacuoles in Tetrahymena pyriformis GL

SYNOPSIS. The following problems concerning food vacuoles were studied by in vivo observations of Tetrahymena: (A) Formation of food vacuoles . The process may be divided into 4 stages. Stage 1—gradual growth of the limiting membrane of the open food vacuole (of short duration). Stage 2—"filling up" of the fully expanded vacuole (of long duration). Stage 3—"closing off" of the vacuole (of brief duration). Stage 4—initial movement of the detached vacuole away from the cy-tostome. The possible role of the oral components (apart from membranellar beating) in the process is discussed. (B) Change of pH in the food vacuole . After ingestion of heat-killed yeast stained with indicator dyes (neutral red, bromcresol purple, bromcresol green, bromphenol blue), the observed color changes indicate that pH is neutral in the forming vacuole as well as in newly formed vacuoles; that a pH value of 6.0–5.5 is reached after ∼ 5 min; and that the lowest pH value between 4.0 and 3.5 is reached after 1 hr. Before egestion the pH again increases. (C) Length of the digestive cycle . A determination of the time required to deplete the cells of labeled vacuoles formed during a short exposure, was attempted. Defecation was observed after 1/2 hr and it was frequent after 2 hr. About 25% and 50% of the labeled vacuoles were removed after 1 hr and 2 hr, respectively; however, labeled vacuoles may still be seen in some cells 6 hr after ingestion. The conclusion is that the digestive cycle lasts ∼ 2 hr and that egestion of undigestible material is a random process.     

Automated image analysis to improve bead ingestion toxicity test counts in the protozoan Tetrahymena pyriformis

AIMS: To improve bead ingestion counts in Tetrahymena pyriformis by automated image analysis as an alternative to direct-counts. METHODS AND RESULTS: Fluorescent latex beads were added to T. pyriformis cultures for ingestion tests. The number of beads ingested by 25 cells was counted directly by epifluorescence microscopy and compared with similar data from image analysis. anova indicated that counts were not significantly different (P < 0.05). The image analysis particularly provided advantages in terms of speed. CONCLUSIONS: The image analysis is superior to direct beads counting in T. pyriformis particularly in terms of speed of analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The image analysis method is very rapid and will allow many more toxicological analyses to be undertaken with less operator error.     

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.     

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.     

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.     

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.     

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.     

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.