cAMP uncouples DNA synthesis and cell division in Tetrahymena

Tetrahymena cells were synchronized by a differential density labeling method. Millimolar concentrations of db-cAMP will cause a delay in the division of the synchronized cells only if added before late S period. The effective concentrations will also cause a precocious initiation of DNA synthesis during G2 just prior to cell division, suggesting that the cyclic nucleotide causes an uncoupling of the program of DNA synthesis and that of karyo- and cytokinesis.     

Changes in calcium and magnesium levels during heat-shock synchronized cell division in Tetrahymena

Calcium and magnesium contents were measured in cells of Tetrahymena pyriformis induced to divide synchronously by a multi-heat-shock procedure. During free-running synchronized cell division in complex proteose peptone medium, significant peaks of both calcium and magnesium were observed at points in the cell cycle just prior to division. No such peaks were detected in cells dividing asynchronously in proteose peptone. When synchronized cell division was followed after transfer to an inorganic medium, cell calcium and magnesium levels were observed to decrease in relation to the corresponding cell number increase, indicating that in concentration terms, calcium and magnesium remain fairly constant. This latter result suggests that neither calcium nor magnesium influxes act as triggers for cell division in Tetrahymena and that the fluctuations of these metals seen during the synchronized division cycle in complex medium represent an effect rather than a cause.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

A method for the immobilization of living Tetrahymena

The surfaces of plastic (polystyrene) Petri dishes from several suppliers were discovered to have the useful property of immobilizing cells of the ciliate Tetrahymena thermophilia upon contact in nutrient-free buffer (10 mM Tris, pH 7.4). The procedure works with cells in both logarithmic and stationary growth phase, so long as they are first transferred to nutrient-free buffer, and then added to dishes already containing buffer to a depth of 2–10 mm. Dish surfaces specially treated for tissue cultures are unsuitable for this purpose. Cells can be released from the dish surfaces by the simple addition of growth medium (1% proteose peptone). Immobilized cells are fully competent to complete conjugation or cell division. The technique offers promise for facilitating experiments requiring microinjection, microsurgery, or simply detailed observation of living protozoan cells.     

Identification and characterization of Tetrahymena myosin

Myosin was partially purified from ciliated protozoan Tetrahymena pyriformis. Tetrahymena myosin has a fibrous tail with two globular heads at one end and contains 220-kDa heavy chains. The tail length of the molecule (200 nm) is longer than that of myosins from other animals (approximately 160 nm). A sample after HPLC column chromatography containing 220-kDa peptide showed a myosin-specific K⁺-/NH₄⁺-EDTA-ATPase activity. Polyclonal anti-crayfish myosin heavy chain antibody reacted with Tetrahymena 220-kDa myosin heavy chain, and monoclonal anti-pan myosin antibody reacted with Tetrahymena 180-kDa peptide. The isolated 180-kDa peptide was identified as a clathrin heavy chain.     

Division and growth kinetics of the division mutant Conical of Tetrahymena : A contribution to regulation of generation time

The recessive mutant conical of Tetrahymena thermophila is characterized by unequal cytoplasmic division resulting in a large anterior (proter) and a small posterior (opisthe) daughter cell with similar macronuclear DNA contents. Opisthes have long and proters short generation times. This gives the opisthes more time to accumulate cell mass, thereby reducing differences in size between sister cells. Growth rates as determined by cytophotometry do not contribute to the reduction and eventual elimination of differences between sisters but rather should increase them, since small cells accumulate mass at a slower rate than large ones. By tracing consecutive generations it is shown that differences between sister cells in generation time as well as in cell size require more than one generation to be regressed to the mean of the whole population. These findings are incompatible with the probabilistic mode of regulation of generation time.     

Tetrin polypeptides are colocalized in the cortex of Tetrahymena

There is a complex system of 2- to 5-nm filaments in the oral apparatus of Tetrahymena. Four major subunit proteins, called tetrins, have been isolated from the filaments. These proteins, showing apparent molecular weights in polyacrylamide gels of 79-89 kDa, will assemble in vitro into 2- to 5-nm filaments. Tetrin filaments in vivo show different packing arrangements in different regions of the oral apparatus. We sought to determine the distributions of tetrin polypeptides within the complex oral structure by obtaining monoclonal antibodies specific for individual tetrins, then mapping their distributions within the oral apparatus using standard fluorescence microscopy, confocal laser scanning fluorescence microscopy, and electron microscopy. The results indicate that the four tetrin polypeptides are colocalized everywhere within the oral apparatus of Tetrahymena. Tetrin-binding proteins or specific nucleating structures may need to be invoked to explain the complex organization of the tetrin network. The 16 monoclonal antibodies obtained were also used to search for evidence of immunological relationships between tetrin and cytoskeletal proteins in multicellular organisms. None was found.     

