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.     

Phospholipase D Activity in the Tetrahymena pyriformis GL

Phospholipase D (PLD) is an enzyme which participates in the signalling mechanism cleaving phosphatidylcholine (PC) to choline and phosphatidic acid (PA). In Tetrahymena pyriformis GL this enzyme activity is enhanced by different kinds of agonists (sodium orthovanadate, sodium fluoride and phorbol 12-myristate 13-acetate), and its activity can be inhibited by inhibitors such as pertussis toxin, calphostin C, genistein, trifluoperazine. These results suggest that the PLD signalling pathway is connected with the tyrosine kinase, phospholipase C, phosphatidylinositol and G-protein coupled signalling pathways. By demonstrating the PLD activity in Tetrahymena our knowledge on the signalling mechanisms at a unicellular level has been extended. The results support our view that most transducing mechanisms that are characteristic of mammalian cells are also in the protozoan Tetrahymena. © 1997 John Wiley & Sons, Ltd.     

Endocytosis and the adaptive acid hydrolases in Tetrahymena pyriformis GL

Endocytosis of yeast cells by Tetrahymena pyriformis GL for a period of 2.5 h produced changes in cellular acid hydrolases. Acid phosphatase, acid deoxyribonuclease and acid proteinase activities were markedly increased, whereas there was a decrease in acid ribonuclease activity and little change in -glucosidase activity. These alterations do not appear to be due to any alteration in the rates of secretion of these enzymes into the milieu. Evidence is presented that the cellular enzyme increases found upon endocytosis of yeast reflect changes in lysosomal enzymes, because it was shown that the acid phosphatase activity increase resulted in an increased amount of latent enzyme within the cell. The results also support the idea that there are at least 3 distinct populations of lysosomes, in addition to phagolysosomes, present in Tetrahymena pyriformis GL, with different modes of formation. There appears to be a large excess of lysosomes, uncombined with phagosomes, present in these fed cells since latency averaged 66% in broken-cell preparations which contained very few intact phagolysosomes. The phagolysosomal acid phophatase activity cannot account for more than 34% of that present in the cell. The endocytosis of yeast in the presence of growth medium resulted in a marked drop in the rate of cell division as compared to cells growing in the growth medium alone. The results are discussed.     

Fluorimetric Analysis of Phospholipase Activity in Tetrahymena pyriformis GL.

The unicellular Tetrahymena enzymatically split the synthetic phosphodiester, 4-methylum-belliferyl phosphocoline substrate. The enzyme activity was completely blocked in vitro and drastically inhibited in vivo by G-protein activating fluorides (NaF; AlF₄ ^– and BeF₃ ^–). The phospholipase A₂ inhibitor, quinacrine, and the protein phosphatase inhibitor, neomycin, inhibited the enzyme activity in vitro and activated it in vivo. Another phospholipase A₂ inhibitor 4-bromo phenacyl bromide was ineffective in vivo and in vitro alike, as well as the cyclooxygenase inhibitor indomethacin. Results of these experiments indicate that some treatments could be specific for a well defined activity (e.g., phospholipase A₂, G-protein) but subject to influence by other enzymes (e.g., phospholipase C, sphingomyelinase). The experiments call attention to the differences in the results of the in vivo and in vitro studies.     

The macronuclear envelope of Tetrahymena pyriformis GL in different physiological states

Summary Macronuclear envelopes were isolated from the ciliated protozoan Tetrahymena pyriformis GL, negatively stained and examined in the electron microscope. The frequency of central granules in the macronuclear pores was evaluated in five different physiological states: (1) stationary phase of growth, (2) exponential phase of growth, (3) heat-synchronized cultures at the end of the heat-synchronization treatment, (4) heat-synchronized cultures at the beginning of the first division, (5) heat-synchronized cultures at the end of the first division.The percentage of pores containing a central granule was markedly enhanced in heatsynchronized cultures at the end of the first division, i.e. a state known for an increase in ribosome formation. Actinomycin D was found to cause a significant decrease in central granule frequency.The observed alterations in central granule frequency seem to confirm the hypotheses which consider the central granule as representing a ribonucleoprotein particle in transit from nucleus to cytoplasm through the nuclear pore.For careful technical assistance I am indebted to Miss Marianne Whiter as well as to Drs. H. Falk, W.W. Franke and P. Sitte for helpful discussions. This work was supported in part by the Deutsche Forschungsgemeinschaft.     

