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.     

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.     

Cortical Patterns in Two Syngens of Glaucoma*

SYNOPSIS. Strains of 2 syngens of Glaucoma (16) were found to have different cortical characteristics. All clones of a particular strain had approximately the same range of corticotypes, approximately the same means and high and very similar variances. The chief differences between the syngens was the corticotypic range. The patterns of variation of cortical elements of syngen 2 appeared to be primarily extensions of the patterns of syngen 1. The range of meridian numbers of different species of Glaucoma overlapped with each other and could not be distinguished by this criterion alone.     

Biochemical Comparison of Some Classical Tetrahymena pyriformis and T. vorax Strains*

SYNOPSIS. The classical Tetrahymena pyriformis and T. vorax strains have been classified in antigenic subgroups based on immobilization and agar double diffusion in the presence of specific antisera. The present study utilized disc electrophoresis of proteins and lactate dehydrogenase isozymes for comparison of 11 strains. In agreement with similarities reported for agar diffusion data, close electrophoretic relationships were observed between strains GL and H, and V₂ and W with aniline black staining. Altho LDH isozyme production was more variable and related to cultural growth phase, similarities were recognized between GL and H, and F and PR.     

The Stability of Cortical Phenotypes in Continuously Growing Cultures of Tetrahymena pyriformis*

Cortical features were analyzed in successive samples of continuously growing stock cultures of amicronucleate strains GL-C and GL-I, and in micronucleate strain WH-6 (syngen 1, mating type I). Thirteen successive samples of strain GL-C, representing a time span of 111 months, 5 samples of WH-6 (43 months) and 2 samples of GL-I (1 month) were examined. The observed range of commonly expressed ciliary row numbers (corticotypes) was 16–20 rows in strain GL-C, 15–20 in strain GL-I, and 16–20 in strain WH-6. These ranges remained constant through time within each strain. The individual samples each included all or a large part of the total range observed in the strain, but the relative abundances of different corticotypes within this range shifted through time. The shifts appeared random, with no discernible trends. Mean contractile vacuole pore (CVP) position and number of CVP meridians were assayed in the 2 “GL” strains. Mean CVP position was an apparently stable character, with only slight fluctuations through time, while the distribution of number of CVP meridians was somewhat less constant. The CVP parameters of strains GL-C and GL-I were considerably different, and both of these strains were very different from the GL strain which had been studied by Nanney. In fact, these 3 “GL” strains have, among them, virtually the entire gamut of known CVP characteristics. The possible significance of these wide differences among strains presumed to be closely related is considered in the Discussion.     

Antigenic Differences Among Some Classical Tetrahymena pyriformis and T. vorax Strains*

SYNOPSIS. Suspensions of whole, killed ciliates were diffused against rabbit antisera for the respective strains to observe cross precipitation. The strains fell into the following groups: I. GL, H, ChS, GP, Aq, L-I, L-II, L-2, and V₁; II. W, L-3, Gl-R, and V₂; III. PR and F; and IV. BF and Lava. T. vorax strains V₁ and V₂ each resembled certain T. pyriformis strains more closely than they resembled each other. The same grouping of strains emerged in comparing antigenic suspensions and in comparing antisera and was confirmed by comparing absorption properties of antigens from the different strains.     

Timing and Regulation of Nuclear and Cortical Events in the Cell Cycle of Tetrahymena pyriformis*

SYNOPSIS. Using continuous flow cultures based on the chemostat principle, we varied the cell generation times of the ciliate Tetrahymena pyriformis strain GL, from 4.9 to 22.2 hr and studied various parameters of the cell cycle at 28 C. These included: the duration of the periods required for oral morphogenesis, macronuclear division, cell division, G₁ S, and G₂. The size of individual cells was also measured. Independent of the growth rate, the period of oral morphogenesis occurred during the last 90 min of the cell cycle. In all cases macronuclear and cell divisions took place during the last part of these 90 min, and the final macronuclear separation occurred just before final cell separation. The S-period increased slightly, while the G₁ and G₂ both increased in roughly the same relative proportion to the increasing generation times. Slowly growing cells (generation time 20.5 hr) were shorter but broader and somewhat larger in volume than quickly growing cells (generation time 4.9 hr).     

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.     

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.     

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.     

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.     

Ionic Resistance in Strains of the Tetrahymena pyriformis Complex1

ABSTRACT. Strains of Tetrahymena thermophila were examined in an attempt to establish what role certain ions (Na⁺, K⁺, Li⁺, Ba⁺⁺, Ca⁺⁺, Mg⁺⁺, Mn⁺⁺, Al⁺⁺⁺, Fe⁺⁺⁺) play in influencing cell survival time in a culture medium. In short-term experiments (20–30 min), cell survival time in a 1% peptone medium is directly related to the valence of the ion employed. Long-term observations (lasting up to five days) in a 1% peptone medium containing lower ion concentrations revealed that the effects on cell-cycle time are not correlated with the valence state of the ion. Comparisons were made among the ionic resistances of strains of T. thermophila, of T. pyriformis sensu stricto, and of two subspecies of T. pigmentosa. Strains within a species are highly correlated in their patterns of ionic response, while marked differences between species occur. The most distinctive group of strains examined came from one of the subspecies (syngen 6) of T. pigmentosa.     

