Sizes of G1 and G2 populations in starved Tetrahymena

Feulgen cytophotometry and autoradiography were used to study DNA content and DNA synthesis in starved and starved-refed Tetrahymena pyriformis GL-C. It was found that (1) the cell population shows a limited increase in cell number during starvation and this increase is restricted to the first 7 h of starvation; (2) at the end of starvation, there is a portion of the cell population whose DNA content is similar to that for standard G2 cells; (3) a significant portion of the dividing cells at the first division following refeeding in the presence of [³H]TdR are unlabeled; (4) these unlabeled cells are among the first to divide and, upon division, generally enter into a cell cycle either lacking a G1 phase or with a shortened G1 phase.     

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.     

Methionine is a regulator of starvation-induced proteolysis in Tetrahymena

The ciliate Tetrahymena thermophila responds to starvation by drastically increasing the rate of proteolysis. The response was reversed by resuspending the cells in a defined growth medium. Among the components of this medium only amino acids were active in inhibiting proteolysis. One amino acid, methionine, accounted for at least 75% of the effect of the complete medium, strongly indicating that in Tetrahymena methionine is the main regulator of step-down proteolysis, a process generally connected with autophagy in eukaryotic cells. The fact that one amino acid has such a drastic effect should make the system well suited for further investigations of the regulation of this process.     

Reactivation of stationary Tetrahymena : A contribution to the question of G0 state

Stationary cells of Tetrahymena were reactivated to exponential growth phase by transfer to fresh medium. The sequence of resuming cell cycle events was analysed by scoring the division index, the labelling index for macro- and micronuclei and the increase in cell number. By long-term labelling it was found that all cells replicate in stationary phase cultures. They also divide eventually. Upon transfer to fresh medium a small fraction of cells (about 3%) divide immediately, whereas the rest divide 3 h later after having replicated their macronuclear DNA. The kinetics of entry into the S phase indicates that these cells have a lag period of about 2 h before they resume progress through the cell cycle. It takes more than 1 h until all cells have begun replication. These data show that in stationary cultures all cells proceed through the events of the cell cycle. The cell cycle phases are extended differentially, G1 taking the largest part. During G2 cells pass very slowly through a certain stage close to division. Under the present conditions there is no indication for cells being in a resting state that is not part of the cell cycle, from which they can be restimulated and which has been called the G0 state. The criteria to demonstrate a resting state of this nature are discussed.     

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.     

Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata

The structure of macronuclear DNA of a hypotrichous ciliate, Stylonychia pustulata, was examined by both electron microscopy and nucleotide sequence analysis. The DNA in the macronucleus consists of small linear molecules with average length of about 3400 base pairs (bp). Most, if not all, of these DNA molecules have identical inverted terminal repeat sequence of 20 nucleotide residues. This sequence is 5'-CCCCAAAACCCC-AAAACCCC.     

Phospholipase D in Tetrahymena: activity and significance

We detected phospholipase D in three species of ciliates: Tetrahymena: T. thermophila, T. pyriformis and T. setosa in nutrient medium supplemented with ethanol in in vivo systems, by the appearance of phosphatidylethanol. The calcium ionophore A23187 increased the synthesis of phosphatidylethanol, as compared with untreated controls. We suggest that Tetrahymena possess a calcium sensitive phospholipase D. Propranolol caused the cells in dense cultures to increase their average generation times or die, dependent on the drug concentration. This inhibition could be overcome by the addition of phospholipids or ethanol. Pure phosphatidylethanol had no effect on growth rates or generation times in cultures at high cell density, but postponed cell death in cultures at low cell density by a factor of 10. We suggest that an important role of phospholipase D in Tetrahymena is to supply the cell with diacylglycerol without which it can not enter the mode of proliferation from the lag phase of the culture.     

A gene function required for cell cycle progression during the G1 portion of the cell cycle and for maintenance of macronuclear DNA synthesis in Paramecium tetraurelia

The ccl mutation in Paramecium tetraurelia reversibly and rapidly blocks cell cycle progression and DNA synthesis at the restrictive temperature. Progression through the cell cycle is blocked during both the G1 and S portions of the cell cycle, while at the restrictive temperature there is neither residual cell cycle progression nor induction of excess delay of subsequent cell cycle events. DNA synthesis activity is reduced to 50% of the normal level in about 5 min and is completely blocked at 30 min after a shift to restrictive temperature. On return to permissive conditions, DNA synthesis is reactivated with similar kinetics.     

