Macronuclear Cytology of Synchronized Tetrahymena pyriformis*

SYNOPSIS. Heat shock and stationary-phase conditions both cause fusion of nucleoli. In both cases the process is reversed when the cell is returned to normal physiological growth conditions. Fusion of nucleoli during the cell cycle of logarithmically growing cells was not observed. Likewise, fusion of nucleoli was not observed when the Padilla and Cameron(8) method of synchronization was used. The macronuclei of cells synchronized by the 1 cold-shock per cycle method(8) more closely resembled macronuclei of log-phase cells than did the macronuclei of cells synchronized by the Scherbaum and Zeuthen(12) heat-shock method.     

Toxicity of Antioxidants to Tetrahymena pyriformis*

SYNOPSIS. The cytotoxicity of 97 antioxidants of various structural types was determined for Tetrahymena pyriformis grown in a peptone medium. Cytotoxicity fo Tetrahymena, generally, was positively associated with acute oral tomicoiciity to rats but not with antioxidant pdency as measured by the Tetrahymena photodynamic assay for antioxidants. Some commonly used hindered-phenol, lipidsoluble anttioxidants, e.g., 2,6-di-tert.-butyl-P-cresol (BHT), had low toxicity (to Tetrahymena but poor antioxidant activity by the photodynamic test. Nmdihydroguahetic acid, with low toxicity to the rat and high Coxicity (to Tetrahymena, had very high activity in the photodynamic test.     

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.     

Properties and partial characterization of the heat-shock factor from Tetrahymena pyriformis

A heat-shock-factor-binding activity was identified in Tetrahymena pyriformis whole-cell extracts and was further purified by sequential heparin-agarose and sequence-specific oligonucleotide affinity chromatography. Tetrahymena heat-shock factor (HSF) was able to bind to the heat-shock elements (HSE) both before and after thermal stress, although heat shock altered both the HSE-binding affinity and the protein.DNA-complex mobility on polyacrylamide gels. The mobility difference was significantly reduced by treatment of the proteins with phosphatase. The HSE-binding proteins, isolated by oligonucleotide-affinity chromatography, migrated on SDS/polyacrylamide gels as a closely spaced doublet to about 70 kDa. Polypeptides with similar molecular mass were recovered from preparative band-shift gels indicating that both are components of the protein.DNA complex.     

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.     

An Ultrastructural Study of Ingestion and Digestion in Tetrahymena pyriformis*

SYNOPSIS. When the structures involved in digestive events in T. pyriformis are examined at the electron microscope level, some information is added to that long known from light microscopy. The food trapping mechanism consists of the three membranelles, undulating membrane, oral ribs, and a “valve” apparently closing the opening to the cytopharynx. Both of the latter structures are supported by microtubules. Fibers extend internally from the cytopharynx and are closely associated with the food vacuole as it forms. Clear vacuoles resembling pinocytic vacuoles appear to arise from differentiated areas of the pellicle and plasma membrane. These vacuoles may fuse with primary lysosomes. Hydrolases are thus contributed to the pinocytic vacuoles which may then fuse with food vacuoles. When first formed food vacuoles contain no hydrolases but may acquire them directly, from primary lysosomes or from pinocytic vacuoles. Digestion proceeds to completion in the food vacuole, at which time soluble food products are released to the cytoplasm. Undigested materials are lost through the cytopyge. In stationary growth phase cells autophagic vacuoles form containing mitochondria and other cellular particulates. Such vacuoles probably contain hydrolases when formed and they may receive others by fusion with primary lysosomes.     

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.     

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.     

Amino Acid Activation in Subcellular Fractions of Tetrahymena pyriformis*

SYNOPSIS. The presence of amino acid activating enzymes was demonstrated in the ciliated protozoan Tetrahymena pyriformis. By employing a sensitive hydroxamate assay procedure, the activation of L-valine was assayed in various subcellular fractions of the ciliate, and some characteristics of the enzyme activity in the most active fraction were determined. Most of the activity resided in pH 5 fractions isolated from high speed supernatants of ciliates disrupted by various physical and chemical methods. No activity could be demonstrated in isolated cilia, in pellicles with attached kinetosomes, in microsomes or in macronuclei, providing these organelles were thoroughly washed. A washed mitochondrial preparation isolated by the Mager and Lipmann procedure activated L-valine; mitochondria isolated by the procedure of Hogg and Kornberg did not. The pH 5 fraction isolated from the 102,000 X g supernatant of digitonin-lysed ciliates was stable for several weeks when stored in 0.1 M Tris buffer, pH 7.6 at – 25 C. The activity of this fraction with respect to L-valine activation was dependent on the presence of ATP¹ and magnesium in the reaction mixture. The optimal concentrations of these components and of L-valine and hydroxylamine were determined, and the linearity of activity with time and enzyme concentration was demonstrated. Valine activation was not modified by dialysis of the pH 5 fraction, or treatment with RNase, or the addition of boiled pH 5 fraction.     

