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.
Tetrahymena pyriformis ingested Escherichia coli for 15–20 min and the fine structure of food vacuoles was analyzed 5, 15, 30, 60, 90, 120, and 180 min after uptake began. From this analysis, eight vacuolar stages could be defined, and three to four stages were found in each sample. Stage 1 represents forming and newly detached vacuoles with a random distribution of bacteria. Stage 2 is the “dehydration” vacuole in which the bacteria are compacted and a few may lyse. Stage 3, corresponding to the acid phosphatase‐positive stage, has an electron‐dense vacuolar matrix revealing components of lysed bacteria and the translucent coat of intact bacteria. Stage 4 is the “halo” stage where centrally located, intact bacteria are surrounded by lysed material being removed by pinocytic activity of the vacuolar membrane. Stage 5 represents lysis of bacteria remaining intact until this stage; the stage is apparently followed by a second stage 4. Stage 6 contains few bacterial profiles in a smeared homogeneous mass. Stage 7 contains numerous vesicular membranous structures which apparently become transferred to the cytoplasm as such. Stage 8 represents defecation vacuoles derived from fusion of smaller vacuoles. The main findings are as follows: I) Bacterial lysis may occur during acidification of the vacuole prior to fusion with lysosomes. II) Digestion of bacteria apparently occurs in “bursts” as indicated by the extended time that vacuoles in stages 4 and 5 are present. III) Bacterial membranous structures seem to be transferred directly to the cytoplasm of Tetrahymena. IV) Mass defecation occurs 2 h after uptake begins.
Selective induction of replication of nucleolar and mitochondrial DNA has been demonstrated in starved-refed cultures of Tetrahymena pyriformis by different techniques.Labeling of starved cells with [3Hithymidine during a nutritional shift-up and analysis of the DNA in isopycnic CsCJ gradients shows that the two initially labeled species of DNA are two species banding on the heavy and light side of the bulk macronuclear DNA. In isolated macronuclei the radioactivity is found only in the high density fraction, which has been shown to be of nucleolar origin. In sucrose gradients the newly replicated mitochondrial and nucleolar DNAs sediment considerably slower than the bulk DNA, as one discrete band corresponding to a molecular weight of about 3 to 4 X 107.Electron microscope autoradiography of cells labeledwith [3Hjthymidine as above shows that the peripheral nucleoli of the macronucleus as well as the mitochondria are labeled before any radioactivity is found in the chromatin granules of the macronucleus. The results clearly indicate that nucleolar and mitochondrial DNA replication are under a control independent of that for the replication of bulk DNA.In Tetrahymena pyriformis the 500-1000 nucleoli are located peripherally in the nucleus (1) and form ribosomes at a rate per nucleolus which is equal to that of HeLa cell nucleoli (2). During starvation the nucleoli fuse into aggregates that again dissociate into multiple peripheral nucleoli during a nutritional shift-up which concomitantly induces rapid acceleration of rRNA synthesis (1).Since morphological studies show that a rearrangement of the nucleolar material occurs during a nutritional shift-up (1), we undertook this study since we felt that nucleolar DNA might show unusual replicative properties during the dissociation of nucleolar aggregates.We have previously studied the replication of the rRNA genes during the nutritional shift-up and have presented evidence from DNA * RNA hybridization experiments (3) suggesting that early during the refeeding period the rRNA genes are replicated preferentially. In the study reported here, the same starvation-refeeding system has been employed and it is shown by EM-autoradiography and labeling with [3H]thymidine that the nucleolar DNA as well as mitochondrial DNA are preferentially replicated during the early times of refeeding. The two species of DNA band in isopycnic CsCl density gradients as two discrete radioactivity peaks on the heavy and light side, respectively, of the bulk DNA. In sucrose density gradients the newly replicated nucleolar and mitochondrial DNAs sediment as one discrete peak with a molecular weight of about 3 to 4 X 107.
MATERIALS AND METHODSCultures of Tetrahymena pyriformis, strain GL, were grown in a complex proteose peptone medium as described earlier (4). Starvation for 24 hr was carried out as described by Cameron and Jeter (5)
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