Patterns of Cortical Stability in Tetrahymena*

SYNOPSIS. A simplified technic is presented for determining clonal stability patterns for numbers of ciliary rows in Tetrahymena. The technic involves plotting the variance of meridian number against the mean number of meridians. By this procedure strains of different syngens, difficult to discriminate on other grounds, may sometimes be shown to be distinctive.     

Relation between Multiplication Rate and Temperature in Tetrahymena pyriformis, Strains HS and GL

SUMMARY. The multiplication rate of Tetrahymena pyriformis HS in proteose peptone medium was measured at 12 temperatures between 18.4°C. and 36.6°C. At the temperature optimum, 32.5°C., the generation time is 2.25 hours. The upper lethal temperature lies between 36.6°C. and 38.0°C. Similarly, a study of Tetrahymena pyriformis GL revealed a temperature optimum for multiplication of 29°C. with a generation time of 3.70 hours. The upper lethal temperature falls between 34.6°C. and 35.4°C. At all temperatures employed the HS strain of organisms multiplies more rapidly than strain GL. Under identical conditions, the two strains have distinctly different growth optima, upper lethal temperatures and growth rates.
As measured by multiplication rate the readjustment to a sudden change in temperature (from 18.4°C. to 27.7°C.) is completed very rapidly, with an effective lag time of about 1 hour. Such a shift in temperature gives rise to a small degree of division synchrony during the first and second population doublings which follow. Subsequently, all traces of division synchrony are lost.     

Survival of Tetrahymena pyriformis and Paramecium aurelia Following Freezing*

SYNOPSIS. Tetrahymena pyriformis strains E, A-136 31C and IMT II survived freezing in 10% dimethylsulfoxide when the temperature was lowered to freezing at 4.5 C/min. Survival was then obtained for at least 128 days by lowering the temperature rapidly to 95°C. Of the 3 strains, T. pyriformis IMT II was most resistant to the effects of freezing. Its volume averaged about half that of either of the other strains and may have contributed to the differences in survival. In addition to differences among strains, a medium relatively low in the concentration of nutrients, a culture nearing peak population, and a rate of cooling of 4.5 C/min, all gave best survival. Paramecium aurelia regained motility after being frozen in 6 to 7.5% dimethylsulfoxide for as long as 7 days at either –27 or –196 C, but cultures were obtained only after storage for 20 min at –27 C. A concentration of 6 to 7.5% dimethyl-sulfoxide, cooling at 4.5 C/min, and culture media containing Aerobacter aerogenes or composed of a commercially available composition were all required for survival of P. aurelia.     

Characteristics of the interaction of legionellae and Tetrahymena pyriformis

L. pneumophila avirulent strains have been shown to lose their capacity for multiplication in T. pyriformis, while the concentration of the virulent strain increases 1000-fold. The loss of the hemolytic activity of L. pneumophila virulent strain leads to the loss of its capacity for multiplication in infusorians.     

Biochemical and Ultrastructural Changes in Tetrahymena pyriformis During Starvation*

SYNOPSIS. Certain of the ultrastructural and biochemical changes occurring during the first 25 hr of starvation in Tetrahymena pyriformis were studied. Ultrastructurally, numerous profiles of degenerating mitochondria were seen in the early stages of starvation. The presence of oxidizable substrate such as glucose and acetate did not prevent this degeneration. Numerous large nucleoli were formed, many of which seemed to be passing into the cytoplasm as forming autophagic vacuoles. There was a transient increase in Oil Red O-positive bodies, presumably lipid (triglycerides). The extent and duration of this increase were pronounced in the presence of acetate. The lipid droplets appeared to arise within the cisternae of the endoplasmic reticulum. Lipid reserves were apparently utilized prior to carbohydrates, as the disappearance of lipid droplets preceded glycogen utilization, both in the presence of acetate and in the absence of exogenous substrate. A considerable loss of cellular protein also occurred. In cells from inorganic medium supplemented with glucose, glycogen occupied much of the cell, leaving only islands of cell organelles. Acid phosphatase was localized, ultrastructurally, mainly in autophagic vacuoles which contained mitochondria and other cell organelles, and in association with small, double-membraned structures which seemed to be sequestering small areas of cytoplasm. Such sequestered areas also appeared within larger autophagic vacuoles. Residual bodies containing concentric whorls of myelin-like membranes surrounding a more solid core accumulated during starvation. Acid phosphatase activity decreased in amount but not in specific activity. The specific activity of cathespin doubled or tripled, but there was little change in total enzyme.     

The Axenic Culture of Strains of the Various Syngens of Paramecium aurelia*

SYNOPSIS Strains of the various syngens of Paramecium aurelia respond differently to the culture media developed for Strain 299 of syngen 8. Not only is there a variety of response between the syngens, but strains belonging to the same syngen respond differently to the 3 growth media.     

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.