Isolation and characterization of dolichols from Tetrahymena pyriformis

Dolichols of Tetrahymena pyriformis were isolated and characterized by TLC, HPLC and mass spectrometry. Four strains of Tetrahymena were studied and found to have relatively small amounts of dolichol, from 0.26 to 2.60 mg dolichol/kg wet weight. All four strains had approximately the same relative proportions of isoprenologs, dolichol-13 (2%), dolichol-14 (74%), dolichol-15 (23%), and dolichol-16 (less than 1%). Tetrahymena dolichols were found mainly in the mitochondrial subcellular fraction (86%). The pellicle fraction contained 9% and the microsomal fraction, 5% of the remaining dolichol. Free dolichol has also been found in the mitochondrial fraction of four other organisms. We were not able to demonstrate dolichyl esters in these organisms, but their presence is inferred, because reduced yields of dolichol were obtained if the lipid extracts were not saponified prior to HPLC assay.     

Isolation of nucleoli from Tetrahymena pyriformis.

A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented.     

Automatic Cell Counting in Continuous Flow Cultures of Tetrahymena pyriformis*

This report describes an electronic cell counter constructed for determining cell number in cultures of the ciliate, Tetrahymena pyriformis. The culture chamber has been equipped with a device which determines the number of cells per unit volume and records the number automatically. As cell multiplication is unaffected by the counting procedure the cells are returned to the culture. Furthermore, keeping the culture volume constant we have arranged a continuous flow of fresh nutrient medium through the culture chamber and thus established conditions under which cell multiplication has continued for months while determinations of cell concentrations have been recorded every 10 min. Since the culture volume has been small, ～25 ml, growth studies utilizing this method require less than one liter of fresh medium per week in spite of the fast multiplication (9 generations per 24 hr) occurring in cultures of Tetrahymena pyriformis under optimal conditions.     

A new fiber-forming protein from Tetrahymena pyriformis

A new fiber-forming protein was isolated from the acetone powder of Tetrahymena pyriformis by co-precipitating with skeletal muscle myosin while trials were made to find actin or actin-like protein in Tetrahymena. It has a molecular weight of 38000 D and forms a tetramer (140000 D, 9 S) in physiological conditions. Its isoelectric point (pH 6.7), amino acid composition and antigenic determinant(s) differ significantly from those of non-muscle actin and skeletal muscle actin. It does not undergo G-F conversion while actin does, and does not activate Mg²⁺-ATPase of skeletal muscle myosin. The protein localizes in the oral apparatus and division furrow as revealed by fluorescent antibody method. The protein can be assembled into 14-nm filaments in a reassembly buffer. The in vitro filaments appear to correspond to some filaments included in the oral apparatus and the contractile ring. The fiber-forming protein from Tetrahymena may play important roles in cell motility including cell division.     

A CO-Binding Hemoprotein Derived from Tetrahymena pyriformis

A CO-binding hemoprotein was purified from Tetrahymena pyriformis and some of its properties were studied.

The hemoprotein possessed protoheme, its molecular weight was about 11,000, and its isoelectric point was at pH 8.2. The oxidized form of the hemoprotein showed the Soret band at 406 nm and had no distinct peaks in the region of α- and β-bands, while the reduced form showed the peaks at 426, 527 and 560 nm. The hemoprotein reacted with CO resulting in shift of the Soret band from 426 to 420 nm. The CO-compound showed a broad band from 537 to 573 nm. The hemoprotein was not autoxidizable or oxygenated either. It did not show either of the cytochrome oxidase, peroxidase and NADH oxidase activities.

It should be carefully determined whether or not cytochrome o is functioning as the terminal oxidase in T. pyriformis, as the CO-binding hemoprotein which does not react with molecular oxygen exists in the organism.