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.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

A high degree of macronuclear chromosome polymorphism is generated by variable DNA rearrangements in Paramecium primaurelia during macronuclear differentiation.

DNA rearrangements in Paramecium lead to the formation of macronuclear chromosomes, the sizes of which range from 50 and 800 kb (1 kb is 10(3) base-pairs). This process does not appear to be a simple size reduction of the micronuclear chromosomes by specific and reproducible DNA sequence elimination and chromosomal breakage followed by chromosomal amplification. On the contrary, this process generates a variety of different, but sequence-related, macronuclear chromosomes from a unique set of micronuclear chromosomes. This paper describes an attempt to understand the nature of the diversity of the macronuclear chromosomes and the mechanisms of their production. The structure of three macronuclear chromosomes, 480, 250 and 230 kb in size, have been determined utilizing chromosome-jumping and YAC-cloning techniques. The two smallest chromosomes correspond roughly to the two halves of the longest chromosome. The main contribution to the diversity arises from the chromosomal ends and is due to variable positions of the telomere addition sites and/or to variable rearrangements of DNA sequences. The 480 kb chromosome contains a region of variable length, which is likely to be due to a variable deletion, located at the position of telomerization seen in the two small chromosomes. A model of chromosomal breakage is proposed to rationalize this result where micronuclear DNA is first amplified, broken and degraded to various extent from the newly formed ends, which subsequently are either telomerized or religated. Potential implications of these processes for gene expression is discussed. Known phenotypes that have a macronuclear determinism could be explained by this type of process.     

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.     

Temperature dependent changes of cell growth parameters in Tetrahymena

Cytophotometric studies revealed gross amounts of RNA and protein to be negatively correlated with incubation temperature. The rate of [³H]-uridine incorporation into TCA-insoluble material of synchronously growing cells followed a cyclic pattern during the cell cycle and was correlated to the event of the S phase.     

Divalent cations and inorganic phosphate metabolism in starved Tetrahymena

Tetrahymena pyriformis maintained under starvation conditions release orthophosphate into the suspension medium. Ca²⁺ and/or Mg²⁺ addition reduced the amount of orthophosphate excreted during a 3-h period. Cellular orthophosphate levels were not altered by divalent cation supplements; however, an increase in the pyrophosphate content was observed which was equivalent in amount to the reduction in orthophosphate efflux. These observations suggest that divalent cations are important not only in the acquisition of phosphorus during growth but also in the conservation of this element during starvation.     

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.     

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.     

Demonstration of a very short post-mitotic G1 period in proliferating PHA-stimulated lymphocytes

Evidence indicating a very short duration of the post-mitotic G1 phase of PHA-stimulated lymphocytes was obtained in experiments where proliferating lymphocytes were pulsed for 1 h with ³H-TdR and then, following different periods of incubation in ‘cold’ medium, again pulsed for 1 h with ¹⁴C-TdR. Cells labelled with ³H-and (³H + ¹⁴C)-TdR were counted in double layer autoradiograms. Cells labelled with ³H-TdR should gradually leave the S phase when incubated in ‘cold’ medium and would consequently not incorporate ¹⁴C-TdR. Thus, the quotient

$\text{\%(}{}^3\text{H}\text{+}{}^{14}\text{C}\text{)}\text{cells}\text{\%}{}^3\text{H}\text{+ \%(}{}^3\text{H}\text{+}{}^{14}\text{C}\text{)}\text{cells}$

should decrease with time in ‘cold’ medium and should be zero after an incubation time corresponding to the length of the S phase. If, however, the sum of the G2 + M + G1 phases is shorter than the length of the S phase, the zero value will not be obtained. This was actually the case, and the quotients obtained agreed well with those that could be calculated on basis of the G1 period being 0 h, whereas they did not agree with those calculated on basis of G1 being 4–6 h.