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.     

Suggestive Structural Evidence for Macronuclear "Subnuclei" in Tetrahymena pyriformis GL

SYNOPSIS Structural changes in the Feulgen-positive material of the Tetrahymena pyriformis GL macronucleus have been observed during the cell cycle. From the finely granulated appearance in the interphase cell it appears as small rods, often arranged in pairs (probably the endomitotic stage) during early morphogenesis and as larger (and fewer) aggregates of granules during the nuclear division. These latter aggregates are also visible in dividing nuclei in the electron microscope where groups of chromation granules are separated by fairly empty nucleoplasm. It is suggested that these Feulgen positive aggregates in dividing nuclei are macronuciear segregation units or "subnuclei." The number per dividing macronucleus may vary from one experiment to another, but the variation seems to be related to cell volume. The distribution of the aggregates among the daughter nuclei is almost equal. The total number per dividing macronucleus is about 80 which is close to the estimated number of "subnuclei" in the T. pyriformis macronucleus (Allen and Nanney, 1958).
Some calculations are made on the polyploidy of the T. pyriformis GL macronucleus. Using published electron micrographs of micronuclei of known age to calculate the total number of chromatin granules per haploid nucleus, the polyploidy of the strain GL macronucleus is about 40. This figure is half of that expected from Allen and Nanney's estimation, since they assumed that the "subnuclei" were diploid; however, it is in agreement with the reported haploid nature of the "subnuclei" as found by Woodard, Gorovsky & Kaneshiro, 1968. Further calculations suggest that each macronuclear "chromosome" is composed of about 40 chromatin granules; an indication of such a chain arrangement of the chromatin granules has been observed in the phase contrast and electron microscope during the earliest macronuclear events, i.e., at the macronuclear "prophase."     

Free ribosomal DNA molecules from Tetrahymena pyriformis GL are giant palindromes

Restriction ondonuclease EcoRI was used to study the structure of the free ribosomal DNA molecules from Tetrahymena pyriformis, strain GL. From the following observations we conclude that the free rDNA molecules from Tetrahymena are giant palindromes³, each containing two genes for preribosomal RNA arranged in rotational symmetry as inverted repeating sequences. Analyses of the sizes of products of partial or complete digestion and quantitative analyses of the products of complete digestion of uniformly ³²P-labeled rDNA yielded an RI endonucleolytic cleavage map which showed that the EcoRI recognition sites are arranged symmetrically about the center of the rDNA molecule.When heat-denatured rDNA was rapidly cooled under conditions in which no renaturation would occur between separated complementary strands of DNA, molecules of half the size of the original rDNA molecule were produced. These were double-stranded DNA molecules as evidenced by their resistance to digestion with S1 nuclease. Moreover, they could be digested with EcoRI to produce fragments of sizes which would be predicted from the assumption that each single strand of the original rDNA molecule had folded back on itself to form a “hair-pin” double-stranded DNA structure. Hybridization experiments between ribosomal RNA and purified rDNA showed that each rDNA molecule contains two genes for rDNA. Hybridization of the isolated EcoRI fragments of rDNA with 25 S or 17 S rRNA suggested that the two structural genes for 17 S rRNA are located near the center of the rDNA molecule and the two genes for 25 S rRNA are found in distal positions.     

Extrusion of DNA from the macronucleus of Tetrahymena pyriformis GL.

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.