Phasing effects of light on cell division in exponentially increasing cultures of Tetrahymena grown at low temperatures

Cultures of Tetrahymena pyriformis (W) grown at 10 ° ± 0.05 °C on proteose peptone-liver extract either in darkness or in relatively low light intensities exhibit marked differences in generation times during the exponential growth phase: about 20 h and 30 h, respectively. If a diurnal LD: 12, 12 or LD: 6, 18 light cycle was imposed on cultures that had been growing in the dark, cell division was phased so that the division bursts (each resulting in an approximate doubling of cell number) occurred once every 24 h and were confined primarily to the dark periods. Microscopic determination of the division index demonstrated that divisions virtually ceased during at least a portion of the light periods; indeed, a decrease in the index often anticipated the actual onset of light. Long trains of 24 h oscillations in apparent cell number could also be obtained in semicontinuous culture in LD: 6, 18. Furthermore, these entrained rhythmic division bursts persisted for at least 6 days with a circadian period if the culture was placed in constant darkness. These results are consistent with the hypothesis that an endogenous, self-sustaining circadian clock mechanism may underlie the observed persisting division rhythmicity as it does in many other microorganisms.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

Hydroxyurea: Morphological effects on the macronucleus of Tetrahymena

Hydroxyurea (10 mM) blocks the exponential growth of populations of Tetrahymena pyriformis (GL-I) by arresting progress through the cell cycle once the cells enter S phase. Fluoromicroscopic examination of euchrysine-stained exponential growth phase cells exposed to HU for a minimum of 90 min show a radical morphological change in the macronucleus. This phenomenon, termed the ‘halo-effect’, is characterized by the formation of apparently membrane-associated aggregates surrounding a constricted chromatin mass. Electron microscopic examination reveals a condensation of chromatin granules away from the surrounding network of membrane-associated nucleoli. Halo-induction by HU is S phase specific. Upon removal of the HU block, the halo remains until the first recovery division. The physiological significance of the halo effect is discussed.     

Heat-sensitive development of the phagocytotic organelle in a Tetrahymena mutant

A laboratory-induced mutant with heat-sensitive development of the phagocytotic organelle has been isolated in Tetrahymena pyriformis, syngen 1; the mutant cells form food vacuoles at 30 °C, but not after incubation at 37 °C. Mutant cells transferred to 37 °C undergo a maximum of 3–5 doublings, but a sizeable fraction remains viable for several days. Results of temperature shift-up experiments reveal that an oral apparatus (OA) constructed at 30 °C remains functional at 37 °C, while one constructed at 37 °C is non-functional with regard to phagocytosis. Preliminary cytological observations reveal severe structural abnormalities of the OA. Thus the mutant appears to be primarily affected in the morphogenesis of the OA. The phenotypic effect of the mutation is reversible by a temperature shiftdown. Changes in phenotype caused by temperature shifts in either direction can occur even in stationary or starved cultures. Cell division is not required for the resumption of phagocytosis after a temperature shiftdown. Null-formers obtained at the first doubling after a temperature shift-up can divide at least once more, indicating that a functional OA is not required for cell division at any stage of the cell cycle. Mutants defective in phagocytosis may prove useful in gaining deeper understanding of this mechanism and its relationship to other cellular processes.     

Deoxyribonucleoside triphosphate and DNA synthesis in synchronized cultures of Chlamydomonas

Pool sizes of dATP, dTTP, dGTP and dCTP were determined during the life cycle of Chlamydomonas using light-dark synchronized cultures. The pools of all four nucleotides were small until the start of the DNA synthesis, when they all increased in close time relationship with the increase in rate of DNA synthesis. The dTTP and dATP pools increased more than 200-fold while the pools of dCTP and dGTP expanded approx. 10 times.     

Parallel evolution of histophagy in ciliates of the genus Tetrahymena

Background  

Species of Tetrahymena were grouped into three complexes based on morphological and life history traits: the pyriformis complex of microstomatous forms; the patula complex of microstome-macrostome transformers; and the rostrata complex of facultative and obligate histophages. We tested whether these three complexes are paraphyletic using the complete sequence of the small subunit rDNA (SSrDNA).